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PubMed1988

Distribution of FMRFamide-like immunoreactivity in the nervous system of the slug Limax maximus.

The distribution of FMRFamide-like immunoreactive neurons in the nervous system of the slug Limax maximus was studied using immunohistochemical methods. Approximately one thousand FMRFamide-like immunoreactive cell bodies were found in the central nervous system. Ranging between 15 micron and 200 micron in diameter, they were found in all 11 ganglia of the central nervous system. FMRFamide-like immunoreactive cell bodies were also found at peripheral locations on buccal nerve roots. FMRFamide-like immunoreactive nerve fibres were present in peripheral nerve roots and were distributed extensively throughout the neuropil and cell body regions of the central ganglia. They were also present in the connective tissue of the perineurium, forming an extensive network of varicose fibres. The large number, extensive distribution and great range in size of FMRFamide-like immunoreactive cell bodies and the wide distribution of immunoreactive fibres suggest that FMRFamide-like peptides might serve several different functions in the nervous system of the slug.

Animals↗
PubMed1989

Distribution of FMRFamide-like immunoreactivity in the brain and neurohypophysis of the lamprey, Lampetra japonica.

Distribution of molluscan cardio-excitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) was determined by means of immunohistochemistry in the brain and neurohypophysis of the lamprey, Lampetra japonica. Many FMRFamide-like immunoreactive neurons were found in the periventricular nuclear region and in a region near the mammillary recess. Neurons situated in the former region were larger. The immunoreactive cell groups were shown to be located at sites differing from those of the AF-positive cell groups. The fibers of immunoreactive neurons extended in all directions within the brain and towards the spinal cord, some reaching the third ventricle and capillaries. Thus, FMRFamide-like immunoreactive peptides appear to function as neurotransmitters or neuromodulators and possibly also as neurohormones. FMRFamide-like immunoreactive material was rarely observed in the posterior neurohypophysis (neural lobe), but was noted to be present to a limited extent in the caudal part of the anterior neurohypophysis (median eminence). It would thus follow that FMRFamide-like immunoreactive neurons may not necessarily be related to the hypothalamo-neural lobe system, but may possibly be associated with the hypothalamo-adenohypophysial system. The pineal body showed no FMRFamide-like immunoreactivity.

Animals↗
PubMedDate not supplied

Regulation of pharyngeal motility by FMRFamide and related peptides in the medicinal leech, Hirudo medicinalis.

The medicinal leech possesses FMRFamide-like immunoreactivity in neural processes and somata associated with the pharynx and pharyngeal ganglia. The pharynx possessed about 25 immunoreactive somata; about half of the approximately 20 neurons of the pharyngeal ganglia were immunoreactive. We provide brief descriptions of several neurons located in the first neuromere of the subesophageal ganglion involved in controlling pharyngeal motility. Double-labeling experiments indicate that one of these cells, named Swallow neuron 1 (SW1), contains a FMRFamide-like peptide. Stimulation of SW1 caused the mouth to open and the pharynx to dilate. Upon termination of SW1 stimulation, the mouth closed, and a peristaltic wave progressed from the mouth down the length of the pharynx. Stimulation of SW1 did not produce 1:1 postsynaptic potentials in pharyngeal muscle cells. Thus, SW1 is apparently not a motor neuron. The pharynx responded to application of FMRFamide and related peptides by producing a series of 20- to 35-s phasic contractions superimposed upon an increase in basal tonus. The peptide-induced response was quantified by measuring increases in basal tonus, peak tension, and integrated area. Although there were some differences in the order of potency depending upon which parameter was considered, the approximate order of potency of RFamide peptides tested was: pQDPFLRFamide > or = FMRFamide approximately YGGFMRFamide > or = YMRFamide approximately FLRFamide approximately GGKYMRFamide approximately YLRFamide > leucomyosuppressin approximately perisulfakinin. Except for differences in potency, each of the RFamide peptides produced similar contractile waveforms. FMRFamide-induced responses were reduced by the protein kinase C inhibitor bisindolylmaleimide I (10 microM), the nonspecific protein kinase inhibitor H-7 (50 microM), and were increased by the protein phosphatase inhibitor okadaic acid (1 microM). However, the FMRFamide-induced response was unaffected by the protein kinase A inhibitor H-89 (1 microM), the phosphodiesterase inhibitor theophylline (1 mM), the phospholipase A(2) inhibitor OBAA (0.1 microM) or the cation channel blocker amiloride (100 microM).

