[Fast and aimed delivery of voltage-sensitive dyes to mammalian brain slices by biolistic techniques].

Fast voltage-sensitive dyes (VSD) are widely used in modern neuroscience for optical recording of electrical potentials at many levels, from single cell compartment to brain areas, containing populations of many neural cells. The more lipophilic a VSD, the better signal-to-noise ratio of the optical signal, but there are no effective ways to deliver a water-insoluble dye into the membrane of live cell. Here we report a new protocol based on rapid biolistic delivery of VSDs, which is optimal for further recordings of optical signals from live neurons of rat brain slices. This protocol allows us to stain locally (150 mkm) neural somata of brain structures with a Golgi-like pattern, and a VSD propagates even to distant neurites of stained cells very quickly. This technique also can be used for rapid local delivery of any lipophilic and water-insoluble substances into live cells, further optical recording of neural activity, and analysis of potential propagation in a nerve cell.

[Serotonergic system of neurons in the CNS of terrestrial snail: morphology, ontogenesis, control of behavior].

The morphology of the serotonergic neurons modulating withdrawal behavior in the CNS of terrestrial snail was studied. It was shown that only one Pd4 neuron projects to the pleural and parietal ganglia. Its intracellular stimulation caused the same effects as the stimulation of the whole group of modulatory neurons. In juvenile snails, the number of serotonergic neurons and their relative sizes are smaller than in adult animals, which can be the reason for the observed age-related differences in the withdrawal behavior.

Immunohistochemical detection of the activation of CREB and c-Fos transcription factors in the nervous system of the terrestrial snail induced by pentylenetetrazole.

Phosphorylation of CREB transcription factor and expression of the immediate early gene c-fos in the brains of vertebrates play a key role in the molecular genetic mechanisms of long-term neuronal plasticity. The present study identifies the conditions for immunohistochemical detection of pCREB and c-Fos in the nervous system of the mollusk Helix lucorum (Pulmonata: Stylommatophora); activation of these transcription factors was demonstrated after administration of the convulsive agent pentylenetetrazole. Basal pCREB and c-Fos levels in the central nervous system of intact animals were low. Injection of pentylenetetrazole at a dose of 600 mg/kg evoked characteristic stereotypical motor responses, along with sharp reductions in the phosphorylation of CREB1 and expression of the immediate early gene c-fos, this also occurring in identified neurons. Double immunofluorescent labeling of pCREB and c-Fos showed that expression of c-Fos transcription factor was seen only in pCREB-immunoreactive neurons. These data provide evidence that activation of pCREB and c-Fos transcription factors can be used as molecular markers for mapping the processes of neuronal plasticity in the nervous systems of mollusks.

Functions of peptide CNP4, encoded by the HCS2 gene, in the nervous system of Helix lucorum.

The aims of the present work were to study the role of neuropeptide CNP4, encoded by the HCS2 gene (which is expressed mainly in parietal command interneurons), in controlling the activity of the respiratory system, and also to study the effects of this neuropeptide on isolated defensive behavior neurons in prolonged culture. The influence of the command interneuron on the pneumostoma included a direct effect consisting of closure and a delayed effect consisting of intensification of respiratory movements. Application of neuropeptide CNP4 produced a pattern similar to the delayed effects seen on stimulation of the command interneuron, i.e., significant increases in the frequency and intensity of pneumostoma movements and strengthening of the rhythmic activity of the pneumostoma motoneuron. Studies of the effects of neuropeptide CNP4 on isolated neurons after prolonged culture showed that neuron process growth correlated with the presence of the neuropeptide in the medium. Identification of the location of the HCS2 precursor protein and neuropeptide CNP4 in isolated command interneurons after prolonged culture showed that that only those parts of the cell showing active process growth were immunopositive. Thus, neuropeptide CNP4 appears to be a secreted neuropeptide controlling respiratory system activity, which may also be involved in rearrangements of the network controlling defensive behavior in Helix snails.

[Immunohistochemical detection of transcription factors CREB and c-Fos activation in the land snail nervous system induced by pentylenetetrazole].

