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PubMed1994

A genetic and molecular analysis of the 46C chromosomal region surrounding the FMRFamide neuropeptide gene in Drosophila melanogaster.

We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes.

Animals↗
PubMed1995

Identification, distribution and physiological activity of three novel neuropeptides of Lymnaea: EFLRlamide 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 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-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.

Amino Acid Sequence↗
PubMed1999

Structure, function, and expression of Drosophila melanogaster FMRFamide-related peptides.

In 1977, Price and Greenberg identified the tetrapeptide FMRFamide as a cardioexcitatory molecule from mollusc. Subsequent to this discovery, FMRFamide-related peptides (FaRPs) have been identified in both invertebrates and vertebrates. Peptides in the FaRP family contain a common RFamide C-terminus and act as modulators and messengers of neural and gastrointestinal functions. Like other organisms, Drosophila melanogaster contains several genes that encode for numerous FaRPs. Elucidating the processing and activities of multiple FaRPs encoded in a single precursor is critical to establishing their roles in physiology. In this manuscript, we describe the distribution of FMRFamide immunoreactive materials in the Drosophila central nervous system and gut, and correlate it with the expression of specific FaRPs and their activities. The unique distributions and biological activities of Drosophila FaRPs suggest that the precursors are highly processed and the structurally related peptides are not functionally redundant. The complete distribution of FaRPs in the central nervous system and gut as detected by FMRFamide antisera is not accounted for by the sum of the individual expression patterns of the known Drosophila peptides. Thus, these data suggest that one or more Drosophila FaRPs or structurally related peptides remain to be discovered.

Amino Acid Sequence↗
PubMed2006

Distribution of neuropeptide FF-like immunoreactivity in the brain of Dermophis mexicanus (Amphibia; Gymnophiona): comparison with FMRFamide immunoreactivity.

Neuropeptide FF (NPFF) is an FMRFamide-related peptide widely distributed in the mammalian brain. NPFF immunohistochemistry labeled cell bodies in a few locations and dense fiber networks throughout the brain. Recently, the distribution of NPFF immunoreactive (NPFF-ir) cells and fibers in the brain of anuran and urodele amphibians was studied and, as in mammals, significant species differences were noted. To further assess general and derived features of the NPFF-containing neuron system in amphibians, we have investigated the distribution of NPFF-ir cell bodies and fibers in the brain of the gymnophionan Dermophis mexicanus by means of an antiserum against bovine NPFF. This distribution was compared to that of FMRFamide immunoreactivity. Major traits shared with anurans and urodeles were the abundant fiber labeling in the ventral telencephalon, hypothalamus, isthmus, ventrolateral medulla and dorsal spinal cord. In addition, in the three amphibian orders the majority of the NPFF-ir cells were located in the preoptic-hypothalamic region. However, distinct particular features were present in the gymnophionan such as the lack of NPFF-ir cells in the telencephalon, brainstem and spinal cord and the absence of NPFF-ir fibers in the hypophysis and the olfactory bulbs. This pattern was distinct from that observed for FMRFamide distribution. Striking differences were noted in the pallium, caudal hypothalamus and midbrain tegmentum where FMRFamide-containing cells were localized. The present results in Dermophis support the idea that data from gymnophionans must be included when stating the amphibian condition of a given system because important variations are obvious when gymnophionans are compared with anurans and urodeles.

Amphibians↗
PubMed1985

Inhibitory influences of FMRFamide on morphine- and deprivation-induced feeding.

Intracerebroventricular administration of 0.01-1.0 microgram of the peptide FMRFamide (Phe-Met-Arg-Phe-NH2) to mice reduced morphine-induced feeding. Administration of FMRFamide also inhibited feeding induced by a 24-hour period of food deprivation. These results suggest that FMRFamide or FMRFamide-like neuropeptides play an important role in the control of opioid-mediated feeding.

Animals↗
PubMed1997

FMRFamide-like peptides in the crayfish (Procambarus clarkii) stomatogastric nervous system: distribution and effects on the pyloric motor pattern.

