Violation of the main regulatory mechanisms at the level of nerve cells can lead to the phenomenon of neurotoxicity/excitotoxicity of glutamate. Such phenomena most often occur under hypoxia/ischemia, inflammatory processes, and activation of immune and autoimmune reactions. Pathological changes in the brain at the early stages of diseases are nonspecific. They differ little from the physiological norm. Such typical pathological processes are characteristic of numerous diseases. They also develop in ischemic and hemorrhagic strokes. The main task of this work was to analyze some physiological and cytochemical processes that are associated with the neurotransmitter glutamate, as well as with highly reactive and highly toxic compounds, reactive forms of nitrogen and oxygen. The reactive forms of nitrogen ( ^∙NO ) and oxygen ( ^∙O_2^ - ) can affect almost all major components of cells and subcellular structures. At low concentrations, they perform a regulatory function. The analysis of the mechanisms of toxic effects of glutamate, reactive forms of nitrogen and oxygen species as a model of stroke allowed us to propose new ways to protect against the damaging effects of the above substances, which can be used in the treatment of ischemic and hemorrhagic strokes.

На основе литературных и собственных данных описаны механизмы возбуждения в нервной системе и нейроглиальные отношения, участвующие в этом процессе. Возбуждение в нервной системе формируется двумя процессами - пассивным и активным. Активный тип возбуждения, требующий энергетических затрат, связан с регулированием мембранных свойств нейронов, что приводит к формированию изменчивой спонтанной импульсации. Порождаемая пассивным процессом спайковая активность очень стабильна и является следствием трансмембранного перемещения ионов Na+ и К+ по градиентам концентраций. Пассивный тип возбуждения обеспечивается глутаматергическими контактами, активный - диффузным выбросом ацетилхолина из холинергических ядер мозга и блокированием К+-проницаемости. Энергетическое снабжение активного процесса возбуждения происходит с участием глии при непосредственной связи глиальных клеток с сосудами мозга, аккумулировании глюкозы в виде гликогена, осуществлении первого этапа энергетического метаболизма - гликолиза и регулирования локального мозгового кровотока, сопряженного с М-холинергическим возбуждением нейронов. При устойчивом снижении скорости М-холинергического процесса (концентрационного, температурного или энергетического) происходит стремительный отток ионов К+ из нейронов, удаление которых из межклеточной среды также является функцией глии.

Excitation mechanisms in the nervous system and neuronal–glial interactions involved in this process are described on the basis of published data and original findings. Two processes, passive and active, form excitation in the nervous system. The active type of excitation requires energy support and is associated with the regulation of the membrane properties of neurons, leading to generation of variable spontaneous pulses. Spike activity generated by the passive process is highly stable and results from transmembrane movement of Na+ and K+ ions along their concentration gradients. The passive type of excitation is due to glutamatergic contacts; the active type of excitation is due to a diffuse release of acetylcholine from cholinergic nuclei of the brain and attenuation of K+ membrane permeability. Energy supply of the active excitation process involves glia. Glial cells directly interact with brain vessels, accumulate glucose in the form of glycogen, realise glycolysis as the first step of energy metabolism, and regulate local cerebral blood flow coupled with M-cholinergic excitation of neurons. A steady decrease in the rate of the M-cholinergic process (in terms of concentration, temperature, or energy) leads to a rapid outflow of K+ ions from neurons, and removing K+ from the intercellular environment is also a function of glia.

The glia–neuron interactions were analyzed in the sensory-motor cortex of guinea pigs and ground squirrels (Spermophilus undulatus) during the active summer months. The glial cells were more concentrated in close proximity (15–25 μm) to neurons (38% in guinea pigs and 22.4% in ground squirrels). A more concentrated distribution of glial cells might be very necessary for spontaneous inactive nerve cells (37.2% in guinea pigs and 23% in ground squirrels), since these neurons are associated with the highest energy demand during their functioning and are most susceptible to disturbances of ion homeostasis. The network structure of glia and the close contact between glial cells and brain capillaries provide additional energy for neurons and stabilize the ion balance in the extracellular medium. Glial density in the sensory-motor cortex of ground squirrels is 3 times higher than that in the cortex of guinea pigs. The high content of glial cells in the ground-squirrel cortex is the most important protective factor for survival of animals during long-term hibernation, when the diffusion of K+ ions from nerve cells drastically increases due to the high temperature sensitivity of the M-cholinergic response.

