fmrfamide and myomodulin

Genes encoding neurohormones and neuropeptide precursors were identified in the genomes of two annelids, the leech Helobdella robusta and the polychaete worm Capitella teleta. Although no neuropeptides have been identified from these two species and relatively few neuropeptides from annelids in general, 43 and 35 such genes were found in Capitella and Helobdella, respectively. The predicted peptidomes of these two species are similar to one another and also similar to those of mollusks, particular in the case of Capitella. Helobdella seems to have less neuropeptide genes than Capitella and it lacks the glycoprotein hormones bursicon and GPA2/GPB5; in both cases the genes coding the two subunits as well as the genes coding their receptors are absent from its genome. In Helobdella several neuropeptide genes are duplicated, thus it has five NPY genes, including one pseudogene, as well as four genes coding Wwamides (allatostatin B). Genes coding achatin, allatotropin, allatostatin C, conopressin, FFamide, FLamide, FMRFamide, GGRFamide, GnRH, myomodulin, NPY, pedal peptides, RGWamide (a likely APGWamide homolog), RXDLamide, VR(F/I)amide, WWamide were found in both species, while genes coding cerebrin, elevenin, GGNG, LFRWamide, LRFYamide, luqin, lymnokinin and tachykinin were only found in Capitella.

Topics: Amino Acid Sequence; Animals; Evolution, Molecular; FMRFamide; Insect Hormones; Leeches; Molecular Sequence Data; Neuropeptides; Neurotransmitter Agents; Polychaeta; Sequence Homology, Amino Acid; Tachykinins

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.

Topics: Animals; Aplysia; Cyclic AMP; Electrophysiology; FMRFamide; Membrane Potentials; Muscle Contraction; Muscles; Neuromuscular Junction; Neurons; Neuropeptides; Serotonin

The Drosophila FMRFamide gene encodes multiple FMRFamide-related peptides. These peptides are expressed by neurosecretory cells and may be released into the blood to act as neurohormones. We analyzed the effects of eight of these peptides on nerve-stimulated contraction (twitch tension) of Drosophila larval body-wall muscles. Seven of the peptides strongly enhanced twitch tension, and one of the peptides was inactive. Their targets were distributed widely throughout the somatic musculature. The effects of one peptide, DPKQDFMRFamide, were unchanged after the onset of metamorphosis. The seven active peptides showed similar dose-response curves. Each had a threshold concentration near 1 nM, and the EC50 for each peptide was approximately 40 nM. At concentrations <0.1 microM, the responses to each of the seven excitatory peptides followed a time course that matched the fluctuations of the peptide concentration in the bath. At higher concentrations, twitch tension remained elevated for 5-10 min or more after wash-out of the peptide. When the peptides were presented as mixtures predicted by their stoichiometric ratios in the dFMRFamide propeptide, the effects were additive, and there were no detectable higher-order interactions among them. One peptide was tested and found to enhance synaptic transmission. At 0.1 microM, DPKQDFMRFamide increased the amplitude of the excitatory junctional current to 151% of baseline within 3 min. Together, these results indicate that the products of the Drosophila FMRFamide gene function as neurohormones to modulate the strength of contraction at the larval neuromuscular junction. In this role these seven peptides appear to be functionally redundant.

Topics: Amino Acid Sequence; Animals; Drosophila melanogaster; Electrophysiology; Enkephalin, Methionine; FMRFamide; Kinetics; Larva; Metamorphosis, Biological; Muscle Contraction; Muscles; Neuromuscular Junction; Neuropeptides; Stimulation, Chemical; Synaptic Transmission

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides.

Topics: Animals; Central Nervous System; FMRFamide; Ganglia, Invertebrate; Immunohistochemistry; Interneurons; Invertebrate Hormones; Isoquinolines; Leeches; Nerve Net; Neuropeptides; Physical Stimulation; Reflex

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 EFLRlamide and 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 immunoreactive 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 cardioexcitatory motoneurons, the Ehe cells (E heart excitors of the visceral ganglion) and penial motoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that these respective 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 central neurons 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-translationa

Topics: Amino Acid Sequence; Animals; Brain Mapping; Central Nervous System; Exons; FMRFamide; Genetic Code; Immunohistochemistry; Lymnaea; Models, Genetic; Molecular Sequence Data; Neurons; Neuropeptides; Neurotransmitter Agents; Protein Processing, Post-Translational

