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.     

Functional morphology of the light yellow cell and yellow cell (sodium influx-stimulating peptide) neuroendocrine systems of the pond snail Lymnaea stagnalis

Neuroendocrine light yellow cells of the pond snail Lymnaea stagnalis express a neuropeptide gene encoding three different peptides. The morphology of the cell system has been studied by in situ hybridization, using two synthetic oligonucleotides encoding parts of light yellow cell peptides I and III, and by immunocytochemistry with antisera to synthetic light yellow cell peptide II and to two fragments of light yellow cell peptide I. One large cluster of light yellow cells was observed in the ventro-lateral protrusion of the right parietal ganglion, smaller clusters lying in the posterior dorsal part of this ganglion and in the visceral ganglion. The cells had an extended central neurohaemal area. Immunopositive axons projected into all nerves of the ganglia of the visceral complex, into the superior cervical and the nuchal nerves, and into the connective tissue surrounding the central nervous system. Axon tracts ramified between the muscle cells of the walls of the anterior aorta and of smaller blood vessels. Peripheral innervation by the light yellow cell system was only found in muscular tissue of the ureter papilla. The antisera to the two peptide fragments of light yellow cell peptide I not only stained the light yellow cells, but also the identified yellow cells, which have previously been shown to produce the sodium influx-stimulating neuropeptide. The latter cells were negative to the in situ hybridization probes and antisera specific to the light yellow cell system. It is therefore unlikely that the yellow cells express the light yellow cell neuropeptide gene. Nevertheless, the cells contain a neuropeptide sharing antigenic determinants with light yellow cell peptide I. Our observations support the hypothesis that light yellow cells are involved in maintaining the shape of the animal via the regulation of ion- and waterbalance processes and blood pressure.     

Light- and electron-microscopic immunocytochemistry of a molluscan insulin-related peptide in the central nervous system of Planorbarius corneus

Summary Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIP_B-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIP_B- and anti-MIP_C-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurohormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes.     

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snailHelix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mountin situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion.In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snailLymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of theHelix family and the light yellow cells ofLymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.     

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

Summary the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.     

Functional morphology of the neuroendocrine sodium influx-stimulating peptide system of the pond snail,Lymnaea stagnalis,studied by in situ hybridization and immunocytochemistry

Summary The functional morphology of the neuroendocrine system producing sodium influx-stimulating (SIS) peptide in the pond snail, Lymnaea stagnalis, was studied by in situ hybridization and immunocytochemistry. The SIS-peptide, which is 76 amino acids long, stimulates sodium uptake from the ambient medium. Two synthetic DNA probes were used for in situ hybridization. The nucleotide sequences were chosen from the cDNA structure; they encode amino acids 8–17 and 64–73, respectively. SIS-peptide sequences 10–20 and 67–76 were synthesized and antibodies were raised to them and affinity-purified. In addition to these antibodies, a monoclonal antibody raised to a bioactive, high-pressure liquid chromatography (HPLC)-purified brain extract was used for immunocytochemistry. Paraffin sections of central nervous systems and of whole snails were studied. The SIS-peptide system could be identified as the previously described yellow cell (YC) system by comparing alternate sections treated with the DNA probes, stained with the antibodies, or stained with alcian blue-alcian yellow. SIS-peptide neurons (45) occur in the ganglia of the visceral ring and in the proximal parts of visceral nerves. Axons run in the nerves of these and in several nerves of other ganglia. Numerous axon branches penetrate the perineurium forming a vast central neurohemal area. The SIS-peptide system innervates the pericardium, the nephridial gland, the reno-pericardial canal, the ureter, the spermoviduct and gonadal acini, the anterior aorta, the ventral buccal artery, and the penis protractor muscle. The morphology of the system is discussed in relation to the process of sodium ion uptake from the ambient medium and from pro-urine, and to that of regulating blood pressure. In the central nervous system and other organs, neurons and axons not labeled with the DNA probes, but immunoreactive to one or two of the antibodies, were observed. It seems unlikely that these elements are functionally related to the SIS-peptide system.     