Animals↗
PubMed2000

Excitation evoked by FMRFamide and FLRFamide in the heart of Buccinum undatum and evidence for inositol 1,4,5-trisphosphate as an RF-tetrapeptide second messenger.

In this study the relative potencies of four established molluscan cardioexcitatory agents were examined on Buccinum heart. The potencies were, in decending order: phenylalanine-leucine-arginine-phenylalanine-NH2 (FLRFamide) > phenylalanine-methionine-arginine-phenylalanine-NH2 (FMRFamide; 80% of maximum) > 5-hydroxytryptamine (5HT; 60% of maximum) > guanosine triphosphate (GTP; 15% of maximum). FMRFamide and FLRFamide had similar dose-response curve patterns with thresholds at 10(-9) mol l(-1) but FLRFamide was more potent than FMRFamide. The superfused atrium was much less sensitive to all agonists than the internally perfused ventricle. FLRFamide and FMRFamide induced small depolarizations (1-2 mV) which triggered a burst of action potentials of about 5 mV which on reaching 4 mV triggered a burst of fast twitch contractions. Lithium, at high concentrations inhibited FMRFamide and 5-HT responses of internally perfused ventricles. Neomycin also inhibited peptide responses, but was without effect on 5-HT responses. Heparin, however, for technical reasons was without effect on ventricular responses to all three agonists. FMRFamide and FLRFamide appear to share a common receptor, the potency difference being due to the substitution of leucine for methionine in FLRFamide. The RF N-terminal sequence appears crucial for receptor activation. The Phospholipase C inhibitor neomycin equally inhibits responses to the two peptides while 5-HT responses are unaffected. This implicates a peptide/receptor interaction which activated inositol 1,4,5-trisphosphate (IP3) as a second messenger.

Animals↗
PubMedDate not supplied

Phe-Met-Arg-Phe-amide (FMRFamide) stimulated growth hormone secretion in conscious OVX rats.

The presence of FMRFamide (Phe-Met-Arg-Phe-amide)-like immunoreactivity in neuronal elements in the hypothalamus suggested a role for this in the hypothalamic control of the anterior pituitary function. We report here the action of FMRFamide on growth hormone release following intracerebroventricular administration to rats. The injection of 200 ng (313.8 picomoles) of FMRFamide (in 2 ul) produced a significantly increased plasma GH 15 min after injection. The GH-increasing effect of 400-800 ng (627-1255 picomoles) of FMRFamide was already developed after 5 min and lasted up to 30 min. No change was detected in the plasma FSH, LH and prolactin levels at any time during the experimental period. The intravenous administration of 10, 30 or 100 ug of FMRFamide had no effect on the plasma GH level. We conclude that FMRFamide can act at low doses to increase GH release through the inhibition of somatostatin release or the stimulation of GRF. We could not exclude a direct site of action in the pituitaries.

Animals↗
PubMed1995

Spatial and temporal analysis of the Drosophila FMRFamide neuropeptide gene product SDNFMRFamide: evidence for a restricted expression pattern.

