Identification of motor neurons that contain a FMRFamidelike peptide and the effects of FMRFamide on longitudinal muscle in the medicinal leech, Hirudo medicinalis

Excitatory motor neurons in the leech are cholinergic. By using a combination of intracellular Lucifer yellow injection and indirect immunofluorescence, we localized FMRFamidelike immunoreactivity to a number of the motor neurons innervating longitudinal and dorsoventral muscle in the leech. All excitatory motor neurons innervating longitudinal muscle (cells 3, 4, 5, 6, 8, L, 106, 107, 108) were labeled with an antiserum to FMRFamide, while the inhibitory motor neurons innervating longitudinal muscle (cells, 1, 2, 7, 9, 102) were not. The excitatory motor neuron innervating medial dorsoventral muscle (cell 117) was labeled, while the excitatory motor neuron innervating lateral dorsoventral muscle (cell 109) was not. The inhibitory motor neuron innervating dorsoventral muscle (cell 101) was also labeled. Nerve terminals along dorsoventral muscle were also labeled with the antiserum. FMRFamide was bath applied to strips of longitudinal muscle while recording tension, and the muscle's response was compared to its response to the previously identified neuromuscular transmitter ACh. Brief applications of FMRFamide caused a contraction approximately one-tenth as large as that caused by an equimolar amount of ACh. The muscle response to FMRFamide was unaffected by curare. During extended exposures, FMRFamide caused a maintained contraction in longitudinal muscle without any apparent desensitization of the FMRFamide receptors and occasionally triggered an irregular myogenic rhythm. This extended exposure to FMRFamide caused a post-exposure potentiation of the longitudinal muscle's response to ACh that shorter applications of FMRFamide did not. Thus FMRFamide may act as a transmitter or modulator in cholinergic motor neurons innervating longitudinal and dorsoventral muscles in the leech.     

FMRF-amide-like substances in the leech. II. Bioactivity on the heartbeat system

In the preceding paper (Kuhlman, J. R., C. Li, and R. L. Calabrese (1985) J. Neurosci. 5: 2301-2309) FMRF-amide-like immunoreactivity was localized to a specific set of neurons in the leech. Three types of these neurons are involved in controlling the animal's heartbeat: HE motor neurons and HA modulatory neurons which directly innervate the hearts, and the swim-initiating interneurons (cells 204) which can accelerate the heartbeat central pattern generator. Application of synthetic FMRF-amide had effects on the hearts and the heartbeat central pattern generator that mimicked the actions of the HA and cell 204 neurons. Bath application of FMRF-amide (10(-7) to 10(-6) M) to the hearts activated their myogenic rhythm and increased their beat tension, thus mimicking the effects of activity in HA cells. Bath application of lower concentrations of FMRF-amide (10(-9) to 10(-8) M) to the isolated central nervous system dramatically accelerated the central motor program for heartbeat, thus mimicking the effects of activity in cell 204. These observations suggest that an FMRF-amide-like substance may be used as a chemical signal by HA and cell 204 neurons. The role of the FMRF-amide-like substance contained in HE motor neurons remains unclear, but it may be released along with the HE cell's neuromuscular transmitter, acetylcholine.     

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.     

FMRF-amide-like substances in the leech. I. Immunocytochemical localization

FMRF-amide-like immunoreactivity (FLI) was localized to approximately 50 neurons in each segmental ganglion of the medicinal leech using immunocytochemical techniques. Although most of these neurons were iterated in each segmental ganglion, some were more restricted in their segmental distribution. The head and tail ganglia likewise contained numerous FMRF-amide-like immunoreactive cells. In addition to cell bodies, many nerve processes and varicosities were also immunoreactive throughout the ganglion. All labeling of FLI was blocked by preabsorption of the anti-FMRF-amide antiserum with synthetic FMRF-amide. Using a combination of Lucifer Yellow cellular injection and indirect immunofluorescence techniques, we identified several of the neurons possessing FLI. Identified neurons included excitatory motor neurons (HE, RPE, LPE, AE, and L), the HA modulatory neuron, interneuron cell 204, and cells of unknown function (AP). The processes of HE motor neurons and HA modulatory neurons which innervate the heart tubes were also immunoreactive. These results indicate a role for FMRF-amide-like substances as neurochemical signals in the leech.     