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 anterior portion of intrinsic buccal muscle (I3a) is innervated by two excitatory motor neurons, B3 and B38, and the newly identified inhibitory motor neuron, B47. We show that B47 is cholinergic while B3 and B38 are not. B3 and B38 have previously been shown to express the neuropeptides FMRFamide and the small cardioactive peptides (SCPs) A and B, respectively. We present evidence here that B47 synthesizes the neuropeptide myomodulin A (Mma). When placed in culture, B3, B38, and B47 continued to synthesize their respective peptides. These peptides were released in a stimulation- and Ca(2+)-dependent manner, suggesting that they are transmitters in these neurons. By using B3-evoked excitatory junction potentials (EJPs) and muscle contractions as assays, we next examined the modulatory effects of superfusion of peptides and stimulation of motor neurons B38 and B47. Superfusing the muscle with low concentrations of the SCPs, FMRFamide, or Mma enhanced B3-evoked EJPs and contractions. Stimulation of B47 simultaneously with B3 reduced the amplitude of B3-evoked contractions. However, when either B47 or B38 was stimulated in extended bursts designed to release their peptide transmitters, subsequent B3-evoked EJPs and contractions were enhanced. We believe that this modulation is due at least in part to the release of peptides from the terminals of B38 and B47. The SCPs potently increase cAMP levels in I3a muscle fibers. Likewise, stimulation of B38 in extended bursts increased cAMP levels in the muscle. This provides independent evidence that the SCPs are released from B38 terminals in the muscle. Therefore, we have described a neuromuscular preparation amenable to the study of both excitatory and inhibitory motor neurons that utilize a variety of conventional and peptide transmitters. Our results suggest that these motor neurons can function in two states. When stimulated in single brief bursts, they primarily release conventional transmitters. When stimulated in a series of prolonged bursts, they release both conventional transmitters and peptide cotransmitters. These dual states are most pronounced in the case of B47, which, depending on the stimulation paradigm, can act selectively to inhibit or enhance the effects of a second motor neuron innervating the same muscle.

Topics: Acetylcholine; Animals; Aplysia; Cells, Cultured; Choline; Electric Stimulation; Evoked Potentials; FMRFamide; Ganglia; In Vitro Techniques; Methionine; Motor Neurons; Muscle Contraction; Neuromuscular Junction; Neuropeptides; Sulfur Radioisotopes; Synaptic Transmission

Involvement of neuropeptides in the regulation of cardiac activity in a prosobranch mollusc, Rapana thomasiana, was studied physiologically as well as immunohistochemically. A catch-relaxing peptide (CARP) showed strong inhibitory effects on the heart with a lower threshold than acetylcholine. The action of CARP was in contrast to that of another neuropeptide, FMRFamide, which has previously been shown to enhance the heart beat. Benzoquinonium blocked the effects of acetylcholine and stimulation of right cardiac nerves 1 and 3 b, but not those of CARP, suggesting that the effects of nerve stimulation are mainly due to the release of acetylcholine. Immunohistochemical examinations demonstrated that FMRFamide-like and CARP-like immunoreactive neurons are distributed in the visceral ganglia. Although a neuron appeared to show weak immunoreactivity to both antisera, evidence for the coexistence of peptides in a single neuron was not exhibited. Positive immunoreactivity to FMRFamide and CARP antisera also appeared in right cardiac nerves 1 and 3. In the heart, FMRFamide- and CARP-like immunoreactive fibers were restricted to the atrium and the aortic end of the ventricle, consistent with the morphological observation of innervation. The present results suggest that FMRFamide- and CARP-like peptides are involved in regulating the heart beat.

Topics: Acetylcholine; Amino Acid Sequence; Animals; Cardiotonic Agents; FMRFamide; Heart; Immunohistochemistry; Invertebrate Hormones; Molecular Sequence Data; Mollusca; Neuropeptides; Oligopeptides; Quaternary Ammonium Compounds; Sequence Homology