The expression of SDNFMRFamide, one of five different FMRFamide-containing peptides encoded by the Drosophila melanogaster FMRFamide gene, has been determined. To study expression, we generated antisera to the N-terminus of SDNFMRFamide to avoid crossreactivity with FMRFamide-containing peptides. The antisera were purified and the specificity characterized. SDNFMRFamide immunoreactive material is present in the central nervous system throughout development. Immunoreactivity is first observed in embryonic neural tissue in a cluster of cells in the subesophageal ganglion and immunoreactive fibers projecting from these cells to the brain and ventral ganglion. This pattern of expression is also observed in neural tissue dissected from larva, pupa, and adult. Double-labelling experiments indicate that cells recognized by SDNFM-antisera are also stained with FMRFamide antisera. Based on position, SDNFMRFamide immunoreactive material is expressed in a limited number of cells that contain the FMRFamide polypeptide precursor. This finding suggests that the Drosophila FMRFamide precursor undergoes differential post-translational processing.

Amino Acid Sequence↗
PubMedDate not supplied

FMRFamide receptors in Helix aspersa.

An in vitro receptor binding assay and an isolated heart bioassay were developed and used to characterize the structure-activity relations (SAR) of FMRFamide receptors in a land snail, Helix aspersa. In the radioreceptor assay, binding of 125I-desaminoTyr-PhenorLeu-Arg-Phe-amide (125I-daYFnLRFamide) at 0 degrees C to Helix brain membranes was reversible, saturable, and specific, with a KD of 14 nM and a Bmax of 85 fmol/mg brain. A lower affinity site was also observed (KD = 245 nM; Bmax = 575 fmol/mg brain). In the heart bioassay, daYFnLRFamide and other FMRFamide analogs increased myocardial contraction force. The SAR of cardiostimulation correlated with the specificity of high affinity 125I-daYFnLRFamide binding to brain and heart receptors. The SAR was also similar to that described for other molluscan FMRFamide bioassays, except for a marked preference for N-blocked analogs. Peptides with N-terminal extensions of desaminoTyr, Tyr, Tyr-Gly-Gly, and acetyl, exhibited the highest potency in both radioligand displacement and cardiostimulation. The endogenous Helix heptapeptide analogs of FLRFamide (pQDP-, NDP, and SDP-FLRFamide) were stimulatory on the heart at low doses, but were inhibitory at moderate to high doses. These peptides were 20 times weaker than FMRFamide in both the brain and heart receptor binding assays, with IC50s about 10 microM. The results suggest that the effects of FMRFamide in Helix are receptor-mediated, and that the heptapeptides do not interact at FMRFamide receptors.

Animals↗
PubMed2000

Comparative immunocytochemical study of FMRFamide neuronal system in the brain of Danio rerio and Acipenser ruthenus during development.

The distribution of FMRFamide-like immunoreactive (ir) neurons and fibers was investigated in the central nervous system of developing zebrafish and juvenile sturgeon (sterlet). Adult zebrafish was also studied. In zebrafish embryos FMRFamide-ir elements first appeared 30 h post-fertilization (PF). Ir somata were located in the olfactory placode and in the ventral diencephalon. FMRFamide-ir fibers originating from diencephalic neurons were found in the ventral telencephalon and in ventral portions of the brainstem. At 48 h PF, the ir perikarya in the olfactory placode displayed increased immunoreactivity and stained fibers emerged from the somata. At 60 h PF, bilaterally, clusters of FMRFamide-ir neurons were found along the rostro-caudal axis of the brain, from the olfactory placode to rostral regions of the ventro-lateral telencephalon. At 60 h PF, numerous ir fibers appeared in the dorsal telencephalon, optic lobes, optic nerves, and retina. Except for ir fibers in the hypophysis at the age of 72 h PF, and a few ir cells in the nucleus olfacto-retinalis (NOR) at the age of 2 months PF, no major re-organization was noted in subsequent ontogenetic stages. The number of stained NOR neurons increased markedly in sexually mature zebrafish. In adult zebrafish, other ir neurons were located in the dorsal zones of the periventricular hypothalamus and in components of the nervus terminalis. We are inclined to believe that neurons expressing FMRFamide originate in the olfactory placode and in the ventricular ependyma in the hypothalamus. On the same grounds, a dual origin of FMRFamide-ir neurons is inferred in the sturgeon, an ancestral bony fish: prior to the observation of ir cells in the nasal area and in the telencephalon stained neurons were noted in circumventricular hypothalamic regions.

