Helix Neurons Research Articles

Invertebrate neurons as a simple model to study the hyperexcitable state of epileptic disorders in single cells, monosynaptic connections, and polysynaptic circuits.

Epilepsy is a neurological disorder characterized by a hyperexcitable state in neurons from different brain regions. Much is unknown about epilepsy and seizures development, depicting a growing field of research. Animal models have provided important clues about the underlying mechanisms of seizure-generating neuronal circuits. Mammalian complexity still makes it difficult to define some principles of nervous system function, and non-mammalian models have played pivotal roles depending on the research question at hand. Mollusks and the Helix land snail have been used to study epileptic-like behavior in neurons. Neurons from these organisms confer advantages as single-cell identification, isolation, and culture, either as single cells or as physiological relevant monosynaptic or polysynaptic circuits, together with amenability to different protocols and treatments. This review's purpose consists in presenting relevant papers in order to gain a better understanding of Helix neurons, their characteristics, uses, and capabilities for studying the fundamental mechanisms of epileptic disorders and their treatment, to facilitate their more expansive use in epilepsy research.

Isolated viagra solution application effects on the electrical activity in Helix neurons

The authors explored effects of applying an isolated solution of Viagra (Sildenafil Citrate) on the electrical activity in unidentified neurons of the Helix visceral ganglion. At a concentration of 0.5×10-2 M, the neurotropic effect of Viagra was present in the form of a persistent hyperpolarization of the neuron membrane, resulting in reduced neuronal impulses, with the membrane potential not reaching its baseline values upon washing out. At a concentration of 10-3 M, the effects were also inhibitory, accompanied by a non-significant cell membrane depolarization. At a concentration of 10-5 to 10-4 M, Viagra produced excitatory effects, with the membrane potential reaching its baseline values upon washing out. The discovered data on the influence and neurotoxicity of Viagra are to be accounted for when administering its specific dosages, to prevent possible negative effects. Further research is needed to expand the knowledge on possible clinical effects of Viagra administering.

Regulation of histone H4 acetylation in the CNS and defensive behavior command neurons of the mollusk Helix mediated by serotonin and neuropeptide FMRFamide

Epigenetic mechanisms are commonly known to underlie memory formation. Presently, scientists’ attention is focused on changes in the levels of histone modifications (mainly acetylation and methylation) in the chromatin of CNS cells tested in various experimental models. Owing to their relatively simple CNSs, mollusks are among the most popular models. Our experiments were con-ducted with the molluskHelix lucorumbecause its CNS had been investigated in detail and most of its neurons had been proven to participate in the formation of different behavior patterns, including the prolonged response to various stimuli. This work concerns the influence of various effectors (serotonin and FMRFamide, associated with CNS activator and inhibitory pathways, respectively) on the acetylation of H4 histone in the subesopha­geal ganglion complex and in defensive behavior command neurons of the right and left parietal ganglia (RPa3/2 and LPa3/2) in the snail. Western blot analysis showed that FMRFamide inhibited histone H4 acetylation induced by serotonin in the subesophageal complex of CNS ganglia. How­ever, serotonin and FMRFamide cooperatively enhanced the induction of histone H4 acetylation in RPa3/2 defensive behavior command neurons. No changes were found in the counterpart LPa3/2. It is a new piece of evidence for functional asym­metry inHelix. The inhibitory pathways mediated by FMRFamide not only inhibit the activatory in­tracellular processes in the entire CNS but can also enhance them, as in RPa3/2 defensive behavior command neurons.

Influence of Membrane Receptor Lateral Diffusion on the Short-Term Depression of Acetylcholine-Induced Current in Helix Neurons.

We have studied how various drugs increasing the rate of nicotinic acetylcholine receptors (nAChRs) lateral diffusion affect the depression of ACh-induced current in land snail Helix lucorum neurons responsible for defensive behavior. The acetylcholine (ACh) iontophoretic application protocol imitated the behavioral habituation protocol for the intact animal. We found that the drugs decreasing cholesterol level in cell membranes as methyl-β-cyclodextrin 1mM and Ro 48-8071 2µM, and polyclonal antibodies to actin-binding proteins as spectrin 5µg/ml and merlin 2.5µg/ml have changed the dynamic of ACh-current depression. The nAChRs lateral diffusion coefficient was obtained by fluorescence recovery after photobleaching. A curve fitting model specially created for analysis of short-term choline sensitivity depression in snail neurons helped us evaluate separately the contribution of nAChRs lateral diffusion, their endocytosis and exocytosis to observed effects during electrophysiological experiments. Taken together, we hypothesize that nAChRs lateral diffusion plays an important role in the cellular correlate of habituation in land snail Helix lucorum neurons.

