Biomedical subjects

I S Zakharov

Publications and source records attributed to I S Zakharov.

At least 19 recordsLinked to original sources

PubMed2006

Regulation of tentacle length in snails by odor concentration.

The upper tentacle of the snail, bearing the olfactory organ, produces complex movements when the snail explores a new environment. Tentacle trajectories were reconstructed in the presence and absence of odors using two simultaneous video recordings. Reconstructions showed that in the absence of odor, snails constantly scanned the surrounding space with the extended tentacles. Presentation of an odor elicited rapid flexion, independent of the odor concentration, accompanied by concentration-dependent tentacle contractions. Activation of identified motoneuron MtC3 is known to elicit tentacle contraction. Recordings made in semi-intact preparations showed that the dynamics and duration of the spike activity of MtC3 produced in response to odors correlated with the degree of tentacle contraction in response to odors. These data suggest that the central motoneuron MtC3, which triggers tentacle contraction, is involved in controlling the margins of the scanning field. Slow contraction or extension of the tentacle, associated with the level of MtC3 activity, may operate to tune the snail's investigative behavior to the conditions of the sensory environment.

Action Potentials↗

PubMed2006

Functions of peptide CNP4, encoded by the HCS2 gene, in the nervous system of Helix lucorum.

The aims of the present work were to study the role of neuropeptide CNP4, encoded by the HCS2 gene (which is expressed mainly in parietal command interneurons), in controlling the activity of the respiratory system, and also to study the effects of this neuropeptide on isolated defensive behavior neurons in prolonged culture. The influence of the command interneuron on the pneumostoma included a direct effect consisting of closure and a delayed effect consisting of intensification of respiratory movements. Application of neuropeptide CNP4 produced a pattern similar to the delayed effects seen on stimulation of the command interneuron, i.e., significant increases in the frequency and intensity of pneumostoma movements and strengthening of the rhythmic activity of the pneumostoma motoneuron. Studies of the effects of neuropeptide CNP4 on isolated neurons after prolonged culture showed that neuron process growth correlated with the presence of the neuropeptide in the medium. Identification of the location of the HCS2 precursor protein and neuropeptide CNP4 in isolated command interneurons after prolonged culture showed that that only those parts of the cell showing active process growth were immunopositive. Thus, neuropeptide CNP4 appears to be a secreted neuropeptide controlling respiratory system activity, which may also be involved in rearrangements of the network controlling defensive behavior in Helix snails.

Animals↗

PubMed2001

Comparison of reactive processes in the rat brain elicited by xenotransplantation of nervous tissues of chicken or pulmonate snail.

It is known that a histocompatibility system is not developed to the same extent in lower invertebrates as in vertebrate animals. We assumed that the xenografts from the newborn invertebrate nervous system would not exert destructive effects on the brain of the vertebrate recipient even without immunosuppressive therapy. In search of brain xenografts (XG) capable to survive in the brain of a recipient without intensive immunosuppression, we transplanted ganglia of terrestrial snails into the rat brain. We compared effects of transplantation of the XG taken from anterior brain of the 18-day embryo chicken (XGC) and from ganglia of a newborn terrestrial pulmonate snail (Helix aspersa L., XGSn). Part of the XGSn were stained by vital fluorescent dyes Bisbenzimid or Fast Blue before grafting. The XGSn were implanted into the neocortex parenchyma in each hemisphere. Rat brains with the XGC were examined 5 days after, and brains with the XGSn - 5 and 28 days after the transplantation. Nonstained sections with the XGSn labeled with fluorescent dyes prior to transplantation were investigated in fluorescent microscope and stained later with tionin and cresyl-violet. Quantitative videoimage analysis of lymphocyte aggregations, reactive gliosis, morphology of the XG areas, and implantation trace was performed. It was found that the XGSn transplantation did not elicit in the rat brain an intensive immunological conflict 5 and 28 days after transplantation. In contrast, the XGC rapidly elicited a strong immune response resulting in massive obliterations in the rat brain and were rejected in 5 days. Labeled snail glia and vessels were observed in the stained XGSn 28 days after transplantation by fluorescence imaging. Putative snail vessels grew into the rat brain from the place of snail tissue transplantation serving the humoral integration of the XG and the host brain. Migration of molluscan glial cells was observed in the brain of recipients.

