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Vol 18, No 4 (2023)

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Reviews

Sphingomyelinases as modulators of synaptic transmission

Gafurova C.R., Petrov A.M.

Abstract

The brain has a high content of sphingomyelin, which is involved in the formation of plasma membranes and myelin, and is also an important for the organization of membrane microdomains (lipid rafts). Lipid rafts, as well as derivatives of sphingomyelin hydrolysis (ceramide, sphingosine, sphingosine-1-phosphate), are vital for synaptic transmission and its regulation. One of the main pathways to control the level of sphingomyelin and its derivatives is cleavage of membrane sphingomyelin by sphingomyelinases.

Sphingomyelinases are localized inside the cell (in association with the plasma membrane, in lysosomes, endosomes, Golgi complex and endoplasmic reticulum) as well as can be secreted into the extracellular space. The levels and activity of sphingomyelinases significantly increase under the action of various stressful stimuli (including inflammation). At the same time, sphingomyelinase activity deficiency causes diseases with severe neurological manifestations.

In the present review, we summarized the data on the currently known effects of acidic and neutral sphingomyelinases on pre- and postsynaptic processes, as well as about the synaptic localization of sphingomyelinases. In addition, a brief analysis of possible synaptic dysfunction due to hypo- or hyperfunction of sphingomyelinases in a number of neurological diseases is given. Thus, sphingomyelinases are considered as important modulators of synaptic transmission at the pre- and postsynaptic levels in normal and pathological conditions.

Genes & Cells. 2023;18(4):255-267
pages 255-267 views

Contribution of 25-hydroxycholesterol to the cross-interaction of the immune and nervous systems

Zakyrjanova G.F., Tsentsevitsky A.N., Giniatullin A.R., Nghomsi S., Kuznetsova E.A., Petrov A.M.

Abstract

25-hydroxycholesterol (25HC) is produced from cholesterol by cholesterol-25-hydroxylase, and its expression, similar to the 25HC level, increases significantly in macrophages, dendritic cells, and microglia during an inflammatory reaction. In turn, 25HC acts on many immune cells; therefore, it can modulate the course of the inflammatory reaction and prevent the penetration of viruses into cells. Data are accumulating about the involvement of 25HC in the regulation of synaptic transmission in both the central and peripheral nervous systems. 25HC production is increased not only during inflammation but in certain neurodegenerative diseases, such as Alzheimer’s disease and amyotrophic lateral sclerosis; thus, this hydroxycholesterol can be important in the adaptation of synaptic activity to inflammatory conditions, pathogenesis of neurodegenerative diseases, and formation of synaptic dysfunctions. The targets of 25HC in the nervous system are glutamate NMDA receptors, liver X-receptors, and estrogen receptors. 25HC can also directly influence the properties of synaptic membranes by changing the formation of membrane microdomains (lipid rafts) where proteins, which are important for synaptic plasticity, are clustered. Current data indicate that the effects of 25HC strongly depend on its concentration and “context” (norm, pathology, and presence of an inflammatory reaction) in which the effect of 25HC is being investigated. This minireview focused on the key aspects of the action of 25HC as both a local regulator of cholesterol homeostasis and a paracrine molecule that realizes the influence of inflammation on neurotransmission processes in the central and peripheral nervous systems.

Genes & Cells. 2023;18(4):269-280
pages 269-280 views

Animal models of epilepsy

Mitina N.N., Kondakova E.V., Tarabykin V.S., Babaev A.A.

Abstract

Animal models of epilepsy are valuable tools for studying the pathogenesis of the disease, developing new methods of treatment, searching for anticonvulsants and evaluating their effectiveness. Rodents, such as rats and mice, are the most popular subjects for research due to the similarity of the human and rodent brain structure. Recent studies include other model species such as dogs, cats, primates, as well as non-mammals such as zebrafish, fruit flies, leeches and planarians.

This review discusses the use of animal models in research and analyzes their advantages and limitations. The classification of models is based on the phenotype of the disorder, with special attention paid to drug-resistant epilepsy. The review also highlights the imperfection of existing models and the need to select the most relevant for specific research purposes. It is also important to remember that animal models cannot fully recreate the complexity of the clinical picture of epilepsy in humans, but they play an important role in understanding the mechanisms of the disease and developing new therapeutic approaches.

In conclusion, the review highlights the need for continuous improvement of existing animal models and the development of new ones to more accurately reflect the diversity of epilepsy phenotypes and provide more effective research and treatment methods. The need for new models of drug-resistant epilepsy, which could help in the development of fundamentally new antiepileptic drugs, remains particularly relevant.

Genes & Cells. 2023;18(4):281-296
pages 281-296 views

Neurophysiological markers that link genes and behavior in humans: examples from rare genetic syndromes associated with autism spectrum disorders

Sysoeva O.V.

Abstract

Rare genetic syndromes associated with autism spectrum disorders have several noninvasive neurophysiological markers that can be linked with molecular genetic characteristics and behavioral characteristics in these diseases. For the recently discovered Potocki–Lupski syndrome associated with disturbances on the 17p11.2 segment, a previously undescribed epileptiform activity was detected, characterized by a saw-like hypersynchronization at a frequency of 13 Hz, which may indicate a certain type of disturbance in the excitation/inhibition balance in neural networks. For a rare case of microduplication in SH3 and ankyrin repeat domains 3 (SHANK3), also associated with the Phelan–McDermid syndrome, we described a pathway from a violation in the functioning of the SHANK3 protein, through a distorted interaction of excitatory and inhibitory neurons, primarily associated with hypofunction of N-methyl-D-aspartate receptors on inhibitory neurons, to reduced temporal resolution in the auditory cortex, reflected in the absence of response following 40 Hz auditory stimulation (40 Hz auditory steady-state response) and underlying problems in speech development. For the Rett syndrome, which is caused by a mutation in methyl CpG binding protein 2 (MECP2), which has a very wide influence on many other genes, the neurophysiological findings were also diverse. Among the most promising are changes in sensorimotor rhythm, potentially associated with a key symptom of the disease, namely, stereotyped hand movements, as well as more delayed latency of the main components of the event-related potentials, which can have a cascading effect on information processing and affect the perception of basic information, including speech.

This review focuses on the presentation of the concept of a neurophysiological profile, the construction of which can help not only to objectify the diagnosis of developmental disorders, but also in the construction of a mechanistic chain from gene to behavior.

Genes & Cells. 2023;18(4):297-307
pages 297-307 views

Glymphatic dysfunction in the pathogenesis of neurodegenerative diseases and pathological aging

Shirolapov I.V., Zakharov A.V., Bulgakova S.V., Khivintseva E.V., Sergeeva M.S., Romanchuk N.P., Pavlova O.N., Kazantsev V.B.

Abstract

Recently, the concept of the glymphatic system as a highly organized perivascular network has been formed, which by hydrodynamic approach, with the key participation of aquaporin-4 as a central molecule, connects the cerebrospinal fluid with the lymphatic vessels of the meninges through the brain interstitium. The latest scientific works demonstrates the potential role of glymphatic dysfunction in the development of neurodegeneration and pathological aging. Although the precise molecular mechanisms of glymphatic pathway function have not yet been fully characterized, the critical processes underlying cerebral solute transport and clearance of amyloid and metabolites have been largely elucidated. The complex interaction between a number of age-associated factors, including cellular aging, disturbances in the sleep-wake cycle with changes in sleep architecture and quality, low-grade systemic inflammation, and the development of concomitant diseases, determines not only life expectancy in general, but also forms the basis of healthy and unhealthy aging the brain in particular. Imbalances in homeostatic functions, changes in the activity of glymphatic clearance and the blood-brain barrier that support the exchange of fluid and solutes in cerebral tissue, which can be observed both normally with physiological aging and with the development of neuropathology, have longitudinal consequences ranging from disruption of synaptic signal transmission to onset of neurodegenerative processes.

This review analyzes the current scientific information in this area of research, details the features of the perivascular glial-mediated transport system, and discusses how its dysfunction plays a fundamental role in the pathological accumulation of metabolites during aging, the development of age-associated changes in the brain, and the progression of neurodegenerative diseases.

Genes & Cells. 2023;18(4):309-322
pages 309-322 views

Original Study Articles

Eview: an open source software for converting and visualizing of multichannel electrophysiological signals

Zakharov A.V., Zakharova Y.P.

Abstract

BACKGROUND: Methods and tools for operating with multichannel electrophysiological signals need to develop and correspond to the speed of data traffic in contemporary experiments. Analyzing and visualizing experimental data with minimal delay and with minimized experimenter effort is a pressing task in the field of neurobiology and requires the use of complex approaches specifically selected for each specific type of experiment. Creating open-source programs that can be promptly adapted for different tasks is one of the approaches that provide the ability to perform complex scientific experiments with high quality.

AIM: This work is aimed at creating open-source software for analytical and visualization support of neurobiological experiments.

METHODS: Software development was performed in MATLAB environment. The program is built on a modular principle and includes an intuitive graphical interface that facilitates control of the signal processing and display.

RESULTS: A software tool was created that allows to optimize and accelerate various stages of electrophysiological research, including preliminary analysis of the quality of the experiment being prepared, in-depth analysis of recorded signals, and preparation of illustrative material for publications.

CONCLUSION: The resulting program has a number of advantages in comparison with similar products in terms of versatility, speed, and availability, and can be used to solve a wide class of research problems.

Genes & Cells. 2023;18(4):323-330
pages 323-330 views

Exploring structural brain changes in children with neonatal brachial plexus palsy: a voxel-based morphometry analysis

Kadieva D.V., Gallo F., Ulanov M.A., Shestakova A.N., Moiseeva V.V., Hodorovskaya A.M., Agronovich O.E.

Abstract

BACKGROUND: Obstetric brachial plexus palsy (OBPP) is paralysis of the upper limb resulting from nerve injury during vaginal delivery. Although current treatment approaches frequently lead to complete reinnervation of the limb, some patients show long-term motor deficits. These impairments may result from upper limb disuse that causes structural brain changes.

AIM: This study aimed to compare deep gray matter volumes between children with OBPP and healthy controls.

METHODS: We analyzed the structural magnetic resonance imaging results of 46 children with OBPP (n=24, mean age — 10.20, of whom 12 were girls) and healthy age-matched controls (n=22, mean age — 9.63, of whom 10 were girls) using a voxel-based morphometry technique in SPM12 package (Statistical Parametric Mapping) in MATLAB R2019b. To minimize false discoveries, we used a stringent procedure to control the family-wise error rate.

RESULTS: We found volumetric brain differences between children with OBPP and healthy controls (all FWE-corrected p <0.005). Children with OBPP had significantly lower gray matter volumes in the left amygdala, bilateral hippocampus, and right entorhinal cortex.

CONCLUSION: Integrating our findings with previous work, we speculate that the amygdala–hippocampus–entorhinal cortex complex might play a significant role in motor disorders.

Genes & Cells. 2023;18(4):331-339
pages 331-339 views

NeuroD2/6 regulate expression balance of transcription factors controlling neurocortical cytoarchitecture

Kondakova E.V., Gavrish M.S., Tarabykin V.S., Yan K.

Abstract

BACKGROUND: Genes of the NeuroD family, including NeuroD1, NeuroD2, and NeuroD6, control neuronal survival, differentiation, maturation, and neurite specification in the nervous system. Deletion of NeuroD1 in the mouse brain results in complete loss of dentate gyrus because of neuronal apoptosis. NeuroD2 is required for neuron survival in the cerebellum and integration of thalamo-cortical connections into neocortex and formation of somatosensory whisker barrel cortex. In NeuroD2/6 double deficient (DKO) mice, callosal axon projections are defective due to abnormal EfnA4 signaling. In order to investigate the NeuroD2/6 controlled molecular cascade, we explored the expression of key transcription factors that control various aspects of cortical development in brains of NeuroD2 and NeuroD6 deficient mutants.

AIM: To investigate possible changes in differentiation programs downstream of NeuroD2/6 transcription factors.

METHODS: Embryos with NeuroD2/6 double deficiency were used in the experiments, and pregnant mice carrying E13.5 embryos were operated for in utero electroporation. We performed in situ hybridization at various stages of embryonic development to study the expression pattern of target genes. Analyzing the activity of a gene promoter, genomic DNA fragments containing NeuroD2/6 motifs were cloned into pMCS-Gaussia Luc vector for luciferase assays. Charts were made with GraphPad Prism software and data were presented as mean ± standard error.

RESULTS: Our findings showed that NeuroD1 expression is ectopically upregulated in postmitotic neurons of NeuroD2/6 DKO neocortex and hippocampus. We detected changes in expression of key transcription factors, Cux1, Tbr1, Lhx2, and Id2. Additionally, Cux1 was shown to be direct target of NeuroD2/6. Moreover, Olig2+ progenitors were increased in NeuroD2/6 DKO neocortex and expression of NeuroD2/6 and Olig2 was mutually exclusive. Thus, NeuroD2/6 regulates the expression of transcription factors in the developing brain.

CONCLUSION: Our findings indicate that cumulative action of NeuroD2 and NeuroD6 is required to initiate and maintain the expression of transcription factors Cux1, Tbr1, Lhx2, and Id2. Additionally, both genes are required to prevent premature differentiation of Olig2 positive glial precursors.

Genes & Cells. 2023;18(4):341-352
pages 341-352 views

Behavioral phenotype of C57Bl/6 mice that endured bullying during infant age period

Kuzmina D.M., Eremeeva N.A., Schelchkova N.A., Mukhina I.V.

Abstract

BACKGROUND: According to UNESCO data for 2018, every third student is involved in bullying. To study the impact of social conflicts on the state of the nervous system the K. Michek model of chronic social stress was used, but the study of the effects of chronic social stress on prepubertal animals has not been conducted.

AIM: To analyze the effect of chronic stress in infant age period to the behavioral phenotype of C57Bl/6 mice in early and long-term periods.

MATERIALS AND METHODS: The objects of the study were male C57Bl/6 mice (n=48). For bullying modeling we chose chronic social stress in infant age period from 20 to 29 postnatal day (P20–P29) according to the “resident–intruder” scheme. Mice were divided into two subgroups to study the early (P31–P35, infant age period) and long-term (P57–P74, adulthood) consequences of chronic social stress. For the behavioral phenotyping we used the following tests: “open field” test, three-chamber social test, object recognition test, passive avoidance task and Barnes maze.

RESULTS: Bullying modeling led to the changes in the behavioral phenotype both in infant age and in adulthood. The behavioral phenotype in infant age period was characterized by increased social activity and recognition, high anxiety, decreased locomotor and exploratory activity, impaired recognition of inanimate objects, but good characteristics of learning, working and long-term memory. In adulthood, the behavioral phenotype of mice retained high anxiety, low level of exploratory activity, good learning and memory characteristics, decline in social recognition in three-chamber test, while the recognition of inanimate objects was preserved at the same level.

CONCLUSION: Chronic social stress in infant age in a mouse model of bullying causes disruption of the behavioral phenotype in infant and adult age. Features of the behavioral phenotype of mice after bullying were an increase in anxiety and social isolation against the background of the ability to learn and good memory.

Genes & Cells. 2023;18(4):353-367
pages 353-367 views

Morphological features of microglial cells in a 5xFAD mouse model of Alzheimer's disease

Okhalnikov A.D., Gavrish M.S., Babaev A.A.

Abstract

BACKGROUND: Aging is an inevitable and irreversible process associated with increased risk of developing various neurodegenerative diseases, one of which is Alzheimer's disease. Currently, the role of glial cells, in particular microglia, in the pathogenesis of Alzheimer's disease is being actively studied. However, only a few studies have correlated the morphological features of microglia and their spatial arrangement in relation to β-amyloid plaques.

AIM: Describe the main morphological parameters of microglia in the 5xFAD mouse model of Alzheimer's disease at a late stage of pathology development.

METHODS: As the studied object, mice were chosen by the age of 15–16 months of the 5xFAD line, as a model of acceleid amyloidosis. The immunohistochemical staining of the study of the morphological diversity of microglia was carried out on the cuts of the cortex of the mouse brain. The obtained confocal images performed an immunogystological analysis of the cuts of the cerebral cortex when analyzed using the Imagej application using the plugins of Skeleton, AnalyzeSkeleton (2D/3D) and FracLac.

RESULTS: During the study, 5xFAD mice were divided into two groups (n=3 each). Carriers of the app and psen1 transgenes were assigned to the “FAD” group, and wild-type mice were assigned to the “Wt” group (control). We analyzed 3–4 sagittal sections (50 µm) of the brain from each mouse. The results showed that microglial cells from mice with signs of Alzheimer's disease have smaller fractal dimension, lacunarity and branching.

CONCLUSION: The presence of β-amyloid plaques contributes to the migration of microglia to the focus of inflammation, its proliferation and transition to the phagocytic and dystrophic subtype. According to fractal analysis, there is a significant (p ≤0.05) decrease in the average branching of microglial processes, a decrease in fractal dimension and lacunarity.

Genes & Cells. 2023;18(4):369-379
pages 369-379 views

Transspinal direct current stimulation with an intensity of 2.5 mA does not affect the corticospinal system excitability and motor skills

Pomelova E.D., Popyvanova A.V., Bredikhin D.O., Koriakina M.M., Shestakova A.N., Blagovechtchenski E.D.

Abstract

BACKGROUND: Noninvasive brain stimulation effectively affects movements, including the spinal cord level. Stimulation effects are very sensitive to montage and protocols of applied stimulation because they can involve different neuronal mechanisms.

AIM: This study aimed to estimate the effect of anodal transspinal direct current stimulation (tsDCS) with an intensity of 2.5 mA applied at the spinal cord level (C7–Th1 segments) with cervical enlargement on the corticospinal system excitability and motor skills.

METHODS: The study involved 54 healthy adults aged 21.19±3.20 years. The effect of tsDCS was assessed using motorevoked potentials from the first dorsal interosseous (FDI) muscle by transcranial magnetic stimulation in the primary motor cortex before stimulation, immediately after stimulation, and after 15 min.

RESULTS: The application of an 11-min anodal tsDCS with a current value of 2.5 mA at the C7–Th1 level did not affect the motorevoked potentials of FDI. Statistically, changes in motorevoked potentials amplitudes did not differ between groups receiving anodal tsDCS and sham stimulation. In addition, anodal tsDCS did not affect motor skills. An individual’s ability to coordinate fingers and manipulate objects effectively (a measure of dexterity) in the nine-hole peg test and pressing a key in response to a visual stimulus in the serial reaction time task did not differ from that with sham stimulation.

CONCLUSION: 2.5 mA anodal tsDCS on cervical enlargement does not affect the corticospinal system excitability or change motor skills associated with precise hand movements.

Genes & Cells. 2023;18(4):381-388
pages 381-388 views

The intensity of transspinal direct current stimulation affects the excitability of the corticospinal system

Popyvanova A.V., Pomelova E.D., Bredikhin D.O., Koriakina M.M., Shestakova A.N., Blagovechtchenski E.D.

Abstract

BACKGROUND: Transspinal direct current stimulation (tsDCS) affects the corticospinal system, one of the central human systems associated with controlling precise voluntary movements. Stimulation effects are very sensitive to montage and protocols of applied stimulation because they can involve different neuronal mechanisms.

AIM: This study aimed to estimate the effects of parameters of anodal tsDCS applied at the level of the spinal cord (C7–Th1 segments) with cervical enlargement to determine the excitability of the corticospinal system and the correction of motor skills in healthy people.

METHODS: The study involved 81 healthy adults aged 21.19±3.20 years. The effect of tsDCS was assessed using motor-evoked potentials from the first dorsal interosseous (FDI) muscle by transcranial magnetic stimulation in the primary motor cortex before stimulation, immediately after stimulation, and after 15 min.

RESULTS: The application of 11-min anodal tsDCS at the C7–Th1 level with a current of 1.5 mA affects the FDI muscle, initially reducing the amplitude of transcranial magnetic stimulation induced motor-evoked potentials immediately after stimulation. The amplitude of the motor-evoked potentials increases after 15 min of stimulation. tsDCS with an intensity of 2.5 mA does not affect the change in the amplitude of motor-evoked potentials. Similarly, no difference was found in the effect of 1.5 mA stimulation on the correction of motor skills in healthy adults at the nine-hole peg test and the serial reaction time task as with 2.5 mA.

CONCLUSION: This study adds information about the optimally appropriate current intensities of stimulation to induce corticospinal system excitability and the ability of tsDCS to influence motor skills in healthy adults.

Genes & Cells. 2023;18(4):389-396
pages 389-396 views

Specificity of frequency-spatial organization of brain activity in coronary heart disease associated with self-assessment of emotion control in men and women

Razumnikova O.M., Tarasova I.V., Trubnikova O.A.

Abstract

BACKGROUND: Deficit in the regulation of emotional stress is considered as an important factor in the development of coronary heart disease (CHD). The functions of assessment and regulation of emotions are performed by the structures of the prefrontal cortex and amygdala, the activation and interaction of which differs in men and women. In this regard, the question of the gender specificity of the cortical mechanisms of emotional regulation associated with coronary artery disease is relevant.

AIM: To find out the significance of self-assessment of emotional control of behavior (EC) in the frequency-spatial organization of brain activity in men and women with CHD.

METHODS: The study was performed in a cardiology clinic involving 56 men (61.2±8.5 years) and 19 women (67.4±4.8 years) diagnosed with CHD. To analyze the frequency-spatial organization of the resting EEG, we used 64-channel EEG recording and calculation of the power of rhythms in six frequency ranges from 4 to 30 Hz using a fast Fourier transform. Spearman's non-parametric correlation analysis was used to determine the correlation of EC as a personality trait according to the questionnaire of emotional intelligence and EEG power indicators.

RESULTS: Correlation analysis of EC and average EEG power indicators revealed positive relationships in the range of 4–13 Hz in the group of men and negative in the group of women (0.19 p <0.030). The regional specificity of the detected effect was characterized by a significant relationship between EC and the power of theta 2, alpha 1, 2, presented in the anterior part of the cortex with the dominance of the left hemisphere in men, but in the posterior part of both hemispheres — in women, and the latter effect was limited by theta 2 and alpha 1 frequency.

CONCLUSION: The results of the performed analysis of the relationship of EC and regional indicators of resting EEG power in the 6–13 Hz range indicate different forms of control of the emotional state in women and men with CHD.

Genes & Cells. 2023;18(4):397-407
pages 397-407 views

Age-related differences in the interhemispheric asymmetry of local cortical responses to abstract and concrete verbs in children: a magnetic mismatch negativity study

Ulanov M.A., Kopytin G.A., Bermudez-Margaretto B., Pomelova E.D., Popyvanova A.V., Blagovechtchenski E.D., Moiseeva V.V., Shestakova A.N., Jääskeläinen I.P., Shtyrov Y.Y.

Abstract

BACKGROUND: The development of brain neural networks that support lexico-semantic processing in children remains a poorly understood topic in neuroscience. Meanwhile, investigations in adults have provided ample evidence regarding the brain circuits underpinning the processing of abstract and concrete semantics. These studies have shown that interhemispheric asymmetry in neural responses across modal and amodal cortical areas might be an important marker that helps in distinguishing these two types of semantics, with more left-lateralized activity patterns for abstract than concrete word comprehension. However, little is known about such distinctions in children; thus, addressing this gap was the goal of this study.

AIM: This study aimed to investigate age-related differences in the lateralization of neural response patterns associated with the processing of abstract and concrete semantics in children.

METHODS: This study employed magnetoencephalography and a mismatch negativity (MMN) paradigm in a group of 41 healthy children aged 5–13 years. The participants were passively exposed to the auditory series of abstract and concrete Russian verbs presented outside the focus of attention. Spatiotemporal patterns of the dynamics of neuromagnetic sources activity were reconstructed using minimum-norm estimate within predefined regions of interest: primary auditory cortex, primary motor cortex, and inferior frontal gyrus of both hemispheres. The magnitudes of MMN responses were further compared statistically between the two hemispheres within two age groups: younger (aged 5–9 years) and older (aged 10–13 years) children.

RESULTS: Regionally specific differences were found in the lateralization of event-related MMN responses to concrete compared with abstract words in motor and inferior frontal cortical areas (paired permutation tests, p <0.05). Moreover, in the younger group (aged 5–9 years), responses to the abstract and pseudoword stimulus were left-lateralized, and this effect was most pronounced in the inferior frontal regions (45 and 47 Brodmann fields) of the left hemisphere. In the older group (aged 10–13 years), no pronounced left-lateralized response was observed in these areas. However, for the concrete hand action verb stimulus, different patterns of the interhemispheric asymmetry of the hand motor area responses were observed: the response in the younger group was right-lateralized, whereas in the older group, the response was bilateral.

CONCLUSION: The present area- and hemisphere-specific dynamics of neuromagnetic responses in the motor cortex and Broca’s area might correlate with the age-related changes in neurocognitive strategies for the comprehension of abstract and concrete language.

Genes & Cells. 2023;18(4):409-420
pages 409-420 views

Machine-learning applications for differentiation across states/stages of creative thinking based on time-series and time-frequency features of EEG/ERP signals

Shemyakina N.V., Velicoborets G.S., Nagornova Z.V.

Abstract

BACKGROUND: The study presents machine-learning (ML) classification approaches for the state/stage differentiation of creative tasks using the “test-control” approach. The control tasks were considered as the initial stages of the creative activity. Time-series and time-frequency electroencephalography (EEG) data analyses were employed in three divergent thinking tasks: 1) creating endings to well-known proverbs (“PROVERBS”, event-related potential [ERP] paradigm); 2) creating stories (“STORIES”, continuous EEG); 3) free creative painting (“viART”, continuous EEG).

AIM: To compare and select effective ML classification approaches for EEG signal separation at different stages or states of creative task performance.

METHODS: In this study, 22 individuals participated in the “PROVERBS” (ERP paradigm), 15 in the “STORIES”, and 1 (a longitudinal case study) in the “viART” tasks. Linear and convolutional neural network (CNN) classifiers were used. EEG data were previous artifacts corrected and converted to current source density (CSD). Continuous EEGs were divided into 4-s intervals and 1500 ms after stimulus presentation, were used in ERPs. The EEG/ERP time-frequency maps (Morlet wavelet transformation) for 3–30 Hz were generated for 4-s intervals with 100 ms shift (continuous EEGs in “STORIES” and “viART”) or for 1500 ms after stimulus presentation (ERPs in “PROVERBS”) and consisted of combined images (224×224 px) for frontal (Fz) and parietal (Pz) brain zones. Image classification was carried out using the modified CNN (ResNet50, ResNet18 architectures).

RESULTS: The offline classification accuracy of the four-class system (description of a picture, inventing a story plot, continuation of story’s plot, and background with open eyes) in the “STORY” creation task was up to 96.4% [±8.3 SD] with ResNet architectures (ResNet50 and ResNet18). The accuracy of the three states discrimination of the artists’ creative painting (resting state with open eyes, painting on canvas, and viewing the painting) was 86.94% for kernel naive bayes and 98.2% for CNN. For the trained and tested samples given for the CNN in consecutive order (neurointerface mode), the accuracy diminished to 70.0% [11% SD] on average. In the ERP paradigm “PROVERBS”, the classification accuracy of the three-class system (creation of “new” ending, naming of semantic synonym, and remembering of the known ending) was 80.5% [±8.7 SD] for the common spatial pattern, followed by rSVM (radial kernel basis support vector machine), compared with 43.2% [±8.8 SD] for CNN.

CONCLUSION: The use of CNNs allowed better classifying of “continuous” long-term states of creative activity. In fast “transient processes” such as ERP, time-series classifiers with spatial filtering proved to be more efficient.

Genes & Cells. 2023;18(4):421-432
pages 421-432 views

Topological data analysis suggests human brain network reconfiguration during the transition from resting state to cognitive load

Ernston I.M., Onuchin A.A., Adamovich T.V.

Abstract

BACKGROUND: Neural networks of the brain continually adapt to changing environmental demands. The network approach in neuroscience, which focuses on the analysis of structural and functional network characteristics related to cognitive functions, is a highly promising avenue for understanding the psychophysiological mechanisms underlying the adaptive dynamics of cognitive processes.

AIM: We aimed to explore how the topological features of functional connectomes in the human brain are linked to different cognitive demands. The focus was on understanding the dynamic changes in brain networks during working memory tasks to identify network characteristics inherent to working memory.

METHODS: We examined the topological characteristics of functional brain networks in the resting state and cognitive load provided by the execution of the Sternberg Item Recognition Paradigm based on electroencephalographic data. Electroencephalogram traces from 67 healthy adults were processed to estimate functional connectivity using the coherence method. We propose that the topological properties of functional networks in the human brain are distinct between cognitive load and resting state, with higher integration in the networks during cognitive load.

RESULTS: The topological features of functional connectomes depend on the current state of cognitive processing and change with task-induced cognitive load variation. Moreover, functional connectivity during working memory tasks showed a faster emergence of homology group generators, supporting the idea of a relationship between the initial stages of working memory execution and an increase in faster network integration, with connector hubs playing a crucial role.

CONCLUSION: Collected evidence suggest that cognitive states, particularly those related to working memory, are associated with distinct topological properties of functional brain networks, highlighting the importance of network dynamics in cognitive processing.

Genes & Cells. 2023;18(4):433-446
pages 433-446 views

Conference proceedings

Effect of long-term social isolation on behavior and brain plasticity in mice with tumor necrosis factor gene knockout

Bazovkina D.V., Ustinova U.S., Adonina S.N., Komleva P.D., Arefieva A.B., Moskaliuk V.S., Kulikova E.A.

Abstract

Prolonged social isolation can disrupt the functional activity of the serotonin (5-HT) neurotransmitter system and neurotrophic support of the brain, activate neuroinflammatory processes, and cause various behavioral disorders [1]. On the contrary, the pro-inflammatory cytokine, specifically tumor necrosis factor (TNF), affects the synthesis of serotonin and the expression of neurotrophins in the brain [2]. Additionally, TNF gene knockout alters the severity of depression-like behavior and cognitive functions in rodents [3].

The objective of this study was to examine the impact of prolonged social isolation on the 5-HT system in the brain as well as the expression of the neurotrophic factors BDNF and NGF in Tnf gene knockout (TNF KO) mice and wild-type C57BL/6 mice. Mice from each strain were divided into two groups: “control”, which were kept in groups, and “experimental”, which were isolated in cages for six weeks. The mice were subjected to a battery of tests including the “open field”, “three-chamber”, and “forced swimming” tests. The expression of genes was gauged in the brain structure of mice through real-time RT-PCR and protein content was analyzed through Western blotting. Serotonin and its metabolite 5-HIAA were quantified using HPLC. The results were analyzed using a two-way analysis of variance followed by Fisher’s multiple comparisons.

The animals’ locomotor activity did not differ between groups. Social isolation in TNF KO mice led to reduced exploratory activity and increased anxiety (p <0.05) in the open field test. In isolated wild-type mice, social object preference in the three-chamber test decreased (p <0.01). Isolation had no effect on depression-like freezing in the “forced swimming” test and cognitive functions in the “new object” test in animals of both strains. Social isolation resulted in decreased expression of the tryptophan hydroxylase 2 gene (synthesizes 5-HT) in the midbrain of wild-type mice (p <0.05) and increased expression of the 5-HT1A receptor gene in this structure in knockout animals (p <0.05). Only the knockout mice exhibited a reduction in 5-HT levels in the hippocampus due to isolation (p <0.05). However, there were no differences observed in the levels of the neurotransmitter and its metabolite 5-HIAA in the frontal cortex and midbrain between groups of both strains of mice. In TNF KO mice exposed to isolation, the mRNA level of the nerve growth factor gene NGF increased in the frontal cortex (p <0.01); additionally, the content of proBDNF protein (a precursor of the BDNF factor) increased in both the hippocampus and frontal cortex (p <0.05). Our findings indicate that Tnf gene knockout modifies the influence of long-term social isolation on behavior, the 5-HT system, and the expression of neurotrophic factors in the brain.

Genes & Cells. 2023;18(4):450-453
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Chronic social defeat stress and glucocorticoid regulation in brain regions: resistance or hypersensitivity?

Bondar N.P., Kisaretova P.E., Reshetnikov V.V., Shulyupova A.S., Ryabushkina Y.A., Salman R.

Abstract

Chronic social stress causes various psychopathologies and is frequently associated with alterations in the HPA axis function. The heightened glucocorticoid hormone levels in the bloodstream instigate an acute bodily response, which fades over time, even with continued glucocorticoid stimulation. It is known that the resistance to elevated hormone levels can affect the effectiveness of therapy in the treatment of stress-induced psychopathologies. Resistance to elevated hormone levels can impact the effectiveness of therapy for stress-induced psychopathologies. To understand the molecular basis of glucocorticoid resistance, we examined the effect of chronic social defeat stress on the transcriptome of two brain regions — the prefrontal cortex and the dorsal raphe nuclei — using an experimental model of depression.

We assessed gene expression levels in C57BL/6 control mice and mice subjected to 30 days of stress, both under basal conditions and following additional stimulation with dexamethasone. The administration of dexamethasone (2 mg/kg) allowed for simulation of the upregulation of glucocorticoids and activation of the glucocorticoid receptor. The results indicate that chronic stress induces gene resistance to glucocorticoid hormones in only 15% of prefrontal cortex genes and 25% of raphe nuclei genes. In stressed animals, there was no response to dexamethasone stimulation, whereas controls showed a reaction. For 66% of the genes in the prefrontal cortex and 40% of the genes in the dorsal raphe nuclei, the response to dexamethasone exhibited a greater intensity in the stressed group as compared to the control group. This set of genes comprises genes linked to immune responses, monoamine conveyance, and synapse establishment. Under stress conditions, as opposed to controls, anti-inflammatory cytokine genes, as well as genes connected to the growth of B- and T-lymphocytes, are downregulated in response to dexamethasone treatment. Furthermore, chronic stress exposure heightens the sensitivity of serotonergic receptor genes Htr1a and Htr5a to dexamethasone. The Htr1a gene exhibited a region-specific response to dexamethasone in stressed animals. Specifically, the expression of the gene increased in response to dexamethasone in the prefrontal cortex, while it decreased in the dorsal raphe nuclei. Additionally, the sensitivity of genes involved in the differentiation of oligodendrocytes changed in the dorsal raphe nuclei of stressed animals.

Thus, our data demonstrate that chronic social defeat stress induces resistance and heightened sensitivity to to glucocorticoid activation, resulting in the development of depression.

Genes & Cells. 2023;18(4):454-455
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Disruption of protein kinase expression in cortical neurons due to the deletion of transcription factor Satb1 leads to neuronal network hyperexcitation and determines hypoxia sensitivity

Celis Suesсun J.C., Gavrish M.S., Turovsky E.A., Varlamova E.G.

Abstract

Neuronal transcription factors regulate the expression of receptors and intracellular signaling molecules involved in excitatory neurotransmission. The transcription factor Satb1 received significant attention for its role in regulating the growth and development of various types of brain neurons in both embryonic and postnatal periods. The depletion of the transcription factor Satb1 can be considered a fundamental mechanism for triggering hyperexcitation in the neural network, given the alterations in the activity and expression levels of proteins, such as glutamate receptors, protein kinases, and cell viability regulators. Although numerous studies were conducted on the role of Satb1 in neurogenesis, none showed any changes in intracellular calcium signaling or protein expression levels following Satb1 deletion in neurons.

Mice with complete (Satb1-null) and partial (Satb1-deficient) deletion of the Satb1 transcription factor were used in this study. Neuroglial cultures were obtained from the cerebral cortex of neonatal mice and were cultivated in-vitro for ten days. The cells were then loaded with a calcium-sensitive Fura-2 fluorescent probe and the dynamics of cytosolic Ca2+ ([Ca2+]i) were recorded using a fluorescent microscope. Spontaneous calcium activity was observed in the cell cultures, and epileptiform activity was modeled using conventional methods, i.e. the medium was depleted of magnesium (magnesium-free model) and 10 µM bicuculline was added (bicuculline model). The control group comprised cerebral cortex cells obtained from normal mice. To analyze expression patterns of genes encoding kinases, total RNA was extracted from cell cultures, and real-time PCR analysis was conducted.

Complete and incomplete deletion of the transcription factor Satb1 had distinct effects on protein kinase expression and genes that regulate cell viability. Satb1-deficient neurons exhibited increased expression of phosphoinositide-3-kinase, protein kinase C, protein kinase B, mitogen-activated protein kinase, as well as Bcl-2, Creb, and Nf-kB genes. The deletion of Satb1 in cortical neurons led to increased expression of only phosphoinositide-3-kinase, while the expression of all other studied protein kinases decreased in the presence of pro-inflammatory factors Nf-kB, Caspase-3, and Tnfα.

At the neurotransmission level, the complete deletion of Satb1 led to heightened spontaneous Ca2+ neuron activity alongside amplified Ca2+ oscillation frequencies and amplitudes when modeling epileptiform activity. Since hyperexcitation of the network is a symptom observed in neuronal networks during hypoxia, and the response of neurons to hypoxia is dependent on the activity of the kinases being studied, experiments were conducted to simulate hypoxia using neurons obtained from mice who lack the Satb1 transcription factor. Concurrently with recording [Ca2+]i, we measured pO2 using a somatic oximeter. The drop in pO2 signified the start of Ca2+ responses in neurons. Initially appearing in 8–10% of cells, hypoxia-triggered the first Ca2+ responses in WT neurons when pO2 dropped to 40 mm Hg. The remaining cells within the microscope field of view did not react. Satb1-null neurons responded to hypoxia at 60 mm Hg, exhibiting high-amplitude Ca2+ oscillations in over 15% of cells. Furthermore, Satb1-deficient neurons exhibited high-frequency Ca2+ oscillations and an increase in [Ca2+]i baseline to a new stationary level when pO2 decreased to 75–80 mm Hg. With over 20% of neurons responding, this suggests that Satb1-null neurons are more sensitive to hypoxia.

Genes & Cells. 2023;18(4):456-459
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Study of the role of chromatin-remodeling factor SWI/SNF in neuronal processes in Drosophila

Deev R.V., Chmykhalo V.K., Stepanov N.G., Lebedeva L.A., Shidlovskii Y.V.

Abstract

SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate the expression of numerous genes through the use of at least 15 subunits. The composition of these complexes is diverse, a result of combinatory assembly of subunits from homologous families with a variety of additional functions. During neurogenesis, researchers have identified distinctive SWI/SNF complexes that are specific to various stages of development. These complexes were detected in stem cells, neuronal progenitor cells, and postmitotic differentiated neurons. Notably, SWI/SNF complexes specific to neuronal progenitor cells are essential for regulating their division. SWI/SNF complexes are critical for adult brain plasticity in neurons and contribute to the regulatory genetic mechanisms that underlie higher neural activity, especially in learning and memory processes. However, the epigenetic regulation of gene expression in neurons remains insufficiently understood.

Mutations in complex subunits result in the development of pathologies. For instance, ARID1A and ARID1B subunit mutations in humans cause Coffin–Siris syndrome, which is a rare congenital multisystem genetic disease primarily characterized by developmental disabilities and intellectual disability. Currently, there are challenges in creating model systems to study Coffin–Siris syndrome. Drosophila has shown promise as a model organism due to its extensive research and suitability in studying neurodevelopmental pathologies. It is a cost-effective species that facilitates tracking of both species-specific features and general patterns for abnormality types. Additionally, Drosophila serves as a model organism due to its abundance of orthologous genes that lead to neuronal developmental abnormalities in humans. Notably, the knockdown of genes that encode subunit complexes in Drosophila produces a noticeable decline in long-term memory formation.

In this study, we examine the involvement of SAYP and osa complex subunits in different molecular mechanisms in Drosophila brain neurons. We use multiple strategies to alter the levels of SAYP and osa factors, such as tissue-specific knockdown, tissue-specific protein degradation, and null allele production. Using the CRISPR/Cas9 technique, we generated novel alleles of the osa and SAYP genes that encode operational proteins which can be selectively degraded at specific times and in specific tissues. We examine the changes in gene expression profiles, chromatin states, and the participation of transcription factors on chromatin, as well as DNA integrity alterations, upon removal of these factors from neurons. We are conducting research on the long-term memory of adult flies and neurogenesis at different developmental stages. Our aim is to gain insight into the role of the epigenetic regulator SWI/SNF in the development of nervous system pathologies and its involvement in the development and functioning of higher eukaryotic nervous systems.

Genes & Cells. 2023;18(4):460-461
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Investigation of hippocampal synaptic plasticity in a mutant mice line predisposed to epileptiform activity

Fedulina A.A., Matveeva M.V., Maltseva K.E., Lebedeva A.V., Tarabykin V.S.

Abstract

The human genetic code is nearly decoded and genetic mutations garnered significant attention from researchers. Investigating mutations that cause disease manifestation and determine genetic predisposition is a priority in this field. The investigation of novel disease manifestation mechanisms represents a contemporary and original strategy that advances the development of treatment and correction methods for multiple neurodegenerative conditions in humans. Epilepsy constitutes one of the prevalent forms of neurological disorders. A full understanding of the complex mechanisms that drive epileptogenesis and seizure onset in temporal lobe and other forms of epilepsy cannot be fully achieved through human clinical trials. Consequently, the use of relevant animal models becomes indispensable.

The aim of this study is to investigate the synaptic transmission and long-term plasticity of the hippocampus in a mutant strain of mice known as S5-1 that exhibits epileptiform activity. The study focuses on S5-1 strain mice that have the tendency to develop epileptiform activity after the induction of ENU-mutagenesis in the DNA molecule. To investigate in vitro activity, researchers use a method that combines electrophysiological and optical techniques to register local field potentials on surviving brain slices.

To evaluate long-term synaptic plasticity, two iterations of the protocol were used: one involved applying small stimulation amplitudes (50 mA), while the other called for large amplitudes (500 mA). In the former case, a high frequency of long-term potentiation was observed within the group with epileptiform activity. In animals with the phenotype, potentiation was between 150–170% of the average response rate to theta-burst stimulation, whereas values in the control group only reached 120–125%. The findings indicate that in a cohort of animals with an epileptiform phenotype, nerve fibers are hyperactivated, which is one of the mechanisms contributing to epileptogenesis. Moreover, in the second scenario, by employing high stimulation amplitudes, a marked decline in synaptic transmission was identified in animals with epileptiform behavioral activity (120–150%) as opposed to the control group (200–250%). A significant reduction in long-term synaptic plasticity in animals displaying epileptiform activity when compared to the control group under high stress stimulation amplitudes may suggest a disruption of synaptic transmission at the molecular and cellular levels, potentially resulting in memory impairment or a decline in cognitive abilities among mutant animals exhibiting epilepsy symptoms.

A plausible mechanism for altering synaptic transmission in the hippocampus of the S5-1 mutant line of mice is that point mutations occur after exposure to mutagen which leads to the development of various pathologies. One of these pathologies results in a disruption of the cytoarchitecture of the cerebral cortex. Consequently, subcortical structures, especially the hippocampus, are indirectly affected leading to the onset of audiogenic seizures. As a consequence, disruptions occur in synaptic transmission and plasticity, potentially resulting in memory impairment and other cognitive impairments. However, additional research is needed to examine the potential mechanism underlying the development of brain disorders in mutant animals.

Genes & Cells. 2023;18(4):462-464
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NeuroD family genes regulate the survival of neurons in the developing hippocampus

Filat’eva A.E., Kondakova E.V., Gavrish M.S., Bormuth O., Bormuth I., Tarabykin V.S.

Abstract

NeuroD 1/2/6 transcription factors belong to the bHLH-containing protein family. They activate transcription and regulate various aspects of neuronal differentiation [1]. In the developing cortex and hippocampus, these three factors express overlapping patterns, indicating a partial redundancy of functions during development [2]. They aid in the creation of the brain’s main commissures, particularly the largest one, the corpus callosum. Agenesis, whether total or partial, is a frequently occurring comorbidity in congenital malformations amongst human beings. It is usually associated with the underdevelopment or absence of the hippocampus [3].

To examine the contribution of these factors in hippocampal formation development, we created allelic genetically modified mouse strains with inactivating mutations in all three genes. The results indicated that the inactivation of NeuroD1 leads to the dentate gyrus’ absence. In contrast, the absence of all three NeuroD genes results in the dentate gyrus’ and other hippocampal formation regions’ absence (CA1, CA2, CA3) at subsequent developmental stages.

To investigate the cellular mechanisms involved in the disruption of hippocampal formation development, it was hypothesized that NeuroD transcription factors may control either neuronal proliferation and differentiation or cell death. To test which of these hypotheses is correct, a series of BrdU injections were performed at the E15 stage of development. We examined several parameters related to proliferation, including the proportion of cells at various differentiation stages, cell cycle exit, cell cycle length, and the number of cells in the S phase of the cell cycle. The results showed no discernible differences in proliferation and cell cycle parameters between triple homozygous embryos and control littermates.

An analysis of programmed cell death, known as apoptosis, was conducted to test the alternative hypothesis. We used two methods to analyze cell death: firstly, we analyzed the activity of caspase-3 which is one of the proteins that activate apoptosis and, secondly, we analyzed the number of double-strand breaks using the TUNEL test. The findings revealed a significant increase in the number of cells positive for both caspase-3 and double-stranded DNA breaks in the developing hippocampus of triple mutants. The amount of apoptotic cells in the developing hippocampal formation relies on the gene dosage of NeuroD 1/2/6 alleles, as evident through their increment as the NeuroD gene dosage decreases. The double KO portrays an intermediate level of cell death, while the triple exhibits the highest level.

To determine the stage in which massive cell death occurs during neuronal differentiation, we modified the BrdU-chase assay. BrdU was injected during the E12 stage, and embryos were surveyed at various intervals (12, 18, and 24 hours) for analysis. This experiment permits estimation of the time period following exit from the mitotic cycle, during which the cell initiates the mechanism of apoptosis.

Genes & Cells. 2023;18(4):465-468
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Sphingomyelinase as modulator of neuromuscular transmission via presynaptic mechanism

Gafurova C.R., Tsentsevitsky А.N., Mukhutdinova К.А., Giniatullin А.R., Petrov А.М.

Abstract

Secretory sphingomyelinases are released in response to various types of stress stimuli, including inflammation [1]. Excessive activity and release of these enzymes can be pathological and accelerate age-related neurological changes, neuroinflammation, neurodegenerative disorders, and muscle dysfunction [1–3]. In contrast, sphingomyelinases were detected in areas close to exocytosis sites and dendritic spines, and their inhibition reduced synaptic transmission at both the presynaptic and postsynaptic levels in hippocampal synapses. Deficiency of acid sphingomyelinase results in neurodegenerative Niemann–Pick disease type A. Knockouts of neutral sphingomyelinases in model mice cause motor defects and disorders that resemble the Alzheimer’s disease phenotype [1, 2]. These findings suggest the existence of a mechanism regulating synaptic transmission, which is dependent on sphingomyelinases.

In this study, we examined the impact of neutral sphingomyelinase on neurotransmission in mouse diaphragm muscles using microelectrode recordings of postsynaptic responses, exo-endocytic FM dyes, and fluorescent probes sensitive to membrane properties.

The researchers discovered that adding sphingomyelinase at a low concentration (0.01 u/ml) for 15 minutes causes disruption of the lipid packaging of selectively synaptic membranes. This disruption leads to ceramide accumulation, a decrease in staining with Alexa Fluor 488 cholera toxin subunit B conjugate (a marker of GM1 ganglioside cluster), and a shift to the green part of the F2N12S fluorescence spectrum (an indicator of the expansion of the lipid-disordered phase). Increased fluorescence of 22-NBD-cholesterol, indicating an increase in membrane fluidity, and enhanced incorporation of exogenous ceramide into the outer monolayer were observed in the synaptic membranes. Increasing sphingomyelinase concentration to 0.1 u/ml intensifies changes in synaptic membranes and leads to corresponding alterations in membrane properties in muscle fiber plasmalemma. Subsequent experiments used a synapse-specific effect on membrane properties by applying sphingomyelinase at a low concentration for 15 minutes.

The application of sphingomyelinase did not alter the amplitude and temporal parameters of miniature postsynaptic responses or the resting membrane potential. Additionally, sphingomyelinase (0.01 u/ml) did not change spontaneous secretion and evoked neurotransmitter release in response to single stimuli. However, the activity of sphingomyelinase led to a notable increase in the release of neurotransmitters and the rate of exocytotic unloading of FM dye from synaptic vesicles during nerve stimulation at 10, 20, and 70 Hz frequencies. The effect of sphingomyelinase on neurotransmission potentiation was irreversible and more significant at 10 Hz activity. Experiments involving intermittent nerve stimulation with short bursts of 60 stimuli each at 10 or 20 Hz frequencies, along with brief 0.5-second rest intervals, indicated that sphingomyelinase elevated the short-term facilitation of neurotransmitter release at the onset of each subsequent episode at 10 Hz (but not at 20 Hz) stimulation.

Experiments with a combination of electrophysiological detection of evoked postsynaptic responses, monitoring of exocytotic release of the FM1-43 dye, and the use of FM1-43 fluorescence quencher that penetrates through the fusion pores have demonstrated that a significant portion of synaptic vesicles release the neurotransmitter through the transient fusion pore (kiss-and-run mechanism) during high-frequency (70 Hz) activity [4, 5]. Treatment with sphingomyelinase inhibited the neurotransmitter release pathway. As a consequence, the majority of neurosecretion events went through exocytosis, completely incorporating the vesicular membrane into the presynaptic membrane.

