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Objectives. To study the structure and formation of Mauthner neuron dendrites in goldfish exposed to neurotoxic β-amyloid fragment 25–35 and prolonged sensory stimulation influencing the afferent inputs to these neurons. Materials and methods. Goldfish Mauthner neurons were studied by light and electron microscopy. Individual dendrites were identified, their volumes were determined, and synapse structure was assessed using virtual 3D images of Mauthner neurons obtained from serial sections of thickness 3 μm. The functional status of Mauthner neurons was evaluated indirectly from the motor lateralization of the fish. Results. Mauthner neurons responded to application of β-amyloid combined with subsequent prolonged sensory stimulation with decreases in the volume of the ventral dendrites, damage to their ultrastructure, and degeneration of a proportion of synapses. Degeneration of more active neurons was more significant than that of less active cells. Newly formed medial dendrites had greater volume and less damaged synapse ultrastructure than ventral dendrites. There were no differences in the sizes of specialized junctions between synapses of the same type on ventral and medial synapses. Conclusions. Medial dendrite formation is a compensatory reaction to dystrophy of the ventral dendrite due to the experimental treatment.

We examined effects resulting from application of aggregated beta-amyloid on Mauthner neurons (MNs) of ambidextral goldfishes and long-lasting rotatory stimulation on lateral motor behavior of such fishes; these individuals were preliminarily adapted to repetitive rotatory stimulation. Using 3D histological reconstruction of the above cells, we measured the volumes of the right and left MNs and examined the ultrastructure of specialized synaptic compartments in excitatory synapses localized on the surface of the soma, lateral dendrite, and ventral dendrite of these cells. In the course of the experiments, control fishes remain ambidextrals, and the mean volumes of the right and left MNs in these individuals were close to each other. In a fish of this group, dimensions of the active zones (AZs) of chemical synapses localized on the lateral dendrite were considerably greater in the right neuron, while those in synapses on the lateral dendrite were smaller in the left neuron. Experimental fishes subjected to the above-mentioned procedures demonstrated clearly expressed motor lateralization; the mean volumes of their right and left MNs were, however, practically equal to each other. Ultrastructural analysis of peculiarities of MNs of such a fish showed that dimensions of the AZs in chemical synapses on the soma and lateral dendrite of the left MN, which became much less active functionally, significantly exceeded the analogous indices in synapses of the same localization on the right, more active, MN. In mixed synapses, the mean dimensions of gap junctions in control and experimental individuals were similar to each other independently of localization of such synapses. Interrelations between functional asymmetry of MNs manifested in lateralization of motor behavior of the fishes and dimensions of synaptic structures on these cells are discussed.

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.

We studied the correlation between functional activity and 3D structure of Mauthner neurons (MNs) of the goldfish in the norm, after isolated application of an aggregated fragment of beta-amyloid 25-35 (Aβ25-35), and a combination of such application with optokinetic stimulation of the fish. It was shown that the clear correlation between the structure and function of MNs observed in the control is strongly disturbed after the action of beta-amyloid aggregates. According to the data of ultrastructural analysis, destruction of the cytoskeleton, vacuolization of the MN cytoplasm, and depletion of the vesicular apparatus of afferent synapses are the main events responsible for such effect of Aβ25-35. Fatiguing sensory optokinetic stimulation of fishes applied against the background of action of Aβ25-35 induces dystrophy of the ventral MN dendrites and the formation, near the latter, of earlier absent hypertrophied medial dendrites; these dendrites were comparable in their dimension with the main MN dendrites in control fishes. The data obtained allow us to suppose that a traumatic mechanical influence of the beta-amyloid aggregates on neurons and their processes is an important factor in the development of amyloidosis-related neurodegenerative diseases, and destruction of the cytoskeleton is one of the key phenomena from this aspect.

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.

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.

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.

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.

We studied the effects of unilateral enucleation of the eye on the motor asymmetry of goldfish fries and morphometric characteristics of their Mauthner neurons, MNs (data of 3D reconstruction using serial slices). Enucleation of the right or left eye in ambidextral fishes resulted in stable preference of turnings during swimming toward the side of visual deafferentation and in a shift of the initial motor asymmetry coefficient (MAC) by 25%, on average. Ipsilateral enucleation of the eye in dextral and sinistral fishes intensified the initial motor asymmetry by 20%. Contralateral enucleation of the eye in dextral and sinistral fishes induced inversion of the motor asymmetry with a decrease in the MAC by 50%, on average. All operated fishes stably (within 3 months or more) preferred to turn toward the side of the enucleated eye. Morphological measurements showed that the size of the ventral dendrite of the MN contralateral with respect to the side of enucleation noticeably decreased. At the same time, this neuron (according to a shift in the motor asymmetry of the fish) became the functionally dominant unit. These data agree with the earlier supposition on a reciprocal relation between the level of functional activity of the MN and the size of its ventral dendrite. Selective changes in the dimension of the ventral dendrite of the MN receiving visual inputs probably resulted from the action of some endogenous trophic factor. The effect of this factor was enhanced after enucleation of the eye and the corresponding dysfunction of the contralateral visual input. A specific local change in the balance of neurotransmitter influences upon the MN ventral dendrite can play the role of such a factor. From the neurophysiological aspect, a decrease in the size of the ventral MN dendrite is a primary link in the chain of events resulting in modification of behavior; it leads to intensification of the integral functional activity of the MN and a shift of the motor asymmetry of the fish compensating, in such a way, the effect of unilateral visual deprivation.

