Ca2+-activated KCa3.1 potassium channels contribute to the slow afterhyperpolarization in L5 neocortical pyramidal neurons.

Layer 5 neocortical pyramidal neurons are known to display slow Ca2+-dependent afterhyperpolarization (sAHP) after bursts of spikes, which is similar to the sAHP in CA1 hippocampal cells. However, the mechanisms of sAHP in the neocortex remain poorly understood. Here, we identified the Ca2+-gated potassium KCa3.1 channels as contributors to sAHP in ER81-positive neocortical pyramidal neurons. Moreover, our experiments strongly suggest that the relationship between sAHP and KCa3.1 channels in a feedback mechanism underlies the adaptation of the spiking frequency of layer 5 pyramidal neurons. We demonstrated the relationship between KCa3.1 channels and sAHP using several parallel methods: electrophysiology, pharmacology, immunohistochemistry, and photoactivatable probes. Our experiments demonstrated that ER81 immunofluorescence in layer 5 co-localized with KCa3.1 immunofluorescence in the soma. Targeted Ca2+ uncaging confirmed two major features of KCa3.1 channels: preferential somatodendritic localization and Ca2+-driven gating. In addition, both the sAHP and the slow Ca2+-induced hyperpolarizing current were sensitive to TRAM-34, a selective blocker of KCa3.1 channels.

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 snail's 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.

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.

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.

Morphological basis for coordination of growth and reproduction processes in the CNS of two terrestrial snails.

The morphology of cells immunoreactive to an antibody against molluscan insulin-related peptide (MIP-IR) was studied in two species of terrestrial snail: Helix lucorum L. and Eobania vermiculata L. Immunocytochemical staining with this antibody to MIP revealed 100-130 cells in the postcerebrum, located in two clusters with common pathways in the dorsal body nerve and the cerebral artery nerve. About 75% of the MIP-IR cells were labeled by backfilling of the dorsal body nerve in Helix and Eobania; the corresponding figures for labeling by backfilling of the cerebral artery nerve were about 60% in Helix and 30% in Eobania. Upon intracellular staining of neurons of the dorsomedial postcerebrum, where most of the MIP-IR cells were located, it was found that they projected either in the dorsal body nerve or in the cerebral artery nerve or in both. The obtained data suggest that growth and reproduction processes (both functions were attributed to the insulin-related peptide-containing neurons) are regulated by the two, partially coinciding subsets of postcerebral MIP-IR neurons with different types of branching.

Helix peptide immunoreactivity pattern in the nervous system of juvenile aplysia.

Distribution of neurons immunopositive to antibody against the small peptides encoded by the Helix Command-Specific 2 (HCS2) gene in the central nervous system of juvenile Aplysia californica was investigated. The HCS2 gene is specifically expressed in the withdrawal behavior neurons of the terrestrial snail Helix lucorum. In Aplysia, 20-25 immunopositive neuronal somata were observed on dorsal surface of each pleural ganglion (including a giant pleural neuron). The HCS2-encoded peptide immunopositive fibers were observed in neuropiles of all ganglia and in many nerves. Functional significance of Aplysia immunopositive cells is discussed.

Selective blockade of gene expression in a single identified snail neuron.

In the present study, the applicability of antisense morpholino oligos for loss-of-function experiments in neurobiology was investigated. The identified withdrawal interneurons of the parietal ganglia expressing helix command neuron-specific 2 (HCS2) gene were pressure injected with HCS2 antisense or control morpholino oligo solution at a final concentration 1-4 microM. No toxic or side effects for the neural functioning were noted immediately or several hours after injection. The changes in the concentration of HCS2-encoded protein in neurons after injection were monitored by two methods, Western blotting and immunostaining of the brain. The amount of the peptide immunoreactive with the HCS2 antibody started to decline in the injected cells at day 2 post-injection, decreased four- to five-fold at day 4, and stayed at this low level thereafter. Similar results obtained by both methods suggest significant selective blockade of production of the HCS2-encoded peptide. In contrast, no substantial decrease of the HCS2-encoded polypeptide was observed after injection with control oligos. Due to the high stability of the morpholino oligos in the cell, they represent a highly efficient tool for a specific long-term blockade of gene expression in molluscan neurons.

