Involvement of intracellular calcium in sensitization of command neurons of defensive behavior in the edible snail (Helix lucorum)

Examinations carried out on command neurons of defensive behavior in the edible snail using electrophysiological methods and a chlortetracycline fluorescent probe revealed that a single sensitizing action alters electrical neuronal activity and the amount of bound calcium in the cells. An initial increase in the amount of bound calcium (the first 15–20 min after the sensitizing action) coincides in time with depolarization, enhancement of plasma membrane excitability, and a decrease of amplitude and duration of the excitatory postsynaptic potentials (EPSP) induced by sensory stimulations. Repeated pronounced increase in the bound calcium level develops 50–60 min after the sensitizing action and correlates with facilitation of neuronal responses to sensory stimuli. Alterations in the bound calcium level in command neurons of defensive behavior in the course of sensitization development differed in dynamics and direction from the previously described bound calcium shifts in the same cells in the course of habituation development.P. K. Anokhin Institute of Normal Physiology, Academy of Medical Sciences of the USSR Moscow. I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 418–427, July–August, 1991.     

Effects of prolonged acclimation to cold on the extra--and intracellular acid-base status in the land snail Helix lucorum (L.)

The aim of this study was to examine the effect of prolonged acclimation to cold on the acid-base status of extra- and intracellular fluids in the land snail Helix lucorum. For this purpose, acid-base parameters in the hemolymph and tissues were determined. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether they change in the snails during acclimation to cold. According to the results presented, there is an inverse pH-temperature relationship in the hemolymph within the first day of acclimation, which is consistent with alphastat regulation. The Pco2 decreased, and pH in the hemolymph (pH(e)) increased by 0.32 U within the first day of acclimation to cold, which corresponds to a change of 0.013 U degrees C(-1). After the first day of acclimation, Pco2 increased in the hemolymph, resulting in a significant drop in pH(e) by 90 d of acclimation to cold. Acclimation of snails to low temperatures did not change the buffer value of the hemolymph. Also, intracellular pH (pH(i)) and intracellular buffer values remained stable during acclimation to cold for prolonged periods. The latter results in conjunction with those obtained by the in vitro determination of the passive component of intracellular fluids indicate an active regulation of pH(i) in H. lucorum during acclimation to cold.     

Postembryonic neuronogenesis 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. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 271–276, 1998     

Growth classes in the adult roman snail (Helix pomatia L.)

Summary a method of dividing adult Helix pomatia into growth classes, which are probably also age classes, is described. Differences in behaviour of individuals previously thought to be associated with their status in a population are probably differences between individuals of different age.     

Evidence for the short-term regulation of glycolytic flux in the isolated perfused ventricle of the land snail Helix lucorum (L.) after treatment with serotonin (5-hydroxytryptamine)

The glycolytic flux and the regulation of phosphofructokinase (PFK) activity by fructose 2,6-bisphosphate and covalent modification was investigated in isolated ventricles of land snail Helix lucorum perfused with or without serotonin. Serotonin evoked a significant increase in the level of glycolytic intermediates and a threefold increase of glycolytic flux. Studies of saturation curves of PFK for the substrate fructose 6-phosphate at pH similar to intracellular pH of heart muscle showed that serotonin increases enzyme sensitivity to activation by fructose 6-phosphate. Moreover, PFK preparations from ventricles perfused with serotonin exhibited lower K _a values for the activators AMP and fructose 2,6-bisphosphate, compared with the enzyme preparations from serotonin-untreated ventricles. The results suggest that PFK was converted to a more active form when exposed to serotonin. In vitro experiments of PFK phosphorylation showed that the conversion of the enzyme to a more active form was possibly due to its phosphorylation by an endogenous cyclic-AMP-dependent protein kinase. The concentration of fructose 2,6-bisphosphate increased in serotonin-treated ventricles and it exerted a synergistic effect with AMP on the activation of PFK. The bound fraction of glycolytic enzymes increased in the serotonin-treated ventricles only after the 4th min of perfusion. The results suggest that the stimulation of glycolytic flux in the ventricles of H. lucorum in the first minutes of perfusion with serotonin was partly due to the activation of PFK via enzyme molecule covalent modification and to increase of fructose 2,6-bisphosphate. Accepted: 8 April 1997     

Synapses identifiable in the parietal ganglia of the snail Helix lucorum

The synaptic plasticity is a background for learning and memory. Identifiable synapses that are the synapses between individually identifiable neurons are a very convenient model for studying plasticity. Synapses between the interoceptive mechanosensory neurons and the command neurons of the withdrawal behavior were identified in the Helix lucorum brain. It was shown that synaptic plasticity estimated by the dynamics of the elementary postsynaptic potentials elicited by single presynaptic spikes differed from the synaptic plasticity estimated by the dynamics of compound synaptic responses of the same neurons to sensory stimulation. Habituation and heterosynaptic facilitation phenomena are discussed in terms of the dynamics of the elementary postsynaptic potentials.     