Subconvulsant doses of pentylenetetrazol uncover the epileptic phenotype of cultured synapsin-deficient Helix serotonergic neurons in the absence of excitatory and inhibitory inputs

Synapsins are a family of presynaptic proteins related to several processes of synaptic functioning. A variety of reports have linked mutations in synapsin genes with the development of epilepsy. Among the proposed mechanisms, a main one is based on the synapsin-mediated imbalance towards network hyperexcitability due to differential effects on neurotransmitter release in GABAergic and glutamatergic synapses. Along this line, a non-synaptic effect of synapsin depletion increasing neuronal excitability has recently been described in Helix neurons. To further investigate this issue, we examined the effect of synapsin knock-down on the development of pentylenetetrazol (PTZ)-induced epileptic-like activity using single neurons or isolated monosynaptic circuits reconstructed on microelectrode arrays (MEAs). Compared to control neurons, synapsin-silenced neurons showed a lower threshold for the development of epileptic-like activity and prolonged periods of activity, together with the occurrence of spontaneous firing after recurrent PTZ-induced epileptic-like activity. These findings highlight the crucial role of synapsin on neuronal excitability regulation in the absence of inhibitory or excitatory inputs.

Synchronization dynamics induced on pairs of neurons under applied weak alternating magnetic fields

Pairs of Helix aspersa neurons show an alternating magnetic field dependent frequency synchronization (AMFS) when exposed to a weak (amplitude B0 between 0.2 and 150Gauss (G)) alternating magnetic field (AMF) of extremely low frequency (ELF, fM=50Hz). We have compared the AMFS patterns of discharge with: i) the synaptic activity promoted by glutamate and acetylcholine; ii) the activity induced by caffeine; iii) the bioelectric activity induced on neurons interconnected by electric synapses. AMFS activity reveals several specific features: i) a tight coincidence in time of the pattern and frequency, f, of discharge; ii) it is induced in the time interval of field application; iii) it is dependent on the intensity of the sinusoidal applied magnetic field; iv) elicited biphasic responses (excitation followed by inhibition) run in parallel for the pair of neurons; and v) some neuron pairs either spontaneously or AMF synchronized can be desynchronized under applied higher AMF. Our electron microscopy studies reveal gap-like junctions confirming our immunocytochemistry results about expression of connexin 26 (Cx26) in 4.7% of Helix neurons. AMF and carbenoxolone did not induce any significant effect on spontaneous synchronization through electric synapses.

Selective modulation of chemical and electrical synapses of Helix neuronal networks during in vitro development

BackgroundA large number of invertebrate models, including the snail Helix, emerged as particularly suitable tools for investigating the formation of synapses and the specificity of neuronal connectivity. Helix neurons can be individually identified and isolated in cell culture, showing well-conserved size, position, biophysical properties, synaptic connections, and physiological functions. Although we previously showed the potential usefulness of Helix polysynaptic circuits, a full characterization of synaptic connectivity and its dynamics during network development has not been performed.ResultsIn this paper, we systematically investigated the in vitro formation of polysynaptic circuits, among Helix B2 and the serotonergic C1 neurons, from a morphological and functional point of view. Since these cells are generally silent in culture, networks were chemically stimulated with either high extracellular potassium concentrations or, alternatively, serotonin. Potassium induced a transient depolarization of all neurons. On the other hand, we found prolonged firing activity, selectively maintained following the first serotonin application. Statistical analysis revealed no significant changes in neuronal dynamics during network development. Moreover, we demonstrated that the cell-selective effect of serotonin was also responsible for short-lasting alterations in C1 excitability, without long-term rebounds.Estimation of the functional connections by means of cross-correlation analysis revealed that networks under elevated KCl concentrations exhibited strongly correlated signals with short latencies (about 5 ms), typical of electrically coupled cells. Conversely, neurons treated with serotonin were weakly connected with longer latencies (exceeding 20 ms) between the interacting neurons. Finally, we clearly demonstrated that these two types of correlations (in terms of strength/latency) were effectively related to the presence of electrical or chemical connections, by comparing Micro-Electrode Array (MEA) signal traces with intracellularly recorded cell pairs.ConclusionsNetworks treated with either potassium or serotonin were predominantly interconnected through electrical or chemical connections, respectively. Furthermore, B2 response and short-term increase in C1 excitability induced by serotonin is sufficient to trigger spontaneous activity with chemical connections, an important requisite for long-term maintenance of firing activity.

