Neural network controlling feeding in Lymnaea stagnalis: Immunocytochemical localization of myomodulin,small cardioactive peptide,buccalin, and FMRFamide- related peptides

This paper investigates the distribution of four classes of neuropeptides, myomodulin, small cardioactive peptide (SCP), buccalin, and FMRFamide, in central neurons forming the network that underlies feeding behavior in the snail Lymnaea stagnalis. Intracellular dye-marking and immunocytochemical analysis, using antisera to the different classes of peptides, were applied to identified neurons of all three levels of the hierarchy of the circuitry: modulatory interneurons (cerebral giant cells, CGC; slow oscillator, SO), central pattern generator (CPG) interneurons (N1, N2, N3), motoneurons (B1–B10), and their peripheral target organs. Myomodulin immunoreactivity was detected in the CGC interneurons, in the SO, and in ventral N2-type CPG interneurons. Several large buccal motoneurons, the paired B1, B2, B3, B7, and neurons located in the dorsal posterior area (putative B4 cluster types) were also myomodulin immunoreactive. Target organs of buccal motoneurons, the buccal mass, salivary glands, and oesophagus contained myomodulin-immunopositive fibers. SCP appeared in N2-type interneurons and was found colocalized with myomodulin in the B1 and B2 motoneurons. SCP-containing neurons in the B4 cluster area were also detected. The buccal mass and salivary glands exhibited SCP-immunoreactive fibers. Buccalin immunoreactivity was scarce in the buccal ganglia and was identified only in N1-type interneurons and three pairs of dorsal posterior neurons. In the periphery, immunoreactive fibers were localized in the oesophagus only. None of the buccal neuronal types examined revealed immunoreactivity to SEQPDVDDYLRDVVLQSEEPLY (“SEEPLY”), a peptide encoded in the FMRFamide precursor protein of Lymnaea. SEEPLY immunoreactivity was confined to a pair of novel ventral neurons with projections to the laterobuccal nerve innervating the buccal mass. Immunoreactive fibers were also traced in this organ. © 1994 Wiley-Liss, Inc.     

Expression of diverse neuropeptide cotransmitters by identified motor neurons in Aplysia

Neuropeptide synthesis was determined for individual identified ventral-cluster neurons in the buccal ganglia of Aplysia. Each of these cells was shown to be a motor neuron that innervates buccal muscles that generate biting and swallowing movements during feeding. Individual neurons were identified by a battery of physiological criteria and stained with intracellular injection of a vital dye, and the ganglia were incubated in 35S-methionine. Peptide synthesis was determined by measuring labeled peptides in extracts from individually dissected neuronal cell bodies analyzed by HPLC. Previously characterized peptides found to be synthesized included buccalin, FMRFamide, myomodulin, and the 2 small cardioactive peptides (SCPs). Each of these neuropeptides has been shown to modulate buccal muscle responses to motor neuron stimulation. Two other peptides were found to be synthesized in individual motor neurons. One peptide, which was consistently observed in neurons that also synthesized myomodulin, is likely to be the recently sequenced myomodulin B. The other peptide was observed in a subset of the neurons that synthesize FMRFamide. While identified motor neurons consistently synthesized the same peptide(s), neurons that innervate the same muscle often express different peptides. Neurons that synthesized the SCPs also contained SCP-like activity, as determined by snail heart bioassay. Our results indicate that every identified motor neuron synthesizes a subset of these methionine-containing peptides, and that several neurons consistently synthesize peptides that are likely to be processed from multiple precursors.     