The nervous system and the mapping of the neurons in the gastropod Helix lucorum L.]

Summarized literature and experimental author's data are presented concerning the structure of the nervous system and identification of individual neurons in the snail Helix lucorum. Information about especially well-known neurons is given in a table, maps of the ganglia are presented altogether with the results of retrograde staining of different cerebral and suboesophageal nerves. Are given the references concerning morphology of the central nervous system of the snail and identifiable neurons.     

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Abstract. The snail Helix lucorum has been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substrate l ‐arginine (l ‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca²⁺, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis in H. lucorum has a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

Effect of serotonin and noradrenaline on the magnitude of the response of the command neurons for defensive behavior in Helix lucorum L

Changes in synaptic responses of identified command neurones of avoidance behaviour to the electric nerve stimulation were investigated in the isolated nervous system of the snail during bath application of serotonin or noradrenaline. Serotonin (10(-5) M) elicited an increase of summary EPSP amplitude in the cells without changes of input resistance and resting potential. Noradrenaline (10(-5) M) application evoked an increase of EPSP amplitude, accompanied by an increase of the input resistance. Mechanisms of serotonin and noradrenaline influence on synaptic responses are discussed.     

Neuronal correlates of changes in the defensive behavior of Helix lucorum L. during ontogeny

In experiments on the isolated nervous system, responses of the same command neurones of avoidance behaviour to electrical stimulation of the intestinal nerve were compared in adult snails (Helix pomatia) and in snails 1-20 days old. Dynamics of spike reactions of the command neurones to rhythmic nerve stimulation differed in adult and newborn snails. In the neurones of young snails, no sensitization, common in the adult animals, was observed. Study of excitatory input in the command neurones during rhythmic nerve stimulation suggests independence of habituation of synaptic input amplitude and spike response sensitization, because dynamics of habituation coincided in the young and adult snails while spike response underwent sensitization only in adult animals.     

Reactions of central neurons to peripheral thermal stimuli in the snail Helix pomatia L

In Helix pomatia L. recordings have been made of 91 single neurons from the cerebral ganglia during peripheral thermal stimulation of the lip region. Temperatures ranged between 12 and 32°C. Different reactions to thermal stimulation could be observed: hyperpolarisation (n=43), depolarisation (n=9), increasing frequency of action potentials (n=10), decreasing frequency of action potentials (n=3). A number of cells did not show any changes in neuronal activity (n=26). The assumption that distinct cell clusters are responsible for the processing of thermal stimuli had to be rejected since cells responding to peripheral thermal stimulation were found in all parts of the ganglia.     

Mechanisms of achromatic vision in snail Helix lucorum L.: intracellular study of light-sensitive cells in retina

Intracellular study of the eye of land snail Helix lucorum L. demonstrated two types of visual cells responding to flashes of white light by slow sustained depolarization (D - type) and by slow sustained hyperpolarization (H - type). Peaks of spectral sensitivity of both types at 465-500 nm coincide with the peak of spectral sensitivity of photopigment 'rhodopsin'. The revealed D- and H - cells in the snail retina are considered as analogous of B- and D - cells, respectively, in the vertebrate visual system.     

Relation between fast and slow elemental synaptic potentials in command neurons in Helix lucorum taurica L

In experiments on a semi-intact snail preparation and a preparation of the snail isolated CNS, after spikes (Sp) evoked in presynaptic neurones by depolarizing current, not only rapid (R) EPSPs emerged in the command neurones of the defensive reaction of closing the pneumostome, but they were also followed by slow (S) EPSPs lasting over 2 min. For each single synaptic contact, the R and S EPSP amplitudes were in a good linear correspondence. In different synapses no direct connection was observed between R EPSP and S EPSP. It is suggested that R and S EPSPs may set in as a result of the action of different substances on the command neurones. Functional significance of S EPSPs with different amplitudes in different command neurones may consist in a prolonged specific preparation of the neurones for the action of stimuli.     

