small-cardioactive-peptide-a and buccalin

Many neurons that contain a classical neurotransmitter also contain modulatory peptides, but it has been difficult to establish unequivocally that these peptides are functional cotransmitters. Here, we provide evidence for functional cotransmission in a neuromuscular system of Aplysia. Using immunocytochemical techniques, we localize members of two peptide families, the small cardioactive peptides (SCPs) and the buccalins (BUCs), to a single subset of dense-core vesicles in the terminals of the cholinergic motorneuron B15. We describe a new preparation and method for the direct detection of released peptides and show that the SCPs and BUCs are released when neuron B15 is intracellularly stimulated. Consistent with their subcellular localization, the SCPs and BUCs are released in a stoichiometric ratio that is constant across conditions that change the absolute amount of peptides released. Peptide release is calcium-dependent but does not require muscle contractions. Thus, the release cannot be attributed to a displacement of peptides that may be present in the extracellular space. In previous studies, we characterized the physiological firing patterns of neuron B15. Here, we simulate these firing patterns and show that peptide release occurs. Additionally, we find that significant quantities of material are released under behaviorally relevant conditions. We find that concentrations of released peptides in the muscle are in the concentration range in which exogenously applied peptides exert characterized modulatory actions on muscle contractions. Together, our findings provide strong support for the hypothesis that peptides contained in neuron B15 are functional cotransmitters.

Topics: Animals; Aplysia; Calcium; Cell Count; Electrophysiology; Immunohistochemistry; Motor Neurons; Muscle Contraction; Muscles; Neurons; Neuropeptides; Neurotransmitter Agents; Staining and Labeling; Time Factors; Tissue Distribution

To gain insights into the physiological role of cotransmission, we measured peptide release from cell B15, a motorneuron that utilizes ACh as its primary transmitter but also contains putative peptide cotransmitters, the small cardioactive peptides (SCPs) and the buccalins (BUCs). All stimulation parameters used were in the range in which B15 fires in freely moving animals. We stimulated neuron B15 in bursts and systematically varied the interburst interval, the intraburst frequency, and burst duration. Both peptides were preferentially released when B15 was stimulated at higher intra- or interburst frequencies or with longer burst durations. Across stimulation patterns, the amount of peptide released depended on the mean frequency of stimulation and was independent of the specific pattern of stimulation. The parameters of stimulation that produce a larger release of peptides correspond to those that evoke larger contractions. Large and frequent contractions are likely to fuse or summate, thus disrupting the rhythmic behavior mediated by the muscle innervated by motorneuron B15. Because the combined effect of the SCPs and BUCs is to accelerate the relaxation and shorten the duration of muscle contractions, these peptides reduce the probability of the disruptive fusion or summation of muscle contractions. Because these cotransmitters regulate an aspect of muscle contractions that is not controlled by acetylcholine (ACh), the primary transmitter of B15, we suggest that peptides and ACh form parallel but functionally distinct lines of transmission at the neuromuscular junction. Both types of transmission may be necessary to ensure that behavior remains efficient over a wide range of conditions.

Topics: Animals; Aplysia; Electric Stimulation; Motor Neurons; Neuropeptides; Reaction Time; Serotonin; Temperature

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.

Topics: Amino Acid Sequence; Animals; Cheek; Feeding Behavior; FMRFamide; Immunohistochemistry; Interneurons; Invertebrate Hormones; Lymnaea; Molecular Sequence Data; Motor Neurons; Nerve Net; Neurons; Neuropeptides; Neurotransmitter Agents

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.

Topics: Amino Acid Sequence; Animals; Aplysia; Artifacts; Central Nervous System; Immunohistochemistry; Invertebrate Hormones; Lymnaea; Molecular Sequence Data; Neuropeptides