Sphingomyelin in the membranes of presynaptic and synaptic vesicles is believed to have varying functions in regulating neurotransmitter release. To investigate this theory, we stimulated the exo-endocytosis processes of synaptic vesicles using sphingomyelinase, which provided access to the synaptic vesicle membranes. Under these circumstances, the enhancing impact of sphingomyelinase on neurotransmitter release and FM1-43 exocytosis rate were notably inhibited.

Based on the obtained data, we hypothesize that sphingomyelinase disrupts the integrity of lipid rafts and selectively forms ceramide accumulations in synaptic membranes within the muscles at low concentrations. This ultimately leads to an increase in the mobilization of synaptic vesicles towards exocytosis sites during 10–70 Hz activity. Presumably, the synaptic vesicles from a distinct pool, which predominantly mediates the neurotransmission at a frequency of 10 Hz, are the most actively involved in exocytosis under these circumstances. Sphingomyelinase modifies membrane properties and can inhibit the release of neurotransmitters through the fusion pore during high-frequency activity, redirecting exocytosis towards the complete fusion pathway. Hydrolysis of sphingomyelin in both plasma and synaptic vesicle membranes reduces the stimulatory effects of sphingomyelinase, indicating a contrasting role of sphingomyelin in presynaptic and vesicular membranes for controlling neurotransmitter release [5].

Genes & Cells. 2023;18(4):469-472
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Changes in neuronal excitability in the rat hippocampus in a prolonged febrile seizures model

Griflyuk A.V., Postnikova T.Y., Amakhin D.V., Soboleva E.B., Zaitsev A.V.

Abstract

Febrile seizures (FSs) are a prevalent neurological disorder among children aged 3 months to 5 years, with the highest incidence observed in the second year of life [1]. Considering that neuronal and glial cell development and synaptic contact formation are ongoing during this period [2, 3], FSs can potentially influence these processes. However, the present data on the effect of FSs on brain development remain inconsistent.

This study aims to examine the impact of extended seizures on the attributes of hippocampal pyramidal neurons in rats of varying ages.

Wistar rats were used in this study. FSs were induced on postnatal day (P)10 through placing pups onto the bottom of a glass chamber for 30 minutes and exposing them to a controlled stream of heated air, causing their body temperature to rise to 39 °C and trigger the occurrence of FSs. Only animals that underwent FSs that persisted for at least 15 minutes were included in the study. Littermates were employed as controls and were placed away from the nest for the same duration but kept at room temperature.

At postnatal days 12, 21–23, and 51–55, the rats were decapitated, and their brains were extracted. Horizontal brain slices (400 micrometers) were sliced. The study examined the biophysical properties of CA1 pyramidal neurons using the whole-cell patch-clamp method. 1.5-second current pulses were injected, and subthreshold membrane properties such as resting membrane potential, input resistance, membrane time constant, and intrinsic firing properties, were assessed. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 stratum radiatum of the hippocampus to assess the efficacy of synaptic neurotransmission at CA3-CA1 pyramidal neuron synapses. The amplitude and fiber volley (FV) amplitude of each fEPSP were measured by applying high amplitude currents to each slice. A sigmoidal Gompertz function was used to evaluate the efficacy of neurotransmission. Paired-pulse stimulation was used to examine potential alterations in short-term synaptic plasticity. Paired pulses were administered at intervals spanning from 10 to 500 ms, and the paired-pulse ratio (PPR) was assessed as the proportion of the amplitude of the second to the first fEPSP for each interval. Maximum electroshock seizure threshold (MEST) was determined two months after FS to assess the animals’ susceptibility to seizures. The lowest current at which tonic hind limb extension was exhibited was determined for every animal.

No significant changes in subthreshold firing properties were observed in P12 or P21 rats following FSs. However, we did observe several changes in intrinsic firing properties. For example, the maximum firing frequency decreased by 23% in P12 rats exposed to FSs, relative to age-matched control rats. Additionally, we noticed that firing frequency adaptation was significantly less pronounced in P12 rats that underwent FSs, compared to control rats of the same age. Seizure-induced alterations in firing characteristics were absent in P21 rats. No significant variations in fEPSP or FV amplitudes were found among the different groups of P12 and P55 rats at different current intensities. However, a significant increase in FV amplitudes and decrease in neuronal input-output (I/O) relationships between fEPSP and FV amplitudes were observed in P21 rats following FSs. At P12, short-term synaptic plasticity was disrupted, evidenced by a significant increase in PPR in rats two days post-FS. However, at P21 and P55, experimental and control groups were not significantly different. MEST test displayed a significant rise in the hind limb extension threshold in rats two months following FS as compared to control animals.

Overall, these findings suggest alterations in neuronal excitability after prolonged FS. Two days after FS, neuronal excitability transiently decreased while PPR increased, indicating a decrease in the probability of presynaptic glutamate release in hippocampal neurons. The efficiency of synaptic neurotransmission in CA3-CA1 was reduced in three-week-old animals. Additionally, the MEST test demonstrated that rats, two months following FS, exhibited a higher hind limb extension threshold in comparison to control animals.

Genes & Cells. 2023;18(4):476-479
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Translational neurobiology of zebrafish (Danio rerio)

Kalueff A.V., Kotova M.M., Galstyan D.S., Kolesnikova T.O.

Abstract

The zebrafish (Danio rerio) is a small freshwater teleost fish species that is increasingly utilized in biomedical research, particularly in neuroscience and biological psychiatry. Currently, zebrafish is the second-most utilized model organism in biomedicine globally, after mice, based on the number of animals tested each year. The model’s significance results from its experimental ease of use, affordability, conservation of fish physiology, relatively high genetic homology with humans (70%), rapid development, and potential for high-throughput bioscreening of drugs and genetic mutations.

Over the past 15 years, our laboratory has conducted extensive experimental work to establish the principles behind using zebrafish to study various brain pathologies, including acute and chronic stress, anxiety, and depression, as well as probing their molecular mechanisms. In addition, existing behavioral models to study the central nervous system (CNS) development and new data on the zebrafish’s critical role in memory research were reviewed. Furthermore, this study will illustrate the effectiveness of integrating zebrafish models with advanced biological research techniques, such as molecular biology, bioinformatics, omics technologies, and chemical biology methods. For example, adult zebrafish experiencing chronic stress exhibit behavioral affective syndromes along with changes in neurochemistry, specifically in the metabolism of serotonin and dopamine in the telencephalon. Moreover, alterations in the expression of genes regulating neurotransmitter receptors, glial biomarkers, cytoskeleton, and pro- and anti-inflammatory cytokines, occur in the brain.

In particular, we will focus on neuroimmune and epigenetic mechanisms of CNS pathogenesis in zebrafish models, including changes in the expression of apoptotic genes in the brain. Additionally, our own findings on using artificial intelligence (AI) systems to study zebrafish behavior after administering various neurotropic drugs, such as anxiolytics, antidepressants, psychostimulants, and hallucinogens will be presented.

In general, zebrafish is a strategic and promising model organism for translational neuroscience research, creating new models of CNS pathogenesis and finding new drugs to treat various human brain diseases. Studies of CNS pathogenesis in zebrafish are critical because of their evolutionary conservatism and ease of laboratory application, revealing novel brain disease biomarkers and potential remediation targets. Meanwhile, certain distinctive aspects of the biology and neurophysiology of zebrafish facilitate the resolution of supplementary experimental issues, thereby enhancing data and discoveries attained in typical animal models using rodents.

Genes & Cells. 2023;18(4):480-482
pages 480-482 views

Deletion of transcription factor Satb1 leads to disturbance in mice behavior through changes in the expression of genes-regulators of the excitive component of neurotransmission

Gavrish M.S., Celis Suescun J.C., Turovsky E.A., Varlamova E.G.

Abstract

The transcription factor Satb1 exhibits widespread expression in multiple tissues and different regions of the brain. Notable levels of expression are observed in the neocortex, the nucleus of the diagonal band, the amygdala, and the tegmental area. In the ventral midbrain, Satb1-positive neurons are only found in a small portion of the substantia nigra, the ventral tegmental area, and the retrorubral field, and none were detected in the inferior colliculus. The transcription factor Satb1 is highly expressed in interneurons labeled as SST+, CR+, and NPY+, but not VIP+ interneurons. In mice with the Satb1 mutation, incomplete eye opening and a constriction reflex were observed.

Behavioral tests revealed that mice lacking Satb1 exhibited deficits in motor coordination, characterized by reduced mobility on flat bars and decreased grip strength on metal wires. In addition, these mice exhibited elevated levels of motor activity in novel environments and heightened anxiety in the light-dark test. Satb1-deficient males demonstrated proficiency in passive avoidance tests, while their female counterparts exhibited no significant difference in entrance time to the dark compartment between the reproduction and learning stages, indicating an impairment in the process of conditioned response formation.

To investigate the expression patterns of genes encoding vital protein kinases and proteins associated with neurotransmission, we extracted total RNA from the cerebral cortex of adult males with an incomplete deletion of Satb1. Real-time PCR analysis was conducted, which revealed a higher expression level of the pik3ca, pik3cb, and pic3cg genes responsible for phosphoinositide-3-kinase isoforms in Satb1-deficient mice compared to wild-type mice. The expression levels of genes encoding protein kinase C (Prkce and Prkcg) as well as Ca2+/calmodulin-dependent protein kinase II (Camk2) were lower in comparison to wild-type mice. Satb1-deficient mice displayed notably higher expression levels of genes encoding NMDA receptor subunits (Grin1, Grin2a, and Grin2b), whereas the expression of Gria1 (encoding the Glua2 subunit of AMPA receptors responsible for Ca2+ receptor conductance) decreased. The influence on excitatory glutamate receptor expression can cause hyperexcitability in animals, particularly when inhibitory neurotransmission is suppressed due to decreased Gabra1 and Gad65/67 expression encoding the GABA(A) receptor and glutamate decarboxylase. In addition, the levels of expression for the Calb1, Calb2, and Pvalb genes responsible for encoding the calcium-binding proteins calbindin, calretinin, and parvalbumin decreased in comparison to the wild-type mice.

Thus, a correlation was found between behavioral disturbances and expression of genes that encode proteins related to brain development and neurotransmission after deleting the Satb1 transcription factor. Satb1-deleted mice exhibited hyperexcitation and impaired motor activity due to impaired gene expression.

Genes & Cells. 2023;18(4):473-475
pages 473-475 views

Effects of intrahippocampal injection of kainate on cytokine expression in cortico-limbic system and the role of cannabinoid system in these effects

Karan A.A., Spivak Y.S., Suleymanova E.М., Gerasimov K.A., Bolshakov А.P., Vinogradova L.V.

Abstract

According to the International League Against Epilepsy, epilepsy is a chronic condition of the brain that is characterized by a predisposition to epileptic seizures along with related neurobiological, cognitive, psychological, and social consequences. This is a general definition that does not take into account the diversity of epilepsies, including different aetiology, symptoms and mechanisms of epileptogenesis, making the development of a unified disease model challenging. The modeling framework addresses this challenge by selecting a distinct type of epilepsy and its associated manifestations, including electrophysiological, morphological (mainly neurodegeneration), and behavioral aspects [1]. In this study, a status epilepticus model is used with intrahippocampal kainic acid administration to reproduce electrophysiological activity and neurodegeneration. The process of reproducing two aspects simultaneously brings the kainate model closer to the disease called “epilepsy”.

Neuroinflammation is a neurobiological process that is associated with a chronic brain condition that is characterized by a persistent susceptibility to epileptic seizures. Specifically, neuroinflammation is the response of the central nervous system (CNS) to various stimuli, including stroke, trauma, infection, autoimmune diseases, stress, and hyperexcitability of the neural network resulting from epileptic seizures. This response involves brain cells, specifically activated microglia and astrocytes, as well as neurons and brain vasculature cells, biosynthesizing and releasing molecules with inflammatory properties [2].

Currently, the study of neuroinflammation in relation to various pathological conditions includes an examination of the influence of the endocannabinoid system (ECS) [3]. However, research on epilepsy primarily focused on the ECS’s effects on network neuronal activity through CB1-mediated changes in synapse function (both excitatory and inhibitory), with only a limited number of studies exploring the interactions between the ECS and neuroinflammation [4].

This study analyzed neuroinflammatory dynamics after kainate administration and the effect of exogenous endocannabinoid receptor modulators on these dynamics. Neuroinflammation was evaluated through the measurement of expression levels of pro- and anti-inflammatory cytokines (IL1b, Il6, Cx3cl1, Ccl2, Tgfb1, Zc3h12a, Tnfa) in various areas including the ipsilateral ventral hippocampus, contralateral dorsal and ventral hippocampuses, neocortex, dura mater, cerebral and hippocampal meninges (undivided arachnoid and pia maters). Expression was quantified using quantitative PCR at 3 and 24 hours following convulsant injection. The study showed that seizures induced by kainate resulted in swift neuroinflammation development in the hippocampus, which resolved nearly entirely after 24 hours. A unique pattern of neuroinflammation was detected in the neocortex, with minor alterations at 3 hours and more pronounced modifications in the expression of inflammatory genes at 24 hours. Using the intrahippocampal kainate administration model, this study was the first to show a significantly delayed neuroinflammatory response in the neocortex compared to the hippocampus across a broad range of genes.

Both activation of the cannabinoid CB1 and CB2 receptors and inhibition of the cannabinoid CB1 receptor increased neuroinflammation. However, cannabinoid receptor activation showed a predominantly proinflammatory effect in the neocortex, while CB1 receptor inhibition had a stronger effect in the hippocampus. Our findings indicate that cannabinoid receptor modulators regulate the kainate-induced neuroinflammatory response in the neocortex and hippocampus differently. Moreover, the well-known anti-inflammatory effect of cannabinoids is evident only within a certain range of cannabinoid concentrations and the timing of drug administration. In some cases, however, cannabinoids may have the opposite effect of increasing neuroinflammation.

Genes & Cells. 2023;18(4):483-486
pages 483-486 views

Recording changes in biochemical parameters in vivo in the ischemic stroke model

Khramova Y.V., Kotova D.A., Ivanova A.D., Pochechuev M.S., Kelmanson I.V., Trifonova A.P., Sudoplatov M.A., Katrukha V.A., Sergeeva A.D., Raevskii R.I., Solotenkov M.A., Fedotov I.V., Fedotov A.B., Belousov V.V., Bilan D.S.

Abstract

Stroke is a significant and socially insidious disease that ranks second among fatal diseases according to the World Health Organization [1]. Understanding the molecular mechanisms behind the pathogenesis of this ailment will enable the development of more effective preventative measures and treatment strategies to minimize the negative consequences of stroke. Despite the abundance of experimental data, most of which were acquired indirectly, the study of the dynamics of biochemical parameters in brain tissue in real time during the acute phase of ischemic stroke is difficult. The use of genetically-encoded sensors creates novel possibilities for monitoring alterations in different biochemical and metabolic parameters in vivo tissues.

In this study, we evaluated pH changes, hydrogen peroxide production (an important type of biologically active ROS), and polysulfide synthesis in various types of brain tissue cells of SHR rats during the development of ischemic stroke in real time using sensors such as SypHer3s (for pH detection), HyPer7 (for H2O2 detection), and PersIc (for polysulfide detection). Middle cerebral artery occlusion was used to simulate an ischemic stroke. The in vivo sensor signals were registered with a fiber optic setup that was created in the laboratory of spectroscopy and nonlinear optics at Moscow State University.

The studies revealed that in the acute phase of stroke, acidosis occurred in the cytoplasm of neurons in the caudate nucleus, the epicenter of ischemia. The pH mutated from 7.25±0.08 to 6.7±0.15 within the first few seconds after arterial occlusion initiation. A gradual increase in pH was observed after the initial drop, which persisted throughout reperfusion but did not return to the original value in all animals. In the penumbra zone, a wave-like shift in sensor signal was detected, whereas no change in sensor signal was noted in the healthy hemisphere. Investigation of the dynamics of H2O2 formation in the mitochondrial matrix of caudate neurons revealed minimal sensor oxidation during ischemia/reperfusion in the acute phase of stroke, indicating low ROS production. Nevertheless, a substantial increase in the sensor signal was detected after 24 hours following the surgery. Thus, the confirmation of oxidative stress development in the affected hemisphere differed from the commonly accepted view in terms of its dynamics. Previously, it was believed that excessive production of H2O2 leading to oxidative stress and related brain cell death occurred primarily in the acute phase. However, a comparison of hydrogen peroxide production dynamics in neurons and astrocytes revealed differences between these cell populations. It was discovered that as early as 12 hours after middle cerebral artery occlusion, the sensor signal in astrocytes increased more intensely than in neurons. This trend persisted until the end of the measurements, 40 hours after surgery. The observed distinctions may stem from glial cells’ protective function in counteracting the harmful consequences of hydrogen peroxide on neurons, along with their contribution to maintaining the myelin structure in the brain. Additionally, the role of astrocytes in neuroinflammation development is noteworthy. Reactive sulfur species, in addition to reactive oxygen species, appear to be significant contributors to the development of pathological processes. The PersIc sensor signal measurement did not show any disparities between the caudate nucleus of the healthy hemisphere and the hemisphere affected by stroke development in terms of polysulfide and persulfide appearance detection. However, the area surrounding the core infarction is noteworthy due to the observed bouts of acidosis using the SypHer3s sensor. Our findings suggest a potential association between these bouts, spreading depolarization, changes in calcium concentration, and the development of neuroinflammation. These reactions may ultimately lead to the synthesis of polysulfides, known modulators of inflammatory reactions.

Thus, our data provides valuable additions to the existing knowledge on metabolic changes that take place during the progression of ischemic brain injury.

Genes & Cells. 2023;18(4):487-490
pages 487-490 views

Chronic social stress alters dexamethasone sensitivity of glucocorticoid receptor target genes

Kisaretova P.E., Shulyupova A.S., Bondar N.P.

Abstract

Glucocorticoids are well-known for their role in adapting to physical and psycho-emotional stress. The prefrontal cortex (PFC) is a crucial target-tissue for glucocorticoid receptors (GR) that coordinates the stress response.

Transcriptome sequencing was conducted on the prefrontal cortex of male C57Bl/6 mice subjected to 30 days of chronic social defeat stress (CSDS). Prior to tissue extraction, the mice were injected with either 2 µg/g dexamethasone or saline, resulting in four groups: CSDS+sal, CSDS+dex, control+sal, and control+dex.

The study sought to identify genes regulated by GR within the differentially expressed genes (DEG) by analyzing five public GR ChIPseq experiments performed on rodent brain tissue. This endeavor aimed to elucidate the role of GR in the PFC stress response. GR binding sites that were situated –5k to +1k bp from tss were categorized as regulatory regions. The closest genes were then identified. For further analysis, 3023 genes recognized as GR-regulated by at least two studies were selected. Of these, 320 genes were demonstrated to be expressed in the PFC based on our RNAseq data.

We found a significant increase in GR sites among PFC DEGs that responded to DEX treatment in both the control group (control+sal vs control+dex: OR=2.17, p <0.001) and CSDS (CSDS+sal vs CSDS+dex: OR=1.86, p <0.001). However, chronic stress alone did not result in enrichment of genes regulated by GR. Notably, genes that responded differently to DEX treatment in CSDS and control showed a higher OR value (dex*CSDS: OR=2.32, p <0.01).

Common GR-target genes between DEX-con and DEX-csds exhibited the same expression change direction, except for the Sft2d2 gene, which encodes a vesicle transport protein. These genes are involved in PDZ domain binding (Fzd2, Mpp3), serine/threonine kinase activity (Rps6ka5, Akt2, Camkk1), and oxidoreductase activity (Prodh, Smox). GR-regulated genes specific to the CSDS group participate in cytokine production (e.g., Ltbp1, P2rx7, Dhx33, Hdac9, Bcl6, Lgr4, etc.) and modulate chemical synaptic transmission (e.g., Arc, Syt12, Cacng3, etc.), including components of the glutamatergic synapse (e.g., Magi2, Erc2, Dnm1, Clstn2, and Itgb1). Changes in expression of structural component genes, including those involved in membrane raft (Cavin1, Smpd2, and Slc2a1) and anchoring junction (B4galt1, Gjb6, Fzd4, and Limk1) genes, indicate the control group’s response to DEX treatment. A total of 14 genes showed differential regulation by GR in both CSDS and control groups. Among these genes are those involved in axon elongation (Link1, Rasgrf1), synaptic morphology (Clstn2), and vesicle endocytosis (Dnm1). Additionally, vital genes for axonal regeneration (Tubb3), neuroprotection (Hspb8), regulation of apoptosis (Bugalt1), and microglia activation (Cavin1) are included.

In conclusion, we aimed to decipher the pathways of GR regulation triggered by social stress and DEX treatment in the PFC. Chronic stress resulted in alterations in GR regulatory networks in the PFC that impacted processes related to synapse function and the inflammatory response.

Genes & Cells. 2023;18(4):491-493
pages 491-493 views

Developmental expression patterns of genes mutated in patients with neurodevelopmental disorders

Kondakova E.V., Gavrish M.S., Filat’eva A.E., Tarabykin V.S.

Abstract

Neurodevelopmental disorders (NDDs) comprise a heterogeneous spectrum of disorders with diverse manifestations, such as microcephaly, structural brain abnormalities, epilepsy, developmental delay, intellectual disability, and autism spectrum disorders [1]. Although relatively rare, each type of NDDs represents a significant population of neurological patients. The global prevalence of NDDs exceeds 15% [2]. NDDs typically arise from molecular cascades that are highly regulated and disrupted by either gene mutations or environmental factors. The genetic basis of a substantial proportion of such disorders is hard to discern given that not all are inherited according to Mendelian principles and involve allelic variations from multiple genes. However, roughly 40% of NDDs are believed to be caused by the disruption of a single gene, indicating monogenic conditions. [3]

Understanding and predicting the physiological function of a protein encoded by a specific gene, determining its interactions with other proteins, and investigating the role of the gene in organ and tissue development requires a close examination of its expression. Thus, a crucial initial step in researching genes associated with neurodevelopmental disorders is to investigate the expression patterns of these genes in the mouse brain at various embryonic developmental stages.

In situ RNA hybridization was used to analyze gene expression patterns in slices of mouse brain tissue. Fixed in 4% paraformaldehyde/phosphate-buffered saline/diethylpyrocarbonate mouse brain samples at embryonic (E12.5, E15.5, E18.5) and postnatal (P1, P21) developmental stages were sectioned using a Leica CM1520 cryostat with 15 µm slice thickness. Next, we conducted in situ hybridization of cellular mRNA using DIG-dUTP-labeled RNA probes that were previously synthesized by PCR with cDNA and gene-specific primers. 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) was used, which produces an insoluble dark blue or purple sediment visible under a light microscope by reaction with alkaline phosphatase, to visualize the localization of mRNA expression in tissues.

The study examined the expression of a member of the CCDC gene family which encodes proteins involved in intercellular transmembrane signal transduction. In situ hybridization was performed on mouse brain slices, revealing significant mRNA expression of the gene in the cerebral cortex. Additionally, mouse knockout experiments are planned to investigate the gene’s role in brain development.

Genes & Cells. 2023;18(4):494-497
pages 494-497 views

Analysis of microRNA expression in rat retina at the early stages of retinopathy development

Kozhevnikova O.S., Kolosova N.G.

Abstract

Age-related macular degeneration (AMD), a neurodegenerative eye disease, is the primary cause of blindness globally, especially affecting the elderly population. MicroRNAs, a type of single-stranded, non-coding RNAs of about 22 nucleotides in length, primarily regulate gene expression negatively. Alterations in the miRNA profile during AMD’s development could potentially aid in early detection and progression monitoring of the disease. However, no data are available on the microRNA expression patterns in the retina during the early stages of AMD. Investigating the molecular mechanisms that underlie age-related retinal degeneration at an early stage, prior to the manifestation of any symptoms, could provide insights into the molecular events that initiate the irreversible stage. In the case of OXYS rats with accelerated aging, dystrophic changes in the RPE, neuroretinal thinning, and impaired choroidal microcirculation — the primary indicators of the dry form of AMD — spontaneously occur by the age of 3 months [1]. A comparative analysis was conducted in this study on microRNA expression in the retinas of OXYS rats at two different stages of retinopathy — 20 days (preclinical) and 3 months (manifestation) — as well as in control Wistar rats. Small RNA-seq sequencing was performed on the DNBSEQ platform.

According to differential expression analysis (DESeq2, q-value <0.05), at 20 days of age, 2 microRNAs exhibited altered expression in OXYS rats compared to Wistar rats. Similarly, at 3 months of age, the expression of 16 microRNAs in OXYS rats was altered compared to Wistars, with 7 miRNAs showing increased levels and 9 miRNAs showing decreased levels. Analysis of age-related changes in miRNA expression demonstrated that in the OXYS rat retina between 20 days and 3 months, the levels of 134 miRNAs altered: 59 miRNAs increased, and 75 miRNAs decreased. Over the course of 20 days to 3 months, 94 miRNAs underwent alterations in the retinas of Wistar rats. Among these, the level of 48 miRNAs increased while the level of 46 miRNAs decreased. The RNAhybrid, miRanda, and TargetScan programs were used to search for target genes of differentially presented miRNAs among different groups. Following this, gene ontologies were studied. In comparison to Wistar rats, “adhesion contacts” and “synaptic vesicle cycle” categories are noticeably populated with target genes of miRNA DE at 20 days in OXYS rats. MiRNA target genes with lower levels at 3 months in OXYS rats compared to Wistar rats are significantly enriched in categories such as vascular branching, extracellular matrix organization, adhesion, and protein deubiquitination. Target genes with increased miRNA levels at 3 months in OXYS rats, compared to Wistar rats, are significantly represented in various signaling pathways, such as mTOR, MAPK, VEGF, and thyroid hormone. Target genes, which serve as common targets for at least 10 microRNAs (miRNAs), were chosen for analysis of the targetome subject to regulation by miRNA molecules exhibiting modified expression. Targetomes containing 400 to 600 genes were obtained for miRNAs that increased or decreased levels in both OXYS and Wistar rats between 20 days and 3 months. Notably, these targetomes were enriched in categories such as axonogenesis, retinal layer formation, visual learning, focal adhesion, and endocytosis. Profiling of 84 miRNAs of the retina of OXYS and Wistar rats at the age of 20 days and 3 months was carried out using PCR panels (Qiagen) to verify the results of small RNA-seq. Our analysis revealed highly convergent results between sequencing and PCR panels. Thus, 16 miRNAs showing altered expression in OXYS retina may serve as potential biomarkers and new targets for studying AMD pathogenesis.

Genes & Cells. 2023;18(4):498-501
pages 498-501 views

Study of the role of evolutionary new enhancers in the development of the corpus callosum

Kustova A.O., Celis Suescun J.C., Rybakova V.P., Tarabykin V.S.

Abstract

One important aspect of the mammalian brain is the exchange of information between neurons located in different hemispheres. This process evolved during the development of mammals. In marsupial (Marsupialia) and monotreme (Monotremata) mammals, the communication between hemispheres is facilitated through an enlarged anterior commissure. In placental (Eutheria) mammals, a new brain structure, the corpus callosum, emerged during the evolutionary process. The corpus callosum, comprising 80% of the brain’s commissural axons, is the largest commissure in the human body. The corpus callosum is a major contributor to the efficiency of higher neural activities, including memory, decision making, social interaction, and language. A possible explanation for the emergence of a novel structure for interhemispheric interaction is a change in the growth direction of neocortical axons during development. Changes in gene expression levels can regulate this process, which involves controlling axon growth and cell migration in the neocortex. A full comprehension of this process will enable the creation of new animal models for studying cortical malformations and the navigation of axons in the cerebral cortex leading to the formation of interhemispheric connections.

Several enhancers were identified for protein-coding genes with differing expression patterns in neocortical cells of placental and non-placental (marsupial) mammals. Throughout the genomes of the house opossum (Monodelphis domestica) and the house mouse (Mus musculus), the acetylation levels of histone H3 on lysine 27 (H3K27ac) were compared. H3K27ac is considered an epigenetic marker for active gene enhancers. Then, a screening of candidate genes was performed to evaluate their localization and expression levels in the cortex during embryonic development. Thus, the Tbr1 gene was identified. Incorrect expression of this gene may result in changes to cortical development.

The CRISPR/Cas9 system was combined with in utero electroporation to completely delete the active Tbr1 gene enhancer in developing neocortical cells of mouse embryos at day 14 of embryonic development. The impact of this enhancer deletion was then analyzed on the expression of Tbr1 in the upper layers of the cortex, as well as the direction of axon growth and neuronal migration on the 18th day of embryonic development.

A significant reduction in Tbr1 expression was observed in the upper layers of the cortex after deletion of the active enhancer. Only 30% of the electroporated neurons retained Tbr1 expression. Moreover, a considerable delay in neuronal migration was observed in the subventricular zone (41% versus 17% in the control group) and in the upper layers of the cortex (20% versus 35% in the control group). However, the direction of axonal growth remained unchanged: callosal axons effectively crossed the midline and created the corpus callosum.

Thus, expression of the evolutionary novel Tbr1 enhancer is important for neuronal migration during corticogenesis. However, its contribution to the development of the corpus callosum is not fully understood. A detailed analysis of the corpus callosum morphology post-enhancer deletion will be the subsequent step.

Genes & Cells. 2023;18(4):502-503
pages 502-503 views

Effects of optogenetic astroglia activation in modulation of synaptic transmission and rhythmogenesis of the hippocampus

Lebedeva A.V., Maltseva K.E., Sokolov R.A., Barabash N.V., Levanova T.A., Rozov A.V.

Abstract

In the field of neuronal signal transduction research, astroglia have become increasingly important. Astrocytes, or astroglial cells, are recognized as the third element in synaptic transmission regulation. This complex is conventionally known as a tripartite synapse. A multitude of mathematical models are being developed to investigate the function of astroglia in the tripartite synapse. Neurobiological experiments were carried out to confirm the dynamics observed in the previously proposed mean field activity mathematical model [1]. The experiments focused on evaluating the effect of optogenetic activation of astrocytes on synaptic transmission in C57BL/6 inbred mouse hippocampal slices. One month prior to the experiments, the AAV GFAP ChR2 eYFP virus was injected into the lateral ventricles of the brains of the experimental mice. This procedure was crucial for the expression of astrocyte-specific photosensitive channels, also known as channel rhodopsin. The patch-clamp method was used to conduct experiments on both the experimental mice and a control group of mice that did not receive virus injections. Spontaneous neuronal activity (local field potentials) and synaptic currents (GABA currents) were simultaneously recorded in the experiments. After activating astrocytes that expressed the photosensitive channel, an increase in GABAergic currents became evident during the transmission of synaptic signals between neurons. This suggests that astrocytes likely modulate synaptic transmission by releasing a gliotransmitter into the synaptic cleft. Thus, the study confirmed the presence of mean field activity patterns in a phenomenological model that describes the dynamics of a population of neurons. This model was developed based on the Tsodyks–Markram model and considers the primary attributes of neuron-glial interaction through a tripartite synapse. The model includes the short-term synaptic plasticity of the Tsodyks–Markram model and the astrocyte potential of synaptic transmission. The activation of astrocytes results in a diverse range of dynamic modes that describe various patterns of network activity under the mean field approach framework.

Currently, multiple experimental hypotheses exist regarding astrocytes’ release of a gliotransfer into the synaptic cleft and its specific type. Alternatively, a complex cascade of sequential activation of glutamateergic and GABAergic receptors may occur. Additional experimental work is necessary to assess the pharmacological contribution of channels and transporters involved in modulating synaptic transmission during astrocyte optogenetic activation.

The obtained results will refine the mathematical model of mean field neuronal activity to increase its biological plausibility. The methodology used involves identifying sparse nonlinear dynamical systems from data by solving for the system’s equations of motion [2]. These equations are reconstructed from noisy measurement data, allowing for a more accurate representation of the dynamic system. The only assumption regarding the arrangement of a dynamical system is that there exist only a handful of significant factors regulating the dynamics, leading to equations that are sparse within the realm of potential functions. Sparse regression is used to ascertain the minimum number of terms required in dynamic equations for precise data representation. This strategy enables the construction of mathematical models that are both as accurate as feasible, and maximally uncomplicated, eliminating the need for retraining. Notably, this method is suitable for parameterized systems and systems subjected to external influences or changes over time. The two approaches were used to forecast the dynamics of the mean field of a neural population. The accuracy of the forecast was subsequently evaluated.

Genes & Cells. 2023;18(4):504-507
pages 504-507 views

Role of RIPK1 kinase in neuronal-glial network adaptation under hypoxic conditions

Loginova M.M., Yarkov R.S., Vedunova M.V., Mitroshina E.V.

Abstract

Cerebral hypoxia is a condition characterized by a reduced oxygen supply to tissues that plays a crucial role in the pathogenesis of numerous neurodegenerative diseases. In hypoxia, intracellular signaling cascades are activated, ultimately leading to various forms of nerve cell death. The initiation of necroptosis under hypoxic conditions is governed by PIPK1 kinase, and its inhibition could potentially offer neuroprotection against hypoxic damage [1–3]. Studies investigating the effects of blocking RIPK1 kinase on the activity of neuronal-glial networks are currently lacking. Consequently, RIPK1 kinase constitutes a promising objective for further exploration. Thus, the aim of this work is to examine the part played by RIPK1 kinase in the adjustment of neuronal-glial networks amid hypoxia.

The study focused on primary cultures of nerve cells in the hippocampus of mouse embryos belonging to the C57Bl/6 line. Hypoxia was induced in vitro on the 14th day of culturing the nerve cells. The RIPK1 kinase inhibitor was administered 20 minutes prior to, during, and after the hypoxia intervention. After 7 days of the stress induction, the calcium and bioelectrical activity of the neuron-glial networks were evaluated. Calcium activity was assessed via the Oregon Green 488 BAPTA-1, AM (Thermo Fisher Scientific, USA) using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss, Germany). The experiments assessed total percentage of oscillating cells in culture, the frequency, and duration of calcium events. The analysis of bioelectrical activity was conducted using the MEA 60 multi-electrode arrays from Multichannel Systems. The registered signal from the arrays was processed through the MEAMAN algorithms in MATLAB (Certificate of State Registration of Computer Program No. 2012611190). The average number of small network packages and spikes was estimated.

Under physiological conditions, spontaneous calcium activity is observed by the 21st day of neuron-glial network development. The percentage of cells exhibiting calcium events is 60.64 ± 3.68%, the frequency of calcium oscillations is 1.52±0.22 osc/min, and the duration is 9.63±0.75 s. During hypoxia modeling, the percentage of cells exhibiting calcium events decreased to 34.77±4.08%, and the frequency of calcium oscillations decreased to 0.64±0.08 osc/min. The inhibition of RIPK1 kinase maintains the percentage of cells exhibiting calcium events at the level of intact cultures, which is 60.38±3.4%.

By the 21st day of culturing primary nerve cell cultures under physiological conditions, spontaneous bioelectrical activity forms. This is indicated by parameters such as the average number of small network clusters and spikes. Hypoxia modeling has a negative impact on the development of spontaneous bioelectrical activity. In the “Intact” group, the average number of small network packs was 36.12±4.27 packs per 10 minutes, while in the cell culture with hypoxia, it was 15.87±3.03 packs per 10 minutes. Additionally, the “Intact” group had an average of 90.22±12.32 spikes, while the cell culture with hypoxia had only 11.58±4.7 spikes. However, blocking RIPK1 kinase during hypoxia preserved the average number of small network packs (23,49±2,14 packs/10 min).

Thus, inhibiting RIPK1 kinase under hypoxic conditions preserves the proportion of cells that exhibit spontaneous calcium events and partially preserves spontaneous bioelectrical activity.

Genes & Cells. 2023;18(4):508-511
pages 508-511 views

Chemogenetic emulation of intraneuronal oxidative stress affects synaptic plasticity

Maltsev D.I., Kalinichenko А.L., Jappy D., Solotenkov M.A., Solius G.M., Mukhametshina L.F., Elesina E.A., Sokolov R.A., Tsopina A.S., Fedotov I.V., Moshchenko A.A., Fedotov A.B., Shaydurov V.A., Rozov A.V., Podgorny O.V., Belousov V.V.

Abstract

Overproduction of reactive oxygen species (ROS) and oxidative cell damage are commonly associated with most brain pathologies [1, 2]. Dysregulation of redox homeostasis in the aging brain is thought to be responsible for impaired synaptic transmission and plasticity, leading to reduced neuronal computational capacity and learning and memory deficits. Studying the contribution of oxidative stress to the development of diseases, such as age-related dementia and Alzheimer’s disease, is complex due to the lack of methods for modeling isolated oxidative damage in individual cell types [3]. We introduce a chemogenetic approach utilizing D-amino acid oxidase (DAAO) from yeast to produce hydrogen peroxide intraneuronally, which is one of the most stable ROS [4]. H2O2 generation was evaluated in primary cultured neurons and acute mouse brain slices through the utilization of a genetically encoded fluorescent biosensor, HyPer7, to validate the methodology [5]. The changes in the fluorescence signal of HyPer7 after treating neurons that expressed DAAO with D-Norvaline (D-Nva), a substrate for DAAO, confirmed the targeted production of H2O2 through chemogenetics. Using electrophysiological recordings in acute brain slices, we demonstrated that intraneuronal oxidative stress induced by chemogenetics did not affect basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals. However, it diminished long-term potentiation (LTP) at the single-cell level.

Astrocytes have the ability to metabolize d-amino acids, rendering the proposed approach ineffective in vivo experiments. Consequently, in vivo testing of the tool was necessary for validation. To achieve this, an optical setup for exciting and detecting the HyPer7 signal was developed and implanted into the mouse brain via optical fibers. By using this approach, we were able to demonstrate the generation of H2O2 in DAAO-expressing neurons in vivo, upon intraperitoneal administration of D-amino acids. The results demonstrate that using a DAAO-based chemogenetic tool, along with electrophysiological recordings, clarifies numerous unanswered queries regarding the part of ROS-dependent signaling in typical brain activities and the impact of oxidative stress on the development of cognitive aging and preliminary neurodegenerative stages. The suggested method is valuable for detecting initial indicators of neuronal oxidative stress. Additionally, it can be used for evaluating probable antioxidants that can effectively combat neuronal oxidative harm.

Genes & Cells. 2023;18(4):512-515
pages 512-515 views

The effect of HSP70 overexpression on the cerebral cortex development

Mitina N.N., Ambrozkevich M.K., Motorina A.O., Tarabykin V.S.

Abstract

Heat shock proteins (HSPs) make up a large family of molecular chaperones, recognized for their role in protein maturation, refolding, and degradation. HSP70 was shown to promote cell survival during several pathological processes in the brain, such as stroke, neurodegenerative diseases, epilepsy, and trauma [1]. In addition, HSPs serve to promote the proper embryonic and postnatal development of many organ systems, such as the central nervous system [2]. Heat shock proteins demonstrate specific expression patterns throughout the development of the nervous system, notably during crucial embryonic and postnatal moments [1].

During embryonic development, neural and glial progenitors must survive within a hypoxic microenvironment while performing energetically demanding actions, such as cell migration and neurite outgrowth. HSPs can activate or inhibit development pathways in the nervous system that modulate cell differentiation, neurite outgrowth, cell migration, or angiogenesis [1].

Indeed, recent studies demonstrated that HSP70 directly regulates the development of the nervous system by modulating signaling cascades involved in cell growth and migration [3]. Additionally, research demonstrated [4] that introducing HSP70 from an external source significantly augments the populations of proliferating cells and differentiated neuroblasts within the mouse hippocampus. Nevertheless, some researchers [1] contend that overexpression of HSPs may negatively impact cell survival. Therefore, the precise role of these chaperones remains largely unexplored.

In our study, we used in utero electroporation to introduce plasmids that controlled HSP70 overexpression into neuron progenitors of mouse embryos on the 14th day of gestation. Additionally, plasmid DNA encoding GFP was used to facilitate subsequent visualization of transformed cells. Brain samples were collected on the 18th day of gestation for immunohistochemical analysis of the sections. Confocal microscopy was used to compare the characteristics of neuronal migration in both control and HSP70 overexpression conditions.

Cells that received plasmids inducing HSP70 were discovered to migrate at a lower pace in comparison to the control. Additionally, it is hypothesized that the induction of HSP70 expression could lead to neuronal malformations and impact the development of neurites.

In the future, the study of cytoarchitectonics in the cortex will continue, examining the identification of electroporated cells in individual neuron populations using Satb2 and Ctip2 markers. Additionally, the differentiation of these cells will be assessed by counting those that have not exited the mitotic cycle, and the hypothesis of apoptosis induction in cells electroporated with HSP70 will be tested.

Genes & Cells. 2023;18(4):516-519
pages 516-519 views

Is alteration of ERK1/2 and p38MAPK signaling pathways activity a general mechanism of Alzheimer’s disease and age-related macular degeneration development?

Muraleva N.A.

Abstract

Age is the primary risk factor for Alzheimer’s disease (AD), the most prevalent progressive senile dementia, and age-related macular degeneration (AMD), the main cause of vision loss in individuals over 60. Effective prevention and treatment of these neurodegenerative conditions are lacking due to incomplete knowledge of their pathogenesis. Risk factors such as smoking, hypertension, hypercholesterolemia, atherosclerosis, obesity, and malnutrition intersect with the pathogenesis mechanisms of both AD and AMD. Oxidative stress, inflammation, mitochondrial dysfunction, and impaired proteostasis maintenance are among the pathological factors associated with the aggregation and accumulation of abnormal extracellular deposits — senile plaques in the brains of AD patients and drusen in AMD patients. Alterations in the regulation of MAPK signaling pathways with age may be associated to the emergence of these abnormalities. Impaired MAPK signaling was confirmed as a significant contributor to the pathogenesis of AD through the results of numerous investigations. MAPK is a potential target for therapeutic interventions. Information on its age-related changes, however, is limited. Virtually no data exist on retinal MAPK activity during AD and AMD development. This study aims to compare changes in ERK1/2 and p38MAPK signaling activity in brain and retina structures with age and AD/AMD progression. Wistar and OXYS rats were used as models of accelerated aging that show concomitant signs of AD and AMD.

At the initial stage of the study, we examined the gene expression related to ERK1/2 and p38MAPK signaling pathways via comparing the transcriptomes (RNA-seq data) of the retina, prefrontal cortex, and hippocampus of OXYS and Wistar rats. The analysis was conducted when the rats were 20 days old and showed no signs of AMD and AD symptoms, during their manifestation period (age 3–5 months) and active progression stage (age 18 months). Changes in gene expression, involved in EPK1/2 [1] and p38MAPK signaling pathways according to the Rat Genome Database, are tissue-specific and dependent on the animal genotype. In the retina, the number of genes associated with ERK1/2 and p38MAPK decreased in both Wistar and OXYS rats with age and was minimal at 18 months of age. The greatest differences in the expression levels of these genes were detected when the rats were 20 days old, at the preclinical stage of AMD-like pathology development in OXYS rats. Both activators and inhibitors of the EPK1/2 and p38MAPK pathways were present among the differentially expressed genes (DEGs) with increased mRNA levels. At 3 months, the number of DEGs decreased, while activator genes predominated among those with increased mRNA levels. At 18 months, the retina of OXYS rats displayed only signs of decreased activity in signaling pathways at the gene expression level.

Age-related changes in gene expression profiles related to the ERK1/2 [2] and p38MAPK [3, 4] signaling pathways in Wistar rats differed between brain structures and the retina. In the prefrontal cortex and hippocampus, the number of genes increased with age, and the expression differed between OXYS and Wistar rats. However, no signs of significant activation of the signaling pathway were observed.

The phosphorylation level of ERK1/2 and p38MAPK signaling pathway kinases serves as an objective indicator of their activity. For protein level evaluation via Western blot analysis, we examined these levels in the retina, prefrontal cortex, and hippocampus. In both rat strains, a significant increase in phosphorylated forms of ERK1/2 and p38MAPK was observed with aging in all examined tissues. Simultaneously, accumulation of the substance became more active in OXYS rats and increased with progression of AD and AMD symptoms.

Therefore, the results indicate that no significant changes in the activity of ERK1/2 and p38MAPK signaling cascades in the retina and brain structures are observed during normal aging of Wistar rats and accelerated aging of OXYS, both at the gene expression and protein level. AD and AMD pathological signs in OXYS rats were found to be associated with increased phosphorylation of ERK1/2 and p38MAPK, suggesting that enhancement of MAPK pathways may be a common mechanism for the early development of AD and AMD.

Genes & Cells. 2023;18(4):520-523
pages 520-523 views

Differentiation therapy as a new multidisciplinary approach to the treatment of human brain glioma

Pavlova G.V., Kolesnikova V.A., Usachev D.Y., Kopylov A.M.

Abstract

Glioblastoma is among the most severe forms of neoplastic disease in the human body, with a highly unfavorable prognosis. The annual incidence of this pathology in the population is 3.5 cases per 100,000. Currently, there are no truly effective treatments for this malignant variety of brain tumor. All known treatment methods, including surgery, radiation therapy, and chemotherapy, provide only a modest extension of the patient‘s lifespan. The heterogeneous structure of glioblastoma, characterized by abnormal regulation of cell proliferation, enables the tumor to withstand diverse therapeutic interventions. Most tumor cells die with radiation therapy or chemotherapy, but a small number of cells are resistant, leading to tumor relapse. Therefore, tumors are able to resist different types of therapy and continue to grow. The discovery of therapy failures highlights the need to search for new approaches in the treatment of glioblastoma. Glioma is comprised of tumor stem cells along with their immature progenitor cells, known as “daughter tumor cells”. All therapeutic approaches that induce cell death to treat this disease may contribute to necrosis in both cancer cells and healthy, actively dividing cells, which may explain treatment failure. Simultaneously, stem cells in poorly dividing tumors resist these effects and survive, ultimately leading to the emergence of a recurrent tumor. In contrast to utilizing cytotoxic effects which is a strategy employed, the alternative approach is to stimulate the „maturation“ of tumor cells, with the goal of losing their ability to proliferate. We propose a new treatment approach for glioma, called “differentiation therapy”. This therapy has a cytostatic effect on tumor cells by using the aptamer biG3T, which blocks their proliferation. Inducer molecules such as SB431542, LDN-193189, Purmorphamine, and BDNF are added subsequently to control neurogenesis pathways. The aptamer bi(AID-1-T) exhibits a cytostatic effect, halting the division of tumor cells without inducing cell death or necrosis. This temporary pause in proliferation sensitizes tumor cells to external influences, promoting their differentiation or maturation. Inductor molecules such as SB431542, LDN-193189, Purmorphamine, and BDNF are commonly used to influence cascades of induced pluripotent cells (iPSCs) for their differentiation into neurons. In cases of differentiation therapy featuring a temporary decrease in tumor cell proliferation levels post-aptamer exposure, inducer molecules possess the ability to steer tumor cells towards maturation. Differentiation therapy was found to be effective in targeting tumor stem cells that are resistant to chemotherapy and radiation therapy, specifically the Nestin and PROM1 (CD133)-positive cells. Studies conducted on cell cultures of gliomas demonstrated the efficacy of this approach in vitro, particularly in patients with high-grade malignancies. In order to achieve an optimal and effective combination of aptamer and factors, we conducted a series of in vivo studies using a rat model implanted with tissue glioblastoma 101/8. When using a combination of differentiation therapy factors in vivo, it‘s imperative to adjust the introduction of such factors to achieve optimal results. The introduction sequence of catheter administration of these therapy factors was found to significantly impact the size of tumors, with either complete tumor disappearance or insignificant size observed. Promising results were shown in animal model pilot studies involving glioblastoma treated with this method.

Genes & Cells. 2023;18(4):524-527
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Genomic studies of neudegeneration in Parkinson’s disease associated with glucocerebrosodase dysfunction on cell and animals models

Pchelina S.N., Bezrukova A.I., Rudenok M.M., Zhuravlev A.S., Rybolovlev I.N., Baydakova G.V., Nesterov M.S., Abaimov D.A., Usenko T.S., Zakharova E.Y., Emelyanov A.K., Shadrina M.I., Slominsky P.A.

Abstract

Mutations in the glucocerebrosidase gene (GBA1), which encodes the lysosomal enzyme glucocerebrosidase (GCase), can cause Gaucher disease, an autosomal recessive disease, and increase the risk of Parkinson’s disease (PD). The risk of developing PD for carriers of homozygous and heterozygous GBA1 mutations increases by 8–10 times, but not all carriers develop PD during their lifetime. Additionally, GBA-associated PD (GBA-PD) represents 10 to 30% of all forms of parkinsonism. The development mechanism of GBA-PD remains unknown. A decrease in GCase activity and accumulation of lysosphingolipids in patients with GBA-PD was shown by us and other researchers [1, 2]. GCase dysfunction is thought to result in impaired autophagy and accumulation of the alpha-synuclein protein, which is a crucial process in neurodegeneration in PD.