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.

Ontogenesis of the snail, Helix aspersa: embryogenesis timetable and ontogenesis of GABA-like immunoreactive neurons in the central nervous system.

Late stages of embryogenesis in the terrestrial snail Helix aspersa L. were studied and a developmental timetable was produced. The distribution of gamma-aminobutyric acid-like immunoreactive (GABA-ir) elements in the CNS of the snail was studied from embryos to adulthood in wholemounts. In adults, approximately 226 GABA-ir neurons were located in the buccal, cerebral and pedal ganglia. The population of GABA-ir cells included four pairs of buccal neurons, three neuronal clusters in the pedal ganglia, two clusters and six single neurons in the cerebral ganglia. GABA-ir fibers were observed in all ganglia and in some nerves. The first detected pair of GABA-ir cells in the embryos appeared in the buccal ganglia at about 63-64% of embryonic development. Five pairs of GABA-ir cell bodies were observed in the cerebral ganglia at about 64-65% of development. During the following 30% of development three more pairs of GABA-ir neurons were detected in the buccal ganglia and over fifteen cells were detected in each cerebral ganglion. At the stage of 70% of development, the first pair of GABA-ir neurons was found in the pedal ganglia. In the suboesophageal ganglion complex, GABA-ir fibers were first detected at about 90% of embryonic development. In the posthatching period, the quantity of GABA-ir neurons reached the adult status in four days in the cerebral ganglia, and in three weeks in the pedal ganglia. In juveniles, transient expression of GABA was found in the pedal ganglia (fourth cluster).

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.

Postembryonic neurogenesis 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.

Nervous system and neural maps in gastropod Helix lucorum L.

The present review summarizes the literature and provides new data concerning nervous system structure and the identification of individual neurons in the snail Helix lucorum. Information about especially well-known neurons is provided in a table, and maps of the identifiable neuron's location in ganglia are correlated with the results of retrograde staining via various cerebral and subesophageal nerves. References concerning the morphology of snail central nervous system and identifiable neurons are given.

Mapping of neurons participating in the innervation of the body wall of the snail.

The results of experiments on the retrograde staining of the left-sided cutaneous nerves of the pedal ganglia of the snail Helix lucorum L. are presented in this paper. The bodies of nerve cells contributing processes to these nerves were identified by means of the precipitation of nickel and cobalt ions by rubeanic acid. A large number of neurons were found, including the parietal command neurons, in all the ganglia, with the exception of the buccal. An especially large number of neurons were stained in the ipsilateral pedal, pleural, and parietal ganglia.

The neuroanatomical basis of feeding behavior in the pteropod mollusc, Clione limacina (Phipps).

1. In order to investigate neural mechanisms underlying the switching from hovering-swimming to hunting behavior in the carnivorous mollusc Clione limacina, the innervation of the major muscle bands was studied. The destination of a majority of nerves was traced. 2. Nervous cells sending their processes in the sensory motor nerves were mapped using the retrograde transport of cobalt ions. 3. The combination of morphological investigation, with local extracellular stimulation of groups of cells in semi-intact preparations, provided a detailed description of the functional involvement of the majority of the neural elements in either type of behavior. As a result, detailed diagrams for intracellular investigation of participation of individual cells and functional groups were obtained.

Functioning of identified snail neurones in electric fields.

In both silent and spontaneously active neurones of the snail Helix lucorum, depolarization and spikes were elicited by low-frequency (0.1 Hz) sinusoidal currents applied to the bath solution. Threshold voltage gradients had a range of 1-10 V m-1, which is less than gradients in the nervous tissue during synchronous activation of the neurones. It is shown that the same neurone can generate spikes in response to opposite directions of polarizing currents. Thresholds of spontaneously active neurones to extracellular currents were significantly lower than thresholds of silent cells. A simple quantitative method for evaluation of the transmembrane voltage drop evoked by an electric field is presented. The role of neuronal branches in the response was studied by electrophysiological and morphological methods.