FMRFamide-immunoreactive structures in the brain of the brown hagfish, Paramyxine atami: relationship with neuropeptide Y-immunoreactive structures.

Localization of the molluscan cardioexcitatory tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2) in the brain and hypophysis of the brown hagfish, Paramyxine atami, was examined by immunohistochemistry specially regarding a possible relationship with neuropeptide Y (NPY). FMRFamide-immunoreactive fibers were demonstrated in many regions of the brain, with the highest density in the diencephalon. However, no immunoreactivity was found in the hypophysis. Labeled cells were chiefly located in the nucleus hypothalamicus of the diencephalon, although a few cells were recognized in the ventrolateral area of the caudal tegmentum. Examination of adjacent sections immunostained alternatively with anti-NPY antiserum and anti-FMRFamide antiserum showed overlapping of the distributional patterns of the immunoreactive structures in the brain. Moreover, the same cells in the nucleus hypothalamicus were immunostained with both antisera. Cross-blocking experiments showed that the FMRF-amide-immunoreactivity is abolished by preabsorption of the antiserum with homologous antigen, but not eliminated completely by pretreatment with appropriate antigens (NPY, avian pancreatic polypeptide and methionine-enkephalin-Arg-Phe). In contrast, the NPY-immunoreactivity was blocked by pretreatment of the antiserum with NPY, pancreatic polypeptide or FMRFamide, although no blocking by enkephalin was observed. Accordingly, the present study shows that, in the brown hagfish, FMRFamide-immunoreactive structures in the brain can be recognized by anti-NPY antiserum.     

Immunohistochemical localization of neuropeptide Y-like substance in the brain and hypophysis of the brown hagfish,Paramyxine atami

The distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the brown hagfish, Paramyxine atami, was examined by use of the peroxidase-antiperoxidase method. Immunoreactive cells were found in two areas of the brain, the nucleus hypothalamicus of the diencephalon and the ventrolateral area of the caudal tegmentum, at the level of the nucleus motorius V–VII. The labeled cells of the nucleus hypothalamicus were loosely grouped and recognized as bipolar neurons. Immunolabeled fibers were widely distributed in the brain, showing the highest density in the diencephalon. They were sparse, or absent, in the olfactory bulb, habenula, primordium hippocampi, neurohypophysis, corpus interpedunculare, and dorsolateral area of the medulla oblongata. The fibers appeared to project exclusively from the ventral hypothalamus to various other portions of the brain: the anterolateral areas of the telencephalon via the basal hypothalamus, the pars dorsalis thalami, the dorsocaudal region of the mesencephalon, and the ventromedial portions of the tegmentum and anterior medulla oblongata. These findings suggest that, in the brown hagfish, NPY-like substance is involved in neuroregulation of various cerebral areas, but it may be of little significance in the control of pituitary function.     

Distribution of FMRFamide-like immunoreactivity in the brain of the elasmobranch fish Scyliorhinus canicula.

The distribution of FMRFamide-like-immunoreactive peptides was investigated in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula using the indirect immunofluorescence technique. FMRFamide-immunoreactive cells and fibers were mainly observed in the telencephalon and the diencephalon, while other brain structures were almost unstained. In the telencephalon, FMRFamide-like-containing neurons were seen in the caudal part of the area periventricularis pallialis, in the posterior area of the nucleus septi medialis and in the nucleus septi caudoventralis. In the diencephalon, numerous FMRFamide-positive cell bodies were observed in the hypothalamus, ventral thalamus and posterior tuberculum. The highest density of immunofluorescent perikarya was found in the nucleus lobi lateralis hypothalami and in the nucleus periventricularis hypothalami. More caudally, the mesencephalon and the caudal brainstem only contained scattered varicose FMRFamide-immunoreactive fibers. Stained fibers were also identified in the median eminence and several FMRFamide-like-positive cells were detected in the dorsal and rostral parts of the neurointermediate lobe of the pituitary. These data indicate that substances related to the molluscan cardioexcitatory peptide FMRFamide are widely distributed in the brain of S. canicula, suggesting their implication in neuroendocrine and/or neuromodulatory functions.     