Age Factors↗
PubMed2001

Development and distribution of FMRFamide-like immunoreactivity in the toad (Bufo bufo) brain.

By using immunohistochemistry, we studied the development and distribution of the FMRFamide-like immunoreactive (ir) neuronal system in the toad brain during the ontogeny. In addition to this, experimental evidence was provided to show that the rostral forebrain-located FMRFamide neurons originate in the olfactory placode and then migrate into the brain along the olfactory pathway. During early development, within the brain, FMRFamide-ir perikarya first appeared in the periventricular hypothalamus. Later in development, FMRFamide-ir cells were visualized in the rostralmost forebrain simultaneously with similar ir cells in the developing olfactory mucosa. Selective ablation of the olfactory placode(s), prior to the appearance of the first FMRFamide-ir cells in the brain, resulted in the total absence of ir cells in the telencephalon (medial septum and mediobasal telencephalon) of the operated sides(s). The preoptic-suprachiasmatic-infundibular hypothalamus-located FMRFamide-ir neurons were not affected by olfactory placodectomy, arguing that they do not originate in the placode. This result points to the placode as the sole source of such neurons in the rostral forebrain.

Animals↗
PubMed1998

The organization of serotonin-, dopamine-, and FMRFamide-containing neuronal elements and their possible role in the regulation of spontaneous contraction of the gastrointestinal tract in the snail Helix pomatia.

The distribution of serotonin-, tyrosine hydroxylase-, and FMRFamide-immunoreactive neuronal elements, as well as the concentrations of serotonin and dopamine in the different parts of the gastrointestinal tract, were studied in the snail Helix pomatia. The sensitivity of the spontaneous contractions of the alimentary tract to serotonin, dopamine, and FMRFamide was also tested. Serotonin-, tyrosine hydroxylase-, and FMRFamide-immunoreactive elements could be demonstrated in each part of the gastrointestinal tract, but they showed different innervation patterns. Serotonin- and tyrosine hydroxylase-immunoreactive elements were dominant in the submucosal layer, whereas FMRFamide-immunoreactive elements were dominant in both the mucosal and submucosal layers. Tyrosine hydroxylase-immunoreactive elements were confined to the longitudinal muscle trabeculae of submucosa, whereas serotonin-immunoreactive elements were distributed throughout the submucosal layer. No serotonin-immunoreactive cell bodies, but only fibers, could be detected in the gastrointestinal tract, and therefore they represent extrinsic elements. Tyrosine hydroxylase- and FMRFamide-immunoreactive cell bodies represent intrinsic elements of the tract. The occurrence and density of the serotonin- and tyrosine hydroxylase-immunoreactive elements showed significant differences in the different parts of the alimentary tract, in accordance with HPLC assays, which revealed a significant frontocaudal decrease in both the serotonin (from 2.11 to 1.21 pM/mg) and dopamine (from 3.28 to 0.52 pM/mg) contents of the different parts of the alimentary tract. Dopamine at 10(-5) M concentration proved to be effective only on the longitudinal muscles by increasing the tone and frequency of contractions, but was ineffective on the circular muscles. Serotonin affected both the longitudinal and circular muscles. Serotonin at 10(-5) M concentration decreased the tone and increased the frequency of low-amplitude contractions of the longitudinal muscles of the esophagus and the gizzard but increased both the tone and frequency of the crop. Serotonin at 10(-9) M concentration slightly decreased the tone and blocked the contractions of the circular muscles in the crop but at 10(-5) M concentration induced contractions of the circular muscles in the gizzard. FMRFamide at 10(-6) M concentration decreased the tone and was shown to block the contractions of both the longitudinal and circular muscles.

Animals↗
PubMed1998

Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the pattern of peptide expression in single neurons resulting from alternative mRNA splicing of the FMRFamide gene.

