The aim of the study was to investigate the relationship between the average size of the active synapse zone and the frequency of synapse-astrocyte membrane contact in the layers of the rat cerebral cortex. The material for the study included 40 μm frontal sections of the primary somatosensory cortex of five outbred male white rats. Astrocytes were labeled for TEM by incubating the sections with primary antibodies to the s100β protein and secondary antibodies conjugated with horseradish peroxidase, followed by label development in a reaction with DAB. Two hundred fifty images with a 25 000× magnification were made for each layer. The length of the synaptic cleft was measured in the images, and the number of synapses forming contact with the astrocyte membrane was counted. The proportion of chemical synapses making contact with the astrocytic membrane in the rat primary somatosensory cortex showed a direct relationship to the average synaptic cleft length only in layers one through four. The data obtained suggest that contact is formed by a combination of a random membrane encounter followed by selective fixation or repulsion of the astrocytic membrane by various factors, only partly determined by the size of the chemical synapse.

Background. Among all the new methods and approaches, virotherapy with oncolytic viruses, both in combination with immunotherapy and without it, shows high efficiency in various phases of clinical trials and good tolerance by patients.Aim. To study the sensitivity of some immortalized cancer cell lines to the R-92 strain of human reovirus with cell characteristics at the ultrastructural level.Materials and methods. The study was carried out on cell lines of HeLa, A549, U87MG. Cells were planted in an amount of 15 thousand per well of a 96-well plate and after adhesion, the virus was inoculated by adding a medium containing virus particles in 4 tenfold dilutions (approximately 10 9 –106 particles per ml). Next, the cells were cultured for 24 h, after which the number of living cells in the wells was determined indirectly using the methyl tetrazolium test, which was carried out according to standard methods. To study the ultrastructure of infected cells, cells were seeded into a T25 flask and inoculated with the virus at the maximum concentration. After 24 h of cultivation, the cells were fixed in a 2.5 % glutaraldehyde solution in phosphate buffer for 1 h, after which they were washed three times in phosphate buffer and samples were processed for TEM according to standard methods.Results. Diluting the virus 1000 times led to a decrease in the cytostatic effect in all three cultures to a level practically no different from the control. HeLa turned out to be the most sensitive culture to reovirus. In the experiment, the number of living cells decreased to 60.4 ± 10.2 % compared to the control during incubation with the maximum number of viral particles and to 63.7 ± 16.2 % with a tenfold dilution of the virus. This indicator was significantly lower than in the other two studied cultures under these cultivation conditions (p <0.001).In addition, at the maximum virus concentration, the A549 culture was less sensitive than the U87MG culture (p <0.01). At lower concentrations of viral particles, the average viability of the studied cell lines did not differ significantly from each other. Analysis of electron diffraction patterns showed that the virus successfully replicates in the cytoplasm of the studied cultures, but is not released from the cell, which is apparently due to the short incubation period. TEM also showed cell damage characteristic of apoptosis or necroptosis, uniformly expressed in all studied cultures.Conclusion. Cell lines A549, HeLa and U87MG, according to the results of the methyl tetrazolium test, demonstrate different sensitivity to the human reovirus strain P-92. The TEM picture of cells from infected cultures showed signs of the development of apoptosis or necroptosis.

