T. A. Korshunova

Postsynaptic calcium contributes to reinforcement in a three‐neuron network exhibiting associative plasticity

We show that activation of a single serotonergic cell is sufficient to trigger long‐term associative enhancement of synaptic input to the withdrawal interneuron in a simple network consisting of three interconnected identified cells in the nervous system of terrestrial snail Helix. 1,2‐bis (2‐aminophenoxy) Ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) injection in the postsynaptic neuron abolishes the pairing‐specific enhancement of synaptic input. Activation of a single modulatory cell that we used to reinforce the synaptic input induced an increase of the intracellular [Ca2+] in the ipsilateral withdrawal interneuron without any changes of its membrane potential or input resistance. Similar changes in intracellular [Ca2+] were observed in the same withdrawal interneuron under bath application of 10−5u2003m serotonin. Responses to repeated glutamate applications to the soma of synaptically isolated withdrawal interneurons increased after 10u2003min of serotonin or thapsigargin bath application, but were absent in conditions of preliminary BAPTA intracellular injection, significantly decreased under heparin injection. Thus, activity of a single modulatory cell may mediate reinforcement via an increase of [Ca2+] in the postsynaptic cell in a simple network consisting of neurons with defined behavioural roles.

Nitric oxide is necessary for labilization of a consolidated context memory during reconsolidation in terrestrial snails

Nitric oxide (NO) is known to be involved in associative memory formation. We investigated the influence of blocking NO function on the reconsolidation of context memory in terrestrial snails (Helix lucorum L.). After a 10 day session of electric shocks in one context only, context memory in snails was observed in test sessions as the significant difference of amplitudes of withdrawal responses to tactile stimuli in two different contexts. After a 1 day rest, a session of ‘reminding’ was performed, preceded by injection in different groups of the snails with either vehicle or combination of the protein synthesis blocker anisomycin (ANI) with one of the following drugs: the NO scavenger carboxy‐PTIO, the NO‐synthase inhibitors N‐omega‐nitro‐L‐arginin, nitroindazole and NG‐nitro‐L‐arginine methyl ester hydrochloride, or the NO donor S‐nitroso‐N‐acetyl‐DL‐penicillamine. Testing the context memory at different time intervals after the reminder under ANI injection showed that the context memory was impaired at 24 h and later, whereas the reminder under combined injection of ANI and each of the NO‐synthase inhibitors used or the NO scavenger showed no impairment of long‐term context memory. Injection of the NO donor S‐nitroso‐N‐acetyl‐DL‐penicillamine with or without reminder had no effect on context memory. The results obtained demonstrated that NO is necessary for labilization of a consolidated context memory.

Synaptic contact between mechanosensory neuron and withdrawal interneuron in terrestrial snail is mediated by l-glutamate-like transmitter

The properties of the monosynaptic input from mechanosensory neurons to withdrawal interneurons were examined in Helix lucorum. The instantaneous I-V relation of the excitatory postsynaptic current in withdrawal interneurons was nonlinear, having a plateau region between -40 and -60 mV. On application of the blocker of vertebrate N-methyl-D-aspartate (NMDA) receptors AP5, or reduction of the Mg(2+) concentration, the current-voltage relation became more linear, suggesting that Mg(2+) may partially block the ion channel underlying the EPSC at voltages ranging from -40 to around -60 mV and the involvement of NMDA-like receptors. DNQX and 6-cyano-7-nitroquinoxaline-2,3-dione, which are known to block the glutamate non-NMDA receptors in mammals, significantly depress in a dose-dependent manner the actions of the natural transmitter. Exogenous L-glutamate applications mimicked the action of the mechanosensory neuron transmitter.

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

Abstract. 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 10–5xa0M 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 10–4xa0M) 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.