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.     

Identification of Aplysia neurons containing immunoreactive FMRFamide

Electrophysiological and immunocytochemical techniques were used in the abdominal ganglion of Aplysia to identify neurons containing immunoreactive FMRFamide. Large numbers of neurons were immunoreactive for FMRFamide, including R2, L2, L3, L4, L5, L6, 2 cells tentatively identified as L12 and L13, and a previously unidentified cluster on the ventral surface of the right lower quadrant. There was also heavy labelling of fibers, often with beaded varicosities, throughout the neuropil, the cell layers, and the sheath overlying the ganglion. This data provides further evidence that FMRFamide is an important neurotransmitter in Aplysia. The demonstration of immunoreactive FMRFamide in the giant cholinergic neurons R2 and LP1(1) suggests that these well-studied and experimentally convenient cells use acetylcholine and an FMRFamide-like peptide as cotransmitters.     

Identification of neurons containing immunoreactive FMRFamide

Electrophysiological and immunocytochemical techniques were used in the abdominal ganglion of to identify neurons containing immunoreactive FMRFamide. Large numbers of neurons were immunoreactive for FMRFamide, including R2, L2, L3, L4, L5, L6, 2 cells tentatively identified as L12 and L13, and a previously unidentified cluster on the ventral surface of the right lower quadrant. There was also heavy labelling of fibers, often with beaded varicosities, throughout the neuropil, the cell layers, and the sheath overlying the ganglion. This data provides further evidence that FMRFamide is an important neurotransmitter in . The demonstration of immunoreactive FMRFamide in the giant cholinergic neurons R2 and LP1₁ suggests that these well-studied and experimentally convenient cells use acetylcholine and an FMRFamide-like peptide as cotransmitters.     

Localization of FMRFamide-like peptides in Caenorhabditis elegans.

The neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) is a member of a large family of related peptides that have been found throughout the animal kingdom. By using an antiserum specific for the Arg-Phe-NH2 moiety, we have found that about 10% of the neurons in the nematode Caenorhabditis elegans are immunoreactive. Most of these neurons, which include sensory, motor, and interneurons, were identified on the basis of their number, position, and projection pattern and by analysis of characterized mutants. Neurons that were immunoreactive in hermaphrodite animals were generally also found in males, but each sex had, in addition, sex-specific immunoreactive cells. Staining of hermaphrodite animals from different larval stages suggests that the onset of FMRFamide-like expression is differentially regulated among the cells. We have found a possible neuromodulatory role for the related peptide FLRFamide (Phe-Leu-Arg-Phe-NH2). In an egg-laying assay, FLRFamide by itself was not active but could potentiate a serotonin effect. The FMRFamide-like immunoreactivity was also used as a marker to examine the differentiation of cells that normally undergo programmed cell death. Cells that are destined to die in the Pn.a lineages appear to differentiate and adopt the fate of lineally equivalent cells before cell death.     

Neuropeptides myomodulin,small cardioactive peptide,and buccalin in the central nervous system of Lymnaea stagnalis: Purification,immunoreactivity, and artifacts

