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PubMed2004

Neuropeptidomics of the grey flesh fly, Neobellieria bullata.

A peptidomics approach was applied to determine the peptides in the larval central nervous system of the grey flesh fly, Neobellieria bullata. Fractions obtained by high performance liquid chromatography were analysed by MALDI-TOF and ESI-Q-TOF mass spectrometry. This provided biochemical evidence for the presence of 18 neuropeptides, 11 of which were novel Neobellieria peptides. Most prominently present were the FMRFamide-related peptides: 7 FMRFamides, 1 FIRFamide, and Neb-myosuppressin. The three putative capa-gene products Neb-pyrokinin and the periviscerokinins Neb-PVK-1 and -2 were detected, as well as another pyrokinin. This Neb-PK-2 was also present in the ring gland along with corazonin, Neb-myosuppressin, and Neb-AKH-GK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained Neb-LFamide, proctolin, and FDFHTVamide, designated as Neb-TVamide. With this study, we considerably increased our knowledge of the neuropeptidome of the pest fly N. bullata, which is an important insect model for physiological research.

Amino Acid Sequence↗
PubMed2005

Mass spectrometric analysis of single identified neurons of an insect.

The combination of retrograde labelling with dextran-tetramethylrhodamine and MALDI-TOF mass spectrometry was used to analyse for the first time the peptidome of a series of morphologically identified single neurosecretory cells of an insect. Eight postero-lateral cells of the metathoracic ganglion of the American cockroach, Periplaneta americana, were used to demonstrate that: (1) the complete dissection procedure can be documented and (2) the mass spectrometric analysis of the dissected somata results in highly reproducible mass spectra. In total, 21 FMRFamide-related peptides were detected in each of the postero-lateral cells which release their neurosecretions via thoracic perisympathetic organs. Direct analysis of these neurohemal organs confirmed the co-storage of FMRFamide-related peptides. Two additional abundant peptides from thoracic perisympathetic organs which were not detectable in the postero-lateral cells were characterized using ESI-Q-TOF MS/MS. De novo sequencing yielded two related peptides (FERL/IEamides) without any similarity with known peptide families of insects.

Animals↗
PubMed2006

A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles.

BACKGROUND: At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. RESULTS: To elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. CONCLUSIONS: Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.

Animals↗
PubMed2003

Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab, Cancer borealis.

Mass spectrometric methods were applied to determine the peptidome of the brain and thoracic ganglion of the Jonah crab (Cancer borealis). Fractions obtained by high performance liquid chromatography were characterized using MALDI-TOF MS and ESI-Q-TOF MS/MS. In total, 28 peptides were identified within the molecular mass range 750-3000Da. Comparison of the molecular masses obtained with MALDI-TOF MS with the calculated molecular masses of known crustacean peptides revealed the presence of at least nine allatostatins, three orcokinin precursor derived peptides, namely FDAFTTGFGHS, [Ala(13)]-orcokinin, and [Val(13)]-orcokinin, and two kinins, a tachykinin-related peptide and four FMRFamide-related peptides. Eight other peptides were de novo sequenced by collision induced dissociation on the Q-TOF system and yielded AYNRSFLRFamide, PELDHVFLRFamide or EPLDHVFLRFamide, APQRNFLRFamide, LNPFLRFamide, DVRTPALRLRFamide, and LRNLRFamide, which belong to the FMRFamide related peptide family, as well as NFDEIDRSGFA and NFDEIDRSSFGFV, which display high sequence similarity to peptide sequences within the orcokinin precursor of Orconectes limosus. Our paper is the first (neuro)peptidomic analysis of the crustacean nervous system.

Animals↗
PubMed1997

Distribution of centrifugal neurons targeting the soma clusters of the olfactory midbrain among decapod crustaceans.

