Two neuropeptides colocalized in a command-like neuron use distinct mechanisms to enhance its fast synaptic connection.

In many neurons more than one peptide is colocalized with a classical neurotransmitter. The functional consequence of such an arrangement has been rarely investigated. Here, within the feeding circuit of Aplysia, we investigate at a single synapse the actions of two modulatory neuropeptides that are present in a cholinergic interneuron. In combination with previous work, our study shows that the command-like neuron for feeding, CBI-2, contains two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Previous studies showed that high-frequency prestimulation or repeated stimulation of CBI-2 increases the size of CBI-2 to B61/62 excitatory postsynaptic potentials (EPSPs) and shortens the latency of firing of neuron B61/62 in response to CBI-2 stimulation. We find that both FCAP and CP2 mimic these two effects. The variance method of quantal analysis indicates that FCAP increases the calculated quantal size (q) and CP2 increases the calculated quantal content (m) of EPSPs. Since the PSP amplitude represents the product of q and m, the joint action of the two peptides is expected to be cooperative. This observation suggests a possible functional implication for multiple neuropeptides colocalized with a classical neurotransmitter in one neuron.

A newly identified buccal interneuron initiates and modulates feeding motor programs in aplysia.

Despite considerable progress in characterizing the feeding central pattern generator (CPG) in Aplysia, the full complement of neurons that generate feeding motor programs has not yet been identified. The distribution of neuropeptide-containing neurons in the buccal and cerebral ganglia can be used as a tool to identify additional elements of the feeding circuitry by providing distinctions between otherwise morphologically indistinct neurons. For example, our recent study revealed a unique and potentially interesting unpaired PRQFVamide (PRQFVa)-containing neuron in the buccal ganglion. In this study, we describe the morphological and electrophysiological characterization of this novel neuron, which we designate as B50. We found that activation of B50 is capable of producing organized rhythmic output of the feeding CPG. The motor programs elicited by B50 exhibit some similarities as well as differences to motor programs elicited by the command-like cerebral-to-buccal interneuron CBI-2. In addition to activating the feeding CPG, B50 may act as a program modulator.

PRQFVamide, a novel pentapeptide identified from the CNS and gut of Aplysia.

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.

Identification and characterization of the feeding circuit-activating peptides, a novel neuropeptide family of aplysia.

We use a multidisciplinary approach to identify, map, and characterize the bioactivity of modulatory neuropeptides in the circuitry that generates feeding behavior in Aplysia. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the cerebral-buccal connective (CBC), a nerve containing axons of many interneurons that control feeding behavior of Aplysia, was used to identify neuropeptides that may participate in generation and shaping of feeding motor programs. Using this functionally oriented search, we identified a novel family of peptides that we call the feeding circuit-activating peptides (FCAPs). Two peptides with masses identical to those observed in the CBCs (molecular weight 1387 and 1433) were purified from buccal ganglia and partially sequenced using mass spectrometry. The amino acid sequence was then used to clone the FCAP precursor, which encodes multiple copies of eight different FCAPs. The two FCAPs present in highest copy number correspond to those observed in the CBC. The distribution of FCAP expression was mapped using Northern analysis, whole-mount in situ hybridization, and immunocytochemistry. Consistent with our initial findings, FCAP-immunopositive axons were observed in the CBC. Furthermore, we found that FCAP was present in some cerebral-buccal and buccal-cerebral interneurons. As their name suggests, FCAPs are capable of initiating rhythmic feeding motor programs and are the first neuropeptides with such activity in this circuit. The actions of FCAPs suggest that these peptides may contribute to the induction and maintenance of food-induced arousal. FCAPs were also localized to several other neuronal systems, suggesting that FCAPs may play a role in the regulation of multiple behaviors.

The enterins: a novel family of neuropeptides isolated from the enteric nervous system and CNS of Aplysia.