Several techniques based on modeling parkinsonism in mice with GCase dysfunction were used to study the impact of GCase dysfunction on DA neuron neurodegeneration [3, 4]. In this study, we evaluated GCase activity, lysosphingolipids level, and the degree of neurodegeneration in DA-neurons of the substantia nigra’s compact (SNc) and reticular part (SNr), as well as the levels of dopamine and alpha-synuclein (total and oligomeric) in the brains of mice with a double “soft” neurotoxic model induced by the introduction of the neurotoxin 1-methyl-4-phenyl-1. This is the first time such an evaluation has been made. The presymptomatic stage of parkinsonism induced by 2,3,6-tetrahydropyridine (MPTP) involved the double administration of 12 μg/kg at a 2-hour interval, in combination with a single injection of the selective GCase inhibitor conduritol-B-epoxide (CBE) at a dose of 100 mg/kg. Additionally, we compared the transcriptomes of primary macrophage cultures from GBA-PD patients [5] with the transcriptome of SN brain tissue in model mice.

We demonstrated that a singular injection of CBE resulted in a 50% decrease in GCase activity in the mouse brain and an elevation in lysosphingolipid levels. Additionally, the introduction of both MPTP and CBE led to an increase in the level of oligomeric forms of alpha-synuclein in the striatum. Simultaneously, degeneration of DA neurons in SNc, assessed by tyrosine hybroxylase (TH) immunohistochemistry 14 days after injection, was comparable to MPTP and CBE (decreasing to 50 and 60%, respectively). The neurotoxic model, when combined, demonstrates a significantly greater reduction in dopamine concentration, accumulation of total alpha-synuclein in the striatum, and more severe neurodegeneration of DA neurons in SNr (70% compared to 45% with MPTP administration).

A comparison of differential gene expression in primary macrophage cultures from patients with GBA-PD and controls revealed a reduction in the expression of genes associated with neurogenesis, such as JUNB, NR4A2, and EGR1. In both the GBA-PD patient group (TRIM13, BCL6) and the MPTP-induced parkinsonism mouse group with GCase dysfunction (MPTP+CBE), genes related to the PI3K-Akt-mTOR signaling pathway, which regulates autophagy, were found to be activated. These genes include Pdk4, Sgk, and Ppp2r3d.

The data obtained indicates that dysfunctional GCase leads to the accumulation of toxic forms of alpha-synuclein and degeneration of DA neurons, similar to the effects of small doses of MPTP. Combining neurotoxins (MPTP+CBE) causes a greater accumulation of alpha-synuclein and a higher degree of neuron degeneration. Transcriptomic analysis conducted on GBA-PD patients’ cells and a combined neurotoxic mouse model (MPTP+CBE) brain revealed modifications in gene expression of autophagy regulation. Approaches focused on enhancing GCase activity and autophagy exhibit potential in developing neuroprotective agents.

Genes & Cells. 2023;18(4):528-531
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Febrile seizures cause a decrease in calcium-permeable AMPA receptors at synapses of rat cortical and hippocampal pyramidal neurons

Postnikova T.Y., Griflyuk A.V., Zhigulin A.S., Soboleva E.B., Barygin O.I., Amakhin D.V., Zaitsev A.V.

Abstract

Febrile seizures (FS) are a prevalent childhood neurological disorder that can result in lasting functional alterations in neural networks, contributing to the onset of epilepsy and cognitive impairment [1]. Higher levels of calcium-permeable AMPA receptors (CP-AMPARs), which lack the GluA2 subunit, are detected in the hippocampus and cortex at an early age. CP-AMPARs are involved in various plastic changes within the central nervous system (CNS), including regular physiological processes (synaptic plasticity) and various pathological conditions. Such changes occur when these receptors are included in the neurons’ membrane, where they are not typically expressed. CP-AMPARs were demonstrated to incorporate into synapses during seizures [2]. The effect of FSs on CP-AMPAR expression is uncertain, and the resultant alterations in neuronal network function are unclear.

The aim of this study was to determine whether the proportion of CP-AMPARs at synapses of pyramidal neurons in the rat entorhinal cortex and hippocampus alters immediately (at 15 min) and 48 h post FS.

Ten-day-old rats were exposed to a stream of warm air (46 °C) for 30 minutes to induce hyperthermia, resulting in the development of FS. Only animals with FS that lasted for a minimum of 15 minutes were included in the study. The control group was comprised of littermates removed from the dam for an equivalent period but kept at room temperature. Isolated pyramidal neurons were used to determine the proportion of CP-AMPARs in the hippocampus. AMPAR-mediated currents were induced by application of 100 μM kainate. Excitatory postsynaptic currents (EPSCs) were evoked by extracellular stimulation in the entorhinal cortex. The antagonist IEM-1460 was used to selectively block CP-AMPARs. The rectification index of AMPA-mediated EPSCs was calculated to better assess the contribution of CP-AMPARs. Neurons expressing CP-AMPARs were visualized using the kainate-induced cobalt uptake method. Brain slices were stimulated with kainate while AR-5 and TTX were present. The DNQX blocker was used for the determination that the influx of Co2+ was mediated by AMPARs. Basal synaptic transmission was assessed by recording field postsynaptic responses in the hippocampus stimulated by Shaffer collaterals at different current strengths.

FS induced a rapid decrease in the levels of CP-AMPARs on the membranes of pyramidal neurons in the hippocampus. As a result, 15 min after FS, IEM-1460 caused significantly less blockade of kainate-evoked current in neurons isolated from rat hippocampus compared to control (22% vs. 14%, p <0.05). A similar finding was observed for EPSCs evoked extracellularly in the entorhinal cortex, with a frequency of 10% in the FS group compared to 3% in the control group (p <0.05). Furthermore, the FS group’s neurons exhibited a significantly greater rectification index of EPSCs compared to the control group’s neurons. However, two days post-FS, no significant differences existed between the two groups. This observation may be attributed to the rapid decrease in the proportion of CP-AMPARs in pyramidal neurons at this stage. The cobalt uptake method supported electrophysiological findings, revealing higher staining levels in the CA1 field of the hippocampus and entorhinal cortex of control rats compared to FS rats. The observed effect surfaced 15 minutes after FS, and no divergences emerged between the groups after two days. Although the proportion of CP-AMPARs was reduced, basal neurotransmission levels in brain slices obtained from rats that underwent FS did not differ from control values.

In summary, the expression of CP-AMPARs in entorhinal cortex and hippocampal pyramidal neurons in young rats decreases significantly with FS. These alterations do not impact the characteristics of basal synaptic transmission in the hippocampus.

Genes & Cells. 2023;18(4):532-535
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Expansion microscopy for visualization of protein clusters in cultured cells and brain tissues

Rakovskaya A.V., Chigriai M.E., Pchitskaya E.I., Bezprozvanny I.B.

Abstract

Many biological studies necessitate high-resolution imaging and further analysis of cellular organelles and molecules. Expansion microscopy (ExM) enables achieving nanometer-level resolution with a standard fluorescence microscope by physically expanding the sample in the gel by multiple factors. In the present study, ExM was used to examine protein clusters of STIM1, a calcium-binding protein, and IP3R, a calcium-gated channel (inositol triphosphate receptor). The endoplasmic reticulum (ER) calcium sensor STIM1 translocates to the ER-plasma membrane junctions, forming clusters to activate store-operated calcium entry (SOCE) upon ER calcium decrease [1]. Expansion microscopy provides the advantage of expanding the sample in all three axes, including the Z-axis, enabling detection of premembrane proteins without relying on TIRF microscopy. STIM1 interacts with end-binding protein 1 (EB1) located at the plus ends of microtubules, which regulates SOCE. This study presents a quantitative approach for analyzing protein clusters using expansion microscopy. STIM1 and its non-EB-binding mutant, STIM1-TR/NN, were used as examples.

In endothelial cells, Ca2+ is released into the cytoplasm from the ER through the main channels of Inositol-1,4,5-triphosphate receptors (IP3R). A previous study demonstrated that these receptors interact with the EB protein through the SxIP amino acid motif, similarly to STIM proteins, regulating clustering and calcium signaling [2]. In hippocampal neurons, the type 1 IP3 receptor forms clusters required for efficient calcium release through the channel in response to stimuli. To investigate the function of IP3 receptor isoform 1 in the brain, IP3R clusters were analyzed in wild-type mice and 5xFAD mice modeling Alzheimer’s disease (AD) since this receptor was observed to exhibit increased activity during this pathological condition [3].

HEK293T cells at 50–70% confluence underwent transfection using mCherry-STIM1 and mCherry-STIM1-TR/NN plasmids to assess the clustering of STIM1 proteins. Cells were fixed and stained with primary mCherry protein antibodies and secondary antibodies conjugated to the Alexa Fluor 594 fluorophore to enhance fluorescence. Next, the cells underwent isotropic expansion in the gel using expansion microscopy. The ExM method was executed following the protocol outlined by Asano et al. [4]. The sample was expanded through dual addition of sterile, distilled water for a period of 20 minutes.

To examine the clustering of IP3R proteins, we obtained frontal slices of the brains of two groups of mice: control and 5xFAD (a mouse model for Alzheimer’s disease), which were 40–50 microns thick. Brain slices were immunohistochemically stained with IP3R1 primary antibody and Alexa Fluor 488 secondary antibody followed by expansion microscopy protocol [4]. The ImageJ and Icy software were used for processing images. Using ImageJ, the neurons’ intensity was determined, leading to the formation of three groups with different fluorescence intensity levels. A certain binarization threshold was implemented in Adaptive3DThreshold, depending on the level. The grouping approach enabled neutralizing the effect of potential disparities in IP3R protein expression levels on the analysis of differences in fluorescence intensity.

According to the literature data, analysis of the results indicates that when the bond with tubulin microtubules is disrupted, STIM1 aggregates more when the calcium store is depleted in comparison to its standard STIM1 variant [5]. Upon evaluation of IP3R cluster morphometric parameters in transgenic mice compared to the control group, we observed an increase in the size and number of IP3R protein clusters in mice of the 5xFAD line within the groups exhibiting the highest neuronal intensity and the groups with the highest and average neuronal intensity. The Western blot method was used to validate the results and demonstrated overexpression of the IP3R protein in 5xFAD mice. This study represents the first time that IP3R protein clusters aggregate more in a mouse model of AD, which is congruent with the literature regarding high IP3R activity in AD [3].

Genes & Cells. 2023;18(4):536-539
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An electronic key to the mysteries of astrocytes

Rogachevsky V.V., Shishkova E.A.

Abstract

Perisynaptic astrocytic processes (PAP) involved in the tripartite synapse respond to local depolarization activation with calcium release from intracellular stores inside astrocytic process nodes, resulting in local and generalized calcium events [1, 2]. Electrophysiology and imaging experiments demonstrate the existence of Ca2+ stores in astrocytic peripheral processes. However, the initial electron microscopic studies suggested that terminal astrocytic processes (TAP), in contact with synapse and located near the axon-spine interface, lack organelles, including the main astrocytic Ca2+ store — the cisternae of the smooth endoplasmic reticulum (sER) [3]. The Ca2+-dependent release of gliotransmitters in vivo is highly doubtful. This is due to several factors. These include the weak electron contrast of sER cisternae which restricts their detection and analysis, the examination of astrocytic processes on single sections, and the limited optical resolution of the equipment used.

Here, we conducted the first comprehensive analysis of TAP in murine hippocampal and cortical synapses, using serial section transmission electron microscopy and 3D reconstructions. The use of alternative approaches to brain tissue fixation and ultrathin staining allowed to increase the contrast of subunit membranes, such as those in sER cisternae of neurons and astrocytes [4, 5]. However, this technique increased the contrast of individual glycogen granules, which obscured cross-sections of sER cisternae due to their similarity in appearance to glycogen granules. To overcome this, we used the rapid disassembly of glycogen during non-anesthetic euthanasia to reveal sER cisternae in astrocyte processes. The removal of glycogen granules from astrocyte sections allowed the clear observation of the long sER cisternae. These cisternae have a diameter ranging from 30 to 60 nm and exhibit similar electron contrast to that of sER cisternae found in neuronal dendrites, including their specialized form in dendritic spines (spine apparatus). In addition to the extended sER cisternae, the PAP cytoplasm contains cross-sections of contrast structures that measure between 10 and 30 nm in diameter. When observed at low magnification, with a final image resolution of 1.7 nm per pixel, these structures appear morphologically and dimensionally indistinguishable from glycogen granules. Analysis at higher magnifications, with a working image resolution of 0.67–0.34 nm/pixel — ten to fifteen times higher than standard scanning electron microscopy resolution — enabled clear identification of membrane-bound organelles.

Serial sections analysis reveals that PAP structures contain two distinct pools of organelles: short (~130–170 nm) “filiform” cisternae of sER with a diameter of 10–30 nm and microvesicles. Additionally, if PAPs with branchlet morphology feature two types of sER cisternae (short “filiform” and extended dilated cisternae) and microvesicles, then TAP membrane organelles are represented only by fragments of short “filiform” sER cisternae and microvesicles. Groups of these slender cisternae and small vesicles are commonly found in close proximity to the active zones of the most active synapses. The non-random distribution of sER cisternae and microvesicles indicates the presence of an active mechanism for directed transport of membrane organelles within highly flattened TAP lamellae. We analyzed our results alongside existing immunoelectron microscopy data from the literature that examines the location of the Ca2+-binding protein calreticulin, a specific sER marker, in PAP. The observed thin sER cisternae serve as an ultrastructural foundation and primary contributor to the development of spontaneous and induced Ca2+ events in the PAPs, as well as a requirement for Ca2+-dependent vesicular release of gliotransmitters located near active synapses.

Despite the well-established belief that organelles are absent in TAP, the data suggest a dynamic regulation of organelle composition and number within PAP lamellae, dependent on synapse activity. These findings open new avenues for investigating neuron-glia interaction and the functional role of astrocytic microenvironments in tripartite synapse plasticity and pathological processes in the brain.

Genes & Cells. 2023;18(4):540-543
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Effect of pioglitazone on the behavior and expression of genes involved in the regulation of epileptogenesis in a lithium-pylocarpine model of temporal lobe epilepsy in rats

Roginskaya A.I., Kovalenko A.A., Zubareva O.E.

Abstract

Epilepsy is a chronic neurological disorder characterized by spontaneous recurrent seizures and various psychoemotional and cognitive impairments [1]. Roughly 30% of patients experience pharmacoresistant epilepsy, and current antiepileptic medications do not prevent the progression of brain epilepsy, necessitating the exploration of novel therapeutics.

Recently, the potential involvement of peroxisome proliferator-activated receptors (PPARs) in the development of epilepsy has been examined. PPARs (α, β/δ, γ) are nuclear transcription factors affecting many intracellular cascades, both in the periphery and in the brain. Their agonists were proposed to restrict neuroinflammation, a crucial factor contributing to the pathogenesis of various neuropsychiatric disorders, such as epilepsy [2].

The aim of this study was to investigate the impact of the PPARγ receptor agonist pioglitazone on the regulation of neuroinflammation and epileptogenesis-associated genes, as well as the expression of disorders in research and social behavior in a lithium-pilocarpine model of epilepsy.

The lithium-pilocarpine model of temporal lobe epilepsy consists of three phases: 1) induction of an acute epileptic status by administering pilocarpine, 2) a latent period lacking seizures, and 3) a chronic period characterized by spontaneous recurrent seizures. The experiments were conducted on male Wistar rats. At seven to eight weeks of age, the research subjects were administered a LiCl solution (w/w, 127 mg/kg). After 24 hours, they were given methylscopolamine (w/w, 1 mg/kg), followed by pilocarpine (w/w, 20–30 mg/kg, 10 mg/kg until convulsions were pronounced) 30 minutes later. Pilocarpine was not administered to the control rats. Pioglitazone was administered using a biphasic course. The first injection was given at a dose of 7 mg/kg, 75 minutes after pilocarpine-induced status epilepticus. Subsequently, the rats were given a dosage of 1 mg/kg, once daily at 24-hour intervals, for a period of 7 days. The open field test and the foreign object test were performed on days 7 and 8 after pilocarpine administration, respectively. The brain was sampled for further biochemical analysis 12 hours after behavioral testing. The temporal cortex underwent analysis using real-time RT-PCR for the expression of Nlrp3, Aif1, Tnfa, Gfap, Il1b, Il1rn, Bdnf, S100a10, Fgf2, and Tgfb1 genes.

The research reveals a surge in the expression of pro-inflammatory proteins and activation markers of glial cell in temporal cortex caused by a lithium-pilocarpine model of epilepsy. This leads to impaired social behavior and hyperactivity in the open field. Pioglitazone successfully decreases the severity of the pilocarpine-induced behavioral disruptions. The PPARg agonist does not have a noteworthy impact on the expression of proinflammatory factors and glia activation markers Aif1 and Gfap. However, it was found to enhance the gene expression of neuroprotective proteins S100A10 and TGFB1 and decrease the expression of growth factors Fgf2 and Bdnf, which worsen epileptogenesis. Overall, these findings suggest that activation of PPARg might provide a protective role in the development of epileptic processes in the brain. Therefore, pioglitazone could be regarded as a possible therapeutic agent for treating epilepsy.

Genes & Cells. 2023;18(4):544-547
pages 544-547 views

Characteristics of murine phenotypes with signs of epilepsy obtained through ENU mutagenesis

Rybakova V.P., Mitina N.N., Babaev A.A., Tarabykin V.S.

Abstract

Epilepsy is a chronic neurological disorder marked by recurring seizures and associated dysfunctions of motor, sensory, autonomic, and mental functions resulting from excessive electrical activity of neurons. Identification and characterization of the mutations that cause this pathology are essential for understanding the mechanisms that control epileptogenesis.

The aim of the study is to characterize the S8-3 mutant mice strain, which shows induced epileptiform activity in response to audiogenic stimulation.

Mutant strains of mice were acquired through induced chemical mutagenesis using N-ethyl-N-nitrosourea (ENU). Twenty-nine male mice were administered three rounds of ENU injections at a dose of 90 mg/kg during the study. On day 21 after birth (P21), identification and selection of mouse mutants with an elevated inclination towards epileptic seizures were conducted using the Krushinsky scale, which considered the intensity of audiogenic seizures. Strains with a recessive mutation were created by selecting animals that exhibited the aberrant phenotype for the second time. The abnormal phenotype was confirmed in the G5 generation, and basic behavioral phenotyping was performed to characterize the resulting epileptic lines. Correct grammar, spelling, and punctuation. This included assessing memory, learning ability, motor reactions, and emotional status. In vitro experiments assessed spontaneous calcium activity with primary neuronal cultures of the cerebral cortex isolated from newborn mice. The Ca2+ indicator used was Oregon Green 488 BAPTA-1 AM.

Upon screening 60 strains of mice for sensitivity to audiogenic stimulation, 12 strains exhibiting epileptiform activity were observed. Subsequently, the S8-3 group was selected for further research, as its offspring (G3) showed a higher frequency of the aberrant phenotype in comparison to the other groups. Results from behavioral studies comparing S5-1 mice to the control hybrid animal group displayed a higher intensity of the acoustic startle response. The open field tests revealed that the motor activity of S8-3 strain mice was higher than that of the control group, based on the average distance traveled, and their anxiety level was lower, as indicated by fewer rears, urinary points, and boluses. When assessing cognitive functions through the CPAR test, mutant individuals exhibited high learning ability.

In vitro experiments showed an increase in the frequency of spontaneous calcium events in primary cell cultures of the cerebral cortex isolated from S8-3 mice.

Genes & Cells. 2023;18(4):548-549
pages 548-549 views

Immunogenic ferroptosis protects against the development of tumour growth

Saviuk М.О., Turubanova V.D., Efimova Y.V., Mishchenko Т.А., Mishchenko Т.А., Vedunova М.V., Krysko D.V.

Abstract

Immunotherapy is a proven and effective anti-tumor strategy, which can be used alongside surgery, radiation therapy, and chemotherapy [1]. Immunogenic cell death (ICD) was identified as a critical factor determining the effectiveness of cancer treatment [2]. The concept of ICD combines the capacity to destroy cancer cells effectively, with the activation of a cancer cell-specific immune response, leading to potent and long-lasting anti-cancer immunity. ICD-inducing agents activate a perilous pathway that triggers the release of ICD mediators called damage-associated molecular patterns (DAMPs). DAMPs encompass a group of naturally occurring molecules that gain immunostimulatory qualities upon exposure to the outer cell membrane or when liberated into the extracellular matrix in a specific spatiotemporal fashion. ATP, the nuclear protein HMGB1, calreticulin (CRT), and type I interferons (IFNs) are among the identified factors [8].

The concept of ICD was initially described for cancer cells undergoing apoptosis, but it was expanded to encompass additional forms of cell death, such as necroptosis, pyrroptosis, ferroptosis, nontosis, etc. [11]. Ferroptosis is a regulated iron-dependent type of cell death that is characterized by the buildup of reactive oxygen species in the cell.

In this study, the immunogenicity of ferroptotic cancer cells in vitro was assessed and their potential as an alternative approach to cancer immunotherapy was tested.

Glioma GL261 and fibrosarcoma MCA205 cells were induced with one of the well-known inducers of ferroptosis, RSL3 (RAS-Selective Lethal 3). After 24 hours of RSL3 stimulation, 80% of GL261 cells and 90% of MCA205 cells showed positivity to Annexin V/Sytox Blue, indicating they were in the late stage of ferroptosis. Similarly, after 3 hours of RSL3 stimulation, 50% of GL261 cells and 45% of MCA205 cells were double positive with Annexin V/Sytox Blue indicating their late-stage ferroptotic state. We evaluated the immunogenic features of early and late ferroptotic cells in vitro, specifically at 3 or 24 hours after RSL3 stimulation. To achieve this, we compared the phenotype of dendritic cells (BMDCs) exposed to late ferroptotic cells with BMDCs exposed to viable cancer cells. Furthermore, immunogenic apoptosis was induced with MTX as a positive control and LPS as a secondary positive control. Late ferroptotic MCA 205 cells surprisingly did not induce phenotypic BMDC maturation, as indicated by the lack of surface activation of costimulatory molecules CD86, CD80, and MHCII. In contrast, a less pronounced phenotypic response compared to MCA205 cells was induced by early ferroptotic glioma GL261 cells. Nonetheless, a decrease in the ability to activate dendritic cells was observed for late ferroptotic glioma cells as well.

The study used the standard tumor prophylactic vaccination model on immunocompetent C57BL/6 J mice to assess the adaptive immune system activation by early ferroptotic cancer cells. Mice were immunized with early or late ferroptosis MCA205 cells. As a negative control, we used PBS or cells that underwent spontaneous necrosis. The mice that were immunized were later confronted with viable MCA205 tumor cells. Protection against tumor growth at the site of infection was deemed indicative of successful activation of the adaptive immune system. Remarkably, mice that received immunization with late ferroptotic MCA205 cells, induced with RSL3 for 24 hours, exhibited conspicuous tumor growth at the infection site, signifying that late ferroptotic cells are not immunogenic in vivo, as per our preliminary observations in vitro.

Genes & Cells. 2023;18(4):550-553
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The significance of photobiomodulation in formation of membrane potential of brain mitochondria in normoxia and after hypoxia in mice

Shchelchkova N.A., Pchelin P.V., Shkarupa D.N., Vasyagina T.I., Bavrina A.P.

Abstract

Photobiomodulation using low-intensity red light (LRL) is considered a safe, non-invasive, and cost-effective method that was proven to possess stimulating, restorative, and rejuvenating effects on body tissues. The therapeutic potential of photobiomodulation was demonstrated in various pathologies such as Alzheimer’s and Parkinson’s diseases and ischemic brain damage [1–3]. The potential photoacceptance of radiation by ETC’s complex IV (CIV) raises concern for the impact of LRL on mitochondria. However, ATP synthesis in mitochondria depends less on their functional state and more on the high electrical potential of coupled mitochondria. This study aimed to investigate the importance of photobiomodulation for the formation of brain mitochondria membrane potential in healthy mice and after hypoxia.

Male C57BL/6 mice were used in the study. The animals were divided into two groups: a healthy control group (n=20) and a group of animals exposed to simulated hypobaric hypoxia (n=20). Half of the control animals (n=10) and half of the animals subjected to hypoxia modeling (n=10) received a single transcranial exposure of LRL (Spectr LC-02, Russia), which had a wavelength of 650±30 nm, for 3 minutes. After 24 hours, the mitochondrial fraction of the left cerebral cortex of the brain was isolated. The resulting fraction was used to examine how the mitochondrial membrane potential (ΔmtMP) dynamically changes by employing the O2k-Fluorescence LED2 amperometric module of the Oroboros Oxygraph-2k respirometer (Oroboros Instruments, Austria) and the fluorescent dye tetramethylrhodamine methyl ester. The collected data were normalized for protein content using the Bradford method. Statistical analysis was conducted with GraphPad Prism 8 and Excel.

When investigating the impact of transcranial administration of LRL on ΔmtMP during CI-supported (CI, NADH-ubiquinone oxidoreductase) oxidative phosphorylation of the left cerebral cortex mitochondria in control animals, an increase of 18% was observed for the parameter. Further, a 40% increase was noted when studying CII-supported (CII, succinate dehydrogenase) oxidative phosphorylation compared to the untreated group. During the evaluation of basal respiration in the untreated control group, the measurement of ΔmtMP was 0.052±0.002 arb. units It was found that the transcranial application of LRL in mice caused a 2-fold increase of ΔmtMP (0.115±0.010 arb. units).

Simulation of hypobaric hypoxia results in a 20% decrease in ΔmtMP during CI-supported oxidative phosphorylation but has no effect on ΔmtMP during CII-supported oxidative phosphorylation. Basal respiration after hypoxia modeling showed a 33% decrease in ΔmtMP compared to control values (0.052±0.002 arb. units and 0.035±0.003 arb. units, respectively).

The transcranial administration of LRL following hypoxia modeling did not alter the dynamics of membrane potential during CI- and CII-supported oxidative phosphorylation, yet considerably amplified ΔmtMP when evaluating basal respiration.

The transcranial LRL irradiation stimulated the healthy control group, resulting in an increase in ΔmtMP for both CI- and CII-supported oxidative phosphorylation and basal respiration. This increase in coupling between oxidation and phosphorylation processes was observed. However, after hypoxia modeling, the photobiomodulation effect of LRL was only observable under basal respiration conditions. The effects of the LRL application align with findings from other studies that suggest an elevation in ΔmtMP and the creation of ATP resulting from the dissociation of NO and the binuclear center of CIV [4].

Genes & Cells. 2023;18(4):554-557
pages 554-557 views

Ultrastructure of neuron-glia interaction in the norm and experimental pathology

Shishkova E.A., Rogachevsky V.V.

Abstract

Long-term experience was gained in analyzing synapses and their glial surroundings in normal, natural, and experimental models of functional plasticity and brain pathology development. However, most studies in this area rely on electrophysiological techniques combined with fluorescent imaging. Notably, fine synapse structure and astrocytic processes cannot be resolved using light or some electron microscopy techniques. Studies on experimental brain pathology using volume electron microscopy methods [1] were previously restricted due to their high labor intensity. However, automated methods for sample preparation and analysis, using machine vision and artificial intelligence, significantly simplified this task.

Using transmission electron microscopy methods and 3D reconstructions, this study examined the Str. radiatum CA1 hippocampal region of rat brains in a chronic lithium-pilocarpine model of epilepsy. The results indicate a decrease in synaptic number along with an increase in their size and a reduction in astrocytic isolation of the active zones. A decrease in glial ensheathment of enlarged active zones and facilitation of neurotransmitter diffusion to active synapses may have a multiplicative effect on epileptiform activity growth and excitotoxicity.

The simplification of the astrocytes meshwork in the somatosensory cortex’s layer 2/3 is comparable to that of layer 1. This decrease in layer 1’s inhibitory action enables pyramidal neurons in layer 2/3 to potentially exhibit epileptiform activity. Thus, the superficial cortical layers’ structural-functional aspects can be used as a natural cellular model in epilepsy development studies.

Reduction of Ca2+ events in astrocyte processes in lithium-pilocarpine induced epilepsy may result from the low buffer capacity of Ca2+ ions in the smooth endoplasmic reticulum (sER) and/or impaired Ca2+ wave transmission through the gap junctions between astrocytic processes. High resolution is necessary to analyze the gap junctions, and special methods are required for sER visualization in perisynaptic astrocytic processes.

We developed original sER staining methods [2] to quantitatively evaluate gap junctions and sER cisternae within astrocytic meshworks in layers 1 and 2/3 of the somatosensory cortex. In layer 1, the area of gap junctions in relation to the volume of an astrocyte was twice as high as in layer 2/3. The proportion of sER volume differed between layer 1 and layer 2/3 tissues. Specifically, the total sER cisternae volume in layer 2/3 was twice as high as in layer 1, relative to the volume of astrocytic processes [3]. Additionally, a doubling of single astrocytic gap junction area concomitant with decreased calcium events was observed.

The results suggest a normal balance between Ca2+ stores (sER) and gap junctions, whose disruption may contribute to the development of seizures.

Genes & Cells. 2023;18(4):558-561
pages 558-561 views

Investigation of the features of immunogenic cell death caused by photodynamic exposure using a photosensitizer from the tetra(aryl)tetracyanoforfirazines group with 9-phenanthrenyl as a side substituent

Sleptsova E.E., Redkin T.S., Saviuk M.O., Kondakova E.V., Vedunova M.V., Turubanova V.D., Krysko D.V.

Abstract

The concept of immunogenic cell death involves the death of tumor cells, leading to the activation of an adaptive immune response in vivo. Such death relies on two significant components: antigenicity and adjuvance of dying cells. The emission of DAMPs achieves adjuvance, which is recognized by antigen-presenting dendritic cell (DC) receptors, resulting in phagocytosis and DC maturation. These cells present antigens of dead cells on their surface to the T-cell population. Antigenicity provides an opportunity to develop adaptive immunity through vaccination with decaying cells targeting a particular tumor pattern (antigen).

Currently, photodynamic therapy (PDT) is recognized as an efficient inducer of immunogenic cell death. In this study, we examined the effectiveness of photodynamic therapy (PDT) using tetracyanotetra(aryl)porphyrazine with a 9-phenanthrenyl group on the periphery of the porphyrazine macrocycle (pz I) as a photosensitizer for inducing immunogenic cell death in tumor cells. The study was conducted on two cell lines: mouse fibrosarcoma MCA205 and mouse glioma GL261.

For photoinduction, cells were loaded with a photosensitizer for four hours. The medium was then replaced with full medium, and the cells were irradiated with a 20 J/cm light dose using an LED light source with an excitation wavelength of 615–635 nm. In all subsequent experiments, cells incubated for 24 hours after photoinduction were used.

A photosensitizer concentration corresponding to 85–90% of dead and dying cells 24 hours after photoinduction was chosen for both cell lines, as it is considered the standard for immunogenic cell death.

The study analyzed the levels of ATP and HMGB1 that were released into the extracellular medium to confirm adjuvanticity. After photodynamic exposure, the level of ATP in the supernatant 24 hours later was significantly higher than the baseline values in the control group prior to PDT for both cell lines. Similar findings were observed for HMGB1 release.

The study aimed to investigate the potential of PDT-killed cells in inducing a persistent immune response. Dying cells of fibrosarcoma MCA205 or GL261 glioma underwent photoinduction using pz I and were used to immunize C57BL/6J mice once or twice a week. After seven days from the last vaccination, viable tumor cells were subcutaneously injected into the opposite side of the mice for observation.

In the MCA205 fibrosarcoma tumor model, 90% of the animals were tumor-free on day 25 of the experiment. In the control group PBS (which comprised mice that received saline solution as immunization), all of the laboratory animals had a tumor by day 12 of observation, and died by day 16. Additionally, on day 16 of the experiment, the volume of tumors in the control group was double the volume of tumors on day 25 of the experiment in the pz I group.

When experimenting with immunization using dying GL261 glioma cells, the pz I group showed an absence of tumor focus in 90% of laboratory animals by day 30. Furthermore, the tumor volume in the group that was immunized with PDT-killed cells was 10 times less compared to the tumor volume of the control group PBS.

Immunization of the Nude strain of bestimus condyles was conducted to evaluate the contribution of adaptive immunity to the manifestation of the antitumor effects of dying cells induced through photodynamic therapy (PDT). Tumor foci appeared and developed similarly in both the experimental and control groups, indicating no significant impact from immunization. Thus, the study demonstrated the critical importance of the T-cell connection in eliciting an effective anti-tumor response. The evidence indicated that if T-cell populations cannot participate in adaptive immune responses, even when immunity is stimulated, a pathological process can develop. Vaccination using photoinduced MCA205 fibrosarcoma cells in immunodeficient Nude mice verified the noteworthy role of the adaptive immune system in executing the antitumor response.

Genes & Cells. 2023;18(4):562-565
pages 562-565 views

The rat brain transcriptome: from infancy to aging and sporadic Alzheimer’s disease-like pathology

Stefanova N.A., Kolosova N.G.

Abstract

Functional traits of the adult brain, which are established early in life, may impact susceptibility to Alzheimer’s disease (AD). Results from prior research conducted on senescence-accelerated OXYS rats, a prominent model for sporadic AD, provide evidence in favor of this hypothesis. The present study examined the transcriptomes of the prefrontal cortex (PFC) and hippocampus in OXYS and Wistar rats (control) during the early postnatal period (at age P3 and P10; P: postnatal day of life) to identify the signaling pathways and processes that contribute to delayed brain maturation in OXYS rats and assess their potential role in the development of AD traits later in life. Next, we compared the differentially expressed genes (DEGs) in the rat PFC and hippocampus throughout the five stages of AD-like pathology, from infancy to the progressive stage. Additionally, we noted conspicuous variations between the strains in the number of DEGs throughout all five ages. Significant differences were found in the number of DEGs between OXYS rats and Wistar rats in both brain structures at both P3 and P10. Notably, changes in gene expression patterns in the PFC and hippocampus of OXYS rats at 3 and 10 days of age are broadly associated with all basic mechanisms involved in Alzheimer’s pathogenesis, which are modified in OXYS rats at different stages of AD. Gene expression changes at P3 and P10 are associated with molecular processes including neuronal plasticity, immune responses, cerebrovascular function, and mitochondrial function. Remarkably, changes in the expression of genes associated with Aβ function were detected. The expression profile of genes linked to APP processing in the brain of OXYS rats is reduced during the early postnatal period. An intriguing finding, with potential significance for the development of AD pathology, is the decreased expression of the Abca7 gene, an important genetic factor of late-onset AD, in both brain regions of OXYS rats during the early postnatal period. The data demonstrated a decrease in Abca7 expression in the brains of OXYS rats at P20 and at 5 and 18 months (p <0.05). Additionally, three genes (Thoc3, Exosc8, and Smpd4) demonstrated overexpression in both brain regions of OXYS rats throughout their lifetimes. In conclusion, we have conducted a comparative analysis of changes in the rat brain transcriptomes from infancy to the advanced stage of AD-like pathology for the first time. The significant and comparable distinctions in gene expression and related processes were noteworthy during the early postnatal timeframe and in the severe stage of the pathology. Our findings indicate that a reduction in the effectiveness of neural network formation in the brain of OXYS rats at an early age is a clear contributor to AD symptomatology. The cause of this phenomenon remains unclear. However, we can identify shortened gestational age, low birth weight, and delayed brain development in infancy as major risk factors for the emergence of a disease-like pathology later in life, as these conditions are typically found in affected rats. Further investigation is needed to determine the causal relation between delayed brain development in infancy and neurodegeneration.

Genes & Cells. 2023;18(4):566-567
pages 566-567 views

The effect of cardarine on the behavior of rats in a lithium-pylocarpine model of temporal lobe epilepsy

Subkhankulov M.R., Sinyak D.S., Zubareva O.E.

Abstract

Epilepsy is a serious neuropsychiatric disorder characterized by the occurrence of spontaneous recurrent seizures and linked cognitive, psychoemotional, and social impairments. Objective research showed that up to 30% of epilepsy patients do not respond to current therapeutic interventions, necessitating the development of novel treatment options. In recent years, researchers actively studied the neuroprotective properties of peroxisome proliferator-activated receptor agonists (PPAR α, β/δ, γ). As nuclear transcription factors involved in inflammatory signaling pathways, PPARs play a role in the pathogenesis of neuropsychiatric disorders, such as epilepsy. Various nervous pathology models were used for the investigations. The neuroprotective properties of PPAR γ agonists were reported in epilepsy models, whereas sufficient investigation into PPAR β/δ agonists’ effects is lacking.

The aim of this study was to evaluate the effectiveness of the PPAR β/δ receptor agonist cardarine in alleviating behavioral disruptions observed in rats exhibiting temporal lobe epilepsy in response to the lithium-pilocarpine model.

The lithium-pilocarpine model is considered one of the most effective experimental models for studying temporal lobe epilepsy in humans. Administration of pilocarpine in animals results in acute seizures, followed by a latent period devoid of seizures. Subsequently, spontaneous recurrent seizures appear, marking the chronic phase of the model. Epilepsy was induced in male Wistar rats that were 7 weeks old, through the administration of pilocarpine, 24 hours after being injected with LiCl (127 mg/kg). The peripheral effects of pilocarpine were negated through the use of methyl bromide-scopolamine (1 mg/kg, administered intraperitoneally 1 hour before pilocarpine). Pilocarpine was administered in fractional doses of 10 mg/kg, every half hour, until the seizures reached at least stage 4 on the Racine scale, with a dose range of 20–40 mg/kg. Following pilocarpine administration, Cardarine was administered intraperitoneally at a dose of 2.5 mg/kg daily for a period of 7 days, with the initial injection taking place 24 hours post-administration. Behavioral tests were conducted during the chronic phase of the model, 2-2.5 months after pilocarpine injection, using several tests, including the Open Field (to measure exploratory behavior, motor activity, and anxiety levels), the Intruder-Resident test (to measure communicative behavior), the Y-maze test (to measure working memory), and the Morris Water Maze (to measure spatial memory in the short and long term).

Untreated experimental animals with temporal lobe epilepsy exhibited heightened vertical and horizontal motor activity, increased anxiety in the Open Field test, reduced communicative activity in the Intruder-Resident test, and impaired memory in the Y-maze test and the Morris Water Maze. Cardarine reduced alterations in vertical movement (time of climbing) and anxiety levels (number of grooming behaviors) in the Open Field, hindered short-term memory in the Y-maze, and restrained communicative conduct in the Intruder-Resident test. However, the medication did not impact the rats’ survival rate, body weight changes, overall activity (distance traveled in the Open Field), and damage to learning and long-term memory in the Morris Water Maze.

Thus, administration of cardarine partially neutralized behavioral disorders developing in rats in the lithium-pylocarpine model of temporal lobe epilepsy.

Genes & Cells. 2023;18(4):568-571
pages 568-571 views

Life after transcription: control of the fate of neurons

Tarabykin V.S., Borisova E.B.

Abstract

The neocortex is a complex structure responsible for higher-order cognitive abilities in mammals. It consists of six cell layers, each comprised of various subtypes of excitatory and inhibitory neurons. These neurons project their axons to specific targets within each layer. All neocortical projection neurons are generated by neural progenitor cells located in the proliferative ventricular zone. In recent decades, significant strides have been made in comprehending the transcriptional programs governing neuronal cell fate specification. Nonetheless, posttranscriptional mechanisms involved in this process remain largely unexplored.

Recently, we found that TrkC-T1, an isoform of the TrkC receptor lacking the kinase domain, determines the fate of corticofugal projection neurons (CFuPN). Our study reveals that the balance between TrkC-T1 and TrkC-TK+, a more widely recognized isoform containing the kinase domain, depends on the type of cell within the developing cortex. Additionally, we demonstrate that two RNA-binding proteins, Srsf1 and Elavl1, work in opposition to establish this balance. Additionally, our data suggests that Srsf1 stimulates the CFuPN fate while Elavl1 stimulates the callosal projection neuron (CPN) fate in vivo by regulating the different TrkC-T1 to TrkC-TK+ ratios.

In this study, we identified a protein translation-dependent mechanism that governs the cell fate switch in the developing neocortex. Our results demonstrate that Ire1α, the Inositol-Requiring Enzyme 1α, regulates global translation rates in the developing neocortex through its dynamic interaction with the ribosome, as well as the regulation of expression of translation elongation factors eIF4A1 and eEF-2. Inactivation of Ire1α leads to decreased protein synthesis rates that are associated with stalled ribosomes and a reduced number of sites for translation initiation. We demonstrate the distinctive sensitivity of neurons determined for the upper layer to translation rates. While eEF-2 is necessary for cortical lamination, eIF4A1 regulates the attainment of upper layer fate in a mechanism of translational control that is dependent on structural elements embedded in the 5’UTR of genes responsible for determining fate downstream of Ire1α. Our findings reveal the developmental control of ribosome dynamics as post-transcriptional mechanisms that coordinate the establishment of neuronal diversity and the assembly of cortical layers.

Genes & Cells. 2023;18(4):572-573
pages 572-573 views

Molecular genetic background for the development of early neurodegenerative processes in retina

Telegina D.V., Kozhevnikova O.S., Kolosova N.G.

Abstract

All neurodegenerative retinal diseases are characterized by decreased metabolic and regenerative processes, impaired microcirculation, and structural abnormalities of the retina. Age is a significant risk factor for age-related macular degeneration (AMD), which is the primary cause of irreversible vision loss in individuals aged over 60 years. Since its pathogenesis is not completely understood, there is currently no effective treatment for AMD. The pathogenesis of age-related retinal changes is still uncertain, despite being grounded on alterations in characteristic features of the retina due to aging. The molecular events preceding and accompanying clinical disease manifestations pose a challenge for human study. The retina shares a uniform basic structure throughout all vertebrate species, enabling the use of animals in exploring the mechanisms responsible for maintaining a healthy physiological structure of the retina and for the pathogenesis of numerous diseases. With the knowledge acquired, novel remedies for these ailments can be developed for humans [1].

The study analyzed the OXYS rat line, known for its premature aging and retinopathy which mimics the dry form of AMD in humans. The aim was to examine how postnatal retinal neurogenesis changes contribute to the development of AMD-like retinopathy in these rats. By approximately 3–4 months of age, all structural components of the retina in OXYS rats demonstrate pathological changes, including vessels (both choroidal and intraretinal), Bruch’s membrane, photoreceptors, ganglion neurons, interneurons, and RPE. This is supported by our research findings [2, 3]. By approximately 3–4 months of age, all structural components of the retina in OXYS rats demonstrate pathological changes, including vessels (both choroidal and intraretinal), Bruch’s membrane, photoreceptors, ganglion neurons, interneurons, and RPE. One hundred percent of OXYS rats exhibit clinical symptoms of retinopathy. As they age, pathological changes intensify and coincide with photoreceptor death, hindered autophagy, and active gliosis [3–5]. Due to its limited capacity for neurogenesis, the adult mammalian retina’s structural and functional properties during its development can exert lasting impacts on ontogeny.

We discovered that OXYS rats exhibited a notable reduction in the population of amacrine neurons during birth, along with an increment in the populations of ganglion and horizontal neurons in the retina as a form of compensation. The postnatal development of the rat retina is finished by the 20th day of life. In OXYS rats, this development is distinct in that it causes a shift in the timing of differentiation of bipolar cells and photoreceptors, leading to the later formation of the outer retinal layer. This layer is composed of synapses between photoreceptors, bipolar cells, and horizontal cells. The delayed onset of synaptogenesis in the OXYS rat retina results in elevated apoptosis levels and a heightened reduction of neurons. Consequently, the processes of photoreceptor differentiation and synaptogenesis remain incomplete by the time of eye opening in OXYS rats. This incomplete development can significantly impact the retina’s structure and functions. These findings indicate that a delay in retinal formation could serve as a predictor of the development of AMD in OXYS rats, and potentially this disease in humans.

Genes & Cells. 2023;18(4):574-577
pages 574-577 views

In vivo study of the role of hydrogen peroxide in the development of ishemic stroke in a model of streptozotocin-induced type I diabetes in rats using genetically encoded biosensor HyPer7

Trifonova A.P., Kotova D.A., Ivanova A.D., Pochechuev M.S., Khramova Y.V., Sudoplatov M.A., Katrukha V.A., Sergeeva A.D., Raevskii R.I., Solotenkov M.A., Fedotov I.V., Fedotov A.B., Belousov V.V., Zheltikov A.M., Bilan D.S.

Abstract

Diabetes mellitus is a significant risk factor for the development of complications following ischemic stroke. However, the precise mechanisms through which elevated glycemic status impacts neuronal metabolism during ischemia remain unclear. One probable reason for the exacerbation of post-ischemic consequences under hyperglycemia is oxidative stress, the indicator of which may be H2O2. In this research, we used the genetically encoded fluorescent biosensor HyPer7 to exhibit the hydrogen peroxide dynamics in the matrix of neuronal mitochondria during ischemic stroke under hyperglycemic and normal glucose conditions.

The research was conducted on SHR rats with normal and high blood glucose levels. Type I diabetes mellitus was induced by injecting streptozotocin, which is toxic to pancreatic β-cells. For the expression of the fluorescent biosensor HyPer7-mito in the neuronal mitochondria of the caudate nucleus region, a suspension of adeno-associated virus particles carrying the sensor gene under the neuronal promoter was administered under stereotactic control. After the rats were injected, optical fibers with a ceramic adapter were implanted directly into their brain striatum, enabling us to register the H2O2 indicator signal using a highly sensitive fluorescence excitation and detection system developed by the Institute of Photonics and Nonlinear Spectroscopy at Moscow State University. The biosensor signal recording was continuously performed in real-time from the moment the animal was anesthetized. While recording, anesthetized rats underwent middle cerebral artery occlusion, which supplies corpus striatum.

Using HyPer7 in the ischemic stroke model described, we observed that the H2O2 concentration dynamics in the affected hemisphere of rats with normal and elevated blood glucose levels were similar during the acute phase of stroke and one day after occlusion. Oxidation of the biosensor was observed in both animal groups during both ischemia and reperfusion. However, HyPer7 exhibited the most marked response one day after the occlusion of the middle cerebral artery, indicating a significant rise in hydrogen peroxide concentration.

Furthermore, the metabolic activity of brain tissue was evaluated through staining slices obtained 24 hours after occlusion with 2,3,5-triphenyltetrazolium chloride. Rats with diabetes exhibited 2.6 times larger brain damage, indicating that hyperglycemia exacerbates the consequences of ischemic stroke. Furthermore, there was a higher mortality rate in the hyperglycemic group following ischemic stroke. This indicates that the consequences of ischemic stroke were more severe under hyperglycemia, with 25% of the animals in the hyperglycemic group dying before the experiment ended, whereas none of the control group animals died.

Our study found that an elevated glycemic status does not impact the creation of H2O2 during the acute phase of stroke or one day after occlusion. However, it significantly intensifies damage to brain tissue and raises mortality rates.

Genes & Cells. 2023;18(4):578-581
pages 578-581 views

The fate of iPSCs-derived low immunogenic dopaminergic neuron precursors after transplantation into the striatum of rats with 6-OHDA-induced parkinsonism

Voronkov D.M., Lebedeva O.S., Stavrovskaya A.V., Bogomiakova M.E., Olshanskiy A.S., Kopylova I.V., Gushchina A.S., Simonova A.V., Ruchko E.S., Illarioshkin S.N., Eremeev A.V., Lagarkova M.A.

Abstract

Parkinson’s disease arises from the demise of dopaminergic neurons in the substantia nigra resulting from both environmental and hereditary factors. Drug interventions can solely delay disease progression, but cannot provide a cure. Therefore, cell replacement therapy may represent a promising treatment approach. Although the United States has already employed this technology, independent clinical and preclinical trials are mandatory for its inclusion in Russia. The iPSC technology enables the acquisition of personalized iPSC lines for each patient, nearly eradicating the immune response. While autologous cell therapy is theoretically ideal, the reprogramming of patient cells into iPSCs and subsequent differentiation for each patient incurs time and cost. Alternatively, differentiated derivatives of low immunogenic iPSCs, lacking HLA class I expression, can serve as a substitute. These cells evade the T-cell immune response, but other minor HLAs and cells from the innate immune system, like macrophages and NK cells, could still participate in the immune response’s development, although not as potent as in allogeneic transplantation. While these cell products don’t escape the immune response entirely, they lessen it significantly, potentially enabling the use of less severe immunosuppressive therapy. In this study, a protocol previously established in the cell biology laboratory was used to differentiate iPSCs lacking HLA class I expression and wild-type iPSCs into midbrain dopaminergic neuron precursors. A thorough analysis of the forerunners was performed and demonstrated appropriate patterning. The progenitors were then implanted into the brains of rats with 6-OHDA-induced Parkinsonism and followed for 6 months to compare their in vivo differentiation to standard differentiation in vitro. In addition, the systemic inflammatory response of the animals to the transplantation and the biodistribution of the injected cells were investigated. The literature inadequately addresses the question of which cells, other than dopaminergic neurons, differentiate during in vivo graft differentiation and potentially cause side effects in cell therapy. The literature suggests that only approximately 3% of transplanted cells differentiate into dopaminergic neurons, which is adequate to improve motor function in model animals. We found that a significant proportion of the progenitors differentiate into glial cells. The dynamics of maturation of transplanted neurons was evaluated. Thus, we approached preclinical testing of the cell product after having characterized in detail the dynamics of maturation and the composition of the graft. Comparison of in vivo and in vitro differentiation will allow evaluation of the quality of cellular material for transplantation.