Phosphorylation of transcription factor CREB and expression of immediate early gene c-fos play a key role in molecular mechanisms of long-term neuronal plasticity in the vertebrate brain. Here, we have defined the procedure of immunohistochemical detection of pCREB and c-Fos in the nervous system of the land snail Helix lucorum (Pulmonata: Stylommatophora) and have shown its activation after the convulsant pentylenetetrazole injection. Baseline pCREB1 and c-Fos levels in the intact snail nervous system appeared to be low. In contrast, injection of pentylenetetrazole (600 mg/kg) produced a rapid induction of CREB phosphorylation and c-Fos expression in a wide range of neurons including a number of identified cells. Double immunofluorescence for pCREB and c-Fos showed that c-Fos was always colocalized with pCREB-immunoreactivity, although the latter had a broader pattern. The results suggest that transcription factors CREB and c-Fos can be used as molecular markers for mapping long-term neuronal plasticity in molluscan nervous system.

[Functional role of neuropeptide CNP4 encoded by gene HCS2 in Helix lucorum nervous system].

Helix Command Specific 2 (HCS2) gene is constantly expressed in parietal premotor (command) interneurons involved in control of the terrestrial snail Helix lucorum withdrawal behavior as a trigger element. It is also expressed under noxious conditions in other neurons presumably involved in withdrawal behavior. In this study we addressed the role of neuropeptide CNP4, encoded by gene HCS2, in the regulation of activity of the respiratory system, and in the influence on growth of isolated neurons in culture. It was shown that activity of the premotor interneuron elicits a direct effect (pneumostome closure), and a delayed intensification of respiratory movements. Application of CNP4 mimicked the delayed effects. Presence of the peptide CNP4 in solution for cultured neurons led to increase of neuronal growth. Immunochemical localization of the protein precursor encoded by gene HCS2 and peptide CNP4 in the cultured premotor interneurons revealed their preferential presence in the growth cones. The obtained results suggest that CNP4 may be secreted and involved in synergic regulation of behavior of a snail.

[Immunocytochemical study of the hcs2 gene products distribution in the command neurons of grape snail]. / Immunotsitokhimicheskoe izuchenie lokalizatsii produktov gena hcs2 v komandnykh neironakh vinogradnoi ulitki.

In the present study the cellular and subcellular distribution of putative protein products of hcs2 gene in the giant command neurons of parietal ganglia of the terrestrial snail Helix lucorum L. were investigated using light- and electron-microscopic immunocytochemistry. The product of hcs2 gene is a hybrid precursor protein belongs to the EF-hand family of the Ca(2+)-binding proteins, whose processed products are neuropeptides. By use of polyclonal antibodys against a synthetic CNP3, CNP4 and C-terminus peptide immunoreactivity was observed in the cytoplasmic secretory granules. The colloidal gold density in the granules for CNP3-4 neuropeptides was twice one for the Ca(2+)-binding protein. These immunocytochemical results point to a specific connection between putative protein products of hcs2 gene and the cell secretory apparatus, that correspond to our early expressed hypothesis that products of hcs2 gene act as neuromodulators or neurotransmitters.

[A comparison of serotonin- and dopaminergic neuronal systems in sexually mature and juvenile terrestrial mollusks of the genera Helix and Eobania]. / Sravnenie serotonin- i dofaminergicheskoi neironnykh sistem u polovozrelykh i iuvenil'nykh nazemnykh molliuskov Helix i Eobania.

The distribution of serotonin- (5-HT) and dopamine- (DA) containing neurons was studied in the brain of adult and juvenile (the first month of life) terrestrial molluscs Helix lucorum and Eobania vermiculata by means of glyoxylic acid fluorescent technique and neurotoxin 5,7-DiHT labelling. The majority of 5-HT-containing cells were grouped in clusters in the pedal ganglia (the median number of cells in a cluster was 213 in Eobania and 155 in Helix). Some cells were described in the cerebral ganglia, visceral and right parietal ganglia. DA-containing neurons were found in the buccal ganglia, cerebral ganglia (there were 6-8 large dorsal cells and about 80 small ventral cells) and pedal ganglia (the median number of cells was 36 in Helix and 12 in Eobania). In juvenile animals of both species the number and distribution of clusters with monoaminergic cells resembled that of the adults. In the juvenile pedal ganglia the number of 5-HT-containing cells was about one fourth of that in adult snails. The number of DA-containing cells in juveniles was the same as in adults in both species. In juvenile snails of both species one big and two medium-sized 5-HT-containing cells were found at the dorsal surface of the cerebral ganglia. Such cells were not revealed in adults. The finding suggests that these cells can change the transmitter phenotype or be transient.