Introduction. The contact point between an astrocyte and a chemical synapse is the location of the signaling and transport processes that play an important role in functioning of the nervous system and in neurological diseases pathogenesis of humans and animals. The goal of the study was to investigate the correlation between the average size of the active zone of the synapse and the frequency of synapse-astrocyte contact formation in the cerebral cortex layers of rats.Materials and methods. The 40 μm frontal sections of the primary somatosensory cortex of 5 outbred male white rats were taken as the material for the study. Astrocytes were labelled for Transmission Electron Microscopy (TEM) by incubation of the tissue sections with primary antibodies to the s100β protein and with horseradish peroxide-conjugated secondary antibodies, followed by the development of the label in the reaction with DAB. For each layer, 250 images were obtained at 25,000х magnification. On the images the length of the synaptic cleft was measured and the number of synapses forming contact with the astrocyte membrane was counted.Results. The value of the fraction of the chemical synapses forming contact with the astrocyte membrane in the primary somatosensory cerebral cortex of rats demonstrated the direct correlation with the average length of the synaptic cleft only in the first to fourth layers. Thus, in the first layer, the value of the fraction of synapses forming contact with the astrocyte was the smallest (PI = 0.27 ± 0.1), as was the length of the synaptic cleft (lI = 329.45 ± 10.45). When moving deeper into the cortex, the fraction of synapses forming contact with the astrocyte and the length of the synaptic cleft increased from the second (PII = 0.48 ± 0.11 and lII = 363.64 ± 11.14) to the third layer (PIII = 0.69 ± 0.09 and lIII = 382.27 ± 9.81), followed by the decrease of both values in the fourth layer (PIV = 0.53 ± 0.09 and lIV = 355.2 ± 8.12). In the fifth layer, the fraction of synapses forming contact with the astrocyte sharply increased again (PV = 0.68 ± 0.08), which, however, was not accompanied by the proportional increase of the average length of the synaptic cleft (lV = 350.79 ± 7.82). At the same time, in the sixth layer of the cortex, on the contrary, the sharp increase in the average length of the synaptic cleft (lVI = 396.03 ± 10.77) was observed, reaching the maximum value through the cortex, with low, compared to other layers, fraction of synapses forming contact with the astrocyte (PVI = 0.44 ± 0.09). Thus, it turned out that the fraction of synapses forming contact with the astrocytic membrane is more related to the density of the astrocytic membranes in the layer (the research we published earlier), rather than to the average length of the synaptic cleft. Discussion and conclusions. The obtained results substantiate that the contact is formed as a result of a combination of random membranes encounter followed by the selective anchoring or repulsion of the astrocyte membrane under the influence of various factors, only partly determined by the size of the chemical synapse.

As the population ages, age-related cognitive impairments are becoming an increasingly pressing problem. Currently, the role of polyamines (putrescine, spermidine, and spermine) in the pathogenesis of cognitive impairments of various origin is actively discussed. It was shown that the content of polyamines in the brain tissue decreases with age. Exogenous administration of polyamines makes it possible to avoid cognitive impairment and/or influence the pathogenetic processes associated with disease progression. There are 3 known ways that polyamines can enter the human body: food, synthesis by intestinal bacteria, and biosynthesis in the body. Currently, one of the most promising approaches to the prevention of cognitive impairment is the use of foods with a high content of polyamines, as well as the use of various probiotics that affect intestinal bacteria that synthesize polyamines. Since 2018, in a number of European countries projects have been launched aimed at evaluation of the impact of a diet high in polyamines on cognitive processes. The review, based on analysis of modern scientific literature and the authors' own data, presents material on the effect of polyamines on cognitive processes and the role of polyamines in the regulation of neurotransmitter processes, and discusses the role of polyamines in cognitive disorders in mental and neurological diseases.

— Numerous data obtained in the last 20 years indicate that all parts of the mature central nervous system, from the retina and olfactory bulb to the spinal cord and brain, contain cells connected by gap junctions (GJs). The morphological basis of the GJs is a group of joined membrane hemichannels called connexons, the subunit of each connexon is the protein connexin. In the central nervous system, connexins show specificity and certain types of them are expressed either in neurons or in glial cells. Connexins and GJs of neurons, combining certain types of inhibitory hippocampal and neocortical neuronal ensembles, provide synchronization of local impulse and rhythmic activity, thalamocortical conduction, control of excitatory connections, which reflects their important role in the processes of perception, concentration of attention and consolidation of memory, both on the cellular and at the system level. Connexins of glial cells are ubiquitously expressed in the brain, and the GJs formed by them provide molecular signaling and metabolic cooperation and play a certain role in the processes of neuronal migration during brain development, myelination, tissue homeostasis, and apoptosis. At the same time, mutations in the genes of glial connexins, as well as a deficiency of these proteins, are associated with such diseases as congenital neuropathies, hearing loss, skin diseases, and brain tumors. This review summarizes the existing data of numerous molecular, electrophysiological, pharmacological, and morphological studies aimed at progress in the study of the physiological and pathophysiological significance of glial and neuronal connexins and GJs for the central nervous system.