MALDI-ToF MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry) has become a fast, reliable and sensitive technique for the identification of neuropeptides in biological tissues. Here, we applied this technique to identified neurons of the cardioregulatory network in the snail Lymnaea that express the FMRFamide gene. This enabled us to study the complex processing of the FMRFamide gene at the level of single identified neurons. In the CNS of Lymnaea, FMRFamide-like and additional peptides are encoded by a common, multiexon gene. Alternate mRNA splicing of the FMRFamide gene leads to the production of two different mRNAs. Type 1 mRNA (exon II) encodes for the tetrapeptides (FLRF/FMRFamide), whereas Type 2 (exons III-V) encodes for the heptapeptides (SDPFLRFamide/GDPFLRFamide). Previous in situ hybridization and immunocytochemical studies indicated that these two transcripts are expressed in the CNS neurons of Lymnaea in a differential and mutually exclusive manner. Two single identified neurons of the cardiorespiratory network, the Ehe neuron and the visceral white interneuron (VWI), were known to express the FMRFamide gene (Ehe, type 1 mRNA; VWI, type 2 mRNA). MALDI-ToF MS analysis of these neurons and other neurons expressing the FMRFamide gene confirmed the mutually exclusive expression of the distinct sets of peptides encoded on the two transcripts and revealed the pattern of post-translational processing of both protein precursors. From the gene sequence it was predicted that 16 final peptide products from the two precursor proteins could possibly exist. We showed that most of these peptides were indeed present in the identified neurons (13) while others were not (three), suggesting that not all of the potential cleavage sites within the two precursors are utilized. In this way, the neuronal expression of the full range of the peptide products resulting from alternative mRNA splicing was revealed for the first time.

Alternative Splicing↗
PubMed1998

Structure of the cDNA encoding the precursor for the neuropeptide FMRFamide in the bivalve mollusc Mytilus edulis.

FMRFamide immunoreactivity is widespread in the tissues of bivalve molluscs, but some of this immunoreactivity may represent distinct related peptides (FaRPs) rather than the exact tetrapeptide FMRFamide. We have cloned the first full-length cDNA encoding the precursor protein for FMRFamide from this class of molluscs to investigate the possibility that additional peptides may be produced. The precursor contains one copy each of NFLRFamide, FLRFamide, ALAGDHFFRFamide and 16 copies of FMRFamide. This precursor is expressed in all three ganglia of the central nervous system. Since the gene encoding the FMRFamide precursor in pulmonate molluscs is alternatively spliced to give two distinct messages, we searched for evidence that the FMRFamide gene of Mytilus is also alternatively spliced. No evidence of alternative splicing was found.

Amino Acid Sequence↗
PubMed1993

Processing of the FMRFamide precursor protein in the snail Lymnaea stagnalis: characterization and neuronal localization of a novel peptide, 'SEEPLY'.

In the pulmonate snail Lymnaea stagnalis, FMRFamide-like neuropeptides are encoded by a multi-exon genomic locus which is subject to regulation at the level of mRNA splicing. We aim to understand the post-translational processing of one resulting protein precursor encoding the tetrapeptide FMRFamide and a number of other putative peptides, and determine the distribution of the final peptide products in the central nervous system (CNS) and periphery of Lymnaea. We focused on two previously unknown peptide sequences predicted by molecular cloning to be encoded in the tetrapeptide protein precursor consecutively, separated by the tetrabasic cleavage site RKRR. Here we report the isolation and structural characterization of a novel non-FMRFamide-like peptide, the 22 amino acid peptide SEQPDVDDYLRDVVLQSEEPLY. The novel peptide is colocalized with FMRFamide in the CNS in a number of identified neuronal systems and their peripheral motor targets, as determined by in situ hybridization and immunocytochemistry. Its detection in heart excitatory motoneurons and in nerve fibres of the heart indicated that the novel peptide may play a role, together with FMRFamide, in heart regulation in the snail. The second predicted peptide, STEAGGQSEEMTHRTA (16 amino acids), was at very low abundance in the CNS and was only occasionally detected. Our current findings, suggestive of a distinct pattern of post-translational processing, allowed the reassessment of a previously proposed hypothesis that the two equivalent sequences in the Aplysia FMRFamide gene constitute a molluscan homologue of vertebrate corticotrophin releasing factor-like peptides.

