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Electrophysiological parameters and bound calcium (Ca(b)) level dynamics during sensitization development or conditioning of food aversion were studied in the command neurons of defense behaviour in the snail Helix lucorum. Responses evoked by a testing sensory stimulus were facilitated 50-60 min after the first sensitizing stimulation, while conditioned responses appeared 80-90 min after the first conditioning. It was the most essential electrophysiological difference between the long-term sensitization and conditioning. Analysis of the Ca(b)) dynamics in the neurons showed significant differences in calcium-dependent metabolism during the sensitization and conditioning, likely underlying the electrophysiological differences.

Changes in the membrane-bound calcium (Ca(b)), polyphosphoinositides (PPI) and cAMP, as well as unit spike activity of the cat brain cortex were studied during 30 min of recovery after 5-min anoxia. The cats with poor recovery revealed low Ca(b) and PPI but high level of the cAMP content. The group of cats with more successful restitution after anoxia revealed a sharp increase in the Ca(b) and PPI and not in the cAMP.

Neurophysiological effects and dynamics of bound calcium (Ca-b) in command neurons of defense behavior (LP11 and RP11) during food aversion conditioning were studied in snail Helix lucorum. It was found that during associative learning both conditioned reaction and nonspecific facilitation of sensory responses were determined in neurons. The nonspecific facilitation of sensory responses was similar to that found during sensitization development in neurons. However, conditioned response appeared 30 min later than long-term sensitization development. Specific dynamics of the Ca-b level in neurons was obtained after three and more conditioning. The dynamics of the Ca-b level correlated with neurophysiological effects and differed from those measured during sensitization development. It is suggested that molecular mechanisms underlying associative learning and sensitization are different in a command neuron of defense behavior in the snail.

By using the methods of vital microscopy and extracellular registration of the neuronal activity the effect of oxymethacil (OM) on microcirculation and NSA in cat brain cortex was studied after grave poisoning by organophosphate paraoxon. The rapid depression of NSA together with the pronounced disturbances of microcirculation in cortical vessels were showed after intravenous injection of paraoxon (0.8 mg/kg). The prophylactic injection of OM (5 mg/kg) induced protective effect on the studied parameters. This effect was more pronounced when OM injection was combined with the M-cholinolytic amyzyl. The present data show the reasonableness of antioxidants using for the correction of a brain functional condition damaged by organophosphates.

The dynamic of the phosphatidylinositol (PI), the phosphatidylinositol 4-phosphate (PIP) and the phosphatidylinositol-4,5-diphosphate (PIP2) contents were studied in the correlation with the neuronal spike activity in cat brain cortex under acute oxygen deficiency caused by cessation of artificial ventilation for 1, 2.5 and 5 min. It was shown that the 1-min anoxia produced the depression of both PIP and PIP2 contents. The depression was followed by the development of the 'asphyxia neuronal activation'. During 2.5 and 5 min of anoxia the decrease of PIP2 content and increase of PIP one were detected against a background of neuronal bioelectrical activity depression. The PI content was constant during all the anoxic period.

Examinations carried out on command neurons of defensive behaviour of snail carried out using electrophysiological methods and a chlorotetracycline fluorescent probe have revealed that a single sensitizing action induces alterations of electrical neuronal activity and bound calcium (Ca-C) level in cells. An initial increase of the Ca-c amount (the first 15-20 min after the sensitizing action) coincides in time with depolarization, enhancement of plasma membrane excitability as well as with a decrease of amplitude and duration of the IPSPs induced by sensory stimulations. Repeated pronounced increase of the Ca-c level develops 50-60 min after the sensitizing action and correlates with facilitation of neuronal responses to sensory stimuli. Alteration of the Ca-c level in command neurons of defensive behaviour in the course of sensitization development differed from the previously described Ca-c shifts in the same cells in the course of habituation development in dynamics and direction.

