[Heterochronies in the Formation of the Nervous and Digestive Systems in Early Postlarval Development of Opistobranch Mollusks: Organization of Basic Functional Systems of the Arctic Dorid Cadlina laevis].

For the first time using laser confocal microscopy and histochemical and immunocytochemical methods (detection of F-actine, catecholamines, acetylcholintransferase, substances of P and FM RFamide) in combination with classical histological methods and electron microscopy of total preparations, the general structure and regularities of formation of the main organs and the nervous, muscular, and digestive systems in early postlarval development (2 to 4 months) in the opistobranch mollusk Cadlina laevis were studied. Heterochronies manifested in positive allometry of the sensory organs, ganglia of the central nervous system, and the pharyngeal region of the digestive system in relation to general body sizes in juvenile individuals compared to adult animals were detected.

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.

[The central pattern generators].

The central pattern generator (CPG) is defined as a set of neurons involved in joint production of patterned motor output. The roundtable discussion on the CPG was a part of the 5th All-Russian Conference on Animal Behavior (Moscow, Nov. 21, 2012). The discussion centred on three core themes: 1) the mechanisms of the organization and reconfiguration of pattern generating neuronal ensembles, 2) extrapolations that extend the CPG concept beyond the motor systems, and 3) evolutionary and developmental aspects of CPG.

[Network, cellular and molecular mechanisms of plasticity in simple nervous systems].

In the present study we will try to single out several principles of the nervous system functioning essential for describing mechanisms of learning and memory basing on our own experimental investigation of cellular mechanisms of memory in the nervous system of gastropod molluscs and literature data: main changes in functioning due to learning occur in effectivity of synaptic inputs and in the intrinsic properties of postsynaptic neurons; due to learning some synaptic inputs of neurons selectively change its effectivity due to pre- and postsynaptic changes, but the induction of plasticity always starts in postsynapse, maintaining of long-term memory in postsynapse is also shown; reinforcement is not related to activity of the neural chain receptor-sensory neuron-interneuron-motoneuron-effector; reinforcement is mediated via activity of modulatory neurons, and in some cases can be exerted by a single neuron; activity of modulatory neurons is necessary for development of plastic modifications of behavior (including associative), but is not needed for recall of conditioned responses. At the same time, the modulatory neurons (in fact they constitute a neural reinforcement system) are necessary for recall of context associative memory; changes due to learning occur at least in two independent loci in the nervous system. A possibility for erasure of memory with participation of nitroxide is experimentally and theoretically based.

[Two-faced nitric oxide is necessary for both erasure and consolidation of memory].

One of ways of nitric oxide (NO) influence on neuronal activity is S-nitrosylation, the covalent attachment of NO group to the thiol side chain of cysteine, changes function of existing proteins, inhibiting their normal role in physiological functions including memory. Influence of NO via GC activates intracellular signaling cascades and triggers increased synthesis ofproteins, influencing the memory. In the present paper we want to express and test the hypothesis that the NO is necessary both for erasure and development of memory. In our experiments in terrestrial snail Helix we tested the idea that NO besides well shown participation in memory development is involved in erasure/lockout of memory during relearning and reconsolidation.

Effects of beta-amyloid (25-35) on learning in the common snail.

The effects of neurotoxic beta-amyloid fragment (25-35) on the formation of behavioral sensitization and a conditioned defensive reflex to food were studied. Administration of beta-amyloid (25-35) to common snails before the start of training led to a significant reduction in sensitization of the defensive reaction, weakening of the formation of the conditioned defensive reflex to food, and impairment of memory. These impairments to behavioral plasticity may be mediated by changes in synaptic plasticity previously observed in the presence of beta-amyloid.

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.

[Beta-amyloid peptide influences behavioral plasticity in terrestrial snail].

Influence of neurotoxic fragment of beta-amyloid peptide (25-35) on Helix lucorum behavioral plasticity (sensitization and food-aversion learning) was investigated. After beta-amyloid peptide (25-35) injection a significant reduction of behavioral long-term sensitization was observed. It was found, that beta-amyloid peptide (25-35) may interfere with associative learning and memory. Our results clearly demonstrate that beta-amyloid peptide (25-35) may play a significant role in behavioral plasticity by chronically eliminating certain underlying forms of synaptic plasticity.

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

1. 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 Ca(2+) 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.

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.

[Functional role of neuropeptide CNP4 encoded by gene HCS2 in Helix lucorum nervous system].

Helix Command Specific 2 (HCS2) gene is constantly expressed in parietal premotor (command) interneurons involved in control of the terrestrial snail Helix lucorum withdrawal behavior as a trigger element. It is also expressed under noxious conditions in other neurons presumably involved in withdrawal behavior. In this study we addressed the role of neuropeptide CNP4, encoded by gene HCS2, in the regulation of activity of the respiratory system, and in the influence on growth of isolated neurons in culture. It was shown that activity of the premotor interneuron elicits a direct effect (pneumostome closure), and a delayed intensification of respiratory movements. Application of CNP4 mimicked the delayed effects. Presence of the peptide CNP4 in solution for cultured neurons led to increase of neuronal growth. Immunochemical localization of the protein precursor encoded by gene HCS2 and peptide CNP4 in the cultured premotor interneurons revealed their preferential presence in the growth cones. The obtained results suggest that CNP4 may be secreted and involved in synergic regulation of behavior of a snail.

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(-5) m serotonin. Responses to repeated glutamate applications to the soma of synaptically isolated withdrawal interneurons increased after 10 min 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.

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.

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.

Place- and behavior-independent sensory triggered discharges in rat hippocampal CA1 complex spike cells.

To test for access of spatial cues to the hippocampus of unrestrained animals, single unit recordings of CA1 complex spike cells were made in thirsty rats as they made alternating visits between the center and corners of an enclosed 60 x 60 cm square arena. At intervals, all lights were turned off in the arena and it was rotated. Although no neuronal discharge activity was found that encoded the rotation angle, 11 of 97 neurons were selectively activated after lights were turned off or on. This activity began at delays of up to several seconds and persisted for tens of seconds. These discharges had no location selectivity or behavioral correlates and continued even as the rat performed several behaviors in different parts of the arena. This confirms similar results in restrained rabbits while showing for the first time that this neuronal activity can be independent of place and behavior. In addition this shows that sensory stimuli can trigger hippocampal discharges even when the rat is not required to make any behavioral responses to them.