[Ultrastructure of afferent synapses on the ventral dendrite of mauthner neurons after goldfish adaptation to optokinetic stimulation].

Using the morphometric techniques, the ultrastructural changes of the afferent synapses on the ventral dendrite of the Mauthner neurons (MNs) were studied after the adaptation of goldfish to long-term fatiguing sensory (visual) stimulation, characterized by the growth of MN resistance. It was shown that after the adaptation, the length of active zones (AZs) in the synapses located on the MN ventral dendrite was significantly reduced by 23%. At the same time, the length the AZs of the excitatory visual synapses was reduced by 29% in comparison with the control, while the length of desmosome-like contacts (DLCs) bordering AZs was increased by 71%. It was also found that the length of AZs in the inhibitory synapses was decreased by 19% after the adaptation, which is consistent with the important role of inhibitory processes in the sensory pathways during the memory formation. Taking into account the actin nature of the DLCs, the basis of the adaptation to the visual stimulation is suggested to be in the presynaptic mechanism of neurotransmitter secretion regulation by actin.

Effect of dopamine on Ehrlich ascites carcinoma cells.

Morphological studies showed that daily intraperitoneal injections of dopamine in doses of 10(-2)and 10(-1)M down-regulates the general number of cells in the Ehrlich ascites carcinoma in 10 and 30 times and decreases their diameter by 27% and 59%, respectively (as compared to the control animals received physiological saline). According to ultrastructural data these injections were followed by the abnormal changes in microvilluses, forming the specific moire fringes in cytosol, thickening of cortical layer, and a significant increase in filament reticulum density (actin fibers) in tumor cells of treatment group specimens. We concluded that the oncocytotoxic effect of dopamine was related to the induced polymerization of cytosol actin.

[Dopamine as a possible substance for oncotherapy and for quantitative evaluation of cytosolic G-actin].

Viability, histology and ultrastructure of normal cells and cells of different degrees of malignancy after interaction with dopamine as well as the ability of these cells and isolated G-actin in model experiments to stain by Falck technique were studied. It is shown that dopamine, virtually having no effect on the viability of the "normal" non-tumorigenic transformed cells, noticeably reduces cell viability of slightly tumorigenic cells, causes a significant reduction in viability of attachable cancerous cells and a very significant decrease in cell viability of cancerous cells growing in suspension. The intensity of fluorescence of the cytosole in cells treated with dopamine, has been very high and varied in different cultures, and that of isolated actin directly depended on its concentration. Common to all cell morphological feature of damage from the action of dopamine and the putative substrate of fluorescence was actodopamine filaments network strands (identified on the structure and size), which appears in the cytosole loci, where they were absent in control. The data show that dopamine can be used as an oncotherapeutic remedy and diagnostic tool interacting with G-actin as a cellular target.

[Ultrastructure of Mauthner neurons after optokinetic stimulation and eye enucleation].

It was previously shown that the contralateral (relative to preferred side of turns) optokinetic stimulation and ipsilateral eye enucleation cause a significant, 2- to 4-fold reduction of the ventral dendrite (VD) volume in one of two goldfish Mauthner neurons (MN) that becomes more active functionally. In this study, we investigated the MN ultrastructure after mentioned unilateral visual effects. In both cases, devastation of the afferent synapses was detected along the full length of the reduced VD, with simultaneous compaction of its cytoskeleton, in contrast to those of VD of the contralateral MN and of lateral dendrites and cell bodies of both neurons. It is suggested that the depleted synapses belong to the excitatory visual afferent input, and both cytoskeletal and synaptic mechanisms are involved in the regulation of MN functional activity through VD.

[Morphological bases of dopamine effect on HEP-2 tumor cells viability].

The purpose of the present investigation was to study the morpho-functional organization of a classical object of cytological research - cultured HEp-2 tumor cells, using dopamine as a penetrating agent, inducing the polymerization of cytosolic actin. It was demonstrated that dopamine introduced into the incubation medium reduced viability and caused morphological disturbances of cultured HEp-2 cells; these effects were proportional to dopamine concentrations (1.0 x 10(-4) M to 1.0 x 10(-3) M) and exposure duration (2 to 3 days). These cells, according to ultrastructural data, underwent fusion and lysis because of the appearance of actin filaments network in the loci of globular actin prevalence in control cells. Dopamine receptors had no effect on cytotoxic effect of dopamine. This was indicated by fluorescent microscopical evidence of dopamine penetration into experimental cells in the presence of haloperidol, as well as destruction of HEp-2 cells under the action of pyrimidinethione, similar to dopamine by characteristics, but lacking its own receptors. It is suggested that cytoplasmic target for dopamine is globular actin and that induced polymerization of this cytoskeletal protein caused injury to tumour cells.