Whole-mount immunocytochemistry was used to map the location of FMRFamide-like peptides in the crayfish (Procambarus clarkii) stomatogastric nervous system. This system contains the pyloric and gastric mill central pattern generators, which receive modulatory inputs from projection neurons with somata located primarily in other ganglia of the stomatogastric nervous system. Our studies revealed stained somata in the commissural and esophageal ganglia. A pair of stained somata was located in the inferior ventricular nerve, and another pair of somata was located in the stomatogastric nerve where it is joined by the two superior esophageal nerves. The stomatogastric ganglion contained no stained somata, but the neuropil was brightly stained and 2-4 axons projected laterally in small nerves directly from the ganglion. These results indicate that FMRFamide or related peptides may act as neuromodulators in the crayfish stomatogastric nervous system. To test this hypothesis, we studied the effects of FMRFamide and four related peptides (DF2, NF1, F1 and LMS) on the pyloric motor pattern. DF2, NF1 and F1 all excited certain pyloric cells, especially the lateral pyloric (LP) and ventricular dilator (VD) neurons, and enhanced pyloric cycling frequency in most actively rhythmic preparations. FMRFamide had no detectable effects on pyloric cells, and LMS had inhibitory effects, causing disruption of the pyloric rhythm in actively cycling preparations and reducing tonic activity in non-rhythmic preparations.

Animals↗
PubMed2001

Three-dimensional computer-aided reconstruction of FMRFamide immunopositive neuron distribution in the ventral ganglion of the barnacle Balanus amphitrite (Cirripedia, Crustacea).

We have implemented a simple program to solve three of the problems related to 3D reconstruction (3D-Rec) of soft tissues: alignment of sections, distortions, and estimation of the spatial position of elements of interest inside the tissues. As a model, we chose the distribution of FMRFamide-like immunopositive neurons in the ventral ganglion of the barnacle Balanus amphitrite collected during different seasonal periods. Images of immunostained sections were acquired by means of a CCD-camera-equipped microscope and a PC and the reference points were taken inside the sections. The FMRFamide-like immunopositive neurons detected in the barnacle ventral ganglion were grouped into four different classes according to size, shape and staining intensity. More numerous FMRFamide-like immunopositive neurons were detected in the autumn-collected barnacle than in the summer counterpart. The two 3D reconstructions obtained from transverse and longitudinal ventral ganglion sections were efficaciously compared after 90 degrees rotation of one of them. Comparison of these two 3D-Rec suggests the presence of at least two groups of FMRFamide-like immunopositive neurons that are seasonally-related and probably involved in reproduction.

Animals↗
PubMedDate not supplied

The distribution of cells containing FMRFamide- and 5-HT-related molecules in the embryonic development of Viviparus ater (Mollusca, Gastropoda).

The timing and spatial distribution of cells containing FMRFamide- and 5-HT-related molecules in the embryonic development of the mollusc Viviparus ater are examined using immunohistochemistry. FMRFamide-like molecules emerge in the early stage E8 (8% of embryonic development) before the 5-HT immunoreactivity, and they are not only found during nervous system ontogeny. As the parts of the digestive tract differentiated, the pattern of the diffuse gut endocrine cells, present in adults, start to be established (E20-E30), and both open and closed cell types are immunoreactive to anti-FMRFamide antibody. From their appearance (E20), cells with a 5-HT-like phenotype are distributed in the central nervous ganglia and progressively assembled during embryonic development. The early occurrence of both these molecules in V. ater embryos reinforces the growing view that neurotransmitters play a regulatory role in embryogenic processes. In particular, the very early presence of FMRFamide-related factors suggests an involvement of these molecules in the regulation of basic, not only neuronal, cell behaviours, while 5-HT seems to be a more specific neural development signal.

Animals↗
PubMed1992

Differential effects of serotonin, FMRFamide, and small cardioactive peptide on multiple, distributed processes modulating sensorimotor synaptic transmission in Aplysia.

At least two processes contribute to the modulation by 5-HT of the connections between sensory neurons and motor neurons in Aplysia. The first involves broadening of the presynaptic spike through modulation of 5-HT-sensitive K+ channels that leads to elevated levels of intracellular Ca2+ and increased release of transmitter. A second process (or set of processes) apparently accounts for the amount of facilitation not produced by presynaptic spike broadening. This spike duration-independent (SDI) process is particularly prominent in depressed synapses. We used a protocol in which spikes were prebroadened into a range of durations in which further spike broadening by itself has little or no effect on facilitation of the EPSP.5-HT produced pronounced facilitation in depressed synapses under these conditions. Another modulatory agent, small cardioactive peptide (SCPb), also broadened spikes in sensory neurons but did not produce facilitation comparable to that produced by 5-HT. These results indicate that 5-HT activates the SDI process whereas SCPb fails to do the same. A 5 min preexposure to the modulatory peptide FMRFamide inhibited 5-HT-induced activation of the SDI process, whereas a 1 min preexposure did not. Another process(es) that may modulate synaptic efficacy in sensorimotor synapses involves changes in the properties of the motor (follower) neuron, such as input resistance. FMRFamide decreased the input resistance of postsynaptic neurons. This action could contribute to the effects of FMRFamide when administered alone, but it did not appear to be responsible for the inhibitory action of FMRFamide on 5-HT-induced facilitation. Neither 5-HT nor SCPb had a clear effect on input resistance. The actions of these three agents, therefore, seem to be differentially distributed among various pre- and postsynaptic processes involved in the modulation of synaptic transmission.