Ca-dependent regulation by Na, K-pump of post-tetanic sensitization of extrasynaptic choline receptors in Helix lucorum neurons

Posttetanic potentiation (by orthodromic stimulation) of cholinosensitivity in LPa3 and RPa3 Helix lucorum neurons that are command in respect to withdrawal behavior was shown earlier (Pivovarov et al., 1999). Now we studied the regulatory role of the Na,K-pump and intracellular free Ga2+ in the posttetanic potentiation (PTP) of cholinosensitivity in command neurons. Semiintact Helix preparation "CNS-visceral bag" was used in experiments. Acetylcholine-induced inward currents were recorded using two-electrode voltage clamp technique. Acetylcholine was applied to somata of the identified LPa3 and RPa3 neurons with a 10-min interval before and after electrical tetanic stimulation of the n. intestinalis (10.5 mA; 0.1 s; 2/s; 2 min). Ouabain (extracellular application, 70 mcM) blocked the PTP. Intracellular injection of BAPTA (1 mM), chelator of Ca2+ ions, prevented the PTP. The PTP was absent after the ouabain application against the background of preliminary intracellular injection of BAPTA. A conclusion war drawn about Ca-dependent participation of Na,K-pump in posttetanic potentiation of cholinosensitivity in command Helix lucorum neurons of withdrawal behavior.     

The potential role of CO2 in initiation and maintenance of estivation in the land snail Helix lucorum

Elevated CO(2) levels are hypothesized to play a role in the initiation and maintenance of estivation in snails through disturbances of acid-base status. The aim of our study was to identify the ambient CO(2) threshold that induces disturbances in acid-base status in the air-breathing land snail Helix lucorum. Acid-base parameters were determined in the hemolymph of snails acclimated to 0.5%, 1%, 2%, 4%, and 8% CO(2) in air for 20 d. In addition, we evaluated the effects of long-term acclimation on metabolic rate and on levels of D-lactate dehydrogenase activity (D-LDH) and of D-lactate in snails after 20 d of exposure to increased CO(2) levels. Helix lucorum proved to be unable to compensate for a decrease in extracellular pH (pH(e)) when acclimated to levels higher than 1% CO(2) in air. The rate of oxygen consumption started to decrease when snails were acclimated to 0.5% CO(2) in air. However, there was no correlation between the drops in pH(e) and in metabolic rate. Long-term acclimation to elevated CO(2) levels induced an increase in the activity of D-LDH with a concomitant accumulation of D-lactate in tissues. This indicates that long-term acclimation to elevated ambient CO(2) levels could reduce the aerobic capacity of land snails and trigger expression of anaerobic pathways of ATP turnover. The threshold levels of ambient CO(2) that induce changes in acid-base status and elicit metabolic depression in adult land snails H. lucorum are higher than the future atmospheric levels that are expected to result from human use of fossil energy resources.     

Selectivity of opioid peptide effects on excitability and various sensory inputs in LPl1 and PPl1 command neurons participating in defensive behavior of the snail Helix lucorum

Opioid peptides effects on neural membrane as well as neural responses evoked by sensory stimuli with different modality and site of application, were investigated in L-RPII command neurones of defensive behaviour of semi-intact preparation in the land snail Helix lucorum. Met-enkephalin (10 uM) application onto the snail CNS increases membrane excitability and produces facilitation of neural responses evoked by quinine solution (0.5%) application onto snail head and depression of reactions evoked by tactile stimulation of the head. Met-enkephalin in dose of 0.1 uM initiates only a depression of neural responses evoked by tactile stimulation of the head. Leu-enkephalin (10 uM) application suppresses neural reactions evoked by tactile stimulation of the head. Membrane excitability and neural responses evoked by quinine application onto the snail head do not change after leu-enkephalin administration. Effects appear 10-20 min after initiation of the peptide application. Initial neural responses were observed 15-30 min after CNS washing with Ringer solution. In addition, facilitation of neural responses evoked by chemical stimulation of the snail head was found 30-50 min after leu-enkephalin washing. Peptides do not change neural responses evoked by tactile stimulation of the snail foot. Neural effects of peptides were prevented by simultaneous naloxon administration (50 uM). Experimental results show selective opioid peptides' effects on excitability and plasticity of L-RPII neural inputs with site- and modality-specifics.