FMRFamide-like substances in the leech. III. Biochemical characterization and physiological effects

FMRFamide-like immunoreactivity has been previously localized to identified neurons in the CNS of the leech, Hirudo medicinalis (Kuhlman et al., 1985a). These leech antigens have been characterized biochemically by reverse-phase high-pressure liquid chromatography (HPLC) followed by radioimmunoassay (RIA). The majority of the FMRFamide-like immunoreactivity recovered by HPLC from extracts of leech nerve cords coelutes with authentic FMRFamide. We have tentatively identified this major leech peptide as authentic FMRFamide. Two neurons that control heartbeat in the leech, the HE motor and HA modulatory neurons, and their neural processes on the heart are FMRFamide-like immunoreactive (Kuhlman et al., 1985a). Single individually dissected HE and HA cells were analyzed by HPLC and RIA. Only 1 FMRFamide-like peptide was found in extracts of HA cells; this peptide was chromatographically indistinguishable from authentic FMRFamide. The FMRFamide-like peptide in HE cells could not be isolated by experimental procedures used. Most of the FMRFamide-like immunoreactivity contained within the neural processes on the heart also coeluted with authentic FMRFamide. HE motor neurons, which are believed to be cholinergic (Wallace, 1981a, b; Maranto and Calabrese, 1984a, b), were examined for their FMRFamide-like effects on the heart. The presence of curare in the bathing medium did not block the ability of FMRFamide to induce myogenic activity in heart muscle, suggesting that FMRFamide and ACh act at different receptor sites on the heart. Prolonged firing in HE cells in the presence of curare also induced myogenic activity in heart muscle. This FMRFamide-like action of the HE motor neurons may be normally masked by their cholinergic actions.     

Fast axonal transport of modulatory neuropeptides from central ganglia to components of the feeding system in Aplysia

The transport of neuropeptides from central ganglia to components of the feeding system was studied in Aplysia. Peptide transport was determined by incubating buccal or cerebral ganglia with 35S-methionine and measuring the appearance of labeled peptides by high-pressure liquid chromatography (HPLC) of extracts of target tissues. Selected nerves were left intact and passed through a Vaseline diffusion barrier separating the ganglia and their targets. Five major labeled peptides were observed to be transported from the buccal ganglia to feeding muscles. They were buccalin, FMRFamide, myomodulin, and 2 small cardioactive peptides. Each of these peptides has been shown to modulate the responses of these muscles to their motor neurons. The peptides were transported by fast axonal transport, as judged by the distance transported and the sensitivity to colchicine. When normalized to correct for differences in total incorporation, the patterns of peptide transport were reproducible between animals. The nature and amount of the peptides transported were different for different muscles. The nature of peptide transport also varied for different nerve groups. These results support the proposition that these 5 peptides act as modulatory transmitters at feeding muscles. No transport of neuropeptides from the cerebral ganglia to feeding muscles was observed, although myomodulin was specifically transported to the buccal ganglia. This suggests that this peptide may play an important role in the previously observed regulation of buccal ganglia activity by neurons in the cerebral ganglia.     

FMRFamide-related peptides,partial serotonin depletion,and osmoregulation in Helisoma duryi (mollusca: pulmonata)

Serotonergic neurons were studied by specific histological methods, and neurons containing Phe-Met-Arg-Phe-NH₂ (FMRFamide)-related heptapeptides were identified with an antiserum specific for these substances in the central nervous system of the freshwater snail Helisoma duryi. Serotonergic neurons and their axons are present in all of the ganglia (paired buccal, cerebral, pedal, pleural, parietal, and single visceral) and major nerves of the central nervous system. Large neurons containing FMRFamide-related peptide immunoreactivity are located in the left parietal and visceral ganglia, whereas a few small neurons are located in the cerebral and pedal ganglia. Both serotonergic and FMRFamide-related peptide-immunoreactive dendrites and varicosities were observed in the kidney. A second antiserum with high affinity for FMRFamide-related heptapeptides was used to measure the levels of the immunoreactive material in various tissues, and such material was found in every tissue analyzed. When snails were exposed to a medium isosmotic to their hemolymph, the levels of immunoreactive FMRFamide-related peptides increased in the hemolymph, central nervous system, mantle, and kidney. Injection of dihydroxytryptamine, which is known to deplete serotonin content in the snail, also reduced the levels of FMRFamide-related-immunoreactive material in the above tissues. Therefore, serotonin may influence the levels of FMRFamide-related peptides in tissues by regulating the rate of their synthesis, axonal transport, or release. Both serotonin and FMRFamide-related peptides could be involved in osmoregulation. J. Comp. Neurol. 393:25–33, 1998. © 1998 Wiley-Liss, Inc.     