Immunocytochemical localization of neurons in the nervous system of the Colorado potato beetle with antisera against FMRFamide and bovine pancreatic polypeptide

Summary Particular neurons in the nervous system of the Colorado potato beetle, Leptinotarsa decemlineata, are recognized by antisera against bovine pancreatic polypeptide and FMRFamide. Both antisera react with the same neurons. Solid phase absorptions showed that antiserum against bovine pancreatic polypeptide cross-reacts with FMRFamide, whereas antiserum against FMRFamide cross-reacts with bovine pancreatic polypeptide. Some of the immunoreactive neurons have axons branching extensively within the neuropile, which suggests that the peptide is used as transmitter. In the corpus cardiacum, a neurohaemal organ in insects, numerous immunoreactive axon terminals are present. Here, the peptide material is presumably released as a hormone.     

Distribution of FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the sphinx mothManduca sexta and colocalization with SCPB-, BPP-, and GABA-like immunoreactivity

Summary Using an antiserum against the tetrapeptide FMRFamide, we have studied the distribution of FMRFamide-like substances in the brain and suboesophageal ganglion of the sphinx mothManduca sexta. More than 2000 neurons per hemisphere exhibit FMRFamide-like immunoreactivity. Most of these cells reside within the optic lobe. Particular types of FMRFamide-immunoreactive neurons can be identified. Among these are neurosecretory cells, putatively centrifugal neurons of the optic lobe, local interneurons of the antennal lobe, mushroom-body Kenyon cells, and small-field neurons of the central complex. In the suboesophageal ganglion, groups of ventral midline neurons exhibit FMRFamide-like immunoreactivity. Some of these cells have axons in the maxillary nerves and apparently give rise to FMRFamide-immunoreactive terminals in the sheath of the suboesophageal ganglion and the maxillary nerves. In local interneurons of the antennal lobe and a particular group of protocerebral neurons, FMRFamide-like immunoreactivity is colocalized with GABA-like immunoreactivity. This suggests that FMRFamide-like peptides may be cotransmitters of these putatively GABAergic interneurons. All FMRFamide-immunoreactive neurons are, furthermore, immunoreactive with an antiserum against bovine pancreatic polypeptide, and the vast majority is also immunoreactive with an antibody against the molluscan small cardioactive peptide SCP_B. Therefore, it is possible that more than one peptide is localized within many FMRFamide-immunoreactive neurons. The results suggest that FMRFamide-related peptides are widespread within the nervous system ofM. sexta and might function as neurohormones and neurotransmitters in a variety of neuronal cell types.Abbreviations AL antennal lobe - BPPLI bovine pancreatic polypeptide-like immunoreactivity - FLI FMRFamide-like immunoreactivity - GLI GABA-like immunoreactivity - NSC neurosecretory cell - SCP _B LI small cardioactive peptide_B-like immunoreactivity - SLI serotonin-like immunoreactivity - SOG suboesophageal ganglion     

High affinity displacement of [(3)H]NPY binding to the crude venom of conus anemone by insect neuropeptides.

The venom from Conus anemone contains a protein, named ANPY toxin, which displayed high affinity (IC(50) in nanomolar range) to neuropeptide Y (NPY), [Leu(31), Pro(34)]NPY, peptide YY, pancreatic polypeptide, the Y(1) antagonist 1229U91, and C-terminal NPY fragments. N-terminal fragments and the free acid form of NPY did not bind to ANPY. The truncated NPY fragments displayed very low affinity to Y(1) receptors and partially inhibited [(3)H]NPY binding to anti-NPY antiserum. Several insect neuropeptides, the sequences of which related to the C-terminal fragments of NPY, were observed to bind with similar affinity or even 20 times higher (Lom-MS and Scg-NPF) affinity than NPY. In contrast, no significant binding of these insect peptides was observed for Y(1) receptors and anti-NPY antiserum. Therefore, ANPY can be viewed as an acceptor that binds with very high affinity to a broad spectrum of vertebrate and invertebrate neuropeptides that share a similar C-terminal amino acid sequence.     