To identify neuropeptides that have a broad spectrum of actions on the feeding system of Aplysia, we searched for bioactive peptides that are present in both the gut and the CNS. We identified a family of structurally related nonapeptides and decapeptides (enterins) that are present in the gut and CNS of Aplysia, and most of which share the HSFVamide sequence at the C terminus. The structure of the enterin precursor deduced from cDNA cloning predicts 35 copies of 20 different enterins. Northern analysis, in situ hybridization, and immunocytochemistry show that the enterins are abundantly present in the CNS and the gut of Aplysia. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry we characterized the enterin-precursor processing, demonstrated that all of the precursor-predicted enterins are present, and determined post-translational modifications of various enterins. Enterin-positive neuronal somata and processes were found in the gut, and enterins inhibited contractions of the gut. In the CNS, the cerebral and buccal ganglia, which control feeding, contained the enterins. Enterin was also present in the nerve that connects these two ganglia. Enterins reduced the firing of interneurons B4/5 during feeding motor programs. Such enterin-induced reduction of firing also occurred when excitability of B4/5 was tested directly. Because reduction of B4/5 activity corresponds to a switch from egestive to ingestive behaviors, enterin may contribute to such program switching. Furthermore, because enterins are present throughout the nervous system, they may also play a regulatory role in nonfeeding behaviors of Aplysia.

Cerebrin prohormone processing, distribution and action in Aplysia californica.

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F- and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda.

Targeting of peptidergic vesicles in cotransmitting terminals.

In the present study, we examined the targeting of neuropeptide-containing vesicles in terminals of neurons that release both neuropeptides and classical transmitters. Single neurons were electrically stimulated with patterns of activity that were recorded in freely behaving animals. The amount of peptide release was measured biochemically using a radioimmunoassay, and the targeting of peptidergic vesicles was quantified using immunoelectronmicroscopy. Repeated electrical stimulation of single neurons produced a very large increase in peptide release. Peptide release is paralleled by a twofold increase in the number of peptidergic vesicles docked at the portion of the terminal membrane that is away from the target muscle. This is in stark contrast to cholinergic vesicles, which aggregate at, and are released from the conventional release sites in close apposition to the muscle. This differential targeting of cholinergic and peptidergic vesicles may play a significant role in the distinct release requirements and spatial and temporal characteristics of the actions of conventional and peptidergic transmitters.

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia.

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.

Serotonergic and peptidergic modulation of the buccal mass protractor muscle (I2) in aplysia.

Plasticity of Aplysia feeding has largely been measured by noting changes in radula protraction. On the basis of previous work, it has been suggested that peripheral modulation may contribute to behavioral plasticity. However, peripheral plasticity has not been demonstrated in the neuromuscular systems that participate in radula protraction. Therefore in this study we investigated whether contractions of a major radula protraction muscle (I2) are subject to modulation. We demonstrate, first, that an increase in the firing frequency of the cholinergic I2 motoneurons will increase the amplitude of the resulting muscle contraction but will not modulate its relaxation rate. We show, second, that neuronal processes on the I2 muscle are immunoreactive to myomodulin (MM), RFamide, and serotonin (5-HT), but not to small cardioactive peptide (SCP) or buccalin. The I2 motoneurons B31, B32, B61, and B62 are not immunoreactive to RFamide, 5-HT, SCP, or buccalin. However, all four cells are MM immunoreactive and are capable of synthesizing MMa. Third, we show that the bioactivity of the different modulators is somewhat different; while the MMs (i.e., MMa and MMb) and 5-HT increase I2 muscle relaxation rate, and potentiate muscle contraction amplitude, MMa, at high concentrations, depresses muscle contractions. Fourth, our data suggest that cAMP at least partially mediates effects of modulators on contraction amplitude and relaxation rate.

Peptide profiling of cells with multiple gene products: combining immunochemistry and MALDI mass spectrometry with on-plate microextraction.