Activity of sensorimotor cortical neurons in the ground squirrel was studied on slices under cooling the incubation medium from 32–34 to 21–26°С. Hypothermia evoked spontaneous firing activity in “silent” neurons and a slight decrease in firing in high-frequency neurons. Changes in the firing rate arose below 27°С and were accompanied by a fall in the spike amplitude. The intensity of hypothermic and post-hypothermic changes in ground squirrels was lower than in guinea pig sensorimotor cortical neurons recorded under the same conditions. In ground squirrels, most hypothermia-resistant were high-frequency (more than 8 spikes/s) neurons, which accounted for 45% of the recorded, while in guinea pigs high-frequency neurons occurred only in 15% of records. By the diameter of cell bodies, the population of sensorimotor cortical neurons was more homogeneous in ground squirrels than in guinea pigs. It is suggested that specific hypothermic changes in sensorimotor cortical neurons of ground squirrels relate to a lower density of K + channels in their plasma membranes, because in the mammalian nervous system the latter open below 27°С due to thermal limitations of the M-cholinergic reaction which blocks these channels.

Olfactory bulbectomy (OBX) in rodents induces a wide spectrum of functional disturbances, including behavioral, neurochemical, and neuromorphological alterations. We have examined the effects of OBX on behavior and the parameters of the cholinergic system in female rats and mice. In rats, OBX resulted in the appearance of some depressive-like behavioral marks, such as the decreased sucrose consumption, hyperactivity, impaired short-term memory and anxiety-like behavioral features, such as shortened presence in the center of the open field arena or open arms of the elevated plus-maze and an enhancement of avoidance behavior. These behavioral abnormalities could be associated with disturbances in hippocampal function, this suggestion being supported by the presence of cellular changes in this brain structure. No effect of OBX on the number of cholinergic neurons in the medial septum-diagonal band as well as on the acetylcholine content and acetylcholinesterase activity in the septum, hippocampus, and neocortex could be detected. In contrast, in mice, OBX impaired spontaneous alternation behavior and decreased the number of cholinergic neurons in the medial septum-diagonal band. These data demonstrate that rats and mice differently respond to OBX, in particular, OBX does not significantly affect the cholinergic system in rats.

In sensorimotor cortical slices of guinea pig in the course of cooling of incubating fluid from 34 to 21-22 delta C it was shown that hypothermia does not influence on the evoked spike reactions to iontophoretic application of glutamate to the soma, but glutamate action on the dendritic locus causes the shot latency somatic spike response during hypothermic increasing of the rate of spontaneous activity and long latency spike response--during hypothermic fall of activity. While the cooling rate of spontaneous activity in the slow firing neurons was mainly increasing and in the high firing neurons (above 4 spikes per second)--decreasing. The changes in spontaneous activity began at 30 degrees C along with the decreased spike reactions to iontophoretic applications of acetylcholine and efficacy of dendro-somatic propagation. At the same temperature the fall of spike amplitude was initiated and increased with further hypothermia. It is proposed that the basis for hypothermic changes of neuronal activity.is the decreased rate of M-cholinergic process at 27-29 degrees C. Neurons of different physiological properties display different sensitivity to hypothermic factor.

