N. R. Tiras

Visual input controls the functional activity of goldfish Mauthner neuron through the reciprocal synaptic mechanism

Goldfish are known to exhibit motor asymmetry due to functional asymmetry of their Mauthner neurons that induce the turns to the right or left during free swimming. It has been previously found that if the less active neuron is subjected to prolonged aimed visual stimulation via its ventral dendrite, the motor asymmetry of goldfish is inverted, testifying that this neuron becomes functionally dominant, while the size of the ventral dendrite under these conditions is reduced 2-3 times compared to its counterpart in mirror neuron. Earlier it has been also revealed that training optokinetic stimulation induces adaptation, a substantial resistance of both fish motor asymmetry and morphofunctional state of Mauthner neurons against prolonged optokinetic stimulation. The aim of this work was to study the cellular mechanisms of the effect of an unusual visual afferent input on goldfish motor asymmetry and Mauthner neuron function in norm and under adaptation. It was shown that serotonin applied onto Mauthner neurons greatly reduces their activity whereas its antagonist ondansetron increases it. Against the background of visual stimulation, serotonin strengthens functional asymmetry between neurons whereas ondansetron smoothes it. Taken together these data suggest the involvement of serotonergic excitatory synaptic transmission in the regulation of Mauthner neurons by vision. Ultrastructural study of the ventral dendrites after prolonged optokinetic stimulation has revealed depletions of numeral axo-axonal synapses with specific morphology, identified by means of immunogold label as serotonergic ones. These latter in turn are situated mainly on shaft boutons, which according to specific ultrastructural features are assigned to axo-dendritic inhibitory synapses. Thus, the excitatory serotonergic synapses seem to affect Mauthner neuron indirectly through inhibitory synapses. Further, it was morphometrically established that adaptation is accompanied by the significant decrease of active zones dimensions in both serotonergic and inhibitory synapses. Finally, it was determined in model experiments that the interaction of globular actin with glycine, a main inhibitory neurotransmitter supposedly directly and chronically affecting the ventral dendrite, results in actin filaments formation. It is assumed that glycine-induced cytosolic actin polymerization is a cause of reduction in the ventral dendrite size under stimulation. Thus, it was established that a rather small group of synapses situated on an individual dendrite of the neuron determines the execution of the important form of animal behavior.

Ultrastructure of Mauthner Cells in Fish Adapted to Long-Duration Vestibular Stimulation and the Effect of Ethanol

Adaptation or resistance of fish Mauthner cells (M-cells) to long duration (2 h) vestibular stimulation (LDS)was produced by daily brief and gradually increasing vestibular stimulation (training). The LDS resistance was accompanied by an increase in the number of desmosome-like junctions in the afferent axosomatic synapses. F-actin, the main component of desmosome-like contacts, has been suggested to be responsible for the increased resistance of M-cells to LDS. The purpose of the present study was to investigate the capacity of M-cells to adapt to LDS under the influence of ethanol, which alters the content of F-actin in cells. The experiments were carried out in goldfish fry. Vestibular stimulation (training and LDS) was performed in special drums that were rotated in two planes. The training time was increased from 1 min on day 1 to 30 min on day 30. For ethanol exposure, fish were immersed daily in a 2% ethanol solution for 20 min. To assess the level of resistance to LDS, motor activity indicating the functional state of M-cells was evaluated before and after LDS. The results show that exposure to ethanol reduces the resistance to LDS in both untrained and trained fish. Electron microscopic data demonstrated some structural changes in the synaptic endings located on M-cell soma in ethanol-exposed fish. Wrapping of boutons by cytoplasmic out- growths and myelin- like structures was observed. Morphometric analysis revealed that exposure to ethanol without training decreases the number of desmosome-like contacts, probably due to ethanol-induced depolymerization of cytoskeletal actin. Ethanol exposure also partly suppressed the increase in the number of desmosome-like contacts that occurs as a result of training. In ethanol-treated trained fish, however, a concomitant increase in the length of desmosome-like contacts was observed. As training alone leads to the formation of additional desmosome-like contacts of standard length, it is possible that although a sufficient amount of such structures cannot be formed in the M-cells of ethanol-exposed trained fish, the existing contacts can be elongated. Thus, possibly changes of the actin state are involved in the adaptation of M-cells to LDS.

Correlation between the Sizes of Individual Parts of Mauthner Neurons in Goldfish and Their Integral Function after Enucleation of the Eye

Three-dimensional reconstruction was used to study the structural correlates of significant increases in the activity of Mauthner neurons (MN) deafferented after unilateral enucleation of the eye evidenced by an irreversible shift in motor asymmetry in goldfish towards the “blind” side. The results showed that in some cases, the dominance of the MN correlated significantly with a decrease in the volume of its ventral dendrite, while in others, it correlated with an increase in the sizes of the body and the lateral dendrite. Both structural signs appeared to result from local redistribution of the neurotransmitter balance due to the tension of the undamaged sensory inputs. Overall, the results demonstrated that prolonged adaptive changes in the behavior of the fish may be regulated by specific morphological rearrangements of MN at the level of individual dendrites acting via feedforward and feedback mechanisms.

