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.

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.

[Effect of polypeptides from scorpion venom on morpho-functional changes of the incubated Mauthner neurons in goldfish]. / Morfofunktsional'nye izmeneniia inkubiruemykh mautnerovskikh neironov zolotykh rybok pod vliianiem peptidov iz iada skorpiona.

Using the electron microscopical method of negative staining it was shown that one of the fractions obtained from scorpion venom directly interacts with monomeric chromatographically pure actin causing its polymerization and transformation from globular into fibrillar form. The effect of long-term orthodromic stimulation on evoked electric activity and on the ultrastructure of Mauthner neurons (MN) in the goldfish hindbrain slices incubated in vitro was studied after the application of this venom fraction. The peptides of this fraction were shown to stabilize the amplitude of MN evoked electric responses to fatigue and to protect the ultrastructure of afferent chemical synapses and MN themselves from stimulation-induced injuries. Increase in morpho-functional resistivity is accompanied by the stabilization of specialized synaptic structures--actin-containing desmosome-like junctions. These data suggest the direct effect of peptides from scorpion venom fraction on actin component of MN cytoskeleton and show the perspective for their application as pharmacological tools capable of penetration into the living cells that may be used for investigation of the role of actin in the mechanisms of adaptation and memory.

[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.

[Morpho-functional changes in mauthner neurons of goldfish adapted to prolonged orthodromic stimulation of acoustic nerve in vitro]. / Morfofunktsional'nye izmeneniia adaptirovannykh mautnerovskikh neironov zolotykh rybok pri dlitel'noi ortodromnoi stimuliatsii slukhovogo nerva in vitro.

In newly-hatched goldfish the adapted state was developed using repeated short-term stimulation of the vestibular apparatus. It differed from the non-adapted state by the absence of fatigue during the turns of fishes produced by unilateral tail fin flicks in ring channel. Electrophysiological study of Mauthner neurons (MN) in adapted goldfish using the slices of medulla oblongata incubated in vitro, has shown the preservation of the amplitude of the extracellular responses after prolonged orthodromic electric stimulation of acoustic nerve, which suppressed the responses of non-adapted MN. Thus, the resistance against fatigue has developed also at the level of MN. Using electron microscopy it was demonstrated that adapted MN following the prolonged electric stimulation in vitro were characterized by the resistance of specialized desmosome-like contacts, similar to what was earlier observed in these neurons in vivo. The data obtained suggest that indirect evaluation of MN functional state in goldfish by counting the number of turns performed by it in narrow ring channel, corresponds to the direct morpho-functional assessment of MN state. This form of innate behavior of freely moving fish seems to be the result of MN excitation.

[Long-term morphofunctional preservation of guinea pig hippocampal slices following short-term treatment with cyclooxygenase inhibitors]. / Dlitel'naia morfofunktsional'naia sokhrannost' srezov gippokampa morskoi svinki posle kratkoi obrabotki ingibitorami tsiklooksigenaz.

Hippocampal slices treated with cyclooxygenase inhibitor indomethacin for three min during the sectioning (45 min) or aspirin (0.5 min) in long term (up to 5 days) preservation in periodic nocturnal hypothermia were studied morphofunctionally using light microscopy and electrophysiological registration of induced population responses of area CAI to stimulation of Schaffers collaterals. Structural disorders were revealed in control slices as early as the third hour of incubation and they were destroyed following the first hypothermal challenge (24 hrs following preparation). The structure in slices treated with blockers remained more stable as compared to control ones and the activity was registered until d 3 (aspirin) and 5 (indomethacin). Morphological changes were not immediately, followed by electric activity decline. On the whole it may be suggested that essential viability increase occurred due to destructive processes inhibition mediated by short living cyclooxygenase metabolites.

Functional stability of hippocampal slices after treatment with cyclooxygenase inhibitors.

