Pedal Peptide Research Articles

Immunological localization of Tritonia peptide in the central and peripheral nervous system of the terrestrial snail Helix aspersa

We report here evidence that the pedal peptides (Peps) first discovered in mollusks may be neurotransmitters with a general role in control of molluscan somatic and visceral muscles. Using Tritonia peptide (TPep) antiserum we obtained morphological evidence for such a role in Helix aspersa. We localized 1,200-1,400 small and medium-sized (5-40 microm) TPep-IR neurons in the central nervous system of Helix and demonstrated the presence of these neurons in each ganglion. Many TPep-immunoreactive (IR) neurons were motoneurons that sent axons to almost all peripheral nerves. TPep-IR fibers innervated the foot, esophagus, hermaphroditic duct, optic tentacles, salivary gland, heart, and proximal and distal aorta. In peripheral tissues TPep-IR fiber ramifications were mostly associated with muscles and with ciliated epithelia. In addition, TPep-IR fibers were in the neuropil of the ganglia, the commissures, and the connectives, and they formed axosomatic terminals in the central nervous system. TPep-IR neurons were found in the esophagus and hermaphroditic duct and as sensory receptors in the bulb of the optic tentacles. These results from Helix, and those reported elsewhere from other mollusks, suggest a general involvement of TPep-like substances in control of muscle- and ciliary-driven motor activities, including perhaps their antecedent sensory and central axosomatic integrative activity.

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.

Differential effects of serotonergic and peptidergic cardioexcitatory neurons on the heart activity in the pteropod mollusc, Clione limacina.

A group of four cardioexcitatory neurons has been identified in the intestinal ganglia of the mollusc Clione limacina. Relatively weak stimulation of the intestinal neurons induced auricle contractions only, while strong stimulation produced initial auricle contractions followed by full-cycle auricle-ventricle contractions. Intestinal cardioexcitatory neurons probably utilized as their transmitter a peptide similar to Tritonia pedal peptide--they showed pedal peptide-like immunoreactivity, and their effects were mimicked by application of the exogenous pedal peptide. The pedal cardioexcitatory neuron was found to produce strong excitatory effects only on the ventricle contractions. Its stimulation induced ventricle contractions in the quiescent heart or significantly accelerated the rate of ventricle contractions in the rhythmically active heart. The pedal cardioexcitatory neuron apparently utilized serotonin as a neurotransmitter, based upon serotonin immunoreactivity, blocking effect of serotonin antagonists mianserin and methysergide, and the observation that exogenous serotonin mimicked its effect. A dense network of pedal peptide-like immunoreactivity was found both in the auricle and ventricle tissue. Serotonin immunoreactivity was densely present in the ventricle, while the auricle contained only a separate serotonin-immunoreactive unbranched axon. Thus, there are two separate groups of central cardioexcitatory neurons with different effects on heart activity, which together might provide a complex cardio-regulatory function in Clione.

Transmitter identification in neurons involved in male copulation behavior in Lymnaea stagnalis.

In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.

Transmitter identification in neurons involved in male copulation behavior inLymnaea stagnalis

In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.

Characterization of a cDNA clone encoding pedal peptide in the terrestrial snail.

We report the isolation of a Helix lucorum cDNA clone encoding a precursor of neuropeptides that are closely related to Aplysia and Tritonia pedal peptides (Pep). The predicted propeptide contains 20 copies of the two variants of Helix Pep interspersed with Lys-Arg endopeptidase cleavage sites. Northern blot hybridization revealed multiple Pep-hybridizing species in the Helix CNS RNA. The Pep gene was expressed by several identified serotonergic neurones in pedal and cerebral ganglia, groups of sensory neurones in procerebrum, peripheral neurones in olfactory bulb, mantle and foot, and group of neurones in pedal ganglia presumably involved in locomotion control. Pep mRNA was detected in several neurones at the early stages of nervous system development.

Modulation of Ciliary Beat Frequency by Neuropeptides From Identified Molluscan Neurons

Prior work in the nudibranch Tritonia diomedea indicated that certain identifiable pedal ganglion neurons (Pd5 and 6) innervating the foot synthesize three novel peptides (TPeps) that resemble Pedal peptide (Pep) identified in the sea hare Aplysia californica. We report here that when TPeps are applied directly to isolated ciliated patches of Tritonia diomedea foot epithelium, there is an increase in ciliary beating that normally drives locomotion. Exposure to TPeps also increases the ciliary beat frequency of cells isolated from the pedal epithelium, suggesting that the observed ciliomotor effects are direct and not mediated by intervening cells. Antibodies to TPep bind to specific cells of the brain and foot and to ciliated peripheral tissues in Tritonia diomedea and in the pulmonate gastropod Lymnaea stagnalis. We suggest, therefore, that TPeps may regulate the activity of ciliated cells responsible for pedal locomotion and other functions in gastropod molluscs.