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.

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.

Morphofunctional Changes in Incubated Mauthner Neurons in Goldfish Treated with Peptides from Scorpion Venom

Electron microscopy with negative contrast showed that direct interaction of one of the peptide fractions of scorpion venom with monomeric chromatographically pure actin led to polymerization of actin, transforming it from the globular form to the fibrillar form. The effects of prolonged orthodromic stimulation on the evoked electrical activity and ultrastructure of Mauthner neurons (MN) were studied in incubated slices of goldfish medulla oblongata in the presence of this actin-polymerizing venom fraction. Peptides in this fraction were found to stabilize the amplitude of the electrical response of MN to exhaustion and to protect the ultrastructure of afferent chemical synapses and the neurons themselves from damage induced by stimulation. Enhancements in morphofunctional resistance were accompanied by stabilization of actin-containing specialized synaptic structures – desmosome-like contacts. The data obtained here provide evidence that peptides of this fraction of scorpion venom have direct actions on the actin component of the MN cytoskeleton and demonstrate potential for its use as a pharmacological tool able to penetrate living cells with value for studying the role of actin in the mechanisms of adaptation and memory.

Structural differences between desmosome-like contacts in afferent chemical and mixed synapses of Mauthner neurons in the goldfish.

the cellular level on the ability of synapses to undergo stable, long-term changes in their conductivity or efficiency. Signal conduction in synapses occurs mainly in the area at which the presynaptic bouton makes contact with the postsynaptic neuron via specialized contact structures. It is generally recognized that signal transmission in synapses of the chemical type takes place within an active zone (AZ) [17, 21]. In mixed-type synapses, where transmission includes chemical and electrotonic components, gap junctions (GJ) are present along with the AZ [12], to provide for electrotonic communication [13]. It has been suggested that long-term changes in synapse conductivity are associated with longterm structural changes in the corresponding conduction zones [8, 19, 20]. These two types of specialized junctions in the contact zones of both chemical and mixed synapses are supplemented by desmosome-like contacts (DLC), also termed puncta adhaerentia ; the structure of these is based, as demonstrated previously [11, 15], on filamentous actin (F-actin) and they have a characteristic distribution in the contact zone. In chemical synapses, they are located beside or around AZ [14, 23]. In mixed synapses, they surround GJ [20, 24]. In terms of their structural organization, the DLC of chemical and mixed synapses are very similar. The presumptive adhesional, mechanical function which they have in synapses and, it seems, their identical compositions, give no reason to expect any differences to exist between the DLC of different synapses. Therefore, the question of the existence of any kind of difference between them has not even been posed to date. However, new experimental data which we have obtained in studies of mixed synapses suggest that the DLC of these synapses perform not only an adhesional function, but may also have a role in the transmission of the transsynaptic electrotonic signal [7, 19]. This suggestion is also supported by data showing that F-actin can conduct an electric current along its molecules both in water [18] and when it is inserted into a bilayer membrane [1, 16]. The association of two functions in a single type of DLC in mixed synapses should be reflected in some way in their overall structure and should lead to differences from the DLC of chemical synapses, which do not have electrotonic transmission [25] and have only one – the adhesional – function. The aim of the present work was to perform comparative electron-microscopic investigations of the two types of DLC in afferent synapses of the chemical and mixed types in Mauthner neurons (MN) in the goldfish.

Induction of Morphological Resistance of Neurons to β-Amyloid

The effects of training with adaptive vestibular stimulation on the function and three-dimensional structure of Mauthner neurons (MN) were studied in goldfish with experimental amyloidosis induced by application of aggregated β-amyloid protein (Aβ25–35). In comparison with controls, adapted MN acquired significant resistance to Aβ25–35. Considering the key role of dopamine in the adaptation of MN to sensory stimuli, its actions on the development of an amyloidosis model were studied; the results showed that application of dopamine to MN, like increases in the dopamine concentration induced by L-DOPA, was followed by protection of MN structure and function from the pathogenic actions of Aβ25–35. Electron microscopic studies showed that the protective action of dopamine on neurons can be explained in terms of its ability to dissociate β-amyloid polymers into short inactive fragments.

Ultrastructure of Afferent Synapses on the Ventral Dendrites of Mauthner Neurons During Adaptation of Goldfish to Optokinetic Stimulation

Morphometric methods were used to study the ultrastructure of afferent synapses on the ventral dendrites of Mauthner neurons (MN) during adaptation of goldfish to prolonged, fatiguing, sensory (visual) stimulation inducing increased resistance of MN. The extent of active zones (AZ) in synapses located on the MN ventral dendrite was found to decrease significantly, by 23%, after adaptation. The extent of the AZ of excitatory visual synapses decreased by 29% as compared with controls, with a simultaneous 71% increase in the extent of desmosome-like contacts (DLC) bordering AZ. There was also a 19% reduction in the extent of AZ of inhibitory synapses after adaptation, which is consistent with the important role of inhibitory processes in sensory pathways during memory formation. Considering the actin nature of DLC, it can be suggested that adaptation to visual stimulation is based on a synaptic mechanism of regulation of neurotransmitter secretion by actin.