Morphofunctional changes in goldfish Mauthner neurons after application of beta-amyloid.

The effects of applying aggregated beta-amyloid peptide fragment 25-35 on the three-dimensional structure and volume of Mauthner neurons (MN) and on motor asymmetry were assessed in goldfish using reconstructions based on serial histological sections. These experiments showed that the motor asymmetry of the fish was stable in the intact state and in controls and correlated tightly with structural asymmetry of neurons. beta-Amyloid produced large changes or inversion in motor asymmetry, which did not coincide with or even contradicted the structural asymmetry of MN. This occurred as a result of marked dystrophy or, conversely, hypertrophy of individual neurons and their individual dendrites, with changes in their proportions. It is suggested that the harmful action of beta-amyloid on MN structure and the discordant ("incorrect") behavior of the fish may result from mechanical deformation evoked by its tape-like fibrils. Overall, the results lead to the conclusion that MN provide a suitable system for studying the structural aspects of amyloidosis.

[Cytochemical and ultrastructural characteristic of BHK-21 cells exposed to dopamine].

BHK-21 cells were incubated in a medium containing dopamine (DA) and then their catecholamine content evaluated by using the Falck cytochemical method. The significant intensification of cell fluorescence as compared to that one in control preparations was detected; this effect was proportional to DA concentration and exposure duration and was more pronounced in cells in suspension than in those attached to the substrate. Simultaneous ultrastructural investigation has shown that an increased intensity of the cytoplasm fluorescence correlated with the appearance of the dense network of fibrils that were morphologically identified as F-actin microfilaments. Prior blockade of dopaminergic receptors by haloperidol did not change the following DA effect both on the fluorescence intensity and cell ultrastructure. The data obtained suggest that DA chronically acting on the living cells was able to penetrate into the cytoplasm, causing actin polymerization and incorporating into the newly formed actin cytoskeleton. Structurally, this may be manifested by cytoskeleton and its derivative hypertrophy, that could have a substantial effect on general morphology of the cell.

[Morpho-functional changes in the goldfish Mauthner neurons after beta-amyloid application].

The influence of aggregated beta-amyloid peptide fragment 25-35 application on three-dimensional structure and volume of Mauthner cells (MCs), as determined by reconstruction from serial histological sections, and on goldfish motor asymmetry was studied. It was shown that in intact and control goldfish motor asymmetry was stable and strongly correlated with structural asymmetry of neurons. But under the influence of beta-amyloid, motor asymmetry appeared to be strongly changed or inverted, did not correlate with structural asymmetry and frequently even was opposite to it. This resulted from strong dystrophy or, on the contrary, hypertrophy of individual neurons and their separate dendrites with the change in the proportions between them. It is suggested that injurious effect of beta-amyloid peptide on MCs structure, discordant with ("irregular") fish behavior, could be the result of mechanical deformation, induced by ribbon-like fibrils of amyloid peptide. These findings collectively suggest that MCs are the adequate object for the study of the structural aspects of amyloidosis.

Changes in the ultrastructure and function of goldfish Mauthner neurons in the presence of 3,4-dihydro-2(1H)-pyrimidinethione.

The behavioral effects of 3,4-dihydro-2(1H)-pyrimidinethione (DPT, a pyrimidine derivative), which is used as a test system for detecting tumor growth, on the ultrastructure and function of Mauthner neurons (MN), were studied in goldfish. Application of DPT to MN was found to lead to increased resistance of neurons to exhaustive stimulation, which was accompanied by increases in the sizes of actin-containing membrane desmosome-like contacts, along with the formation of bundles of actin stress fibers; these effects are similar to those previously reported with dopamine. The similarity of the morphofunctional changes in MN on exposure to an artificial chemical substance for which there are no membrane receptors and dopamine itself suggests that they have trophic effects on the stabilization and polymerization of cytoskeletal actin due to direct penetration into postsynaptic neurons.

Effect of the inhibitory neurotransmitter glycine on slow destructive processes in brain cortex slices under anoxic conditions.