The glomeruli of the olfactory bulb of mammals are the primary coding elements of olfactory information. The excitation pattern produced by individual glomeruli in response to olfactory stimulation is stable and specific to certain odors. First of all, this is due to the structure of the neural circuits of the olfactory bulb. Nevertheless, there is reason to believe that auxiliary cells of the nervous system—astrocytes—play a role in the organization of the primary processing of the olfactory signal. It is known that astrocytes not only have a direct effect on synaptic activity and plasticity, but coordinate the joint work of neuronal circuits and the vascular component, forming so-called “neuro-glio-vascular ensembles” as well. In this study, we carried out a morphological study of peripheral processes and gap junctions of astrocytes in order to study the structure of neuro-glio-vascular ensembles at the level of organization of the olfactory bulb glomeruli neuropil. The study showed that the main part of the astrocytic processes inside the glomeruli is located in the area of dendro-dendritic connections of the interneurons and projection neurons of the olfactory bulb, while in the zone of the primary switching of the olfactory signal, the astrocytic processes are practically absent. We also found a pronounced imbalance in the expression of the main astroglial connexins between the different functional poles of the neuro-glio-vascular ensembles of the olfactory bulb and the presence of heterotypic contacts formed by Cx30. The functional significance of the observed features of connexins expression in the olfactory bulb has yet to be studied.

Functional cortical columns and nuclei of the ventral thalamus play a key role in processing of sensory information; therefore, detailed studies on formation of neuron-to-neuron gap junctions in these areas are of great theoretical and practical importance. In the present study, we applied electron-microscopy methods to examine the structure and specific distribution of interneuronal gap junctions in the cortical layer IV and thalamic nuclei, including VPM, RTN, Pom, and VPL. In the cortex, we found more interneuronal gap junctions than in thalamic nuclei. In all structures studied we revealed and described axo-dendritic, dendrodendritic, and “mixed” synapses. We report on the axo-dendritic gap junctions for the first time. It is suggested that this type of contacts plays some functional role in local synchronization of neuronal activity within one ensemble on the presynaptic level.

Complex morphological study of gap junctions (GJs) in the blood–brain barrier in the rat cortical barrel columns using light and transmission electron microscopy and immunohistochemistry showed that astrocytes united by GJs in a single network can act as the main mediator between neurons and the vascular bed, forming a complex of neurogliovascular ensembles. The possibility of using such complexes to determine the functional organization of cortical columns is discussed.

AIM:to conduct an electron microscopic study of intercellular communication in the samples of gemistocytic astrocytoma, oligodendroglioma, and glioblastoma.MATERIAL AND METHODS:Surgically resected tumor tissue fragments were fixed in 2.5% glutaraldehyde solution, afterfixed in 1% OsO4 solution, dehydrated, and embedded in epoxy resin. Ultrathin sections were examined using a Jem 1011 electron microscope (Jeol, Japan).RESULTS:Solitary and closely spaced gap junctions (GJs) formed by the thin processes that have the ultrastructure of an astroglial processes were identified in the astrocytoma samples. In this case, chemical synapses were noted to be completely absent in gemistocytic astrocytoma and glioblastoma. The identified GJs had a small length and deformed nexuses. The oligodendroglioma samples exhibited intact astroglial processes around the chemical synapses; however, interglial GJs were not found.CONCLUSION:The investigation showed the presence of intercellular GJs with some ultrastructural differences in the samples of low- and high-grade astroglial tumors. According to current data, astrocytomic GJs are able to create a stable self-sustaining network that promotes tumor progression and provides resistance to a therapeutic intervention. At the same time, the noticeable reduction in the number of GJs, which is most pronounced in the oligodendroglioma sample, can accelerate tumor cell migration into the surrounding parenchyma. The investigation of GJs should be, of course, continued using a group of a larger number of glial tumors to confirm the intercellular communication features revealed in this study.