1. The electrophysiological properties of sensory neurons that mediate withdrawal reflexes of Aplysia can be modulated by a variety of neurotransmitters. We compared the known excitatory actions of serotonin (5-HT) with the actions of FMRFamide (Phe-Met-Arg-Phe-NH2) and myomodulin (Pro-Met-Ser-Met-Leu-Arg-Leu-NH2) on the durations of action potentials and excitability. In addition, with the use of voltage-clamp and pharmacological separation techniques, we characterized the membrane currents that were modulated by each of the three agents. 2. Application of 5-HT produced an increase in the duration of action potentials and an enhancement of excitability in somata of the tail sensory neurons. FMRFamide and myomodulin reversed these excitatory effects and decreased the duration of action potentials and excitability. These results indicated that FMRFamide and myomodulin exerted inhibitory effects on the electrophysiological properties of the sensory neurons. properties of the sensory neurons. 3. FMRFamide appeared to modulate three K+ currents. The first current, which was increased by FMRFamide, had properties closely resembling those of the S-K+ current (IK,S). These properties include slow activation, little inactivation, and relative insensitivity to the K+ channel blockers 4-aminopyridine (4-AP) and tetraethylammonium (TEA). The second current, which was reduced by FMRFamide, had kinetic and pharmacological properties similar to those of a component of the Ca(2+)-activated K+ current (IK,Ca). Finally, at large depolarizations, FMRFamide appeared to increase a third current that was attenuated by 4-AP, suggesting that FMRFamide also modulated the delayed or voltage-dependent K+ current (IK,V). 4. Myomodulin appeared to modulate two of the currents modulated by FMRFamide, because it increased both IK,S and IK,V. Unlike FMRFamide, however, myomodulin did not appear to modulate IK,Ca. 5. Arachidonic acid mimicked the modulation of IK,S, IK,Ca, and IK,V by FMRFamide. Because myomodulin did not modulate IK,Ca, it appears that a second messenger other than arachidonic acid or its metabolites mediates the modulatory effects of myomodulin. 6. These results indicate that both FMRFamide and myomodulin can inhibit the tail sensory neurons by increasing IK,S. FMRFamide, but not myomodulin, also reduces IK,Ca, which suggests that under some conditions FMRFamide may also have excitatory actions. Finally, these results suggest that the effects of FMRFamide and myomodul

Topics: 4-Aminopyridine; Action Potentials; Animals; Aplysia; Arachidonic Acid; Cell Membrane; FMRFamide; Ganglia; In Vitro Techniques; Kinetics; Neurons, Afferent; Neuropeptides; Serotonin; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Time Factors

An examination of the cellular properties and synaptic outputs of mechanoafferent neurons found on the ventrocaudal surface of the cerebral ganglion of Aplysia indicated that the cerebral mechanoafferent (CM) neurons are a heterogeneous population of cells. Based on changes in action potential duration in response to bath applications of 5-HT in the presence of TEA, CM neurons could be divided into 2 broad classes: mechanoafferents whose spikes broaden in response to 5-HT (CM-SB neurons) and mechanoafferents whose spikes narrow in response to 5-HT (CM-SN neurons). Morphological and electrophysiological studies of the CM-SN neurons indicated that they were comprised of previously identified interganglionic cerebral-buccal mechanoafferent (ICBM) neurons and a novel set of sensory neurons that send an axon into the LLAB cerebral nerve and have perioral zone receptive fields that are similar to those of ICBM neurons. Changes in spike width due to 5-HT were correlated with changes in synaptic output as indicated by the magnitudes of EPSPs evoked in postsynaptic neurons. Electrical stimulation of cerebral nerves and connectives also produced spike narrowing or broadening, and the sign of the effect was a function of the parameters of stimulation. Both heterosynaptic facilitation and heterosynaptic depression of EPSPs evoked in follower cells could be demonstrated. A variety of putative neuromodulators other than 5-HT were also found to affect the duration of action potentials in both classes of CM neurons. FMRFamide had effects opposite to that of 5-HT. SCPB and a recently characterized Aplysia neuropeptide, buccalin, broadened the spikes of both CM classes. Another neuropeptide, myomodulin, decreased the duration of CM-SB neuron spikes but had no effect on CM-SN spikes. Since the CM neurons appear to mediate a variety of competing behaviors, including feeding, locomotion, and defensive withdrawal, the various neuromodulator actions may contribute to the mechanisms whereby behaviors are selected and modified.

Topics: Action Potentials; Animals; Aplysia; Brain; FMRFamide; Ganglia; Mechanoreceptors; Neural Pathways; Neurons, Afferent; Neuropeptides; Neurotransmitter Agents; Reaction Time; Serotonin; Synapses