Animals↗

PubMed2001

A single serotonergic modulatory cell can mediate reinforcement in the withdrawal network of the terrestrial snail.

A cluster of 40 serotonergic cells in the rostral part of pedal ganglia of the terrestrial snail Helix lucorum was shown previously to participate in the modulation of withdrawal behavior and to be necessary during the acquisition of aversive withdrawal conditioning in intact snails. Local extracellular stimulation of the serotonergic cells paired with a test stimulus elicited a pairing-specific increase (the difference between paired and explicitly unpaired sessions was significant, p <.01) of synaptic responses to test stimulation in the premotor interneurons involved in withdrawal. This result suggested participation of serotonergic cells in mediating the reinforcement in the withdrawal network. Intracellular stimulation of only one identified Pd4 cell from the pedal group of serotonergic neurons paired with a test stimulus also significantly increased (the difference between paired and explicitly unpaired sessions was significant, p <.05) synaptic responses to paired nerve stimulation in same premotor interneurons involved in withdrawal. Morphological investigation of a cluster of pedal serotonergic neurons showed that only the Pd4 cell had branches in the parietal ganglia neuropile where the synapses of premotor withdrawal interneurons and of presynaptic neurons are located. The data suggest that a single serotonergic cell can mediate the reinforcement in the withdrawal network of the terrestrial snail. Patterns of responses of the Pd4 cells to tactile and chemical stimuli conform to the suggestion.

Animals↗

PubMed2001

Participation of GABA in establishing behavioral hierarchies in the terrestrial snail.

GABA-immunoreactive fibers were observed in the neuropile of each ganglion of Helix lucorum, while GABA-immunoreactive neural somata were found only in the buccal, cerebral, and pedal ganglia. Bath application of 10(-5) M GABA to the preparation "buccal mass-buccal ganglia" elicited a sequence of radula movements characteristic of feeding behavior. Corresponding bursts of activity were recorded in the buccal nerves under GABA application and in the buccal neurons recorded optically. In preparations of isolated central nervous system, the bath applications of GABA (10(-5) to 10(-4) M) elicited no changes in synaptic input of the premotor interneurons involved in the withdrawal behavior. However, a significant decrease in amplitude of the synaptic input and in the number of spikes in responses elicited by the test nerve stimulation was observed in metacerebral serotonergic neurons involved in modulating the feeding behavior. GABA application inhibited the spontaneous spike activity in some pedal serotonergic neurons involved in the network underlying withdrawal responses and evoked bursting activity in the other neurons of this functional group. The effects of GABA application on mechanically isolated serotonergic neurons suggest that the primary effect of GABA is inhibition. Thus, our results give evidence of the putative role of GABA in activating the feeding behavior and in the synergistic suppression of serotonergic modulation of the withdrawal behavior and serotonergic modulation of feeding, which has corresponded to the observed behavioral suppression of withdrawal reactions during feeding.

Animals↗

PubMed2001

Up- and down-regulation of Helix command-specific 2 (HCS2) gene expression in the nervous system of terrestrial snail Helix lucorum.

A novel gene named Helix command-specific 2 (HCS2) was shown to be expressed predominantly in four giant parietal interneurons involved in withdrawal behavior of the terrestrial snail Helix lucorum L. and several single neurons in other ganglia. Decrease in spontaneous electrophysiological activity of neurons in the isolated CNS by 24h incubation in saline with elevated Mg(2+) concentration significantly decreased the number of HCS2-expressing neurons. Five short-term serotonin applications (each of 10microM), during a 24h incubation of the nervous system in saline induced expression of the HCS2 gene in many cells in cerebral, parietal, pleural and pedal ganglia. Dopamine applications under similar conditions were not effective. Application of anisomycin or cycloheximide, known to block protein synthesis, did not prevent the induction of HCS2 expression under serotonin influence. Skin injury elicited a significant increase in the number of HCS2-expressing cells 24h later in pleural and cerebral ganglia. Incubation of the isolated nervous system preparations for three days in culture medium elicited close to a maximum increase in number of HCS2-expressing cells. Elevation of the normal Mg(2+) concentration in the culture medium significantly decreased the number of cells demonstrating HCS2 expression. Application of the cAMP activator forskolin (10microM) increased the expression under Mg(2+), indicating that cAMP was involved in the up-regulation of HCS2. Application of thapsigargin (10microM), known to release Ca(2+) from intracellular stores, was also effective in increasing expression, suggesting participation of Ca(2+) in regulation of HCS2 expression. Cellular groups expressing the HCS2 gene under different conditions seem to be functionally related since it was demonstrated earlier that some neurons constituting these clusters are involved in the withdrawal behavior and the response of the organism to stress stimuli. From these results we suggest that the HCS2 pattern of expression can be down-regulated by a decrease in synaptic activity in the nervous system, and up-regulated by external noxious inputs, as well as the application of neurotransmitters and second messengers known to be involved in the withdrawal behavior and maintenance of isolated ganglia in culture medium. When up-regulated, the HCS2 expression appears, at least in part in neurons, to be involved in the withdrawal behavior.