AIM:Тo conduct an immunohistochemical (IHC) study of the expression of connexin 43 in the samples of glial tumors of various grades: gemistocytic astrocytomas (Grade 2), oligodendrogliomas (Grade 2) and glioblastomas (Grade 4).MATERIAL AND METHODS:The material investigated was fragments of human brain glial tumors (grade 2 gemistocytic astrocytomas (n=2), grade 2 oligodendrogliomas (n=2), and grade 4 glioblastomas (n=14) and those of tumor-surrounding tissue (n=4). The material was fixed in 10% buffered formalin, dehydrated, and embedded in paraffin according to the standard technique. IHC studies of the slices applied primary rabbit polyclonal antibodies against connexin 43 ('Spring Bioscience', USA) and the Dako EnVision + Peroxidase (DAB) visualization system ('Dako', Denmark). After the immunohistochemical reaction, the cell nuclei were stained with Mayer's hematoxylin.RESULTS:Immunohistochemistry showed the changing pattern of connexin 43 expression as compared with intact tissue in the glial tumors. Instead of the fine-granular expression in the thin cellular processes in the neuropil, the tumors mainly displayed a coarse-grained cytoplasmic and even nuclear reaction. The morphology and localization of positive structures depended on the variant of an examined tumor. In addition, the most malignant brain gliomas generally exhibited a reduction in the expression of connexin 43, i.e. its quantity is inversely proportional to the degree of malignancy of the tumor.CONCLUSION:The low connexin 43 expression levels may reflect both a reduction in astroglial functional gap junctions and semicanals and a decrease in the amount of the protein itself that has independently antioncogenic properties. The observed cytoplasmic and nuclear expression of connexin 43 is most likely to be associated with the aberrant activity of a number of kinases, such as proto-oncogene tyrosine-kinase Src or protein kinase C (PKC).

The aim of this work was an immunohistochemical study of the expression of neuronal and glial proteins, and of gap junctions proteins (connexin 36, connexin 43) in ventral posteromedial (VPMN), ventral posterolateral (VPLN) and reticular (RТN) nuclei of the thalamus in rats. It was found that VPMN and VPLN of the thalamus were characterized by a homogeneous distribution of synaptophysin, grouped arrangement of astrocytes, horizontal orientation of somatostatincontaining myelinated and unmyelinated nerve fibers, forming the bundles, and running through the barreloid septum, expression of connexin 36 and 43 as well as of parvalbumin revealing barreloids in 4 μm-thick sections. In RTN the content of myelin basic protein, neurofilaments, parvalbumin, and somatostatin was increased, while the amount of glial fibrillary acidic protein and connexin 43 was moderate, and synaptophysin and connexin 36 were absent.

Despite a growing interest in gap junctions (GJs) of mammalian brain, their distribution and role in cell ensembles of thalamus remains unknown. The aim of this work was ultrastructural and immunoelectron study of glial GJs in ventral posteromedial (VPM) and posteromedial (POM) thalamic nuclei and thalamic reticular nucleus (RTN) of rats. GJs were identified by standard techniques of transmission electron microscopy and by pre-embedding immunohistochemistry protocol using anti-connexin-43 antibodies with Dako EnVision System + Peroxidase (DAB) detecting system. It was found that glial cells surround thalamocortical axons and axo-spiny synapses and form numerous elongated gap junction plaques located near chemical synapses. A single axon-spiny chemical synapse can be surrounded by several (up to 4) gap junctions that seem to form peculiar networks of glial cells united by GJs. Closely adjacent gap junctions disposed at an angle from 30° to 140° to each other were revealed. Immunoelectron labeling demonstrated that gap junction plaques located around chemical synapses have an astroglial origin. Despite the accumulation of osmiophilic material in the contact zone, ultrastructural signs of GJs were clearly identified. Due to the formation of intercellular glia-glial GJs astroglia may acquire a function of spatial buffer to regulate extracellular concentration of potassium and other ions, providing intracellular and extracellular ion homeostasis. We believe that astroglial processes joined into a network by GJs play a key role in the circulation of information and can modulate subcortical neuronal ensembles. We suggest that a close spatial location of astroglial GJs and asymmetrical chemical synapses is reflected in the functional organization of specific and nonspecific thalamic nuclei, which are the main centers of the afferent and efferent inputs of the cerebral cortex.

