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In this paper, we present a study of the effects of reducing serotonin levels by para-chlorophenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, the limiting enzyme in serotonin biosynthesis, on two forms of behavioral plasticity in the terrestrial snail, Helix lucorum: the conditioned defensive reflex food aversion and long-term sensitization. We found that the snails injected with PCPA did not learn the conditioned reflex food aversion, while saline solution injection did not affect the learning process. The latency of consuming one kind of food in these snails was significantly reduced compared to the active control. We also found that PCPA injection prevents the formation of long-term sensitization.

It was previously shown that both associative learning and the formation of long-term sensitization led to the increase in excitability of premotor interneurons of the defensive behavior of terrestrial snail Helix lucorum. In the present study, we analyzed the role of intracellular calcium ions in the maintenance of increased excitability in premotor interneurons of terrestrial snail after the formation of a conditioned defensive reflex. It was shown that the increase of the intracellular Ca2+ concentration after adding caffeine to the solution washing the nervous system of the mollusk led to a decrease of the threshold of action potential and to an increase of the critical level of depolarization without a change of the membrane potential of premotor interneurons in both intact and trained snails. The decrease of the intracellular Ca2+ concentration in premotor interneurons by the intracellular injection of (ethylene glycol-bis (2-aminoethylether)-N, N, N, N-tetraacetic acid) (EGTA) resulted in a significant increase of the threshold of generation of the action potential in intact snails. But the values of threshold of generation of the action potential in trained snails after injection of EGTA did not significantly differ from the values of studied parameters before injection. After application of the membrane-penetrating chelator, BAPTA-AM, the changes in the membrane and threshold potentials of premotor interneurons of intact and trained snails were not observed. Our results demonstrated that both the increase and decrease of intracellular Ca2+ concentration were not involved in maintaining the changes of membrane characteristics of premotor interneurons observed after associative learning.

A variety of physicochemical methods were used to examine the self-organization, physicochemical, UV absorption, and fluorescent properties of diluted aqueous solutions (calculated concentrations from 1·10−20 to 1·10−2 M) of the membrane voltage-dependent potassium channels blocker 4-aminopyridine (4-AP). Using the dynamic light scattering method, it was shown that 4-AP solutions at concentrations in the range of 1·10−20–1·10−6 M are dispersed systems in which domains and nanoassociates of hundreds of nm in size are formed upon dilution. An interrelation between the non-monotonic concentration dependencies of the size of the dispersed phase, the fluorescence intensity (λex 225 nm, λem 340 nm), specific electrical conductivity, and pH has been established. This allows us to predict the bioeffects of the 4-AP systems at low concentrations. The impact of these diluted aqueous systems on the electrical characteristics of identified neurons of Helix lucorum snails was studied. Incubation of neurons in the 4-AP systems for which the formation of domains and nanoassociates had been established lead to a nonmonotonic decrease of the resting potential by 7–13%. An analysis of the obtained results and published data allows for a conclusion that a consistent change in the nature and parameters of the dispersed phase, as well as the pH of the medium, apparently determines the nonmonotonic nature of the effect of the 4-AP systems in a 1·10−20–1·10−6 M concentration range on the resting membrane potential of neurons. It was found that the pre-incubation of neurons in the 4-AP system with a concentration of 1·10−12 M led to a 17.0% synergistic decrease in the membrane potential after a subsequent treatment with 1·10−2 M 4-AP solution. This finding demonstrates a significant modifying effect of self-organized dispersed systems of 4-AP in low concentrations on the neurons’ sensitivity to 4-AP.

It was shown by electron paramagnetic resonance (EPR) spectroscopy that one day after bilateral occlusion of the common carotid arteries (to simulate ischemia) in Wistar rats (group 1, n = 15) under ketamine-xylazine-acepromazine anesthesia (55.6, 5.5, 1.1 mg/kg, respectively, intraperitoneally), the content of nitric monoxide (NO) in the olfactory bulbs decreased, and recovered after two days. A similar trend was observed in Wistar rats (group 2, n = 15), which, simultaneously with ischemia modeling, were implanted intranasally with mesenchymal stem cells (MSCs). An intact group of rats (group 3, n = 15) was kept in the usual conditions of the vivarium and did not undergo surgical interventions. The content of copper in the olfactory bulbs of rats (for assessing the activity of superoxide dismutases 1 and 3) after ischemia modeling (group 1) tended to increase and maintain the increase for two days of observation. Intranasal administration of MSCs during the ischemia modeling was accompanied by a more significant increase in copper content on the first day after brain ischemia modeling, but after two days, a tendency towards restoration of the initial copper level was noted.