Genes & Cells. 2023;18(4):582-584
pages 582-584 views

Post-stress expression of genes involved in neuroplasticity in the hippocampus and blood of rats with different level of nervous system excitability

Vylegzhanina A.E., Shalaginova I.G., Zachepilo T.G., Dyuzhikova N.A.

Abstract

The causes and mechanisms of most mental disorders remain unclear. While stress is known to trigger depressive and anxiety disorders, the role of genetically determined individual differences in forming vulnerability to post-stress neurochemical disorders, including those that affect neuroplasticity, remains unresolved.

Early studies suggest that acute and chronic stress lead to elevated glucocorticoid levels in the bloodstream and alter the expression of glucocorticoid and mineralocorticoid receptors. Additionally, both forms of stress can decrease the levels of brain-derived neurotrophic factor (BDNF), which plays a role in neuronal growth, development, and synapse building [1]. However, current studies insufficiently explain the complete mechanism of the stress response as they only explore variations in post-stress alteration of gene expression for proteins related to differing nervous system functional characteristics.

The strains of rats from the Biocollection of the Pavlov Institute of Physiology, RAS, are a convenient model for studying the impact of genetically determined nervous system features on stress reactivity. The strains, selected based on the threshold of nervous system excitability [2], include HT (high threshold of excitability, low excitability) and LT (low threshold of excitability, high excitability) strains. These strains display contrasting responses to extended emotional and painful stress and exhibit unique changes in brain structures implicated in emotional regulation and stress reactivity at the molecular, cellular, and epigenetic levels [2].

The aim of this study is to examine the mRNA levels of genes encoding glucocorticoid and mineralocorticoid receptors (NR3C1, NR3C2) and brain-derived neurotrophic factor (BDNF) in the hippocampus and blood of rat strains that exhibit contrasting nervous system excitability. The study investigates interstrain variations in animals under non-stressful conditions and changes in response to prolonged exposure to emotional and painful stimuli.

The experiment utilized 96 animals, with 6 animals in both the control and experimental groups at each time point. The stress model utilized was long-term emotional and painful stress as per K. Gecht [2]. The study assessed the expression of genes for glucocorticoid receptors and neurotrophic factor by isolating RNA from the hippocampus and blood of rats of two strains decapitated at 1, 7, 24, and 60 days post-stress. Gene expression analysis was conducted through real-time PCR and underwent further data processing using the ΔΔCt method. The statistical tests employed included the Kruskal–Wallis and Mann–Whitney tests.

The analysis of the expression of the observed genes in intact animals demonstrates that highly excitable LT rats have a significantly lower level of expression of the nr3c2 gene, which encodes the mineralocorticoid receptor in the hippocampus, when compared to the low excitable HT strain. When studying the short-term and long-term effects of stress on the expression of the observed genes (nr3c1, nr3c2, bdnf), a decline was observed in the level of bdnf mRNA in the hippocampus of the LT strain 60 days after stress.

The study suggests that the genetically determined characteristics of highly excitable animals affect the weakened adaptive abilities of their nervous system. One of the markers of this weakening is the reduced level of mRNA for the nr3c2 gene in the hippocampus under normal conditions, as well as a decrease in the expression of the bdnf gene in response to stress.

Genes & Cells. 2023;18(4):585-588
pages 585-588 views

Oxysterol-dependent pathway of regulation of synaptic transmission in the neuromuscular junction of mice

Zakyrjanova G.F., Tsentsevitsky A.N., Giniatullin A.R., Kuznetsova E.A., Petrov A.M.

Abstract

Cholesterol plays a crucial role in maintaining the stiffness, flexibility, and porosity of the bilipid layer. Moreover, cholesterol has a strong attraction to several membrane proteins, allowing it to modify their function, thereby influencing intracellular processes. Through enzymatic and oxidative reactions, cholesterol produces different types of oxysterols. One of the oxysterols synthesized is 25-hydroxycholesterol (25HC), formed with participation from the cholesterol-25-hydroxylase enzyme predominantly in macrophages, dendritic cells, and microglia.

25HC effectively regulates cholesterol homeostasis within individual cells, ensuring that cholesterol concentration remains at or below nanomolar levels. However, similar to the paracrine agent 25HC, macrophages and microglia produce it at significantly higher concentrations during inflammatory reactions.

Extensive research has focused on the role of 25HC in immune response during inflammation. 25HC appears to exert a variety of effects on the immune response. It promotes the secretion of inflammatory cytokines and chemokines such as IL-1 beta, IL-6, IL-8, CCL5, and macrophage colony-stimulating factor, and inhibits inflammation by blocking inflammasome activity. Increased production of 25HC via Toll-like receptor 4 activation reduces B-cell proliferation. 25HC has the ability to prevent viral penetration by integrating into the membrane and modifying its properties.

Macrophages play a significant role in both adaptive and innate immunity and are found in large numbers in skeletal muscle, suggesting the possible functional importance of 25HC in the interaction between the immune system and skeletal muscles. We discovered a concentration-dependent effect of 25HC on the neuromuscular synapse of mice: high concentrations (1–10 µM) enhance, while low concentrations (0.01–0.1 µM) inhibit the process of neuromuscular transmission [1].

25HC is a ligand for LX-receptors, which we observed to be expressed in the synaptic region of motor neuron axons. The high concentration of 25HC modulates synaptic transmission through an LX-receptor-dependent pathway. Furthermore, 25HC activates LX-receptors that are likely associated with estrogen receptors α, leading to the activation of the Gi-protein/βγ-dimer of G-protein/phospholipase C/Ca2+ protein kinase C signaling pathway. The potentiating effect of 25HC, which activates the LX-receptor/estrogen receptor α complex, depends on lipid rafts. This is because both receptors are localized in lipid microdomains, and the destruction of lipid rafts precludes the stimulating effects of 25HC. In addition, the study identified the contribution of reactive oxygen species (ROS) in the 25HC-dependent enhancement of synaptic transmission. Indeed, treatment with 25HC (1 µM) leads to an elevation in both ROS production in the synaptic region and hydrogen peroxide concentration in the extracellular environment. This phenomenon is dependent on an increase in intracellular calcium ions concentration. It is noteworthy that ROS, in this instance, act as signaling molecules since the level of lipid peroxidation is not impacted by 25НС.

Expression of 25HC increases in various neurodegenerative conditions. The concentration of 25HC escalates in amyotrophic lateral sclerosis, marked by progressive muscle atrophy resulting in death. High concentrations of 25HC above 5–30 µM can reduce the survival rate and trigger apoptosis of motor neurons. Nonetheless, low concentrations, 1 µM or lower, have the opposite effect, boosting the neurons’ survival rate.

The pivotal role of lipid rafts in ALS pathogenesis is evident. For instance, a decrement in caveolin-1 level in ALS brings about lipid raft disruption, thus accelerating disease progression. Moreover, in SOD1G93A ALS model mice during the pre-onset phase, alterations in membrane properties were observed, including lipid raft destabilization, lipid bilayer ordering, and heightened membrane fluidity [2]. One possible causal factor for this phenomenon could be muscle ceramide increase, which causes raft destabilization during motor unloading [3–5]. In fact, we observed elevated ceramide levels in the membrane of ALS model mice during the pre-onset stage. An increase in extracellular choline levels was detected in ALS, likely the result of heightened non-quantum secretion of the neurotransmitter, potentially due to lipid rafts disruption. Unregulated elevation of acetylcholine in ALS is a significant contributor to motor dysfunction and age-related morphological changes in the neuromuscular junction. An example of such changes would be the disruption of clustering of nicotine acetylcholine receptors in the postsynaptic membrane. Furthermore, disruption of lipid rafts in amyotrophic lateral sclerosis (ALS) was associated with elevated levels of hydroperoxides in muscle homogenates and lipid peroxidation in ALS model mice. Consequently, it can be inferred that the membrane properties are altered in ALS.

Furthermore, it was observed that 25HC can prevent the initial alterations of membrane properties by promoting the stabilization of lipid rafts in neuromuscular synapses in pre-onset ALS mouse models. Additionally, 25HC averted the accumulation of ceramide in the neuromuscular synapse. We observed that 25HC can inhibit synaptic alterations at the neuromuscular synapse in ALS. These alterations include elevated lipid peroxidation, increased extracellular choline levels, and disrupted clustering of nicotinic acetylcholine receptors [2].

Thus, 25HC has a multidirectional impact on neuromuscular transmission, hindering the recruitment of synaptic vesicles at low concentrations and enhancing the mobilization of vesicles at higher concentrations. Furthermore, 25HC exhibited a favorable impact on ALS, as it prevented the early manifestation of differences in the properties of the neuromuscular synapse in the ALS model. 25HC has the ability to relieve synaptic anomalies including increased membrane fluidity, ceramide accumulation, decreased membrane ordering, and lipid peroxidation. Furthermore, it decreases the heightened level of extracellular choline, which could potentially lead to neuromuscular synapse fragmentation in ALS.

Genes & Cells. 2023;18(4):589-592
pages 589-592 views

Pharmacological impact on the expression of microglial and astroglial proteins involved in the regulation of epileptogenesis as a possible new strategy for epilepsy therapy

Zubareva O.E., Roginskaya A.I., Kovalenko A.A.

Abstract

Epilepsy is a debilitating neurological disorder. While current antiepileptic medications can alleviate acute seizures, they do not typically prevent long-term brain damage caused by the chronic condition. Consequently, there is a pressing need for novel therapies that directly target epileptogenesis to address the comprehensive course and etiology of epilepsy.

For a considerable amount of time, research on the origin of epilepsy has chiefly concentrated on anomalies in the performance of neurons. As per typical theories, this is rooted in the unevenness between the performance of the excitatory (glutamate) and inhibitory neurotransmitter systems within the brain. Nevertheless, significant evidence has emerged in recent times that suggest the contribution of glial cells in the onset of epilepsy and the creation of neurological disorders following a seizure [1]. The astrocyte-produced excitatory amino acid transporter 2 (EAAT2) was found to regulate glutamatergic system activity, aiding the elimination of extracellular glutamate at central nervous system synapses. Astrocytes and microglia produce proteins that protect neurons from damage during epileptogenesis. In addition, astrocytes and microglia regulate neuroinflammation, which can worsen the development of epilepsy. Moreover, research demonstrated that astrocytes and microglia can have distinct functional states, with the A1 and M1 phenotypes generating mainly proinflammatory factors, while the A2 and M2 phenotypes produce anti-inflammatory factors. Drugs that stimulate polarization of glial cells from M1/A1 to M2/A2 phenotypes were proposed to be a successful strategy for treating inflammatory diseases, including epilepsies [2].

Numerous scientific findings indicate that peroxisome proliferator-activated receptor agonists (PPARs) exhibit similar properties [3]. PPARs (α, β/ and ) are nuclear transcription factors that are integral to the mechanisms of gut-nerve interactions, primarily regulating lipid and energy metabolism. However, PPAR agonists are capable of regulating inflammatory and oxidative signaling pathways involved in the development of various neuropsychiatric disorders, such as epilepsy. Furthermore, prior research described the neuroprotective qualities of certain PPAR agonists in a temporal lobe epilepsy (TLE) model.

In this study, the effects of pioglitazone, a PPARγ agonist, and Bifidobacterium longum, a probiotic that stimulates PPARγ expression in the brain [4], on astroglial and microglial mRNA protein production in brain structures that may impact epileptogenesis were investigated.

The study used the lithium-pilocarpine model of temporal lobe epilepsy in male Wistar rats. This model is highly regarded as an effective experimental approach for studying different stages of epileptogenesis from the earliest phases of the disease, prior to seizure manifestation, to the chronic phase accompanied by the development of spontaneous recurrent seizures [5].

During epileptogenesis in experimental animals, we observed an increase in the expression of proinflammatory proteins (Nrlp3, Il1b, and Tnfa) and markers of microglial and astroglial cell activation (Aif1 and Gfap) in the temporal cortex and hippocampus. This was demonstrated through the use of reverse transcription and real-time polymerase chain reaction during both the latent and chronic phases of the model. The development of neurodegenerative processes in the brain and formation of behavioral disorders, which are characteristic of the lithium-pilocarpine model, accompany these changes.

Pioglitazone administration, at a dosage of 7 mg/kg via intraperitoneal injection, was performed 75 minutes after induction of the TLE model. This was followed by a daily dose of 1 mg/kg for 7 consecutive days. Brain sampling for biochemical analysis was conducted after the seven-day treatment period. Findings indicated the upregulation of the interleukin-1 receptor antagonist gene, Il1rn, as well as expression of neuroprotective proteins S100a10 and Tgfb1.

Over a 30-day period, rats in the lithium-pilocarpine TLE model were orally administered a dose of 109 CFU/rat of Bifidobacterium longum. This resulted in an enhanced expression of the IL1rn gene in the temporal cortex, ventral hippocampus, and amygdala, as well as a reduction in the severity of neurodegenerative and behavioral disorders associated with the TLE model. The sample was taken 24 hours after the final administration.

The results suggest that therapy targeting the expression of astroglial and microglial proteins could be a promising approach for developing new and comprehensive treatments for epilepsy. Further research on the effects of PPARα and PPARβ/δ agonists on the expression of these genes is necessary, as it remains poorly understood.

Genes & Cells. 2023;18(4):593-596
pages 593-596 views

The role of mirror system in influencing the valence evaluation of words: a TMS study

Behera S.K., Nieto Doval C.M., Lyusin D.V., Feurra M.

Abstract

The mirror neuron system (MNS) has been a captivating area of research since its discovery in macaque monkeys [1]. Researchers have established the role and presence of the human auditory mirror system in both empathy [2] and musical creativity [3]. As well, the mirror system allows to learn new behaviors by observation, facilitating knowledge acquisition and action understanding. In addition, research has indicated the influence of stimulus valence on the human MNS [4]. The human brain is sensitive to the positive valence of words, and this reactivity may vary with the strength of the valence compared to negative words. Additionally, it can perceive differences in valence for negative stimuli and words. However, what about emotional words that are auditory? Do these words’ valence, whether positive, negative, or neutral, impact auditory mirror neurons and cortical excitability? The study examines how auditory words of different valences affect the MNS and cortical activity. This novel approach uses auditory stimuli to examine the role of mirror neurons in neuromodulation through a transcranial magnetic stimulation (TMS) protocol, which has not been done in previous investigations. The results of this study suggest that both positive and negative words will induce greater excitability [4], as evidenced by motor evoked potentials. The results of this study will have broad applications. Firstly, it will offer a new contribution to the investigation of mirror neurons through TMS protocol. Secondly, inferences can be drawn and applied in the field of therapeutics, where language is used as a tool to mediate the process. In general, it will increase personal awareness of language in daily communication, leading to an enhanced communication process. Furthermore, the study’s findings imply the existence of auditory mirror neurons and their pivotal role in comprehending the emotional valence of different words, thereby influencing our feelings. This will advance our understanding of the human brain and empower us to make informed decisions.

The study is being conducted with the HSE Automated system for non-invasive brain stimulation, which allows for simultaneous recording of brain activity and eye movement.

Genes & Cells. 2023;18(4):598-601
pages 598-601 views

Neural mechanisms of associative cortical plasticity in cognitive domain: Magnetoencephalographic studies

Chernyshev B.V., Pultsina K.I., Tretyakova V.D., Razorenova A.M., Pavlova A.A., Stroganova T.A.

Abstract

Acquisition of language semantics is widely believed to be facilitated by biological mechanisms of associative learning [1]. However, current understanding of brain mechanisms underlying learning and memory is primarily based on simple models, including animal models, with mechanisms of learning in the cognitive realm largely unknown. Semantic language learning serves as a compelling example of these cognitive processes, whose brain mechanisms pose a challenge to explanation at the level of basic neural networks. Particularly, many processes underlying such learning still lack clear understanding, including the mechanisms that enable correlational coincidence of neuronal representation required for Hebbian plasticity, the reverberatory replay of the to-be associated representations in working memory, and the involvement of consolidation, among others.

We conducted a series of studies modeling the acquisition of semantic knowledge of action-related vocabulary to investigate these questions. We implemented a unique trial-and-error-based procedure for semantic acquisition, in which participants obtained associations between newly introduced pseudowords and actions through active learning. The experimental procedure comprised the delivery of acoustically equivalent pseudowords that were previously unknown to the subjects. Participants were instructed to execute movements of their hands and feet in response to specific pseudowords. Following each trial, participants received either positive or negative feedback depending on their performance. During the learning phase, MEG was recorded, as well as during passive presentations of the same pseudowords before and after learning to control for attention and motor preparation effects. Our analysis involved event-related fields and beta oscillations.

Our experiments showed that cortical plasticity related to word learning could be detected immediately after learning, without the need for prolonged consolidation time [2].

We observed two effects during learning that could contribute to Hebbian plasticity by linking auditory speech representations of pseudowords with associated movements.

First, we observed the reactivation of auditory speech representations during the initialization of movement. This mechanism apparently creates the conditions for temporal coincidence of activations in two representations, specifically an auditory speech representation and a representation of a motor program.

During the learning process, we observed a considerable rise in beta oscillations that occurred during and after action execution. This effect comprised a typical beta-rebound in the sensorimotor regions and a significant surge in beta activity in various associative cortical regions. We postulate that these beta oscillations constitute a reverberation mechanism that reinforces the associative connection and guards it against potential interference [3].

In further experiments, we administered the learning process over two consecutive days with an overnight interval between recordings. We evaluated beta power dynamics during the acquisition of novel pseudowords on day 1 and during task rehearsal following overnight sleep on day 2. Beta event-related synchronization in the frontal regions emerged upon the attainment of the pseudoword learning rules on day 1, and remained unchanged after sleep and subsequent rehearsal on day 2. The prefrontal beta rhythm’s dynamics mirrored the corresponding behavioral changes: subjects executed the task with minimal errors on day 2 upon meeting the learning criterion on day 1. Beta event-related synchronization augmented continuously in the posterior temporal and parietal cortices on day 2 and potentially contributed towards the assimilation of newly-acquired associations in long-term memory.

In summary, our research offers fresh perspectives on the intricate neural mechanisms underlying plasticity in the human brain within the cognitive domain.

Genes & Cells. 2023;18(4):602-605
pages 602-605 views

Neurophysiological mechanisms of exploration and exploitation in high-functioning autism: Magnetoencephalographic study

Chernyshev B.V., Pultsina K.I., Tretyakova V.D., Miasnikova A.S., Prokofyev A.O., Kozunova G.L., Stroganova T.A.

Abstract

Intolerance of uncertainty and high sensitivity to the threat of failure are believed to contribute to chronic anxiety in individuals with high-functioning autism. This study examines the influence of these personality traits on the brain processes involved in decision-making in a probabilistic setting among autism patients. Furthermore, we explore the causal relationship between these personality traits and chronic anxiety in individuals with high-functioning autism.

Twenty-one high-functioning autistic individuals who are intolerant to uncertainty, aged 19 to 46, and 21 neurotypical volunteers of the same age, carried out a probabilistic choice experiment with two alternatives. One option yielded a monetary profit of 70% of the time, while the other brought only a 30% gain. Following each selection, the subjects received feedback on their decision and gradually learned, through trial and error, to prefer the more advantageous option. From this point forward, we consider frequent selections of a beneficial stimulus to align with the internal utility model, specifically the exploitation strategy. On the other hand, sporadic choices of a detrimental stimulus correspond to an exploration strategy, which may prove disadvantageous in a stable environment but enables adaptation to unforeseen changes in the surroundings.

We hypothesized that there are characteristic differences in brain activity reflecting exploration and exploitation strategies between groups, which would emerge during the decision-making period and after internal feedback evaluation regarding advantageous and disadvantageous choices [1]. Beta oscillations (16–30 Hz) were analyzed in the magnetoencephalographic recordings. The decrease in beta oscillation power below baseline during the period between stimulus presentation and subjects’ response indicates activation in brain regions related to decision-making strategy. Conversely, an increase in beta oscillation power following feedback signaling a disadvantageous choice reflects a mechanism that reinforces internal value models in current task conditions [1]. The cortical sources of beta oscillations in 448 cortical areas were estimated using the sLoreta technique at the single-trial level. Mixed linear models (LMM) were used for statistical analysis with correction for multiple comparisons via the FDR method for the number of cortical areas analyzed. The study concentrated on the time interval for decision-making (–900 to –300 ms prior to the motor action of choice) and the post-feedback period (500 to 900 ms after the commencement of the feedback presentation) [1].

Based on questionnaire results, individuals with high-functioning autism exhibited a significantly lower tolerance for uncertainty and a greater intolerance for uncertainty when compared to neurotypical participants.

The study generated two primary findings. Initially, the extent of brain activity during decision-making differed in control subjects and subjects with autism based on the type of choice, with exact opposition. Opting for an advantageous vs. disadvantageous choice was linked with reduced activation of the inferior temporal, parietal, and medial frontal cortex regions in control subjects and elevated activation in these regions for subjects with autism spectrum disorder. These findings suggest that neurotypical individuals utilize fewer neural resources and exhibit decreased emotional response while deciding in favor of a known profitable outcome compared to opting for an uncertain choice — one that is more likely to result in a negative outcome based on previous experiences. Individuals with autism allocate disproportionate amounts of attention and emotional resources towards planning actions that are deemed safe and offer only a probable (though not definite) advantage. Conversely, the prospect of failure due to risky behavior induces a comparatively muted response in their brains.

Second, our internal evaluation of feedback focused on variations in functional activity within the orbitofrontal and lateral prefrontal cortical areas during exploratory (disadvantageous) choices between individuals with autism and control subjects. As previously reported, neurotypical subjects demonstrated significant beta synchronization following negative feedback after disadvantageous choices [1]. Contrary to control participants, individuals with autism spectrum disorder exhibited a lack of synchronization in frontal beta oscillations following losses incurred from unfavorable selections. This observation may indicate insufficient reinforcement of the internal utility model, which typically strengthens in response to negative outcomes that align with predicted results [1].

Overall, our study found that individuals on the autism spectrum with a high intolerance for uncertainty exhibit significantly increased activation of brain decision-making systems in situations that are perceived to be low-risk and with a high probability of a successful outcome. These findings clarify the reasons behind the puzzling rise in anxiety and autonomic reactivity among individuals in situations where they anticipate rewards, which are not inherently aversive unlike fear of punishment [2].

Genes & Cells. 2023;18(4):606-609
pages 606-609 views

Analysis of heart rate indices at different levels of sleepiness

Demareva V.A.

Abstract

The objective detection of sleepiness through physiological indices is crucial for ensuring transportation and industrial safety. As gadgets become more commonplace for measuring heart rate, it is important to identify valid indices that correlate with the level of sleepiness. Research indicates that time-domain and frequency-domain heart rate indices align with levels of alertness and sleepiness [e.g., 1, 2]. The aim of this paper is to identify heart rate indices that correlate with varying degrees of evening sleepiness in vivo, whether low or high.

The data analyzed in this paper was obtained from 32 recordings from the SSDD (Subjective Sleepiness Dynamics Dataset) collected at the UNN Cyberpsychology Laboratory since 2022. The experiment was designed in the following manner: participants put on a heart rhythm sensor (Polar H10) at 7:40 PM in their homes and connected it to a smartphone app. Participants then completed a self-reported questionnaire in the electronic system. Starting at 8:00 PM, and every 30 minutes thereafter, the participant recorded data on the subjective level of sleepiness via the Karolinska (KSS) and Stanford (SSS) sleepiness scales up until the time of the participant’s bedtime. The selection of 32 recordings met specific criteria: all participants went to bed between 10:30 and 11:00 PM and completed both the KSS and SSS on time at each time point (08:00, 08:30, 09:00, 09:30, and 10:00 PM).

For each time point, an integral sleepiness score was calculated according to the formula:

sl=(KSS/10+SSS/7)/2.

The 4-minute rhythmogram recordings corresponding to the time points of KSS and SSS filling were chosen for analysis. Data were processed in Jupyter Notebook. Heart rhythm indices, including time-domain, frequency-domain, and nonlinear indices, were computed for each time point from the rhythmogram — NN-interval sequence — using the “hrv-analysis” package. The study employed Student’s t-test to compare indices for varying levels of sleepiness based on integral scores, where “low level” was defined as sl <0.45 and “high level” was defined as sl >0.55. Additionally, the Pearson criterion was used to evaluate the correlation between sl and heart rate indices at each time interval.

The indices responsible for the variability of the NN-interval sequence, namely NNi_50 (p=0.027), NNi_20 (p=0.007), and pNNi_20 (p=0.024), exhibited lower values during periods of “low” sleepiness (N=54) as compared to “high” sleepiness (N=58). Moreover, we observed that the autonomic balance index, i.e., the ratio of the power of the heart rate variability spectrum in the low-frequency band to that in the high-frequency band, was higher during ‘high’ sleepiness (p=0.015). Analysis revealed that correlations between integral sleepiness score and heart rate indices were present only for the 08:30 PM time point. Correlations of sl with the NN-interval range (R=–0.388; p=0.028), the severity of sympathetic regulatory circuit activity (R=–0.383; p=0.031), and the nonlinear cardiovagal index (R=–0.359; p=0.043) were found.

Thus, it can be inferred that increased sleepiness is associated with decreased heart rate variability indices, along with decreased functioning of the autonomic nervous system overall.

Genes & Cells. 2023;18(4):610-613
pages 610-613 views

Neural oscillatory correlates of motor vigor: an magnetoencephalographic study

Ivanova M.D., Germanova K.G., Herrojo Ruiz M.

Abstract

Previous studies have shown that the anticipation of reward enhances motor performance, which reduces movement time and increases velocity [1]. In our recent study [2], we observed that when participants are required to infer the changing probabilities of a reward in a dynamic and uncertain setting, heightened expectations are consistently associated with faster motor performance. The study showed that performance time sensitivity to prediction strength remained consistent among both young and older healthy adults, as well as those with Parkinson’s disease. While the effects of dynamic motor strength have been observed, the neurological processes involved remain to be determined [3].

The study examined the neural oscillatory connections to motor vigor in dynamic and unpredictable settings. We used magnetoencephalography (MEG) and individual structural magnetic resonance imaging (MRI) to record readings from 25 healthy human participants (18 females) during the execution of our newly developed reward-based motor decision-making task [2]. This study used a reversal learning paradigm with shifting stimulus-outcome relationships. Participants were required to deduce which of two stimuli was linked to a reward on each trial, and indicate their choice through one of two finger press sequences, each with a distinct auditory response. The task was conducted in an unstable context, leading to fluctuations in the probability of reward associated with each response over time.

First, we examined decision-making behavior using the validated Hierarchical Gaussian Filter (HGF, [4]). The model that most accurately described the behavioral data was the three-level “extended” HGF for binary categorical inputs, which is paired with a response model where decisions are dependent on the trial-wise estimate of volatility. This study allowed for the generation of reward probability trajectories on a trial-by-trial basis. Subsequently, applying Bayesian linear mixed models, we found a relationship between belief strength regarding reward contingencies and performance tempo on a trial-by-trial basis.

The analysis of MEG signals is centered on reconstructing oscillatory activity sources using Linearly Constrained Minimum Variance beamforming [5]. Currently, we use convolution models in the source space to identify neural oscillatory correlations that differentiate motor performance and decision making. Next, we will evaluate connectivity patterns between frontal and motor regions that underlie the effects of motor invigoration. Identifying particular patterns of oscillatory connectivity that modulate motor vigor can provide insights into motor deficits observed in neurological and neuropsychiatric conditions associated with behavioral apathy.

Genes & Cells. 2023;18(4):614-617
pages 614-617 views

Emotion regulation: a study of electroencephalographic correlates

Kosonogov V.V., Ntoumanis I., Hajiyeva G., Jaaskelainen I.

Abstract

Effective emotion regulation is associated with improved subjective well-being, mental health, and social goal attainment. Poor psychosocial well-being, depression, and psychiatric disorders are often associated with the inability to regulate emotions. Cognitive reappraisal, which focuses on events, and expressive suppression, which is reaction-oriented, have both garnered significant attention in psychophysiological studies as effective strategies for emotion regulation. Cognitive reappraisal focuses on changing the meaning of a situation, while suppression occurs later in the emotional experience, when efforts are made to suppress the behavioral and physiological responses associated with ongoing emotions [1]. Suppression regulates emotions for a shorter period, while reappraisal has a long-term effect on emotion regulation.

While somatovegetative markers of emotion regulation have been thoroughly investigated [2, 3], further research is needed to explore the brain correlates of this process. Our aim was to examine the electroencephalographic (EEG) correlates of emotion regulation. For this study, we computed correlations between subjects or inter-subject correlation (ISC; an indicator of attention, engagement, and tension [4]) and EEG valence and arousal indices [5] (64 leads in total). Sixty participants (average age=26.0) either suppressed their emotional responses, employed reappraisal, or viewed neutral 1-minute videos with negative content (36 trials in total). Both suppression and reappraisal elicited higher levels of ISC compared to viewing negative or neutral videos (F(3, 168)=25.23, p <0.001, η²=0.10). The pairwise comparisons revealed that viewing neutral videos resulted in lower ISC than viewing negative videos (t(56)=4.26, p <0.001, d=0.47), suppression (t(56)=9.04, p <0.001, d=0.78), and reappraisal (t(56)=11.0, p <0.0001, d=0.77). Both suppression and reappraisal resulted in higher ISC compared to watching negative videos (t(56)=3.38, p=0.002, d=0.40 and t(56)=2.96, p=0.005, d=0.39, 1-β=0.83, respectively). It suggests a need for task engagement and feedback processing to manage emotion. Additionally, the arousal index was greater in all negative conditions, suggesting that regulation necessitated a specific level of arousal (χ2(3)=12.8, p=0.005, W=0.075). Wilcoxon’s tests revealed a significant decrease in arousal index when viewing neutral videos compared to negative ones (V=465, p=0.012, r=0.053). Conversely, suppressing (V=499, p=0.019, r=0.057) and reappraisal (V=395, p=0.004, r=0.096) elicited higher levels of arousal. Thirdly, the EEG valence index exhibited elevated levels in both emotions regulation states compared to the neutral state (χ2(3)=10.5, p=0.015, W=0.061). Furthermore, the Wilcoxon tests revealed that the valence index was reduced when watching neutral videos in comparison to suppression (V=525, p=0.048, r=0.136) and reappraisal (V=500, p=0.048, r=0.165). This suggests an upsurge in the positive emotional aspect when regulating emotions.

Overall, various EEG measurements reflect distinct aspects of emotion regulation, although both suppression and cognitive reassessment induced greater brain resource allocation than passive viewing.

Genes & Cells. 2023;18(4):618-621
pages 618-621 views

Cognitive decline and affective alterations in SCA2-58Q mice

Marinina K.S., Bezprozvanny I.B., Egorova P.A.

Abstract

The cerebellum is traditionally recognized as a brain region dedicated to motor control and movement coordination. Yet, the growing body of research has recently highlighted the cerebellar role in cognitive processes, learning, and emotional states. Patients with damage to this particular region of the brain exhibit deficiencies in verbal communication, impaired speech, executive functions, and memory, along with various affective disorders [1]. In the field of neurodegeneration research, the cerebellum is primarily recognized for its role in the onset of spinocerebellar ataxias (SCAs). SCA2 is an autosomal dominant polyglutamine-related neurological disorder resulting from a notable escalation in the number of CAG triplet repeats that encode the amino acid glutamine in the ATXN2 gene. The mutated protein ataxin-2 fails to perform its molecular functions, disrupting calcium homeostasis and cellular activity, ultimately resulting in widespread Purkinje neuron death in the cerebellar cortex [2]. In our research, we used SCA2-58Q transgenic mice that express the mutant human ataxin-2 gene under the action of the Purkinje-specific promoter (PCP2/L7). Each of these tests were aimed at evaluating specific aspects of the mice’s behavior. This model enabled to investigate the direct involvement of Purkinje cells in the cognitive and affective symptoms associated with SCA2. Our team conducted three behavioral tests to determine the anxiolytic behavior: the open field test, the novelty suppressed feeding test, and the light-dark place preference test. The novel object recognition and fear conditioning tests were used to assess recognition and contextual memory, respectively. The Morris water maze test was conducted to evaluate spatial learning and memory. The researchers assessed depression and anhedonia levels using the forced swimming, tail suspension, and sucrose preference tests. The study revealed anxiety, spatial memory impairment, and affective decline in 7–8-month-old SCA2 mice. Previous results of the beam walk test [3, 4] indicated a delay in motor skill development in SCA2-58Q mice, which began at 8–10 months of age. This indicates that cognitive decline and affective changes occur before the primary motor impairment in spinocerebellar ataxia type 2. Currently, there is no disease-modifying treatment for SCA2, and patients are typically given supportive therapy and treatment for individual symptoms. In conclusion, our research provides a more comprehensive clinical picture and takes a step towards optimizing the diagnosis and selecting more effective therapy options for SCA2.

Genes & Cells. 2023;18(4):622-624
pages 622-624 views

Neurophysiology of creativity in conditions of competitive social interaction: data of EEG hyperscanning study

Nagornova Z.V., Shemyakina N.V.

Abstract

Brain activity changes significantly across various social interaction conditions. However, the impact of social interaction context on neurophysiological correlates of cognitive and creative activity per se remains insufficiently explored [1]. Two distinct types of interactions can be identified when it comes to resolving tasks: cooperation or competition. The aim of this study was to evaluate how competition conditions affect the amplitudes of event-related potentials (ERP) when solving creative and non-creative problems.

Subjects (26 men and 18 women) performed two types of tasks both individually and in pairs of the same gender. The tasks consisted of creative and non-creative activities. In the creative tasks, participants were asked to describe unusual uses of everyday objects, such as a brick, newspaper, or paper clip. In the non-creative tasks, participants were requested to list items from various proposed categories, such as transport, furniture, or sports. A total of over one hundred samples of each type were provided for both individual and joint performance. The order of task performance, whether individual or joint, was randomized between pairs of participants. When tasks were performed in pairs, the first responding participant’s answer created competitive conditions and was considered the first response.

During the tasks, we recorded EEG/ERP data synchronously using a Mitsar-202 electroencephalograph (LLC Mitsar, St. Petersburg) and the WinEEG software package (Ponomarev V.A., Kropotov Yu.D., No. State Registration 2001610516 dated 08.05.2001). The recording was monopolar from 15 leads with a referent — combined ear electrode. We modified the 10/20 system with a sampling rate of 500 Hz, and a recording band of 0.53–150 Hz. We analyzed the data using a band of 1.6–30 Hz and a 50 Hz cutoff filter. ERPs for each task (creative or non-creative) were compared between the competition and individual performance conditions by conducting a repeated-measures ANOVA for the STATE factor (individual performance/competition conditions) and STATExZONE factor interaction (15 EEG leads) in the selected time intervals showing differences.

Competition conditions resulted in decreased amplitudes of both sensory components, P1 and P2, and semantic components with N400 and P600 latency in both creative and non-creative activities. This suggests the presence of difficulty in finding an answer. The proportion of correct responses was significantly lower in the competitive conditions than during individual task performance. The median correct response rate was 45% (31–55) compared to 56% (49–74) in the creative task, and 87% (82–93) compared to 66% (60–76) in the non-creative task, during competition and individual performance, respectively. Probably, a significant portion of the resources used when performing the task in social interaction conditions were aimed at evaluating the partner’s reactions and responses. This assessment was reflected in the reduction of earlier components associated with attention (P1, P2) as well as later components associated with semantic processing of stimuli (N400, P600). The conditions of the competition had a more noticeable impact on the ERP in the creative task as compared to the non-creative task.

Genes & Cells. 2023;18(4):625-628
pages 625-628 views

Temporal dynamics of the mirror neurons effect and its stimuli dependent modulation: TMS study

Nieto Doval C., Ragimova A.A., Feurra M.

Abstract

The study of mirror neurons (MN) has made significant progress since human studies commenced. However, when using transcranial magnetic stimulation (TMS), there are inconsistencies in the literature regarding stimulus presentation, duration of presentation, and timing of TMS pulses.

The study assessed the effects of stimuli presentations, using both pictures and videos of hand movements. To accomplish this, single-pulse TMS was applied to the dominant primary motor cortex (M1) during varying time frames (0, 320, 640 ms). Motor evoked potentials were then recorded from the FDI (index finger) and ADM (little finger) muscles of 29 healthy participants via adhesive electrodes. Subjects’ hands were positioned perpendicular to each other, and visual stimuli were presented under three varying conditions. The TMS coil was accurately repositioned using an Axilum Cobot robotic arm and navigation stimulation system to maintain consistency throughout the experiment.

The aim of this study is to provide a comprehensive analysis of stimulus presentation and stimulation timeframes to achieve optimal settings. This paper describes the two most commonly used stimulus modalities, namely, picture and video [1–4], and the frequently employed timeframes for TMS: from movement initiation (picture and video condition) to offset (post-video condition), with different timings (0, 320, and 640 ms) [1, 2, 5]. Notably, the stimulation at the offset of the movement is a novel concept in literature. We conducted three distinct three-way repeated measures ANOVAs employing independent variables. The collected data indicate that the two types of stimulation during the onset of movement, i.e., photograph and video, display varying changes over time. At 320 ms, MEPs increase for the related muscles while nonrelated muscles exhibit inhibitory effects at 640 ms. In the condition of stimulation during movement offset (post-video), this double dissociation is present across all stimulation time frames. Hence, the majority of mirror response can be attributed to inhibition of nonrelated muscles. This study displays the temporal progression of the mirror effect and its impact on both related and unrelated muscles throughout time.

The obtained data illuminates unresolved inquiries in human mirror neuron research and details the impacts of diverse stimuli presentations and TMS stimulation durations. With this information, an ideal protocol can be established to examine the human mirror neuron system tailored to specific research needs. Furthermore, these outcomes can foster the creation of enhanced rehabilitation protocols for patients with movement disorders in clinical settings.

Genes & Cells. 2023;18(4):629-632
pages 629-632 views

Working memory: what does research say about oscillation and functional connectivity?

Otstavnov N.S., Voevodina E.A., Fedele T.

Abstract

Working memory is a crucial cognitive function for processing and storing information in short-term memory during purposeful activities [1]. Numerous studies using EEG, MEG, and stereo-EEG were conducted to identify the neurophysiological correlates of working memory, including oscillations in specific brain regions in the absence of a memorized stimulus. The studies explored multiple aspects such as encoding, maintenance, processing, ordering, updating, and inhibition and various working memory modalities. Additionally, the studies highlighted the significance of examining the functional connectivity between different brain regions. As a result, researchers have amassed a large amount of data that is often contradictory and lacks organization [2].

The aim of this study is to perform a methodical review of literature published from 1980 up to the present period, cataloged in the Scopus, Web of Science, and Pubmed databases. The systematic review follows the PICo structure and addresses three main questions: which neuronal networks are involved in different working memory modalities and detect synchronizations between and within frequencies in cognitively safe subjects; are there disparities in the characteristics of neuronal networks between adults and the elderly; and can various interventions be employed to align the characteristics of neuronal networks in the elderly with those of adults [3]. The systematic review follows PRISMA-P 2015 protocol [4]. Main inclusion criteria focus on investigating working memory, analyzing visual, verbal and nonspecific modalities, using EEG/MEG/stereo-EEG, assessing adult and elderly subjects, and providing quantitative results from behavioral and neurocognitive research. Exclusion criteria: The study excluded emotional stimuli and assessments of long-term memory as well as the use of fMRI. Additionally, unigender studies and studies with subjects who had cognitive or other impairments were not included. In addition, the study required a sufficient number of subjects and time-frequency analysis. Moreover, physical or dietary interventions were excluded, and experimental research was necessary for inclusion. The study did not assess working memory in a foreign language for the subjects. A systematic review was conducted on the platform nested-knowledge.com.

Based on the given criteria, a literature search was performed, yielding 2,828 articles meeting the inclusion criteria. These were further screened by two experts, resulting in the identification of 234 relevant articles, and upon full text review, 89 articles were deemed relevant. The resulting pool of articles is characterized by the following parameters: 69 articles describe adult subjects, 5 articles describe elderly subjects, and 12 articles compare both groups. Additionally, 42 articles focus on oscillation, 15 on functional connectivity, and 32 on both metrics. Concerning working memory, 22 articles describe all stages, 8 focus on encoding, 11 on encoding and maintenance, 4 on maintenance and recall, 23 on maintenance, 9 on maintenance and processing, 7 on processing, 1 on recall, and 3 without separating stages. Furthermore, 28 articles pertain to verbal memory, 51 to visual memory, 8 articles contain comparisons of modalities, and 2 articles discuss modally non-specific tasks.

Preliminary findings from the systematic review indicate distinct functions of various frequency bands for working memory. Specifically, theta rhythms (4–8 Hz) in the frontal lobe were predominantly associated with information maintenance, while demonstrating a propension for increased synchronization during information processing. The alpha rhythm (8–14 Hz) was associated with inhibiting irrelevant data and protecting the current content of working memory, aligning with the widely accepted paradigm [5]. The beta rhythm (14–28 Hz) was most frequently noted to occur when maintaining and recalling information, and its power was associated with memory accuracy indicators. The gamma rhythm (28 Hz and higher) was seen during the process of encoding and maintaining information, and exhibited greater power for more complex stimuli.

The analysis of connectivity revealed that encoding visual and verbal stimuli involves interhemispheric frontal-temporal and frontal-central connections that interact through theta rhythms. Additionally, storing information relies on the interplay of theta and gamma rhythms between the frontal and parietal networks. Notably, when dealing with verbal stimuli, the connectivity of the frontal and temporal brain lobes through theta rhythm enhances with load during storage. The alpha rhythm facilitates communication across posterior and frontal divisions during information storage, whereas beta rhythm is associated with frontal-temporal connections. During visual information storage, theta rhythm mediates communication across frontal-postcentral connections. As storage load increases, theta rhythm leads to strengthening of frontal-parietal and frontal-frontal connections until the threshold of working memory capacity is reached, after which these connections weaken.

When processing information, connectivity of the right frontal-occipital network, right prefrontal and left occipital regions, right frontal and occipital-parietal areas for visual memory, and frontal-parietal regions for verbal memory were observed in the theta band. Connectivity of the occipital brain regions primarily occurred in the alpha spectrum, and temporal regions exhibited high frequencies.

No specific differences were observed between the elderly and adults, aside from the suppression of low frequencies and the prevalence of high frequencies in the connectivity analysis.

Genes & Cells. 2023;18(4):633-636
pages 633-636 views

Model of cognitive activity of the human brain based on the mathematical apparatus of quantum mechanics

Petukhov A.Y., Petukhov Y.V.

Abstract

Describing the transmission and processing of information by an individual is a fundamental issue in modern cognitive science. Previously, unique scientific models for information transfer between individuals [1], as well as models for cognitive activity [2], were developed. However, numerous presented models lack scalability and formalization, hindering their ability to provide a comprehensive explanation for information transfer processes and their distortion due to interaction with the external communicative environment.

One aspect of human cognitive activity is that individuals think not based on a code, as a computer does, but through the interaction of various mental images. Despite the fact that these images have a specific material foundation in the form of electrical and chemical activity in the human brain, describing them using conventional mathematical models presents several difficulties [3].

Therefore, this paper suggests novel techniques for describing information images/representations’ functioning, which simulate an individual’s cognitive activity. These methods rely on the mathematical models of self-oscillatory and conventional quantum physics (including potential wells and virtual particles conventionally used in the physical domain) for characterizing basic interactions [4]. The authors adopt a phenomenological perspective and do not regard cognitive systems as quantum.

The aim of this study ject is to establish a model for cognitive function in the human brain using the mathematical principles of self-oscillating quantum mechanics from the perspective of information imaging and representation.

Methods. The theory proposes [5] that information images/representations (IR) share characteristic properties with virtual Feymann particles and other elementary particles. The human mind is presented as a one-dimensional potential well with finite walls of varying sizes and an internal potential barrier that simulates the boundary between consciousness and subconsciousness. The authors have applied parametrization based on this theory.

As a result, we propose the foundations of the mathematical apparatus based on classical quantum mechanics, followed by the mathematical apparatus of self-oscillating quantum mechanics. The latter, though little known, may allow to predict certain cognitive functions of the human brain by modifying and applying it to non-quantum settings. An equation is derived for the state function of the information image of an individual engaging in cognitive activity.

Primary calculations were conducted on the state functions of information images using a computer model. The movement patterns of the information image within and outside were then deduced.

Genes & Cells. 2023;18(4):637-639
pages 637-639 views

Neural tracking of natural speech listening in children: temporal response function (TRF) approach

Rogachev A.O., Sysoeva O.V.

Abstract

Speech development is crucial for a child’s mental growth. Moreover, speech development significantly impacts a child’s educational and professional achievements. It enables the child to interact with the external environment and develop self-awareness and behavioral skills. Thus, the study of the mechanisms of speech development disorders and the development of diagnostic and remediation strategies is essential.

Numerous cognitive and neurophysiological investigations into speech and its associated disorders among children are presently being conducted. Electroencephalography (EEG) studies demonstrated constant evoked reactions in response to auditory and visual stimuli associated with speech, including individual phonemes and syllables. Moreover, alterations in these reactions were detected among children with diagnosed speech ailments. The debate surrounding the neurophysiological predictors and correlates of specific speech development disorders continues. The use of isolated “ideal” stimuli and multiple repetitions of a single stimulus, as required by the method of evoked potentials, may create peculiarities in experimental techniques. Thus, brain responses to prolonged, “natural” stimuli may differ from those obtained with isolated stimuli. This could potentially reduce the ecological validity of such studies.

In recent years, the temporal response function has become increasingly popular in speech research. This method enables estimating neurophysiological responses to continuous, natural, and ecologically valid stimuli [1–3]. When applied to speech research, this method allows for the study of the brain’s response to changes in acoustic, linguistic, and semantic characteristics present in natural narrative speech [1].

The mathematical basis of the temporal response function (TRF) is the solution of the equation:

w=(STS+λE)–1·STR,

It is calculated from the stimulus characteristics, represented by the matrix S, the neurophysiological signal corresponding to the stimulus, represented by matrix R, and the temporal response function, represented by matrix w, a matrix of linear transformation coefficients from stimulus space to response space [1]. The TRF serves as a “bridge” between the stimulus and the neurophysiological response as it reflects the neural operations that occur between the two. The S and R matrices are matrices with time lags, enabling estimation of the brain’s response to the presented stimulus within a specific time period.

The TRF has been utilized extensively in speech studies [2, 3]. Nevertheless, few studies have used this approach in research that involves children [4, 5]. The use of ecologically valid speech stimuli in child studies simplifies their performance in experimental paradigms and enables the evaluation of brain responses to speech as it occurs in real-life situations, not only in experimentally created conditions. The TRF has various applications to both linguistic and acoustic features of speech, which attracts particular interest in studying the psychophysiological mechanisms of speech development in children with various developmental trajectories. This approach is applied in our study of speech development in children aged 3 to 8 years.

Fifty-six children, consisting of 33 boys and 23 girls aged between 3 and 8 years, participated in this study with a mean age of 5.64 (SD=1.33 years). Participants were required to listen to three audio stories, including a children’s story about hedgehogs and adapted versions of the tales “Brick and Wax” and “The Golden Duck”, all of which were recorded by a female voice. All audio stimuli were accompanied by video to maintain children’s attention. The total duration of the stimuli was 15 minutes. The audio stories were presented using Presentation® software from Neurobehavioral Systems, Inc. in Berkeley, CA. The comprehension of the stories was assessed by asking children 8 “yes/no” questions after each story. Furthermore, on a different day of the study, the Preschool Language Scales Fifth Edition (PLS-5) method was used to examine the child’s current level of receptive and expressive speech development.

A 32-channel EEG was obtained using a Brain Products actiCHamp (Brain Products GmbH, Gilching, Germany) with reference electrodes positioned at the FCz location. EEG pre-processing was completed with the MNE library for Python, which entailed data filtering between 1 and 15 Hz, visually examining record for any noisy channels, interpolation of deficient channels (as needed), removal of oculomotor artifacts using independent component analysis, and re-referencing the EEG recording to an average electrode. The EEG and stimulus were synchronized by labeling at the start of the stimulus. They were subsequently aligned during specific epochs. Processing was carried out with MATLAB (version 2021b) using the mTRF Toolbox [1]. The Toolbox’s functions were employed to assess the speech stimulus envelope, which was then introduced as input to the TRF. The stimulus and EEG sampling rate were reduced to 128 Hz, and the analysis used a time window ranging from –200 to 800 ms. The TRF prediction coefficient, representing the correlation coefficient between actual data and data predicted by the model post-training and cross-validation, was selected for analysis.

The mean value for prediction coefficients across the entire sample was 0.041 (range: –0.002 to 0.106). These coefficients were significantly different from zero (t(55)=13.1, p <0.001). Additionally, a significant positive correlation was found between the prediction coefficients averaged intraindividually across all EEG channels and the age of the participants (r=0.379, p=0.004). The linear model underlying the TRF was able to predict the EEG signal better as the age of the child increased.

A significant positive correlation was observed between the prediction coefficient values and the values on the receptive speech scale of the PLS-5 (r=0.33, p=0.026). In addition, PLS-5 scores were strongly correlated with age (r=0.596, p <0.001).

There was a positive correlation observed between the model prediction coefficient and the scores obtained from the listening comprehension questionnaire (r=0.39, p=0.012). Additionally, the questionnaire scores were found to be significantly associated with scores from the PLS-5 receptive speech scale (r=0.82, p <0.001) as well as with the age of study participants (r=0.51, p=0.001).