The aim of the study was laminar morphometric study of immuno-labeled parvalbumin containing (PA+) neurons of cortical somatosensory area SI in outbred albino rats (n = 10). The study of frontal and tangential sections 60 μm and 4 μm thick demonstrated a considerable diversity in cell body shape and size as well as in branching of the processes in PA+ neurons in all the layers of the cortex. The greatest number of PA+ neurons (47.1%) was found in layer IV of the cortex, in the zone of barrel formation. The study of tangential sections has shown that the largest number of PA+ neurons was localized in the barrel septa (43%). In layer IV, their greatest density was detected in the walls of the barrel, making it possible to clearly identify their outlines. Quantitative predominance of PA+ neurons in the septa may be associated with the direction of their dendrite course into the inner part of the barrel and the formation of dendro-dendritic gap junctions that, in turn, could be a morphological basis of individual local pacemaker rhythmogenesis and regulation of the functional state of the cortical columns.

The last decade is characterized by development of such technologies as RNA-sequencing and biochips which resulted in discovery of new perspective biomarkersfor personalized diagnostic of cancer. Among them, the long non-coding RNA (IncRNA) are of special interest because according the recent studies they are positioned as important regulators of gene expression on epigenetic, transcription and post-transcription levels. The review considers the role of long non-coding RNA in cancerogenesis. The corresponding of their application in diagnostic is evaluated. A number of examples ofperspective diagnostic and prognostic markers. Their degree of implementation in oncological practice is discussed.

The aim of the present work was to identify columns in neocortical field S1 on frontal brain sections from white mongrel rats (n = 10) using immunocytochemical methods and antibodies to neuron proteins (synaptophysin, neurofilaments) and glial cells (glial fibrillary acidic protein (GFAP), myelin basic protein). Analysis of the expression of the main neurospecific antigens showed that on thin sections (4 μm), columns could be identified on the basis of groupings of astrocytes and neuron processes, i.e., axons and dendrites. Analysis of GFAP expression also showed that cortical layer I generally contained large numbers of large astrocytes with branching processes, as well as numerous small processes with high expression intensity. The synaptophysin content was high in all layers of the cortex, though the most intense reaction was seen in the molecular layer, as for reactions for GFAP. Expression of myelin basic protein identified radially distributed myelinated neuron processes in the cortex.

OBJECTIVE:To reveal the expression of neuronal connexin 36 (Cx36) in gliomas and then to analyze the ratio of expression of Cx36 to that of neuroglial antigens (synaptophysin, neurofilaments, and glial fibrillary acidic protein).MATERIAL AND METHODS:Varying grade human glioma samples and tumor-adjacent tissue fragments were used for immunohistochemical examination.RESULTS:A procedure for immunohistochemical detection of Cx36 in brain tissue was tried out. It was shown that the decreased level of the examined neuronal proteins was accompanied by the impaired coexpression of synaptophysin/neurofilaments and Cx36 in the series of astrocytomas--anaplastic astrocytomas--glioblastomas. The immunohistochemical heterogeneity of glioblastomas was found.CONCLUSION:The findings suggest that it is promising to include anti-Cx36 antibodies in immunohistochemical panels when examining brain tumors. Data on the lower levels of the examined neuronal proteins, on the specific features of their distribution and impaired coexpression expand their idea on the pathogenesis of a brain tumor process and determine an area for further investigations.

Electron microscope studies of gap junctions (GJ) on serial sections of the barrel cortex in rats showed that GJ make contact with one or both of the processes forming chemical synapses, though these associations could not be demonstrated on single sections. On serial sections, a single field could show two GJ close together, and each GJ could be followed through 2–3 sections in the series. Given the variants described for the distribution of GJ in the cortex, it is suggested that GJ may provide the structural basis for local synchronization of bioelectrical activity not only at the postsynaptic, but also at the presynaptic level; formation of GJ occurs both before and after development of chemical synapses.