Immunocytological localization of pedal peptide in the central nervous system and periphery of Aplysia

Immunocytology using antisera raised to conjugated pedal peptide (Pep) was used to localize the peptide in the CNS and periphery of Aplysia. A total of over 200 neurons in the CNS exhibited Pep-like immunoreactivity. As expected from results presented in the previous paper, immunoreactive neurons were heavily concentrated in the pedal ganglia, primarily in a broad ribbon comprised of about 60 large contiguous neurons on the dorsal side of each ganglion. Smaller and less numerous immunoreactive neurons were found in the other ganglia. A number of neurons primarily located in the abdominal ganglia had dense networks of immunoreactive varicose fibers surrounding their cell bodies. Many immunoreactive axons were observed in peripheral nerves, particularly those nerves leaving the pedal ganglia. Analyses of sections of body wall indicated that Pep-like immunoreactivity was localized to a series of varicose axons that appeared to be associated with vascular spaces, muscle fibers, and other large cells. These axons likely arise from pedal ganglion nerves that were shown to transport large amounts of 35S-labeled Pep to the periphery. These results suggest that Pep is a transmitter-like neuropeptide that is likely to have a number of important physiological actions in Aplysia.     

Distribution of myomodulin-like and buccalin-like immunoreactivities in the central nervous system and peripheral tissues of the mollusc,Clione limacina

The distribution of the myomodulin-like and buccalin-like immunoreactivities in the central nervous system and peripheral tissues associated with feeding was examined in the pteropod mollusc Clione limacina by using wholemount immunohistochemical techniques. Immunoreactive neurons and cell clusters were located in all central ganglia except the pleural ganglia, with approximately 50 central neurons reactive to myomodulin antiserum and 60 central neurons reactive to buccalin antiserum. All central ganglia contained a dense network of myomodulin- and buccalin-immunoreactive processes in their neuropil regions and connectives. In the periphery, the primary attention was focused on the tissues associated with feeding, especially feeding structures unique to Clione, such as hook sacs and buccal cones, which are used for prey capture and acquisition. All of these feeding structures contained myomodulin-immunoreactive and buccalin-immunoreactive fibers, with each peptide family showing specific innervation fields that were common in buccal cones and were totally different in the hook sacs. The specific central and peripheral distribution of myomodulin-like and buccalin-like immunoreactivities as well as specific effects of the exogenous peptides on identified neurons involved in the control of feeding behavior and swimming suggest that neuropeptides from myomodulin and buccalin families act as neurotransmitters or neuromodulators in a variety of central circuits and in the peripheral neuromuscular systems associated with feeding in Clione limacina. J. Comp. Neurol. 381:41-52, 1997. © 1997 Wiley-Liss, Inc.     

Interganglionic axonal transport of neuropeptides in Aplysia

The transport of neuropeptides between central ganglia was studied in Aplysia. Peptide transport was determined by incubating ganglia with 35S-methionine and measuring the appearance of labeled peptides in connected ganglia. Selected interganglionic connectives were left intact and passed through a diffusion barrier separating the ganglia. Labeled peptides transported between ganglia included FMRFamide, myomodulin, and pedal peptide. Each of these peptides has been shown to be physiologically active in Aplysia. In addition to these previously characterized neuropeptides, a number of other as yet uncharacterized labeled peptides were also transported. All the peptides were transported by fast axonal transport as judged by the distance transported and/or the sensitivity to colchicine. Overall, FMRFamide and several unidentified peptides were the predominant transported peptides. However, the nature and amount of the peptides transported differed for each ganglia. These results support the proposition that the labeled peptides have transmitterlike actions and suggest that there are a number of neuropeptides that are likely to have central actions that have not yet been characterized in Aplysia.     