Ontogeny of the FMRFamide-immunoreactivity in the rat forebrain and diencephalon

Ontogeny of the FMRFamide (molluscan cardioexcitatory neuropeptide)-containing structures in the forebrain and diencephalon of the rat was investigated by employing immunohistochemical methods. FMRFamide-like immunoreacted (FMRF-IR) fibers first appeared in the borders of the periventricular zone and the preoptic area at embryonic day 18 (E18). Toward birth, the FMRF-IR fibers gradually increased both in immunoreactivity and in number in these areas. A pronounced increase in FMRF-IR was also found in the septum, the arcuate nucleus, the median eminence, the paraventricular nucleus and the amygdaloid complex. A few FMRF-IR fibers appeared at the prenatal stage in the caudate nucleus, the bed nucleus of the stria terminalis, the dorsomedial nucleus and the cortex. The first FMRFamide-immunoreactive neurons were seen in the caudate-putamen and the amygdaloid complex at E21. These FMRF-IR cells increased in immunoreactivity and a significant number of cells was noted in these nuclei in the adult rat. The highest density of FMRF-IR neurons, especially in the amygdala and tuberal hypothalamic area, was detected at postnatal two weeks (P15). FMRFamide-like immunoreactivity in the forebrain and diencephalon appeared in the cell fibers prior to that observed in the cell bodies. This may suggest that some of the immunoreacted fibers may have originated from the lower areas of the rat brain. High densities of FMRF-IR cells present in the embryonic and early postnatal stages may indicate that FMRFamide is an important factor involved in developmental organization of the central nervous system. These results also indicate a differential genesis of FMRF-IR neuronal groups in different regions.     

Co-localization of peptides in the Brockmann bodies of the cod (Gadus morhua) and the rainbow trout (Oncorhynchus mykiss)

Endocrine cells exhibiting immunoreactivity to FMRFamide-like, LPLRFamide-like, neuropeptide Y(NPY)-like and peptide YY(PYY)-like peptides were found in the periphery of the Brockmann bodies of the cod, Gadus morhua, and rainbow trout, Oncorhynchus mykiss. No immunoreactivity or very weak labelling was found with antisera to pancreatic polypeptide (PP). Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was found in nerve fibres, whereas labelling with VIP antiserum in endocrine cells disappeared after preincubation with nonimmune serum. There were always more immunoreactive cells in the rainbow trout than in the cod. No immunoreactivity could be seen with antisera to gastrin/cholecystokinin (CCK) or enkephalin. Double-labelling studies were performed to study the colocalization of the peptides in peripheral endocrine cells. Cells immunoreactive to NPY were also labelled with antisera to FMRFamide, LPLRFamide and PYY. The co-localization pattern of NPY varied; in some Brockmann bodies, a population of the immunoreactive cells showed co-localization and others contained NPY-like immunoreactivity only, whereas in other Brockmann bodies, all NPY-labelled cells also contained FMRFamide-like, LPLRFamide-like and PYY-like immunoreactivity. Cells immunoreactive to PYY similarly contained FMRFamide-like, LPLRFamide-like and NPY-like immunoreactivity, comparable to the patterns observed with NPY. Glucagon-like immunoreactivity was found at the periphery of the Brockmann bodies. A subpopulation of the glucagon-containing cells contained NPY-like immunoreactivity. PYY-like immunoreactivity was also found co-localized with glucagon-like immunoreactivity, as were FMRFamide-like and LPLRFamide-like immunoreactivity. Therefore, either NPY-like and PYY-like immunoreactivity together with FMRFamide-like and LPLRFamide-like immunoreactivity occur in the same endocrine cells of the Brockmann body of the cod and rainbow trout, or a hybrid NPY/PYY-like peptide recognized by both NPY and PYY antisera is present in the Brockmann body.     

