N. I. Bravarenko

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.

Caspase-like activity is essential for long-term synaptic plasticity in the terrestrial snail Helix.

Although caspase activity in the nervous system of mollusks has not been described before, we suggested that these cysteine proteases might be involved in the phenomena of neuroplasticity in mollusks. We directly measured caspase‐3 (DEVDase) activity in the Helix lucorum central nervous system (CNS) using a fluorometrical approach and showed that the caspase‐3‐like immunoreactivity is present in the central neurons of Helix. Western blots revealed the presence of caspase‐3‐immunoreactive proteins with a molecular mass of 29 kDa. Staurosporin application, routinely used to induce apoptosis in mammalian neurons through the activating cleavage of caspase‐3, did not result in the appearance of a smaller subunit corresponding to the active caspase in the snail. However, it did increase the enzyme activity in the snail CNS. This suggests differences in the regulation of caspase‐3 activity in mammals and snails. In the snail CNS, the caspase homolog seems to possess an active center without activating cleavage typical for mammals. In electrophysiological experiments with identified snail neurons, selective blockade of the caspase‐3 with the irreversible and cell‐permeable inhibitor of caspase‐3 N‐benzyloxycarbonyl‐Asp(OMe)‐Glu(OMe)‐Val‐Asp‐(OMe)‐fluoro‐methylketone prevented development of the long‐term stage of synaptic input sensitization, suggesting that caspase is necessary for normal synaptic plasticity in snails. The results of our study give the first direct evidence that the caspase‐3‐like activity is essential for long‐term plasticity in the invertebrate neurons. This activity is presumably involved in removing inhibitory constraints on the storage of long‐term memory.

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.

Long-term sensitization and environmental conditioning in terrestrial snails

The hypothesis that a long-term increase of behavioural responses in snails (over a period of days) might be due to environmental conditioning was examined. Training consisted of delivering electric shocks non-contingently with test stimuli twice per day for 5 days to freely moving snails on a ball floating in water. After training, a significant difference in amplitude of a withdrawal reaction to tactile test stimulation appeared between shocked and control snails. Responses were significantly facilitated in shocked animals for up to 12 days after training, but only if the animals were tested in the environment used for training. Testing of the same groups of animals crawling freely on the glass lid of a tank in which they lived between experimental sessions revealed no difference in responses to the same stimuli between shocked and control snails. Injection of the neurotoxin 5,7-dihydroxytryptamine, which selectively impairs serotonergic cells, eliminated the differences between shocked and control animals. Changing the pH of the water in which the ball floated, by addition of citric acid, led to a significant selective increase of responsiveness in snails sensitized in this environment relative to the responsiveness of the same snails with normal water in the tank. The results suggest that the long-term sensitization of withdrawal reactions observed is at least in part a manifestation of an associative process, namely environmental conditioning.

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.

Postsynaptic induction of long-term synaptic facilitation in snail central neurones.

&NA; Long‐term facilitation in molluscs is believed to be induced due to purely presynaptic activations. We recorded excitatory postsynaptic potentials (EPSPs) simultaneously from two identified neurones of snail parietal ganglia. We report a non‐decrementing facilitation induced by intracellular tetanization with concomitant presynaptic activation. The mean EPSP amplitude measured in 10 neurones 30‐50 min after tetanization was 17% greater than in the non‐tetanized control neurones. Only short‐lasting (5‐10 min) postsynaptic changes were found (post‐tetanic hyperpolarization and resistance decrease). The facilitation was especially prominent (34%, 10 min post‐tetanus) but decreased within 15 min in preparations with rapid wash‐out of the external media. The data suggest that induction of long‐term enhancement in molluscs depends on postsynaptic events and, like in mammals, may involve increased postsynaptic Ca2+ and subsequent release of retrograde messengers.

Cannabinoid regulation in identified synapse of terrestrial snail

In the terrestrial snail a direct monosynaptic glutamatergic connection between the primary sensory neuron and a premotor interneuron involved in withdrawal behaviour can be functionally identified using electrophysiological techniques. We investigated the involvement of cannabinoids in regulation of this synaptic contact. The results demonstrate that the specific binding sites for agonists to mammalian type 1 cannabinoid receptors (CB1Rs) exist in the snails nervous system. Application of a synthetic cannabinoid agonist anandamide selectively changed the efficacy of synaptic contacts between the identified neurons. A decrease in the long‐term synaptic facilitation of the synaptic contact elicited by high‐frequency nerve tetanization in the presence of cannabinoid agonist anandamide was observed, suggesting a possible role of endocannabinoids in regulation of plasticity at this synaptic site. The selective antagonist of CB1Rs [N‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide] AM251 bath application was changing the efficacy of the synaptic contact only when the postsynaptic neuron had been intracellularly activated before its application. This observation implies an involvement of endocannabinoids in plasticity phenomena induced by activity in the postsynaptic target. Additional support of endocannabinoid involvement in synaptic function at this site was given by experiments in which AM251 blocked the short‐term suppression of synaptic excitation evoked by low‐frequency nerve tetanization, a phenomenon qualitatively similar to cannabinoid‐dependent synaptically evoked suppression of excitation demonstrated in the mammalian nervous system. The results of the present study suggest an involvement of cannabinoids in the regulation of synaptic efficacy. Further, anandamide could be a candidate for an endogenous neuromessenger involved in plasticity processes.

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.

Effects of serotonin levels on postsynaptically induced potentiation of snail neuron responses.

Studies on identified neurons in the common snail were performed to investigate potentiation of EPSP arising after intracellular tetanization of the post-synaptic neuron. These experiments showed that high-frequency intracellular tetanization of a command neuron leads to biphasic long-term increases in the amplitude of synaptic responses to test stimulation. The role of serotonin in forming potentiation was studied. It was suggested that the presence of particular serotonin concentrations in the intercellular fluid is required for forming the second phase of the increase in synaptic responses, while the first (transient) phase is insensitive to CNS serotonin levels.

Dependence of synaptic facilitation postsynaptically induced in snail neurones on season and serotonin level.

THE problem of stability of long-lasting synaptic facilitation postsynaptically induced by intracellular current pulses without concomitant presynaptic activation was addressed. A short (15–20 min) phase of synaptic facilitation induced by intracellular tetanization in identified snail neurones was stable and present in all experiments, while a long-term phase (lasting ≥ 50 min) was observed only some experiments. Data analysis revealed dependence of the long-term phase on season. Dependence of the long-term phase of facilitation on serotonin concentration in hemolymph, which is known to change with season, was shown using the selective neurotoxin 5,7-dihydroxytriptamine. Dependence of habituation rate in the same synaptic connection on season and serotonin level was shown.