Due to the intracellular chemical complexity and a wide range of transmitter concentrations, the detection of the complete set of peptide transmitters in a single cell is problematic. In the current study, a multidisciplinary approach combining single-cell MALDI-MS peptide profiling, northern analysis, in situ hybridization, and immunocytochemistry allows characterization of a more complete set of neurotransmitters than individual approaches in the Aplysia californica B1 and B2 motor neurons. Because different results were obtained using both in situ and immunohistochemical techniques compared to previous reports, MALDI-MS assays have been used to examine CP1-related gene products in these cells. However, MALDI with standard sample preparation does not detect the presence of the CP1 gene products. A novel on-plate microextraction approach using concentrated MALDI matrix 2,5-dihydroxybenzoic acid with a mixture of acetone and water as the solvent has been developed to allow the detection of trace-level gene expression products. Both neuropeptide precursors in the B1 and B2 neurons-the SCP and CP1 prohormones-end with large peptides that have multiple cysteine residues. For SCP, MALDI-MS verifies the presence of a novel 9325 Da SCP-related peptide. In the case of CP1, a disulfide-bonded homodimer is detected and the disulfide bonding pattern elucidated using MALDI-MS coupled with on-plate enzymatic digestion.

Peptide cotransmitter release from motorneuron B16 in aplysia californica: costorage, corelease, and functional implications.

Many neurons contain multiple peptide cotransmitters in addition to their classical transmitters. We are using the accessory radula closer neuromuscular system of Aplysia, which participates in feeding in these animals, to define the possible consequences of multiple modulators converging on single targets. How these modulators are released onto their targets is of critical importance in understanding the outcomes of their modulatory actions and their physiological role. Here we provide direct evidence that the partially antagonistic families of modulatory peptides, the myomodulins and buccalins, synthesized by motorneuron B16 are costored and coreleased in fixed ratios. We show that this release is calcium-dependent and independent of muscle contraction. Furthermore, we show that peptide release is initiated at the low end of the physiological range of motorneuron firing frequency and that it increases with increasing motorneuron firing frequency. The coordination of peptide release with the normal operating range of a neuron may be a general phenomenon and suggests that the release of peptide cotransmitters may exhibit similar types of regulation and plasticity as have been observed for classical transmitters. Stimulation paradigms that increase muscle contraction amplitude or frequency also increase peptide release from motor neuron B16. The net effect of the modulatory peptide cotransmitters released from motorneuron B16 would be to increase relaxation rate and therefore allow more frequent and/or larger contractions to occur without increased resistance to antagonist muscles. The end result of this modulation could be to maximize the efficiency of feeding.

The bovine mu-opioid receptor: cloning of cDNA and pharmacological characterization of the receptor expressed in mammalian cells.

The cDNA coding for the bovine mu-opioid receptor has been cloned and sequenced. Conserved sequences from murine delta-receptor cDNA were used as primers in polymerase chain reaction (PCR) to amplify cDNA, prepared by reverse transcription of bovine brain mRNA. This cDNA was used to probe a bovine brain library. The partial sequence obtained was extended to provide the full length clone by PCR. The cDNA has an open reading frame of 1203 base pairs (bp) with a 3'-untranslated region of 1900 bp and a 5'-untranslated region of 265 bp. The protein contains 401 amino acids and has 94% amino acid identity with the human and 91% with the rat mu-opioid receptor. It has the putative seven transmembrane domains, characteristic of G protein-coupled receptors and contains 5 potential N-linked glycosylation sites near the N-terminus. Several potential phosphorylation sites and a putative palmitoylation site are also present. The receptor was stably expressed in HEK293 cells. The binding profile was found to be that of a typical mu receptor, i. e., mu agonists and antagonists, but not delta and kappa ligands, bound with high affinity. Functional assays, namely, opioid stimulation of [35S]GTPgammaS binding and inhibition of forskolin-activated adenylyl cyclase, were also found to be highly specific for mu-opioid agonists. The receptor was downregulated by chronic exposure to mu agonists but not delta or kappa agonists. Evidence is presented indicating that the cloned receptor is the same as the bovine mu receptor previously purified to homogeneity in our laboratory. No evidence was found for genes for multiple mu-type opioid receptors.

The Aplysia mytilus inhibitory peptide-related peptides: identification, cloning, processing, distribution, and action.

Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.

Modulation of radula opener muscles in Aplysia.