Animals↗

PubMed2001

Development of neuronal ganglion xenografts from gastropoda in rat brain.

Survival of neuronal ganglia from newborn snail (Helix aspera L.) in the brain of adult rats was studied. Snail ganglion survived in the brain of warm-blooded animals for 6 months without inducing immune conflict. At early stages (5 days) after transplantation, xenografts increased in size and were several times larger than native ganglia from 10-day-old snails, thereafter (on days 28 and 180) they became smaller still surpassing the sizes of ganglia from snail of the corresponding age. Rapid enlargement of the xenograft was due to cell reactive processes in the ganglion. Deep penetration of large vessels from xenografts to rat brain was observed.

Animals↗

PubMed1998

Postembryonic neurogenesis in the procerebrum of the terrestrial snail, Helix lucorum L.

Neuronogenesis during posthatching development of the procerebrum of the terrestrial snail Helix lucorum was analyzed using bromodeoxyuridine immunohistochemistry to label proliferating cells. Comparison of the distribution of labeled cells in a series of animals which differed in age at the time of incubation with bromodeoxyuridine, in survival time after incubation, and in age at sacrifice reveals a clear pattern and developmental sequence in neuron origin. First, the proliferating cells are located only at the apical portion of the procerebrum. Second, cells which are produced at any particular age remain, for the most part, confined to a single layer in the procerebrum. Third, as development proceeds, each layer of previously produced neurons is displaced toward the basal part of the procerebrum by the production of additional neurons. Our results suggest that the vast majority of the neurons (probably about 70-80%) of the snail procerebrum are produced during the first 1-2 months of posthatching development.

Animals↗

PubMed1998

Putative neuropeptides and an EF-hand motif region are encoded by a novel gene expressed in the four giant interneurons of the terrestrial snail.

Nine giant interneurons located in the pleural and parietal ganglia of the terrestrial snail Helix lucorum L. were reported to be a key element in the network controlling withdrawal behaviour of the animal. Using a combination of complementary DNA subtraction cloning and differential screening approaches we have isolated a novel gene named HCS2 which is expressed predominantly in a subset of these interneurons. The predicted amino acid sequence of the HCS2 protein contains at the N-terminus a hydrophobic leader sequence and four putative neuropeptides, and at the C-terminus a perfect match to the consensus motif of the EF-hand family of the Ca2+-binding proteins. All four predicted neuropeptides bear a C-terminal signature sequence Tyr-Pro-Arg-X (where X is Ile, Leu, Val or Pro), and three of them are likely to be amidated. Physiological action of three synthetic peptides corresponding to the predicted mature HCS2 peptides mimics fairly well the described action of parietal interneurons on follower motoneurons controlling pneumostome closure. In situ hybridization experiments demonstrated that the HCS2 gene is selectively expressed in the four parietal giant interneurons, as well as in several small unidentified neurons. The onset of the HCS2 transcription during embryogenesis coincides temporally with the time-point when the first withdrawal responses of the embryo to tactile stimulation appear. We propose that the HCS2 gene encodes a hybrid precursor protein whose processed products act as neuromodulators or neurotransmitters mediating the withdrawal reactions of the snail, and in addition may participate in the calcium regulatory pathways or calcium homeostasis in command neurons.

Amino Acid Sequence↗

PubMed1998

A novel neuropeptide precursor gene is expressed in the terrestrial snail central nervous system by a group of neurons that control mating behavior.