To determine the distribution of two systems of centrifugal neurons innervating the soma clusters of the olfactory midbrain across decapod crustaceans, brains of the following nine species comprising most infraorders were immunostained with antibodies against dopamine and the neuropeptides substance P and FMRFamide: Macrobrachium rosenbergii, Homarus americanus, Cherax destructor, Orconectes limosus, Procambarus clarkii, Astacus leptodactylus, Carcinus maenas, Eriocheir sinensis and Pagurus bernhardus. One system consisting of several neurons with dopamine-like immunoreactivity that originate in the eyestalk ganglia was present in the four crayfish but not in any other species. These neurons project mainly into the lateral soma clusters (cluster 10) comprising the somata of ascending olfactory projection neurons and innervate very sparsely the medial soma clusters (clusters 9 and 11) containing the somata of local interneurons. In the innervation pattern of the lateral cluster, the dopamine-immunoreactive neurons showed large species-specific differences. The other system comprises a pair of giant neurons with substance P-like immunoreactivity. These neurons have somata in the median protocerebrum of the central brain and major projections into the lateral clusters and the core of the olfactory lobes, the neuropils that are the first synaptic relay in the central olfactory pathway of decapods; minor arborizations are present in the medial clusters. The system of substance P-immunoreactive giant neurons was present and of great morphological similarity in all studied species. Only in one species, the shrimp Macrobrachium rosenbergii, evidence for co-localization of FMRFamide-like with substance P-like immunoreactivity in these neurons was obtained. These and previously collected data indicate that the centrifugal neurons with dopamine-like immunoreactivity may be associated with the presence of an accessory lobe, a second-order neuropil that receives input from the olfactory lobe and only occurs in spiny lobsters, clawed lobsters and crayfish. The pair of centrifugal giant neurons with substance P-like immunoreactivity, on the other hand, appears to be a constitutive component of the decapod crustacean brain that most likely is functionally associated with the olfactory lobe. Both systems apparently exert modulatory functions on olfactory information processing by preferentially targeting the somata of the projection neurons. Thus, in the olfactory projection neurons, the somata seem to be more directly involved in information processing than in most other neurons of the arthropod CNS.

Animals↗
PubMed1998

The muscular contractions of the midgut of the cockroach, Diploptera punctata: effects of the insect neuropeptides proctolin and leucomyosuppressin.

We have previously shown differential expression of leucomyosuppressin (LMS) mRNA in apparent endocrine cells in the anterior region of midguts of the cockroach Diploptera punctata, using in situ hybridization. In contrast, other FMRFamide-related peptides, as revealed by immunohistochemistry, have been found most abundantly in the posterior region in both apparent endocrine cells and nerve tracts. Here, we partially purified extracts of anterior and posterior cockroach midguts, using HPLC coupled with radioimmunoassay, and found, among multiple FMRFamide-like immunoreactive fractions, one fraction co-eluting with LMS in both regions. The presence of a co-eluting fraction in the posterior region, in the absence of LMS mRNA positive endocrine cells suggests that LMS might therefore be present in nerve tracts running along the length of the midgut. Using a circular muscle contraction assay from different portions of midgut, we determined the effects of LMS, proctolin and a variety of other midgut peptides on contractions of the midgut of Diploptera. Proctolin caused a sustained tonic contraction in the anterior midgut, the amplitude of which was dose-dependent. In contrast, LMS, and its relative SchistoFLRFamide, reduced the amplitude of these contractions. LMS and SchistoFLRFamide also inhibited spontaneous phasic contractions, which were elicited by proctolin application in only a few preparations. Other postulated midgut peptides did not induce or inhibit contractions, nor augment the proctolin-induced contractions. The C-terminal truncated sequences of LMS, HVFLRFamide and VFLRFamide, were sufficient to reduce the amplitude of the proctolin-induced contractions. This work illustrates a possible physiological role for LMS in Diploptera midguts, in the passage of food along the alimentary canal.

Animals↗
PubMed2002

The terminal nerve ganglion cells project to the olfactory mucosa in the dwarf gourami.

Single- and double-label immunocytochemical studies were conducted using antisera to salmon gonadotropin-releasing hormone (sGnRH) and molluscan cardioexcitatory peptide (FMRFamide) to determine whether terminal nerve ganglion cells project to the olfactory mucosa in the dwarf gourami, Colisa lalia. Both peptides were present in terminal nerve ganglion perikarya and fibers in brain and nasal cavity. Labeled fibers were present in the olfactory nerve and could be traced to the olfactory mucosa. All terminal nerve ganglion cells contained both sGnRH and FMRFamide-like peptides. This study suggests that the terminal nerve ganglion cells can influence both brain and chemoreceptive structures.