Роль серин/треониновых и тирозиновых протеинфосфатаз командных нейронов виноградной улитки на клеточном аналоге привыкания

Effects of some inhibitors of serine/threonine and tyrosine protein phosphatases on the depression and spontaneous recovery of the acetylcholine-induced inward current (ACh-current) in command Helix neurons of defensive behavior at the cellular correlate of habituation were investigated. The following drugs were used: okadaic acid (reduces activity ofphosphatases PP1 and PP2A), endothall (PP2A), cyclosporine A and cypermethrin (PP2B), CCT007093 (PPM1D), dephostatin (blocks tyrosine phosphatases). All used inhibitors modify the depression flow, and endothall reduces spontaneous recovery of ACh-current also. Obtained results indicate that changes in cholinosensitivity of command neurons depend on activity of all investigated protein phosphatases. Mathematical model considers the possibility of different localizations of receptors in a neuron and regularity of transitions between them. This model makes it possible to conclude participation indicated phosphatases in mobility of membrane cholinoreceptors ensuring the ACh-current modification at the cellular correlate of habituations. Comparison of experimental and calculated curves of ACh-current change allows to conclude that the main target of protein phosphatases is the transport system of a neuron--cytoskeleton and motor proteins.

Mechanisms Underlying the Effects of Salicylic Acid and Its Salts on Helix Neurons

In experiments on Helix neurons, we found that calcium is not involved in the neurotropic effects of salicylic acid and cobalt and zinc salicylates. This conclusion is based on the fact that the addition of BaCl2 to the applied solutions of salicylates did not change the effects of the latter. Extracellular application of adenosine triphosphate (ATP) significantly modified the effects of salicylic acid and its salts. We believe that the inhibitory effect of salicylic acid on Helix neurons depends on the ability of salicylic acid to suppress ATP synthesis, while the activating effect of cobalt and zinc salicylates on neurons is related to the opposite effects of these salts on ATP synthesis.

My first 20 years in neuroscience

Intriguing facts were obtained in the first electrophysiological investigations (1964) that the action potentials (AP) produced by direct depolarization of the cell membrane in different species of mollusks showed specific relations to changes in external ionic composition. In Helix neurons, the generation of AP was well maintained in sodium-free solutions with high calcium or barium content. The amplitude of the spike overshoot in the case was linearly related to the logarithm of calcium concentration. It is interesting that increase in external calcium ions decreased the ionic conductance of the resting membrane (R0) also in linear relation to the logarithm of Ca2+ or Ba2+ concentration. It was found for the first time (1965) that addition of Ba2+ to the external solution produced in the neurons well-developed prolonged (protracted) APs in all cases. However, the excitability of Planorbis and Limnea neurons was rapidly (during 3 min) reversibly depressed in sodium-free solutions. We found that, after injections of horse radish peroxidase (HRP) in the spinal cord of cats, the enzyme was transported retrogradely to brain stem neurons in the bulbar medial reticular formation, the vestibular complex, and the red nucleus. We obtained (1975) intriguing facts in our investigations: we recorded the labeled neurons in the locus coeruleus and subcoeruleus, as well as in the paraventricular hypothalamic nucleus. The existence of straight pathways from hypothalamus to the spinal cord has not been demonstrated earlier. The next step of our study was to determine the corresponding spinal funiculi where descending fibers (from various brain stem cell groups) are located. Indeed, in our pioneer studies (1977), we found that the fibers from the hypothalamus, which descend throughout the spinal cord, are located mainly in the lateral funiculus, ipsilaterally.

Neurotoxins produced by the Cylindrospermopsis raciborskii (Cyanobacteria) isolated from Lake Balaton (Hungary)

