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.

Involvement of actin in the electrotonic conductivity of mixed synapses in Mauthner neurons in the goldfish.

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.

Ultrastructure of desmosome-like contacts of mixed synapses of Mauthner neurons in long-term potentiation.

Electron microscopic morphometry was used to study the effects of long-term potentiation on the structure of fibrillar "cross-bridges" in the clefts of desmosome-like contacts in mixed synapses in Goldfish Mauthner neurons. These experiments showed that the number of bridges increased as the level of potentiation of electrotonic transmission increased. The structure of bridges changed after potentiation, which did not occur in controls (incubation). Double bridges appeared, which could have an altered (from control) organization within the cleft. The results obtained here and previously suggest that the bridges may be made of actin. Bridges are evidently a channel in which actin is organized as in nanotubules or plasmodesmata, and this may explain the stability of the bridge structure to treatment with cytochalasin and other external damaging factors which we have observed.

[The ultrastructure of desmosome-like contacts in mixed synapses of Mauthner cells followed long-term potentiation]. / Ul'trastruktura desmosopodobnykh kontaktov smeshannykh sinapsov mautnerovskikh neironov pri dolgovremennoi potentsiatsii.

Using electron microscopic morphometry, the effect of long-term potentiation on the structure of transverse fibrillar bridges localized in a cleft of desmosome-like contacts in mixed synapses of goldfish Mauthner neurons, was studied. The number of bridges was shown to increase with the augmentation of the potentiation level of electrotonic conductivity. In contrast to control (incubation), the structure of bridges was also changed after potentiation. The paired bridges appeared that could have an arrangement within the cleft different from that one in control. On the basis of the present results in conjunction with earlier findings, the possible actin nature of these bridges was suggested. The bridge seems to represent a channel containing actin, arranged in a pattern similar to that one in nanotubules or plasmodesmas, what can, possibly, explain the stability of bridge structure observed after application of [symbol: see text] D and other extrinsic damaging factors.

[Involvement of actin in electrotonic conductivity in the mixed synapses of goldfish Mauthner neurons]. / Uchastie aktina v élektrotonicheskoi provodimosti smeshannykh sinapsov mautnerovskikh neironov zolotoi rybki.

The effect of highly specific and selective actin-polymerizing and labelling agent, phalloidin, on electrotonic conductivity and structure of the mixed synapses of goldfish Mauthner neurons (MN) was studied. It was shown that the paired subthreshold electrostimulation of afferent input against a background of phalloidin application resulted in the average 80% increase of the amplitude of MN response to the second stimulus. In control group it increased by only 10% and was observed only after suprathreshold stimulation, while subthreshold stimuli were ineffective. We interpret these data as the manifestation of increased conductivity of the mixed synapses, induced by actin polymerization. At the ultrastructural level, phalloidin application at MN and their mixed synapses increased the size and number of actin-containing desmosome-like junctions, as well as the number of fibrillar bridges crossing their cleft. Using the phalloidin-colloid gold marker, the actin nature of these bridges was demonstrated. Interdependent morpho-functional changes found in the mixed synapses, provide the indication of actin involvement in the conduction of electrotonic signal through the mixed synapse. The bridges crossing the cleft of desmosome-like junction could be the structural substrate of this process.

[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.

[Structural differences between the desmosome-like contacts in the chemical and afferent synapses of Mauthner neurons in the goldfish]. / Strukturnye razlichiia mezhdu desmosomopodobnymi kontaktami v afferentnykh khimicheskikh i smeshannykh sinapsakh mautnerovskikh neironov zolotoi rybki.

Comparative ultrastructural investigation of the desmosome-like contacts at chemical and mixed afferent synapses of the goldfish Mauthner neurons was carried out. It was revealed that these contacts at mixed synapses differed from those at chemical ones by thin transverse fibrillar bridges which cross the gap and connect two adjoining membranes of the junction. We suppose that these crossbridges together with gap junctional connexons may serve as a substrate for electronic coupling at mixed synapses demonstrated earlier.

In vitro long-term potentiation of electrotonic responses of goldfish mauthner cells is accompanied by ultrastructural changes at afferent mixed synapses.

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.

[The ultrastructure of Mauthner's neurons in the surviving medulla oblongata of goldfish]. / Ul'trastruktura mautnerovskikh neironov v perezhivaiushchem prodolgovatom mozge zolotykh ryb.

Ultrastructure of Mauthner neurons (MN) was studied in fragments of myelencephalon incubated for 0.5-1.5 hours obtained from immature gold fish. Incubation terms increase along with destruction extent and reorganization of cytoplasmic organelles and nucleus with structure of MN afferent synaptic apparatus less disturbed. Specialized synaptic contacts-active zones, cleft contacts and desmosome-like structure are well retained. The data obtained indicate MN viability during the incubation terms studied, which allows to use surviving grafts with MN as a model for studying ultrastructural bases of certain functional changes of neurons and other neurobiological problems.