In goldfishes, we studied (i) manifestations of functional activity of Mauthner neurons (MNs) reflected in motor behavior and (ii) changes in 3-D morphometry (ratio of volumes) and ultrastructure of MNs after applications of an actin-polymerizing peptide obtained from scorpion venom and after vestibular rotational stimulations (trainings) inducing natural modification of functions of the MNs (adaptation). In MNs subjected to application of the peptide, the increase in the functional resistance and morphological stability caused by long-lasting stimulation directly depended on the dose of the applied peptide or on the effectiveness of trainings, whereas in intact and control MNs such stimulation resulted in significant decreases in the activity and volumes of these cells. At the ultrastructure level, both applications of the peptide and trainings caused the formation of extensive bundles of actin filaments (“stress-fibers”) in the cytoplasm of MNs and led to an increase in the dimension of desmosome-like contacts (DLCs) in afferent synapses. At chemical synapses, the effect looked like a reciprocal decrease in the length of the active zones (a structural sign of long-term depression, LTD), while at mixed synapses this was manifested in an increase in the number of fibrillar bridges in the gaps of DLCs (a structural sign of long-term potentiation, LTP). The data obtained allow us to hypothesize that LTD of the efficacy of transmission through chemical synapses is involved in the formation of the adaptation state of the MNs and that polymerization of actin in the cytoplasm and DLCs underlies the mechanism of LTD and adaptation. The development of ultrastructural manifestations of LTP at mixed synapses after polymerization of actin by the peptide, which is related to a reciprocal increase in the efficacy of “mixed” afferent inputs, explains the maintenance of a high integral level of activity of the MNs, despite a drop in the functional activity of “ chemical” afferent inputs. Therefore, the actin cytoskeleton plays a clearly significant role in the maintenance of the balance of excitation at the neuronal level.

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.

We examined the morphological peculiarities of Mauthner neurons, MNs, in goldfishes with a phenotypically different or an experimentally modified preference to perform rightward vs leftward turnings in the course of motor behavior; this preference was characterized by values of the motor asymmetry coefficient (MAC). 3D reconstruction of MNs was performed based on several histological sections; volumes of the soma, lateral and ventral dendrites (LD and VD, respectively), initial segment of the axon, as well as full volumes of the right and left neurons, were calculated. Differences between the above parameters were expressed as structural asymmetry coefficients (SACs). It was shown that clear orientation asymmetry of motor behavior of the fish is accompanied by differences in the dimensions of MNs and their compartments; MNs localized contralaterally with respect to the preferred turning side were considerably bigger than ipsilateral neurons. Experimental influences inducing inversion of the motor asymmetry of fishes inverted structural asymmetry of their MNs. In fishes with no phenotypical preference of the turning side and in individuals whose motor asymmetry was smoothed due to experimental influences (rotational stimulations), structural asymmetry of the MNs was also smoothed. Changes of the structural proportions developed, as a rule, due to decreases in the dimensions of one or both MNs and their compartments. The MAC value was in direct correlation with the value of SAC of the MNs and with values of this coefficient for the soma and the sum soma + LD. At the same time, reciprocal relations were found for the MAC and structural asymmetry of the VD; the decrease in the volume of VD was related to an increase in the preference of the contralateral turning side by the fish, and vice versa. In general, the results of our study demonstrate that both morphological and functional peculiarities of MNs correlate to a significant extent with such a form of motor behavior of fishes as realization of spontaneous turnings.

We studied changes in the motor asymmetry of the goldfish induced by single-session long-lasting vestibular stimulations (clockwise and counter clockwise rotations around the rostro-caudal body axis) and repetitive everyday short sessions of such stimulation (training); the latter mode led to the development of adaptation (resistance to fatigue). Rotational stimulation of different durations and directions elicited effects of different patterns and intensities. Such stimulation enhanced or, vice versa, smoothed the motor asymmetry in “dextral” and “ sinistral” fishes, up to full symmetry or even a change of the preferred turning direction. Adaptation to unilateral rotational stimulation allows an experimenter to selectively and gradually induce the resistivity of the left-or right-ward asymmetry to fatigue effects. Earlier, we found that the motor asymmetry in the goldfish, which is determined by the functional asymmetry of the brain, correlates with the morphological asymmetry of Mauthner neurons localized in the medulla in a mirror manner and playing a crucial role in the control of turnings in the course of locomotion (swimming). Experimental rotational stimulation-induced gradual modification of the motor asymmetry in the goldfish can serve as a physiological model for more detailed studies of the structural base of the functional brain asymmetry and some mechanisms of adaptation on the level of single neurons.