Substantively, the predictive coefficient of the temporal response function illustrates the cortical tracking process of the stimulus currently receiving attention and is significantly associated with listening comprehension [2, 3]. Our research indicates a significant and positive correlation between children’s age, their comprehension of speech as measured by the PLS-5 method, and the results of the listening comprehension questionnaire conducted immediately after the experimental task. The prediction coefficient supports this finding. Thus, the use of the temporal response function enables the evaluation of the cerebral cortex’s capacity to follow the acoustic signal of speech in children. Additionally, this approach yields neurophysiological markers of speech reception and comprehension processes. It is feasible to apply an experimental framework to identify neurophysiological correlations of receptive speech across various age groups and participants with varying levels of language and speech skills. The experimental paradigm presented here is a component of research carried out by the Neurobiology of Oral and Written Speech in Developmental Disorders division at the Center for Cognitive Sciences, Sirius University. The authors extend their gratitude to the study participants and project team.

Genes & Cells. 2023;18(4):640-644
pages 640-644 views

Effects of transcranial magnetic stimulation on cortical structures during motor imagination performance in the brain-computer interface

Savosenkov A.О., Grigorev N.A., Udoratina A.M., Kurkin S.A., Gordleeva S.Y.

Abstract

The aftermath of a stroke can frequently result in impaired motor functions causing problems performing habitual limb movements. These movement disorders stem from damage to the cerebral cortex and disruptions to neuronal connections in the central pyramidal pathways [1]. Restoring motor skills following a stroke is a time-consuming and challenging process, requiring resources from both external medical sources and the patient. Despite these challenges, it is still feasible for patients to regain control over their limb movements. The common approach for stroke neurorehabilitation consists of therapeutic physical activities and kinesiotherapy. These methods rely on afferent information during motor tasks to repair connections between intact brain areas [2]. Through exercising the impacted limbs, synaptic rearrangement happens in the cortex, awakening dormant neurons and increasing cortical areas adjacent to inactive regions. Although these techniques can partially restore movement, a significant number of stroke patients still face impairments. It is crucial to acknowledge the limitations of the current interventions and explore new approaches for better outcomes. Traditional rehabilitation methods often do not fully restore movement control, prompting researchers to explore alternative approaches. Motor-imagery-based brain-computer interfaces (BCIs) have gained attention recently [3, 4]. They allow for integrating various feedback mechanisms, which can be combined with upper and lower limb exoskeletons. The level of control a subject has over a BCI system directly affects the recovery process [5]. Introducing transcranial magnetic stimulation (TMS) into motor-imagery BCIs shows promise for developing a unified and highly efficient approach to post-stroke rehabilitation.

Twenty-nine healthy adult participants (21 females) with a mean age of 20.93±2.14 years were recruited for this experiment. All participants were right-handed and had no prior experience with brain-computer interfaces. Ethical approval was obtained from the local ethics committee of Lobachevsky State University of Nizhny Novgorod (ethical approval No. 2, dated 03/19/2021), and written informed consent was obtained from all participants. Subjects were randomly assigned to receive either a sham intervention (n=15) or active TMS (n=14). The experiment displayed tasks on a 24-inch LCD screen placed 2 meters away. Participants sat in a reclining chair with hands on adjustable armrests for EEG signal recording. Motor-imagery-based BCI training took place over two days with four daily tasks: motor performance, quasi-motor, and two motor imaginations of the dominant hand. EEG activity was recorded prior to and after completing the tasks. Each task comprised of 20 trials, each lasting 10 seconds. TMS was administered in between two motor imagination tasks, and a rest period of 2 minutes followed the stimulation. The researcher objectively monitored electromyography (EMG) in real-time during motor tasks. Certified NVX 52 amplifiers with 32 Cl/Ag electrodes positioned according to the international 10-10 system recorded electroencephalography (EEG) signals. The EEG was digitized at a sampling rate of 1000 Hz and filtered with a 50 Hz Notch filter. Disposable electrodes were used to record EMG data from musculus flexor digitorum superficialis on the right hand. EMG signals were digitized at 1000 Hz and filtered using a 50 Hz Notch filter. TMS was applied to the dorsolateral prefrontal cortex (dlPFC) using a figure-8 coil connected to a Neuro MS/D magnetic stimulator. Sham stimulation was performed with the same parameters, except the coil was tilted 90 degrees to simulate the sound of real stimulation. A nonparametric permutation test was conducted to assess the statistically significant dissimilarities between rest periods and periods subsequent to conducting motor imagery after TMS. Significant clusters of the highest event-related desynchronization (ERD) were observed. The analysis demonstrated a significant negative cluster in the theta rhythm (0–6 Hz) that was distinct from the magnetic stimulation site. Similar significant ERD clusters were identified during the first and second series of imaginary movements.

The study examined alterations in motor cortex function following targeted rTMS. The results demonstrated that TMS using particular parameters resulted in the pre-activation of brain regions comparable to those stimulated during motor imagery. The implementation of this stimulation protocol increased activity in cortical regions related to motor imagery. The outcomes propose the probable effectiveness of TMS in elevating motor cortex activation during the rehabilitation process.

Genes & Cells. 2023;18(4):645-648
pages 645-648 views

How can quasi-movements be useful in the examination of voluntary movements? An external perspective integrating neuroscience, psychology, and philosophy

Yashin A.S., Vasilyev A.N., Shishkin S.L.

Abstract

In 2008, V. Nikulin et al. [1] identified quasi-movements (QM), a type of motor task relying on voluntary movements. QM become noticeable when a person reduces a movement to the point where its associated muscle activity is undetectable through electromyography (EMG) signals. Like overt movements (OM) and kinesthetic motor imagery (imagined movements, IM), QM prompt event-related desynchronization (ERD) of the sensorimotor rhythms of the electroencephalogram (EEG). Drawing upon M. Jeannerod’s [2] claim concerning the existent line of motor system states between OM and IM, Nikulin et al. postulated that QM could fall under a classification of actions in the intermediate section of said range. Notwithstanding, their findings indicate that, as the magnitude of motion dwindles, QM will continue to manifest an agent’s inclination towards executing a physical action. How does the difference in intention relate to the range of possible motor system states across tasks?

In this study, we evaluated the continuum hypothesis based on QM’s intermediate position between physical and mental actions. We developed two versions of this hypothesis. One pertains to the brain mechanisms responsible for executing motor tasks and predicting their sensory outcomes. According to this hypothesis, the operation of these mechanisms continuously shifts between full-fledged OM and IM. Another version of the hypothesis posits the agent’s awareness [3] of action and proposes a continuum of the agent’s mental states ranging from OM to IM. The second version of the hypothesis suggests that the agent perceives certain actions situated between OM and IM (such as QM) as intermediate ones.

If the first version of the continuum hypothesis is correct, a correlation between ERD power and residual EMG in QM would have been expected. This assumption was made based on the realization of the continuum hypothesis. In the case of OM, high muscle activity and desynchronization of the μ-rhythm occur, whereas in the case of IM, there is a significantly lower ERD with almost complete absence of muscle activity. Since muscle tension is a direct result of motor system function, variations in the power of sensorimotor rhythms within a certain range may regulate EMG amplitude. In order to examine the second version of the continuum hypothesis, we have opted to survey participants to gain insight into how individuals subjectively differentiate between QM and OM in addition to IM when viewed from a first-person perspective.

Twenty-three healthy participants took part in our study. The motor tasks in our experiment were based on the thumb abduction method used in Nikulin et al.’s study. This method involves tensing the m. abductor pollicis brevis muscle, enabling accurate measurement of muscle activity. The experiment was conducted over two days, with participants receiving training on the first day for performing thumb abduction, QM, and kinesthetic motor imagery. On the second day, participants replicated their learned skills under three conditions, each corresponding to a different motor task. They rhythmically performed OM, QM, or IM following rhythms consisting of three tones in each condition. To facilitate EEG analysis, the motor tasks were compared to a visual attention task that required participants to count the elements in a picture. On the second day, the participants’ EMG and 128-channel EEG were recorded.

Using more sensitive processing methods than in previous studies [1, 4, 5], we analyzed the disparity between ERD in QM and IM and explored the correlation between EMG parameters and ERD power in QM. Furthermore, we administered a survey to the participants, focusing on their perception of motion in QM and its realism. The respondents provided affirmative or negative responses. We asked participants to provide in-depth reports on the subjective distinctions between QM and OM/IM. Our aim was to examine whether judgments about QM were impacted by residual EMG by comparing these responses with EMG data.

The average EMG values obtained in both QM and IM were comparable to those found by Nikulin et al. Nevertheless, a thorough examination of the EMG data demonstrated heightened peak muscle activity in QM trials. While the contralateral component of the ERD μ-rhythm was independent of the EMG amplitude, it exhibited greater strength in QM as opposed to IM. This result indicates that QM strictly defined and those accompanied by increased muscle activity have a stable pattern of motor system activity that differs significantly from that of IM. Therefore, QM is more likely to constitute a distinct motor phenomenon.

In addition to EEG analysis, we examined subjective reports from participants. We categorized the free-form reports into common descriptors of variations in motor tasks and examined the connection between the descriptors, question responses, and the proportion of trials with elevated EMG in the QM condition. There was no correlation between the proportion of trials with elevated EMG and the participants’ reports on the feeling of movement or the perceived reality of QM. In addition, the descriptors distinguishing QM from IM were not influenced by the residual EMG. Analysis of the free-form reports revealed that participants had comparable intentions in both the OM and QM conditions, which differed from those in the IM condition. The intention to execute a movement in the QM condition correlated with References to “sending a command” to the muscles. Additionally, the perceived reality of QM correlated with mentions of muscle tension during QM, indicating sensory feedback.

The obtained results are not well-matched with either version of the continuum hypothesis. The lack of correlation between EMG and the contralateral element of μ-rhythm desynchronization suggests that the simplest realization of the first version is incorrect. Although QM acts as an intermediate between OM and IM, and EEG presents it as a steady independent phenomenon instead of being part of a continuous range of actions. Subjectively, QM is experienced as an OM with insufficient feedback. This reduces the agent’s confidence regarding the movement’s reality. The agent’s awareness when performing QM and IM differs in quality as they are distinct actions, despite potential imagined feedback in QM. Our study has some limitations, specifically regarding the range of actions between overt and imagined movements, which may not necessarily involve QM. The states within the continuum may possess a complexity greater than the previously assumed arrangement. Another limitation is associated with the experiment’s relatively small sample size. It would be advantageous to use a larger sample size to gain better insights into studying QM.

Genes & Cells. 2023;18(4):649-652
pages 649-652 views

Neurorehabilitation of post-stroke patients using a noninvasive spinal neuroprosthesis

Ananyev S.S., Moshonkina T.R., Zharova E.N., Shandybina N.D., Vershinina E.A., Lyakhovetsky V.A., Grishin A.A., Gerasimenko Y.P.

Abstract

Neurorehabilitation of post-stroke patients with motor impairments is a significant and yet unresolved issue in restorative medicine. We propose a novel approach to rehabilitating such patients using transcutaneous electrical spinal cord stimulation (scTS), which targets the neural locomotor networks of the lumbar enlargement of the human spinal cord [1]. The Spinal Neuroprosthesis device was developed to control stimulation, providing noninvasive and phase-dependent activation of motoneuronal pools of flexors and extensors during a certain phase of the stepping cycle, combined with the activation of neuronal locomotor networks [2].

The aim of this study is to assess the efficacy of Spinal Neuroprosthesis in regulating locomotor functions among post-stroke patients with motor disorders. The study is designed to provide an objective evaluation of the medical device’s effectiveness.

The study was conducted at the Russian Research Institute of Neurosurgery named after Prof. A.L. Polenov. The study enrolled 20 patients who had been experiencing severe motor disorders of the lower extremities in the form of hemiparesis. The duration of stroke among these patients ranged from 3 to 12 months. They were divided into two groups: control and experimental. The control group underwent sham (scTS-) stimulation during a motor rehabilitation session, while the experimental group received real scTS, establishing the difference between the groups. The rehabilitation program comprised 15 sessions of stimulation to the spinal cord. The treatment protocol comprised an initial evaluation of patients’ neurological and rehabilitation status and an investigation of spatial-temporal and kinematic parameters of walking. Subsequently, patients participated in rehabilitation sessions, which entailed walking on the treadmill and over-ground stepping with scTS. Finally, patients underwent a follow-up examination that included a re-evaluation of their neurological and rehabilitation status, as well as an investigation of spatial-temporal and kinematic parameters of walking.

At the beginning of the program, the distance traveled by patients in the control and experimental groups during a six-minute walk test, according to the study results, did not differ significantly. However, following the treatment, patients in the experimental group demonstrated a substantially lengthier distance covered during the 6-minute test than the control group. Both groups of patients in the 10-meter walk test demonstrated an increase in distance walking speed, although the patients in the experimental group had a greater increase in speed compared to those in the control group. These improvements were more pronounced in patients from the experimental group. The results from neurological scales indicated an increase in muscular strength, improvement in balance functions, and an increase in functional independence in both groups.

The findings provide evidence that the Spinal Neuroprosthesis effectively regulates stepping movements and restores locomotor function in patients post-stroke. Clinically significant improvements are observed within two weeks of neuroprosthesis use. Additionally, training increases patients’ exercise tolerance while walking, speed of movement, and functional independence.

Genes & Cells. 2023;18(4):654-657
pages 654-657 views

Current strategies for regenerative therapy of spinal cord injury

Baklaushev V.P.

Abstract

Spinal cord injury (SCI) is a leading cause of death and severe disability amongst young people. The incidence of SCI is 0.6–1.0 per 10,000 individuals. Unfortunately, there are no effective methods of restoring locomotor function for individuals with severe SCI. To address this issue, exoskeleton technology controlled using BCI is actively being developed for prosthetic locomotion. Despite the lack of encouraging data for severe spinal cord injuries, regenerative technologies continue to hold promise for spinal cord repair. The limited options for regenerating the central nervous system in humans necessitate creating new sources of neural stem cells for regeneration. Reprogramming autologous somatic cells neurologically can effectively serve as such a source [1]. Nevertheless, the constitution of neuroglial progenitors, which are necessary for regenerating damaged axons of the pyramidal tract, still requires clarification [2]. Some interesting efforts are underway to directly reprogram glial cells in situ using a variety of biotechnological approaches [3]. Overcoming or preventing the formation of a harsh scar tissue at the injury site is the key obstacle to successful regeneration therapy for SCI. Various scaffolds are being developed to facilitate axon regeneration, and several gene therapy agents are being tested to either knock down scar formation factors or activate extracellular matrix remodeling and reparative regeneration.

Neuromodulation shows promise for SCI treatment. Studies indicate that epidural stimulation of the L2-S1 spinal cord in humans and mammals activates SPG neurons, aiding spinal walking generator functions. A potentially successful treatment approach involves scaffolds with reprogrammed cells and neuromodulation [4].

Genes & Cells. 2023;18(4):658-660
pages 658-660 views

Multifractal characteristics of neuronal activity of the globus pallidus in patients with dystonia

Dzhalagoniya I.Z., Semenova Y.N., Gamaleya A.A., Tomsky A.A., Shaikh A., Sedov A.A.

Abstract

Dystonia is a movement disorder characterized by involuntary muscle contractions resulting in anomalous postures, often accompanied by dystonic tremors. Among the indicative dystonic postures is an involuntary tonic tilt or rotation of the head. Currently, the etiology and pathogenesis of the disease remain theories. Currently, only low-frequency (theta-alpha) oscillations in the globus pallidus are regarded as the possible biomarker of pathological activity in dystonia. In many patients with dystonia, the absence of rhythmic neuronal activity in the basal ganglia renders this biomarker unusable for surgical treatment of the disease. Additionally, the mechanisms underlying the rearrangement of temporary patterns of neural activity that result in pathological symptoms remain unclear.

We propose that the appearance of these pathological rhythms is associated with the reduction of dynamic complexity in the pattern of neural activity within the globus pallidus. We aim to use multifractal analysis in this study to evaluate changes in the globus pallidus neural activity pattern’s dynamic complexity and their correlation with the clinical manifestations of dystonia.

The study examined the electrical activity of the globus pallidus in 23 patients receiving deep brain stimulation (DBS) treatment. We used the microelectrode registration technique and local field potential (LFP) registration during intraoperative neuron monitoring to precisely identify the specific locations of the external and internal segments of the globus pallidus (GPe and GPi). In four patients, an additional postoperative recording of local field potentials (LFP) was conducted while performing DBS and vibrational impact on the neck muscles. A neurologist evaluated the patients to determine the severity of the disease using the Burke-Fahn-Mardsen Dystonia Rating Scale (BFMDRS).

The LEAD DBS software package was employed for postoperative reconstruction of the DBS stimulating electrode positions and for simulating the optimal stimulation zone, selected by the neurologist (i.e., volume of tissue activated, VTA). Subsequently, we identified the region that presented the most significant variation in multifractal spectrum parameters following DBS to understand its correlation with the optimal VTA.

A total of 745 local potential records were analyzed, with 433 in GPe and 312 in GPi. The severity of dystonia was found to be correlated with the parameters of the multifractal spectrum (rho: –0.5630882, p=0.005149 for GPe and rho: –0.5180609, p=0.01133 for GPi). Moreover, as the dystonia severity increased along the BFMDRS scale, the multifractal spectrum became narrower and more asymmetric. In post-surgery recordings, we observed changes in the shape of the multifractal spectrum during DBS stimulation or vibration. In particular, when exposed, the multifractal spectrum significantly widened, and its symmetry was restored. We identified the region with the greatest alteration in the width of the multifractal spectrum following DBS and discovered that this region considerably intersected or fully matched the neurologist’s chosen zone for optimal DBS stimulation.

We demonstrated that multifractal analysis can serve as an ancillary approach to evaluate the extent of proprioceptive feedback impairment and as a biomarker of dystonia. The noteworthy and substantial correlation with dystonia severity underscores the considerable potential of multifractal spectrum features as a biomarker of dystonia. Additionally, the overlapping areas with the most significant DBS impact and the widest variation in the multifractal spectrum in DBS can be used as a tool for identifying the most favorable region for DBS stimulation.

Genes & Cells. 2023;18(4):661-663
pages 661-663 views

Electrical resistance of brain tissue during terminal ischemia

Mingazov B.R., Vinokurova D.E., Zakharov A.V., Khazipov R.N.

Abstract

Terminal ischemia is characterized by several electrophysiological processes, including hyperpolarization, spreading depolarization (tSD), and negative ultraslow potentials (NUP) [1]. tSD is knowin to induce depolarization of neuron membranes to roughly 0 mV, accompanied by the influx of positive ions into cells, which results in the displacement of extracellular fluids into neurons, leading to cell swelling. In addition to the movement of extracellular fluid, cerebrospinal fluid flows into the perivascular region of the penetrating vessels. These processes result in a reduction of extracellular space and brain edema [2], which can elevate the likelihood of patient mortality up to 80% [3]. The decrease in extracellular space may lead to an upsurge in the electrical resistance of the tissue, enabling it to function as an important indicator of the anatomical and functional state of the brain during traumatic injuries and hemorrhages [4].

Our goal was to assess the resistance of the extracellular space in the rat’s barrel cortex by measuring voltage step amplitudes caused by current injection between the V1 cortex and the tail vein of an animal. We used 16-channel probes with iridium electrode sites and glass microelectrodes containing Ag/AgCl conductors filled with NaCl solution. We used a metal Ag/AgCl electrode as a reference, implanted in the cerebellum. The experimental animals were euthanized via inhalation of isoflurane at a lethal concentration.

Isoflurane-induced respiratory arrest led to the subsequent development of a sequence of electrophysiological processes that included hyperpolarization, terminal spreading depression, and negative ultraslow potential. The NUP persisted throughout the entirety of the recording period (30–90 minutes), with a significant increase in extracellular space resistance occurring during this time. The rise in resistance began concurrently with the end of respiration. The resistance increased by 40 [23–57]% (median [25th–75th percentile], p=0.002, n=11) after 30 minutes of breath cessation, and by 46 [(–15)–64]% (p=0.109, n=8) after 60 minutes. The resistance demonstrated equivalent changes in all cerebral cortex depths. Additionally, the NUP amplitude following respiratory arrest had a correlation with the increase of voltage step amplitudes at 30 minutes (R=–0.713, p=0.014). The resistances measured using signals from both Ag/AgCl- and Ir electrodes did not vary in any of the experiments conducted (p=1, n=5).

Thus, the rise in electrical resistance in brain tissue was shown to initiate at the point of respiratory arrest in the animal and progresses alongside the terminal processes in the cerebral cortex. This correlation is specifically linked to the fluctuations in the negative ultraslow potential. In tDS and NUP, a significant increase in tissue edema occurs due to the movement of water from the extracellular spaces into the cells, which leads to a decrease in extracellular space volume and an increase in resistance. The process can last for several minutes and is characterized by a continuous increase in voltage step amplitudes. These results suggest that tissue resistance increases during focal ischemia, as SD and NUP are also present during the formation of the ischemic focus [5].

Genes & Cells. 2023;18(4):667-670
pages 667-670 views

Differences in neuronal activity of subthalamic nuclei in asymmetric manifestation of Parkinson’s disease

Pavlovsky P.N., Gamaleya A.A., Sedov A.S.

Abstract

Parkinson’s disease (PD) is a widely prevalent condition that has spurred extensive research into identifying its biological markers. Different models posit that various patterns of subthalamic nucleus (STN) activity may constitute such markers, but as yet, none have been established definitively. One major limitation of human studies in this area is the lack of a reliable control group.

Therefore, patients with asymmetric motor symptoms in Parkinson’s disease are of great interest. I.I. Koloman and A.Sh. Chimagomedova [1] demonstrated that motor malfunctions’ asymmetry is indicative of asymmetry in the degenerative process within the substantia nigra. Additionally, while the disease normally manifests unilaterally, only a limited number of patients maintain notable distinctions in motor symptom severity as the condition advances.

In our study, we analyzed the single-unit activity (SUA) and local field potentials (LFP) recordings obtained duringdeep brain stimulation (DBS) surgeries in 12 patients. Neurologists assessed the severity of bradykinesia, rigidity, and tremor using the UPDRS 3 scale to meet the inclusion criteria, and the difference between hemibodies for each symptom had to be at least 25%. The median disease duration at the time of surgery ranged from 6 to 22 years. The subthalamic nucleus that exhibits more pronounced disturbances on the contralateral side of the body is labeled as “affected”, while the opposite STN, acting as the conditional control, is labeled as “nonaffected”.

For analysis, we used a hierarchical clustering method following Ward’s algorithm [2] to classify the recorded neurons into three groups based on their activity type: tonic, burst, and pause cells. We observed no significant differences in neuron activity between hemispheres, as well as the predicted hyperactivity of affected STN neurons outlined by the classical model [3] of basal ganglia functioning. However, the research demonstrates a significant increase in pause neurons and decrease in burst neurons within the affected nucleus. Furthermore, both of these types were found to be situated in the upper half of the STN, which is recognized as the motor region of the nucleus. Our hypothesis is that as the disease advances, some STN neurons (specifically burst neurons in our sample) modify their activity patterns towards a more rhythmic activity (pause neurons in our sample). This supports the notion that rhythmic STN neuron activity is linked to Parkinson’s disease [4].

LFPs were analyzed by estimating oscillation synchrony (o-scores) in multiple spectral bands following 1/f correction. Parkinson’s disease is typically associated with boosted oscillation power in the beta range (13–30 Hz). Our findings established that the power of oscillations in the low-frequency beta range (13–20 Hz) were indeed increased in the affected nucleus, but, in contrast to expectations, the power in the high-frequency part (20–30 Hz) of the band was significantly lower.

This is in line with the modern theory of distinct physiological functions of oscillations in these subbands [5]. Additionally, we noticed a rise in frequency spectrum in the alpha range (8–12 Hz) and a decrease in the gamma range (30–60 Hz) in the impacted nucleus.

Our findings indicate that a more rhythmic burst neuron activity mode and an increased number of pause neurons in the motor zone of the STN can serve as a marker for PD. Increased power oscillation in alpha and low-frequency beta bands, along with reduced synchronization in high-beta and gamma bands, may also be linked to this disorder. However, further investigation is necessary to determine their association with disease symptoms.

Genes & Cells. 2023;18(4):671-674
pages 671-674 views

Efficacy of antitumor vaccines based on photoinduced GL261 glioma cells using photosensitizers from the group of tetra(aryl)tetracyanoporphyrases with different aryl substituents

Redkin T.S., Sleptsova E.E., Savyuk M.O., Kondakova E.V., Vedunova M.V., Turubanova V.D., Krysko D.V.

Abstract

Glioblastomas are solid tumors in the brain that pose a challenge for traditional treatments like surgery, radiation therapy, and chemotherapy. Complete cures are not guaranteed, and these treatments cause numerous side effects. First-line treatment approval has only been granted to Temozolomide (TMZ), the sole chemotherapy drug available. Using TMZ increases the median overall survival from 15 to 17 months. In general clinical practice, innovative interventions have not demonstrated efficacy due to glioma heterogeneity and their immunosuppressive microenvironment.

Immunogenic cell death (ICD) activates an immune response against cancer cells by emitting damage-associated molecular patterns (DAMPs) upon cell death/dying. The DAMPs activate an anti-tumor immune response. Photodynamic therapy (PDT) can induce ICD.

Integrating the principles of immunogenic cell death into glioma immunotherapy could elicit a targeted immune response against the diverse tumor. Vaccination with dendritic cells represents a promising approach for immunotherapy.

The goals of this research are to evaluate the efficacy of a prophylactic vaccine using immunogenically-photoinduced glioma GL261 cells and a dendritic cell vaccine in an orthotopic in vivo model.

The glioma cell line (GL261) is cultured in RPMI medium supplemented with 10% serum, L-glutamine, 1% penicillin, and 1% streptomycin in a CO2 incubator. Photodynamic treatment involves using photosensitizers from the tetra(aryl)tetracyanoporphyrazine group with 9-phenanthrenyl (pz I) or 4-(4-fluorobenzyloxy)phenyl (pz III) as side substituents. The GL261 cell line is incubated in a serum-free solution containing a selected porphyrazine for four hours. Subsequently, the solution is exchanged with complete medium and the cells are activated through photodynamic treatment at a dose of 20 J/cm2. The cells are then further incubated for 24 hours in a CO2 incubator.

Immunizing mice includes subcutaneously injecting photoinduced GL261 glioma cell lysates twice with a 7-day interval. One week after the final immunization, viable GL261 glioma cells are injected into the brain using a stereotaxis frame. Measuring the neurological status of the animals occurs for 25 days, and tumor localization and volume are detected on day 23 by MRI. The survival rate of the experimental groups was 100% (pz III) or significantly higher (pz I) than that of the control groups. Additionally, neurological symptoms were either not identified (pz III) or were significantly lower (pz I) compared to the control animal groups experienced.

The dendritic cell vaccine was based on photoinduced GL261 glioma cells. The femur and tibia bones of C57BL/6J mice were used and differentiated for 9 days in RPMI medium to isolate bone marrow stem cells. The medium was supplemented with 5% fetal bovine serum, 20 ng/ml GM-CSF, 1% L-glutamine, 1mM sodium pyruvate, 50 µM β-mercaptoethanol, 100 units/ml penicillin, and 100 µg/L streptomycin. The culture medium was renewed on days 3 and 6 to maintain stability. Protein levels are quantified in cell lysates collected. 2 mg of protein is added to a dendritic cell suspension, and after 90 minutes, 0.5 µg/ml lipopolysaccharide is added for 24 hours. Intraperitoneal dendritic cell vaccine immunization on animals is conducted, with injections 7 days apart. One week post-last immunization, viable GL261 cells are intracranially injected via a stereotactic frame. The neurological status of the animals was monitored for 25 days, and an MRI was conducted on day 23 to evaluate the brain tumor. The survival rate of animals in the experimental groups did not significantly differ from those in the control groups. However, the experimental groups exhibited significantly lower neurological symptoms, and the tumor was visualized without any areas of necrosis, with a smaller volume.

Genes & Cells. 2023;18(4):675-678
pages 675-678 views

How STN activity analysis could help to improve DBS stimulation in Parkinson’s disease

Sayfulina K.E., Gamaleya A.A., Sedov A.S.

Abstract

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease globally, associated with the degeneration of dopaminergic neurons in the substantia nigra. The key motor symptoms of PD comprise bradykinesia (voluntary movement slowness and difficulty), rigidity (muscle stiffness), and tremor.

The primary methods to alleviate the extent of indicators in PD are drug therapy using the dopamine precursor levodopa and deep brain stimulation (DBS). The targets for DBS in PD are the nuclei found within the basal ganglia system, specifically the subthalamic nucleus (STN) or the internal segment of the globus pallidus.

There remains an issue of selecting the optimal stimulation program. Identifying the most efficient contact for DBS is a complex and time-consuming process made through clinical observations. One possible way to optimize this process is by analyzing neurophysiological activity of the nucleus and identifying patterns characteristic of the contacts that hold the greatest promise for clinical improvement.

Several studies have investigated the association between LFP and clinical improvements, yet a widely accepted method for choosing stimulation parameters based on STN activity has not been established. The method proposed by Strelow et al. for selecting stimulation contacts based on LFP in the STN is one example [1]. The group’s developed method showed the same efficiency as clinical-driven selection, using only oscillation power in the broad beta range as the activity parameter. Our hypothesis suggests that including more parameters in the analysis could enhance the accuracy of predicting the most effective contacts.

Thus, the study aimed to identify the subthalamic nucleus activity parameters associated with clinical enhancement post-DBS.

The investigation enrolled six patients with PD aged from 44 to 62 years of age (mean 52.8 years, std. 8.2 years) who exhibited akinetic-rigid disease symptoms, namely bradykinesia and rigidity. A total of 12 STN were studied. Implantation of DBS electrodes was carried out in the STN using directional 8-contact electrodes made by St. Jude Medical (USA) with externalization of temporary leads.

LFP recordings were obtained from the implanted electrodes on the first and fifth post-operative days. We examined periods of wakeful rest prior to and following levodopa administration, which we refer to as the OFF-state and ON-state, respectively.

The neurologist assessed clinical symptoms using the UPDRS III scale, evaluating rigidity and bradykinesia on both the left and right sides of the body. The assessment occurred one day prior to surgery and six months after the DBS system was implanted (with consistent stimulation programming during that time). The analysis used the mean improvement in rigidity and bradykinesia. The formula used to calculate the improvement parameter, or the effect of stimulation, is as follows:

(r0–r1)/r0+(g0–g1)/g0)/2.

In this equation, r1 represents the rigidity score after stimulation, r0 represents the preoperative rigidity score, g1 represents the bradykinesia score after stimulation, and g0 represents the preoperative bradykinesia score. All scores were measured during the OFF state.

Recordings were preprocessed using scripts based on the MNE Python software package. We computed 15 bipolar signals using signals from 8 contacts on each electrode. Only those within the stimulation area were used in the analysis.

Spectral analysis was conducted on the bipolar signals, where the power spectral density (PSD) was determined using the Welch method on frequencies ranging from 1–49 Hz with a 1 Hz increment for each bipolar contact. The obtained spectra underwent an aperiodic component subtraction through the fooof method [2], following which the average PSD was obtained for various frequency bands, including theta (4–7 Hz), alpha (8–12 Hz), low (13–19 Hz) and high (20–30 Hz) beta subbands, and the low gamma range (31–49 Hz).

Additionally, bursts within each frequency band were analyzed by extracting periods of increased power spectral density (twice the median value of the recording) from the local field potentials. Various parameters were then calculated for these bursts, including mean burst duration, standard deviation of burst duration, and burst percentage.

Statistical analysis was conducted with R software. The regression analysis aimed to identify parameters linked to clinical improvement. Total improvement in bradykinesia and rigidity served as the dependent variable for the model. Selected LFP parameters for each frequency range and medication state (ON and OFF) were used as predictors in the linear regression model. Factors that did not contribute to model improvement and had high cross-correlations (r >0.8) were excluded to simplify the model.

Analysis uncovered three frequency bands associated with clinical improvement: alpha, low beta, and high beta. A correlation between STN activity parameters and improvement was found in both the ON and OFF states.

The final model (R2=0.55, p <0.001) revealed a noteworthy direct link between clinical improvement and the subsequent factors: decreased beta PSD in the OFF state (p <0.001), and increased alpha PSD in the ON state (p=0.03).

Factors associated with lower clinical improvement included high beta PSD in the ON-state (p=0.018), percentage of high-beta bursts in the ON state (p=0.01), standard deviation of alpha burst duration in the OFF state (p=0.006), and percentage of alpha bursts in the ON state (p <0.001).

Low beta-band power’s association with clinical improvement is consistent with prior literature [3], and beta burst activity parameters have previously been linked to improvement [4]. However, we discovered differential associations with improvement for both low and high beta oscillations, and an additional association with improvement for alpha activity. Based on our findings, we suggest that several STN LFP parameters beyond broad beta power, such as bursts and PSD within alpha, low-beta, and high-beta bands, are associated with clinical improvement. These parameters could be used to determine the most effective stimulation contacts. The evaluation of these parameters requires further investigation.

Genes & Cells. 2023;18(4):679-682
pages 679-682 views

Neuronal signatures of abnormal globus pallidus activity in patients with Parkinson’s disease and dystonia

Sedov A.S., Dzhalagoniia I.Z., Filiushkina V.I., Usova S.V., Semenova Y.N., Gamaleya A.A., Tomskiy A.A.

Abstract

Parkinson’s disease (PD) is a hypokinetic movement disorder. It is characterized by bradykinesia, rigidity, tremor, and postural unsteadiness. Dystonia is a hyperkinetic movement disorder characterized by involuntary sustained or intermittent muscle contractions that cause abnormal, often repetitive movements, postures, or both. The traditional functional model posits that there is greater neuronal activity in the internal segment of the globus pallidus (GPi) in PD, a hypokinetic movement disorder, and reduced neuronal activity in dystonia, a hyperactive movement disorder [1, 2]. Deep brain stimulation (DBS) of this structure was shown to have a positive clinical effect in both cases. This paradox remains unresolved, necessitating further research on the neuronal processes in the basal ganglia, enhancing motor control models, and analyzing the impact of DBS stimulation on brain activity. Increased synchronization of beta (13–35 Hz) rhythms in patients with PD indicates an excessive amount of antikinetic inhibitory motor signals. Meanwhile, increased synchronization of theta-alpha (4–12 Hz) rhythms is associated with dystonia pathology [3, 4]. Although frequency and spatial synchronization have been actively studied, the functional role of these rhythms in the organization of motor control in normal and pathological states remains unknown.

The study compared the activity of the internal and external segments of the globus pallidus (GPi and GPe) in patients with PD and patients with generalized (GD) and cervical (CD) dystonia. The analysis was conducted at both the single-neuron and neuronal population levels. Microelectrode recording was performed on single-unit activity of the globus pallidus during neurosurgical procedures. The purpose was to implant DBS electrodes into the internal segment of the GPi in studied patients. A total of 20 procedures were conducted. Multichannel recording of local field potentials (using 16 channels) from the globus pallidus was obtained postoperatively through temporary externalized electrodes in 8 patients. The study’s ethical approval was obtained from the committee at the N.N. Burdenko Center for Neurosurgery. Neuronal activity analysis used a previously established method separating neurons’ activity into tonic, burst, and pause patterns through hierarchical clusterization [5]. We analyzed the distribution of patterns and their main quantitative characteristics, such as the average firing rate, coefficient of variation of interspike intervals, asymmetry index, burst and pause indices, percentage of bursting discharges, oscillation scores, and other relevant parameters. Spectral analysis and quantitative assessment of oscillatory processes in various frequency ranges were used to analyze the local field potentials (LFP). An examination of periodic and aperiodic (1/f) constituents of local field potentials was performed using the neural signal spectrum parametrization algorithm as a combination of aperiodic elements and periodic oscillatory crests [6].

The study demonstrates comparable firing rates of GPi cells across all groups examined. However, the PD group exhibited a higher level of tonic neuronal activity with decreased theta-alpha oscillations. The analysis of GPi neuron distribution reveals a significant increase in tonic cells (11% GD, 15% CD, 49% PD) and a decrease in paused neurons (28% GD, 25% CD, 11% PD). The patient group with Parkinson’s disease exhibited a markedly elevated firing rate of tonic neurons (33 imp/sec GD, 51 imp/sec CD, 91 imp/sec PD) compared to the control groups, as well as a reduced firing rate of burst neurons (62 imp/sec GD, 68 imp/sec CD, 56 imp/sec PD), with statistical significance at p <0.001. Multifactor analysis using machine learning algorithms demonstrated the significance of non-linear characteristics in neuronal activity for classifying patients by disease type, including differential entropy, theta oscillations, pause index, among others. Activity of neurons in the GPe and GPi activity in the DBS stimulation area did not exhibit significant differences between the patient groups studied.

Spectral analysis of local field potentials revealed a marked elevation of theta activity and a decline in alpha, low-, and high-beta activity in both segments of the globus pallidus of patients with dystonia compared to those with PD. The random forest algorithm indicated that the most crucial factors for identifying the patients under study were oscillations in the high-frequency beta range and the slope of the aperiodic component. The area of stimulation via DBS exhibited increased theta activity and decreased low beta activity and an aperiodic component in patients with dystonia. No significant differences were found in the area of DBS stimulation and beyond for patients with PD. Significant differences were observed in theta and beta activity when comparing the DBS stimulation area activity among the examined patients.

The study revealed the neural organization of globus pallidus as heterogeneous, displaying diverse activity patterns. Multidirectional changes in neuronal activity, differences in activity pattern distribution, and non-linear characteristics are supported by both firing rate and firing pattern models of the basal ganglia. The analysis of local field potentials revealed a shift in both periodic and aperiodic components of globus pallidus activity in movement disorders. The interplay of these components determines the pathology of movement disorders, rather than heightened oscillations in a single frequency range. The lack of variance in neural activity in the stimulated area provides partial resolution to the enigma of the GPi-DBS’s efficacy in hypo- and hyperkinetic conditions. To accurately anticipate the area of stimulation and clinical outcomes, a comprehensive strategy is required, which integrates a blend of linear and non-linear components of single unit activity, along with periodic and aperiodic elements of local field potentials of the globus pallidus.

Genes & Cells. 2023;18(4):683-686
pages 683-686 views

Quasi-movements and attempted movements: a possible alternative to motor imagery in BCI-based neurorehabilitation

Shishkin S.L., Berdyshev D.A., Yashin A.S., Zabolotniy A.Y., Ossadtchii A.E., Vasilyev A.N.

Abstract

Motor imagery (MI) is a frequently used “mental trigger” for non-invasive brain-computer interfaces (BCI). Numerous studies have examined the effectiveness of MI-BCI for post-stroke rehabilitation. However, the results remain inconclusive. A potential obstacle to the effectiveness of this method could stem from an ongoing debate between the internal focus of mental activity (i.e., modeling of reality) inherent in MI and the perceived significance of sensory feedback from the actual physical environment in BCI-facilitated therapy. The requirement to allocate attentional resources to both internal actions and external consequences may contribute to the low accuracy of MI-BCI classifiers in most users. Moreover, internal focus of attention in MI may partially account for the consistent failures in combining MI-BCI with eye tracker-based interaction technologies, since external focus of attention is crucial for gaze control.

A potentially effective replacement for motor imagery in BCIs is attempted movements (AMs). Studies have shown that BCIs are more successful in decoding AMs than MI (e.g., [1]). AMs involve attempted, but unrealized movements caused by paralysis or amputation. Despite their potential, AMs have received little attention, possibly because of modeling challenges with healthy participants and the widespread popularity of MI-BCIs. One approach to modeling them in healthy subjects is to use quasi-movements (QM), which are voluntary movements that are minimized by the subject to such an extent that they eventually become undetectable by objective measures [2]. However, QM has been studied even less than AM since its discovery by V.V. Nikulin and colleagues [2]. This may be due to the inadequate understanding of the difference between QM and MI.

We recently proved that the sensorimotor rhythm’s event-related desynchronization in QM does not rely on the residual electromyogram (EMG), indicating that strict EMG control, which is often impossible, may not be necessary for QM contrary to prior views. As a result, QM can be embraced more frequently as an alternative to MI in BCIs [3]. Moreover, we substantiated and refined earlier findings [2] that QM possesses a striking similarity to actual movements [4]. Here, we present our initial findings on the asynchronous classification of QM. These findings may serve as a foundation for a real-time QM-BCI system.

We used the EEG data recorded from 23 participants who synchronized their QM and MI with rhythmic sound triplets. A convolutional neural network called SimpleNet [5], with high interpretability, was trained on a subset of individual data separately for QM and IM, compared to a referential non-motor task. The network was applied offline and was unaware of sound timing, to another subset in 1.5-second windows with 0.1-second steps. QM/IM were detected when four consecutive positive windows occurred with a refractory period of three seconds.

Due to the high variability in MI-BCI performance among untrained individuals, we only assessed classifier performance amongst participants exhibiting a TPR (true positive rate) greater than 0.5. We identified 7 such participants in QM and 5 in MI. QM showed better intention detection than MI, though not significantly, according to the Mann-Whitney test. The mean values ± standard deviation for TPR were 0.81±0.12 in QM and 0.77±0.12 in MI, while the false alarm rate (s–1) was 0.03±0.02 in QM and 0.04±0.03 in MI, and the response time (s) was 2.81±0.06 in QM and 2.86±0.10 in MI.

Our initial findings on asynchronous BCI modeling are consistent with previous studies that have demonstrated superior QM classification in synchronous paradigms as compared to MI [2]. Notably, only minor hyperparameter optimization was performed for SimpleNet, leaving ample room for improvement in classification. Given the superior classification of AM over MI [1] and the promising preliminary findings presented here, the use of AM by end-users appears to be a viable option. Utilizing QM in studies that model AM could likely lead to further promotion and development of this technique. Additionally, the comparable nature of QM, AM, and overt movement implies their applicability in conveying intention via gaze-controlled interfaces. Although overt motor confirmation is suitable, MI-BCIs have shown inadequacies in this regard.

Genes & Cells. 2023;18(4):687-690
pages 687-690 views

Dynamics of functional impairments during focal transient ischemia in three-dimensional cortical space

Vinokurova D.E., Zakharov A.V., Mingazov B.R., Khazipov R.N.

Abstract

Ischemic injury in the cerebral cortex results in decreased electrical activity across all frequency bands and the emergence of abnormal electrophysiological patterns, including spreading depolarization (SD) and negative ultraslow potential (NUP) [1, 2]. Despite this, the specific dynamics of these changes in electrical activity within the three-dimensional cortical space during ischemia remain incompletely described and poorly understood.

To simultaneously investigate changes in electrical activity across the layers of the cerebral cortex and in the horizontal cortical space, we used two linear 16-channel silicone probes (Neuronexus, USA) in combination with a flexible transparent 60-channel matrix of subdural electrodes (MIPT, Russia) and intrinsic optical signal imaging (IOS, 665 nm, transillumination mode) during focal ischemia induced by intracortical injection of the potent vasoconstrictor endothelin-1 (ET-1, 1 μL, 1 μM). These experiments were carried out in head-restrained rats under urethane anesthesia (1.5 g/kg).

Formation of the ischemic lesion following ET-1 administration was correlated with clusters of SDs that demonstrated considerable variability in their propagation patterns within both vertical and horizontal planes. The initial SDs originated from the injection site of ET-1 and diffused across all cortical layers. Subsequently, the initiation point of the ensuing SDs gradually shifted towards the deeper layers while the electrical activity showed inadequate recovery between SDs within the injection site. SDs originating in the surrounding cortex did not invade the area near the injection point. Instead, they tended to spread around, often compartmentalizing in the superficial layers of the cortex. Some SDs were observed deep in the cortex, while others were detected on the surface via superficial electrodes and IOS, without leaving typical intracortical electrode traces. Electrographic activity was significantly depressed, especially in the superficial layers around the injection site, three hours after ET-1 administration. However, it returned to pre-ET-1 levels at the remote site, with a spatial gradient observed in subdural electrodes. Functional impairments corresponded to the histological lesion observed in coronal brain sections. Recently discovered NUPs were initiated by SD and were most prominent in the electrodes closest to the ET-1 injection site. These NUPs reached their maximal amplitude at one hour and subsided three hours after ET-1 injection.

Our research indicates intricate dynamics in the creation of an ischemic focal point. The data gathered suggest that the emergence of cerebral harm during focal ischemia is associated with the growth of a focus extending both horizontally and vertically across cortical dimensions. This growth is fueled by the generation of SDs within the ischemic penumbra.

Genes & Cells. 2023;18(4):691-693
pages 691-693 views

Blockade of histone deacetylase activity affects transcription and splicing of neuronal and glial genes

Borodinova A.A., Beletskiy A.P., Balaban P.M.

Abstract

The study of the molecular mechanisms underlying plastic processes in the nervous system is of great interest in modern neuroscience. It is important to understand that epigenetic modifications, which are crucial for the development and cellular differentiation, can also be involved in plastic processes in the adult nervous system.

In our early work, we provided evidence that the expression of important memory-related genes, such as Prkcz and Prkci, can be regulated epigenetically [1]. In the current study, we extended the previous work to the systemic level by applying the RNA sequencing approach to evaluate global changes in the expression patterns of various genes during the induction of epigenetic rearrangements. Rat cortical neuron cultures were incubated with one of the nonselective histone deacetylase inhibitors (trichostatin A, TSA; sodium butyrate, NaB) to change the level of epigenetic regulation. Next, the total RNA was extracted and subjected to RNA-Seq libraries preparation and subsequent NGS-sequencing.

Bioinformatics analysis of transcriptomic data revealed substantial overlapping of differentially expressed genes (DEGs) in NaB-treated and TSA-treated groups, indicating that different histone deacetylase (HDAC) inhibitors induce transcriptional changes in primary neuron cultures through common regulatory pathways irrespective of chemical structure of applied inhibitor. We found that histone deacetylase blockade is accompanied by a transition from proliferative processes to cellular differentiation. Gene Ontology (GO) analysis of DEGs datasets revealed that the upregulated genes were engaged in cell differentiation and specialization, tissue and embryonic morphogenesis, and the development of various peripheral tissues and organs. On the contrary, genes that reduce the expression under induction of epigenetic rearrangements were involved in biological processes associated with cell proliferation and, most interestingly, the specialization of various brain cells (neurons, astrocytes, oligodendrocytes). It was shown that the expression of a number of glial markers typical for astrocytes and oligodendrocytes was significantly reduced after application of HDAC inhibitors, which was also confirmed by quantitative PCR using specific primer pairs on selected target genes. During the data analysis, we also found a significant decrease in the expression of various neuronal markers associated with the cytoskeleton, the organization of pre- and postsynaptic endings, synaptic transmission.

It is known that fine-tuning of various processes in the central nervous system is due to the production of different isoforms of proteins from the same gene due to the process of alternative splicing of the resulting mRNA. According to the literature, epigenetic rearrangements create a certain environment for the regulation of alternative splicing of different genes [2, 3]. It is shown that production of alternative isoforms can play an important role in various plastic processes [4, 5]. Therefore, using IsoformSwitchAnalyzeR and DEXSeq packages we analyzed the possibility of alternative splicing during the induction of epigenetic rearrangements in rat cortical neuron cultures by evaluating the abundance of various transcripts based on exon usage. We found that some glial genes and a large number of neuronal genes, especially those associated with postsynaptic organization and cell communication, were alternatively spliced after application of histone deacetylase inhibitors. Inhibition of HDAC activity in cortical neuron cultures mainly affected the choice of alternative transcription starts (ATSS) and terminators (ATTS), and to a lesser extent alternative splicing of exons. Obtained results were selectively confirmed by the quantitative PCR using specific pairs of primers for individual exons of different transcript isoforms.

Thus, within this study, it was found that histone deacetylases play an important role in the specialization of various brain cells, and the suppression of their activity affects the expression and alternative splicing of various glial and neuronal marker genes. We do not exclude that global transcriptome changes caused by alternative splicing will lead to qualitative rearrangements of the neuron network, and this is the direction of future research.

Genes & Cells. 2023;18(4):698-701
pages 698-701 views

Benzopyran derivative penetrates the blood–brain barrier, eliminates synaptic deficiency and restores memory deficit in 5xFAD mice

Popugaeva E.A., Zernov N.I., Veselovsky A.V., Poroikov V.V., Melentieva D.M., Bolshakova A.V.

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disease that primarily affects older individuals and is characterized by gradual memory loss. Effective drug therapy for the disease is currently lacking. The clinical study findings of the medicinal properties of amyloid-binding antibodies demonstrate a multifaceted and intricate pathogenic mechanism of AD. Consequently, there is a crucial necessity for the exploration of novel molecular targets that can be subjected to pharmacological intervention to impede the disease progression.

The calcium hypothesis of AD posits that the pathogenesis of AD is rooted in the aberrant performance of calcium-permeable proteins that disrupt Ca2+ homeostasis. Such proteins include plasma membrane ion channels (NMDAR, AMPAR, and VGCC), intracellular ER ion channels (RyanR and IP3R), TRPC6-dependent store operated channels, and the mitochondrial pore-forming protein (mPTP). In support of the calcium hypothesis, the only pharmaceutical medication that offers temporary relief of AD symptoms is the memantine, an NMDAR blocker.