In sensorimotor cortical slices of guinea pig in the course of cooling incubating fluid from 34 to 21-22°C it was shown that hypothermia exerted both increase and decrease of spontaneous activity in different neurons. On hypothermic increase of firing level spike responses of soma to iontophoretic application of glutamate to dendritic locus appeared with shorter latencies and with longer latencies – on hypothermic decrease of spontaneous activity. At the same time hypothermia did not influence on the evoked spike reactions to iontophoretic application of glutamate straight to the soma. It means that hypothermic disorders of neuronal activity are not connected with changes in sensitivity to glutamate but determined by changes of amplitude of glutamatergic excitation while propagating along dendritic branches. The changes in spontaneous activity began at 30°C along with the decreased spike reactions to iontophoretic applications of acetylcholine and efficacy of dendro-somatic propagation. At the same temperature the fall of spike amplitude was initiated and increased with further hypothermia. It is proposed that the basis for hypothermic changes of neuronal activity is the decreased rate of M-cholinergic process at 27-29°C which leads both to attenuation of conductive function of dendrites and imbalance of K+ ion homeostasis. Peculiarities of hypothermic regulation of neuronal spike activity depend on individual functional properties of cortical neurons.

Studies using living slices of guinea pig sensorimotor cortex showed that functional differences in the regulation of the level of spontaneous activity in different neurons coincide with the heterogeneous distribution of glial satellite cells. The greatest increases in spike activity in response to acetylcholine (up to 36 spikes/sec) were seen in “silent” neurons, which accounted for 37.2% of all neurons in layer V. Morphometric analysis showed that the same proportion of neurons (38.6%) had glial satellites. Spontaneously active neurons showed only small (5–22 spikes/sec) increases in spike activity over baseline. It is suggested that the maximal development of M-cholinergic reactions regulating the level of spontaneous activity in inactive neurons requires a supplementary energy source, which is provided by the contacts of neurons with their surrounding glial satellite cells.

Проведено исследование морфологических изменений и экспрессии маркеров астроглии и микроглии в сенсомоторной коре и гиппокампе крыс, перенесших хроническое невротизирующее воздействие. Установлено, что адаптация к хроническому стрессу сопровождается возникновением большого числа поврежденных нейронов как в слое V сенсомоторной коры, так в пирамидном слое гиппокампа, причем наиболее выражены эти изменения в поле СА3. Неврозоподобное состояние у крыс характеризуется увеличением экспрессии маркеров микроглии в гиппокампе, которое сохраняется в течение месяца после окончания стрессорного воздействия. Экспрессия астроцитарного маркера GFAP снижается в поле СА3 гиппокампа. Введение производного пантотеновой кислоты – пантенола, способствует стабилизации глиального ответа на хронический стресс, но его эффекты не длительны и не могут предотвратить активацию микроглии в постстрессорный период.

We studied the morphological changes and expression of the astroglial and microglial markers in the sensorimotor cortex and hippocampus of rats that were subjected to chronic stress. Chronic neurotization was associated with the appearance of a large number of damaged neurons in layer V of the sensorimotor cortex and the pyramidal layer of the hippocampus. These changes were considerably expressed in the CA3 field. A neurosis-like state in rats was accompanied by an increased expression of microglial markers in the hippocampus and this effect was evident even 1 month after the end of stress. The expression of the astroglial marker GFAP decreased in the CA3 hippocampal field. Treatment of animals with the pantothenic acid derivative panthenol stabilized the glial response to chronic stress; however, its effects were not long-lasting and did not prevent activation of microglia during the period after stress.

Cross-correlation analysis was used to study the interaction of neurons in the sensorimotor and visual areas of the cortex in rabbits with cryptic foci of excitation formed in the representation area of the forelimb; the role of sensorimotor cortex neurons responding to light in this interaction was also studied. The results showed that in rabbits with cryptic foci of excitation, sensorimotor cortex neurons responding to light stimuli showed correlational relationships with cells in the visual cortex significantly more frequently than neurons not responding to light, while visual cortex neurons significantly more frequently formed correlational relationships with sensorimotor cortex neurons not responding to the stimulus.

Using surviving slices of guinea pig somatosensory cortex, it was shown that functionally different regulation of spontaneous firing activity in different neurons corresponded to irregular distribution of glial satellites. Maximal increase of spike activity induced by acetylcholine (up to 36 spikes per second) was detected in "silent" neurons which account for 37.2% of nerve cells in layer V. According to the morphometric analysis, the same relative number of neurons (38.6%) were surrounded with glial satellites. In spontaneously active neurons only a small elevation of firing activity (5-22 spikes per second) above the basal level was recorded. The results allow to suggest that M-cholinergic reaction, controlling the spontaneous activity level, requires the additional energy supply for its maximal expression in inactive neurons. This is achieved by contacts of neurons with the surrounding glial satellites.