We report the isolation of a cDNA clone encoding a neuropeptide precursor named preproGFAD from the central nervous system (CNS) of the snail Helix lucorum. Analysis of the expression of this gene shows that it is neurospecific and expressed in several groups of CNS neurons. Most notable is the expression of preproGFAD gene in the right mesocerebrum, where the neurons controlling mating behavior are located. The expression in this particular region is observed in adult animals but not in juvenile ones. The preprohormone is 108 amino acids long and contains a hydrophobic leader peptide and eight Lys-Arg recognition sites for endoproteolysis. The post-translational processing of the prohormone may lead to the generation of seven tetrapeptides, Gly-Phe-Ala-Asp-COOH (GFAD). This peptide has the same sequence as two previously isolated peptides from a related snail, Achatina fulica. The first of them (achatin-I) contains D-Phe; the second (achatin-II) is its L-Phe-containing stereoisomer. Injection of synthetic D-GFAD in nanomolar concentrations into intact animals caused an increase of the heartbeat rate and opening of the genital atrium. In preparations containing CNS with intact innervation of reproductive organs, bath application of D-GFAD caused extensive movements of the penis but not of other reproductive organs. Intracellular activation of individual neurons expressing the preproGFAD gene also elicited penis movements. D-GFAD also suppressed activity of neurons modulating feeding behavior. Our data therefore indicate that the preproGFAD gene encodes the precursor of a neuropeptide that participates in the regulation of male mating behavior.

Amino Acid Sequence↗

PubMed1997

Characterization of a cDNA clone encoding pedal peptide in the terrestrial snail.

We report the isolation of a Helix lucorum cDNA clone encoding a precursor of neuropeptides that are closely related to Aplysia and Tritonia pedal peptides (Pep). The predicted propeptide contains 20 copies of the two variants of Helix Pep interspersed with Lys-Arg endopeptidase cleavage sites. Northern blot hybridization revealed multiple Pep-hybridizing species in the Helix CNS RNA. The Pep gene was expressed by several identified serotonergic neurones in pedal and cerebral ganglia, groups of sensory neurones in procerebrum, peripheral neurones in olfactory bulb, mantle and foot, and group of neurones in pedal ganglia presumably involved in locomotion control. Pep mRNA was detected in several neurones at the early stages of nervous system development.

Amino Acid Sequence↗

PubMed1996

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS.

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snail Helix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mount in situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion. In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snail Lymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of the Helix family and the light yellow cells of Lymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.

Amino Acid Sequence↗

PubMed1993

Neurophysiological correlates of tactile stimulus-induced whole-body eversion, a novel type of behavior in the snail Helix pomatia L.

Repeated weak tactile stimuli to the exposed skin of the foot of withdrawn snails (Helix pomatia L.) result in a rapid whole-body eversion response not previously described. We studied the neurophysiological correlates of this novel type of behavior in semi-intact preparations consisting of the foot, pneumostome and mantle collar attached to the CNS. The pneumostome opening component of the eversion response is retained in semi-intact preparations and can be triggered by a series of weak tactile stimuli to the foot. The same stimuli also strongly excite a giant neuron (LPd7) in the pedal ganglion. This cell has axon branches in the anal and left pallial nerve which innervate the pneumostome and mantle collar. Intracellular stimulation of LPd7 causes the pneumostome to open. Bursts of spikes in LPd7 also lead to contractions of a specific group of muscles in the mantle collar innervated by the left pallial nerve. Both responses are retained when the CNS is bathed in high Mg2+/0 Ca2+ saline but the mantle response has a much shorter latency than the opening of the pneumostome. We conclude that LPd7 is an efferent neuron which causes opening of the pneumostome via a peripheral pathway in the mantle collar and may also be a motoneuron of muscles in the mantle collar. Besides its pneumostome-opener and putative mantle motoneuron function, the LPd7 cell also triggers movements of the foot which are also part of the eversion sequence in intact snails. The LPd7 cell receives excitatory input from neurons which trigger withdrawal reactions in active snails. The excitation is due to peripheral interactions between the two cell types and leads to opening of the closed pneumostome following brief aversive stimuli. We suggest that LPd7 is part of a novel putative neuronal network underlying the described eversive reaction to tactile stimuli.

Animals↗

PubMed1992

The neuroanatomical basis of feeding behavior in the pteropod mollusc, Clione limacina (Phipps).