Animals↗
PubMed1996

Receptor inactivation by dye-neuropeptide conjugates: 2. Characterization of the quantum yield of singlet oxygen generated by irradiation of dye-neuropeptide conjugates.

Different neuropeptide analogues of the neurotransmitter FMRFamide were covalently attached to a tethered dye, forming dye-neuropeptide conjugates capable of stably binding to the FMRFamide receptors. Singlet oxygen (1 delta O2) generated by laser irradiation of the conjugates bound to this receptor should inactivate it if (a) the distance 1 delta O2 must diffuse to reach the photo-sensitized receptor is less than 1000 A, (b) the conjugate binds the receptor with the same affinity as the indigenous neurotransmitter, and (c) the quantum yield (phi) of 1 delta O2 is sufficient. Previous studies determined that the first two constraints are satisfied. The results of the present study confirm that the third constraint is also satisfied, as the phi of 1 delta O2 resulting from the laser irradiation of the conjugates were uniformly large, exceeding those for the dye itself, ranging from 0.25 at pD 6.0 to 0.93 at pD 9.0.

Animals↗
PubMed2000

Immunohistochemical cell types in the terminal nerve ganglion of the cloudy dogfish, Scyliorhinus torazame, with special regard to neuropeptide Y/FMRFamide-immunoreactive cells.

Previous studies showed immunoreactivities for neuropeptide Y (NPY), molluscan cardioexcitatory tetrapeptide (FMRFamide), and gonadotropin-releasing hormone (GnRH) in the terminal nerve of elasmobranchs. The present immunohistochemical study demonstrated two types of cells, i.e. GnRH- and NPY/FMRFamide-positive cells, in the terminal nerve ganglion of the elasmobranch Scyliorhinus torazame. The second cell type (non-GnRH element) contained a substance with a common structure or epitope recognized by anti-NPY and anti-FMRFamide antibodies. The NPY/FMRFamide-like immunoreactivity was associated with granules 70-130 nm in diameter, found in the cell bodies, axons, and axon endings.

Animals↗
PubMed1999

Centrifugal Phe-Met-Arg-Phe-NH2-like immunoreactive innervation of the retina in a non-teleost bony fish, Lepisosteus osseus.

In all teleosts studied, Phe-Met-Arg-Phe-NH2- (FMRFamide-) like immunoreactive fibers originating from structures related to the olfactory system project to the retina. A complete report on this olfacto-retinalis projection in fish that are phylogenetically older than teleosts is still missing. We have visualized FMRFamide-like immunoreactive fibers in the optic nerve, the optic chiasm and in the retina of the longnose gar, Lepisosteus osseus. They terminate on amacrine or horizontal cells in the internal nuclear layer. Additionally, some of them appear to contact retinal ganglion cells.

Animals↗
PubMed1999

Distinct responses of osphradial neurons to chemical stimuli and neurotransmitters in Lymnaea stagnalis L.

1. In Lymnaea stagnalis L. (Pulmonata, Basommatophora) the neurons in the osphradium were visualized by staining through the inner right parietal nerve by 5,6-carboxyfluorescein (5,6-CF). Three types of neurons were identified: three large ganglionic cells (GC1-3; 80-100 microns), the small putative sensory neurons (SC; 20 microns) and very small sensory cells (3-5 microns). 2. The ganglionic and putative sensory neurons were investigated by whole cell patch-clamp method in current-clamp condition. The three giant ganglionic neurons (GC1-3) located closely to the root of osphradial nerve, had a membrane potential (MP) between -30 and -70 mV and showed tonic or bursting activities. The small putative sensory cells (SCs) scattered throughout the osphradial ganglion, possessed a MP between -25 and -55 mV and showed an irregular firing pattern with membrane oscillations. At resting MP the GC1-3 cells were depolarized and increased the frequency of their firing, while the SCs were hyperpolarized and inhibited by NaCl (10(-2) M) and L-aspartate (10(-5) M) applied to the osphradium. 3. 5-Hydroxytryptamine (5HT, 10(-6) M), gamma-aminobutyric acid (GABA; 10(-6) M) and the GABAB agonist baclofen (10(-6) M) depolarized the neurons GC1-3 and increased their firing frequency. In contrast, on the GC1-3 neurons, acetylcholine (Ach; 10(-6) M) and FMRFamide (10(-6) M) caused hyperpolarization and cessation of the firing activity. The 5HT effect was blocked by mianserin (10(-6) M) but picrotoxin (10(-5) M) failed to block the GABA-induced effect on the GC1-3 cells. 4. The small putative sensory neurons (SCs) were excited by Ach (10(-6) M) and 5HT (10(-6) M) but were inhibited by GABA (10(-6) M). FMRFamide (10(-6) M) had a biphasic response. The Ach effect was blocked by hexamethonium (10(-6) M) and tetraethylammonium (10(-6) M), indicating the involvement of nicotinic cholinergic receptors. 5. The distinct responses of the two populations of osphradial neurons to chemical stimuli and neurotransmitters suggest that they can differently perceive signals from environment and hemolymph.