FMRFamide- and adipokinetic hormone-like immunoreactivity in the nervous system of the mosquito, Aedes aegypti

As demonstrated with immunocytochemistry, specific cells and axons in the nervous system of female Aedes aegypti contain antigens immunologically related to FMRFamide (phenylalanine-methionine-arginine-phenylalanine-amide) and locust adipokinetic hormone I (AKH). In the supra-esophageal ganglion, including some medial neurosecretory cells, and in all ganglia of the ventral nerve cord, there are 100-120 cells immunoreactive to a FMRFamide antiserum. The same cells cross-react with a bovine pancreatic polypeptide antiserum, but when the latter antiserum is preabsorbed with FMRFamide, immunoreactivity is lost. However, immunoreactivity is maintained when FMRFamide antiserum is preabsorbed with pancreatic polypeptide, suggesting that the immunoreactive peptide is more closely related to FMRFamide. There are 6-12 cells in the supra- and subesophageal ganglia immunoreactive to an AKH antiserum, and some of the same cells are reactive to the FMRFamide antiserum. As well, unpaired cells in each of the abdominal ganglia are positive for both AKH and FMRFamide. Although the function of the FMRFamide- and AKH-like peptides in mosquitoes is unknown, this study, combined with previous reports on the localization of FMRFamide-like peptides in midgut endocrine cells, supports the concept of a brain-midgut neuroendocrine axis in this insect.     

Immunocytochemical localization of pedal peptide in the central nervous system of the gastropod mollusc Tritonia diomedea

Tritonia pedal ganglion peptides (TPeps) are a trio of pentadecapeptides isolated from the brain of the nudibranch Tritonia diomedea. TPeps have been shown both to increase the beating rate of ciliated cells of Tritonia and to accelerate heart contractions in the mollusc Clione limacina. Here we examine the immunocytochemical distribution of TPeps in the Tritonia central nervous system. We found the brain and buccal ganglia to be rich sources of TPep immunoreactivity. Specific cells in both structures, some of them previously identified, were immunoreactive. Moreover, immunoreactive fibers were seen connecting ganglia and exiting almost all the major nerves. In the brain, we found that the paired, ciliated statocysts apparently receive TPep innervation. In addition, we observed unstained cell bodies in each buccal ganglion with extensive TPep immunoreactive projections surrounding their somata and primary neurites. Similar projections were not observed in the brain. We also compared the TPep immunoreactivity with that of SCP(b) in the buccal ganglia. We observed many neurons and processes that were immunoreactive to both peptides. One neuron that contains both TPep- and SCP(b)-like peptides (B12) has an identified role in the Tritonia feeding network. Together, these findings suggest that TPeps may play an active role in the central nervous system of Tritonia as neurotransmitters modulating orientation, swimming, and feeding.     

SCPB- and FMRFamide-like immunoreactivities in lobster neurons: Colocalization of distinct peptides or colabeling of the same peptide(s)?

Virtually all of the SCPB-like immunoreactive neurons (ca. 60 cells) in the lobster Homarus americanus also contain FMRFamide-like immunoreactivity. Control experiments reveal that SCPB-and FMRFamide-like immunoreactivities are successfully preadsorbed with their specific antigens, while the normal staining pattern is retained following preadsorption of each antibody with the alternate peptide. These experiments potentially lead to the conclusion that the anti-SCPB and anti-FMRFamide antibodies are labeling distinct compounds that are colocalized in lobster neurons. The lobster nervous system does not, however, contain authentic FMRFamide, but rather several FMRFamide-like compounds (Trimmer et al., J. Comp. Neurol. 266:16-26, 1987). The most abundant of these is the octapeptide TNRNFLRFamide. Experiments demonstrate that SCPB-like immunoreactivity is completely preadsorbed with synthetic TNRNFLRFamide, while there is a significant or complete loss of staining after preadsorption of the FMRFamide antibody with this molecule. Met-enkephalin-Arg-Phe-amide (YGGFMRFamide), an extended opioid peptide containing the FMRFamide sequence, also preadsorbs SCPB- and FMRFamide-like immunoreactivities, while Met-enkephalin-Arg-Phe (YGGFMRF) has no effect on the staining properties of these antibodies. These results suggest that the SCPB antibody can bind to extended forms of FMRFamide-like molecules, and that anti-SCPB and anti-FMRFamide antibodies may be colabeling one or more FMRFamide-like molecules in lobster neurons.     