Slow destructive processes in brain cortex were studied under deep hypoxia (anoxia). Study of the character and dynamics of DNA destruction showed that apoptosis and necrosis run in parallel under the experimental conditions. These processes typically develop in tens of hours. A similar conclusion was reached from electron microscopic study of the tissue ultrastructure. More detailed study revealed that a relatively rare type of apoptosis not involving cytochrome c release from the intermembrane space of mitochondria and not associated with opening of the mitochondrial nonspecific pore occurs under the experimental conditions. As this is occurring, the process can be slowed by high concentrations of glycine, an inhibitory neurotransmitter. The study of DNA destruction demonstrated that high concentrations of glycine selectively slow apoptosis but have almost no effect on necrosis. Glycine also drastically decreases changes in the tissue ultrastructure, particularly of mitochondria, arising under anoxia. Glycine does not notably influence the mitochondrial oxidative phosphorylation system. Study of impairment of mitochondrial function demonstrated that the oxidative phosphorylation system is not disturbed for 1 h, which is several times longer than the inhibition time of brain function under deep hypoxia. The mitochondrial respiratory system is preserved for a relatively long time (24 h). Malate oxidase activity is deactivated after 48 h. The succinate oxidase fragment of the mitochondrial respiratory chain proved especially resistant; it retains activity under anoxia for more than 72 h. A possible mechanism of the effect of high glycine concentrations is discussed.

[Changes of the goldfish Mauthner neuron ultrastructure and function under the influence of 3,4-dihydro-2(1H)-pyrimidinethione].

The effects of pyrimidine derivative 3,4-dihydro-2(1H)-pyrimidinethione, (DPT) used as a test-system for detection of tumor growth, on the goldfish Mauthner neurons (MN) ultrastructure and function, as manifested in behavioral changes, were studied. The results of investigations demonstrated that an application of DPT on MN had the effects similar to those of dopamine application, as established earlier, causing the enhancement of MN resistance to fatigue stimulation, accompanied by an increase of the dimensions of the actin containing desmosome-like afferent admembranous synaptic contacts, and formation of the cytoplasmic bundles of actin stress-fibers. Similarity of morpho-functional changes of MN, induced by DPT, an artificial chemical substance, which has no receptors on the neuronal membrane, and by natural neurotransmitter dopamine, allows us to suggest possible trophic stabilizing and polymerizing effects of both substances on cytoskeletal actin due to their direct penetration into postsynaptic neuron.

The structure of mixed synapses in Mauthner neurons during exposure to substances altering gap junction conductivity.

The aim of the present work was to study the effects of dopamine, ecdysone, and chlorpromazine, substances which alter the conductivity of gap junctions (GJ), on the ultrastructure of mixed synapses in goldfish Mauthner neurons. These studies showed that dopamine, which increased the electrical conductivity of mixed synapses, appeared to target desmosome-like contacts (DLC). Hypertrophy of DLC, along with increases in the numbers of bridges within their clefts, showed that the mechanism by which dopamine increased electrical conductivity involved neuronal actin. This was indicated by the transformation of isolated monomeric muscle actin into polymerized actin in the presence of dopamine. Conversely, GJ were degraded by dopamine. Ecdysone, which also increased GJ conductivity, altered GJ structure, increasing the numbers of GJ at the attachment zone and decreasing the sectional length. but had virtually no effect on DLC structure. Ecdysone also showed no interaction with DLC in in vitro conditions. The mechanism of action of ecdysone is thus associated primarily with GJ function. Chlorpromazine, which decreased GJ conductivity, partially or completely degraded the fibrillar juxtamembrane material of DLC, preventing actin polymerization, with corresponding in vitro effects, but produced no changes in GJ. The mechanism of its action therefore appears to be based on changes in the state of neuronal actin.

Localization of calcium ions in mixed synapses of Mauthner neurons during exposure to substances altering the conductivity of gap junctions.

The pyroantimonate method was used to study the distribution of calcium ions in the mixed synapses of Mauthner neurons after exposure to substances altering the electrotonic conductivity of these synapses mediated by gap junctions (GJ). Ecdysone, an agent which increases GJ conductivity, produced precipitates of calcium pyroantimonate coating the whole postsynaptic surface of the GJ area, making them strongly asymmetrical. Precipitate granules were also seen to appear in the clefts of desmosome-like contacts (DLC). Chlorpromazine, which decreases GJ conductivity, produced precipitates in GJ clefts and on the pre- and postsynaptic membranes. No precipitate formed in DLC clefts. These results demonstrate that ecdysone acts as an agent selectively increasing GJ conductivity without affecting DLC function. Chlorpromazine had a double action, blocking conduction through both GJ and DLC. Thus, studies of agents altering GJ permeability require consideration of the possibility that they may interact with actin-containing structures also involved in the transport of the electrotonic signal.