It has been found in in vitro experiments that fluorescence intensity of deionized solution containing a chlorotetracycline fluorescent probe increases insignificantly at the addition of calmodulin of S-100 proteins. Subsequent introduction of Ca2+ into the medium results in the pronounced fluorescence increase depending on Ca2+ concentration. Addition of specific protein blockers--W7 (calmodulin inhibitor) and antibodies to S-100 brought about a decrease of fluorescence. In in vivo experiments on chlorotetracycline-stained neurons of Helix Pomatia ganglia subesophageal complex it has been shown that bringing of antibodies to S-100 and calmodulin significantly decreases the fluorescence intensity of these cells. These data suggest that the chlorotetracycline probe is an indicator of calcium ions binding with calcium-binding proteins both in in vitro and in vivo systems.

The chlorotetracycline fluorescent probe technique has been for the first time applied to in situstudy based on the contact microscopy of the vital cortex preparation. Changes of calcium bound with membranes included a rapid early decrease of the bound calcium in 5-min anoxia. The process revealed specific features for different layers of the cortex. The role of the early disturbance of intracellular calcium metabolism in mechanisms initiating neural responses to hypoxia, is under discussion.

Seven min after anoxia lasting for 2.5 and 6 min, changes in neurons and neuropil of the cat cerebral cortex sensomotor area have been studied electron microscopically. By means of a microscope MCCO having a long focus, the state of blood microcirculation in the pia mater vessels has been visually observed. During early postanoxic period, peculiar changes are mainly observed in neuronal bodies and degree of their manifestation depends on duration of the proceeding oxygen deficiency. In neuronal cytoplasm clearance of matrix, disorganization of the endoplasmic reticulum cisterns and formation of lamellar bodies are observed, as well as hypertrophy of Golgi complex and increasing number of free ribosomes. In the bodies of large pyramidal neurons of V layer, conglomerates of lipofuscin granules appear. In dendrites and myelinated axons destruction of membrane complexes and formation of myelin-like bodies are noted. In neuroglia glycogen granules precipitate. The most pronounced changes are revealed near microvessels which are filled with cellular blood elements. When estimating the ultrastructural changes of the cerebral cortex during early postanoxic period, it is necessary to take into account both duration of oxygen deficiency and the state of the blood microcirculation in various cortical areas.

By using the complex of techniques on the basis of life-time microscopy and spectrophotometry of the structures of II, III layers of the cat brain motor cortex there was studied the effect of ionophoretically delivered corazole on the dynamics of integral redox-state of pyramidal cells bodies, the surrounding neuropil as compared to the changed bioelectrical activity. The development of the convulsant activity of neurons coincided with the shift of their redox-state towards the accumulation of intracellular reducing equivalents. Both in the bodies of neurons and in the structures of neuropil a stable pronounced increase of restoration of intracellular redox-systems was preceded by the process of wave-like fluctuating changes in the content of reducing equivalents. The role of the revealed disturbances of oxidation-reduction processes in the mechanisms of corazole-induced lesions of neurons is discussed.

Using the complex methodical approach on the basis of in vivo microscopy of the cat motor cortex preparation the common principle of reaction of both the "silent" and the spontaneously active pyramidal neurons was revealed: their "seizure discharges" activation in hypoxia only occurred in a certain rather obvious disturbance of the intracellular redox-systems condition. At the initial stages of the firing changes in the spontaneously active neurons, no noticeable increase in their redox-systems restoration was observed.

Pyramidal neurons stained with methylen blue (from layers III and V of the cat brain cortex) are revealed in vivo after a special preparation of the contact objectives in the reflecting light. Small pieces of the brain tissue with identified neurons were separated under visual control, after which an oriented piece placed in a special grid-basket appeared to be stabilized by the borders of the grid. This method permits investigating the same neurons after fixation by means of histological light or electron microscope techniques.

A mathematical model describing the dynamics of oxygen transport in constructions with capillaries and pyramidal cells of the cat brain sensomotor cortex, aided to calculate the transitional processes of O2 tensions in neurons, capillaries and surrounding tissues during changes of the blood flow velocity in the capillaries and of the neurons respiration intensity. The model is represented in the form of a system of differential equations in particular derivatives, the solution of which was accomplished by the digital computer with the nets technique. Changes of pO2 levels within the space of modelled constructions in sharp shifts of the above parameters occurred within 2-5 sec. The time delays between the changes of these physiological parameters and the pO2 levels in neurons depended on peculiarities of neuron-capillary interrelationship, intensity of O2 consumption, blood flow velocity in the capillaries and their density.