Biochemical and immunocytological localization of the neuropeptides FMRFamide, SCPA, SCPB, to neurons involved in the regulation of feeding in Aplysia

The localization of the neuropeptide FMRFamide in the buccal ganglia and buccal muscles of Aplysia was studied by immunocytology and high-pressure liquid chromatography (HPLC) combined with either a sensitive bioassay or 35S-methionine labeling. Immunocytology with an antiserum directed to FMRFamide stained a large number of fibers, varicosities, and neuronal somata. Two groups of stained neurons were of particular interest. One was the S cells, a group comprised of many small neurons, the majority of which were stained. HPLC of pooled labeled S cells confirmed that at least some of these neurons synthesize FMRFamide. The other group of stained neurons were in the ventral cluster, a group comprised of a small number of large neurons, many of which are motor neurons that innervate the buccal muscles involved in producing biting and swallowing movements. Several of the ventral neurons were previously shown to contain 2 other neuropeptides, the small cardioactive peptides SCPA and SCPB. These neurons are sufficiently large to permit HPLC analyses of the neuropeptides synthesized by individual neurons. This procedure confirmed that individual ventral neurons synthesized FMRFamide, or the SCPs, or all 3 peptides. The coexistence of FMRFamide and the SCPs in the same neuron was confirmed by simultaneous staining of sections from the buccal ganglia with a monoclonal antibody to the SCPs and an antiserum to FMRFamide. The coexistence of the 3 peptides in the same neuron was surprising in light of the observations that these peptides often have opposite biological activity. The ventral neurons are large and potentially identifiable as individuals. Thus, these neurons may be particularly useful for studying the physiological and behavioral roles of neuropeptides in generating complex behaviors.     

FMRFamide peptides in Helisoma: identification and physiological actions at a peripheral synapse

Previous reports have demonstrated powerful neuromodulatory actions of the molluscan tetrapeptide FMRFamide in both the central and peripheral nervous systems of the freshwater snail Helisoma. The present study was designed to examine both the nature of the FMRFamide-like peptides in Helisoma and to define their physiological actions at a peripheral synapse. We report that, as determined by HPLC/RIA and mass spectrometry, Helisoma contains both FMRFamide and 2 of its analogs, FLRFamide and GDPFLRFamide. Whereas whole animals contain about 100 pmol/gm of these peptides, they were enriched in the nervous system (3000 pmol/gm) and in a peripheral target organ, the salivary glands (500 pmol/gm). For histochemical and physiological studies we examined the salivary glands, which are known to be innervated by neuron 4 of the buccal ganglion. We confirmed the presence of FMRFamide-like fibers on the salivary gland by immunohistochemistry using a polyclonal antiserum. These fibers appear to be largely derived from somata located in the central ring ganglia. For physiological tests we examined the neuron 4-gland synapse, at which presynaptic action potentials normally evoke a suprathreshold EPSP in gland cells. Bath application of FMRFamide, FLRFamide, or GDPFLRFamide at micromolar concentration to a buccal ganglia/salivary gland preparation completely suppressed spontaneous rhythmic activity. The sites of action of these peptides were examined by iontophoretic application of FMRFamide to neuron 4 or the salivary gland. Application of the peptide to the soma of neuron 4 caused a hyperpolarization that suppressed spontaneously generated action potentials. When applied to the salivary gland, FMRFamide caused a hyperpolarization that reduced the EPSPs evoked by neuron 4 to below spike threshold. The latter observation implies a postsynaptic locus of action for FMRFamide, and this possibility was tested by direct depolarization of the gland with iontophoresis of ACh (the putative transmitter of neuron 4). Such depolarizations were also reduced by FMRFamide. We conclude that Helisoma contains FMRFamide and 2 of its analogs, these peptides being enriched in the nervous system and salivary glands. Furthermore, these peptides can suppress activation of the salivary glands by actions both directly on gland cells and on the effector neuron.     