[The mapping of the neurons that participate in the innervation of the body wall of the edible snail]. / Kartirovanie neironov, uchastvuiushchikh v innervatsii stenki tela vinogradnoi ulitki.

In the paper are presented the results of the experiments with the retrograde staining of the left-side cutaneous nerves of the pedal ganglia of the snail Helix lucorum. The somata of neurons which sent processes forming the studied nerves were revealed with the help of nickel and cobalt ions precipitation by the rubeanic acid. A lot of nerve cells including some command neurons were found in all the ganglia excluding the buccal ones. Especially large amount of neurons was stained in the ipsilateral pedal, pleural, and parietal ganglia.

[The nervous system and the mapping of the neurons in the gastropod Helix lucorum L]. / Nervnaia sistema i kartirovanie neironov briukhonogogo molliuska 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.

Role of the cerebral ganglia in the organization of alimentary behavior of the pteropod mollusc Clione limacina.

The role of the cerebral ganglia in the switching of the behavior of a mollusc from free swimming to hunting behavior was studied. For this purpose the bodies of neurons providing processes to sensory and motor nerves were stained by means of retrograde axonal transport of Co2+ ions, and the behavioral reactions occurring during extracellular stimulation of particular regions of the ganglia were investigated. Comparison of the morphological data with the behavioral reactions showed that the cerebral neurons participate on the basis of sensory information in the switching of quiet swimming to one of three modes: hunting behavior (including the seizure of food), active avoidance, and passive-defensive behavior.

[The role of the cerebral ganglia in the organization of feeding behavior in the pteropod mollusk Clione limacina]. / Rol' tserebral'nykh gangliev v organizatsii pishchevogo povedeniia krylonogogo molliuska Clione limacina.

The role of cerebral ganglia in switching from free swimming over to hunting behaviour in Clione limacina (Phipps) was studied. The cell bodies of central neurones giving processes to the sensory and motor nerves were mapped by retrograde axonal transport of Co2+ ions, and behavioural reactions under the extracellular electric stimulation of different ganglia areas were investigated. Comparison of morphological and behavioural data suggests main role of cerebral ganglia neurones in switching of behaviour from free swimming over to any of the three possible states: hunting (including food uptake), active or passive defensive behaviour.

[Localization of areas of action potential generation in neurons of the edible snail]. / Lokalizatsiia zon generatsii potentsialov deistviia v neironakh vinogradnoi ulitki.

Site of origin of action potentials in silent and spontaneously active unipolar neurons of the snail Helix lucorum was established by stimulation of neurons isolated together with the initial part of the process from the neuropile by the sinusoidal extracellular current flowing along the soma-axon direction in the saline. The source of the spontaneous activity was in the neuronal process in all investigated neurons and one trigger zone usually dominated. Threshold of the action potential generation in response to the extracellular current in silent neurons was also connected with the axonal trigger zone activation. The low-threshold zone of action potential generation in the soma was found only in 20% of studied neurons.

[Magnitude of the potential generated in the nerve cell of molluscs in a low-frequency electric field]. / Velichina potentsiala, navedennogo na nervnoi kletke molliuskov v nizkochastotnom élektricheskom pole.

The distribution of potential in CNS neurones under the action of extracullular electric current was studied in snails. The voltage drop occurred mainly on the cellular membrane. The contribution of processes to the potential induced in the cell is evaluated.

[Low-threshold area of action potential generation in the somatic membrane of mollusk neurons]. / Nizkoporogovaia oblast' generatsii potentsialov deistviia v somaticheskoi membrane neironov molliuskov.

The excitability of somatic membrane of Helix lucorum neurons was studied by means of extracellular electric current of various direction (frequency 0.1 Hz). Inhomogenous excitability of somatic membrane was shown and the existence of the local low-threshold area of spike generation in the soma was demonstrated.