The influence of cyclooxygenase inhibitors on functional stability of hippocampal slices, determined by electrophysiological criteria of recovery after slicing and long-term maintainence of population activity, was studied. Transient (3 min) treatment of slices during slicing with indomethacin (45 microM) or aspirin (0.5 mM) allowed registration of the population responses from the second minute. The activity reached 100% after 15 min incubation and could be registrated for 3 days under conditions of overnight hypothermia. The presence of the same drugs for the entire incubation period had the same effect. The present findings suggest that slicing is a crucial point for triggering of pathological events mediated by cyclooxygenase products and that blockade of cyclooxygenase provides for the further high longterm functional stability of brain slices.

Preparation of isolated perfused ground squirrel brain.

This paper describes preparation of the isolated brain of hibernating ground squirrel maintained by intraarterial perfusion. This technique allows a long-term survival (about 3 days) of the isolated brain of adult animals. The preparation viability was assessed by extracellular investigation of stability of structure-specific electrical activity. The areas investigated include neocortex, hippocampus, thalamus, hypothalamus, and brain stem.

[The excitability of neurons of the medial septal area in brain slices from hibernating and active Citellus undulatus susliks]. / Vozbudimost' neironov medial'noi oblasti septuma v srezakh mozga giberniruiushchikh i aktivnykh suslikov Citellus undulatus.

Spontaneous and evoked neuronal activity of the medical septum-diagonal band complex (MS-DB) has been investigated in slices from the brain of hibernating and active ground squirrels, as well as guinea pigs. In all experimental groups, the majority of the MS-DB neurones exhibited high regular of rhythmic burst spontaneous activity which persisted even after synaptic blockade in half of the neuronal population. Under the same conditions, the activity of the surrounding structures was completely suppressed. The density of the spontaneously active neurones in the slices, as well as the mean frequency of discharges in the MS-DB of hibernating ground squirrels, were significantly higher than in active ground squirrels and guinea pigs. Stimulation of the medial forebrain bundle evoked initial suppression of the activity in the majority of MS-DB units; in many of them, the suppression was followed by a burst discharge. Neurones with background rhythmic burst activity always reacted by resetting the spontaneous bursts. In total, 50-60% of the MS-DB neurones in active ground squirrels and guinea pigs reacted by post-inhibitory bursts, whereas in hibernating animals these responses were observed nearly in all neurones. Threshold values of the stimulating current were lower in hibernating animals; the intraburst density of spikes was increased.

[A comparative analysis of the functional stability (based on electrophysiological criteria) of slices of the suslik and guinea pig brains maintained under deep hypothermia]. / Sravnitel'nyi analiz funktsional'noi sokhrannosti (po élektrofiziologicheskim kriteriiam) srezov mozga suslikov i morskikh svinok pri soderzhanii v glubokoi gipotermii.

The effect of prolonged deep cooling has been investigated in hippocampal and septal slices from the brain of hibernating and active ground squirrels, as well as of the guinea pigs. The slices were kept at low temperatures (2-4 degrees C) for various periods of time (from several hours to 6 days) and were periodically tested in a warm (31 degrees C) incubation medium. Hippocampal field potentials (mainly of the field CA1) and spontaneous activity of single neurones of the medial septum were recorded. Significant differences were observed in the recovery of functional activity of the slices after their preparation as well as after cooling between experimental groups of animals. Slices from hibernating ground squirrels retained their activity for 7-9 days, those from active ones--for 6-7 days, whereas slices from guinea pigs did not recover their functional activity after cooling for more than 1-2 days.

[The effect of prolonged hypothermia on the structure and function of the neurons of surviving slices of guinea pig and suslik hippocampus]. / Vliianie dlitel'noi gipotermii na strukturu i funktsiiu neironov perezhivaiushchikh srezov gippokampa morskoi svinki i suslika.

Hippocampi of the guinea pig and ground squirrel were used to examine the neuronal structure of CA1 and CA3 field in surviving slices incubated in warm chamber to rehabilitate the functional activity; then the slices were subjected to a prolonged cooling under zero temperatures. It was shown that the structure of the guinea pig hippocampal slices, first of all of the neuronal cytoskeleton, was disturbed at 1.5 h warm incubation. A further 1 h cooling in a refrigerator resulted in a significant swelling of synapses, dendrites and their organelles. In 24 h the structure was severely damaged. It was determined for the first time that the ground squirrel hippocampal slices revealed in the same conditions a high tolerance to both warming and long-term cooling. Even after 5 days in a refrigerator the neurons and dendrites could be clearly seen in slices, nearly intact ultrastructure of their cytoskeleton, synapses and organelles was preserved. A correlation was found between the structural integrity and the functional activity of hippocampal slices. It was suggested that the cytoskeleton may play a crucial role in maintaining the neuronal structure under conditions of both heating and cooling of the hippocampal slices. These data are useful to further elaborating appropriate methods for prolonging the term of incubation and to neurobiological investigation of surviving brain slices.

