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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 <rs <0.28 and –0.20 <rs <–0.40, respectively; 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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].

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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].

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.

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.

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.

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.

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].

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.

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.

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.

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.