Diversity in cell specific co-expression of four neuropeptide genes involved in control of male copulation behaviour in Lymnaea stagnalis

We report here the neuron-specific co-expression of four genes coding for neuropeptides involved in the control of male behaviour. These neurons are located in the anterior lobe of the right cerebral ganglion in the central nervous system of Lymnaea stagnalis and project via the penis nerve to the penial complex. In order to accomplish optimal assurance we applied in situ hybridization, immunocytochemistry and matrix-assisted laser desorption ionization mass spectrometry. The anterior lobe neurons express the gene encoding the amidated tetrapeptide APGWamide. Subsets of these cells are now shown to co-express the APGWamide gene exclusively with one of three other neuropeptide genes, encoding Lymnaea neuropeptide Y, conopressin or pedal peptide, respectively. All four genes are also expressed in other neurons in other centres projecting to the penial complex, but in these cells co-expression was not observed. The neuropeptides encoded by the genes could be identified in the anterior lobe cell bodies on the basis of immunocytochemistry and mass spectrometrical analysis. The neuropeptides APGWamide and Lymnaea neuropeptide Y, which are co-localized in the anterior lobe cells as well as in axons innervating the penis retractor muscle, do not induce muscle contraction but have a modulatory action by affecting the relaxation rate and amplitude of the contraction. APGWamide and conopressin had earlier been suggested to modulate peristalsis of the vas deferens. Thus, it seems that the neurons co-expressing the various combinations of neuropeptide genes in the anterior lobe represent functional units, each acting in the fine tuning of different muscles involved in specific aspects of male copulation behaviour.

Further mapping of the Achatina giant neurone types sensitive to the neuroactive peptides isolated from invertebrates

1. 1. The effects of the 10 synthetic neuroactive peptides originally isolated from invertebrates, applied locally to the neurone tested by the brief pneumatic pressure ejection on the identifiable neurone types of Achatina fulica Férussac were examined. 2. 2.Achatin-1 (Gly-D-Phe-Ala-Asp), an Achatina endogenous tetrapeptide having a D-phenylalanine residue, ejected locally, showed the depolarizing effects on nearly half of the number of neurone types tested. 3. 3. ACEP-1 (Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH 2), isolated originally from Achatina atria, and pedal peptide (Pro-Leu-Asp-Ser-Val-Tyr-Gly-Thr-His-Gly-Met-Ser-Gly-Phe-Ala) and buccalin (Gly-Met-Asp-Ser-Leu-Ala-Phe-Ser-Gly-Gly-Leu-NH 2), found in Aplysia neurones, showed excitatory effects on some Achatina neurone types. 4. 4. Myomodulin (Pro-Met-Ser-Met-Leu-Arg-Leu-NH 2), found in Aplysia neurones, produced a hyperpolarization on nearly half of the number of Achatina neurone types tested. The two FMRFamide-like peptides, <EDPFLRFamide (<Glu-Asp-Pro-Phe-Leu-Arg-Phe-NH 2), isolated from Helix heart, and AF1 (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH 2), from Ascaris head, also showed hyperpolarizing effects on more than half of the number of Achatina neurone types. 5. 5. SALMFamide I (Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-NH 2), isolated from Asterias nervous system, CCAP (Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH 2), from Carcinus pericardial organ, and corazonin (<Glu-Thr-Phe-Gln-Tyr-Ser-Arg-Gly-Trp-Thr-Asn-NH 2), from Periplaneta cardiac corpus, had no effect on Achatina neurones.

Release of pedal peptide from Aplysia neurons in primary culture

Pedal peptide (Pep) is a modulatory neuropeptide that is predominantly synthesized in a group of neurons on the dorsal surfaces of the pedal ganglia of Aplysia. Following the determination that Pep is the major peptide selectively present in these neurons in situ, primary cell culture of single Pep-neurons was used to study the release of this neuropeptide. Individual Pep-neurons were grown in culture where they extended many branched neurites with large varicosities. Immunocytology revealed that these newly grown varicosities were intensely Pep immunoreactive. Cultured Pep-neurons, grown in a medium containing radiolabeled methionine, synthesized labeled Pep and transported it into their regenerated neurites. Finally, these neurons released radiolabeled Pep in a calcium- and stimulation-dependent fashion. These results, taken together with previous findings, strongly support the proposition that Pep is a transmitter in Aplysia.