Neuronal interactions between neuropeptide Y (NPY) and catecholaminergic systems in the rat arcuate nucleus as shown by dual immunocytochemistry

Several recent studies have suggested interactions between catecholamine (CA) and neuropeptide Y (NPY) neuronal systems in the rat brain. In order to obtain morphological evidence for such CA/NPY interactions in the arcuate nucleus, we have used a double immunostaining procedure using an anti-tyrosine hydroxylase (TH) antiserum as a marker for catecholamine neurons and an anti-NPY antiserum. This double staining, where the first staining is silver-gold intensified, was detectable at both light and electron microscopic levels. In semi-thin sections, a substantial overlap and close proximity of TH-immunopositive neurons and NPY neuronal elements could be seen within the arcuate nucleus. At the electron microscopic level, direct appositions between TH- and NPY-immunoreactive structures could be detected. These appositions were of axosomatic, axodendritic or axoaxonic types without any synaptic membrane differentiation. Moreover, direct appositions between NPY-immunoreactive structures have also been observed. This morphological study showing appositions between TH and NPY neuronal systems suggest direct interactions between these two systems in the arcuate nucleus.     

Identical immunoreactivity of afferents to the rat suprachiasmatic nucleus with antisera against avian pancreatic polypeptide,molluscan cardioexcitatory peptide and neuropeptide Y

Summary Avian pancreatic polypeptide (APP)-like, molluscan cardioexcitatory peptide (FMRF)-like and neuropeptide Y (NPY)-like immunoreactivities were studied in a secondary visual pathway in rat brain. The cell bodies of this pathway are located in the lateral geniculate nucleus and its terminal plexus is found in the suprachiasmatic hypothalamic nucleus (SCN). The neurons and terminal plexus demonstrated by antiserum to each peptide are identical, and immunoreactivity is blocked by preabsorption of each antiserum with a low concentration of the antigen against which it was raised. Immunoreactivity is also blocked by preabsorption of each antiserum with either NPY or APP. In contrast, APP- and NPY-like immunoreactivities are blocked only partially when these antisera are preabsorbed with concentrations of FMRF as high as 100 M. Since NPY is the only one of these peptides that has been isolated from mammalian brain, we conclude that NPY is the endogenous CNS peptide produced by neurons of the lateral geniculate-SCN projection.     

Immunohistochemical localisation of natriuretic peptides in the brains and hearts of the spiny dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa

Summary The avidin-biotin peroxidase technique was used to determine the distribution of natriuretic peptides in the hearts and brains of the dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa. Three antisera were used: one raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide, but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); and the third raised against rat atrial natriuretic peptide (termed rat atrial natriuretic peptide-like immunoreactivity). Only natriuretic peptide-like immunoreactivity was observed in the heart ofS. acanthias which was most likely due to the antiserum cross-reacting with C-type natriuretic peptide. No immunoreactivity was found in theM. glutinosa heart. In the brain ofS. acanthias, natriuretic peptide-like immunoreactive fibres were located in many areas of the telencephalon, diencephalon, mesencephalon, rhombencephalon, and spinal cord. Extensive immunoreactivity was observed in the hypothalamo-hypophyseal tract and the neurointermediate lobe of the hypophysis. Natriuretic peptide-like immunoreactive perikarya were found in ventromedial regions of the telencephalon and in the nucleus preopticus. Most perikarya had short, thick processes which extended toward the ventricle. Another group of perikarya was observed in the rhombencephalon. Porcine brain natriuretic peptide-like immunoreactive fibres were observed in the telencephalon, diencephalon, mesencephalon, and rhombencephalon, but perikarya were only present in the preoptic area. In theM. glutinosa brain, natriuretic peptide-like immunoreactive fibres were present in all regions. Immunoreactive perikarya were observed in the pallium, primordium hippocampi, pars ventralis thalami, pars dorsalis thalami, nucleus diffusus hypothalami, nucleus profundus, nucleus tuberculi posterioris, and nucleus ventralis tegmenti. Procine brain natriuretic peptide-like immunoreactive perikarya and fibres had a similar, but less abundant distribution than natriuretic peptide-like immunoreactive structures. Although the chemical structures of natriuretic peptides in the brains of dogfish and hagfish are unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic peptide or porcine C-type natriuretic peptide. The presence of natriuretic peptides in the brain suggest they could be important neuromodulators and/or neurotransmitters. Furthermore, there appears to be divergence in the structural forms of natriuretic peptides in the hearts and brains of dogfish and hagfish.     