We observed fibers immunoreactive (IR) to serotonin (5-HT), the myomodulins (MMs), and FMRFamide on the I7-I10 complex in the marine mollusk Aplysia californica. The I7-I10 muscle complex, which produces radula opening, is innervated primarily by one motor neuron, B48. B48 is MM-IR and synthesizes authentic MM(A). When B48 is stimulated in a physiological manner, cAMP levels are increased in opener muscles. cAMP increases also are seen when the MMs are applied to opener muscles but are not seen with application of the B48 primary neurotransmitter acetylcholine (ACh). Possible physiological sources of 5-HT and FMRFamide are discussed. When modulators are applied to resting opener muscles, changes in membrane potential are observed. Specifically, 5-HT, MM(B), and low concentrations of MM(A) all depolarize muscle fibers. This depolarization is generally not sufficient to elicit myogenic activity in the absence of neural activity under "rest" conditions. However, if opener muscles are stretched beyond rest length, stretch- and modulator-induced depolarizations can summate and elicit contractions. This only occurs, however, if "depolarizing" modulators are applied alone. Thus other modulators (i.e., FMRFamide and high concentrations of MM(A)) hyperpolarize opener muscle fibers and can prevent depolarizing modulators from eliciting myogenic activity. All modulators tested affected parameters of motor neuron-elicited contractions of opener muscles. MM(B) and 5-HT increased contraction size over the range of concentrations tested, whereas MM(A) potentiated contractions when it was applied at lower concentrations but decreased contraction size at higher concentrations. FMRFamide decreased contraction size at all concentrations and did not affect relaxation rate. Additionally, the MMs and 5-HT increased muscle relaxation rate, decreased contraction latency, and decreased the rate at which tension was developed during motor neuron-elicited muscle contractions. Thus these modulators dramatically affect the ability of opener muscles to follow activity in the opener motor neuron B48. The possible physiological significance of these findings is discussed.

Insulin prohormone processing, distribution, and relation to metabolism in Aplysia californica.

The first Aplysia californica insulin gene is characterized and its proteolytic processing from prohormone to final peptides elucidated using a combination of biochemical and mass spectrometric methods. Aplysia insulin (AI) is one of the largest insulins found, with a molecular weight of 9146 Da, and an extended A chain compared with other invertebrate and vertebrate insulins. The AI prohormone produces a series of C peptides and also a unique N-terminally acetylated D peptide. AI-producing cells are restricted to the central region of the cerebral ganglia mostly within the F and C clusters, and AI is transported to neurohemal release sites located on the upper labial and anterior tentacular nerves. The expression of AI mRNA decreases when the animal is deprived of food, and injections of AI reduce hemolymph glucose levels, suggesting that the function of insulin-regulating metabolism has been conserved.

Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli.

Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) are homologous amidated peptides that were originally identified on the basis of similarity to the molluscan neuropeptide FMRF-amide. They have been hypothesized to have wide-ranging functions in the mammalian central nervous system, including pain modulation, opiate function, cardiovascular regulation, and neuroendocrine function. We have cloned the NPFF gene from human, bovine, rat, and mouse, and show that the precursor mRNA encodes for all three of the biochemically identified peptides (NPFF, NPAF, and NPSF). We demonstrate that NPFF precursor mRNA expression by Northern analysis and map sites of expression by in situ hybridization. We confirm the validity of the in situ hybridization by showing that its distribution in the brain and spinal cord matches the distribution of NPFF and NPSF immunoreactivity. We go on to show that the mRNA levels (as measured by in situ hybridization) in the spinal cord can be up-regulated by a model for inflammatory pain (carrageenan injection), but not by a model for neuropathic pain (lumbar nerve ligation). Our results confirm the evolutionary conservation of NPFF, NPAF, and NPSF neuropeptide expression in mammalian brain. They also provide a context for the interpretation of the pain-sensitizing effects of injections of these peptides that have been previously reported. Our results support a model for the role of these peptides in pain regulation at the level of the spinal cord.

Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP.

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs.

In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

Costorage and corelease of modulatory peptide cotransmitters with partially antagonistic actions on the accessory radula closer muscle of Aplysia californica.

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.

Release of peptide cotransmitters in Aplysia: regulation and functional implications.

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.