NPY- and C-PON-immunoreactive substances in the central nervous system of Helix pomatia

The distribution of neuropeptide-tyrosin (NPY)- and C-flanking peptide of neuropeptide-tyrosine (C-PON)-immunoreactivities in the central nervous system of the pulmonate gastropod, Helix pomatia, was investigated. NPY- and C-PON-like substances were localized in neuronal somata and neuntes, but were not co-localized within the same cells. NPY-immunoreactive substances were also found in endocrine/paracrine like cells located in the epineurium. C-PON and NPY, both reduced serotonin activated isometric contractions of Helix aorta, suggesting that they may act as modulators in the control of the vascular system.     

Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.     

Satellite glial cell responses to neuronal firing in the nervous system of Helix pomatia

Patch clamp experiments were conducted on satellite glial cells attached to the cell body of neurons in place within the nervous system of the snail Helix pomatia. The glial cells were studied using cell-attached and whole-cell patch clamp configurations while the underlying neurons were under current or voltage clamp control.The resting potential of the glial cells (–69 mV) was more negative than that of the underlying neurons (–53 mV), due to their high K⁺ selectivity. Densely packed K⁺ channels were present, some of which were active at the cell resting potential. Neuronal firing elicited a cumulative depolarization of the glial cells. Large K⁺ currents flowing from V-clamped neurons depolarized the glial layer by up to 30 mV. The glial depolarization was directly correlated with the size of the neuronal K⁺ current. The glial cells recovered their resting potential within 2–5 sec. The neuronal depolarization induced a delayed (20–30 sec) and persistent (3–4 min) increase in the glial K⁺ channel opening probability. Likewise, pulses of K⁺ (20–50 mM)-rich saline activated the glial channels, unless the underlying neuron was held hyperpolarized. In low Ca²⁺-high Mg²⁺ saline, neuron depolarization and K⁺-rich saline did not activate the glial K⁺ channels.These data indicate that a calcium-dependent signal released from the neuronal cell body was involved in glial channel regulation. Neuron-induced channel opening may help eliminate the K⁺ ions flowing from active neurons.I. Gommerat is recipient of a fellowship from the Ministère de la Recherche et de la Technologie.This work was supported by the CNRS and by a grant from the Fondation pour la Recherche Médicale. We would like to thank Mrs. M. André and Mr. G. Jacquet for technical assistance and Mrs. J. Blanc for improving the English.     

Ultrastructure of the bimodal pacemaker neuron in the central nervous system of Helix pomatia L

Fine structure of the identified (RPal) giant neuron of Helix pomatia has been investigated after isolation. The following findings have been established: 1. The cytoplasm of the RPal neuron is electron-microscopically characterized by a strongly invaginated nuclear envelope, a highly-developed rER system, a large number of free ribosomes and by 110-250 nm-sized neurosecretory-like granules with finely-granulated inner content. 2. The fine-structural arrangement of the cytoplasm exhibited a seasonal variation, viz. in winter it was found to be layered, while in spring it showed a homogeneous structure. 3. On the cell surface free axon profiles containing vesicles were sometimes found, which might be afferent connections to the cell body.     

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 m); (ii) large neurons (80–100 m); (iii) medium-sized neurons (40–70 m); (iv) small neurons (12–30 m). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.     

Distribution of tyrosine-hydroxylase-immunoreactive and dopamine-immunoreactive neurons in the central nervous system of the snail Helix pomatia