Immunocytochemical distribution of FMRFamide-like substance in the brain of the cloudy dogfish,Scyliorhinus torazame

Summary The distribution of the molluscan cardioexcitatory tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH₂) in the brain of the cloudy dogfish, Scyliorhinus torazame, was examined by immunocytochemistry. FMRFamide-like immunoreactivity was demonstrated to occur extensively in various regions of the dogfish brain, except for the corpus cerebelli. Immunoreactive neuronal perikarya were located in the ganglion of the nervus terminalis, the preoptic area, and the hypothalamic periventricular gray matter consisting of the nucleus medius hypothalamicus, the nucleus lateralis tuberis, and the nucleus lobi lateralis. some of the immunoreactive cells in the hypothalamus were identified as cerebrospinal fluid-contacting neurons. The bulk of the immunostained fibers in the nervus terminalis penetrated into the midventral portion of the telencephalon and ran dorsocaudally toward the basal telencephalon and hypothalamus, showing radial projections or ramifications. The labeled fibers were abundant in the midbasal part of the telencephalon and in the hypothalamus, where some fibers were found in loose networks around the cell bodies of the nucleus septi and hypothalamic periventricular nuclei. The fibers demonstrated in the hypothalamus terminated around the vascular wall of the primary capillary plexus of the median eminence or penetrated deeply into the pars intermedia of the hypophysis. These results suggest that, in the dogfish, an FMRFamide-like substance participates in the regulation of adenohypophysial function. This molecule may have a role as a neurotransmitter and/or neuromodulator in the central nervous system.     

Interrelation between alpha 2-adrenoreceptor system and neuropeptide Y in rat nucleus tractus solitarii.

In several systems, alpha 2-adrenoreceptor agonists and neuropeptide Y (NPY) potentiate one another. We reported recently that NPY is a potent depressor agent in the nucleus tractus solitarii (NTS). The purpose of this study is to investigate the possible modulation of the agonist effect by NPY in this site. Microinjection (60nl) of NPY, anti-NPY antiserum, the alpha 2 agonist alpha-methylnorepinephrine (alpha-MNE), clonidine, and the alpha 2 antagonists idazoxan and yohimbine were made into the NTS. Administration of idazoxan (0.2 nmol) prior to the injection of NPY (2.3 pmol) attenuated the potent depressor and bradycardic effect of NPY. There was a similar attenuation of yohimbine's effect. Similarly, prior administration of the anti-NPY antiserum attenuated the depressor effect of the central antihypertensive agents, alpha-MNE and clonidine, whereas inactivated antiserum or control normal rabbit serum were not able to attenuate these effects. Even a subdepressor dose of NPY (47 fmol) could potentiate the effect of alpha-MNE. These results demonstrate a reciprocal potentiation of NPY and alpha 2 agonists in the brainstem, and suggest that NPY and catecholamines interact in central cardiovascular regulation.     

An FMRFamide antiserum differentiates between populations of antigens in the ventral nervous system of the locust,Schistocerca gregaria

Summary The distribution of FMRFamide immunoreactive neurones in the ventral nerve cord of the locust, Schistocerca gregaria, is described. These neurones are found only in the suboesophagael and thoracic ganglia, although immunoreactive processes are found in the neuropils of the abdominal ganglia. Many of these neurones also react with an antiserum raised against bovine pancreatic polypeptide (BPP), but this antiserum also reveals another population of cells in the abdominal ganglia. The staining obtained with the BPP antiserum is blocked by preabsorption of the antiserum with FMRFamide; the converse is not true: FMRFamide-like immunoreactivity is not suppressed by preincubation with BPP. These results suggest that there are at least two endogenous peptide antigens in the locust nerve cord: one is found in cells of the suboesophageal and thoracic ganglia, and the other is found in cells of the abdominal ganglia.     