Effect of β-amyloid peptide on behavior and synaptic plasticity in terrestrial snail

A large body of evidence implicates beta-amyloid peptide (betaAP) and other derivatives of the evolutionarily highly conserved amyloid precursor protein (APP) in the pathogenesis of Alzheimers disease. However, the functional relationship of APP and its proteolytic derivatives to synaptic plasticity is not well known. We demonstrate that 30 min exposure to the 25-35 fragment of betaAP do not markedly change the dynamics of synaptic responses in identified neurons of terrestrial snail while a significant decrease of long-term sensitization was observed after 180 min betaAP bath application. In the behavioral experiments, a significant reduction of sensitization, and decreased ability to develop food-aversion conditioning was observed after betaAP injection. Our results clearly demonstrate that the neurotoxic 25-35 fragment of betaAP may play a significant role in behavioral plasticity by chronically eliminating certain underlying forms of synaptic plasticity. The study also proposes a novel invertebrate model to Alzheimers disease.

Olfactory experience modifies the effect of odour on feeding behaviour in a goal-related manner

Natural food odours elicit different behavioural responses in snails. The tentacle carries an olfactory organ, and it either protracts toward a stimulating carrot odour or retraces in a startle-like fashion away from a cucumber odour. The tentacle retraction to cucumber was still present after the snails were fed cucumber during inter-trial periods. Also, snails without any food experience displayed a longer latency to the first bite of cucumber than of carrot and rejected cucumber more often. After tasting these foods, the latency to carrot was not affected while the latency to and number of rejections of cucumber decreased. These results suggest that initial repulsive features of food odour can be only partially compensated by olfactory learning and feeding experience. In the present study, we demonstrated that an invertebrate can be repulsed or attracted by the same natural odour at the same time and that these behavioural responses are likely aimed at achieving different physiologically relevant goals.

Nitric oxide is necessary for long-term facilitation of synaptic responses and for development of context memory in terrestrial snails.

Correlated electrophysiological and behavioral experiments in the snail Helix lucorum were conducted to investigate the contribution of nitric oxide (NO) to synaptic plasticity during withdrawal reflex and aversive context memory development. Time, stimulation frequency and number of tetani/electrical shocks were determined in vitro and in vivo. In isolated brain preparations, nerve tetanization accompanied by bath application of serotonin induced long-term facilitation (LTF) of the excitatory postsynaptic potential (EPSP) in withdrawal interneurons. Bathing with either the NO-synthase inhibitor N-omega-nitro-L-arginin (L-NNA) or the NO-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO) before the tetanization prevented tetanus-induced long-term increase of EPSP. Withdrawal interneurons are key elements in the network underlying aversive behavior, with LTF considered the basis for aversive learning. We hypothesized that L-NNA injections in free-behaving snails could influence aversive learning. Snails were trained for 1 or 5days to remember the context in which they were shocked. In one-day training experiments, the snails received 5 electrical shocks in one context. Different groups of snails were sham-injected or L-NNA-injected before or after training. After training, the sham-injected groups demonstrated a significant increase in behavioral responses compared to the L-NNA-injected groups. On the following day, only sham-injected snails demonstrated altered behavioral responses, but no associative context differences were observed. These results correlated with the electrophysiological results. In another series of experiments, the snails received electrical shocks for 5days. Testing on the second day after training demonstrated that the sham-injected group maintained selective aversive context memory, whereas the L-NNA-injected snails were not different between the two contexts. Together these results demonstrated that inhibition of NO synthesis prevents memory formation and influences synaptic plasticity in the withdrawal interneurons that underlie the behavioral changes. This suggests that NO influences the behavior via regulation of synaptic plasticity.

Central Pattern Generators

A central pattern generator (CPG) is defined as a set of neurons whose members work together to generate organized motor output activity. A round-table discussion on central pattern generators was held on November 21, 2012 as part of the Fifth All-Russian Conference on Animal Behavior in Moscow. The main topics of discussion were: 1) the mechanisms of the organization and rearrangement of pattern-generating neuron ensembles; 2) the possibility that structures of the central pattern generator type have a role in controlling non-motor brain functions; and 3) the evolutionary and ontogenetic aspects of central pattern generators.

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

Intracellular localization of the HCS2 gene products in identified snail neurons in vivo and in vitro.

SUMMARY1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca2+ concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy.2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system.3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons.4. The immunogold particles were practically absent in the bodies of cultured neurons.5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.