E. N. Bezgina

Morphofunctional effects of applications of glutamate and dopamine on the goldfish Mauthner neurons

In the goldfish, we studied the effects of intramedullar applications of glutamate (Glu), dopamine (DA), and of long-lasting rotational stimulation on the functional activity, dimensional characteristics, and ultrastructure of Mauthner neurons (MNs). Applications of Glu, especially when combined with rotational stimulation, were found to result in suppression of the function of MNs, in a decrease in their dimensions and lengths of desmosome-like contacts (DLCs, whose structure is determined by filamentous actin) in afferent mixed and chemical synapses, and in destruction of actin microfilaments in the cytoskeleton of MNs. Applications of DA, vice versa, induced an increase in the resistance to the effects of long-lasting stimulation and stabilized the dimensions of MNs; the length of DLCs increased in afferent synapses of both the above types, and the number of fibrillar actin bridges in the DLC cleft of mixed synapses also increased. Bundles of the actin filaments, which were preserved after stimulation, appeared in the cytoskeleton of MNs. Testing of the action of neurotransmitters on actin preparations in vitro showed that Glu entirely depolymerizes filamentous actin, while DA, vice versa, polymerizes monomeric actin. Thus, the Glu-and DA-induced reactions are similar in their types and are of a reciprocal nature both in the actin cytoskeleton of MNs in situ and in purified actin in vitro; these effects correlate with suppression of the functional state of MNs under the influence of Glu and with stabilization of this state under the influence of DA. These results agree with the concept on the roles of depolymerization and polymerization of actin in changes of the morphofunctional state of MNs and show that actin of the cytoskeleton of MNs is a cellular target for the actions of Glu and DA. The similarity between the effects of tested neurotransmitters on actin in MNs in situ and in cell-free preparations in vitro allows us to hypothesize that these transmitters can penetrate into the neuron.

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 specifi c moiré fringes in cytosol, thickening of cortical layer, and a signifi cant increase in fi lament reticulum density (actin fi bers) in tumor cells of treatment group specimens. We concluded that the oncocytotoxic effect of dopamine was related to the induced polymerization of cytosol actin.

Ultrastructure of Mauthner Neurons in Optokinetic Stimulation and Enucleation of the Eye

Previous studies have shown that contralateral (to the preferred turning side) optokinetic stimulation and ipsilateral enucleation of the eye lead to significant (by a factor of 2–4) decreases in the volume of the ventral dendrite (VD) in one of the two Mauthner neurons (MN) of the goldfish, which becomes functionally more active. We report here our studies of MN ultrastructure after these unilateral influences from the visual system. In both cases, the whole length of the shrunken VD showed emptying of synapses and compaction of its cytoskeleton as compared with the cytoskeleton of the VD of the contralateral MN and the cytoskeleton of the lateral dendrites and bodies of both neurons. It is suggested that emptied synapses are part of the excitatory visual input and that the control of the functional activity of MN via VD involves both cytoskeletal and synaptic mechanisms.

Dopamine as a possible substance for oncotherapy and for quantitative valuation 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 cytosol 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 cytosol 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.

Cytochemical and Ultrastructural Characteristics of BHK-21 Cells Exposed to Dopamine

BHK-21 cells were incubated in medium containing dopamine (DA) and catecholamine contents were then measured using the Falck cytochemical method. As compared with controls, significant increases were seen in the fluorescence of cells and these were proportional to the concentration and duration of exposure to DA and more marked in cells in suspension than in attached cells. Parallel electron microscopic studies showed that the increased fluorescence intensity of the cytoplasm correlated with the presence of dense networks of fibrils which were identified on the basis of morphological characteristics as microfilaments consisting of F-actin. Prior blockade of dopaminergic receptors with haloperidol did not alter the subsequent effects of DA on fluorescence intensity or cell ultrastructure. These data suggest that, in conditions of chronic exposure, DA can penetrate into the cytoplasm, inducing actin polymerization and becoming bound to the newly formed actin cytoskeleton. Structurally, this can be apparent as hypertrophy of the cytoskeleton and its derivatives, with significant influences on the overall structure of the cell.

Morphofunctional Changes in Goldfish Mauthner Neurons after Application of β-Amyloid

The effects of applying aggregated β-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. β-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 β-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.

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.

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.

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.

The Neuroprotective Effect of the Thr–Ser–Lys–Tyr Peptide in a Goldfish Mauthner Cell Model in vivo

The effects of the Thr–Ser–Lys–Tyr peptide, which was shown to display neuroprotective activity in cell cultures in vitro, were studied in the model of paired Mauthner cells of goldfish. It was found that intracerebral injections provided the peptide to be applied into the zone of the right Mauthner cell under the fourth ventricle of the hindbrain lead to a dose-dependent decrease in the number of spontaneous turns of the goldfish to the left. It was shown that this effect is not eliminated under long-lasting optokinetic stimulation when the fish instinctively follow stimuli with a low spatial frequency that are moving in the nasal-to-temporal direction. We used the method of three-dimensional reconstruction by serial histological sections to study the dendrite morphology of the Mauthner cells in control and experimental goldfish. It was found that optokinetic stimulation of control goldfish evokes the dystrophy of the ventral dendrite of the right Mauthner cell, which is the target of this type of stimulation. Conversely, the peptide stabilize the size of the ventral dendrite of the right Mauthner cell under stimulation. These data could be interpreted as evidence of the neuroprotective effect of the Thr–Ser–Lys–Tyr peptide in vivo.