Distribution of calcium ions in the mixed synapses of Mauthner neurons in the goldfish in normal conditions, in exhaustion, and in conditions of adaptation to exhaustion.

The aim of this study was to investigate the structure of large myelinated club terminals of Mauthner neurons (MN) in the goldfish at different levels of functional activity and the distribution within these synapses of calcium ions as assessed using a modified pyroantimonate method. In intact preparations, calcium pyroantimonate precipitates were not seen in gap junctions (GJ) or desmosome-like contacts (DLC). Fibrillar bridges in DLC clefts were not contrasted. After natural stimulation, which induces long-term adaptation in MN, GJ showed electron-dense precipitates lining the whole cleft. Granules and clumps of precipitate were also seen in DLC clefts, with intense deposition on bridges. Increases in calcium ion concentrations to and above the levels detectable by the pyroantimonate method are known to block electrotonic transmission; filamentous actin is known to conduct the electrotonic signal as a cation current. The staining of DLC bridges with calcium pyroantimonate is therefore evidence for an association between calcium ions and actin molecules, as DLC bridges consist of actin, i.e., we have obtained evidence for the functioning of bridges as electrotonic transsynaptic shunts at the moment of fixation. These data lead to the conclusion that DLC in mixed synapses, apart from the known adhesive functions, also have a communication function. This appears in extreme conditions, allowing the synapse to maintain or change its conductivity according to ongoing need.

[Calcium ions localization in mixed synapses of mauthner cells after exposure to substances changing the gap junction conductivity]. / Lokalizatsiia ionov kal'tsiia v smeshannykh sinapsakh mautnerovskikh neironov pri deistvii veshchestv, izmeniaiushchikh provodimost' shchelevykh kontaktov.

Using the pyroantimonate method, the distribution of calcium ions in Mauthner cell mixed synapses was studied following their treatment with substances changing gap junction (GJ) synapse electrotonic conductivity. Ecdyson, the drug known to increase GJ conductivity, induced the appearance of calcium pyroantimonate precipitates covering the whole postsynaptic area of GJs, making them highly asymmetric. Formation of precipitate grains was also observed in the cleft of desmosome-like contacts (DLCs). Chlorpromazine, substance that decreases GJ conductivity, induced the formation of precipitates in GJ cleft and on both pre- and postsynaptic sides of their surfaces. No precipitates were seen in DLC cleft. These results show that ecdyson acts as a substance selectively increasing the GJ conductivity with no effect on DLC function. Chlorpromazine has dual effect, by blocking the communication through both GJ and DLC. Thus, using chemicals that change GJ permeability, the possibility of their interaction with actin-containing structures which also participate in the electrotonic signal transfer, should be taken into consideration.

[Substances affecting gap junction conductivity and Mauthner neuron mixed synapses]. / Struktura smeshannykh sinapsov mautnerovskikh neironov pri vozdeistvii veshchestv, izmeniaiushchikh provodimost' shchelevykh kontaktov.

The aim of this work was to study the effect of dopamine, 20-hydroxyecdysone and chlorpromazine, drugs changing the conductance of gap junctions (GJs), on the ultrastructure of goldfish Mauthner neurons mixed synapses. It was shown that desmosome-like contacts (DLCs) were the presumable targets for dopamine, that increased the electrotonic conductance of mixed synapses. Their hypertrophy, as well as an increase in the number of bridges in their clefts suggests that neuronal actin is involved in the mechanism of dopamine-induced increase of electrotonic conductance. This assumption is further supported by the transformation of extracted monomeric muscular actin into polymeric actin in the presence of dopamine. On the contrary, GJs were shown to be damaged by dopamine treatment. Ecdysone, which is also known to increase the conductance of GJs, changed their structure, increasing their number in the zone of apposition and reducing the profile length, but practically not affecting the DLC structure. It also does not interact with isolated actin in vitro. Therefore, the mechanism of ecdysone action is mainly associated with GJ function. Chlorpromazine, which reduces the conductance of GJs, was shown to damage, partially or completely, DLC admembranous fibrillar material, thus preventing actin polymerization, as shown by in vitro experiments, but had no effect on GJs. Therefore, the mechanism of its action, appears to be based on the changes in the state of neuronal actin.