Synaptic loss in the brains of AD patients is associated with cognitive impairments. Drugs that reduce synaptic loss show promise as pharmacological agents. The transient receptor potential cation channel, subfamily C, member 6 (TRPC6), regulates excitatory synapse formation. Positive regulation of TRPC6 leads to increased synapse formation, enhances learning and memory in animal models. Therefore, TRPC6 channels constitute an attractive molecular target. We have previously demonstrated that TRPC6 channels regulate store-operated calcium entry (nSOCE) in hippocampal neurons [1]. TRPC6-dependent nSOCE is essential for support of neuronal spines and protection against amyloid toxicity in vitro. Research indicated that TRPC6 positive regulators can restore long-term potentiation deficits in brain slices obtained from AD transgenic mouse models. However, none of the TRPC6-targeting compounds tested in our study have successfully crossed the blood-brain barrier (BBB).

Recently, a novel selective TRPC6 agonist, 3-(3-,4-dihydro-6,7-dimethoxy-3,3-dimethyl-1-isoquinolinyl)-2H-1-benzopyran-2-one (C20), was identified [2]. The aim of this study is to investigate the therapeutic profile of the newly discovered selective TRPC6 positive modulator. Our findings indicate that C20 binds to TRPC6 extracellularly at the agonist binding site. Additionally, C20 exhibits synaptoprotective properties in in vitro experiments and recovers synaptic plasticity in brain slices of aged 5xFAD mice. C20 effectively passes through the BBB without causing acute or chronic toxicity in dosages ranging from 10–100 mg/kg. In a 14-day treatment of intraperitoneal injections with a 10 mg/kg dosage of C20, hippocampus-dependent context and hippocampus-independent cued fear memory improved in 5xFAD mice [3]. Therefore, C20 is a promising TRPC6-specific compound that may aid in reducing cognitive decline.

Genes & Cells. 2023;18(4):716-719
pages 716-719 views

Activity of hippocampal CA1 field neurons during aversive memory formation and reactivation in mice in vivo

Roshchina M.A., Roshchin M.V., Borodinova A.A., Aseyev N.A., Zuzina A.B., Balaban P.M.

Abstract

According to modern concepts, the dorsal hippocampus, specifically the CA1 field, plays a crucial role in the formation and reactivation of contextual fear conditioning (CFC) memory [1–5]. However, the extent to which the neurons of the dorsal hippocampus participate in CFC learning or memory reactivation remains poorly understood. The aim of this study was to examine the in vivo activity of neurons in the hippocampal CA1 field during CFC memory training and testing. The study conducted experimentations on male mice of the C57Bl/6 line (N=4). Miniature fluorescence microscopes, also known as miniscopes, were used to monitor neuronal activity in the CA1 field. The CA1 field in the hippocampus was injected with an AAV vector carrying the GCaMP6s calcium sensor, and implanted with a GRIN lens in the same area as the miniscope lens. The mice underwent CFC task training and the duration of freezing was then measured.

After the training session, the mice exhibited a notable increase in freezing duration, suggesting the formation of context aversive memory. Throughout the training, a total of 591 active neurons were recorded (147.8±74.9 neurons per mouse), while 512 (128.0±40.6 neurons per mouse) neurons were recorded. The average frequency of calcium events per second during the complete duration of training session was 0.037±0.003, while for the testing, it was 0.042±0.015 events/second. Around 46% of the registered neurons remained active throughout the complete training procedure. The mean frequency of calcium events in these neurons surged considerably following the application of an electric shock (from 0.035±0.007 events/sec to 0.086±0.013 events/sec). Using k-means clustering, certain neurons showed increased activity after electric shock exposure, while others showed decreased activity. However, the type of activity change did not affect subsequent neuronal dynamics during memory retrieval. During memory retrieval, we observed that an average of 30–40% of neurons were reactivated. The number of active neurons notably decreased during episodes of freezing and almost all registered neurons were activated during episodes of movement. The average frequency of calcium events in the reactivating neurons did not change from the training to testing session.

Thus, new data was obtained on the activation of neurons in the hippocampal CA1 area during memory formation and retrieval in CFC.

Genes & Cells. 2023;18(4):720-722
pages 720-722 views

Role of heterosynaptic plasticity in the modification of sensory responses of mouse visual cortex neurons

Smirnov I.V., Osipova A.A., Simonova N.A., Smirnova M.P., Borodinova A.A., Malyshev A.Y.

Abstract

Synaptic plasticity is a critical factor in neural network function during development, perception, learning, and memory. However, current ideas on the role of synaptic plasticity in cortical network mechanisms are mainly correlative because cellular and molecular mechanisms are predominantly studied in reduced preparations. Currently, the majority of research on synaptic plasticity mechanisms is focused on studying homosynaptic (associative, Hebbian) plasticity. This type of plasticity involves modifying the same synapses that are directly involved in the induction process. However, heterosynaptic plasticity, which occurs in synapses that were inactive during induction, plays a crucial role in the function of neural networks, in addition to the more extensively researched homosynaptic plasticity [1, 2]. In this study, we examined how heterosynaptic plasticity, triggered by intracellular tetanization of pyramidal neurons in the visual cortex of mice, affects their response to visual stimulation in vivo.

In the initial stage of the experiment, we recorded intracellularly the visual cortex neurons of anesthetized mice by means of the whole-cell patch clamp approach. We employed an intracellular tetanization procedure to assess the effect of heterosynaptic plasticity on visual responses. Specifically, we applied ten bursts of 5 action potentials each second at a frequency of 100 Hz during the tetanization process in the recorded neuron, repeating the procedure five times at 60-second intervals. Previous studies in brain slices have shown that this protocol leads to substantial plastic changes in the synaptic inputs of a given neuron, including potentiation, depression, and no change (see review [3]). As visual stimuli, we used vertical and horizontal bars moving in opposite directions on the computer screen. A small hyperpolarizing current was continuously applied to the cell to prevent the generation of action potentials. As a result, the changes in the cell membrane potential represented the responses to the visual stimuli. Intracellular tetanization led to a noteworthy amplification in the amplitude and area of the response to the optimal stimulus, which was based on the orientation and movement direction. The reaction to other stimuli, on the other hand, did not encounter any substantial change. A consequence of this was an elevation in the simplified index of directional selectivity of tetanized neurons, computed as the ratio of the optimal stimulus response amplitude to the response amplitude in the opposite direction (null direction).

To minimize the influence of intracellular perfusion, which unavoidably arises during whole-cell patch clamp recordings, and to extend the duration of cell response recording after tetanization, we conducted additional experiments involving extracellular recording of cell activity and optogenetic stimulation through an optical fiber inserted into the recording microelectrode. Two weeks prior to the experiment, the pyramidal neurons in the 2/3 layer of the mouse visual cortex underwent viral transduction to express the fast channel rhodopsin oChiEF. During the experiment, visual responses of neurons were recorded for 15–40 minutes. Subsequently, we induced bursts of action potentials with a frequency of 75 to 100 Hz in the recorded neuron using otptogenetic tetanization and continued to record the visual responses for at least 40 minutes. In this series of experiments, we recorded action potentials induced in neurons by visual stimulation, which was similar to that used in experiments with intracellular recording. Finally, we calculated total post-stimulus histograms from the responses. Intracellular tetanization was found to cause a significant reduction in response amplitude to the optimal stimulus, while responses to stimuli with other orientations and directions of movement remained unaffected. As a result, the index of cell directional selectivity decreased in this series of experiments, indicating a direct opposition to the results obtained through intracellular recording experiments. To clarify this discrepancy, we conducted theoretical simulations using the Leaky Integrate and Fire (LIF) model neuron. We used a model in which orientation is determined by different peak positions of inhibitory and excitatory components of visual responses when moving in optimal and opposite orientations [4]. Simulations were carried out at two different resting potentials: –90 mV, which simulated experiments involving intracellular recordings and injections of hyperpolarizing currents, and –65 mV, which simulated experiments involving extracellular registrations of spiking cell responses in the UP-state mode. Our findings suggest that tetanization causes potentiation of both excitatory and inhibitory components of the responses, leading to the observed situation.

During our experiments on visual cortex slices, we discovered that intracellular tetanization of pyramidal neurons in layer 2/3 of the visual cortex causes balanced heterosynaptic changes in excitatory inputs to dendritic regions distant from the soma. This means the net change in all inputs after tetanization equals zero, balancing potentiation and depression. Conversely, it causes unbalanced potentiation of excitatory perisomatic inputs. Furthermore, previous research has demonstrated that the occurrence of high-frequency action potentials in layer 5 pyramidal neurons within the neocortex results in the strengthening of their inhibitory perisomatic inputs originating from adjacent parvalbumin interneurons [5]. Based on the above-cited work, our experiment suggests that intracellular tetanization of the pyramidal neuron in the 2/3 layer of the visual cortex may result in the potentiation of perisomatic inhibitory inputs and the development of simultaneous perisomatic excitatory inputs through heterosynaptic plasticity mechanisms. If the changes in excitatory and inhibitory inputs are balanced, our model experiments show that this can lead to changes in the directional selectivity of the observed cells.

Thus, high-frequency spike activity in the absence of specific sensory activation, such as during sleep, may decrease the directional selectivity of visual cortical neurons. This prepares the neurons to adjust their visual responses more finely to new scenes during wakefulness. The potentiation of perisomatic excitatory and inhibitory synaptic inputs, through heterosynaptic plasticity, may be the mechanism responsible for such tuning.

Genes & Cells. 2023;18(4):723-726
pages 723-726 views

Effects of spreading depolarization induced by amygdala micro-injury on fear memory in rats

Smirnova M.P., Pavlova I.V., Vinogradova L.V.

Abstract

Spreading depolarization (SD) is a wave of intense neuroglial depolarization that is widely recognized as a component of the acute brain response to various types of injury. Clinical studies conducted on patients with different types of stroke and traumatic brain injury which used intracranial recordings of cortical activity revealed a frequent incidence of cortical SD [1]. Site-specific intracerebral microinjections of drugs in experimental animals, or functional stereotactic surgery in patients, cause damage to both the neocortex and subcortical structures in the local area. Currently, most studies on SD are limited to the neocortex, but research shows that SD can be triggered in nearly all brain structures, albeit with varying levels of effectiveness [2]. The underlying mechanisms that render deep brain structures more vulnerable to SD remain unclear. Our recent research found that local injury to the amygdala can also lead to SD, although with a reduced likelihood compared to the neocortex [3]. The amygdala, a structure located in the temporal lobe, is highly susceptible to brain injury and plays a significant role in emotional behavior [4]. Specifically, it serves as a pivotal hub for fear memory and post-traumatic stress disorder pathogenesis. The post-injury neurobehavioral impairments are attributed to the limbic structure. Patients suffering from traumatic brain injury and stroke often exhibit cognitive dysfunction, generalized anxiety disorder, posttraumatic stress disorder, and other neuropsychiatric states. The role of SD in the pathogenesis of the behavioral deficit and its underlying mechanisms are not well understood.

In this study, we examined the impact of amygdala micro-injury and associated SD on fear memory in rats. Neuronal tissue damage was induced using the standard method of intracerebral injection of substances through a thin 30 G needle inserted into a guide cannula (23 G) previously implanted in the amygdala. We used the classical Pavlovian fear conditioning paradigm to assess animal behavior. Twenty-four hours following the acquisition of fear, fear memory was evaluated during the first test. An hour after the first test, bilateral microinjury of the amygdala was performed. The influence of amygdala microinjury on aversive memory was assessed during the second test 24 hours later. Following the second test, rats underwent a two-day fear extinction process one to two days later.

We found that bilateral micro-injury to the amygdala resulted in bilateral SD, unilateral SD, or no SD. Neither the injury nor the SD induced significant changes in fear conditioning during test 2, but they did affect subsequent fear extinction. The effect depended on whether the SD was triggered by the injury. If no SD occurred, fear extinction was disrupted, and rats exhibited high levels of freezing in response to sound. However, if bilateral SD was triggered by amygdala damage, fear memory was extinguished successfully and rapidly. The results of the present study suggest a strong involvement of SD induced by amygdala micro-injury in fear memory extinction.

This information is crucial in comprehending the fundamental mechanisms behind post-traumatic stress disorder and exploring novel therapeutic approaches for treating this condition. The discoveries could be valuable in designing and interpreting experiments that involve local intracerebral microinjection.

Genes & Cells. 2023;18(4):727-730
pages 727-730 views

Developing and testing a method of remotely improving younger students executive functions, volitional attention and auditory memory

Tomenko T.R., Bogdanova M.A.

Abstract

During primary school age, children undergo a quick progression of voluntary actions that indicate cognitive predictors for academic triumph [1]. Modern research indicates that the effectiveness of educational activities heavily relies on the development of working memory, cognitive flexibility, and self-control, all of which are closely associated with brain control functions. Additionally, the level of auditory-speech memory development has a direct influence on reading skills development and the ability to articulate and convey thoughts [2, 3]. According to various sources, between 15 to 40% of schoolchildren in primary grades fall behind, which can cause significant long-term cognitive and social difficulties and lead to various psychosocial consequences. Thus, the importance of taking preventive measures for developing these functions in primary school has increased to prevent the formation of mental defects, which are major factors in a child‘s maladaptation in society.

The current study aims to analyze the effectiveness of the author‘s remote program “Kind Elephant” in improving the auditory-speech memory, voluntary attention, and control functions of younger elementary school students.

The program is designed to complete a range of tasks over a seven-week period, following standardized methods while also accounting for the neurodynamic parameters of children in this age group.

Seventy-six students between the ages of 7 and 9, in grades 1 and 2, were enrolled in the study. Of these, 44 completed the Kind Elephant development program, while the remaining 32 participants served as the control group.

With the assistance of the auditory-speech test and computer techniques, data was obtained on the development of control functions, voluntary attention, and auditory-speech memory in schoolchildren in two sections that were seven weeks apart. The techniques used were Analysis of Sentence Understanding, Bourdon‘s Proof-Reading Test, Hands-Legs-Head, and Understanding of Similar Sounding Words [4]. To investigate the assessment of children‘s programming and control functions, parents completed a BRIEF questionnaire (Behavior Rating Inventory of Executive Function) before and after the study [5]. An analysis was conducted to determine the significance of differences in indicators between the two groups‘ results.

This study demonstrates the potential for remote development techniques to enhance the ability to regulate behavior, maintain focus on specific activities, reduce fatigue, increase productivity, and improve auditory-speech memory volume.

Thus, younger schoolchildren‘s cognitive development can occur remotely, resolving access challenges for parents and enabling more children to keep pace with peers and thrive in social contexts. The authors maintain that using technology within reasonable limits can be acceptable for children‘s daily life if tailored to their mental maturation.

Genes & Cells. 2023;18(4):731-734
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Weak cued fear memory strengthening by re-activating the engram

Toropova K.A., Ivashkina O.I., Yurin A.M., Anokhin K.V.

Abstract

One of neurobiology’s primary objectives is to discover the mechanisms by which past experiences influence current behavior and learning. While it is acknowledged that previous cognitive experiences can facilitate the creation of new memories in both humans and animals, investigations into the behavioral and neuronal aspects of the phenomenon of new memory being dependent on past cognitive experiences remain to be explored. The aim is to evaluate the theory that the facilitating effects of prior experiences on the creation of new memories are solely evident if these occurrences involve shared groups of neurons in the brain. To this end, a novel method was devised to lightly instruct mice to instinctively freeze when presented with a cue by exposing them to a conditioned signal for a brief (5 seconds) period, followed immediately by a gentle electrodermal stimulation. We discovered that weak memory can be strengthened through repeated weak learning in cumulative learning, despite the lack of formation of long-term memory caused by weak learning itself. Strengthening of memory only occurs when the animal is trained on the same conditioned signal during both weak training times. On the other hand, if the conditioned signals used during the first and second weak training are insignificantly different, no memory formation will occur for either. However, memory reinforcement in cumulative learning depended on context, fully manifesting only when multiple training sessions occurred in the same environment. We examined if long-term memory formation of the conditioned signal during cumulative learning relied on the animals’ past experiences, which were subject to alteration through repeated training. We discovered that successful long-term memory formation for the conditioned signal occurred when two weak learnings were separated by more than 30 minutes (up to 30 days), but not when the interval between learnings was 30 seconds or 5 minutes. Thus, weak memory reinforcement in cumulative learning occurs only when the second training is separated from the first by a time interval sufficient to form a latent memory of the first. The data obtained suggest that weak training of the conditioned reflex freeze results in long-term plastic rearrangements in the brains of mice. These changes can be strengthened through repeated weak training to the same conditioned signal, resulting in the behavioral manifestation of memory. We then investigated the activity of different brain regions during cumulative learning. We demonstrated that repeated weak training elicits selective activation of mouse brain regions that are crucial for long-term memory formation, including associative cortical regions, amygdala, and hippocampus. Conversely, such activation was absent after a single weak training, where the brain activity was equivalent to the mice that received the conditioned stimulus without reinforcement and remained untrained. This study suggests that a weak single training can form a memory trace in the brain, which can be strengthened through repetition. However, identifying this trace at the level of entire brain structures appears to be unattainable. Therefore, we directly evaluated the main hypothesis of this study by examining the overlap of neuronal populations in transgenic Cre-lineage mice. We labeled neurons involved in the first weak learning with fluorescent protein expression and cells active in the second learning with immunohistochemical staining for the native Arc protein. It was demonstrated that during cumulative learning, over 30% of neurons in the prelimbic cortex, auditory cortex, and amygdala were reactivated, while only 10% of cells were reactivated when two weak learnings were performed on different conditioned signals that did not lead to the formation of memories. Consequently, the present study examines the phenomenon of newly formed memory dependence on individual experience history at both a behavioral and neuronal level. The study demonstrated that the enhancing effect of previous experiences on memory creation relies on the repeated activation of identical neurons.

Genes & Cells. 2023;18(4):735-738
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Histone acetylation increase rescues a weak remote fear memory in rats

Vinarskaya A.K., Zuzina A.B., Balaban P.M.

Abstract

According to multiple studies, inhibitors of histone deacetylases (HDACs) hinder the activity of HDACs, thus enhancing histone acetylation [1, 2]. This process can promote long-term memory and potentiation [3, 4], and even alleviate memory deficits [5]. Our recent findings demonstrate that sodium butyrate (an HDAC inhibitor) improves rat’s recent fear memory and serves as a cognitive enhancer.

The aim of this study was to investigate the potential of HDAC inhibitor sodium butyrate to improve the weakening of remote fear memory in rats. The experiment was conducted on both male and female Wistar and Long Evans rats. The animals were routinely handled for a week prior to the start of the experiment. They underwent contextual fear conditioning, where the unconditioned stimulus (US) — an electrical shock to the foot — was connected with a conditioned stimulus (CS), a particular context. The duration of their freezing behavior was assessed during the test session T0. At 24 hours after conditioning (test session T1), the animals that were conditioned froze when in the conditioning environment, providing evidence of successful learning. Subsequently, their freezing responses were measured during retrieval sessions six months after conditioning (test session T2). Following T2, the control groups were administered sham injections of saline, while the experimental groups were given injections of sodium butyrate. Freezing duration was then evaluated in all groups during a subsequent trial (T3) 24 hours later.

After completing the training, all groups of rats displayed a longer duration of freezing, indicating the formation of context fear memory during the test session T1 compared to the test session T0. After six months (test session T2), all groups demonstrated a significant decrease in the duration of freezing compared to the T1 test session. The experimental groups were intraperitoneally injected with sodium butyrate immediately after T2, while the control groups received a vehicle injection of sterile saline. 24 hours after the third test session, results showed that rats treated with sodium butyrate had significantly higher freezing responses to the context compared to those receiving vehicle treatment. Additionally, the sodium butyrate-treated groups in the third test session exhibited similar levels of freezing as seen in the first test session. Male and female groups injected with sodium butyrate showed similar levels of freezing and did not display any differences throughout all test sessions. Thus, retrieving weakened remote contextual fear memory followed by immediate administration of the HDAC inhibitor sodium butyrate resulted in a significant improvement of memory. Our findings demonstrate that the reactivated remote fear memory was enhanced when exposed to the HDAC inhibitor sodium butyrate. This aligns with previous studies suggesting the role of HDAC inhibitors in facilitating contextual fear.

Genes & Cells. 2023;18(4):739-741
pages 739-741 views

Highly stable long-term memory in a mouse model of post-traumatic stress disorder

Zamorina T.A., Toropova K.A., Ivashkina O.I., Anokhin K.V.

Abstract

The retention of information represents a defining trait of cognitive systems. The primary function of an organism’s memory is to retain engrams of unique experiences for extended periods, without interrupting memory trace during new learning processes. One of the cardinal unsolved issues in neuroscience lies in unveiling the mechanisms responsible for long-term memory. Despite this, a trustworthy experimental animal model for single-trial long-term memory remains undeveloped. To address this challenge, we aimed to create an animal model to study lifelong memory formation with a single trial. We used a mouse model of post-traumatic stress disorder (PTSD) for this purpose [1]. In this model, mice exposed to a powerful electrical foot shock develop highly persistent traumatic memories, which results in long-term behavioral changes [1]. Our hypothesis was that this model could uncover the mechanisms related to lifelong memory.

The primary rationale for adopting the PTSD model as a model of lifelong memory pertains to the heightened durability of traumatic memories in comparison to conventional aversive memories [2]. Protein synthesis inhibitors, which are needed for long-term memory consolidation, can be used as amnesic agents to evaluate memory stability [3]. In a PTSD model using predator scent, the administration of a protein synthesis blocker prior to a traumatic experience disrupts the development of PTSD in mice [4]. However, it remains unclear how traumatic memory impairment affects PTSD development in the footshock model and whether the impairment persists long-term.

Based on the evidence that the formation of PTSD necessitates the consolidation of associative memory, which is reliant on protein synthesis and coincides with changes in the stress response system, Siegmund and Watzhek [1] present a two-part proposal concerning the onset of PTSD. The two-part hypothesis regarding PTSD posits that PTSD formation involves sensory conditioning and sensitization processes that mutually reinforce one another. In line with this theorem, we considered the effects of studying context and exposure timing on the development of post-traumatic stress disorder, as it could potentially interfere with sensory conditioning formation [5].

The aim of this study is to create an experimental approach for developing enduring long-term memory through a single trial event on adult mice. We methodically analyzed behavioral expressions and the endurance of normal and traumatic fear memory, as well as their sensitivity to protein synthesis inhibition. Additionally, we investigated the effects of separating the timing of the associative and aversive elements of traumatic memory on PTSD development in a mouse model.

The experiment involved male C57Bl/6 mice, aged between 3–4 months and 15–18 months (for an investigation into aged mice), which were placed in an electrified chamber. After 170 seconds, the mice experienced either one footshock (1.5 mA, 2 s) to elicit fear memory or three footshocks (1.5 mA, 10 s) for PTSD induction. After receiving the footshock, the mouse was held in the chamber for 60 seconds before being returned to its home cage. A memory test was conducted seven days later by placing the mice back in the same context. To assess the existence of standard PTSD symptoms, like sensitization and generalization, the creatures were exposed to an unfamiliar context and an unexpected auditory stimulus, respectively. In the experiment, animals were placed in unfamiliar or familiar safe contexts that differed from their previous ones. The duration of freezing was measured to assess fear and evaluate memory retention, sensitization, and generalization. Anxiety levels were evaluated using the elevated plus maze test.

In the process of constructing a highly stable long-term memory model, we evaluated the behavioral performance of PTSD-induced (PTSD), fear-conditioned (FC), and active control (AC) groups of animals at 7 days, 1 month, and 3 months after exposure. As a result of PTSD induction, mice displayed increased fear levels for up to 3 months in the training context, along with heightened fear sensitization and generalization at 7 days following exposure, relative to the FC and AC groups. The PTSD group exhibited heightened freezing behavior within a month and a decreased number of entries to the closed arms during the initial three months following exposure when contrasted with the FC and AC groups. We additionally noted that the FC group displayed raised fear levels in contrast to the control animals up to 3 months post-exposure, albeit lower in magnitude than that of the PTSD group. The FC group, similar to the PTSD group, showed increased sensitization and generalization compared to control animals at 1 week post-exposure, but to a lesser extent than we observed in the PTSD group. The findings suggest that traumatic memory remains present for a minimum of three months, whereas fear memory in the fear conditioning paradigm diminishes in intensity during this time. Hence, PTSD induction effectively functions as an animal model of profoundly stable long-term memory, in opposition to the fear conditioning paradigm.

As the high stable long-term memory model is designed for testing in senior animals, the impact of aging on traumatic memory formation and storage becomes an important inquiry at six months and one year following exposure. We analyzed the formation of both aversive and traumatic memory in mice aged 15–18 months, one week after footshock exposure. In the study involving older mice, the PTSD group exhibited increased levels of fear memory and sensitization when compared with both the FC and AC groups, in addition to displaying higher levels of generalization and anxiety when compared with the AC group. Furthermore, mice that underwent rear-conditioning showed heightened levels of fear during learning, fear sensitization, and generalization, although not anxiety. These findings demonstrate the formation of traumatic and aversive memory in aged mice, indicating that conducting memory tests six months and a year after exposure is an appropriate approach.

To evaluate the durability of traumatic and aversive memory, we administered a protein synthesis inhibitor called cycloheximide (Chm) to interfere with memory formation. Mice were given a cycloheximide solution (95 mg/kg) via intraperitoneal injection 30 minutes before footshock (Chm-PTSD and Chm-FC groups) while control animals received a saline injection (Sal-PTSD, Sal-FC). After training, the Chm-FC group displayed a decrease in freezing rate when compared to the saline-injected group at the 7- and 30-day marks. The Chm-PTSD group exhibited fear levels comparable to those of the Sal-FC group in the training context, and did not display sensitization or generalization of fear. These findings suggest that inhibiting protein synthesis during memory formation resulted in complete amnesia for standard aversive memory and merely weakened traumatic memory. As a result, only the associative component of traumatic memory remained intact, while nonspecific symptoms of PTSD were absent. The strong resilience of memory in the PTSD model to severe disruptions, such as protein synthesis blockade, also confirms its stability, which facilitates exploration of lifelong memory mechanisms in the PTSD model.

To examine the timing effect of the associative and aversive component of PTSD development, we evaluated traumatic memory formation when contextual memory formation was absent, and contextual exploration occurred three days before shock cessation. A week after exposure to immediate and intense footshock, mice in the experimental group exhibited lower levels of fear, reduced fear sensitization, and less anxiety compared to those in the PTSD-induced group. If an intense foot shock was preceded by a contextual study three days earlier, the animals exhibited lower levels of fear in an unfamiliar, safe context, and reduced anxiety compared to the mice induced with PTSD. In both cases, the fear level after the shock cessation was identical to that of the mice who experienced an immediate, moderate shock. Based on our findings, we concluded that aversive memory forms in the absence or prior formation of contextual memory in relation to traumatic exposure. However, PTSD induction does not occur under such circumstances. This indicates that contextual memory needs to be formed simultaneously with the traumatic event for the development of traumatic memory in the mouse model of PTSD.

In summary, our findings indicate that aversive memory tends to fade over time, while traumatic memory remains stable for a minimum of 3 months following its induction. Therefore, PTSD proves to be a fitting candidate laboratory model for high, stable, and long-term memory. Aged mice aged between 15–18 months display the formation of traumatic memory and experience PTSD symptoms in a manner similar to adult animals aged between 3–4 months in the PTSD model. For the induction of PTSD in mice, contextual memory formation about the traumatic environment and the traumatic event occurrence may coincide, which is crucial.

Genes & Cells. 2023;18(4):742-747
pages 742-747 views

Central pattern generators for biomorphic robotics

Zharinov A.I., Potapov I.A., Kurganov D.V., Lobov S.A.

Abstract

Typically, the structure of the robot fish frame significantly differs compared to the real organism. One significant difference is in the number of body segments. While live fish can have between 16 (moon fish) to 400 belt fish [1] segments, robots usually have only 5–6 segments since substantial precision is unnecessary when simulating movement. At the same time, this method limits a significant portion of the control circuit’s structure compared to a fish’s nervous system because it only requires control over a smaller number of body segments.

Control systems using different oscillators can simulate the functioning of fish central generators [2–4]. Typically, each half-center of the fish’s CPGs is interconnected with and mutually inhibitory towards the others, with each being responsible for the antagonist muscles. In this case, the generator’s pattern characteristics stem from the mutual influence of oscillator-antagonist pairs connected to each other. The half-centers’ interaction mechanism with each other is designed to match the movement pattern’s desired final parameters.

This “artificial” approach is unsuitable for working with spike neurons because the mechanisms of cellular interaction are well-defined. Altering how cells interact with each other when creating a biologically relevant model is also undesired. Here we present evidence that incorporating select physiological traits of fish into the design of a CPG structure utilizing spike neurons can enhance the system’s functional capacity.

Previously, we demonstrated a half-center CPG model using Izhikevich neurons [5]. This model can serve as a control loop for a tuna robot. Although this development aligns with the fundamental principles of CPG organization in fish, reproducing the typical generator mode of operation for pike on it proved challenging. This is due to the fact that the anguliform type of locomotion implies the presence of a moving wave, which means a phase lag in the activation of half-centers.

One potential solution to the problem lies in the physiology of fish, specifically the structure of their muscle fibers. Fish have muscle segments called myomeres, which correlate with the number of vertebrae and spinal centers that create the CPG. A distinguishing feature of these myomeres is their zigzag shape. To achieve body bending at a single point, it requires a collaborative action between multiple myomeres and corresponding CPG segments.

While our model assumes control of the entire fish with only 5 segments of the CPG, the actual pike includes 56–65 segments. To attain the necessary difference in activation phase between generator segments, we propose increasing the number of generator nodes responsible for operating a single propulsion unit.

Indeed, increasing the number of transmission segments resulted in a steady divergence in activation phases among successive segments of the CPG.

However, the incorporation of supplementary segments fails to address the challenge of managing the frequency of the CPG’s operation and shifting between patterns. Consequently, we intend to integrate CPG neurons of diverse types outlined in the model, along with introducing feedback to rectify its modes of operation in the future.

Genes & Cells. 2023;18(4):748-751
pages 748-751 views

Study of the influence of ionizing radiation on the scattering properties of the brain white matter

Achkasova K.A., Kuhnina L.S., Moiseev A.A., Bogomolova A.Y., Gladkova N.D.

Abstract

Radiation therapy is a vital part of combined treatment for brain neoplasms. In order to enhance treatment effectiveness during irradiation, ionizing radiation is used to expose both the tumor and nearby tissues. This leads to pathological changes within the surrounding brain tissues, particularly affecting the white matter [1]. This can create challenges when attempting to differentiate between tumor and normal brain tissues during surgical resection, ultimately resulting in potential postoperative complications. Therefore, the development of novel techniques for identifying white matter alterations caused by radiation therapy during surgical procedures is required.

The aim of this study was to examine the impact of ionizing radiation on the scattering characteristics of the brain’s white matter and assess the feasibility of using optical coherence tomography (OCT) to identify potential changes.

The study analyzed ex vivo rat brain samples, examining a control group and a group exposed to ionizing radiation at a 15 Gy dose to the right hemisphere. Subsequently, the animals were euthanized at seven different times throughout the study (2–14 weeks). An OCT study and an immunohistochemical study of the frontal sections of the brain followed. The attenuation coefficient was calculated and en-face color-coded optical maps were constructed to quantitatively process OCT data. The corpus callosum was selected as the region of interest.

As a result of the study, acute changes in the white matter were observed 2 weeks after irradiation, along with early delayed changes occurring at 6 and 12 weeks post-irradiation. These changes were characterized by reversible edema of the corpus callosum. Two weeks after irradiation, a moderate edema only occurred in the irradiated hemisphere. However, at 6 and 12 weeks, edema was also present in the contralateral hemisphere, with significant severity, indicating the spread of the process along myelinated nerve fibers. The analysis of OCT data showed changes in the attenuation coefficient values. We observed statistically significant reductions in the attenuation coefficient values at all time points with edema of the corpus callosum in different brain hemispheres compared to the control group (p <0.05).

During this study, the corpus callosum demonstrated structural changes from ionizing radiation exposure. These changes were identified by a reduction in its scattering properties, which can be observed using OCT.

Genes & Cells. 2023;18(4):753-755
pages 753-755 views

Imaging of mouse somatosensory cortex neurons in vivo using miniscope

Bukov G.A., Gerasimov E.I., Pchitskaya E.I., Vlasova O.L., Bezprozvannyi I.B.

Abstract

Visualizing neuronal activity in the brain in vivo is a crucial task in modern neurobiology. Imaging changes in the neuronal networks of various brain regions in neurodegenerative diseases, such as Alzheimer’s disease, can uncover functional aberrations in neuronal connections during their early stages. One current method of obtaining in vivo neural activity data is the miniscopic fluorescence microscopy technique, which facilitates in vivo registration of excitation within the neural networks of brain areas, followed by subsequent analysis [1]. The Miniscope, a miniature fluorescence microscope, enables researchers to work with freely moving laboratory animals, which sets it apart from other in vivo imaging methods, such as two-photon microscopy.

In this study, we injected an adeno-associated virus containing the GCaMP6f gene, which encodes a fluorescent calcium-sensitive protein, into the somatosensory cortex region of the brain at coordinates AP-2.1, ML+2.1, DV-0.05). We conducted the in vivo recording of calcium level changes in 3-month-old C57BL/6J mice by placing a 5×5 mm clear glass cranial window over the injection site of the virus. After 4 weeks, we placed and fixed a Baseplate over this cranial window to hold the Miniscope V4 over the clear cover glass for efficient recording.

In future studies, researchers will introduce the adeno-associated virus carrying the GCaMP6f protein gene into the somatosensory cortex of 3-month-old 5xFAD mice with Alzheimer’s disease to compare the activity of somatosensory cortex neural networks in free-ranging wild-type mice and 5xFAD mice, and detect differences in the functioning of these neural networks. Additionally, they will place a clear glass cranial window over the somatosensory cortex to install Miniscope V4. As these studies advance, Miniscope V4 data on somatosensory neuron activity in wild-type (C57BL/6J line) and Alzheimer’s disease transgenic mice (5xFAD line) will be utilized to evaluate somatosensory neural network states during assorted behavioral tests. Future studies will analyze the somatosensory cortex neuron activity during vibrissae stimulation, as abnormally high neuronal population activity in the somatosensory cortex has been observed in 5xFAD line mice with Alzheimer’s disease [2]. These findings will be crucial in the pharmacological assessment of potential therapeutic agents for Alzheimer’s disease treatment.

Genes & Cells. 2023;18(4):756-758
pages 756-758 views

Crosstalk of CGMP and CAMP in the vertebrate phototransduction cascade

Chernyshkova O., Erofeeva N., Meshalkina D., Belyakov M., Firsov M.

Abstract

The conventional phototransduction cascade suggests that cyclic guanosine monophosphate (cGMP) functions as the chief secondary messenger while intracellular calcium concentration dominates as the central feedback modulator. Countless years and several scientific institutions have led to this conclusion, and it stands as the most extensively researched and explicit among all sensory transduction schemes. However, experimental evidence suggests that our understanding of the phototransduction cascade mechanisms remains significantly incomplete [1]. According to the canonical cascade scheme, all transients should be completed within a second once the light stimulus is no longer present. However, our data indicates that there are long-lasting changes in cell sensitivity and dark current parameters after the stimulus, which can last for more than 10 s. Phenomena that deviate from the standard phototransduction cascade behavior can potentially be clarified by an alternative regulatory mechanism that is based on cyclic adenosine monophosphate (cAMP). Previous research has provided convincing evidence that the intracellular levels of cAMP can significantly impact the functioning of the phototransduction cascade on both slow (day) [2] and relatively fast (minutes) [3] time scales. Additionally, there is phenomenological evidence indicating the existence of other regulatory signaling pathways in the phototransduction cascade without a corresponding mechanism in the classical phototransduction scheme, including inositol triphosphate (IP3) and diacylglycerol (DAG). We investigate whether cAMP, IP3, and DAG regulate the phototransduction cascade during photoresponse. For this regulatory effect to occur, there must be a change in the signaling molecule concentration during the process. These processes occur in less than a second, and it is crucial for the presence of the regulatory effect. Given that traditional fluorescence methods cannot measure the concentration of any signaling molecule in the retina, a hardware-software setup has been developed that allows cryofixation of retinal samples at the required speed. The setup permits fixing up to six samples in a series with a delay of no more than 80 milliseconds after light stimulation. The concentration of signal molecules is assessed using high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry.

The results demonstrate a 4.5-fold elevation in cAMP concentration 1.1 s after switching on a light with an intensity close to saturation. The concentration of cAMP is directly proportional to the intensity of the stimulating light; there is no increase in cAMP at lower light intensities. No noteworthy changes in IP3 and DAG concentration were detected in response to light stimulation. The findings align with existing literature on the kinetics of light-triggered protein kinase A (PKA) activity [3], which indicated an initial decrease followed by an increase in PKA activity. These results could potentially inform the revision and expansion of the phototransduction cascade model.

Genes & Cells. 2023;18(4):759-762
pages 759-762 views

Analysis of the hippocampal neural network activity in vivo by miniature fluorescence microscopy in neurological pathologies

Gerasimov E.I., Mitenev A.V., Pchitskaya E.I., Chukanov V.S., Bezprozvannyi I.B.

Abstract

Miniature fluorescent microscopy is a method that enables neurobiologists to visualize and record neuronal activity of a specific brain region in vivo in freely moving mice [1]. The use of miniscope presents a novel approach for acquiring extensive data on the structure, function, and organization of the neuronal network in the region of interest at the in vivo level [2, 3]. In this way, the use of miniscope could also identify changes caused by pathological conditions, such as seizures, neurodegenerative diseases, and neurological complications resulting from past viral infections, like influenza virus. Data obtained through miniature fluorescent microscopy contains information on the functioning properties and original connections of hundreds of simultaneously recorded neurons. Our group developed an open-source toolbox to move from qualitative to quantitative analysis of recorded data, providing neurobiologists with statistical metrics from miniscope processed recordings through “Minian” [4]. Neuronal network state was defined in open-field test conditions under normal circumstances using a self-developed toolbox in the current study.

In this study, 5-month-old wild B6SJL mice were injected with AAV-GCaMP6f virus in the hippocampus. After 3 weeks, a gradient lens was implanted over the hippocampus with baseplate fixation. Changes in calcium levels were measured using Miniscope v3 in the “open field” test. A software package was created for quantitative analysis of the neuron activity data. As a result, the study concluded that the most consistent data over the course of five days were the Pearson correlation coefficient for the active spike method (based on binary results from active phase segmentation) and the network degree level (the ratio of interconnected neurons depending on the presence of a connection). These measures displayed a high level of stability throughout the recordings. Furthermore, the PCA method applied to the calculated statistics indicated a close relationship between the coordinates that described the activity of the hippocampal neuronal network during the five-day testing period.

The miniscope technique appears to be an effective tool for identifying shifts in neuronal networks during the progression of neurodegenerative diseases, such as Alzheimer’s disease [5]. It may also aid in detecting possible changes following neurological complications related to viral infections.

Genes & Cells. 2023;18(4):763-766
pages 763-766 views

Molecular targets for optogenetic stimulation of astrocytes for recovering cognitive functions in neurological complications

Gerasimov E.I., Erofeev A.I., Bolshakova A.V., Bezprozvannyi I.B., Vlasova O.L.

Abstract

Recently, mounting evidence suggests that cognitive impairment may accompany traditional neurological diseases, such as neurodegenerative disorders, as well as result from previous infections (COVID-19, influenza). One approach to mitigating the neurological pathological state involves regulating abnormal neural activity. Nevertheless, addressing this issue directly may not always be feasible due to neuronal overexcitation or inadequate stimulation, leading to unfavorable outcomes. Meanwhile, astrocytes adapt their activation levels exclusively to the group of neurons requiring activation, boosting cognitive functions as an example [1]. Optogenetics was employed in this study to selectively stimulate metabotropic astrocyte receptors in acute hippocampal slices of mice with an Alzheimer’s model. The aim was to examine the effect on electrophysiological function of neurons, strength of synaptic contacts ex vivo, and cognitive performance in vivo. Several fundamentally different approaches exist for optogenetic stimulation of cells, including the use of molecular targets such as ionotropic receptors (e.g., ChR2) or metabotropic receptors (e.g., OptoGq). Our studies have shown enhanced activity of hippocampal pyramidal neurons and potentiated field excitatory potentials (fEPSP) following optogenetic activation of astrocytes expressing the metabotropic construct OptoGq. Conversely, the use of ChR2 resulted in an opposite effect [2]. For this reason, all subsequent investigations used a metabotropic construct. Astrocytes are known to respond to external stimuli via intracellular calcium [Ca2+] waves. The propagation of this wave results in the release of D-serine, cytokines, and lactate, subsequently modulating the activity of neurons. The role of astrocytes in regulating the function of NMDA receptors by releasing or removing glutamate from the extracellular environment is critical in modulating neural network excitation. Given the association of astrocytes with the pathogenesis and pathological mechanisms involved in neurodegenerative disorders, controlling their activity becomes a pressing and indispensable aspect of therapy.

In the present investigation, optogenetic stimulation of hippocampal astrocytes transduced by AAV5_GfaABC1D_opto-a1AR-EYFP virus (which encodes a Gq-coupled metabotropic receptor) resulted in enhanced electrophysiological activity of hippocampal pyramidal neurons. This was evidenced by increased sEPSC of pyramidal neurons and the potentiation of field excitatory postsynaptic potentials (fEPSP) in the hippocampal region, following light activation of astrocytes [2]. A significant activation of early gene expression (cRel, Arc, Fos, JunB, and Egr1) was detected in hippocampal slices [3]. Additionally, optogenetic activation of the metabotropic receptor during behavioral tests in vivo restored cognitive functions in mice with an Alzheimer’s disease model.

The activation of the Gq-coupled metabotropic receptor was found to be a molecular target that promotes positive changes in neuronal functioning at ex vivo and in vivo levels in both wild type mice and a mouse model of Alzheimer’s disease. Expression of OPTO-α1AR in astrocytes could potentially have a beneficial impact on other neuropathological conditions. In the future, alternative less-invasive methods, such as chemogenetics, could be employed to specifically activate astrocytes in distinct brain regions.

Genes & Cells. 2023;18(4):767-770
pages 767-770 views

Infragranular excitatory projection to granular neurons in neonatal rodent somatosensory neocortex

Idzhilova O.S., Simonova N.A., Minlebaev M.G., Malyshev A.Y.

Abstract

Though the principles of central nervous system development are genetically encoded, the cortical activity is also critically involved in these processes. While this question is quite important, up to now we are limited in our understanding of the role that neuronal activity plays in formation of functionally linked cellular ensembles in the developing cortex. Recently, transient inhibitory neuronal projections were shown in the barrel cortex during the critical period of its development. Expression of interneuronal connections from infragranular to other cortical layers exactly during the period of barrel formation suggests their critical role in establishment of adult-like columnar organization of the barrel cortex. While the inhibitory connections were demonstrated, the question remains, whether the transient connectivity is restricted by emergence of inhibitory projections, or both types (including excitatory connections) could be expressed during the critical period of the barrel cortex development.

Here, we aimed to answer this question using in vitro optogenetic stimulation of the neurons in the infragranular layers of the neonatal mouse barrel cortex. A viral vector of serotype AAV.PHP.eB containing channelrhodopsin-2 along with a fluorescent Venus tag sequence under the hSyn promoter was delivered via intraventricular injection into the neonatal mouse brain at P0. This transduction protocol resulted in neuron-specific expression of the construct primarily in the L2/3, L5 and L6 cortical layers at P7. At P7, acute coronal brain slices containing the barrel cortical field were prepared. For a given cortical column, the infragranular layers were optogenetically mapped while simultaneous whole-cell electrophysiological registration of a pyramidal cell in the barrel was performed. Holding potential was varied to discriminate between light-evoked EPSCs and IPSCs.

The results of our preliminary recordings in the neonatal somatosensory cortex showed presence of neuronal projections from infra- to granular layer, which is in agreement with the already demonstrated data. However, L4 EPSCs evoked by infragranular layer stimulation were also recorded, suggesting the expression of the excitatory connections from infra- to granular layers early in development. Though we require to continue our recordings, our findings suggest an even more complex network interactions that shape the barrel cortex L4 during the early postnatal stages.

Genes & Cells. 2023;18(4):771-773
pages 771-773 views

Calcium activity of hippocampal CA1 neurons during memory formation and retrieval in young and old mice

Rogozhnikova O.S., Ivashkina О.I., Toropova K.А., Sotskov V.P., Plusnin V.V., Anokhin K.V.

Abstract

It is known that in the process of memory formation for new experiences, neurons in many regions of the brain are activated. In particular, neurons of the CA1 area of the hippocampus are activated during memory formation when animal first observe a new context [1]. However, it is not quite clear how the activity of CA1 neurons changes during memory formation and retrieval. Moreover, the question remains what changes in neuronal activity are observed in old animals during memory formation and retrieval.

In our work, we investigated changes in the calcium activity of hippocampal CA1 neurons during the formation and retrieval of associative memory of the context model of context preexposure facilitation effect in young and aged mice.

Calcium activity of individual neurons was recorded using a miniature microscope (miniscope), which allows optical detection of active neurons through the fluorescence of the calcium sensor. For this purpose, the mice underwent stereotactic surgery in which the calcium fluorescent sensor NCaMP7 was injected into the CA1 area of the hippocampus [2]. Then, a 0.5-mm-diameter GRIN lens was implanted into the studied area, and miniscope mounts were placed on the mouse head. The experiment was performed one week after the surgery. On the first day, the procedure of pretraining was performed: the mice were placed in a new context for 5 minutes free exploration, as a result of which the mice formed a spatial perception of the context. Three days later, the mice were briefly placed in the same context and immediately received footshock for 2s (1.5mA). Thus, the previously formed perception of the context was associated with the animal's state of fear. We tested associative memory three days after shock application: mice were placed in the same context for 5 minutes.

A measure of formed associative memory was their level of freezing behaviour in the context. Young mice showed a low level of freezing on the first visit of the context. We also observed a low level of freezing in older mice on the first day of the experiment. At the same time, the retrieval of a previously formed memory significantly increased the level of the freezing in older mice, compared with the first day, indicating that this animal had formed an associative memory of the context. To analyze changes in calcium activity during memory formation and retrieval, we assessed changes in the level of neuronal activity in each individual animal freezing act. Calcium activity of individual CA1 area neurons was recorded during the first visit to the environment and during memory retrieval. In each mouse, about 20 neurons were recorded in the two groups under study in two sessions of the experiment. However, we did not find any significant changes in the number of active neurons in young and old animals at the moments of their fading into the environment.

The results indicate that the processes of associative memory formation and retrieval do not manifest themselves in changes in the number of active cells at the moment of the animal freezing behaviour. Possibly, the studied processes of memory formation and retrieval are reflected in other forms of brain activity, such as cognitive maps of the context, which represent a network of cognitively specialized neurons (place fields).

Genes & Cells. 2023;18(4):774-777
pages 774-777 views

Nitrogen-doped carbon nanotubes for self-powered memristive systems

Il'ina M.V., Soboleva O.I., Polyvianova M.R., Il'in O.I.

Abstract

Memristive devices are one of the promising candidates for creating neuromorphic systems due to the possibility of multilevel switching, low operating voltages and high scalability. However, as with any passive element, the memristor requires an external bias voltage to operate, which requires the inclusion of a power source in the circuit. In this regard, of great interest are works on the creation of self-powered memristive systems consisting of connecting in series a memristor and a nanogenerator that converts the energy of the external environment into electrical energy [1, 2]. Such a memristive system has a high potential for applications in aerospace and implantable electronics. At the moment, the first self-powered memristive and sensor systems based on metal oxides and piezoelectric nanogenerators (PENG) have already been developed [2]. The main problems in this area are to reduce the size of the nanogenerator and to match the output parameters of the nanogenerator and the input parameters of the memristor. In the framework of this work, these problems are being resolved by creating a self-powered memristive system based on nitrogen-doped carbon nanotubes (N-CNTs).

Previously, we studied the memristive properties of N-CNTs and showed that nanotubes demonstrate reproducible multilevel switching with a resistance ratio in the high- and low-resistance states (HRS/LRS) of about 4 ⋅ 105 [3, 4]. It was found that the memristive effect in N-CNTs is due to the incorporation of nitrogen atoms into the nanotube structure and the formation of an internal piezoelectric field [4]. As part of further studies, it was found that an array of vertically aligned N-CNTs is a promising material for creating PENG: the generated output voltage is hundreds of mV and the current generated by single nanotube reaches hundreds of nA [5]. The results obtained allow us to speak about the possibility of developing a self-powered memristive system by connecting in series a memristor and PENG based on N-CNTs.