Acetylcholine↗
PubMed1989

Direct modulation of Aplysia S-K+ channels by a 12-lipoxygenase metabolite of arachidonic acid.

Lipoxygenase metabolites of arachidonic acid have recently been shown to modulate the activity of ion channels in nerve and muscle cells. The mechanism of action of these metabolites is, however, unknown. In sensory neurons of Aplysia, the S-K- channel is under the dual modulatory control of 5-hydroxytryptamine (5-HT), which decreases the number of active S channels through cyclic AMP-dependent phosphorylation, and the neuropeptide FMRFamide, which increases the probability of S-channel opening through the 12-lipoxygenase metabolite 12-hydroperoxyeicosatetraenoic acid (12-HPETE). Here we report that the increase in the probability of S-channel opening with FMRFamide is mimicked by application of 12-HPETE to cell-free membrane patches that lack ATP and GTP. Thus, 12-HPETE can act directly to modulate S-channel activity, independently of protein phosphorylation or dephosphorylation, G-protein activation or cyclic nucleotides.

Adenosine Triphosphate↗
PubMed1999

Antisera to multiple antigenic peptides detect neuropeptide processing.

Peptides act as critical messengers of essential physiological function. Frequently, several peptides are encoded in the same precursor and, often, there is structure relatedness among the gene products. The complexity of protein precursors and presence of homologous peptides raises issues about regulation of gene expression and function of structurally-related peptides. We have determined the cellular location of DPKQDFMRFamide and SDNFMRFamide encoded in the Drosophila FMRFamide gene. We raised antisera that distinguish between the two peptides and conducted double label immunostaining utilizing antisera raised in the same host species. We found that DPKQDFMRFamide and SDNFMRFamide are present in distinct distribution patterns. We also established that the peptides are present in cells stained by FMRFamide antisera. Thus, our data are consistent with the conclusion that Drosophila contains cell-specific proteolytic processing enzymes capable of posttranslationally cleaving a polypeptide protein precursor to yield unique expression patterns of neuropeptides that may have diverse activities.

Animals↗
PubMed1991

Age-related differential expression of neuropeptide mRNAs in Aplysia.

The basis of aging in the Aplysia nervous system is unknown. We now report age-related changes in mRNA expression of phe-met-arg-phe-NH2 (FMRFamide) and egg-laying hormone (ELH) in the abdominal ganglion, a part of the CNS, in young, mature and old Aplysia. Northern blot analysis revealed two mRNA species of 1.4 and 3.2 kb for RMRFamide and a single mRNA species of 0.8 kb for ELH. FMRFamide mRNA level increased 1.5-fold from young to mature and then decreased 3-fold in old animals. ELH mRNA gradually increased between young and mature animals and then escalated 25-fold in old animals. Age differentially affected the mRNA of these two peptides, which may contribute to behavioral changes previously reported.

Aging↗
PubMed1999

Neuropeptide gene families in the nematode Caenorhabditis elegans.