Event Abstract Back to Event Neurotoxins produced by the Cylindrospermopsis raciborskii (Cyanobacteria) isolated from Lake Balaton (Hungary) Agnes Vehovszky1*, András Ács2, Attila W. Kovács1, Henriette Szabó1, János Gyori1 and Anna Farkas1 1 Hungarian Academy of Sciences, Balaton Limnological Research Institute, Hungary 2 University of Pannonia, Hungary The membrane effects of the crude extracts (100 mg dry weight/ml) and solid phase extracted fractions (1 mg dry weight/ml) of the cyanobacteria Cylindrospermopsis raciborskii (ACT 9502, ACT 9504, ACT 9505 strains) isolated from Lake Balaton, Hungary were characterized on identified central neurons of snails (Lymnaea stagnalis and Helix pomatia). The membrane effects were also tested on the purified fractions of Oscillatoria formosa (PCC 6506) strains used as reference for a neurotoxin (anatoxin-a) production. The purified fraction of Cylindrospermopsis raciborskii strains evoked strong intracellular (either depolarizing or hyperpolarizing) membrane responses on the identified neurons of both Helix and Lymnaea, similar to the locally applied acetylcholine, iontophoretically injected near the surface of the cell body. Moreover, cyanobacterial extracts applied by perfusion reversibly blocked the acetylcholine responses, suggesting inhibition of the cholinergic receptors. A similar reversible, dose-dependent inhibition of acetylcholine responses was recorded in the presence of the neurotoxin anatoxin-a or the PCC 6506 extracts. Liquid chromatography-mass spectrometry (LC-MS) carried out on solid phase extracted fractions of the ACT 9502, ACT 9504, ACT 9505 and PCC 6506 strains confirmed the presence of the cholinergic neurotoxins: anatoxin-a and homoanatoxin-a in the extracts of PCC 6506 strain, and trace amounts of the same toxins in the extracts of Cylindrospermopsis raciborskii strains isolated from Lake Balaton. Moreover, we cannot rule out the presence of other, still unidentified neurotoxic substances, as suggested by the compound electrophysiological responses of the crude cyanobacterial extracts and the purified fraction of the ACT 9504 strain. This work was supported by the Hungary OTKA grant K63451. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Cellular neuroscience Citation: Vehovszky A, Ács A, Kovács AW, Szabó H, Gyori J and Farkas A (2010). Neurotoxins produced by the Cylindrospermopsis raciborskii (Cyanobacteria) isolated from Lake Balaton (Hungary). Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00174 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Apr 2010; Published Online: 30 Apr 2010. * Correspondence: Agnes Vehovszky, Hungarian Academy of Sciences, Balaton Limnological Research Institute, Budapest, Hungary, agnes@tres.blki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Agnes Vehovszky András Ács Attila W Kovács Henriette Szabó János Gyori Anna Farkas Google Agnes Vehovszky András Ács Attila W Kovács Henriette Szabó János Gyori Anna Farkas Google Scholar Agnes Vehovszky András Ács Attila W Kovács Henriette Szabó János Gyori Anna Farkas PubMed Agnes Vehovszky András Ács Attila W Kovács Henriette Szabó János Gyori Anna Farkas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Voltage-dependent blockade of delayed rectifier K+ current by nitroprusside and ferricyanide

NO donor nitroprusside (NP) is a biologically active drug with hypotensive and neurotropic properties. Its effects are due to NO as well as to other derivates, specifically ferricyanide (FC) and ferrocyanide (F(2+)C) ions. In the present work we studied effects of NP, FC, and F(2+)C on delayed rectifier K+ current. The experiments were conducted on isolated neurons of land snail Helix using two-microelectrode voltageclamp technique. Delayed rectifier K+ current often displayed abnormal rectification. NP (1 mM) caused voltage-dependent reduction of K+ current, which looked like enhancement of the abnormal rectification. FC (1 mM)—but not F(2+)C—acted likewise; the effects of NP and FC were not additive. Dibutiryl cGMP (dbcGMP) had an opposite effect, weakening abnormal rectification of the K+ current. Protein kinase inhibitor H-8 (10 μM) caused voltage-dependent reduction of the K+ current, as NP and FC did. The effects of H-8 and NP and effects of H-8 and FC were not additive. The results suggest that: (i) NP effects on delayed rectifier K+ current are mediated by FC and (ii) the effects of these drugs involve phosphorylation processes.

Helix neuronal ensembles with controlled cell type composition and placement develop functional polysynaptic circuits on Micro-Electrode Arrays

Neuronal cell cultures on Micro-Electrode Arrays (MEAs) provide an essential experimental tool for studying the connectivity and long-term activity of complex neuronal networks. MEA studies are generally based on the analysis of mixed neuronal populations constituted by a large number of cultured cells with cell type composition and connectivity patterns which are quite unpredictable a priori. In this work, we propose a different approach which consists of assembling on MEAs neuronal circuits formed by individually identifiable C1, C3, and B2 Helix neurons. Cells were plated under conditions of controlled number and position to form neuronal networks of defined composition. We performed multi-site electrophysiological recordings, and we characterized the firing dynamics. By means of cross-correlation analysis, we studied the electrophysiological properties of MEA-coupled microcircuits and characterized their activity patterns. We showed how the synaptic connectivity, actually observed in polysynaptic circuits of C1, C3 and B2 neurons, correlates well with the expected connectivity of C1–B2, B2–B2 and B2–C3 cell pairs as previously reported in conventional electrophysiological studies in culture.