GABA‐like immunoreactivity in Biomphalaria: Colocalization with tyrosine hydroxylase‐like immunoreactivity in the feeding motor systems of panpulmonate snails

The simpler nervous systems of certain invertebrates provide opportunities to examine colocalized classical neurotransmitters in the context of identified neurons and well defined neural circuits. This study examined the distribution of γ‐aminobutyric acid‐like immunoreactivity (GABAli) in the nervous system of the panpulmonates Biomphalaria glabrata and Biomphalaria alexandrina, major intermediate hosts for intestinal schistosomiasis. GABAli neurons were localized in the cerebral, pedal, and buccal ganglia of each species. With the exception of a projection to the base of the tentacle, GABAli fibers were confined to the CNS. As GABAli was previously reported to be colocalized with markers for dopamine (DA) in five neurons in the feeding network of the euopisthobranch gastropod Aplysia californica (Díaz‐Ríos, Oyola, & Miller, 2002), double‐labeling protocols were used to compare the distribution of GABAli with tyrosine hydroxylase immunoreactivity (THli). As in Aplysia, GABAli‐THli colocalization was limited to five neurons, all of which were located in the buccal ganglion. Five GABAli‐THli cells were also observed in the buccal ganglia of two other intensively studied panpulmonate species, Lymnaea stagnalis and Helisoma trivolvis. These findings indicate that colocalization of the classical neurotransmitters GABA and DA in feeding central pattern generator (CPG) interneurons preceded the divergence of euopisthobranch and panpulmonate taxa. These observations also support the hypothesis that heterogastropod feeding CPG networks exhibit a common universal design.     

Matching neural and muscle oscillators: control by FMRFamide-like peptides

Stomatogastric nervous systems of the shrimp, Palaemon serratus, were stained with antisera raised against the peptide FMRFamide. FMRFamide-like immunoreactivity was found in fibers in the input nerve to the stomatogastric ganglion (STG), in several STG somata, in dense neuropil in the STG, in the motor nerves that innervate the dilator muscles of the pyloric region, but not in the pyloric dilator (PD) motor neurons. FMRFamide and several FMRFamide-like peptides elicited sequences of rhythmic depolarizations and contractions of the pyloric dilator muscle. As peptide concentrations were increased, a discrete threshold for contraction was found, above which contractions were initiated with a decreasing latency in an all-or-none fashion. Muscles stopped rhythmically contracting after many seconds to several minutes of activity; the duration of spontaneous oscillatory activity in peptide was proportional to the concentration of applied peptide. In the absence of peptide, each motor neuron discharge evoked small graded muscle contractions. During peptide-induced oscillations, motor neuron activity did not always entrain muscle oscillations. After spontaneous oscillations had stopped, when the motor neurons were stimulated in the presence of the peptide, each motor neuron burst evoked large amplitude contractions as a result of the peptide-induced regenerative properties of the muscle membrane.     

