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.

Animal models mimicking human transient ischemic attack (TIA) and cerebral microinfarcts are essential tools for studying their pathogenetic mechanisms and finding methods of their treatment. Despite its advantages, the model of single arteriole photothrombosis requires complex experimental equipment and highly invasive surgery, which may affect the results of further studies. Hence, to achieve high translational potential, we focused on developing a TIA model based on photothrombosis of arterioles to combine good reproducibility and low invasiveness. For the first time, noninvasive laser speckle contrast imaging (LSCI) was used to monitor blood flow in cerebral arterioles and reperfusion was achieved. We demonstrate that irradiation of mouse cerebral cortical arterioles using a 532-nm laser with a 1-mm-wide beam at 2.4 or 3.7 mW for 55 or 40 s, respectively, after 15 mg/kg intravenous Rose Bengal administration, induces similar ischemia-reperfusion lesions resulting in microinfarct formation. The model can be used to study the pathogenesis of spontaneously developing cerebral microinfarcts in neurodegeneration. Reducing the exposure times by 10 s while maintaining the same other parameters caused photothrombosis of the arteriole with reperfusion in less than 1 h. This mode of photodynamic exposure caused cellular and subcellular level ischemic changes in neurons and promoted the activation of astrocytes and microglia in the first day after irradiation, but not later, without the formation of microinfarcts. This mode of photodynamic exposure most accurately reproduced human TIA, characterized by the absence of microinfarcts.

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.

Using electron microscopy, we studied ultrastructural changes in neurons and glia cells of ganglia abdomibal 4 and 24 hours after transection of interganglionic connectives. Experiments on such model objects as ganglia of invertebrates provide information on the general biological mechanisms of the nervous system's reactions to mechanical damage. In the control samples, the bodies and nuclei of neurons had a rounded shape, the nuclear chromatin of neurons differed in the degree of condensation. The cytoplasm contained numerous Nissl bodies, the Golgi apparatus, located mainly in the perinuclear region, various elements of the cytoskeleton, ribosomes, elongated and rounded mitochondria with a moderately dense matrix and normal cristae filling the entire volume of mitochondria. Glial cells surrounded the bodies of neurons or unmyelinated axons of the neuropil and formed a multilayer sheath tightly attached to the neuron soma or axons. 4 hours after axotomy, disorganization of Nissl bodies and mitochondrial cristae was observed in the cytoplasm of neurons. 24 hours after the axotomy, ultrastructural changes in neurons intensified: compression of nuclei and compaction of nuclear chromatin were observed. There was a further disorganization of Nissl's substance, the appearance of voids and the loss of organelles. The internal structure of neuropil axons was completely destroyed. However, the ultrastructure of glial cells was found to be more preserved, and the presence of a certain amount of intact mitochondria in them indicated the continuation of synthetic processes in them. Some of the described ultrastructural changes in the ganglia of the abdominal nerve cord of crayfish indicate early stages of necrosis, but taking into account such changes as the contraction of nuclei and condensation of chromatin, these changes should be attributed to a mixed type of cell death.

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.

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.

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.

Non-thermal effects of direct electric fields and alternating electromagnetic fields (EMF) have been successfully used in a number of studies and applications in agriculture and biotechnology. Among different kinds of high strength EMF generators, the Tesla transformer (TT) is known as a widely applied, low cost, and troubleproof device, which generates EMF in the range of 2–8MHz. Despite of a number of developed and perspective applications of high strength EMFs in agriculture and biotechnology, the EMFs generated by TT, as well as the 1–50MHz range of high strength EMF still remain unexplored in the fields of plant physiology, ultrastructure studies and biochemistry. In this work, we have shown that TT-EMFs (4MHz) induced fast stem and petiole bending, disappearance of cell organelles, vacuolar membranes, and increase of a non-photochemical chlorophyll fluorescence quenching in petioles. It is intriguing that such fatal effects can be evoked in plants by EMFs which are well known as harmless for man at the applied strength and frequency.

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.

Autophagy in flower petal epidermal cells is a little studied process. However, it is of interest regarding the physiology of chlorophylless plant tissues. We show that the utilization of organelles in the Petunia hybrida L. flower petal epidermis occurs via the autophagic-like organelle targeting into vacuole during the particular process of vacuole fragmentation and growth of secondary vacuoles (ATVF). It differs from macroautophagy (formation of autophagosome) and microautophagy. Peroxisomes were shown to be subjected to both macroautophagy (as single microbodies and their assemblies) and to ATVF when they are in complexes with mitochondria and chromoplasts. In addition, we have demonstrated that ATVF in flower petals is accompanied with the sequestration of the cytoplasmic sectors located between the neighboring cell wall ingrowths, the ultrastructure of which has been reported in the present work for the first time. It has been shown there are more numerous mitochondria, a more pronounced ATVF, and a thicker cytoplasmic layer in the blue tissues, possibly reflecting more intensive metabolic processes in them compared to white tissues. We assume that these differences may be of interest for the further studies and may be discussed including in terms of anthocyanin-dependent photochemical effects.

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.

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.

Electron microscopic investigation of gap junctions (GJ) on serial sections of rat barrel cortex has shown that GJ were in contact with one or both processes that formed chemical synapses, however, these connections could not traced in single sections. In the serial sections, it was possible to observe two GJ in the immediate proximity to one another, in a single field of vision, thus, each GJ was traced in two or three successive sections in a series. Considering the described variants of GJ arrangement in the cortex, it is suggested that GJ could be a structural basis for local synchronization of the bioelectrical activity not only at postsynaptic, but also at presynaptic level, and the formation of GJ occurs both before, and after the development of chemical synapses.

: Immunohistochemical investigation of the regional distribution of neuronal and glial elements in the barrels of somatic cortex was carried out in rats (n=10). High level of synaptophysin protein expression was detected in the in barrel walls together with the accumulation of astroglial cells in their central areas. Ultrastructural investigation of chemical synapse structure showed the predominance of asymmetrical perforated axospinous contacts, presumably of excitatory type. The symmetric inhibitory synapses were more frequently located in the barrel walls in the areas of the denser neuronal perikarya distribution, as well as on the large dendritic processes. The number of vertically oriented myelinated axons within the barrels was found to be significantly greater than the number of the horizontally oriented interneuronal circuits.