We examined changes in the ultrastructure of afferent mixed synapses on the membrane of Mauthner neurons (M cells) of the goldfish, which were related to two functional states, long-term potentiation (LTP) of the electrotonic response (a model form of the memory trace) and adaptation (resistivity to fatigue resulting from long-lasting motor training and considered a natural form of the memory trace manifested on the neuronal level). LTP was induced in medullary slices using high-frequency electrical stimulation of the afferent input. Adaptation was produced using natural vestibular stimulation (everyday motor training, which modified motor behavior of the fish and function of the M cell). It was supposed that if the LTP phenomenon is involved in the formation of natural memory, both the adaptation and the LTP states should be accompanied by similar specific structural modifications. Indeed, it was found that in both cases the number of fibrillar bridges in the gaps of desmosome-like contacts (DLC) in the mixed synapses on the M cell surface demonstrated an about twofold increase. These bridges are known to include actin filaments, which function as conductors of cationic signals; thus, the LTP-related increase in the density of bridges corresponds to increased efficacy of electrotonic coupling via mixed synapses. Such a structural correlate of LTP, which probably has the same functional significance in mixed synapses of the “adapted” M cells, allows us to suppose that LTP is a natural property of the nervous system. The LTP-type intensification of the relay function of mixed synapses, which corresponds to adaptation, is probably a compensatory rearrangement allowing M cells to maintain some balance of the synaptic influences and, at the same time, to remain in a stable and plastic state; this is necessary for stable functioning under changing environmental conditions.

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.

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.

The effects of phalloidin, a preperation which highly specifically and selectively polymerizes actin and which binds to actin, on the electrotonic conductivity and structure of mixed synapses were studied in goldfish Mauthner neurons (MN). These experiments showed that paired subthreshold electrical stimulation of the afferent input in the presence of phalloidin led to increases in the amplitude of MN responses to the second stimulus by an average of 80%. In controls, this amplitude increased by only 10% and only when suprathreshold stimuli were used, while subthreshold stimuli were ineffective. We regard these results as demonstrating increases in the conductivity of mixed synapses, this being induced by polymerization of actin. At the ultrastructural level, application of phalloidin to MN and their mixed synapses induced increases in the sizes and numbers of actin-containing desmosome-like contacts, and in the numbers of fibrillar bridges in the clefts of these contacts. Use of colloidal gold as a label for phalloidin demonstrated that bridges were made of actin. The interdependent morphofunctional changes seen in mixed synapses provide grounds for suggesting a role for actin in the conduction of the electrotonic signal through mixed synapses. The structural substrate for this process may be provided by bridges in the clefts of desmosome-like contacts.

Three-dimensional computer reconstruction working from serial histological sections was used to study the morphology of the right and left Mauthner neurons (MN) in goldfish fry showing marked preferences to turn stably to the right or left in a narrow water channel or showing no asymmetry in their choice of side during turns. Visually, fish with left-sided motor asymmetry had larger MN on the right side, while fish with right-sided motor asymmetry had larger MN on the left side. Fish with symmetrical turns to the right and left showed no differences in MN size. Quantitative assessment of the MN of fish with preferences for turns to one side or the other revealed significant differences in the sizes of the somatic part, the axon hillock, and the axons of neurons located on the contralateral side of the medulla oblongata. Analysis of the statistical relationships between the functional (motor) asymmetry of fish and the morphological asymmetry of the somatic parts of MN in the same fish revealed a stable correlation (0.69) between these measures. Given that MN initiate unilateral turns of the body in free movement, the data obtained here lead to the conclusion that the larger neuron is more frequently activated in natural conditions as compared with the smaller, contralateral, neuron.

The potentiated afferent mixed synapses of the Mauthner cells of fry and adult goldfish in stumps of the medulla oblongata incubated long-term in vitro were studied by electrophysiological and electron microscopic methods. It was shown that brief high-frequency stimulation of posterior branches of the eighth nerve induced a long-term potentiation of electrotonic transmission at large and small mixed club endings. It was about 135% upon subthreshold stimulation and about 200% upon suprathreshold stimulation. The ultrastructural analysis of ultrathin sections of potentiated mixed synaptic endings revealed an increase in the dimensions of desmosome-like contacts which was proportional to the degree of potentiation, about 135% or 200%, depending on the type of stimulation. The dimensions of gap junctions remained unchanged. The dimensions of active zones at potentiated synapses were reduced two-fold as compared with their unpotentiated counterparts, irrespective of the type of stimulation. Considering that desmosome-like contacts consist predominantly of F-actin, a molecule which possesses electroconductivity, it can be assumed that this cytoskeletal protein is involved in the process of potentiation. The increase in the synapse electrical conductivity can be mediated either directly, by shunting the synaptic junction with polymer actin filaments in the region of desmosome-like contacts, or indirectly, via the interaction of actin with gap junction connections situated nearby.