FMRF -amide-like immunoreactivity in brain and pituitary of the hagfish Eptatretus burgeri (Cyclostomata)

Summary Paraffin sections of brain and pituitary of the hagfish Eptatretus burgeri were immunostained with an antiserum to FMRF-amide. Immunoreactivity was visible in a large number of neurons in the posterior part of the ventromedial hypothalamus and in long neuronal processes extending cranially from the hypothalamus to the olfactory system and caudally to the medulla oblongata. FMRF-amide-like immunoreactivity was also found in cells of the adenohypophysis. These observations suggest that the hagfish possesses a brain FMRF-amide-like transmitter system and pituitary cells containing FMRF-amide-like material.Antisera to ACTH, -MSH and pancreatic polypeptide gave no immunoreaction in hagfish brain or pituitary. D aspartic acid - F phenylalanine - L leucine - M methionine - R arginine; - W tryptophan - Y tyrosine     

Mapping of neurons in the central nervous system of the guinea pig by use of antisera specific to the molluscan neuropeptide FMRFamide

Summary Immunoreactive neurons were mapped in the central nervous system of colchicine-treated and untreated guinea pigs with the use of two antisera to the molluscan neuropeptide FMRFamide ¹. These antisera were especially selected for their incapability to react with peptides of the pancreatic polypeptide family. Only one group of perikarya was stained by both antisera; this group was mainly located in the nucleus dorsomedialis hypothalami and extended to the nucleus paraventricularis and nucleus periventricularis hypothalami. The perikarya were found to project fibers to all regions of the hypothalamus, to the septum, nucleus proprius striae terminalis, nucleus paraventricularis thalami, nucleus centralis thalami, nucleus reuniens, medial, central and basal amygdala, area praetectalis, area tegmentalis ventralis of Tsai, substantia grisea centralis mesencephali, formatio reticularis mesencephali, nucleus centralis superior, locus coeruleus, nuclei parabrachiales, nucleus raphe magnus, A 5-region, vagus-solitarius complex, ventral medulla, nucleus spinalis nervi trigemini, and substantia gelatinosa of the spinal cord. In many brain regions FMRFamide-immunoreactive processes were found in close contact with blood vessels.Abbreviations of Amino Acids D aspartic acid - F phenylalanine - G glycine - H histidine - L leucine - M methionine - P proline - R arginine - V valine - W tryptophan - Y tyrosine     

Recognition of neuropeptides FMRFamide and LPLRFamide by chicken cerebellum avian pancreatic polypeptide binding sites

Binding isotherms were constructed for the binding of synthetic tetrapeptide and pentapeptide fragments to membranes prepared from chicken cerebellar tissue. Both the tetrapeptide (FMRFamide), which was originally isolated from ganglia of mollusks, and the pentapeptide (LPLRFamide) previously isolated from chicken brain are known to increase blood pressure and modulate brain neurons in rats. The C-terminal dipeptide sequences of the two peptides are identical and both show similarity to the dipeptide sequence established for the pancreatic polypeptide (PP) family. Specific high-affinity binding sites exist for the latter peptide, sites which are competed for (though with less affinity) by neuropeptide Y (NPY). Affinity for cerebellar membranes was virtually equivalent for the synthetic peptide LPLRFamide and FRMFamide; the binding affinities (IC50) of all fragments tested (C-terminal pentapeptides of avian PP and NPY, and FMRFamide and LPLRFamide) fell in the same approximate range. Since the N-terminal residues of FMRFamide and LPLRFamide are not homologous with equivalent residues of APP or NPY, our results indicate that only Arg-Tyr-NH2 or Arg-Phe-NH2 sequences are necessary for binding of the carboxy terminus peptides of the PP family. In this respect, these sequences are functionally equivalent.     

Pancreatic beta cells synthesize neuropeptide Y and can rapidly release peptide co-transmitters

Background

In addition to polypeptide hormones, pancreatic endocrine cells synthesize a variety of bioactive molecules including classical transmitters and neuropeptides. While these co-transmitters are thought to play a role in regulating hormone release little is known about how their secretion is regulated. Here I investigate the synthesis and release of neuropeptide Y from pancreatic beta cells.