It was shown that the application of methiothepin, the antagonist of serotonin (5-HT) receptors, caused a decrease in the membrane potential of the high-threshold premotor interneurons LPa3 and RPa3 of the intact snails and its increase to the previous level after the subsequent application of 5-HT in vitro. The application of methiothepin also led to a decrease in the membrane potential of these neurons, but the subsequent application of 5-HT did not lead to the return of the membrane potential to the previous level in animals trained in defensive reflex. It can be assumed that conditioning of the defensive reflex causes a state change of the 5-HT receptor system of premotor interneurons of defensive behavior.

HIGHLIGHTS The injection of p-chlorophenylalanine, specific blocker of 5-HT synthesis 3 days before reminder with anisomycin administration prevented forgetting. It is known that the reminder cause reactivation of the long-term memory and it leads to reconsolidation of memory. We showed earlier that the disruption of the reconsolidation of contextual memory in terrestrial snail was caused by anisomycin, the inhibitor of protein syntheses (Gainutdinova et al., 2005; Balaban et al., 2014). In this paper we investigated the possible changes of the memory reconsolidation under the conditions of serotonin deficit, caused by administration of p-chlorophenylalanine, the inhibitor of tryptophan hydroxylase synthesis (intermediate stage of the synthesis of serotonin). It was shown that the forgetting process for contextual memory after reminder and inhibition of protein synthesis did not occur if the serotonin transmission in nervous system was impaired. This effect was significantly different from the direct action of anisomycin, which blocked the reconsolidation of contextual memory. We concluded that the serotonin system was included to the process of memory reconsolidation.

Long-term changes in membrane potential after associative training were described previously in identified premotor interneurons for withdrawal of the terrestrial snail Helix. Serotonin was shown to be a major transmitter involved in triggering the long-term changes in mollusks. In the present study we compared the changes in electrophysiological characteristics of identifiable premotor interneurons for withdrawal in response to bath applications of serotonin (5-HT) or serotonin precursor 5-hydroxytryptophan (5-HTP) in preparations from naïve, neurotoxin-injected or associatively trained snails. It was found that 5-HT or 5-HTP applications caused a significant decrease of membrane potential in premotor interneurons of naïve snails, associatively trained snails and snails with impaired serotonergic system by injection of a selective neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) 1 week before the experiments. Applications of 5-HT or 5-HTP did not cause significant changes in the action potential (AP) threshold potential of these neurons in naïve snails. Conversely, applications of 5-HT or 5-HTP to the premotor interneurons of previously trained or 5,7-DHT-injected snails caused a significant increase in the firing threshold potential in spite of a depolarizing shift of the resting membrane potential. Results demonstrate that responsiveness of premotor interneurons to extracellularly applied 5-HT or 5-HTP changes for days after the associative training or serotonin depletion. Similarity of the effects in trained and 5,7-DHT-injected animals may be due to massive release of serotonin elicited by 5,7-DHT injection. Our results suggest that serotonin release due to aversive conditionining or elicited by the neurotoxin administration triggers similar changes in resting membrane potential and AP threshold in response to bath applications of 5-HT or its precursor 5-HTP.

It is shown that after the elaboration of a conditioned reflex in snails, a reliable decrease can be observed in the membrane potential (Vm) of the premotor interneurons at 4 mV, daily injection of serotonin (5-HT) causes a decrease in Vm at 4.5 mV, the same change is observed for Vm in the snails trained after the injection of 5-HT. A single injection of 5-HT causes a depolarization shift of Vm at 5 mV. After the initial stage of training (10–12 pairs) the snails, injected by 5-HT, there is a depolarization at 4.5 mV.

It has been shown that after 5 h of spinal cord injury, there is a decrease of nitric oxide (NO) production in the spinal cord. Seventy-two hours after the spinal cord injury, the level of NO production in the spinal cord and the heart increases by 2.5 times, and in the liver, it increases threefold. In the chronic period of a traumatic spinal cord disease in the spinal cord tissue, the level of NO production was significantly higher than at the control level.

Experimentally, it was shown that the action potential threshold in neurons of the trained snails increases in response to serotonin (5-HT) application, in contrast to intact snails. This result demonstrates a decrease of excitability of the premotor interneurons in response to extracellular 5-HT after the training.