Co-existence of immunoreactivity to anti-dopamine,anti-serotonin and anti-vasotocin in the cerebral giant neuron of the pond snail Lymnaea stagnalis

Summary Consecutive sections of certain neurons in the central ganglia of the pond snail Lymnaea stagnalis appear to be immunoreactive to anti-dopamine and anti-serotonin. The Cerebral Giant Neurons stain in addition with antivasotocin. The observations indicate the presence of two biogenic amines within the same neuron and in addition their co-existence with a biologically active peptide.     

On the issue of innervation of dorsal longitudinal musculature of <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> with inferior cervical nerve

Using the method of the anterograde dextran tetramethylrhodamin transport, there is obtained the topographic picture of branching of inferior cervical nerve axons on fibers of the dorsal longitudinal muscle in Lymnaea stagnalis (L.). Using the retrograde staining, the neuronal bodies sending their processes into this nerve are marked. Manifestations of asymmetry in distribution of neurons stained through the right and left nerves are described. The electron microscopic studies have shown that the main number of the inferior cervical nerve axons is represented by thin fibers presumably belonging to the sensory cells. A part of the nerve fibers and their endings show imunoreactivity to serotonin and acetylcholine. The serotoninergic fibers predominate quantitatively over the cholinergic ones and account for a half of the fibers stained with dextran. A possible functional role of the serotoninergic and cholinergic innervation of the dorsal longitudinal muscle in Lymnaea stagnalis is discussed.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 40, No. 6, 2004, pp. 569–578.     

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.     

Neurosecretion in the basommatophoran snail Bulinus truncatus (Gastropoda,Pulmonata)

Summary The neurosecretory system of the freshwater snail Bulinus truncatus was investigated. With the Alcian blue-Alcian yellow (AB/AY) staining method at least 10 different types of neurosecretory cells (NSC) were distinguished in the ganglia of the central nervous system. The differences in staining properties of the NSC — with AB/AY the cells take on different shades of green and yellow — are borne out at the ultrastructural level: the NSC types contain different types of neurosecretory elementary granules.The neurosecretory system of B. truncatus is compared to that of Lymnaea stagnalis, the species which has received the most attention among the pulmonates. It appears from the comparison that the systems of both species show many similarities, although some differences are also apparent.     

Ultrastructural immunocytochemical evidence for peptidergic neurotransmission in the pond snail Lymnaea stagnalis

Summary Three neuronal systems of the pond snail Lymnaea stagnalis were immunocytochemically investigated at the ultrastructural level with the unlabeled peroxidase-antiperoxidase technique. Preliminary electrophysiological and cell-filling investigations have shown that a cluster of neurons which reacts positively with an antiserum against the molluscan cardio-active peptide FMRFamide, sends axons to the penis retractor muscle. In this muscle anti-FMRF-amide (aFM) positive axons form neuro-muscular synapses with (smooth) muscle fibers. The morphological observations suggest the aFM immunoreactive system to be involved in peptidergic neurotransmission. In the right parietal ganglion a large neuron (LYAC) is penetrated by aFM positive axons which form synapse-like structures (SLS) with the LYAC. The assumption that the SLS represent the morphological basis for peptidergic transmission is sustained by the observation that iontophoretical application of synthetic FMRFamide depolarizes the LYAC. The axons of a group of pedal anti-vasopressin (aVP) positive cells run in close vicinity to the cerebral ovulation (neuro-)-hormone producing cell system (CDC system) Synapses or SLS between the two systems were not observed. The fact that (bath) application of arg-vasopressin induces bursting in the CDC, may indicate that the vasopressin-like substance of the aVP cells is released non-synaptically.     

Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization

Summary The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material.A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as ectopic CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral gangion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed. The nerves emanating from this ganglion contained many more immunoreactive axons than those from the left pedal ganglion. A polyclonal antibody raised against the synthetic fragment of CDCH stained, in addition to the neurons and axons revealed with the monoclonal antibody and the cDNA probe, three other major groups of neurons. Two are located in the cerebral ganglion, the other in the left pedal ganglion.The present findings suggest the presence of a system of neurons that contain CDCH or CDCH-like peptides. The role this system may play in the control of egg-laying and egg-laying behaviour is discussed.     