Functional stability of the brain slices of ground squirrels, Citellus undulatus, kept in conditions of prolonged deep periodic hypothermia: electrophysiological criteria.

The brain of hibernating animals, controlling the physiological functions during the hibernation cycles, is itself subject to deep cooling during bouts of hibernation. This suggests its high tolerance to deep hypothermia. Effects of prolonged deep cooling were investigated in hippocampal and septal slices, taken from the brains of three groups of animals: hibernating ground squirrels, actively waking ground squirrels, and guinea-pigs. The slices were kept at a low temperature (2-4 degrees C) for various periods of time (from several hours up to six days) and periodically tested in warm (31 degrees C) incubation medium. The hippocampal field potentials (mainly of field CA1), as well as spontaneous activity of single neurons of hippocampus and medial septum were recorded. For comparative purposes mean amplitudes of population spikes and mean frequency of spontaneous neuronal discharge were used. Significant differences between the experimental groups were observed in recovery of functional activity of the slices after their dissection from the brain, as well as after deep cooling. In both cases re-establishment of neuronal activity in ground squirrels occurred more rapidly, than in guinea-pigs. The most dramatic was the difference in maximal time of survival of the slices under conditions of deep cooling. Independently of periodicity of the electrophysiological testing in warm medium, the slices taken from hibernating squirrels retained their activity for seven to nine days, the slices of waking ground squirrel hippocampus survived up to six to seven days, while those of guinea-pis did not recover their functional activity after cooling for more than one to two days.(ABSTRACT TRUNCATED AT 250 WORDS)

Paradoxical state-dependent excitability of the medial septal neurons in brain slices of ground squirrel, Citellus undulatus.

Spontaneous and evoked neuronal activity of the medial septum-diagonal band complex was investigated extracellularly in slices, taken from the brain of the three groups of animals: hibernating ground squirrels, waking ground squirrels, and guinea-pigs. All slices were incubated at 31-32 degrees C. The slices of the ground squirrels' brain were retested after keeping them for 15-36 h in the refrigerator at 2-4 degrees C. In all experimental groups the majority of the medial septum-diagonal band complex neurons had high regular or rhythmic burst spontaneous activity, which in half of the neuronal population persisted in conditions of synaptic blockade. The low-frequency irregular activity of the surrounding structures (lateral septum, caudate, accumbens, medial preoptic area) was completely suppressed in these conditions. The density of the spontaneously active neurons in the slices, as well as the mean frequency of discharges in the medial septum-diagonal band complex of hibernating ground squirrels, was significantly higher than that in waking ground squirrels and guinea-pigs. Stimulation of the medial forebrain bundle evoked initial suppression of activity in majority of the medial septum-diagonal band complex units; in many of them the suppression was followed by a burst discharge. The neurons with background rhythmic burst activity always responded by resetting the spontaneous bursts. In total, about 50-60% of the medial septum-diagonal band complex neurons of waking ground squirrels and guinea-pigs responded by post inhibitory bursts to the stimulation of medial forebrain bundle, while in hibernating ground squirrels such responses were observed in nearly all neurons. The threshold values of the stimulating current were significantly lower in the hibernating ground squirrels' group, the mean duration of the initial suppression was shorter, the intraburst density of spikes and/or duration of the bursts was increased. Thus, evaluation of spontaneous and evoked activity on the basis of various criteria revealed surprising similarity between the two groups of active animals, while the activity and excitability of the medial septum-diagonal band complex neurons was approximately doubled in the hibernating animals. This difference between active and hibernating ground squirrels was preserved during retesting after deep and prolonged cooling of the slices. The experiments demonstrate paradoxical stable increase of activity and excitability of the medial septum-diagonal band complex neurons in the hibernating ground squirrels.(ABSTRACT TRUNCATED AT 250 WORDS)

Some unusual characteristics of the nucleus anterodorsali thalami: neurophysiological and ultrastructural investigation.