Neuropeptides have diverse roles in the function and development of the nervous system. With the completion of the sequencing of the C. elegans genome, rapid identification of nematode neuropeptide genes is possible. To date, 41 C. elegans neuropeptide genes have been identified. Of these genes, 20 genes, named flp (FMRFamide-like peptide) genes, encode FMRFamide-related proteins (FaRPs). Deletion of one of the flp genes, flp-1, results in several behavioral defects, suggesting that at least one flp gene is not functionally redundant with other flp genes. Twenty-one genes, named neuropeptide-like protein (nlp) genes, encode peptides distinct from the FaRP family. The predicted nlp-1 and nlp-2 neuropeptides have modest similarity to buccalin and myomodulin, respectively. Cellular expression patterns and genetic analysis of flp and nlp genes suggest that neuropeptides in nematodes also have widespread and varied roles in nervous system function.

Amino Acid Sequence↗
PubMed1999

Comparison of FaRP immunoreactivity in free-living nematodes and in the plant-parasitic nematode Heterodera glycines.

The family of FMRFamide-related peptides (FaRPs) is widely distributed among invertebrates, where the peptides serve as neuromodulators. Published reports indicate that numerous FaRP sequences exist in free-living and animal parasitic nematodes. Using a FMRFamide ELISA, FaRP immunoreactivity was detected in extracts of the soybean cyst nematode, Heterodera glycines, in both sexes and at all developmental stages. HPLC-ELISA results revealed a number of immunoreactive components in H. glycines preparations, and a comparison with extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus showed significant qualitative differences in FaRP immunoreactivity between the plant parasite and the two free-living nematodes. Total and specific immunoreactivities varied during H. glycines development, with the highest specific activity in juveniles and males, and the highest total activity in mature females. Total female immunoreactivity was located primarily within the mature eggs. A significant portion, however, was associated with the female body, perhaps with egg laying.

Animals↗
PubMed1998

Disruption of a neuropeptide gene, flp-1, causes multiple behavioral defects in Caenorhabditis elegans.

Neuropeptides serve as important signaling molecules in the nervous system. The FMRFamide (Phe-Met-Arg-Phe-amide)-related neuropeptide gene family in the nematode Caenorhabditis elegans is composed of at least 18 genes that may encode 53 distinct FMRFamide-related peptides. Disruption of one of these genes, flp-1, causes numerous behavioral defects, including uncoordination, hyperactivity, and insensitivity to high osmolarity. Conversely, overexpression of flp-1 results in the reciprocal phenotypes. On the basis of epistasis analysis, flp-1 gene products appear to signal upstream of a G protein-coupled second messenger system. These results demonstrate that varying the levels of FLP-1 neuropeptides can profoundly affect behavior and that members of this large neuropeptide gene family are not functionally redundant in C. elegans.

Animals↗
PubMed1999

Differential modulation of motor neurons that innervate the same muscle but use different excitatory transmitters in aplysia.

The medial portion of intrinsic buccal muscle 3 (I3m) is innervated by two excitatory motor neurons, B3 and B9. B3 uses glutamate as its fast transmitter and expresses the neuropeptide FMRFamide, whereas B9 uses acetylcholine as its fast transmitter and expresses the neuropeptide SCP. This preparation was used to study peptidergic modulation of muscles innervated by neurons that use different fast excitatory transmitters. First, we determined the effects of the application of the neuropeptides expressed in these neurons on excitatory junction potentials (EJPs) and contractions. FMRFamide increased the amplitude of EJPs and contractions evoked by B3 while decreasing those evoked by B9. This is the first observation in buccal muscle of a substance that modulates two excitatory neurons innervating the same muscle in opposite directions. SCP increased EJPs contraction amplitude, and the rate of muscle relaxation for both motor neurons. We determined that SCP potently increased cAMP levels in I3m as it does in other buccal muscles. Stimulation of B9 also caused increased cAMP levels in I3m providing independent evidence for SCP release. Finally, stimulation of B9 increased both the contraction amplitude and relaxation rate of B3-evoked I3m contractions in a manner similar to that observed using exogenous SCP. By inhibiting B9's cholinergic transmission with an antagonist, we were able to observe modulatory effects of B9 in the absence of fast excitatory effects. We found that the magnitude of the modulation was dependent on the firing frequency and did occur at frequencies and patterns of firing recorded previously for B9 during ingestive-like motor programs.

Acetylcholine↗