Methodology/Principal Findings

NPY appears to be an authentic co-transmitter in neonatal, but not adult, beta cells because (1) early in mouse post-natal development, many beta cells are NPY-immunoreactive whereas no staining is observed in beta cells from NPY knockout mice; (2) GFP-expressing islet cells from an NPY(GFP) transgenic mouse are insulin-ir; (3) single cell RT-PCR experiments confirm that the NPY(GFP) cells contain insulin mRNA, a marker of beta cells. The NPY-immunoreactivity previously reported in alpha and delta cells is therefore likely to be due to the presence of NPY-related peptides. INS-1 cells, a beta cell line, are also NPY-ir and contain NPY mRNA. Using the FMRFamide tagging technique, NPY secretion was monitored from INS-1 beta cells with high temporal resolution. Peptide release was evoked by brief depolarizations and was potentiated by activators of adenylate cyclase and protein kinase A. Following a transient depolarization, NPY-containing dense core granules fused with the cell membrane and discharged their contents within a few milliseconds.

Conclusions

These results indicate that after birth, NPY expression in pancreatic islets is restricted to neonatal beta cells. The presence of NPY suggests that peptide co-transmitters could mediate rapid paracrine or autocrine signaling within the endocrine pancreas. The FMRFamide tagging technique may be useful in studying the release of other putative islet co-transmitters in real time.     

Distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the cloudy dogfish,Scyliorhinus torazame

Summary Using a specific antiserum raised against synthetic neuropeptide Y, we examined the localization of immunoreactivity in the brain and hypophysis of the cloudy dogfish, Scyliorhinus torazame, by the peroxidase-antiperoxidase method. Immunoreactive perikarya were demonstrated in the ganglion of the nervus terminalis, the dorsocaudal portions of the pallium dorsale, the basal telencephalon, and the nucleus lateralis tuberis and the nucleus lobi lateralis in the hypothalamus. Labeled perikarya were also found in the tegmentum mesencephali, the corpus cerebelli, and the medulla oblongata. Some of the immunoreactive neurons in the hypothalamus were of the CSF-contacting type. The bulk of the labeled fibers in the nervus terminalis ran toward the basal telencephalon, showing radial projections and ramifications. Large numbers of these fibers coursed into the nucleus septi caudoventralis and the nucleus interstitialis commissurae anterioris, where they became varicose and occasionally formed fine networks or invested immunonegative perikarya. In the diencephalon, immunoreactive fibers were observed throughout the hypothalamus, e.g., in the pars neurointermedia of the hypophysis, the subependymal layer of the lobus inferior hypothalami, and in the neuropil of the posterior (mammillary) recess organ. Labeled fibers were scattered throughout the rest of the brain stem and were also seen in the granular layer of the cerebellum. These results suggest that, in the dogfish brain, neuropeptide Y or a related substance is involved in a variety of physiological processes in the brain, including the neuroendocrine control of the hypophysis.     

Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon

An immunocytochemical method was used for localization of pancreatic polypeptide (PP) immunoreactive substances in the eyestalk of Penaeus monodon using anti-C-terminal hexapeptide of PP (anti-PP6) antiserum. Approximately 200 neuronal cell bodies were recognized in the ganglia between the medulla interna (MI) and medulla terminalis (MT) and surrounding MT in conjunction with the neuronal processes in medulla externa (ME), MI, MT and sinus gland. About half of the PP immunoreactive neurons were also recognized by a combination of three monoclonal antibodies raised against FMRFamide-like peptides. Isolation of the PP immunoreactive substances from the eyestalk was performed using 7500 eyestalks extracted in methanol/acetic acid/water (90/1/9) followed by five to six steps of RP-HPLC separation. Dot-ELISA with anti-PP6 antiserum was used to monitor PP-like substances in various fractions during the purification processes. Four new sequences of one hexapeptide; RARPRFamide, and three nonapeptides; YSQVSRPRFamide, YAIAGRPRFamide and YSLRARPRFamide were identified, and named as Pem-PYF1-4 due to their structural similarity to the PYF found in squid Loligo vulgaris. Each of the new peptides shares four to seven common residues with the C-terminus of the squid PYF and with the NPFs found in other invertebrates. The NPY/PP superfamily as well as the FMRFamide peptide family may be present throughout vertebrates and invertebrates.