The distribution and characterization of dopamine-containing neurons are described in the different ganglia of the central nervous system of Helix on the basis of the distribution of tyrosine hydroxylase immunoreactive (TH-ir) and dopamine immunoreactive (DA-ir) neurons. Both TH-ir and DA-ir cell bodies of small diameter (10–25 m) can be observed in the buccal, cerebral and pedal ganglia, dominantly on their ventral surface, and concentrated in small groups close to the origin of the peripheral nerves. The viscero-parietal-pleural ganglion complex is free of immunoreactive cell bodies but contains a dense fiber system. The largest number of TH-ir and DA-ir neurons can be detected in the pedal, and cerebral ganglia. The average number of TH-ir and DA-ir neurons significantly differs but all the identifiable groups of TH-ir neurons also show DA-immunoreactivity. Therefore, we consider the TH-ir neurons in those groups as being DA-containing neurons. The amounts of DA in the different ganglia assayed by high performance liquid chromatography correspond to the distribution and number of TH-ir and DA-ir neurons in the different ganglia. The axon processes of the labeled small-diameter neurons send thin proximal branches toward the cell body layer but only rarely surround cell bodics, whereas distally they give off numerous branches in the neuropil and then leave the ganglion through the peripheral nerves. In the cerebral ganglia, the analysis of the TH-ir pathways indicates that the largest groups of labeled neurons send their processes through the peripheral nerves in a topographic order. These results furnish morphological evidence that DA-containing neurons of Helix pomatia have both central and peripheral roles in neuronal regulation.     

Monoclonal antibodies to individual antigens of the nervous system of the snail, Helix pomatia

Seven positive hybridoma clones were chosen by immunoenzyme analysis amons 103 clones obtained by hybridization of NSO plasmocytoma cells and splenocytes from BALB/C mice, immunized with snail's nervous system antigens. Specific binding of Mabs with neuron cytoplasmic antigens was indicated on cryostat sections of visceral, pedal and cerebral ganglia. The Mabs obtained could be used for the study of physiological role of antigens identified.     

Synaptic organization of a multifunctional interneuron in the central nervous system of Helix pomatia L.

Summary The morphology, axonal arborization and ultrastructure of synaptic connections of the V21 giant neuron in the visceral ganglion of the snail Helix pomatia has been investigated following intracellular labelling with horseradish peroxidase. The V21 neuron establishes several afferent and efferent axo-axonic connections, mainly along the first half of the primary axon. Collaterals of 200–300 m length originate from the primary axon, which shows further arborization, and both afferent and efferent synaptic contacts are formed on these fine axon profiles. Afferent and efferent contacts of the cell occur within very short distances of a few micrometers. On the basis of ultrastructure and vesicle and granule content, several afferent terminals can be distinguished on V21 labelled axon profiles. The majority of these afferent terminals resembles peptidergic-(neurosecretory)-like terminals. This finding supports the possible transmitter role of neuropeptides in the central nervous system of gastropods. Our results are consistent with and provide morphological evidence for recent electrophysiological observations suggesting that, in addition to integrating input, the V21 neuron functions as an interneuron in Helix central nervous system.     

Diurnal rhythms in cyclic nucleotide metabolism in Helix nervous system

Concentrations of cAMP (cyclic adenosine 3′,5′-monophosphate) and cGMP (cyclic guanosine 3′,5′-monophosphate), in ganglia from the garden snail Helix pomatia, vary considerably over the course of the day. There is a maximum in the concentration of both cyclic nucleotides between 08:00 and 12:00 (lights on 06:00 to 18:00), with the cAMP maximum occurring slightly later than that in cGMP. In addition there can be several smaller maxima in cAMP and cGMP levels; the timing of these can be markedly different from experiment to experiment, with cAMP and cGMP sometimes in and sometimes out of phase with each other. This pattern is observed in Helix which had been activated from the dormant state 4–6 days earlier, but is not present in dormant or in long-active animals. The cyclic nucleotide rhythm can be seen in ganglia maintained in organ culture, and persists for at least 24 hours after removal of the tissue from the animal. There appears to be little change in the level of basal or Na Fstimulated adenylate cyclase activity in Helix ganglia over the course of the day. On the other hand, both cAMP and cGMP phosphodiesterase activities exhibit rhythms which are consistent with the rhythms in cAMP and cGMP concentrations.     