The distribution of neurones immunoreactive for β-tyrosine hydroxylase,dopamine and serotonin in the ventral nerve cord of the cricket,Gryllus bimaculatus

The cellular localization of the biogenic amines dopamine and serotonin was investigated in the ventral nerve cord of the cricket, Gryllus bimaculatus, using antisera raised against dopamine, -tyrosine hydroxylase and serotonin. Dopamine-(n<-70) and serotonin-immunoreactive (n<-120) neurones showed a segmental arrangement in the ventral nerve cord. Some neuromeres, however, did not contain dopamine-immunoreactive cell bodies. The small number of stained cells allowed complete identification of brain and thoracic cells, including intersegmentally projecting axons and terminal arborizations. Dopamine-like immunostaining was found primarily in plurisegmental interneurones with axons descending to the soma-ipsilateral hemispheres of the thoracic and abdominal ganglia. In contrast, serotonin-immunostaining occurred predominantly in interneurones projecting via soma-contralaterally ascending axons to the thorax and brain. In addition, serotonin-immunoreactivity was also present in efferent cells and afferent elements. Serotonin-immunoreactive, but no dopamine-immunoreactive, varicose fibres were observed on the surface of some peripheral nerves. Varicose endings of both dopamine-and serotonin-immunoreactive neurones occurred in each neuromere and showed overlapping neuropilar projections in dorsal and medial regions of the thoracic ganglia. Ventral associative neuropiles lacked dopamine-like immunostaining but were innervated by serotonin-immunoreactive elements. A colocalization of the two amines was not observed. The topographic representation of neurone types immunoreactive for serotonin and dopamine is discussed with respect to possible modulatory functions of these biogenic amines in the central nervous system of the cricket.     

Structure of visual pathways in the nervous system of freshwater pulmonate molluscs

Central nervous system of freshwater pulmonate molluscs Lymnaea stagnalis and Planorbarius corneus was stained using retrograde transport of neurobiotin in the optic tract fibers. In both species, perikarya and fibers of the stained neurons are found in all ganglia except the buccal ones. Afferent fibers of the optic nerve form dense sensory neuropil located in relatively small volume of cerebral ganglia. Typical neuronal groups sending their processes into the optic nerves of ipsilateral and contralateral body halves are described. Among them, neurons of visceral and parietal ganglia innervating both eyes concurrently as well as sending projections into peripheral nerves are revealed. These neurons, supposedly, have a function to integrate sensory signals, which may be a basis for regulation of light sensitivity of retina and functioning of peripheral organs. Bilateral links of the molluscan eye with the pedal ganglia cells and statocysts are found, which is, likely, a structural basis of certain known behavioral patterns related to stimulation of visual inputs in the studied gastropod molluscs.     

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.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.     

Neurons in a variety of molluscs react to antibodies raised against the VD1/RPD2 α-neuropeptide of the pond snail Lymnaea stagnalis