The neuropeptides myomodulin, small cardioactive peptide (SCP), and buccalin are widely distributed in the phylum Mollusca and have important physiological functions. Here, we describe the detailed distribution of each class of peptide in the central nervous system (CNS) of the snail Lymnaea stagnalis by the use of immunocytochemical techniques combined with dye-marking of electrophysiologically identified neurons. We report the isolation and structural characterization of a Lymnaea myomodulin, PMSMLRLamide, identical to myomodulin A of Aplysia californica. Myomodulin immunoreactivity was localized in all 11 ganglia, in their connectives, and in peripheral nerves. In many cases, myomodulin immunoreactivity appeared localized in neuronal clusters expressing FMRFamide-like peptides, but also in a large number of additional neurons. Double-labelling experiments demonstrated myomodulin immunoreactivity in the visceral white interneuron, involved in regulation of cardiorespiration. SCP-like immunoreactivity also appeared in all ganglia, and double-labelling experiments revealed that in many locations it was specifically associated with clusters expressing distinct exons of the FMRFamide gene that are differentially expressed in the CNS. Characterization of the two types of SCP-antisera used in this study, however, suggested that they cross-reacted with both FMRFamide and N-terminally extended FMRFamide-like peptides. Selective preadsorption with these cross-reacting peptides resulted in elimination of the widespread staining and retention of bona fide SCP immunoreactivity in the buccal and pedal ganglia only. Buccalin immunoreactivity was limited to the buccal and pedal ganglia. It did not coincide with the distribution of either myomodulin or SCP. Most immunoreactive clusters were found in the pedal ganglia. © 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- 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.     

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.     

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 of FMRFamide-like immunoreactivity in the brain of the viviparous skink (Chalcides chalcides)

Neuroanatomical distribution of FMRFamide-like immunoreactivity was investigated in the brain and olfactory system of the viviparous skink, Chalcides chalcides. In the adult brain FMRFamide immunoreactive (ir) perikarya were observed in the diagonal band of Broca, medial septal nucleus, accumbens nucleus, bed nucleus of the anterior commissure, periventricular hypothalamic nucleus, lateral forebrain bundle, and lateral preoptic, subcommissural, suprachiasmatic and lateral hypothalamic areas. This pattern was seen in both male and female brains. Though all major brain areas showed FMRFamide-ir innervation, the densest ir fiber network was observed in the hypothalamus. During development, ir elements were observed for the first time in embryos at mid-pregnancy. FMRFamide perikarya were located along the ventral surface of the vomeronasal nerve, in the olfactory peduncle mediobasally, as well as in the anterior olfactory nucleus and olfactory tubercle. Furthermore, some ir neurons were observed in the rhombencephalic reticular substance; however, the ir fiber network was poorly developed. Later in development FMRFamide-ir neurons appeared also in the bed nucleus of the anterior commissure as well as the rhombencephalic nucleus of solitary tract and the dorsal motor nucleus of vagus nerve. In juveniles, the distribution profile of FMRFamide immunoreactivity was substantially similar to that of the adults, with a less widespread neuronal distribution and a more developed fiber network. Ontogenetic presence of FMRFamide immunoreactivity in the nasal area has been linked to the presence of a nervus terminalis in this reptile.     

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.     

Molluscan neuropeptide-like and enkephalin-like material coexists in octopus nerves

Antibodies against the opioid peptide enkephalin and the molluscan neuropeptide FMRFamide immunostained identical nerve terminals and neurosecretory granules in a neuropil layer of the octopus vena cava. Enkephalin—like immunoreactivity was significatly enhanced by enzymatic digestion of the sections with trypsin or with carboxypeptidase-B, prior to incubation with antibodies; FMRFamide-like immunoreactivity was prevented by tryptic digestion. Coexistence of the two different immunoreactive substances and the effects of enzymatic cleavages indicate occurrence of a common enkephalin-Arg-Phe precursor. The same population of nerve terminals also contained immunoreactive α-melanotropin-like material.     

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.     

Cranial motor neurons contain either galanin- or calcitonin gene-related peptidelike immunoreactivity

The demonstration of coexistence of a peptide or peptides in neurons that produce a small molecule neurotransmitter has become increasingly frequent. The calcitonin gene-related peptide (CGRP) is known to be colocalized in the cholinergic neurons of both cranial and spinal motor nuclei. The present study demonstrates that all somatic motor cranial nerve nuclei contain CGRP- and galaninlike immunoreactivity. The perikaryal content of both peptides is increased by colchicine pretreatment and by transecting axons arising from the perikarya, and both peptides are found in nerve fibers innervating striated musculature. CGRP- and galaninlike immunoreactivity appear to be present in different populations of neurons. In contrast to CGRP, galaninlike immunoreactivity was not detected in spinal motor neurons. These observations suggest that galanin and CGRP participate in the process of synaptic transmission at the neuromuscular junction of cranial motor neurons.     