Method of microscopic study of living nervous elements of the brain stained with vital dyes in the falling light was developed. Investigation of the morphology of neurons, glial cells and vasculo-nervous relations in the cat's cerebral cortex in living state in situ became possible thanks to the proposed method. Conditions were made to observe the patterns of dynamic changes in these structures under effects of different stimuli.

The biologically active substances induced mostly similar changes of the restorative equivalents level whereas the pattern of equivalents varied in neuronal activity of the mammal brain cortex. The effects of angiotensin II and cAMP on neuronal activity were different. The data obtained suggest that the angiotensin II effect on neurons is actualized both through the cAMP system and through other molecular mechanisms.

The native dynamics of Ca2+ binding with the membrane components in functionally different neurons of Helix pomatia when producing habituation to tactile stimuli are studied by fluorescent chlortetracyclin method. In the command neurons of the defensive behavior the weakening of the electrophysiological response characteristic for habituation and a decrease in the Ca2+ content were found. An increase in the Ca2+ content was found in supposed sensory neuron and in motoneurons in case of tactile stimulation, but according to electrophysiological criteria the habituation development in these cells was impeded. It is supposed that production of habituation is followed by hydrophobic-hydrophilic translocations of Ca2+-binding molecules in nerve cells.

The ultrastructural changes in the cat sensorimotor cortex during 30- to 60-minute recovery after a 2.5- to 6-minute anoxia have been studied. The most prominent changes were hypertrophy of Golgi apparatus, reorganization of the neuronal endoplasmic reticulum (including the formation of lamellar bodies), increase of the number of lysosomes within nerve cell bodies, formation of deep invaginations of the neuronal cytoplasm into the nucleus, intensification of endocytosis and phagocytosis in the dendrites, appearance of the ultrastructural heterogeneity of the synapses (from normal synapses to depleted ones), normalization of ultrastructure of the part of mitochondrial pool. Glycogen granules were revealed in glial cell processes. The ultrastructural changes after a 6-minute anoxia followed by a 30- to 60-minute recovery were more expressed than after a 2.5-minute one.

Contact microscopy of vital brain preparation revealed changes in bioelectric activity of the cat brain cortex during short--time anoxia following cAMP microionophoretic administration. cAMP at that acted as a protector of single pyramidal neurons as manifested in a decrement of "anoxia excitation" frequency and in an increasing number of previously "silent" neurons which could not be made active with an ordinary anoxia. The intracellular reductive equivalent accumulation rate also decreased essentially during anoxia. These effects depended on time interval after cAMP administration.

Changes in membrane-bound calcium (Ca2+(b)) content in the brain cortex membrane structures were studied on subcellular fractions (synaptosomes, microsomes, mitochondria) during in vitro anoxia. The changes in Ca2+ content in hydrophobic domains of intracellular membranes were assessed, using chlorotetracycline fluorescent probe. It has been found that membranes of different neuronal compartments are not equally vulnerable to anoxia. A decrease in Ca2+9(b) content in response to anoxia occurs in synaptosomes and microsomes much sooner than in mitochondria. Therefore, Ca2+ release from intracellular membrane compartments, preceding the massive inward flow of extracellular Ca2+, seems to be one of those mechanisms initiating a complex range of intracellular reactions to disturbed oxygen supply in brain cortex neurons.

The dynamics of calcium ions bound within intracellular membranes (Ca2+b), changes of redox--state and bioelectrical activity were studies in vital brain preparation after kainic acid (KA) application either to the micro--zones iontophoretically or to the whole surface of the preparation. The Ca2+b dynamics depended on KA dose and included release of Ca2+b as well as bounding of calcium attracted from extracellular space. The early period of the reaction consisted of an increased intracellular reduction equivalents and a release of Ca2+b. Theme correlating processes were followed by "seizure" activity specific for the KA effect. The calcium free bounding effect started to prevail with increasing KA dose.