Processing of the FMRFamide Precursor Protein in the Snail Lymnaea stagnalis: Characterization and Neuronal Localization of a Novel Peptide, 'SEEPLY'

In the pulmonate snail Lymnaea stagnalis , FMRFamide-like neuropeptides are encoded by a multi-exon genomic locus which is subject to regulation at the level of mRNA splicing. We aim to understand the post-translational processing of one resulting protein precursor encoding the tetrapeptide FMRFamide and a number of other putative peptides, and determine the distribution of the final peptide products in the central nervous system (CNS) and periphery of Lymnaea. We focused on two previously unknown peptide sequences predicted by molecular cloning to be encoded in the tetrapeptide protein precursor consecutively, separated by the tetrabasic cleavage site RKRR. Here we report the isolation and structural characterization of a novel non-FMRFamide-like peptide, the 22 amino acid peptide SEQPDVDDYLRDWLQSEEPLY. The novel peptide is colocalized with FMRFamide in the CNS in a number of identified neuronal systems and their peripheral motor targets, as determined by in situ hybridization and immunocytochemistry. Its detection in heart excitatory motoneurons and in nerve fibres of the heart indicated that the novel peptide may play a role, together with FMRFamide, in heart regulation in the snail. The second predicted peptide, STEAGGQSEEMTHRTA (16 amino acids), was at very low abundance in the CNS and was only occasionally detected. Our current findings, suggestive of a distinct pattern of post-translational processing, allowed the reassessment of a previously proposed hypothesis that the two equivalent sequences in the Aplysia FMRFamide gene constitute a molluscan homologue of vertebrate corticotrophin releasing factor-like peptides.     

Localization and differential expression of FMRFamide-like immunoreactivity in the nematode Ascaris suum

By immunocytochemical and immunohistochemical methods, FMRFamide-like immunoreactivity (FLI) was localized to many neurons and processes in the Ascaris nervous system, including the head, tail, and lateral lines. Some of these cells were identified; they included sensory neurons, interneurons, and motor neurons. FLI was also present in the pharyngeal neurons and in their varicosities near the surface of the pharynx. By HPLC analysis of extract, only a subset of the FMRFamide-like peptides (FLPs) expressed in Ascaris heads, and heads from which the pharynx had been removed, were expressed in the pharynx. Furthermore, FLPs appeared to be differentially expressed in female heads and tails and male heads and tails. Acetone and acid methanol differentially extracted subforms of FLI from Ascaris heads and from C. elegans. © 1993 Wiley-Liss, Inc.     

Tachykinin-related neuropeptides in the central nervous system of the snail Helix pomatia: an immunocytochemical study

The distribution of neurons reacting with an antibody raised against an insect neuropeptide, locustatachykinin I, was investigated in the CNS of the snail Helix pomatia. The localization of the neurons was compared with that of the substance P-like immunoreactive (SPLI) neurons in the different ganglia. Altogether, there are 800–1000 locustatachykinin-like immunoreactive (LomTKLI) neurons in the Helix CNS, occurring with an overwhelming dominancy (83.5%) in the cerebral ganglia. Within the cerebral ganglia, the majority of LomTKLI neurons were localized in the procerebrum. The number of SPLI neurons was high; 2000 SPLI nerve cells were found in the Helix CNS. The majority (44.5%) of SPLI neurons was also found in the cerebral ganglia and they were also concentrated in the procerebrum. The neuropils of all ganglia were densely innervated by both LomTKLI and SPLI fibers except the medullary mass of the procerebrum where only SPLI elements form an extremely dense innervation. In addition to the neuropil processes, LomTKLI neurons sent axon processes to the peripheral nerves. SPLI fibers also formed a dense network of varicose fibers in the connective tissue sheath around the ganglia where they innervated the blood vessel walls too. Immunolabeling on alternating cryostat sections revealed that LomTKLI and SPLI neurons are localized near each other in most cases; co-localization of the two immunoreactive materials could be seen in a very small number of neurons of the pedal and pleural ganglia. The present results show that the Helix CNS possesses distinct neuronal populations using different tachykinin-related peptides. It is suggested that the differential distribution of these neuropeptides also implies a diversity in their central and peripheral functions.