Distinct localization of FMRFamide- and Bovine Pancreatic Polypeptide-like material in the brain, retrocerebral complex and suboesophageal ganglion of the cockroach Periplaneta americana L.

One bovine pancreatic polypeptide (BPP) antiserum and two FMRFamide antisera were applied in the peroxidase-antiperoxidase (PAP) immunohistochemical technique on a complete series of sections of brains, suboesophageal ganglia (SOG), corpora cardiaca (CC) and corpora allata of Periplaneta americana L. Double immunohistochemical staining demonstrated that the same perikarya and processes were stained by both the BPP and FMRFamide antisera. This was caused by cross-reaction of the BPP and FMRFamide antisera with common antigenic determinants as was shown by a number of solid-phase absorptions. Application of a third FMRFamide antiserum, which was especially selected for its inability to react with bovine and avian pancreatic polypeptide, showed that more than half of the structures that were stained with the ‘unspecific’ BPP and FMRFamide antisera, contained material which was genuinely FMRFamide-like. This peptide material was located in cerebral neuronal structures, in the SOG, in the storage site of the CC and in numerous nerve fibres throughout the neuropile regions, which suggested a neurotransmitter/modulator as well as a neurohormonal role. The FMRFamide-like peptide was also found to be present in the same brain sites as an adipokinetic hormone-like peptide, but double labelling revealed that these two substances were never located in the same perikarya or fibres.     

Identification, Distribution and Physiological Activity of Three Novel Neuropeptides of Lymnaea: FLRlamide and pQFYRlamide Encoded by the FMRFamide Gene, and a Related Peptide

We are interested in analysing the detailed modulation of defined neuronal systems by multiple neuropeptides encoded in the FMRFamide locus of the snail Lymnaea. Cloning of the FMRFamide gene has predicted the existence of two novel peptides previously unknown from biochemical analysis, the pentapeptides EFLRlamideand QFYRlamide. These peptides may form part of a new family of peptides sharing the sequence motif –FXRlamide. In this paper we adopt a novel approach to first identify and characterize –FXRlamide-like peptides in extracts from the central nervous system of Lymnaea. By a combination of high-performance liquid chromatography (HPLC) and continuous-flow fast atom bombardment mass spectrometry, we identify three novel peptides: EFLRlamide, pQFYRlamide and pQFLRlamide. The first two are those predicted in exon II of the FMRFamide locus whereas the last is, interestingly, a product which cannot be derived from post-translational modification of the predicted peptides but must be encoded by as yet unidentified nucleotide sequences. A specific antibody raised to EFLRlamide, and immuno reactive to all three peptides, revealed EFLRlamide-like expression throughout the central nervous system in the same cells where exon II is transcribed and the peptide SEEPLY (a post-translational product of exon II) was localized. Additional cells, however, were also identified. Immunoreactivity was mapped in a number of identified neurons in the central nervous system, including two heart cardio excitatory motoneurons, the E_he cells (E heart excitors of the visceral ganglion) and penialmotoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that the serespective classes of neurons innervate also exhibited EFLRlamide immunoreactivity. The central and peripheral localization of EFLRlamide-like immunoreactivity suggested that EFLRlamide/pQFYRlamide may have an important physiological role in both these peripheral systems as well as in the central nervous system. This was confirmed by physiological experiments that showed that EFLRlamide and pQFYRlamide inhibited many centralneurons and in particular the Bgp neurons in the right parietal ganglion. EFLRlamide had complex biphasic effects on the frequency of heart-beat: an initial inhibitory response was followed by a long-lasting increase in the rate of beating. Taken together with earlier work, this study now completes the analysis and localization of the full set of post-translational products of the FMRFamide precursor in Lymnaea and supplies further evidence towards the characterization of the physiological systems which such peptides may modulate in concert.