The VD₁ and RPD₂ neurons of Lymnaea stagnalis innervate other central neurons, certain skin areas, the pneumostome area, and the auricle of the heart. Recently, a set of four (, , , ) neuropeptides produced by these giant neurons and by certain other central neurons has been characterized. Although alternative splicing of the preprohormone of these neurons yields at least 10 different neuropeptides, an affinity-purified antiserum directed against a domain common to all neuropeptides has previously been shown to be highly selective in staining VD₁, RPD₂ and other neurons that produce the preprohormone. Since the gene encoding the neuropeptides is structurally similar to that expressed in R15 of the marine opisthobranch Aplysia californica, we have used the affinity purified antiserum as a marker for VD₁/RPD₂-related systems in other molluscs. Immunopositive neurons and fibers are observed in the central nervous systems of all species studied (Achatina fulica, Anodonta sp., Aplysia brasiliana, A. californica, Bulinus truncatus, Cepea sp., Eobania vermiculata, Helix aspersa, H. pomatia, Limax maximus, Mytilus edulis, Nassarius reticulatus, Viviparus viviparus). Several medium-sized and small neurons and 1–4 giant neurons are found in the pulmonates and opisthobranchs. The giant neurons in pulmonates have locations in the subesophageal ganglion, axonal branching patterns, and terminal arborizations in the auricle of the heart; all these characteristics are similar to those of VD₁ and RPD₂. Double-labelling (Lucifer yellow injection, immunocytochemistry) confirms that the two giant neurons in Helix pomatia are Br and Br. The immunoreactive cells in A. fulica appear to include the VIN and PON neurons. The antiserum also stains cells that appear to be the R15 neurons in two Aplysia species. The small and medium-sized neurons are distributed widely over the central ganglia of opisthobranchs and pulmonates. In prosobranchs and bivalves, small neurons are found in the cerebral and abdominal ganglia. No evidence has been found for innervation of the heart in these latter groups but in M. edulis, immunoreactive terminals can be observed in the gill. The results suggest the evolutionary conservation of immunoreactive peptides and the neurons that produce them, and thus support and extend previous hypotheses regarding the homology of certain giant neurons across molluscan species.     

A study of the cerebral region of the visual system in the pulmonata

The distribution of central axons of receptor cells of the eyes and the locations of neurons sending axons into the optic nerves were studied in the cerebral ganglia of the pulmonate mollusksLymnaea stagnalis andHelix sp. by the method of axonal transport of cobalt chloride injected via the optic nerves. Afferent fibers of these nerves form terminal ramifications (chiefly dorsally) in the middle part of the cerebral ganglion. Some of them pass through the commissure to the symmetrical region of the opposite cerebral ganglion. Neurons innervating the eyes are located in several regions of both cerebral ganglia. InLymnaea they are distributed near the point of entry of the optic nerve, in the region of the commissure, the mesocerebrum, and the posterior part of the ganglion. InHelix these neurons are found in the same regions except in the posterior part of the ganglion. In electrophysiological experiments responses of neurons in these parts of the cerebral ganglion to adequate stimulation of the eye were recorded. Differences in the character of responses and also the presence of neurons indifferent to stimulation of the eye are evidence of the functional heterogeneity of these areas. This suggests that morphologically separate visual centers do not exist in the cerebral ganglion of the Pulmonata. Neurons giving specific responses to stimulation of the eye and evidently belonging to different levels of the visual system (afferent or efferent divisions) are closely connected both with each other and with cells of other functional systems.A. A. Ukhtomskii Physiological Research Institute, A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 179–184, March–April, 1982.     

Neuroanatomical and immunocytochemical studies of the head retractor muscle innervation in the pond snail, Lymnaea stagnalis L

Axonal tracing and immunocytochemical techniques were used to study the innervation of the head retractor muscle (HRM) in the pond snail Lymnaea stagnalis L. Fibers of both the superior and inferior cervical nerves which innervate the HRM form endings that comply with the structure of chemical synapses. The somata of neurons with axons in these nerves are located in all except the buccal ganglia of the central nervous system, and this seems to be a special feature of the HRM motor system. By staining the filamentous actin with Oregon-green conjugated phalloidin, we demonstrated that the HRM has a multiterminal innervation and one muscle fiber can contain several synaptic endings which appear to be both morphologically and physiologically different. The morphological diversity of synaptic vesicles suggests a multiplicity of neurotransmitters acting on these nerve-muscle junctions. Immunocytochemical evidence was found for a strong serotonergic and FMRFamidergic innervation of muscle fibers through axons of the inferior cervical nerve. The thin fibers of the inferior cervical nerve possess immunoreactivity to glutamate, gamma-aminobutyric acid (GABA) and choline-acetyltransferase, and form sparser innervation patterns in the muscle. Our results indicate that several neurotransmitters are present in the nerves innervating the Lymnaea HRM and may therefore participate in the control of this muscle. The possible behavioral significance of such different neurotransmitter sets involved in the regulation of contractions is discussed.