To optimize the output characteristics of the PENG, in particular, the amplitude of the generated voltage, and the input switching voltage of the N-CNT-based memristor, studies were carried out to increase the piezoelectric response and reduce the switching voltage of the N-CNT resistance by changing the concentration of the dopant nitrogen in the nanotube growth process. It was found that it is necessary to grow N-CNTs with a doping nitrogen concentration of up to 12% and a high aspect ratio of length to diameter (more than 60) to create PENG with an output voltage of up to 2 V. These N-CNT parameters are provided at a low growth temperature (500–550 C°) and high ratio of acetylene and ammonia flows (1:5 - 1:6). On the contrary, the N-CNTs with a small aspect ratio (less than 30) and doping nitrogen concentrations of 4–6% are required for the manufacture of memristors with a minimum switching voltage (about 2 V), These N-CNT parameters are provided by increasing the growth temperature to 615 C° and reduction in growth time. The obtained results can be used in the development of self-powered memristive and sensor systems based on nitrogen-doped carbon nanotubes.

Genes & Cells. 2023;18(4):810-813
pages 810-813 views

Investigation of the functioning of a neurohybrid system based on the FitzHugh–Nagumo radio generator and mouse hippocampal neurons

Matveeva M.V., Fedulina A.A., Beltyukova A.V., Maltseva K.E., Gerasimova S.A., Mishchenko M.A., Mikhaylov A.N., Kazantsev V.B., Lebedeva A.V.

Abstract

Currently, in the treatment of neurodegenerative diseases that are difficult to respond to drug therapy, electrical stimulation of the brain is performed through invasive intervention in damaged structures of the nervous tissue. The development and development of invasive technologies of closed-loop neural interfaces have made it possible to achieve great success in restoring neural connections, since they have finer and more precise stimulation settings that respond to changes in the physiological state, which is important in the process of restoring the functions of the nervous tissue. Advances in these areas open up prospects for the treatment of a wide range of diseases of the motor system and neurodegenerative diseases of the brain.

In this study, a neurohybrid closed system was used, consisting of a FitzHugh-Nagumo radio generator and live surviving slices of the mouse brain hippocampus. For the preparation of surviving slices of the hippocampus, sexually mature males aged 2-3 months of the C57BL/6 mouse line were used. For the preparation and incubation of slices of the hippocampus, a solution of artificial cerebrospinal fluid (ACSF) was used, composition in (mM): 126 NaCl; 3.5 KCl; 1.2 KH2PO4; 26 NaHCO3; 1.3 MgCl * 6H2O; 2 CaCl2 * 6H2O; 10 D-glucose at constant carbogen saturation (95% O2 and 5% CO2). Registration of the electrical activity of brain neurons was carried out using optical and electrophysiological methods.

In experiments on pairing a neuron-like FitzHugh-Nagumo generator and biological nerve cells in a closed circuit, an effect was obtained when the activity of brain nerve cells switched the generator to a self-oscillating mode. The evoked oscillations in the neuron-like generator provided an effective stimulus for the activation of nerve fibers in the perforant pathway of the hippocampus. As a result, it was possible to fix a decrease in the frequency of the generator impulses, which was provoked by the responses of living neurons to the incoming stimulus from the neuron-like generator. These results show the ability of live neural networks to control an artificial signal by adjusting its parameters by changing their own activity and confirm the efficiency of using closed loop systems when combining live and artificial neurons. The present study requires further experiments to create more physiological conditions for the functioning of the proposed neurohybrid system. In addition, this neurohybrid system will be improved and have adaptive properties through the use of memristive devices. Advances in this direction will help solve the urgent problem of restoring lost brain functions at the cellular and network levels.

Genes & Cells. 2023;18(4):818-820
pages 818-820 views

Optogenetic stimulation suppresses ictal activity in a 4-aminopyridine model of epileptic activity in vitro

Zaitsev A.V., Proskurina E.Y., Trofimova A.M., Postnikova T.Y., Ergina Y.L., Amahin D.V., Kim K.K., Tiselko V.S., Chizhov A.V.

Abstract

The WHO estimates that nearly 1% of the population suffers from epilepsy. Despite advances in the development of new antiepileptic drugs, seizures cannot be completely eliminated in nearly one-third of patients.

One promising approach to the treatment of epilepsy may be gene therapy. Because epileptic activity is caused by an imbalance between excitation and inhibition, researchers have focused primarily on regulating neuronal excitability. Initially, the main approaches were based on the hyperexpression of inhibitory peptides such as galanin or NPY, or the suppression of neuronal excitability by the hyperexpression of potassium channels in neurons. However, these effects should be well calculated and strictly dosed, as it is difficult to correct the expression later. If the expression is too low, the anticonvulsant effect will not be achieved, and if the expression is too high, the neuronal networks will be impaired due to strong inhibition.

For this reason, methodological approaches to treatment in which the effect on the neuronal excitability in the epileptic focus can be controlled are of great interest. Optogenetic methods offer such an advantage. Optogenetics uses light to alter the excitability of specific neuronal populations and can also be used in a biofeedback paradigm in which the light source is activated only at the risk of generating seizure activity. A number of optogenetic tools have now been developed, including light-activated cationic (e.g., ChR2) and anion channels (ACR), metabotropic receptors, pumps (NpHR, Arch), and enzymes. However, the optogenetic approach has a number of technical difficulties in delivering the light source and the risk of developing an immune response to the expression of rhodopsins.

This report reviews the experience of practical application of optogenetic tools in the use of 4-aminopyridine in vitro model of epileptiform activity in experiencing slices of the entorhinal cortex. We tested the efficacy of suppressing ictal activity using several variants of optogenetic stimulation: (1) activation of excitatory and inhibitory neurons (in Thy1-ChR2-YFP mice), (2) activation of inhibitory interneurons only (in PV-Cre mice after virus injection with the channelodopsin-2 gene), hyperpolarization of excitatory neurons after expression of (3) archaeodopsin or (4) a light-dependent sodium pump. We found that ictal activity was successfully suppressed when low-frequency optogenetic stimulation induced regular interictal activity. Usually, interictal activity was induced by rhythmic synchronous activation of the principal neurons of the entorhinal cortex. In other cases, the ictal activity was preserved, although its characteristics may have changed. We determined the parameters of optogenetic stimulation that were most effective in suppressing ictal activity.

The availability of a wide range of gene therapy approaches for epilepsy that have demonstrated efficacy in preclinical studies suggests that clinical trials of some of these approaches will begin in the next few years.

Genes & Cells. 2023;18(4):786-788
pages 786-788 views

Design of spiking neural network architecture based on dendritic computation principles

Mavrin I.A., Ryndin E.A., Andreeva N.V., Luchinin V.V.

Abstract

The paper presents the hardware architecture design of a spiking neural network (SNN) based on dendritic computation principles. The integration of active dendritic properties into the neuronal structure of SNN aims to minimize the number of functional blocks required for hardware implementation, including synaptic connections and neurons. The available memory on the neuromorphic architecture imposes limitations on implementation, hence the need to reduce the number of functional blocks.

As a test task for the SNN based on dendritic computations, we selected the image classification of eight symbols, consisting of digits one through eight. These symbols are depicted as 3×7 pixel, 1-bit images.

Active dendritic properties were analyzed using the “delay plasticity” [1] principle, which introduces the mechanism of adjusting input signal delays in spiking neuron inputs. We designed an SNN model with complementary delay inputs, referred to as the active dendrite SNN, as a principle implementation. Input spikes arriving at the primary inputs are duplicated to the delay inputs after a modifiable time delay. For convenience, each delay input was set at a single value.

The input images were scanned sequentially. The neural network received three direct and three inverse inputs from the six main inputs that were coded with spikes corresponding to three pixels of a string. An “on” pixel was coded with a spike arriving at a direct input, while an “off” pixel was coded with a spike arriving at the corresponding inverse input. The line scanning time was 10 μs, input width was 1 μs, and delay time was 5 μs.

The optimization of spiking neuron parameters was performed through a stochastic search algorithm based on simulated annealing. The parameters optimized for the Leaky-Integrate-and-Fire (LIF) neurons included the leakage time constant (22.8 μs), firing threshold (1150 arbitrary units), and refractory period (1 μs).

The active dendrite SNN training employed the tempotron learning rule [2]. The training optimized the following parameters: the maximum change in synaptic weight on potentiation and depression (0.7 and –3 arbitrary units, respectively) and the synaptic weight’s upper bound (195 arbitrary units).

Complementary delayed inputs facilitated the learning of the order in which input patterns arrived for SNN neurons during training.

The paper compares an SNN architecture based on dendritic computations to our previously designed two-layer SNN with a hidden perceptron layer and an output layer consisting of LIF neurons [3].

Using the same LIF neuron design, input image coding, and LIF neuron layer structure as in the proposed architecture, our two-layer SNN with a hidden perceptron layer and output layer of LIF neurons successfully recognized 3×5 images of three symbols with only 10 neurons and 63 synapses. Alternatively, the active dendrite SNN was able to recognize 3×7 images of eight symbols with four neurons and 48 synaptic weights.

In conclusion, incorporating active dendrite properties into the SNN architecture for image recognition resulted in optimized functional block usage, lowering the number of neurons and synapses used by 60 and 24%, respectively.

Genes & Cells. 2023;18(4):821-824
pages 821-824 views

Neuroelectronics as neuromorphic and neurohybryd systems enabled by memristive technology

Mikhaylov A.N.

Abstract

Due to its distinctive capability to replicate vital functions of synapses and neurons, memristive devices and arrays enable both the hardware implementation of neural networks and a significant advancement towards integrating artificial electronic and biological systems to address pressing issues in artificial intelligence (AI), robotics, and medicine. This research area is still nascent and can be viewed as part of the broader field of neuroelectronics. The aforementioned is a fusion of analog and digital solutions used for diverse computational tasks inspired by biology. Notably, analog neuromorphic systems that employ memristive components are distinctive features of this arena and they can significantly enhance throughput and energy efficiency in contrast to existing AI accelerators. Designing neuromorphic systems grounded on this fresh component base mandates coordinated and interdisciplinary research and development. The foundation of this scientific and technological field lies in the cross-cutting technology of memristive devices and circuits. This technology enables the development of a novel brain-like information and computing system infrastructure that can be applied in a diverse range of fields. Current perspectives include the seamless integration of memristive devices and arrays with CMOS circuits, and the co-optimization of materials, devices, and architectures to create prototypes for computing and information systems. These systems replicate computational features present in biological neural networks capable of solving cognitive tasks that are typically either intractable by traditional AI or highly time-consuming. Neuroelectronic solutions can integrate with the brain or living neuronal cultures to form neurohybrid systems. In this presentation, we discuss two distinct strategies for connecting memristive systems with biological neural networks both in vitro and in vivo. These include a perceptron using an array of programmable memristive weights represented by metal-oxide resistive-switching devices and a methodology leveraging memristive stochastic plasticity and neural synchrony, which is part of the brain’s spiking architecture. Finally, the concept of a memristive neurohybrid chip is presented to create a compact, multifunctional, bidirectional interface between biological neural networks and memristive electronics, combined with microelectrode and microfluidic systems on a single chip. The technological advancements in component base and the development of memristor-based neuroelectronic systems will diversify hardware for the continuous evolution and mass application of artificial intelligence technologies. This will enable the creation of hybrid intelligence based on the symbiosis of artificial and biological neural networks and allow for the establishment of novel tasks at an unprecedented level.

Genes & Cells. 2023;18(4):825-826
pages 825-826 views

The use of memristive devices in machine vision systems

Shchanikov S.A.

Abstract

The comparison results of processing units with memristive devices versus modern hardware accelerators of artificial neural networks (ANNs) based on traditional electronic components, as presented in the review [1], demonstrate numerous advantages across all major indicators such as throughput, energy efficiency, accuracy, and others. This report analyzes the current state of memristive devices in addressing machine vision issues. Special attention is paid to the concept of [2] neuromorphic machine vision systems (MVS) based on memristive devices. This concept’s distinct feature lies in its fully analog system, commencing from information input to its output. It encompasses sensory and neural components. The sensory part is responsible for gathering visual information and transferring it to the neural segment for processing through the ANN model algorithm.

A specific instance for implementing the input channel of the sensor component involves connecting a photodiode (PD) and a memristor in a single circuit. When the circuit is flipped in reverse bias and light falls on the PD, a photocurrent flows through it from the cathode to the anode. Depending on the light intensity and exposure time, this photocurrent alters the resistance of the memristor, thereby converting illumination into resistance. If visual information doesn’t require encoding with memristor resistances, they can be replaced with a load resistance of the same nominal value for all channels. Irrespective of the input channel variant, the signal encoding visual information is fed into the neural part without digitization. Memristors act as synapses as part of the neural part. They can be used to implement synapses in both traditional formal ANN architectures, where input information is multiplied by a pre-programmed weight, and synapses for spiking neural networks, where a memristor exhibits synaptic plasticity mechanisms similar to those in biological neural networks [3].

If the output from the suggested MVS input channel variants is connected to a device that operates on the “integrate and fire” principle, the device can be deemed as not only an input for a structured ANN, but also a presynaptic neuron for a spiking ANN. The neuron’s frequency of spikes will depend on the light intensity; the brighter the light, the higher the frequency of spikes and vice versa. Faster charge accumulation occurs in channels with low resistance. The complete analog machine vision system will function as a spike neural network, without incorporating any analog-to-digital or digital-to-analog converters. Compared to digital machine vision systems, this approach will significantly decrease energy consumption while producing wearable and on-board electronics with distinct tactical and technical features. This design can be tailored to the size of modern matrices of photo and video fixation devices and employed as a hardware accelerator for the ANN models currently used to process images, and it can serve as a foundation for advancing this area.

Genes & Cells. 2023;18(4):831-834
pages 831-834 views

Cyclops states in repulsive theta-neuron networks

Bolotov M.I., Munyayev V.O., Smirnov L.A., Osipov G.V., Belykh I.

Abstract

Networks of phase oscillators have become a widely established paradigmatic model for studying emergent collective behavior across several real-world systems, including neuronal networks, populations of chemical oscillators, and power grids. The Kuramoto model, involving one-dimensional or two-dimensional phase oscillators, demonstrates the potential for networks to showcase exceptional collective dynamics. This encompasses various outcomes such as full, partial, explosive, and asymmetry-induced synchronization, clusters, chimeras, solitary states, and generalized splay states. Notably, increasing all-to-all coupling in the classical Kuramoto model induces full synchronization as the most probable outcome and dominant rhythm. Kuramoto networks with repulsive coupling usually display splay, generalized, and cluster splay states, but the conditions under which a certain rhythm can arise and prevail are not entirely understood.

Equally important for connecting Kuramoto networks to practical physical systems is understanding the function of higher-order coupling terms. These terms display a Fourier decomposition of a general 2π-periodic interaction function [1]. Previous studies have demonstrated that the inclusion of higher-order terms in the classical Kuramoto model of oscillators with all-to-all attractive coupling can lead to multiple synchronous states and switching between synchronization clusters. However, the impact of higher-order coupling modes on rhythm generation in repulsive networks remains unexplored.

In this work, we present significant progress in addressing the critical issue related to repulsive Kuramoto–Sakaguchi networks of phase oscillators with phase-lagged first-order and higher-order coupling. We demonstrate that weakly repulsive networks of even and odd numbers of oscillators with first-order coupling are dominated by two-cluster and three-cluster splay states, respectively. The three-cluster splay states consist of two distinct coherent clusters and one solitary oscillator. These tripod states can be considered a fusion of a two-body chimera and a solitary state. We have dubbed these patterns of three oscillators as “Cyclops states” in reference to the Greek mythological giant with a single eye. The solitary oscillator and synchronous clusters respectively represent the Cyclops’ eye and shoulders. We present a remarkable discovery that the inclusion of higher-order coupling modes leads to worldwide stability of cyclops states across almost the entire range of the phase-lag parameter controlling repulsion [2].

Beyond the Kuramoto oscillators, we demonstrate the robust presence of this effect in networks of canonical theta-neurons with adaptive coupling. Furthermore, our results provide insight into identifying dominant rhythms within repulsive physical and biological networks.

Genes & Cells. 2023;18(4):844-846
pages 844-846 views

WaveNet vocoder for prediction of time series with extreme events

Gromov N.V., Levanova T.A.

Abstract

Extreme events are typically defined as rare or unpredictable events that deviate significantly from typical behavior. Despite this, objective criteria for extreme events have yet to be established. Rareness may be characterized by certain scales or spatial and temporal boundaries, while intensity is an indication of an event’s potential to cause a significant change. One of the most prominent occurrences of extreme events in both neuroscience and medicine is in the case of epileptic seizures [1].

In speech synthesis, vocoder networks like WaveNet [2] generate audio. The model is a multi-layer convolutional neural network that functions as a causal filter and doesn’t predict the future. Due to this quality, the vocoder may have potential in time series prediction. Audio time series can be regarded as a dynamic system characterized by unpredictable switching regimes. For instance, transitioning from one letter to another can result in significant deviations in amplitude, similar to extreme events. This network receives r previous input counts known as a receptive field, and uses them to predict the next sample. The network is tree-like in structure, with exponentially increasing distances between subsequent layers of inputs. This is a necessary feature since the receptive field r is usually quite large, on the order of one or two thousand. Without this exponential increase in distance, the number of layers would depend linearly on r. Recurrent neural networks pose a challenge in optimizing the loss function when predicting time series sequences, as they tend to predict samples very similar to the previous one, causing the network to converge towards the mode. However, in a convolutional network, the output to the model will be longer due to the large receptive field. In the case of sound analysis, for instance, multiple oscillations occur within a given timeframe and the network does not elevate any specific sample.

The study used artificial data generated from two coupled Hidmarsh–Rose neurons with chemical synaptic couplings. The observed variable was determined by the biological significance of the system, specifically the total membrane potential. The results exhibited extreme events across various coupling parameter values. Based on prior research [3], a numerical standard was selected for the events. The WaveNet vocoder model exhibits a 91% accuracy rate and 82% recall rate when forecasting extreme events of the same width as the prediction. It is noteworthy that recall is crucial in the forecast of extreme events since it identifies instances where the model predicted falsely that an extreme event would not occur.

Genes & Cells. 2023;18(4):847-849
pages 847-849 views

Neural activity of the subthalamic nucleus during voluntary movements in patients with Parkinson’s disease

Filyushkina V.I., Belova Е.М., Usova S.V., Tomskiy A.A., Sedov A.S.

Abstract

Microelectrode recording (MER) during electrode implantation for deep brain stimulation (DBS) can determine the boundaries of the subthalamic nucleus (STN), and track neural activity during rest and voluntary movement in Parkinson’s disease (PD) patients. However, the functional role of STN in motor control remains unclear, despite its effectiveness in stimulation.

Single unit activity of STN was recorded in 16 patients with PD during implantation of deep brain stimulation electrodes. Electromyograms of forearm muscles and a phonogram featuring verbal commands were simultaneously recorded with MER. The patients were instructed to perform motor tests involving clenching their hand into a fist. Out of the 560 neurons studied, 93 (16.6%) responded to motor tasks. The registered neurons were classified into three patterns using the hierarchical clustering method of histograms of interspike interval density — namely, tonic, burst, and pause patterns.

Sensitive neurons were classified according to their responses, with activation (76.9%) and inhibition (23.1%) being the two identified types. In 90% of cases, neuron activation preceded movement. Of the responses, 53.8% were classified as tonic and 46.2% as phasic. Two thirds of inhibitory responses were advanced, occurring 0.2–0.3 seconds before movement onset. One third of neurons were activated after movement initiation with a delay of 0.05–0.2 seconds. 83.3% of the inhibitory neurons responded tonically, while the remaining 16.7% responded phasically. Notably, all phasic reactions occurred before the onset of movement.

A comparison of the parameters of sensitive and non-sensitive neurons revealed several differences. The findings suggest a potential physiological distinction between the two neuron types. All three patterns of activity were present in both types of neurons, but sensitive neurons exhibited a wider representation of pause neurons (57.9% vs. 49.5%). Additionally, burst and pause neurons responded to movements that featured significantly lower variance of multiple activity parameters, such as firing rate, burst index, mean burst length, and oscillation scores in the 8–12 and 12–20 Hz range. The distribution of sensitive neurons along the electrode trajectory through the subthalamic nucleus was analyzed. Sensitive neurons were located significantly more dorsally compared to non-sensitive neurons, and no sensitive pause cells were observed in the ventral half of the STN.

Based on our findings, it is presumed that the pause pattern plays a critical role in both motor control and its related disorders in Parkinson’s disease. A diverse range of neuronal responses suggests a high degree of heterogeneity among STN neurons implicated in motor control. The presence of both delayed and advanced neural responses suggests that STN involvement in motor control encompasses both the preparation and initiation of movement, as well as the regulation of ongoing movements via afferent feedback. The range of neural responses aligns with the dynamic model of basal ganglia, which suggests that STN can perform distinct functional roles during different stages of movement, including initiation, control, and completion.

Genes & Cells. 2023;18(4):664-666
pages 664-666 views

Interhemispheric connectivity dynamics of brain activity indused by cortical spreading depolarization in rats

Medvedeva T.M., Suleymanova E.M., Vinogradova L.V.

Abstract

Growing experimental and clinical evidence indicates the crucial role of network dysfunction in neurological disorders’ pathogenesis, including migraine, one of the most prevalent chronic brain diseases. Episodic headache attacks, frequently unilateral, accompanied by an aura, are associated with migraine. Migraine aura is a neurological condition characterized by the temporary development of unilateral sensory, motor, and/or speech disturbances. The symptoms are thought to indicate transient cerebral dysfunction in the cerebral cortex resulting from cortical spreading depolarization (SD), a wave of strong cellular depolarization that gradually spreads through the cortex at a rate of 3–5 mm/min. Electrophysiologically, the cortical SD wave is revealed by a high-amplitude slow negative shift in the extracellular potential and temporary suppression of the electrical activity of the cortex (EEG depression). The change in extracellular potential is associated with strong neuroglial depolarization and disruption of local ion homeostasis, which lasts for 1–2 min in healthy neuronal tissue. The SD results in a momentary suppression of the spontaneous electrical activity within the cortex, which is preceded by a brief excitation of the neurons.

The neurological symptoms of the aura suggest a unilateral impairment of interhemispheric interactions during the early phase of a migraine attack. Our study investigated the effect of unilateral SD (a probable pathophysiological mechanism of migraine aura) on interhemispheric functional communication in freely behaving rats using local field potentials of the visual and motor cortex. Two methods were used to examine connectivity: mutual information function, computed using the method proposed in [1], and phase synchronization, calculated through the method [2], for four frequency bands: delta (1–4 Hz), theta (4–10 Hz), beta (10–25 Hz), and gamma (25–50 Hz). This was done by performing calculations on non-overlapping twenty-second intervals. Functional connectivity evolution was analyzed using local field potential records collected from homotopic points of the motor and visual cortex of two hemispheres in freely moving rats after inducing a single unilateral cortical SD in the somatosensory cortex.

Cortical SD caused a significant wide-band decline (3–4 times) in interhemispheric functional connectivity in both the visual and motor cortex areas. Following the depolarization wave, the functional decoupling of the hemispheres began and progressively intensified, concluding by 5 min after the induction of the cortical SD wave. The network impairment displayed region- and frequency-specific features, with greater prominence observed in the visual cortex than in the motor cortex. The decline in functional connectivity was concurrent with abnormal animal behavior and aberrant activity in the ipsilateral cortex that appeared after the SD wave had ended.

The study indicated that unilateral SD leads to a reversible decline in the functional interhemispheric connectivity in the awake animal cortex. Given the crucial role of synchronizing cortical oscillations for processing sensory information and integrating sensorimotor functions, the intracortical functional interactions disruption resulting from a unilateral SD wave, which we discovered in our present study, could contribute to the neuropathological mechanisms of migraine aura and sensory processing dysfunction during a migraine attack.

Genes & Cells. 2023;18(4):862-865
pages 862-865 views

Associations of neuro-glial network calcium activity with mice movements in vivo

Krivonosov M.I., Varekhina A.V., Anokhin K.V., Ivanchenko M.V.

Abstract

Calcium imaging of nerve activity in the mouse hippocampus provides insights into cell-to-cell interactions [1]. Over time, fluctuations in cellular calcium levels encode distinct brain states during mouse brain imaging. Although synapses connect cells and transmit information, it is challenging to detect the spatiotemporal transmission pathway via calcium imaging of multiple cells due to the complexity of the spatial structure and imaging in a separate focal plane [2]. Reconstruction of the dynamic graph of connections between cells was proposed to overcome this problem.

A dynamic graph comprises of individual graphs for each time point. A graph linked to a particular time t is composed of vertices representing cells and edges that depict the transmission of signals between cells at that moment. This paper puts forth 3 approaches for creating networks. The first method considers the overlapping time intervals of calcium events in individual cells [3]. The link is established between the cell with the earlier event and the cell with the later start of the event during the moments when the events occurred simultaneously in separate cells. Alternatively, time intervals between the start of events were taken into account. Potentially connected events that began no later than 2 s were linked, with the edge drawn from the cell depicting the earlier starting event to the second cell. The third technique involved linking sequentially occurring events in distinct cells. The edge is drawn from all active cells at the previous time step to newly activated cells at the subsequent time step.

Data analysis is based on an experiment in which a mouse moved along a circular track while the fluorescence of neuronal calcium activity and the mouse’s position were recorded at a frequency of 20 frames per second [4]. A red dot was marked on the mouse’s head to track its position. The reconstructed dynamic graph was compared to the angular coordinate of the mouse on the ring to look for repeating patterns of activity. The racetrack was divided into 20 overlapping sectors, each spanning 36 degrees. The reconstructed networks were then assigned to sectors that aligned with the mouse’s angular position on the track.

Next, we estimated the frequency of individual edge repetitions within each network group. Only edges that occurred at least three times were chosen for further analysis. We found repeated activations of various cell pairs that corresponded to clockwise and counterclockwise movement within the same sector. Furthermore, we identified the presence of alternating activation, where activity occurred in the first cell, then the second cell, and then again in the first cell. In addition, we identified complex sequences of 5–6 non-sequential activations, represented as a digraph without cycles, which is typical for single sectors.

Genes & Cells. 2023;18(4):850-853
pages 850-853 views

Bursting activity in the reduced mean-field model of neuron-glial interaction

Olenin S.M., Levanova T.A., Stasenko S.V.

Abstract

The study of neural activity synchronization in the brain is a central focus of modern neurobiology and neurodynamics. In a healthy brain, cognitive functions necessitate the accurate integration of neural activity at specific spatiotemporal scales. Some neurological and psychiatric disorders exhibit observable variations in patterns of synchronous population activity. One of the most significant and compelling synchronous population activity patterns is the bursting activity that participates in various informational and physiological processes, including epilepsy. Several mathematical models have been proposed to explain the mechanisms of formation of the bursting activity. The most intriguing and scientifically sound models enable incorporation of astrocytic modulation of neural activity [1].

In this paper, we present a novel phenomenological model for accurately replicating the bursting neural population activity. Our model builds upon the Tsodyks-Markram [2] model and incorporates vital aspects of neuron-glial interaction through a tripartite synapse. The presented model is a simplified version of the previously proposed model [3]. During astrocyte activity (which lasts seconds), we can set the probability of neurotransmitter release, u, which is determined in fractions of a second. This enables the proposed model to be written in the following manner:

r=–E+α ln(1+exp((Ju(Y)xE+I0)/α)),

=(1–x)/τDu(Y)xE,

=–y/τY+βσy(X).

Here, E(t) represents the average neuronal activity of the excitatory population, while x(t) denotes the quantity of available neurotransmitter. Additionally, y(t) describes the concentration of gliotransmitters, which are released as a result of biochemical reactions during neuron-astrocyte interactions. The function describes the alteration in the likelihood of neurotransmitter release when gliotransmitters are present

u(y)=u0+(∆u0)/(1+exp(–50(yythr)).

Here u0 represents the probability of neurotransmitter release without astrocytic influence; ∆u0 indicates the change in the probability of neurotransmitter release caused by the interaction of the gliotransmitter with the presynaptic terminal, and ythr represents the threshold value that determines the change in the probability of neurotransmitter release due to the effect of a gliotransmitter. The function describes the influence of neurotransmitter on the concentration of the gliotransmitter

σy(x)=1/(1+exp(–20(xxthr)),

where xthr is the astrocyte activation threshold.

Note that the proposed model does not account for synaptic depression’s mechanism. Therefore, various dynamical regimes’ formation in the model is solely controlled by astrocytic dynamics.

In the study, control parameters I0 and u0 were selected while the remaining parameters were held constant. Specifically, τ=0.013, τD=0.08, α=1.58, J=3.07. Changes in the concentration of neurotransmitters and gliotransmitters were characterized by parameters ∆u0=0.305, τν=3.3, β=0.3, xxthr=0.75, ythr=0.4. The research was conducted using computer modeling and numerical methods in nonlinear dynamics.

The proposed model exhibits a diverse range of population activity patterns, such as spiking and bursting regular and chaotic activity. Chaotic dynamics zones are identified in the control parameter plane. An account is given of the mechanisms behind the emergence of these activity patterns through bifurcation analysis. It is shown that the occurrence of chaotic activity in the system can be associated both with the cascade of period doubling bifurcations and with the further development of chaos, as a result of which a homoclinic attractor appears in the system according to the Shilnikov scenario. It was also shown that multistability is observed in the system in a certain range of parameters.

Note that the emergence of multistability and bursting activity in the model is independent of the intricacy of neuron and glial cell dynamics. These dynamics are instead influenced by the feedback loop between the presynaptic neuron and the glial cell. The effects of neuron-like dynamics and neural-glial interaction demonstrated are generally applicable, without specifying particular characteristics of neural-glial interaction, network architecture, or single-cell neuron dynamics. It should be noted that our proposed model solely concerns the potentiation of synaptic transmission by astrocytes.

In summary, the proposed phenomenological model for population activity can replicate various patterns of neuron activity found in a wide range of dynamic memory and information processing studies. This research can potentially lead to the development of new, effective treatment methods for neurological diseases that involve neuron-glial interaction. Another potential application for these results is in the development of an optimized live chip that has preset functions. This requires a deeper understanding of rhythmogenesis in neural networks and brain function.

Genes & Cells. 2023;18(4):870-873
pages 870-873 views

Development of the bioinspired propulsion system for a robotic fish

Mitin I.V., Korotaev R.A., Mironov V.I., Lobov S.A., Kazantsev V.B.

Abstract

Biomimetic robots aim to replicate the movement principles of living creatures, which have been continuously perfected by nature to ensure survival. Technically, survival comprises two factors: optimizing movement efficacy, such as speed, distance traveled, or acceleration, and minimizing energy consumption during movement. Researchers have made several attempts to develop a biomimetic fish robot [1–3].

Tunas are a group of fish with numerous evolutionary adaptations that make them exceptional swimmers, including tail shape, lateral peduncle keels, pectoral fin shape, and finlets. Thunniform locomotion is characterized by limited undulation, typically restricted to the rear one-third of the body, with maximal amplitude reached at the end of the tail peduncle.

A bioinspired propulsion system was developed for a robotic fish model. It is based on the combination of an elastic cord with a tail fin that is firmly attached to the cord. Two symmetric movable thrusts that simulate muscle contractions connect the tail fin to a servomotor. The propulsion system provides oscillatory tail movement that can be controlled for amplitude and frequency. This movement translates to the movement of the robotic fish, which executes the thunniform principle of locomotion.

The body and tail fin of the robotic fish were designed using a computational model that simulates a virtual body in water. Subsequently, we constructed a prototype of the robotic fish and tested it under experimental conditions.

Experiments were conducted to investigate the relationship between the robot’s kinematics and the dynamic parameters of the propulsion system. The results showed that increasing the frequency of tail fin oscillations led to an increase in the robot’s speed. At fixed frequencies, there was an interval of energetically efficient travel speeds up to a threshold velocity. Movement at higher speeds was achievable; however, it was accompanied by greater power consumption. The conclusion aligns with the data from COT studies on living tunas, indicating qualitative agreement. However, the robot’s values were quantitatively higher. Our robotic fish reached a maximum speed of approximately 0.4 BL/s, exceeding speeds in other works where a simplified tail section was used (0.22 BL/s [4], 0.254 BL/s [5]).

Our study demonstrated a correlation between the efficiency of robot swimming and amplitude, which was previously unexplored in addition to previous findings on the relationship between tail beat frequency and swimming speed, as well as the dependence of frequency and swimming speed. The results showed that increasing the oscillation amplitude of the propulsion system, at a fixed frequency, only led to a rise in swimming speed up to a certain threshold. However, further increases in amplitude resulted in minimal speed increases at higher energy costs.

An evaluation of energy efficiency was conducted, assessing its dependence on dynamic parameters of tail oscillation. The results show that the transport cost rises as the tail amplitude rises beyond the threshold. Furthermore, it was found that for a fixed frequency, an interval of energetic preferred speeds up to a threshold exists. Although moving at a higher speed is possible, it consumes more power. In general, it is preferable to increase caudal fin oscillation frequency rather than amplitude to increase swimming speed. These findings are in qualitative accordance with the outcomes of the numerical simulation of thunniform swimming.

Genes & Cells. 2023;18(4):866-869
pages 866-869 views

Automated analysis of animal behavior and its relation to key aspects of the environment reveals new cognitive specializations of neurons

Plusnin V.V., Pospelov N.A., Sotskov V.P., Dokukin N.V., Rogozhnikova O.S., Toropova K.A., Ivashkina O.I., Anokhin K.V.

Abstract

A thorough analysis of animal behavior is essential for examining the relationship between specific neuron activations with the elements of the external environment, behavior, or internal state. Machine learning techniques have made some advancements in automatic segmentation of animal behavior based on data concerning the location of animal body parts [1–3]. At present, these methods cannot achieve the level of segmentation accuracy desired or make correlations between an animal’s behavioral acts and key environmental factors. To address this issue, the authors have created a software package that can extract a variety of behavioral variables from video recordings of animals in experimental settings, enabling mathematical analysis of a behavioral act continuum.

The identification of specific aspects of an animal’s anatomy is crucial for extracting a vast array of behavioral variables. In order to accomplish this task, our team employed DeepLabCut, an accessible toolkit for tracking experimental animal behavior that operates on the principle of transfer learning through deep neural networks. We have devised a technique to ascertain the positions of animal body parts in diverse behavioral situations, resulting in a body parts collection meeting two criteria: offering superior responsiveness to small motor movements of the animal and delivering a high percentage of correct body part locations. In scenarios employing camera shooting from above, such a collection encompasses the nose, ears, tail base, body center, forelimbs, hind limbs, and both flanks of the animal’s body.

Next, we created software tools to extract and annotate behavioral variables from data on animal kinematics in various cognitive tasks. Our automated system comprises two main scripting modules: CreatePreset and BehaviorAnalyzer. The CreatePreset module interacts with users to select the type of arena geometry, object location, and necessary temporal and spatial parameters for analysis. The script’s result saves as a mat-file for analyzing the behavior of all experiment videos, assuming a constant relative position of the arena and the video camera alongside the experiment’s design. The BehaviorAnalyzer module conducts initial processing on time series data consisting of coordinates of an animal’s body parts. This results in the formation of a kinematogram, which details the kinematics of the body parts. The module then isolates individual behavioral acts of the animal and annotates its behavior based on motivational and environmental factors.

Using mutual information-based methods, we analyzed the specialization of hippocampal CA1 neurons in animals as they explored arenas with varying degrees of novelty. Through the analysis, we have identified neurons that exhibit selectivity in relation to specific continuous kinematic parameters governing the posture and trajectory of the animal. These parameters include the animal’s location in the arena space (X and Y coordinates), as well as the speed and angle of rotation of the animal’s head (i.e. absolute orientation in the arena). Neurons specialized in discrete acts of behavior were identified, including rests, locomotions, freezing, rears, and acts of interaction with objects. Furthermore, a selective activation of neurons was found with regard to an additional set of distinct parameters, which combine the animal’s location in the arena and its speed.

Genes & Cells. 2023;18(4):874-877
pages 874-877 views

A reservoir computing system with volatile and non-volatile organic memristors as a promising hardware architecture

Matsukatova A.N., Prudnikov N.V., Kulagin V.A., Trofimov A.D., Emelyanov A.V.

Abstract

In recent years, many scientific groups have been working on hardware implementation of the artificial neural networks to approach the computational efficiency of their biological counterpart. Memristors may play the role of synapses in such networks [1]. Varieties of memristive structures and materials have already been tested in different neural network architectures, but still no memristor is considered ideal for hardware synapse implementation [1]. One of the most significant problems is the presence of inherent stochasticity distinctive for all memristive devices, which complicates the training of the neural networks [1]. Several approaches were proposed to partially mitigate this problem, e.g., a reservoir computing system (RCS) [2] and spiking neural networks (SNN) [3] as well as defect engineering for memristive characteristics improvement. In this work, we propose to combine RCS with SNN and create a bio-inspired neuromorphic system based on two types of organic memristors with specifically designed structures and advanced characteristics.

The RCS consists of two main parts: the reservoir and the readout [2]. The reservoir layer extracts some representative features from the input data due to its internal nonlinear dynamics. The readout layer then uses these features to classify the input data. Typically, a conventional fully connected neural network is used as a readout layer in the RCS. The training process occurs only in the readout layer, while a reservoir is not trainable. This decrease in trainable parameters considerably reduces the memristive stochasticity impact on the training process.

The use of different types of memristors for the RCS is essential. The reservoir layer should consist of memristors with short-term memory, i.e., volatile memristors. This way, memristors can process each input sample individually. Volatile polyaniline-based memristors were chosen for this layer implementation. They can operate within a biologically plausible time range, which is essential as we aim to mimic biological systems [4]. In contrast, the reservoir layer should consist of memristors with long-term memory, i.e., non-volatile memristors, because the readout layer should preserve the trained synaptic weights. Non-volatile parylene memristors with incorporated MoO3 nanoparticles were chosen for the readout layer.

The reservoir computing system adopts some essential principles of brain function, as both short- and long-term memory are significant in biological systems. However, traditional neural networks are commonly used as a readout layer in the RCSs [2]. Their training requires global weight updates, making them vulnerable to memristive stochasticity. In contrast, the SNNs allow local training, e.g., using bio-inspired learning rules, which makes them more effective and robust [3]. Consequently, we presume that a fully organic RCS with an SNN readout layer is a promising hardware memristive architecture.

The work consists of two parts: hardware and software. First, the polyaniline- and parylene-based memristive devices were fabricated and tested. Hardware polyaniline reservoir demonstrated an ability to extract characteristic features from the input data. Nanocomposite parylene memristors were suitable for the role of synapses in the readout layer due to the unique combination of high switching speed, high stability, low power consumption and the possibility of crossbar implementation. Next, the traditional and spiking readout layers were compared in simulation. It was shown that the SNN readout layer is more adaptive and sustainable to noise in image classification tasks as well as memristive stochasticity [5].

Genes & Cells. 2023;18(4):814-817
pages 814-817 views

Searching for cognitive specializations of neurons using mutual information framework

Pospelov N.A., Sotskov V.P., Plusnin V.V., Rogozhnikova O.S., Toropova K.A., Ivashkina O.I., Anokhin K.V.

Abstract

Of particular interest for researching the cognitive specializations of neurons is their correlation with environmental variables and animal behavior. Mutual information (MI) is a preferable method for measuring such correlations, as it allows for the assessment of non-linear relationships between variables, detects synchronization, and provides both significance and strength quantification. However, calculating MI for real data is significantly challenging. In this study, we used updated MI calculation techniques to analyze the connection between calcium fluorescence signaling and behavioral variables. Our approach encompasses novel strategies which we compiled into a software program known as INTENS (Information-Theoretic Evaluation of Neuronal Specializations), and it enabled to identify specialized neurons in mice hippocampal calcium activity data while they explored the arena with varying levels of novelty.

Numerous methods exist for analyzing the relationship between neuron spikes and behavioral variables, including information-theoretical approaches [1]. Extracting information about the relationship between calcium fluorescent signals and behavior is of particular interest due to the signal’s ability to provide crucial information about subthreshold activations of the neuron. In this study, we use the GCMI Gaussian copula entropy method to calculate mutual information [2]. This method relies on the fact that mutual information between two random variables is independent of their marginal distributions and only depends on the type of copula used (a multidimensional distribution where each marginal distribution is uniform).

The actual MI was compared to its corresponding values computed on the time-shifted signals for assessing the statistical significance of the computed information association between the calcium signal and the behavioral variable. Additionally, we devised a technique for gauging the strength of the coupling effect. This involved normalizing the mutual information between the fluorescence signal and the behavior with the entropy value of both variables, previously calculated as random variables. Importantly, the approach outlined earlier is effective for analyzing continuous variables such as calcium signal and animal speed, as well as pairs of continuous and discrete variables such as calcium signal and the presence or absence of grooming.

The analysis of calcium signals recorded from the CA1 region of the hippocampus revealed neuronal specializations related to the animal’s external environment, such as place cells, and specializations related to its behavioral activities, including neurons activated during running, rearing, and freezing. Some neurons selectively activated in response to discrete parameters included the animal’s location within the arena (center, walls, and corners) and its speed (rest, slow, and fast). A total of 781 specializations were detected across 472 neurons throughout all four sessions of the experiment. Notably, a single neuron could have several specializations. However, more than half (55%) of the neurons were found to have only one specialization.

Genes & Cells. 2023;18(4):878-881
pages 878-881 views

Neurophysiology of creativity and machine learning applications for creative process’ stages differentiation through assessment of EEG/VP signals

Shemyakina N.V., Nagornova Z.V.

Abstract

Of particular interest for researching the cognitive specializations of neurons is their correlation with environmental variables and animal behavior. Mutual information (MI) is a preferable method for measuring such correlations, as it allows for the assessment of non-linear relationships between variables, detects synchronization, and provides both significance and strength quantification. However, calculating MI for real data is significantly challenging. In this study, we used updated MI calculation techniques to analyze the connection between calcium fluorescence signaling and behavioral variables. Our approach encompasses novel strategies which we compiled into a software program known as INTENS (Information-Theoretic Evaluation of Neuronal Specializations), and it enabled to identify specialized neurons in mice hippocampal calcium activity data while they explored the arena with varying levels of novelty.

Numerous methods exist for analyzing the relationship between neuron spikes and behavioral variables, including information-theoretical approaches [1]. Extracting information about the relationship between calcium fluorescent signals and behavior is of particular interest due to the signal’s ability to provide crucial information about subthreshold activations of the neuron. In this study, we use the GCMI Gaussian copula entropy method to calculate mutual information [2]. This method relies on the fact that mutual information between two random variables is independent of their marginal distributions and only depends on the type of copula used (a multidimensional distribution where each marginal distribution is uniform).

The actual MI was compared to its corresponding values computed on the time-shifted signals for assessing the statistical significance of the computed information association between the calcium signal and the behavioral variable. Additionally, we devised a technique for gauging the strength of the coupling effect. This involved normalizing the mutual information between the fluorescence signal and the behavior with the entropy value of both variables, previously calculated as random variables. Importantly, the approach outlined earlier is effective for analyzing continuous variables such as calcium signal and animal speed, as well as pairs of continuous and discrete variables such as calcium signal and the presence or absence of grooming.

The analysis of calcium signals recorded from the CA1 region of the hippocampus revealed neuronal specializations related to the animal’s external environment, such as place cells, and specializations related to its behavioral activities, including neurons activated during running, rearing, and freezing. Some neurons selectively activated in response to discrete parameters included the animal’s location within the arena (center, walls, and corners) and its speed (rest, slow, and fast). A total of 781 specializations were detected across 472 neurons throughout all four sessions of the experiment. Notably, a single neuron could have several specializations. However, more than half (55%) of the neurons were found to have only one specialization.

Genes & Cells. 2023;18(4):882-885
pages 882-885 views

Dynamics of two neuron-like generators with memristive connection

Bolshakov D.I., Mishchenko M.A., Belov A.I., Matrosov V.V., Mikhaylov A.N.

Abstract

At present one of the most urgent tasks of interdisciplinary science is the design and research of neuromorphic devices. Such devices are most often used to create systems for processing various kinds of information with algorithms similar to the data processing algorithms of the human brain or the brain of animals. The development of such neuromorphic electronics will allow computing devices and information processing systems to be built based on new principles and with a high level of parallelism [1].

Neuromorphic devices require the development of electronic components: neurons and synapses.

The paper [2] proposed a phase-locked loop system with a bandpass filter in the control circuit. A more detailed study of the mathematical model of such a system has shown missing equilibrium states corresponding to the synchronization mode of the phase-locked loop system, but there are self-oscillating modes of varying complexity. Self-oscillations observed in such system are similar to spike and burst oscillation of the neuron’s membrane potential.

Hardware implementation [3] of the considered neuron like generator in the form of an electronic device demonstrated the possibility of reproducing the same dynamic modes as in the mathematical model [2].

A fundamental disadvantage of the proposed model [2] and its experimental implementation [3] – the absence of an excitable mode (by excitable we mean a dynamic system with a stable equilibrium state and a periodic pseudoorbit of large amplitude, passing in the vicinity of the equilibrium state), when pulse generation would only respond to external disturbance. At the same time, the vast majority of brain neurons are in the excitable subthreshold mode, and their generation is primarily caused by presence of multiple connection.

One of the tasks of this work was to modification the existing model of the neuron-like generator in order to preserve the known dynamics and add a mode of excited oscillator.

While solving this problem, a modification of the neuron like generator based on the phase-locked loop system with a band-pass filter in the control circuit was proposed and implemented as an electronic circuit. The modification eliminates the basic drawback of the initial model – inability to work in the excitable mode. The new dynamic mode with the absence of self-oscillations was obtained by adding an electronically controlled switch between the low- and high-pass filters in the control loop.

Existence of the excitable mode and the existence of previously known self-oscillating modes of varying complexity was demonstrated experimentally: spike, burst, and chaotic modes was confirmed [4].

Another task in this work was to explore the dynamics of two neuron-like generators with memristive coupling.

A second-order memristor model based on Chua's memristor was used as a model of synaptic connection.

When solving this problem, nonlinear frequency dependences of the conductivity of the memristive element were found. This dependence has the same character for self-oscillating modes of varying complexity: spike and burst.

In addition, it was demonstrated synchronization of two neuron-like generators connected through a memristive element. Synchronization of two coupled neuron-like generators is interim in nature and strongly depends on the current state of the memristive element [5].

Genes & Cells. 2023;18(4):790-793
pages 790-793 views

Principles of analog neuromorphic computing: from components to systems and algorithms

Demin V.A., Emelyanov A.V., Nikiruy K.E., Surazhevsky I.A., Sitnikov A.V., Rylkov V.V., Kashkarov P.K., Kovalchuk M.V.

Abstract

This report presents the current state of affairs in the implementation of artificial intelligence hardware accelerators based on practically successful neural network algorithms of the first and second generations based on formal artificial neural networks (ANNs). The shortcomings of existing solutions are noted and ways to overcome them using analog neuromorphic architectures are outlined.

The latter are created on the principles of the structuring and functioning of a living nervous system, using artificial neurons and models of synaptic contacts - the so–called memristors, electrically rewritable nanoscale elements of non-volatile memory [1-3]. With the use of these elements, it is possible to significantly increase the performance and energy efficiency of algorithm accelerators based on the ANNs [4-6], as well as the formation of promising computing systems based on bioplausible 3rd generation neural network algorithms - Spiking Neural Networks (SNNs) [7-9].

The original method of substantiating the optimal rules for local tuning SNNs with frequency encoding and the possibility of their implementation in the form of the Spike-Timing-Dependent Plasicity (STDP) are discussed [10]. The results of SNN learning stability to a variability of analog memristors, as well as the use of noise as a constructive factor in the fine-tuning and maintenance of SNN memristive weights are demonstrated [7, 11].

Also, approaches to the implementation of local plasticity rules with dopamine-like modulation as a type of SNN reinforcement learning are discussed. The latter is necessary for the formation of imitative "needs" of an agent in the process of its autonomous functioning [12, 13, 14]. The first results on the creation of a prototype of a memristive implantable neuroprosthesis of the motor activity are considered [15, 16].

Finally, possible hardware solutions for both neuronal elements and synaptic connections based on suitable memristive devices are demonstrated. The concept and first results on the creation of an analog neuromorphic computing system based on the above components are presented.

Thus, an attempt is made to systematize the existing and original methods of implementing energy-efficient and compact analog neuromorphic computing systems for real-time and life-learning artificial intelligence.

Genes & Cells. 2023;18(4):794-797
pages 794-797 views

Dynamics of space coding by mouse hippocampal CA1 field neurons in a free navigation task in different environments

Sotskov V.P., Plyusnin V.V., Dokukin N.V., Pospelov N.A., Anokhin K.V.

Abstract

The development of persistent cognitive adaptations in neurons is a key area of focus in contemporary neuroscience. A significant illustration of such adaptations occurs through spatial specializations in place cells with one or more receptive fields that are sensitive to spatial cues (place fields) [1].

In prior research, we extensively examined the dynamics of place field formation in granular layer neurons of the CA1 field in the hippocampus of mice during arbitrary free navigation in a circular track [2]. As the primary factor for maintaining stability in the spatial specialization of place cells, we have introduced dynamic selectivity. This approach enables us to monitor the latency of each place field’s formation and the selectivity dynamics of place cells to their respective fields throughout one or multiple shooting sessions. However, in one-dimensional settings, like the circular track referenced previously, place fields can demonstrate direction-specificity with regards to animal movement [3]. This factor considerably complicates the analysis for arbitrary movement trajectories of animals. In this study, additional experiments were conducted to capture the neural activity of mice in two-dimensional environments. The experiments were conducted in both a rectangular field with objects and a circular arena with a varying number of obstacles.