Reflections

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Characterization and role ofHelixcontactin‐related proteins in culturedHelix pomatianeurons

We report on the structural and functional properties of the Helix contactin-related proteins (HCRPs), a family of closely related glycoproteins previously identified in the nervous system of the land snail Helix pomatia through antibodies against the mouse F3/contactin glycoprotein. We focus on HCRP1 and HCRP2, soluble FNIII domains-containing proteins of 90 and 45 kD bearing consensus motifs for both N- and O-glycosylation. Using the anti-HCRPs serum, we find secreted HCRPs in Helix nervous tissue isotonic extracts and in culture medium conditioned by Helix ganglia. In addition, we demonstrate expression of HCRPs on neuronal soma and on neurite extensions. Functionally, in Helix neurons, the antisense HCRP2 mRNA counteracts neurite elongation, and the recombinant HCRP2 protein exerts a strong positive effect on neurite growth when used as substrate. These data point to HCRPs as novel neurite growth-promoting molecules expressed in invertebrate nervous tissue.

Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons.

The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of co-localization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity.

Involvement of Na,K-pump in SEPYLRFamide-mediated reduction of cholinosensitivity in Helix neurons

SEPYLRFamide acts as an inhibitory modulator of acetylcholine (ACh) receptors in Helix lucorum neurones. Ouabain, a specific inhibitor of Na,K-pump, (0.1 mM, bath application) decreased the ACh-induced inward current (ACh-current) and increased the leak current. Ouabain decreased the modulatory SEPYLRFamide effect on the ACh-current. There was a correlation between the effects of ouabain on the amplitude of the ACh-current and on the modulatory peptide effect. Ouabain and SEPYLRFamide inhibited the activity of Helix aspersa brain Na,K-ATPase. Activation of Na,K-pump by intracellular injection of 3 M Na acetate or 3 M NaCl reduced the modulatory peptide effect on the ACh-current. An inhibitor of Na/Ca-exchange, benzamil (25 μM, bath application), and an inhibitor of Ca 2+-pump in the endoplasmic reticulum, thapsigargin (TG, applied intracellularly), both prevented the effect of ouabain on SEPYLRFamide-mediated modulatory effect. Another inhibitor of Ca 2+-pump in the endoplasmic reticulum, cyclopiazonic acid (applied intracellularly), did not prevent the effect of ouabain on SEPYLRFamide-mediated modulatory effect. These results indicate that Na,K-pump is responsible for the SEPYLRFamide-mediated inhibition of ACh receptors in Helix neurons. Na/Ca-exchange and intracellular Ca 2+ released from internal pools containing TG-sensitive Ca 2+-pump are involved in the Na,K-pump pathway for the SEPYLRFamide-mediated inhibition of ACh receptors.

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.

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.

In vitro formation and activity-dependent plasticity of synapses between Helix neurons involved in the neural control of feeding and withdrawal behaviors

Short-term activity-dependent synaptic plasticity has a fundamental role in short-term memory and information processing in the nervous system. Although the neuronal circuitry controlling different behaviors of land snails of the genus Helix has been characterized in some detail, little is known about the activity-dependent plasticity of synapses between identified neurons regulating specific behavioral acts. In order to study homosynaptic activity-dependent plasticity of behaviorally relevant Helix synapses independently of heterosynaptic influences, we sought to reconstruct them in cell culture. To this aim, we first investigated in culture the factors regulating synapse formation between Helix neurons, and then we studied the short-term plasticity of in vitro-reconstructed monosynaptic connections involved in the neural control of salivary secretion and whole-body withdrawal. We found that independently of extrinsic factors, cell–cell interactions are seemingly sufficient to trigger the formation of electrical and chemical synapses, although mostly inappropriate—in their type or association—with respect to the in vivo synaptic connectivity. The presence of ganglia-derived factors in the culture medium was required for the in vitro reestablishment of the appropriate in vivo-like connectivity, by reducing the occurrence of electrical connections and promoting the formation of chemical excitatory synapses, while apparently not influencing the formation of inhibitory connections. These heat-labile factors modulated electrical and chemical synaptogenesis through distinct protein tyrosine kinase signal transduction pathways. Taking advantage of in vitro-reconstructed synapses, we have found that feeding interneuron–efferent neuron synapses and mechanosensory neuron–withdrawal interneuron synapses display multiple forms of short-term enhancement-like facilitation, augmentation and posttetanic potentiation as well as homosynaptic depression. These forms of plasticity are thought to be relevant in the regulation of Helix feeding and withdrawal behaviors by inducing dramatic activity-dependent changes in the strength of input and output synapses of high-order interneurons with a crucial role in the control of Helix behavioral hierarchy.