Analysis of Variance↗
PubMed1991

Neuropeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-amide (GDPFLRFamide) and Ser-Asp-Pro-Phe-Leu-Arg-Phe-amide (SDPFLRFamide) are encoded by an exon 3' to Phe-Met-Arg-Phe-NH2 (FMRFamide) in the snail Lymnaea stagnalis.

Biochemical analysis has shown the pond snail Lymnaea stagnalis to contain 2 main classes of Phe-Met-Arg-Phe-NH2 (FMRFamide)-like neuropeptides: the tetrapeptides FMRFamide and Phe-Leu-Arg-Phe-NH2 (FLRFamide), and the heptapeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDP-FLRFamide) and Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFFRFamide). By genomic mapping and DNA sequencing, we show here that the GDP/SDPFLRFamide coding region lies 3' to the FMRFamide coding region. The absence of an initiating start methionine and the presence of good-concensus 3' and 5' splice sites suggests that the GDP/SDPFLRFamide coding region makes up 1 exon of a larger gene. In addition to 7 copies of GDPFLRFamide and 6 copies of SDPFLRFamide, the exon encoding the heptapeptides also encodes 3 novel peptides, Glu-Phe-Phe-Pro-Leu-NH2 (EFFPLamide), Ser-Asp-Pro-Tyr-Leu-Phe-Arg-NH2 (SDPYLFRamide), and Ser-Asp-Pro-Phe-Phe-Arg-Phe-NH2 (SDPFFRFamide). In contrast to the tetrapeptide FMRFamide precursor protein, the GDP/SDPFLRFamide peptides are encoded contiguously, being separated only by single basic amino acids.

Amino Acid Sequence↗
PubMed1991

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.

Animals↗
PubMed1986

Modulation of cholinergic transmission by opiate peptide and FMRFamide on identified neurons of Helix pomatia L. (Gastropoda, Mollusca).

Identified neurons regulating visceral functions in Helix pomatia reacted selectively to FMRFamide peptide applied to the membrane, some becoming depolarized others hyperpolarized. The effect of morphine and leu-enkephalin was similar to that of FMRFamide. The ACh-induced depolarization was decreased by FMRFamide, morphine, leu-enkephalin and naloxone on the neuron LPa5, while this modulatory effect was not observed on other neurons involved in the regulation of visceral functions. The opiate peptides and FMRFamide may play a modulatory role in molluscan CNS.

Animals↗
PubMed1988

The voltage-dependent, slow inward current induced by the neuropeptide FMRFamide in Aplysia neuron R14.

The effects of the peptide FMRFamide (Phe-Met-Arg-Phe-NH2) on the soma of neuron R14 in the abdominal ganglion of Aplysia californica and A. brasiliana were characterized. Pressure-ejected FMRFamide caused 3 types of responses, (1) a fast outward current (duration, less than 30 sec), (2) a fast inward current (duration, less than 20 sec), and (3) a slow inward current (peak at 0.5-1 min; duration, 2-3 min). The slow inward current, the chief object of this study, arises from a voltage-dependent conductance increase. The FMRFamide-elicited slow inward current is largest between -40 mV and -20 mV, the region of a negative slope resistance in the normal current-voltage relationship for R14. The slow FMRFamide-induced inward current is largely carried by Na+. This current is independent of external [K+] but depends inversely on external [Ca2+] and [Cl-]. The concentrations of the latter ions may influence the voltage dependence of the response. The slow inward current has many properties in common with inward currents induced in other molluscan neurons by applications of neuropeptides or intracellular injections of cyclic nucleotides.

Animals↗
PubMed1988

FMRFamide-like immunoreactivity in brain and pituitary of Xenotaca eisenii: (Ciprinidontoformes, Teleostei).