Distribution and characterization of pedal peptide immunoreactivity in Aplysia

Pedal peptide (Pep) is a very abundant neuropeptide in Aplysia. A radioimmunoassay (RIA) was developed to quantify Pep-like immunoreactivity (IR-Pep) in tissue extracts. IR-Pep was present in very high concentrations in the central nervous system (CNS) and two peripheral tissues: the large hermaphroditic duct (LHD) and the foot. RIA of fractions from high-pressure liquid chromatography (HPLC) indicated that Pep itself was the predominant immunoreactive species in each of these tissues. Lower concentrations of Pep were found in a number of other peripheral tissues. Incorporation of labelled amino acid indicated that Pep was synthesized in the LHD, whereas Pep in the foot was synthesized primarily in central neurons and transported to the foot. IR-Pep was further localized by immunocytology. All peripheral IR-Pep appeared to be associated with neuronal fibers, most commonly varicose axons. Immunoreactive innervation of the LHD and foot was particularly dense but positive staining was also observed in other tissues including tegument, gill, gut, and heart, IR-Pep innervation in all tissues including the LHD appeared to be localized predominantly in muscular portions of the tissue. Spontaneous contractions of isolated LHD were accelerated by the application of Pep. Pep appears to act as a transmitter or neuromodulator at a number of different sites in Aplysia.

Involvement of pedal peptide in locomotion in Aplysia: Modulation of foot muscle contractions

Pedal peptide (Pep) is a 15-amino-acid neuropeptide that is localized within the Aplysia central nervous system (CNS) predominantly to a broad band of neurons in each pedal ganglion. Pep-neurons were identified by intracellular staining and immunocytology or by radioimmunoassay (RIA) of extracts from identified neurons. RIA reveals that 97% of all Pep-like immunoreactivity (IR-Pep) in pedal nerves is found in the three nerves that innervate the foot. Nearly every Pep-neuron sends an axon out at least one of these three nerves. Application of Pep to foot muscle causes an increase in the amplitude and relaxation rate of contractions driven by nerve stimulation or intracellular stimulation of pedal motor neurons. The increase in relaxation rate was the predominant effect. Intracellular recording in "split-foot" preparations reveals that Pep-neurons increase their overall firing rates and fire in bursts with each step during locomotion. Recovery of IR-Pep from foot perfusate following pedal nerve stimulation increases in a frequency-dependent fashion. Thus it appears that one function of Pep-neurons is to modulate foot muscle contractility during locomotion in Aplysia.

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.

Sequence of pedal peptide: a novel neuropeptide from the central nervous system of Aplysia

We report the identification of a novel neuropeptide from Aplysia nervous tissue. The peptide was termed Pedal peptide (Pep) because it was predominantly synthesized in the pedal ganglia. Pep was purified and sequenced from pooled extracts of pedal ganglia. The following sequence was proposed: Pro-Leu-Asp-Ser-Val-Tyr-Gly-Thr-His-Gly-Met-Ser-Gly-Phe-Ala. Enzymatic hydrolysis procedures indicated that Pep had a free carboxyl terminal. A peptide with the proposed sequence was synthesized and compared with the native peptide. Chromatographic properties of the 2 peptides under 3 different conditioned were compared and found to be identical. Electrophysiological responses to the 2 peptides were compared on an identified neuron in the abdominal ganglia and found to be qualitatively and quantitatively very similar. Both peptides produced net inward currents that were associated with a decrease in membrane conductance. The results from these 2 procedures confirmed that the proposed Pep sequence was correct. Quantitative measurements of the incorporation of 35S-methionine into Pep suggest that cell bodies that synthesize Pep were present predominantly in the pedal ganglia but should also be found in other central ganglia as well. Pep-like immunoreactive neurons are found predominantly in the pedal ganglia and less frequently in the other ganglia (Pearson and Lloyd, 1989). Quantitatively, Pep constitutes one of the predominant peptides in the nervous system of Aplysia. Pep does not appear to be a member of any other previously identified invertebrate or vertebrate peptide family.

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.