Preconditioning of the brain induces tolerance to the damaging effects of ischemia and prevents cell death in ischemic penumbra. The development of this phenomenon is mediated by mitochondrial adenosine triphosphate-sensitive potassium (K+ATP) channels and nitric oxide signalling (NO). The aim of this study was to investigate the dynamics of molecular changes in mitochondria after ischemic preconditioning (IP) and the effect of pharmacological preconditioning (PhP) with the K+ATP-channels opener diazoxide on NO levels after ischemic stroke in rats. Immunofluorescence-histochemistry and laser-confocal microscopy were applied to evaluate the cortical expression of electron transport chain enzymes, mitochondrial K+ATP-channels, neuronal and inducible NO-synthases, as well as the dynamics of nitrosylation and nitration of proteins in rats during the early and delayed phases of IP. NO cerebral content was studied with electron paramagnetic resonance (EPR) spectroscopy using spin trapping. We found that 24 hours after IP in rats, there is a two-fold decrease in expression of mitochondrial K+ATP-channels (p= 0.012) in nervous tissue, a comparable increase in expression of cytochrome c oxidase (p = 0.008), and a decrease in intensity of protein S-nitrosylation and nitration (p = 0.0004 and p = 0.001, respectively). PhP led to a 56% reduction of free NO concentration 72 hours after ischemic stroke simulation (p = 0.002). We attribute this result to the restructuring of tissue energy metabolism, namely the provision of increased catalytic sites to mitochondria and the increased elimination of NO, which prevents a decrease in cell sensitivity to oxygen during subsequent periods of severe ischemia.

Highlights Daily injection of serotonin before a training session accelerated defensive reflex conditioning in snails. Daily injection of 5-hydroxytryptophan before a training session in snails with a deficiency of serotonin induced by the “neurotoxic” analog of serotonin 5,7-dihydroxytryptamine, restored the ability of snails to learn. After injection of the “neurotoxic” analogs of serotonin 5,6- and 5,7-dihydroxytryptamine as well as serotonin, depolarization of the membrane and decrease of the threshold potential of premotor interneurons was observed. We studied the role of serotonin in the mechanisms of learning in terrestrial snails. To produce a serotonin deficit, the “neurotoxic” analogs of serotonin, 5,6- or 5,7-dihydroxytryptamine (5,6/5,7-DHT) were used. Injection of 5,6/5,7-DHT was found to disrupt defensive reflex conditioning. Within 2 weeks of neurotoxin application, the ability to learn had recovered. Daily injection of serotonin before a training session accelerated defensive reflex conditioning and daily injections of 5-HTP in snails with a deficiency of serotonin induced by 5,7-DHT restored the snail's ability to learn. We discovered that injections of the neurotoxins 5,6/5,7-DHT as well as serotonin, caused a decrease in the resting and threshold potentials of the premotor interneurons LPa3 and RPa3.

We investigated the influence of the protein synthesis blocker anisomycin on contextual memory in the terrestrial snail Helix. Prior to the training session, the behavioral responses in two contexts were similar. Two days after a session of electric shocks (5 d) in one context only, the context conditioning was observed as the significant difference of behavioral response amplitudes in two contexts. On the day following testing of context learning, a session of “reminding” was performed, immediately after which the snails were injected with anisomycin or vehicle. Testing of long-term context memory has shown that only anisomycin injections impaired the context conditioning. In control series, the snails were injected after the training session with anisomycin/saline without reminding, and no impairment of the long-term context memory was observed, while injection of anisomycin during the training session completely abolished the long-term memory. No effects of anisomycin on the short-term memory were observed. Surprisingly, injection of anisomycin after the reminding combined with reinforcing stimuli elicited no effect on the context memory. Differences between single-trial and multisession learning are discussed.

It has been found that application of a nitric oxide donor—the sodium nitroprusside—causes the hyperpolarization shift of the membrane potential of the premotor interneurons of defensive behavior of terrestrial snails. It is assumed that the response of a neuron to NO depends on its location in the neural network.

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.

The effects of antibodies to calcium-binding S100B protein diluted to 10 − 12 (LAS100B) on the long-term sensitization in the Helix lucorum snail (neurobiological model of the anxious-depressive state) were evaluated. The administration of LAS100B prior to conditioning of long-term sensitization in the terrestrial snail 10 min prior to the first electric stimulus) prevents strengthening of the defensive reaction of withdrawing the ommatophores (eye tentacles) and the defensive reaction of closing the pneumostome. This effect is termed “protective”, as it prevents the conditioning of long-term sensitization. At the same time, snails given an injection of saline developed long-term sensitization with a significant strengthening of the defensive reactions of withdrawing the ommatophores and closing the pneumostome. When LAS100B was administered before long-term sensitization in advance, the membrane and threshold potentials of premotor interneurons, which regulate defensive behaviour, decreased to a significantly lesser extent compared to the long-term sensitization arm. It is possible that the “protective” effect is linked to the mechanisms of maintaining the membrane potential and changes in extra- and intracellular balance of calcium-binding S100B protein.