Amino Acid Sequence↗
PubMed2000

Cloning and expression of a FMRFamide-gated Na(+) channel from Helisoma trivolvis and comparison with the native neuronal channel.

We have cloned a cDNA encoding a Phe-Met-Arg-Phe-NH(2) (FMRFamide)-gated Na(+) channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amino acid sequence of the protein expressed by HtFaNaC is 65 % identical to that of the FMRFamide-gated channel cloned from Helix aspersa (HaFaNaC). HtFaNaC expressed in oocytes was less sensitive to FMRFamide (EC(50) = 70 microM) than HaFaNaC (EC(50) = 2 microM). The two had a similar selectivity for Na+. The amplitude of the FMRFamide response of HtFaNaC was increased by reducing the extracellular concentration of divalent cations. The conductance of the two channels was similar, but the mean open time of unitary events was shorter for expressed HtFaNaC compared to expressed HaFaNaC. Each channel was susceptible to peptide block by high agonist concentrations. In marked contrast to HaFaNaC and other amiloride-sensitive Na(+) channels, amiloride, and the related drugs benzamil and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), enhanced the FMRFamide response in oocytes expressing HtFaNaC cRNA. The potentiating effects of EIPA and benzamil were greater than those of amiloride. Unitary current analysis showed that with such drugs, there was channel blockade as well as an increased probability of channel opening. The similar permeability of the oocyte-expressed HtFaNaC and the Helisoma neuronal channel, and the susceptibility of both to agonist blockade and blockade by divalent cations, suggest that the channels are the same. However, neuronal channels were less susceptible to enhancement by amiloride analogues and in some patches were more sensitive to FMRFamide than expressed HtFaNaC.

Amiloride↗
PubMed1993

Distribution of FMRFamide-immunoreactive nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana in corresponding differential interference-contrast (Nomarski) images.

Immunoreactivities for FMRFamide and substance P (SP) in the carotid labyrinth of the bullfrog were detected using the peroxidase-antiperoxidase method, and the results compared with corresponding differential interference-contrast (Nomarski) images. Colocalization of both peptides was determined by the indirect double immunofluorescence method. Immunoreactivities for FMRFamide and SP were found in nerve fibers distributed in the intervascular stroma of the labyrinth. The FMRFamide-immunoreactive fibers were less numerous than the SP-immunoreactive fibers. In the Nomarski image, FMRFamide-fibers were recognized in relief, with most of them located near the walls of blood vessels. All FMRFamide-fibers coexisted with SP. The results suggest that FMRFamide-immunoreactive fibers are also involved in local vascular regulation of the carotid labyrinth.

Animals↗
PubMed1999

Distribution of FMRFamide-like immunoreactivity in the amphibian brain: comparative analysis.

FMRFamide is a small neuropeptide present in particular neurons of the basal forebrain and midbrain of the vertebrate groups studied, especially fishes and mammals. In order to assess interspecies variation, the distribution of FMRFamide-like immunoreactivity was studied in the brains of 13 species of amphibian. Although FMRFamide-immunoreactive (IR) terminals occurred throughout much of the brain, IR cell groups were noted in circumscribed regions of the CNS. In the eight anuran species studied, two major populations of labeled perikarya were observed: one in the septopreoptic area and another one in the caudal portion of the diencephalon. The rostrocaudal extent of both and the number of labeled somata in each neuronal group displayed species-specific differences. In urodeles and gymnophiones, labeled perikarya were located in the diencephalon, but there were remarkable species differences in the number of such cells. It is discussed whether sex or season of collection may account for some of the differences observed. The distribution of FMRFamide-IR perikarya, fibers, and pathways in the brain of anurans, urodeles, and gymnophiones was compared. The existence of FMRFamide perikarya in the anterior preoptic neuropil and medial septum appeared to be a feature common to all anurans; labeled neurons in the dorsal thalamus, however, may be present only in the (viviparous) gymnophione Typhlonectes compressicauda. Cerebrospinal fluid contacting FMRFamide neuronal cell bodies and fibers were observed in each of the three taxonomic orders. The data are compared with those previously obtained for other groups of vertebrates.