Effects of a chronic combined unpredictable stress on activities of two cell death-related proteases, calpain and cathepsin B, were studied along with indices of nitrergic system in rat brain structures. Male Wistar rats were subjected to a 2-week-long combined stress (combination of unpaired flash light and moderate footshock associated with a white noise session). Stress resulted in a significant loss in the body and thymus weight and increased defecation in the open field test, though neither motor and exploratory activity, nor plasma corticosterone differed from the respective control levels. Decreased calpain activity and increased cathepsin B activity were demonstrated in the hippocampus of stressed rats (previously we have shown that caspase-3 activity was significantly suppressed in the brain of rats subjected to same type of stress). A significant reduction in the number of NOS-containing neurons was accompanied by a chronic stressinduced decline in NOS activity in the neocortex. Similar changes were observed in the hippocampus. However, levels of NO metabolites were elevated in both structures. Thus, stress-induced structural modifications in the brain may be mediated by disturbances in the nitrergic system and increased lysosomal proteolysis.

Studies using living slices of guinea pig sensorimotor cortex showed that changes in temperature from 24°C to 37°C produced stepwise increases in neuron spike frequency at two temperature zones: 27–29°C and 34–36°C. Changes in spontaneous activity were accompanied by decreases in spike amplitude at t < 27°C and t > 34°C. After cooling to 24°C, spike amplitude generally recovered completely when the temperature was increased to 32–34°C. The decrease in spike amplitude at t > 35°C could not be restored by decreasing the temperature. It is suggested that these effects are associated with the K+ permeability of neuron membranes.

Mnemotropic effect of Piyavit®-a biologically active compound of natural origin—was studied on conditioned avoidance in rats. Morphological changes in neocortex and hippocampus (neuron-glia complex and brain capillaries) were significant and oppositely oriented. We assume that the improvement of rats’ memory under the Piyavitis associated with metabolic changes in the nervous tissues.

The interaction between neurons of sensorimotor and visual cortices was investigated by cross-correlation analysis. In this interaction, we examined the role of sensorimotor neurons responding to light. In rabbits with a hidden focus of excitation, neurons of the sensorimotor cortex responding to light significantly more often formed correlation joints with cells of the visual cortex than neurons not responding to light. On the other hand, neurons of the visual cortex significantly more often formed correlation joints with neurons of the sensorimotor cortex not responding to light.

Behavioral changes and accompanying morphological neuron-glia reorganization in the rat brain were analyzed after long-term immobilization. Wistar rats (n = 23) were stressed by interruptive immobilization, which was carried out within three week daily for 7-8 h. Behavioral immobilization of rats was accompanied by a decrease in the locomotor and exploratory activity in "open field" test and increase in the number and duration of freezing episodes. The morphometric studies revealed a statistically significant threefold increase in the density of hypoxic neurons in the motor neocortex of both hemispheres and CA3 field of the hippocampus in the experimental animals as compared to control. The number of glia cells in the motor cortex did not change. The increase in the density ofglial cells and multi-nucleolar neurons in CA3 region of the hippocampus are indicative of the compensatory processes in the brain. The hypoxic changes in neurons were of the functional character.

Behavioral and neuronal-glial changes after emotional stress induced by discontinuous (7–8 h per day for one week) immobilization were compared in Wistar rats (n = 20). Immobilization led to increases in horizontal and vertical activity and the duration of “comfort” grooming in the open field test. Morphometric measurements demonstrated significant increases in the density of hypoxic neurons in the motor area of the right hemisphere of experimental animals as compared with measures in controls. Hypoxic changes in neurons were functional in nature. Experimental rats can be regarded as a model of the redistribution of brain functional activity with a preferential increase in the role of the left hemisphere.