Cobalt (Co2+) influence on calcium bound (Cab2+) dynamics detected by a fluorescent chlorotetracyclin probe (CTC), as well as bioelectric activity were studied in the feline vital brain cortex preparation with contact microscopy and microelectrode technique. It has been found that Co2+ applied on the preparation surface or injected ionophoretically into microzones under microscopy produced a distinct decrease of CTC-Cab2+ fluorescence intensity. This indicates a decrease of Cab2+ content in the hydrophobic membrane loci. The effect is reversible and depends on Co2+ concentration. Neuronal excitation appeared after the fluorescence decrease up to 5-12% to the start level. It has been concluded that Cab2+ content dynamics plays an important role in the Co2+ "seizure" discharges mechanisms in the cortex.

Effects of a vital dye--methylene blue--on brain neuronal structures were investigated in the alive preparations of mammal brains by means of contact optical microscope in falling polarization light. Tinctorial properties of neuronal cortical structures were studied under the influence of a number of experimental factors (illumination conditions, dye concentration, temperature regime and oxygen supply to the preparation). A certain dependence in the character of dyeing and bleaching neuronal cells on the factors mentioned above was stated. Conditions for appearance of the alternating photodynamical effect and measures to prevent it were clarified. Practical recommendations were given for application methylene blue in prevital morphological investigations of mammal central nervous system.

The ultrastructure of cat sensomotor cortex has been studied during a 15-minute recovery after 2.5-6-minute oxygen supply cessation. An increase of osmopholia of free and endoplasmic reticulum bound ribosomes was detected in addition to a great number of altered mitochondria with longitudinal crystal arrangement. Besides, numerous activated synapses, local destructive changes of membrane complexes in dendrite and myelinated axons cytoplasm and glycogen granule accumulation in neuroglia were noticed. During the recovery period more prominent changes were shown after a 6-minute anoxia than after a 2.5-minute one.

The time course of redox systems of some identified Helix pomatia neurons and their bioelectrical activity during exposure to cAMP and prostaglandin E2 under normoxia and anoxia was studied by means of a complex technique on the basis of contact vital microscopy. It was discovered that neuronal response to the substances was uncertain under varying conditions of neuronal oxygen supply. In the presence of preliminary exposure to cAMP and prostaglandin E2 the character of shifts in redox systems and bioelectrical activity occurring in the neurons during oxygen deprivation was to be changed.

The ultrastructure of the cat sensorimotor cortex was studied following 2.5 and 6 minutes of anoxia, resp. In the 6 minutes anoxia, the phenomenon of phagocytosis of synaptic buttons by dendrites was elucidated. Under anoxic condition, the newly formed thin dendritic protrusions at synaptic junctions encircle the synaptic buttons like amoeboid pseudopodia. The phenomenon of dendritic phagocytosis was demonstrated in dendrites with a dark cytoplasm only. In the 6 minutes anoxia, a rather large amount of axo-dendritic synapses was observed having the normal ultrastructure.

Microiontophoretic application of H+ and H+ + HCO-3 to optically monitored multipolar neurons in the caudal and intermediate bulbar chemosensitive areas increased their firing rate and induced discharges in previously "silent" neurons. A reverse effect occurred in some cases. An interrelationship was revealed between changes of the neurons redox-state and the parameters of their spike activity.

Changes of morphological and neurophysiological characteristics of pyramidal neurons in response to complete ischemia have been studied by means of vital microscopy of cat and rabbit cortex neurons in addition to some other cytological methods. These responses were found to be complex involving phasic changes in the neuron size, membrane potential and redox potential. Neurons of particular types responded to normo- and hypothermic ischemia differently. Pyramidal neurons retained their vital ability for the whole prolonged period of the complete ischemia (up to 40--60 min).

Using the complex methodical approach on the basis of in vivo microscopy and chlortetracycline fluorescent probe of the cat brain cortex preparation the authors studied the changes in the content of the bound calcium in the structures of II-III motor cortex layers in response to influence by metrasol. Correlation between rapid decrease of some part of the bound calcium and development of neuron seizures was discovered. Moreover process of calcium absorption by intracellular membranes in response to influence of metrasol in high doses was revealed. Apparently functional different intracellular membranes are involved in this process.