In this paper, we conduct a comparative analysis to examine the critical parameters governing the development of spatial specializations in a one-dimensional circle track and two-dimensional arenas. These parameters include specialization latency, average dynamic selectivity, initial increase in selectivity, and the proportion of “immediate” place fields that remain stable from the first animal visit. The study discovered that the mean selectivity of the place fields escalated in each session, with higher rates observed in subsequent sessions versus the initial session in a novel setting. Additionally, there was a significant occurrence of “immediate” place fields, representing 11% of all place fields in the rounded arena with obstacles, and 25% of all place fields in the circular track.

Additionally, a population analysis of neural activity was performed on the first session in a circular track and a round arena with obstacles. Using Laplace eigenmaps for the dimension reduction of population vectors, the trajectory of animals was reconstructed, and the accuracy of this reconstruction agreed with the average dynamic selectivity for each animal included in the analysis. Thus, dynamic selectivity was verified as a measure of the spatial coding quality of the entire registered population of neurons.

Genes & Cells. 2023;18(4):778-781
pages 778-781 views

Classification of dendritic spiking neurons’ shape through clustering and machine learning techniques

Vasiliev P.I., Smirnova D.S., Chukanov V.S., Bezprozvannyi I.B., Pchitskaya E.I.

Abstract

A synapse is a specialized region between two adjacent neurons that allows information to be transmitted from one cell to another. The formation of these connections and transfer of signals via electrical impulses is a critical characteristic of neuronal cells. The synapse is formed by the axonal bouton on the axon side and the dendritic spine, a specialized outgrowth of the dendritic membrane, on the dendrite side. Dendritic spines exhibit diverse shapes and sizes, differing significantly across brain regions, cell categories, and animal species. Changes in dendritic spine morphology occur in neurodevelopmental and neurodegenerative ailments while also responding to external stimuli. Although deemed to facilitate synaptic plasticity, further investigation is essential for establishing the correlation between spine construction and its function. To address the issue in modern neurobiology of characterizing synapse morphology on 3D neuron images, the development of effective analytical methods is necessary. Our team has produced an open-source software solution for precise dendritic spine segmentation using 3D dendrite images. This software calculates the 10 most widely used 3D-adapted morphological features [1, 2] and enables classification and clustering of dendritic spine data sets to determine their shape. In addition to numerical features for describing the shape of a dendritic spine, researchers proposed using a histogram of chord lengths, known as the chord length distribution histogram (CLDH). This involves generating a set of random chords within the dendritic spine’s volume, connecting its outer boundaries and forming a histogram. By setting n=30 000, the probabilistic fluctuations of the histogram become insignificant. The derived metrics were then used for clustering and classifying the dataset.

Classification based on predetermined morphological groups is a frequently used approach for analyzing the morphology of dendritic spines. This method involves categorizing spines into established groups such as thin, mushroom, and stubby. Experimenters generally perform classification in a semi-automated manner, leading to considerable error. We have created a spine categorization tool using a machine learning algorithm. The tool classifies spines based on a consensus reached by eight experts who manually labeled the training dataset. The accuracy of this method surpasses 77% when using classical morphological features and is comparable to expert labeling. The implementation of this approach reduces classification bias and complexity.

Recent studies, including those using live microscopy in vitro and in vivo, indicate that dendritic spine shapes exhibit a continuum rather than distinct categories [3]. Therefore, it is essential to establish a dependable methodology for evaluating and examining the morphology of dendritic spines. We have created a clustering tool that establishes both the number of groups and their content based on data, rather than the experimenter’s discretion. This tool leverages the k-means and DBSCAN algorithms for its representation. Three clustering methods are presented to determine the number of clusters: the silhouette method, the elbow method, and a novel method, developed by the authors, based on the max class divergence criteria. The authors assume that cluster quality improves as clusters differ significantly in the number of mushroom/thin/stubby spine classes, as marked by experts. The benefit of this approach is that it considers the particular data type for clustering. Without this knowledge, assessing the quality of clustering is challenging. The use of the CLDH metric for clustering yielded a consistent and stable number of clusters (n=5) across all three methods described. These clusters contain dendritic spines that share similar shapes and have been validated by experts. In contrast, using classical metrics resulted in a variable cluster count ranging from n=4 to n=14. These findings suggest that the CLDH metric, with its complexity, provides enough information about synapse shape to enable precise clustering.

Genes & Cells. 2023;18(4):890-893
pages 890-893 views

Novel neuromorphic architectures based on crossbar arrays of (Co-Fe-B)x(LiNbO3)100−x nanocomposite memristors

Emelyanov A.V., Matsukatova A.N., Iliasov A.I., Демин V.A., Rylkov V.V.

Abstract

Memristor-based neuromorphic computing systems (NСSs) provide a fast, high computational and energy efficient approach to neural network (NN) training and solving cognitive problems (pattern recognition, big data processing, prediction, etc.) [1]. Memristors could be organized in large crossbar arrays to perform vector-matrix multiplication (VMM) in a natural one-step way by the weighted electrical current summation (according to the Ohm’s and Kirchhoff’s laws) [1]. In contrast, being the most massively parallel operation in NN learning and inference, VMM is extremely time- and energy-expensive in traditional von Neumann architectures. Owing to this difference, memristor-based NCSs are of high interest. Memristors have already been successfully implemented for diverse NCS realizations, and such schemes as multi-layer perceptron (MLP) [2], long short-term memory and others have been demonstrated. Most of these NCSs are usually trained by various types of gradient descent learning algorithm, the hardware realization of which is challenging due to unreliable cycle-to-cycle (c2c) and device-to-device (d2d) variations of memristive devices. Several approaches have been proposed to partially mitigate these problems, including reservoir computing [3] and fine feature engineering [4]. The general idea of such approaches is to reduce the number of required weights (i.e. memristors) compared with fully connected NNs. In this respect, such novel architectures as convolutional NN (CNN) and MLP-mixer are of high interest as they provide significant weight reduction without classification efficiency drop. Although CNN based on memristors was already demonstrated, different aspects of its realization (such as hybrid hardware-software co-design) have yet to be studied. MLP-mixer was realized only in software. Therefore, in this work we have studied the possibility of hardware realization of CNN and MLP-mixer networks based on crossbar arrays of memristors. For this purpose, we studied (Co-Fe-B)x(LiNbO3)100−x nanocomposite (CFB-LNO NC) memristors, which operate through a multifilamentary resistive switching (RS) mechanism, demonstrate high endurance, long retention and possess multilevel RS [5].

Crossbar array of memristors was fabricated using laser photolithography for patterning electrode buses and ion-beam sputtering on the original facility for active layer deposition (~10 nm thick LiNbO3 and ~290 nm thick CFB-LNO NC with x ≈10–25 at.%). Details of the fabrication process could be found elsewhere [5].

I-V curves of the fabricated memristors showed small c2c and d2d variations, plasticity with 16 different resistive states and endurance of more than 105 cycles. Using the nanocomposite based crossbar arrays, we implemented a hybrid CNN, consisting of a hardware feature extractor with one/two kernels and a software classifier. Additionally, we have demonstrated in simulation that the usage of the memristors under study in the accurately adapted MLP-Mixer architecture results in high classification accuracy that is resilient to memristive variations and stuck devices.

Genes & Cells. 2023;18(4):798-801
pages 798-801 views

Application of low-frequency photostimulation of parvalbumin interneurons to control epileptiform activity in the hippocampus

Trofimova A.M., Postnikova T.Y., Proskurina E.Y., Zaitsev A.V.

Abstract

Low-frequency electrical stimulation of the brain is used to suppress seizure activity in people with resistant forms of epilepsy [1]. Low-frequency stimulation of certain cell types, such as optogenetic activation of inhibitory parvalbumin (PV) interneurons, can be considered as a promising method for treatment of resistant forms of epilepsy [2]. In this work, we investigated the effect of PV interneuron photostimulation on epileptiform activity in the mouse hippocampus and entorhinal cortex.

This work was performed on 4-month-old B6.129P2-Pvalbtm1(cre)Arbr/J (JacksonLab) mice expressing Cre recombinase in PV interneurons. Adenoassociated viral construct (AAV9-EF1a-DIO-hChR2(H134R)-mCherry) carrying the canalorhodopsin type 2 gene (ChR2) was injected into the CA1 field of the hippocampus at the border with the entorhinal cortex using stereotactic coordinates (AP: -4 mm, ML: 3.5 mm, DV: -3.5 mm). Experiments were performed after 4-5 weeks on surviving brain slices. ChR2-expressing interneurons were activated by 470-nm wavelength light using a laser diode-fiber light source. Epileptiform activity was induced in the slice by application of pro-epileptic solution with 4-aminopyridine (100 μM). Biophysical properties of neurons were recorded by the patch-clamp method in a whole-cell configuration. Epileptiform activity in the slice was recorded by the field potential withdrawal method.

We determined the optimal frequency and duration of photostimulation affecting epileptiform activity in the hippocampus of mice. We tested how PV interneuron photostimulation affects pyramidal neurons in the hippocampus using the patch-clamp method. The following parameters were assessed: intensity, duration, and frequency of PV interneuron photostimulation under normal conditions. Thus, at low photostimulation frequency we observed synchronized responses of the pyramidal cells. And the optimal duration of photostimulation should not have exceeded 25 ms. Then we decided to check how the selected photostimulation parameters affect the seizure activity in the slice. For this purpose, we used the method of recording field potentials. In the CA1 field of the hippocampus, photostimulation with a frequency of 1 Hz and a light flash duration of 10 ms induced regular interictal activity. This induced interictal activity completely suppressed the occurrence of ictal discharges in the brain slice. After cessation of photostimulation, the frequency of intrinsic epileptic-like events in the CA1 field of the hippocampus decreased compared with the pre-stimulatory level.

We found that photostimulation of PV interneurons results in discharges in response to light turn off, indicating synchronous activation of pyramidal neurons. Low-frequency photostimulation of PV interneurons is more effective in modulating epileptiform activity in the CA1 field of the hippocampus. The use of low-frequency optogenetic stimulation of PV interneurons seems to be a promising approach in the control and suppression of seizure activity.

Genes & Cells. 2023;18(4):782-785
pages 782-785 views

Dynamics of oscillator populations globally coupled with distributed phase shifts

Smirnov L.A., Pikovsky A.S.

Abstract

Globally coupled populations of oscillators are exemplary models for synchronization and the emergence of collective modes. In many cases, the nature of the global coupling is predetermined by the setup. Nevertheless, the oscillators can possess distinct properties and intrinsic noise, leading to non-identity of the system elements. The variation in natural frequencies among the oscillators is the primary source of non-identity. This feature has already been extensively studied. Our research focuses on the impact of phase shift coupling disorder. These phase lags naturally occur where the global force must propagate to reach spatially distributed elements of the population.

First, we develop a phase model in the Kuramoto–Sakaguchi form for a group of quadratic integrate-and-fire neurons that are inter-connected by a synaptic current transmitted with different time delays. This occurs when each cell receives input from other cells of the ensemble with an inherent delay. From a mathematical perspective, a global force affects oscillators with different time delays, resulting in a spread of phase lags in the corresponding phase model. Assuming a weak interaction between system units, we transform to slow-varying phases and use the standard time-average procedure.

Secondly, we demonstrate that a distribution of phase shifts in coupling may result from local oscillator properties, particularly when synaptic coupling of neurons incorporates a “low-pass filter” of an incoming, globally averaged synaptic field. For this purpose, we examine the classic θ-neuron model, which is frequently used to analyze the collective dynamics of a Type I neuron population. Here, we posit that neurons interact through chemical synapses, with each corresponding synaptic current forcing on the neuron satisfying the relaxation equation while having an individual, disordered value of the relaxation constant. Under this assumption of weak coupling and through the use of multiple timescale analysis, we obtain the Kuramoto-Sakaguchi model of phase oscillators which exhibit distributed phase lags.

Next, we will consider the characteristics of the phase model. In the thermodynamic limit, the one-particle probability density function characterizes the continuum of phase oscillators. It evolves according to the continuity equation and possesses an exact solution in the Ott–Antonsen ansatz form at each α phase lag value. This manifold is attractive and represents a special ansatz for the expansion of a Poisson kernel in a Fourier series with respect to the phase variable, as demonstrated in other papers. Using this analytical approach, we obtain a low-dimensional depiction of the collective behavior of the system, which indicates that in the equations for macroscopic complex fields, the redefined order parameter Q(t,α) depends solely on the phase shifts α through the initial conditions. However, its dynamics remain independent. Using stability analyses in the linear approximation and reduced equations, we argue that during the dynamics process, the memory of the initial state is lost and Q(t,α)→Q(t). After Q(t) converges, the population dynamics with a distribution of phase shifts reduces to a single dynamical equation for the auxiliary order parameter Q(t), and the original order parameters are connected to it through circular moments of the phase shift distribution g(α).

All theoretical concepts are confirmed through numerical calculations performed directly within the oscillatory population models under consideration.

Genes & Cells. 2023;18(4):886-889
pages 886-889 views

Subthalamic nucleus neuronal activity entropy in the effective stimulation area in patients with Parkinson’s disease

Zakharov N.I., Belova E.M., Gamaleya A.A., Tomskiy A.A., Sedov A.S.

Abstract

One of the most successful and promising treatments for Parkinson’s disease today is deep brain stimulation (DBS). Accurate localization of the stimulation area is critical for the outcome of surgical DBS electrode implantation in the subthalamic nucleus (STN). To achieve this, microelectrode recording is used during surgery to precisely locate the STN borders [1], while test stimulations with a macroelectrode are widely employed to enhance DBS electrode placement accuracy. Thus, through testing multiple trajectories, the most clinically effective one can be selected. However, a comprehensive and reliable description of the specific single neuron activity associated with successful DBS electrode implantation is currently lacking.

A study was conducted using microelectrode recordings (MER) of single neuron activity in the STN of 21 Parkinson’s disease patients (UPDRS III off/on=46.9/12.8) to identify the neuronal activity features associated with the most favorable clinical outcome of test stimulation. A comparative analysis of 29 activity parameters, such as firing rate, oscillatory activity intensity in different frequency ranges (Oscores), coefficient of variation, burst, pause index, among others [2], was conducted for 618 identified neurons. In addition, the approach of computing the entropy of the change in interspike intervals (ISI) of the pulse sequence [3] and the process of identifying patterns of neural activity using hierarchical clustering [4] were implemented.

A comparative analysis of single neuron activity indicated notable (p <0.05) variations between disregarded and selected paths for the ultimate implantation of DBS electrode, solely relying on entropy parameters. Trajectories that delivered optimal test stimulation outcomes demonstrated reduced entropy of interspike intervals. Only the patterns of neuronal activity characterized by extended periods of steady firing and brief pauses, referred to as tonic and pause activity, respectively, were found to contribute to the difference in entropy between trajectories. In contrast, there were no statistically significant differences detected in entropy values associated with neurons demonstrating burst activity. Furthermore, we observed a positive and significant correlation between entropy values and the degree of improvement in disease presentation before and after administration of medication.

A comparative analysis was conducted on the activity of single neurons along different trajectories with varied test stimulation responses. The results showed a weak effectiveness of linear parameters in determining the optimal electrode insertion path for improved clinical outcomes. However, the use of non-linear activity parameters, specifically entropy, in single neurons effectively differentiated trajectories with significant versus absent/insignificant test stimulation results. Furthermore, the significance of entropy in establishing the basal ganglia functions and describing information transfer processes in movement control is emphasized by interpreting this parameter as a measure of uncertainty or unpredictability of the information system in relation to the findings of other studies [5].

Genes & Cells. 2023;18(4):894-897
pages 894-897 views

Design of a memristor-based neuron for spiking neural networks

Ostrovskii V.Y., Druzhina O.S., Kamal O., Karimov T.I., Butusov D.N.

Abstract

The primary objective of neuromorphic system design is to surpass limitations in energy efficiency and scaling of classical von Neumann computing systems, through the emulation of animals’ nervous systems. This is achieved by conducting calculations in memory and encoding information in impulse signals, ultimately leading to enhanced adaptability. Adhering to these principles allows for improved energy efficiency and computational speed when solving machine learning problems, encompassing biomedical applications, embedded systems, and cyber-physical systems. Functional blocks modeling the main elements of the central nervous system, namely neurons and synapses, offer an advantage in implementing learning on a chip. The use of memristive electronic components, capable of altering their resistance based on the charge flowing through them, opens new doors for hardware implementation of neuromorphic systems. These devices offer advantages over conventional transistor electronics with respect to power consumption, component density, and performance. To achieve optimal results, the architecture of neuromorphic systems should be optimized at the device level.

Memristive components are utilized to create neurons and synapses. This thesis is specifically focused on producing memristive neuron-like spike signal generators. Previously, memristive neurons were crafted using a locally active element comprised of vanadium dioxide VO2, which incorporated a negative differential resistance section of the IV-curve. One of the recent advancements in this field is a spiking neuron with frequency adaptation [1]. Its drawbacks, however, involve separating the memristive and locally active elements physically, resulting in higher energy consumption and decreased integration quality. In [2], models of memristive neurons with minimal complexity are introduced, which incorporate the Leaky Integrate-and-Fire principle. However, the circuits presented require the application of negative voltage pulses to a DC battery to reset the memristor to its initial high-resistance state. This limitation restricts its sphere of application in neuromorphic systems. This paper proposes a model of a neuron that overcomes these limitations by using the negative differential resistance of the memristor to generate spikes, along with integrating supplementary circuit components to sustain the resistive switching cycles of the memristor.

The neuron model under consideration is implemented using the NI Multisim 14.2 SPICE environment and has been verified in the NI LabVIEW 2022 tool environment. The equations of the modified model of the generalized mean metastable switch of the memristor with self-directed channel [3] represent the current in the memristor branch of the neuron equivalent circuit. The simplicity of the equivalent circuitry of the neuron is attained by merging all the nonlinear features necessary for spike generation into one memristor model. The experimental phase of the study employed obtainable memristors from Knowm Corporation and the laboratory prototyping platform NI ELVIS III. The investigation of the proposed neuron model was accomplished through the application of sinusoidal and rectangular input signals. The refractory time of the neuron model was calculated.

The chosen stack of computer simulation and semi-natural modeling technologies is applied within the research-driven design concept of electronic devices. This approach considers the importance of refining the properties and identification of the design object or its components during the development cycle.

Genes & Cells. 2023;18(4):827-830
pages 827-830 views

Control of nitrogen defects in carbon nanotubes for self-powered memristive systems

Il’in O.I., Homlenko D.N., Khubezhov S.A., Rudyk N.N., Il’ina M.V.

Abstract

Recent studies show that the additional introduction of heteroatoms into the structure of CNTs makes it possible to change their electronic and physical properties [1]. Of great interest is the process of doping CNTs with nitrogen atoms [2]. The introduction of nitrogen defects into a lattice of carbon atoms makes it possible to modify the CNT structure up to the demonstration of anomalous properties that are not appropriate for this material [3]. It has been shown that multi-walled N-CNTs can exhibit memristive and piezoelectric properties [4].

The parameters of CNTs during synthesis can be controlled by the plasma enhanced chemical vapor deposition (PECVD) method. The addition of ammonia (NH3) to the carbonaceous gas in the PECVD process allows CNTs to be doped directly during growth. At the same time, the dopant concentration and the type of nitrogen defects have a significant effect on the properties of CNTs. The memristive properties of CNTs have already been sufficiently studied [5], however, for their application in self-powered systems, additional studies of the parameters of the piezoelectric module of N-CNTs are required. The aim of this work is to study the effect of ammonia flow on the concentration, type of nitrogen defects, and the value of the piezoelectric modulus during growth of CNTs by the PECVD.

Silicon (100) substrates were used as samples with films of a buffer (Mo, 100 nm) sublayer and a catalytic layer (Ni, 15 nm). CNTs were grown at a temperature of 550 °C in an atmosphere of acetylene (C2H2, 35 sccm) and NH3. The C2H2 flow was kept constant, while the NH3 flow was changed in the C2H2:NH3 ratio from 1:1 to 1:10.

Based on the obtained SEM images, it was found that with an increase in the ratio of C2H2:NH3 flowes, an increase in the density of nanotubes in the array were observed. This occurs due to more active growth of N-CNTs on small nickel catalytic centers due to the accelerated process of hydrogen desorption and its binding with ions in ammonia plasma, which leads to an increase in the growth rate of nanotubes on smaller catalytic centers. Thus, the area of the catalytic center is one of the limiting factors of the growth rate and allows one to control the aspect ratio and density of CNTs in the array. An analysis of the XPS spectra showed that with an increase in the ratio of C2H2:NH3 flows from 1:1 to 1:10, a nonlinear change in the concentration of the nitrogen dopant in N-CNTs from 8.4 to 12 at % is also observed. This led to a nonlinear change in the piezoelectric modulus of nanotubes from 8.7 to 20.6 pm/V and a change in their memristive properties. It has been established that an increase in the concentration of doping nitrogen leads to an increase in the piezoelectric modulus of N-CNTs, which is the source of the memristive effect. The obtained results can be used in the development of energy-efficient piezoelectric nanogenerators based on an array of vertically aligned N-CNTs for autonomous memristive systems.

Genes & Cells. 2023;18(4):806-809
pages 806-809 views

A positive allosteric modulator of TRPC6 promotes neuroprotective effects in vitro

Zernov N.I., Melentieva D.M., Ghamaryan V.S., Makichyan A.T., Hunanyan L.S., Popugaeva E.A.

Abstract

Alzheimer’s disease is a neurodegenerative disorder and the primary cause of dementia among elderly individuals. Unfortunately, there is no known cure for Alzheimer’s disease. Recently, a TRPC6-mediated intracellular signaling pathway was discovered, which plays a vital role in memory formation by regulating dendritic spine stability. Knockdown of TRPC6 expression was found to prevent store-operated calcium entry. The overexpression of TRPC6 or its pharmacological activation restores store-operated calcium entry in hippocampal neurons affected by Alzheimer’s disease [1, 2]. TRPC6 overexpression rescues mushroom spine loss in presenilin and APP knock-in mouse models of Alzheimer’s disease [1] and protects neurons from ischemic brain damage. Mice that overexpress TRPC6 in the brain exhibit improved cognitive function and increased excitatory synapse formation. These findings propose TRPC6 as a promising molecular target for the treatment of synaptic deficiency. We recently demonstrated that compound 51164 (N-(2-chlorophenyl)-2-(4-phenylpiperazine-1-yl) acetamide), a piperazine derivative, enhances TRPC6 channels and induces an upregulation of postsynaptic neuronal store-operated calcium entry. Furthermore, it increases mushroom spine percentage and recovers synaptic plasticity in mouse models of Alzheimer’s disease that have an amyloidogenic nature [2]. However, additional investigations of 51164 have demonstrated that the compound is unstable in plasma and cannot penetrate the blood-brain barrier (unpublished data). As such, the aim of this study is to discover a new piperazine derivative that functions as a positive modulator for TRPC6-specific and showcases neuroprotective qualities.

In this study, we present in silico and in vitro investigations of a novel TRPC6 specific modulator. Based on our in silico research, we narrowed down the selection to 14 compounds through the piperazine derivative 51164 that met all drug-lead likeness criteria and showed a high affinity for the active center of TRPC6. Calcium imaging technique was usedto establish that the compound z12_30 triggered the activation of TRPC6 but not the structurally linked TRPC3 channel. In addition, a molecular dynamics approach revealed that z12_30 forms a stable complex with the TRPC6 active site. As a result, z12_30 was selected as the lead compound for further investigation. Studies showed that z12_30 safeguards hippocampal mushroom spines from amyloid toxicity in vitro and effectively restores synaptic plasticity in brain slices from aged 5xFAD mice. Preclinical trials demonstrate that z12_30 remains stable in both human and mouse plasma samples.

We suggest that z12_30 is a promising prototype of a TRPC6-selective drug suitable for treating synaptic deficiency in hippocampal neurons affected by Alzheimer’s disease.

Genes & Cells. 2023;18(4):694-696
pages 694-696 views

Development of a microelectrode for simultaneous in vivo calcium and electrophysiological recording of hippocampal neuronal activity

Erofeev A.I., Vinokurov E.K., Vlasova O.L., Bezprozvannyi I.B.

Abstract

Calcium (Ca2+) imaging is a commonly utilized neuroscience technique for in vivo recording of neuronal activity. It involves the optical measurement of calcium concentration using genetically encoded calcium indicators (GECI) [1]. However, the kinetics of changes in the fluorescence of GECI are relatively slow and limited by the biophysics of the calcium binding [2]. In response to single action potentials (APs) in pyramidal neurons, most of the widely used GECI have a fluorescence half-life of approximately 100 ms [3]. As a result, GECI cannot provide complete information about the dynamics of neural ensembles. To address this issue, new variants of GECI, such as jGCaMP7 [3], and jGCaMP8 [4], have been developed, or genetically encoded voltage indicators (GEVI), such as JEDI-2P [5], have been utilized. However, the speed of GECI or GEVI is still lower than that of electrophysiological registration methods. Thus, we have designed a microelectrode that can be utilized with a gradient lens for in vivo calcium imaging with a miniscope.

The miniscope is a miniature microscope for single photon epifluorescence Ca2+ imaging, which enables recording of neuronal activity in freely moving laboratory animals, unlike the traditionally used two-photon imaging technique. The miniscope utilizes gradient-index (GRIN) lenses that are implanted directly into the brain of a laboratory animal, instead of a conventional lens. The gradient lens is a transparent cylinder with a diameter of 1.8 mm and a length of 3.8 mm. To facilitate electrophysiological recording, we developed a microelectrode that can be aligned with a GRIN lens.

The microelectrode is a three-layer structure consisting of: 1) a polyimide film, 2) conductive copper tracks deposited through thermoforming, and 3) a polyimide film with cutouts for pads. On one side of the microelectrode, there are 12 gold-plated conductive contacts for registering local field potentials, while on the other side, a similar number of conductive tracks are present for connecting to a connector that transmits data to the processing board. The flexible microelectrode is wrapped around a gradient lens and fixed using thermoforming, after which it is implanted in the animal's brain.

Using the developed microelectrode, our aim is to perform a comparative analysis of the calcium and electrophysiological activity of hippocampal neurons in freely moving wild-type mice and in a mouse model of Alzheimer's disease. This study will enable the identification of any abnormalities in Alzheimer's disease at the level of neural ensembles and may suggest new treatment approaches or mechanisms for the development of the progressive memory loss pathology associated with this disease. We would like to express our gratitude to Anastasia Viktorovna Bolshakova for her administrative assistance, and to the staff of the Laboratory of Molecular Neurodegeneration for their invaluable help and advices.

Genes & Cells. 2023;18(4):802-805
pages 802-805 views

Development of a model to study visual categorization learning in chickens (Gallus gallus domesticus)

Diffine E.A., Tiunova A.A., Anokhin K.V.

Abstract

Categorization is a cognitive process that enables individuals to recognize similar yet distinct stimuli as equivalent [1–3]. To categorize an object, agents must identify key features of the new object by applying what they have learned from previous interactions with objects in that category [4]. Thus, categorization eliminates the need for the agent to repeatedly investigate each new object, thereby significantly expanding its adaptive capabilities. However, the nervous mechanisms that regulate this process are still not well understood. The primary aim of the present study is to establish an experimental behavioral model that will facilitate the investigation of the neurobiological mechanisms underlying visual categorization learning.

Chickens (Gallus gallus domesticus) were selected as a visual learning model. The study of categorization used the chickens’ innate tendency to peck at new small objects and remember their characteristics. For this purpose, a chick was placed in a cage that resembled a house cage with beads affixed to the floor. The “bead floor” consisted of more than 100 beads of different colors and food scattered throughout [5]. In the developed model, pecking solely on the beads was deemed inaccurate as opposed to pecking on the food. For training purposes, the chick was given 80 peckings and evaluated based on how many nibbles it used to create categories for “beads” vs. “food”. If the chick did not make at least 5 cues or was unable to make 80 cues within 10 minutes during the training session, it was excluded from the study.

First, the study investigated the effect of simultaneously presenting two new categories of “food” and “beads” on chicks’ behavior. The group that formed both “beads” and “new food” categories made more errors during the learning period, but performed as well as the group that only formed the “bead” category during the subsequent test. The study analyzed whether chicks could categorize objects (beads) of different sizes into a unified group. The chicks were trained with a floor of small beads and later tested with a different floor of larger beads. Results revealed that the chicks did not transfer their categorization ability from small to large beads. In the opposite scenario, where the chicks were presented with large beads during their training, and then offered small beads during the test, they refrained from pecking the smaller ones.

In the next stage, we examined how the object’s color affected the development of a novel category. The results showed that when the chicks were presented with the floor containing an additional color of beads, specifically yellow, they made the majority of errors while pecking the “new type of beads”. Then, the study evaluated whether the chicks could transition the classification of “beads” between color set № 1 (blue, pink, green, yellow, and black silver) and color set № 2 (light green, beige, blue, red, gold, and white), and vice versa. The findings indicate that the chickens do form the categories of “beads” in both versions of the task, and after training, do not peck at beads of the new color. Chickens are capable of categorizing “beads” and “food”, and can generalize within these categories.

Thus, this experimental study established the formation of categories in chickens during fast learning. Future application of the model will enable the investigation of the underlying neurobiological mechanisms in categorization learning.

Genes & Cells. 2023;18(4):702-705
pages 702-705 views

Neuroplasicity and the developmental dyslexia intervention

Dorofeeva S.V.

Abstract

A growing body of literature suggests that timing plays a critical role in neuroplasticity processes and the molecular mechanisms necessary for learning and memory [1, 2]. Of particular significance to remedial education is identifying the time parameters for primary stimulation that are necessary and sufficient, the time frame relevant for transitioning to long-term memory, and the appropriate periods for restimulation. The translation of short-term stimulation into long-term memory is regulated by diverse processes that are mechanistically distinct and activated by synaptic activity [1], while also relying on protein and glycoprotein synthesis [3] and myelination processes.

The current research explores the potential benefits of incorporating neuroscience research on the timing of neuroplasticity mechanisms in designing intervention programs for individuals with developmental dyslexia, specifically focusing on enhancing cognitive functions and skills crucial for reading. Based on available evidence, we have determined optimal training and break time periods for a 10-year-old child with developmental dyslexia resulting from multiple deficits.

During the 21-day intervention program, 12 training sessions were conducted each day, commencing at 8 or 9 am and held hourly thereafter. Each session comprised a brief, targeted training exercise ranging from 3 to 7 minutes, depending on the child’s aptitude, followed by a playing session or computer game lasting up to 15 minutes. A 40-minute break followed each session. Brief training sessions were required due to the swift exhaustion of the subject child. The sessions were selected based on the evidence that brief stimulation can still result in a high level of CERB phosphorylation, even if it lasts only a few minutes [4]. Playing sessions were necessary for supporting the child’s motivation throughout the lengthy and intensive intervention program, and the activities performed during these sessions were pertinent to developing specific skills. The duration of the breaks was determined by evidence indicating the time required for primary memory consolidation processes and protein synthesis necessary for long-lasting synaptic plasticity. We have made an effort to eliminate any potential sources of emotional engagement or significant new information during breaks, allowing the initial stage of memory consolidation to occur without any unnecessary disruption. During the training sessions, various tasks were used to target specific types of processing such as phonological, visual, speech, and multimodal processing (e.g., visual-motor, audio-visual, or reading). Each session exclusively focused on one type of exercise. In our prior study [5], we discussed the linguistic aspects of the program and the exercises employed.

Significant progress was achieved as a result of the 21-day intervention, surpassing the progress achieved in three years of schooling and during traditional remediation programs with speech therapists that lasted 1–3 sessions per week for 40–120 minutes. Following the intensive intervention, supportive training was continued for one year while considering the crucial timing for neuroplasticity. Afterward, the child reached a normative level of reading, and the effect was maintained throughout their entire period of school education. Based on the timing of neuroplasticity processes, this is the first intensive intervention program experience for dyslexia that we are aware of.

Intensive remediation programs, based on relevant findings regarding the mechanisms of memory consolidation, may enhance neural memory trace reinforcement. However, further research is necessary to optimize the timing and length of sessions and identify the most effective combination of linguistic and neurophysiological aspects for intervention.

Genes & Cells. 2023;18(4):706-709
pages 706-709 views

Engram for the complex signals in the mouse brain: distinct neuronal ensembles for compound conditioning stimulus and its components

Ivashkina O.I., Toropova K.A., Anokhin K.V.

Abstract

Natural learning involves multiple sensory modalities receiving complex stimuli. Elemental learning theories suggest separate encoding and association of each component in a compound signal. Configural theories predict the formation of a representation of the entire complex signal, which is associated with the second event. We developed a mouse model of fear conditioning to a compound tone-light cue or its separate components to test these alternative theories. First, we investigated the memory dynamics of compound cues and their individual components and discovered that they mature at varying times following conditioning. We demonstrated that the memory of the components matures at different intervals after training: memory of the auditory stimulus and the auditory component of CCS is demonstrated behaviorally right after training, whereas memory of the light stimulus and the light component of CCS matures within three days. The memory of CCS, its components, and discrete conditioned stimuli persists for an extended period, up to one month. A similar dissociation was observed in extinction experiments, revealing that the extinction of memory for one CCS component did not affect the memory of the other component when the extinction procedure began a day after training. In addition, when the extinction procedure began seven days after training, while the memory was fully mature, the extinction of one component of contextual conditional stimuli led to the extinction of the other component. Next, c-Fos imaging was conducted to examine cellular activity across multiple brain regions, including the frontal, prelimbic, cingulate, retrosplenial, parietal, primary and secondary visual, primary and secondary auditory cortices, as well as the hippocampus and amygdala. This examination was performed following conditioning using either the entire compound cue or its individual components. We discovered different cortical activation patterns between compound-cue and single-cue conditioning. Conditioning to the compound cue activated prelimbic and frontal associative cortices, whereas single cues did not. Third, we demonstrated that retrieval of memory only through the entire compound cue, and not through single cues, activated the parietal cortex, primary visual cortex, mediolateral secondary visual cortices, and hippocampal CA1. Fourth, through in vivo two-photon imaging, we examined retrieval-induced c-Fos expression in the parietal cortex of fos-EGFP transgenic mice and identified at least three distinct neuronal populations with differential response specificity to the compound signal and its components. Taken together, our data suggest that intricate signals have the potential to establish both integral and elemental neuronal representations. These representations can be used separately in behavior and have different long-term memory dynamics.

Genes & Cells. 2023;18(4):710-712
pages 710-712 views

“Time windows” for switching kinase-phosphatase balance during inhibition of long-term potentialization of hippocampal synapses by amyloid aggregates

Maltsev A.V., Balaban P.M.

Abstract

Phosphorylation and dephosphorylation of target proteins are crucial mechanisms for regulating cellular functions. These processes are mediated by protein kinases and phosphatases, respectively. Changes in the kinase-phosphatase balance, known as K-P balance, are well documented during the progression of amyloidosis. The overactivation of kinases or the failed activity of phosphatases is the root cause of hyperphosphorylation of the structural tau protein leading to the formation of fibrillar tangles. The data suggests bidirectional regulation of phosphorylation/dephosphorylation processes during amyloidosis-related states is consistent across different animal models and many protocols. Therefore, it is crucial to identify the “time windows” during which the K-P balance can be altered.

Field excitatory postsynaptic potentials (fEPSPs) were recorded from the stratum radiatum located in area CA1. Baseline synaptic responses were generated by paired-pulse stimulation of the Schaffer collaterals at a frequency of 0.033 Hz using a bipolar electrode. Four 100 Hz trains were delivered 5 minutes apart to induce long-term potentiation (LTP). Serine and threonine phosphatase activity was assessed using a non-radioactive molybdate dye-based assay kit (Promega, USA) based on the manufacturer’s instructions. The procedure relies on the creation of a colored complex of phosphates with a dye containing molybdenum, followed by the measurement of optical densities of supernatants that were incubated against a control at 640 nm. The activity of protein kinase C (PKC) will be measured directly via an enzyme immunoassay (ELISA) on a plate reader, using a commercial Abcam kit (Abcam, USA). The technique relies on the binding of engineered antibodies to activated PKC, and optical densities of incubated supernatants are measured against a control at 450 nm.

In this study, we identified two important time periods during the application of amyloid Aβ25-35 aggregates that affect plasticity in the CA3-CA1 synapses. Specifically, the PKC isoforms are activated during early neurochemical events (0–20 min of incubation of slices in the presence of Aβ25–35 aggregates). If the hippocampal CA3-CA1 synapses are tetanized during this period, we did not observe any inhibition of synaptic plasticity either in the early or late phase development of LTP. Biochemical analysis indicates that hippocampal slices pretreated with Aβ25–35 aggregates for 20–30 minutes exhibit increased PKC activity, whereas no such effect is observed after one hour of incubation. Simultaneously, the hour-long incubation of hippocampal slices in the presence of Aβ25–35 aggregates resulted in the disappearance of long-term potentiation and the return of responses to the pre-tetanic level of synaptic transmission during the late-LTP phase (3 hours after tetanus induction). Inhibitory analysis revealed that an abrupt rise in the activity of stress-induced phosphatase 1α (PP1α) interrupts kinase-mediated signals after LTP induction, leading to the suppression of fEPSPs in the CA3-CA1 synapses. These findings suggest that exposing hippocampal slices to Aβ25–35 aggregates for 20–30 minutes may alter the K-P balance and enhance kinase activity through PKC isoform induction. The majority of PKC isoforms exhibit a tendency to desensitize following activation. Probably, the depletion of the pool of PKCs, which occurs within the first 20–30 minutes after incubation of hippocampal slices with Aβ25–35 aggregates, along with a notable elevation in the activity of stress-induced phosphatase PP1α, is accountable for the second time window for switching the kinase-phosphatase balance, stabilized after an hour of incubation of hippocampal slices in the presence of Aβ25–35 aggregates.

Incubating hippocampal slices with Aβ25–35 aggregates (for 0–30 minutes) leads to a shift in the kinase-phosphatase balance towards the activity of kinases due to the activation of PKC isoforms. After this stage, there is a significant rise in phosphatase activity, resulting from the induction of PP1α. This induces a shift in the balance between kinase and phosphatase towards dephosphorylation processes.

Genes & Cells. 2023;18(4):713-715
pages 713-715 views

Extreme synchronization events in a model neuron-astrocyte network

Tsybina Y.A., Kastalskiy I.A., Andreev A.V., Frolov N.S., Hramov A.E., Gordleeva S.Y.

Abstract

The examination of synchronization in neural networks is essential to comprehend how the brain functions in normal and pathological states. The synchronization of signals between groups of neurons is paramount for brain activity and associated with various brain functions, including memory, movement, sleep, and attention. However, a balance between synchronization and desynchronization must be maintained for proper brain function. During epilepsy, spontaneous transitions occur between two states of brain activity: synchronous and asynchronous. These transitions can be induced by various factors, including imbalances in chemical signals, changes in network activity, and other mechanisms that are not yet fully understood. Understanding these spontaneous transitions and the underlying mechanisms is a crucial aspect of epilepsy research. It will lead to the development of novel treatment approaches and strategies for inhibiting synchronized activity and averting seizure episodes.

A neuron-astrocyte network model was constructed in this study, which displayed spontaneous and induced transitions between asynchronous and synchronous states triggered by external stimuli. The network model utilized 1000 Izhikevich neurons [1] interconnected with excitatory synapses and organized according to a scale-free graph. Each neuron had a bidirectional interaction with a single astrocyte, resulting in a total of 1000 astrocytes in the network. The Ullah model [2] simulated intracellular calcium ion concentration dynamics in a single astrocyte. The action potential generated by a neuron causes the release of neurotransmitters into the synaptic cleft, which triggers the corresponding astrocyte to release calcium ions from its endoplasmic reticulum into the cytoplasm, generating a calcium impulse. The calcium pulse within the astrocyte led to the release of a gliotransmitter. This gliotransmitter has the potential to regulate the synaptic transmission efficiency of both presynaptic and postsynaptic terminals associated with the respective astrocyte. The study simulated the effect of astrocytic suppression on neurotransmitter release. The strength of the synaptic input connection to the neuron that interacts with the astrocyte decreased in proportion to the amplitude of the calcium pulse in the astrocyte.

Within the context of the implemented model study, we analyzed the global order parameter’s dependence on the maximum synaptic weight. Our bifurcation analysis showed the presence of hysteresis within a specific range of parameter values for connection weight maximums. In this range, the system under consideration is capable of displaying both synchronous and asynchronous regimes. However, an asynchronous regime was the only observed outcome below the lower boundary of the range, and only a synchronous regime was observed above the upper boundary. A statistical analysis was carried out on the duration of the model’s asynchronous states, specifically for the parameter value of the maximum synaptic weight near the upper boundary of the hysteresis region. The network dynamics were simulated for a prolonged period, and a histogram of the asynchronous state durations was plotted on a double logarithmic scale. The resulting data points were approximated using linear regression, which yielded a power-law exponent of –3/2.

Based on the obtained results, the neuron-astrocyte network can exhibit spontaneous transitions between two states: synchronous and asynchronous, similar to pathological processes in the brain. More precisely, the power-law exponent of –3/2 aligns with values discovered in experimental recordings of epileptic activity in rodent brains [3–5]. Detailed modeling of biophysical processes revealed the spontaneous emergence of global order in the network induced by noise. Astrocytic effects mediated the disruption of synchronization in the neural network by reducing the efficiency of synaptic transmission.

Genes & Cells. 2023;18(4):835-838
pages 835-838 views

Synchronization in the two time-scale multiplex network with symplectic interactions

Laptyeva T., Jalan S., Ivanchenko M.

Abstract

Mounting experimental evidence suggests a significant role for glial cells, particularly astrocytes, in shaping neuronal activity in both cell cultures and the brain, driving interest in exploring multi-component network dynamics [1]. These systems are modeled at varying levels of physiological detail, ranging from the Hodgkin-Huxley and Ullah models for neurons and astrocytes, respectively, to simple integrate-and-fire models or phase oscillators. Multiplex networks are commonly used to model generic dynamical phenomena, with an emphasis on network connectivity effects rather than the complexity of an individual cell’s behavior. This model is particularly advantageous due to its use of a two-layer network structure [2]. This structure accurately describes neural connectivity through the long-range random coupling, while glial cells interact locally through the diffusion of mediatory molecules. Furthermore, the oscillatory timescales between neural and glial cells differ by at least one order of magnitude.

In this context, the analysis of synchronization is a focal point as it is a crucial process that underpins information processing, decision making, and movement control in living neural systems [3]. Network topology is known to be essential, where all-to-all coupling and random Erdos–Renyi connectivity support the second-order phase transition to global phase coherence. In contrast, regular local coupling such as a 2D lattice only permits frequency and phase locking. Additionally, symplectic networks are capable of demonstrating a first-order phase transition, which is commonly referred to as “explosive” synchronization [4].

Recently, studies have shown that combining model neural (random) and glial (regular lattice) oscillatory layers can lead to a variety of outcomes [5]. Specifically, this combination was found to induce a second-order phase transition in the regular glial layer. In addition, synchronization in both layers may be preceded by desynchronization as the coupling between the layers becomes stronger.

Here we investigate synchronization in the multiplex neural-glial network, where the neural layer is symplectic and contains triadic interactions. We demonstrate that symplectic coupling in the neural layer can induce the first order transition in the regular (glial) layer. Additionally, we show that such a transition can be induced by strengthening the glial and interlayer coupling, even if the symplectic neural layer alone is below the synchronization threshold. Desynchronization in the neuronal layer, resulting from the moderated coupling to the glial layer, never occurs abruptly.

Genes & Cells. 2023;18(4):854-857
pages 854-857 views

Phase-locked states in a spiking neural network model with a context-dependent connectivity

Makovkin S.Y., Kastalsky I.A.

Abstract

The investigation of nonlinear processes in brain systems based on the oscillatory-wave approach is currently one of the most consequential domains of researching signal generation and processing mechanisms in the brain. These investigations may offer insights into various phenomena, such as the mechanism of associative memory, based on network models of biologically relevant neurons. A neural network architecture is proposed in this paper to solve applied problems, such as signal filtering, processing, and recognition of information images. Mechanisms for incomplete fragments were implemented, enabling the extraction and complete restoration of objects from memory. Each neuron in this neural network comprises a Hodgkin–Huxley biophysical model with a Mainen modification, the dynamics of which most plausibly reproduce the processes in the neural cells of the brain.

In the research on individual neurons, a constant external current with varying amplitudes was applied. The study determined the average periods of self-oscillations and identified parameter values for the Andronov–Hopf bifurcation. In particular, a stable limit cycle is created and destroyed depending on different scenarios when the bias current is increased or decreased. A stable limit cycle arises via the Andronov–Hopf bifurcation and is terminated via the saddle-node bifurcation on the cycle.

The study identified parameter regions whereby two neurons with excitatory and inhibitory synaptic connections synchronize. The neurons were all in self-oscillating mode and exhibited a stable limit cycle in the phase space.

A three-layer neural network was constructed, comprising of a reference neuron, sensory, control, and interneurons. The control and intermediate layer connections are arranged based on the Hebbian learning rule.

Areas of excitation for various types of neurons were found.

Experiments were conducted using the neural network architecture to distinguish binary information patterns encoded through signal phase. Excitatory and inhibitory synaptic connections were employed to encode the images stored in memory. The dynamic patterns are determined through the in-phase or anti-phase mode of phase locking, i.e., phase synchronization relative to the global rhythm. Before reaching the output layer of neurons, the signals are summed on the interneuron layer with the signal of the reference neuron, resulting in the filtering out of a certain information segment (in-phase or anti-phase). The network accurately recognized the patterns after parameter adjustments were made.

Areas of neuron phase synchronization were discovered, enabling control of network activity modes. The confirmation of the viability of phase capture and pattern recognition modes using Hodgkin–Huxley–Mainen neurons was achieved.

In total, as a result of the implementation of the spike neural network modeling project:

1) two-parameter diagrams of regions of neuronal excitation in various modes are calculated;

2) the effect of excitatory and inhibitory coupling in synaptic currents is used to represent the input signal;

3) an abstract mathematical algorithm for calculating the Hebbian matrix has been transferred to the implementation of synaptic currents between layers of neurons;

4) the effect of phase clusters is used to represent the output pattern;

5) the neurons’ topology in the visual-cerebral department served as a working model for recognizing graphic images through the Hopfield network on a spike neural network, using the Hebbian rule.

Genes & Cells. 2023;18(4):858-861
pages 858-861 views

Period addition cascade in a model of neuron-glial interaction

Barabash N.V.

Abstract

We analyze a system of four differential equations that describe the dynamics of a neuron-glial network using the mean field approximation [1, 2]:

(τ=–E+α ln(1+e1/α(JuxE+I0)),

=(1–x)/τDuxE,(1)

=U(y)–u/τF+U(y)(1–u)E,

=–y/τy+βσ(x),

where E(t) is the average activity, x(t) is the fraction of available neurotransmitter released into the synaptic gap with a probability u(t); y(t) is the fraction of the gliatransmitter released by the astrocyte. Sigmoidal functions U(y) and σ(x) correspond to changes in the base probability level u(t) during the release of the gliatransmitter and activation of astrocytes during neurotransmitter release, respectively. The input inhibitory current corresponds to a bifurcation parameter with a negative value of I0 <0, while the remaining parameters have positive and fixed values. The rest of the parameters are positive and fixed. For a detailed description of the model, including information on the type of functions and parameter values, refer to works [1, 2].

For a constant value of U(x)=const, the first three equations in system (1) represent the Tsodyks–Markram model, which explains the short-term synaptic plasticity phenomenon [1]. The model was enhanced with a fourth equation for y in [2], incorporating the influence of astrocytes via the concept of a tripartite synapse [3].

Model (1) illustrates a wide range of dynamic behavior including quiescence, regular tonic activity, and chaotic bursting activity. These behaviors correspond to various sets in the phase space, such as stable equilibrium states, limit cycles of period 1, limit cycles of any period n∈N, and chaotic attractors. Changing the I0 l parameter causes sets to bifurcate, resulting in the loss of stability of certain attractors and the emergence of others, leading to a shift in the dynamic regime. Therefore, in terms of dynamics, the conditions for bifurcation and the characteristics of the newly formed attractors are crucial.

In this presentation, we have obtained a series of numerical bifurcations in system (1) that correspond to the shift from tonic activity to burst activity, resulting in subsequent modifications to the bursts. Specifically, our findings demonstrate that an increase in the number of spikes per burst is determined by a period adding cascade where the aforementioned limit cycle of period n becomes unstable, allowing for a previously established stable cycle of period n+1 to occupy the position of the “main” attractor. This process culminates in the vanishing of the orbit with an endless period due to the saddle-node bifurcation of cycles, followed by the creation of a dependable cycle with a period of 1.

The main properties of the cascade were reproduced in our model one-dimensional piecewise-smooth map

z¯=1z6,                      for z < 0,μ1μ(z-1)6,for z > 0,

where z∈R1, μ is a bifurcational parameter. The map’s results suggest that an increase in current I0 i in model (1) may lead to the emergence and disappearance of quasi-strange attractors (quasi-attractors), implying chaotic behavior in connection with burst variation.

Genes & Cells. 2023;18(4):840-843
pages 840-843 views

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