FMRFamide-immunoreactive neurons were determined immunocytochemically by means of the PAP-technique for unlabeled antibodies. FMRFamide-containing perikarya were found in the frontoventral telencephalon, in the valvula and corpus cerebelli, and in the rostral and caudal medulla oblongata. FMRFamide immunoreactive cells were localized in the anterio lobe of the pituitary. FMRFamide fibres occurred in the bulbus olfactorius, in the anterior, medial and lateral parts of the telencephalon, in the commissura anterior, beneath the nucleus praeopticus, beneath the nucleus dorsomedialis, ventrolis and anterior tuberis as well as in the caudal hypothalamus, surrounding the nucleus recessus lateralis and posterior. In the mesencephalon, FMRF-amide-containing fibres appeared in the dorsal tegmentum, in the torus semicircularis and in the deep layers of the tectum opticum. In the rhombencephalon, only few fibres were found in the cerebellum but a strong innervation occurred in the ventrolateral and dorsolateral medulla oblongata. The present results are discussed with those of other teleost species.

Animals↗
PubMed1993

Presence and distribution of immunoreactive FMRFamide- and bovine pancreatic polypeptide-like peptides in a protochordate ascidian.

FMRFamide and bovine pancreatic polypeptide (BPP) immunoreactivities were detected in the cerebral ganglion and in the alimentary tract of the protochordate ascidian Styela plicata. Neurons expressing immunostain to FMRFamide were mainly found in the cortical zone of the ganglion and a network of immunoreactive beaded nerve fibers was present in the neuropil. Co-localization of FMRFamide- and BPP-like materials was demonstrated in few nerve cell bodies and both peptides likely play a neurotransmitter role. Parallel, endocrine-like cells containing FMRFamide-like materials were distributed among the esophageal and gastric epithelia, whereas no PP immunostaining was found. The tetrapeptide could be involved in the regulation of the digestive processes by means of endocrine and/or paracrine mechanisms.

Animals↗
PubMed1997

A slow outward current activated by FMRFamide in heart interneurons of the medicinal leech.

The endogenous neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) can accelerate the oscillation of reciprocally inhibitory pairs of interneurons that pace heartbeat in the medicinal leech. A model based on all available biophysical data of a two-cell heart interneuron oscillator provides a theoretical basis for understanding this modulation. Previously observed modulation of K+ currents by FMRFamide cannot account for this acceleratory effect in the model. This observation prompted the present reexamination of K+ currents in heart interneurons. We devised better methods for separation of the various components of K+ current and more accurately measured their activation and deactivation kinetics. Moreover, we demonstrated that FMRFamide activates a previously undetected K+ current (IKF), which has very slow activation and deactivation kinetics. Addition of physiologically measured amounts of IKF to the model two-cell oscillator can account for the acceleratory effect of FMRFamide.

Animals↗
PubMed1994

Colocalization of octopamine and FMRFamide related peptide in identified heart projecting (DUM) neurones in the locust revealed by immunocytochemistry.

Immunocytochemical techniques are employed to reveal colocalization of octopamine with FMRFamide related peptide in the locust ventral nervous system. In each unfused pregenital abdominal ganglia (A4-A6) there are 3 octopamine-like immunoreactive neurones. By combining intracellular Lucifer yellow staining with subsequent immunocytochemistry these are individually identified as the efferent dorsal unpaired median (DUM) neurones DUM-1 and DUM-2, which innervate abdominal tergal and respectively sternal skeletal muscles, and DUM heart-1, an FMRFamide-like immunoreactive neurone which projects to the heart and associated alary muscles. Colocalization of octopamine- and FMRFamide-like immunoreactivity in DUM heart-1 is verified by alternate staining of consecutive sections. With respect to locust ventral ganglia, this investigation shows that colocalization of octopamine with an FMRFamide related peptide is restricted to a single DUM cell occurring in each abdominal ganglion 2-7, which most likely corresponds to segmental homologues of DUM heart-1.

Abdomen↗
PubMed1978

FMRFamide increases the adenylate cyclase activity and cylic AMP level of molluscan heart.

FMRFamide (phenylalanyl-methionyl-arginyl-phenylalanine amide) is a cardioexcitatory peptide recently isolated and identified in molluscan ganglia. Both FMRFamide and 5-hydroxytryptamine (5HT), the cardioexcitatory neurotransmitter in molluscs, were tested on the ventricle of the bivalve Mercenaria mercenaria. Both agents increased myocardial contractility, the intracellular cyclic AMP concentration of intact hearts and the adenylate cyclase activity of a myocardial membrane fraction. FMRFamide was 5--10 times more potent than 5HT. All of the effects of 5HT, and none of those of FMRFamide, were blocked by methysergide, a specific 5HT antagonist.

Adenylyl Cyclases↗