Amphibians↗
PubMed1992

Identified FMRFamide-immunoreactive neuron LPL16 in the left pleural ganglion of Aplysia produces presynaptic inhibition of siphon sensory neurons.

The gill- and siphon-withdrawal reflex of Aplysia undergoes transient inhibition following noxious stimuli such as tail shock. This behavioral inhibition appears to be due in part to transient presynaptic inhibition of the siphon sensory cells, which can be mimicked by application of the peptide FMRFamide. Although FMRFamide is widespread in the Aplysia nervous system, an FMRFamide-containing inhibitory neuron has not previously been identified. We have searched for such a neuron by combining FMRFamide immunofluorescence with fluorescent dye backfilling from the abdominal ganglion, the location of the siphon sensory cells. These methods localized a neuron in the left pleural ganglion, which we have named LPL16. LPL16 is FMRFamide immunoreactive; it is excited by tail shock; and stimulation of LPL16 produces inhibition of siphon sensory cell-to-motor cell postsynaptic potentials and narrowing of action potentials in the sensory cells in tetraethylammonium solution. These results indicate that LPL16 participates in the inhibitory effects of tail shock, and support the idea that FMRFamide plays a physiological role in the inhibition.

Animals↗
PubMed1990

Interspecific comparison of a Drosophila gene encoding FMRFamide-related neuropeptides.

In order to identify functionally important regions of a neuropeptide gene in Drosophila melanogaster, we have studied its occurrence in related species and have characterized the structure of a homologous gene in Drosophila virilis. The melanogaster gene encodes a precursor that contains 13 neuropeptides related to the molluscan tetrapeptide FMRFamide (Nambu et al., 1988; Schneider and Taghert, 1988). Using the melanogaster gene as a probe in Southern blot analysis, related sequences were detected in DNA from each of 7 species tested. D. virilis, which is estimated to have diverged from D. melanogaster between 60 and 80 million years ago (Throckmorton, 1975), was chosen for more detailed study. Immunocytochemical staining using an antibody to authentic FMRFamide revealed a similar set of immunoreactive neurons in the CNS of larvae from the 2 Drosophila species. Using a melanogaster gene probe, overlapping clones were isolated from a virilis genomic library; DNA sequence analysis indicated the presence of a homologous gene. Comparisons of the genes and deduced proteins between the 2 species revealed the following points. (1) Both genes are divided into 2 exons: in D. melanogaster the exons are 106 and 1352 bp long; in D. virilis, they are 169 and at least 1232 bp long; in both species, the intron is approximately 2.5 kb long. (2) The sequence of exon I has largely diverged, and in neither species are exon I sequences translated. In this vicinity of the gene, sequence conservation is limited to a 67 bp region that spans the TATA box and the RNA start site. (3) The deduced neuropeptide precursors have very similar sizes (347 vs 339 amino acids) and the presumed signal sequences are perfectly conserved. (4) While the melanogaster precursor contains 13 FMRFamide-related peptides, the virilis precursor contains only 10. (5) The sequences of some but not all of the FMRFamide-like peptides are perfectly conserved. (6) In the rest of the precursor, significant sequence conservation is found only in the N-terminal portion; immediately downstream of the final FMRFamide-like peptide, the protein sequences are highly divergent. (7) 5' to the RNA start sites (1.2 kb of melanogaster DNA and 1.8 kb of virilis DNA), 17 small (9-52 base pairs) regions are evolutionarily conserved (greater than 80% sequence conservation). We discuss neuropeptide biosynthesis, the functions and evolution of FMRFamide-like neuropeptides in insects, and the cell-specific regulation of neuropeptide gene expression in the contexts of these results.

Animals↗