Single-unit activity and ultrastructural characteristics were investigated in anterodorsal nucleus of thalamus (AD) in rabbits, guinea pigs and rats. Spontaneous activity of the AD units consisted of long high-frequency trains of spikes alternating with prolonged periods of silence. The typical thalamic "inactivation bursts" were never observed in AD. The AD neurons were highly reactive to sensory stimuli of various modalities. The stimuli evoked sustained discharges with or without initial short-latency on-effects. The AD neurons in incubated slices responded to electrical stimulation of afferent inputs by high-frequency sustained discharges (up to several s). The neurons had long period of facilitation, which allowed summation of effects of repetitive stimuli following with intervals up to 20-30 s, and equally long refractory periods after tonic discharge. Ultrastructural investigation revealed highly developed Nissl bodies and extensive multilocular Golgi complex in large AD neurons. Specialized standard combinations of elements of rough and smooth endoplasmic reticulum ("doublets") were widely distributed in their cytoplasm. Large aggregates of finely granular or filamentous material (nematosomes) were present in AD neurons. The perivascular glial endfeet in some areas of the AD were very thin or absent altogether, with direct apposition of neuronal somata to the capillary basal membrane, which was characterized by high pinocytotic activity. All functional and structural features of AD listed above were absent in adjacent anteroventral nucleus. It is suggested that the AD neurons possibly have some specialized neurosecretory activity, which, regarding the role of this nucleus in relaying multimodal sensory information to the highest levels of limbic system (hippocampal formation and retrosplenial cortex), may have some special significance for memory functions.

[Comparative characteristics of the neuronal activity of the anterodorsal and anteroventral nuclei in thalamus slices from guinea pigs]. / Sravnitel'nye kharakteristiki aktivnosti neironov anterodorzal'nogo i anteroventral'nogo iader v srezakh talamusa u morskikh svinok.

Extracellular investigation of neurons of the anterodorsal (AD) and anteroventral (AV) nuclei in the incubated slices of the thalamus of guinea pigs and rabbits revealed unusual properties of the AD units. They responded only to electrical stimuli of considerable duration (1-5 ms) by multispike initial bursts or by prolonged (1-15 s) high frequency phasic discharges. Summation of effects of successive stimuli following with intervals of 2-30 s was necessary for development of phasic discharges. Prolonged refractory state (up to 2-15 s) followed the discharges. They were blocked in low Ca2+, high Mg2+ medium and facilitated in a medium with high concentration of K+. The nature of unusual properties of AD neurons, not present in the neighbouring AV nucleus, are discussed.

[Effect of electric stimulation of cortical portions of the limbic system on the activity of neurons of the anterior thalamic nuclei in the rabbit]. / Vliianie élektricheskoi stimuliatsii korkovykh otdelov limbicheskoi sistemy na aktivnost' neironov perednykh iader talamusa u krolikov.

Effects of electrical stimulation of the subiculum (SB) and posterior limbic cortex (PLC) were studied extracellularly in the anteroventral (AV) and anterodorsal (AD) limbic thalamic nuclei of awake chronic rabbits. Stimulation of SB and PLC evoked in some AV neurones discharges of 1-2 spikes. Gradual potentiation and low frequency of following (up to 10-15 Hz) were characteristic of these responses. Activity of the majority of AV cells was suppressed by stimulation with appearance of inactivation bursts, "neuronal spindles" and modulation on delta-frequencies. Spike responses were evoked by SB and (rarely) by PLC stimulation only in a certain class of AD neurones which tentatively are regarded as relay cells. The neurones with high-frequency, low-amplitude discharges (putative inhibitory interneurones) reacted to stimulation of PLC and to a lesser extent of SB by prolonged series of spikes (150 ms--2s). Stimulation of PLC exerted prolonged influence upon neuronal responses to sensory stimuli.