[Calcium ions distribution in mixed synapses of the mauthner neurons of goldfish in the norm, fatigue, and adaptation state]. / Raspredelenie ionov kal'tsiia v smeshannykh sinapsakh mautnerovskikh neironov zolotoi rybki v norme, pri utomlenii i pri adaptatsii k nemu.

The aim of this investigation was to study the structure of giant myelinated club-shaped terminals (afferent mixed synapses) of goldfish Mauthner (M-) cells in different functional states and to demonstrate calcium ion localization in them using modified pyroantimonate method. It was shown that in intact preparations calcium pyroantimonate precipitate was detected neither in gap junctions (GJ) nor in desmosome-like junctions (DLJ). The fibrillar bridges within DLJ cleft were not contrasted. After natural stimulation, which elaborated a long-term adaptation of M-cells, electron dense precipitate was found in GJ, lining all the cleft. Simultaneously fine granules and aggregates of precipitate appeared in DLJ gap and were intensely deposited over the bridges. It is known that the increase of calcium ion concentration up to and above the level demonstrable by pyroantimonate method blocks the electrotonic coupling and that filamentous actin is able to conduct electrotonic signal as a cationic current. Therefore calcium pyroantimonate staining of DLJ bridges, which were earlier shown to contain actin, indicates the association of calcium ions with filamentous actin, i.e. the functioning of bridges as transsynaptic electrotonic shunts at a moment of fixation. The data obtained allow to make a conclusion that DLJ in mixed synapses have not only a known adhesive function, but also a communicative one. The latter is manifested in extreme conditions, thus permitting synapse to maintain or change their conductivity in accordance with environmental demands.

[Morphofunctional features of microtubule ultrastructure and MAP2 protein phosphorylation in hippocampal neurons of rats with predisposition to audiogenic epilepsy]. / Morfofunktsional'nye osobennosti ul'trastruktury mikrotrubochek i fosforilirovaniia belka MAP2 v neironakh gippokampa krys, predraspolozhennykh k audiogennoi épilepsii.

It has been shown that modification of microtubule (MT) ultrastructure are accompanied by functional changes in microtubule-associated protein MAP2 in the hippocampus of Krushinsky--Molodkina rats (KM), which are prone to autogenic seizures. The morphogenetic analysis revealed that contrary to Wistar rats, which are insensitive to sound stimulation, in KM the middle length of microtubule fragments in the apical dendrites of pyramidal neurons in CA3 hippocampal area was reduced. Using immunoblot and autoradiography methods, we found that the level of MAP2 and the rate of its cAMP = and Ca(2+)-calmodulin-dependent phosphorylation were increased in hippocampus of KM, in comparison with Wistar rats. Daily repeated sound stimulation for 20 days (audiogenic kindling) induced a further decrease in length of MT fragments, and an increase of their density in the proximal part of apical dendrites of KM. Moreover, audiogenic kindling induced additional increase in MAP2 phosphorylation state, but did not change the level of MAP2 in KM hippocampus. We suppose that the obtained alteration of MAP2 phosphorylation state exerted influence on kinetic parameters of microtubule assembly, serving as part of genetically determined predisposition of KM to audiogenic epilepsy.

[Primary culture of epithelial secretory cells from venom gland of the common adder Vipera berus]. / Pervichnaia kul'tura kletok sekretornogo épiteliia iadovitoi zhelezy gadiuki obyknovennoi Vipera berus.

A primary culture of epithelial secretory cells from the venom gland of Vipera berus was obtained. The cells adhered to collagen 1 and to a mixture of adhesion proteins (Matrigel), proliferated and retained the features of differentiation. Electron microscopy demonstrated the presence of all ultrastructures typical of these cells in vivo, a full complex of intercellular junctions, and cellular membrane polarity. The immunohistochemistry confirmed the capacity of secretory cells to synthesize venom in culture. We have studied the role of carbochole, an agonist of M-cholinoreceptor, in the initiation of the secretory cycle in cells in vitro. We propose that M-cholinoreceptors may play an important role in the initiation of the secretory cycle in vivo.