1. In order to investigate neural mechanisms underlying the switching from hovering-swimming to hunting behavior in the carnivorous mollusc Clione limacina, the innervation of the major muscle bands was studied. The destination of a majority of nerves was traced. 2. Nervous cells sending their processes in the sensory motor nerves were mapped using the retrograde transport of cobalt ions. 3. The combination of morphological investigation, with local extracellular stimulation of groups of cells in semi-intact preparations, provided a detailed description of the functional involvement of the majority of the neural elements in either type of behavior. As a result, detailed diagrams for intracellular investigation of participation of individual cells and functional groups were obtained.

Animals↗

PubMed1987

Effect of 5,7-dihydroxytryptamine on the food-aversive conditioning in the snail Helix lucorum L.

The effects of 5,7-dihydroxytryptamine (5,7-DHT), a drug which selectively ablates serotonergic terminals, were examined on acquisition of food-aversive conditioned reflex in the snail Helix lucorum. The percent of feeding reactions decreased from 80 to 15% in the conditioned group of animals after 5-8 pairings of food and electric shock. The behavioral performance of 5,7-DHT-injected animals after the same training session coincided with the data received from the unpaired control group: the percent of feeding reactions remained the same as before the training. Conditioning was carried out on the semi-intact 'lip-CNS' preparations as well. Intracellular recordings from the neurons responding to the withdrawal reaction confirmed the results of the behavioral experiments. Elaboration of associative changes was effective on preparations made from normal snails, whereas no changes were noted in 5,7-DHT-treated and pseudoconditioned animals. In 5,7-DHT-treated animals some components of the feeding behavior and withdrawal reaction changed as well. The appetitive phase duration of feeding lengthened significantly, moreover the sensitization of the withdrawal reaction evoked by rhythmic tactile stimulation disappeared in preparations made from drug-treated snails.

5,7-Dihydroxytryptamine↗

PubMed1987

Neural mechanisms of age-dependent changes in avoidance behaviour of the snail Helix lucorum.

In order to study the emergence of different components of learning and memory, we investigated developmental changes in behavioural, neurophysiological and histochemical experiments in newborn and adult terrestrial snails (Helix lucorum L.). The absence of sensitization and inability to develop avoidance-conditioned reactions were revealed in behavioral experiments in juvenile snails under 1 month old. Investigation of neural mechanisms of this behavioral deficit showed the absence of sensitization in spike reactions to repeated nerve stimulation in command neurons for the avoidance behaviour in juvenile snails. The same dynamics of response amplitude, as in juvenile snails, was seen in adults only after selective impairment of serotonergic neurons by treatment with 5,7-dihydroxytryptamine. During 1 month after hatching, the serotonin content investigated with fluorescence techniques was very low in the nervous system. This result suggests that the absence of sensitization, as well as inability to be aversively conditioned is related to 5-hydroxytryptamine level, which changes during postnatal development in the snail.

Action Potentials↗

PubMed1986

[Role of serotonin in the development of the defensive reflex to food in the snail].

9-16 days after injection of 5,7-dihydroxytryptamine which elicits degeneration of serotonergic nerve cell terminals, pairing of food and electrical shock had no effect on responses of injected animals to food, while definite food-aversive reactions were observed in control animals. In neurophysiological experiments applications of serotonin in the chamber containing the nervous system was used as a reinforcing stimulus. The amplitude of synaptic responses to nerve stimulation increased significantly in preparations in which stimulation was paired with serotonin application. In neurons involved in defensive reactions after 3-7 pairings of a drop of juice to the chemoreceptive part of the skin with serotonin application a new spike response to food has appeared. Unpaired presentations of the same stimuli were not effective. It is concluded that serotonin is an important factor in formation of conditioned adverse reactions in the snail.

Animals↗

PubMed1982

[Polymodal sensory input in command neurons of snails].

Putative command neurones of snail's escape behaviour reacted with excitatory postsynaptic potentials and spikes to tactile, vestibular, light-off and thermal stimuli evoking behavioural escape reactions. Presented data confirm the command function of identified neurones. No monosynaptic connections have been revealed between intracellularly recorded primary cells of the statocyst and command neurones, but it has been shown that command neurones reacted to strong stimulation of the acoustic nerve. Presence of second-order sensory neurones in the vestibular pathway was assumed.

Acoustic Stimulation↗