Effect of the low-frequency impulse magnetic field on the autonomic nervous system in animals

The effect of weak (up to 3.5 mT) low-frequency (up to 100 Hz) impulse magnetic field on the state of the vegetative nervous system of animals has been studied by analyzing the variability of the heart rate. The effect of the magnetic field was estimated by a specially designed complex for recording cardiac signals of animals. Several specially selected regimes of impulse magnetic fields were studied. It was shown that the impulse magnetic field possesses a high biological activity at all regimes used, and the indices of the vegetative nervous system after the exposure to the impulse magnetic field approach the values typical for normotonic animals. This makes it possible to use magnetic fields at these regimes in magnetotherapy.     

Effect of x-rays on the pacemaker neurons of Helix pomatia

The effect of superlethal doses of ionizing radiation on exciting and electrical properties of giant neurons of the central nervous system of Helix pomatia has been investigated. At early times following irradiation the excitability does not significantly change whereas the membrane potential, resistance and pump-induced hyperpolarization increase. At later times, a stabilization of these parameters is followed by a diminution of resistance, a decrease of membrane potential and pump-induced hyperpolarization, and even the neuron death.     

Diurnal rhythms in cyclic nucleotide metabolism in Helix nervous system.

Concentrations of cAMP (cyclic adenosine 3',5'-monophosphate) and cGMP (cyclic guanosine 3',5'-monophosphate), in ganglia from the garden snail Helix pomatia, vary considerably over the course of the day. There is a maximum in the concentration of both cyclic nucleotides between 08:00 and 12:00 (lights on 06:00 to 18:00), with the cAMP maximum occurring slightly later than that in cGMP. In addition there can be several smaller maxima in cAMP and cGMP levels; the timing of these can be markedly different from experiment to experiment, with cAMP and cGMP sometimes in and sometimes out of phase with each other. This pattern is observed in Helix which had been activated from the dormant state 4-6 days earlier, but is not present in dormant or in long-active animals. The cyclic nucleotide rhythm can be seen in ganglia maintained in organ culture, and persists for at least 24 hours after removal of the tissue from the animal. There appears to be little change in the level of basal or NaF-stimulated adenylate cyclase activity in Helix ganglia over the course of the day. On the other hand, both cAMP and cGMP phosphodiesterase activities exhibit rhythms which are consistent with the rhythms in cAMP and cGMP concentrations.     

Pigment-dispersing hormone-like immunoreactive neurons in the central nervous system of the gastropods, Helix pomatia and Lymnaea stagnalis

By using an antiserum raised against a crustacean #-pigment-dispersing hormone (PDH), the distribution and chemical neuroanatomy of PDH-like immunoreactive neurons was investigated in the central nervous system of the gastropod snails, Helix pomatia and Lymnaea stagnalis. The number of immunoreactive cells in the Helix central nervous system was found to be large (700-900), whereas in Lymnaea, only a limited number (50-60) of neurons showed immunoreactivity. The immunostained neurons in Helix were characterized by rich arborizations in all central ganglia and revealed massive innervation of all peripheral nerves and the neural (connective tissue) sheath around the ganglia and peripheral nerve trunks. A small number of Helix nerve cell bodies in the viscero-parietal ganglion complex were also found to be innervated by PDH-like immunoreactive processes. Hence, a complex central and peripheral regulatory role, including neurohormonal actions, is suggested for a PDH-like substance in Helix, whereas the sites of action may be more limited in Lymnaea.     

Effects of the hypnotic drug etomidate in a model nervous system (Buccal ganglia, Helix pomatia).

1. Effects of the hypnotic drug etomidate were studied with intracellular recordings of the identified neurons B1 to B4 of the buccal ganglia of Helix pomatia. 2. At threshold doses of 10 mumol/l, etomidate mainly affected interneuronal networks. 3. In concentrations above 200 mumol/l, the drug induced typical epileptic activities (paroxysmal depolarization shifts, PDS). Neurons B1 to B4 generated epileptic activities in differential concentration ranges. PDS were synchronized via electrical contacts. PDS could be blocked by the "calcium antagonist" verapamil but not by a block of chemical synaptic transmission. 4. In comparison with the epileptogenic drug pentylenetrazol, effective doses of etomidate to induce PDS were about 100 times lower.