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.     

Immunocytochemical distribution of neuropeptide F (NPF) in the gastropod mollusc,Helix aspersa,and in several other invertebrates

The distribution of neuropeptide F (NPF) immunoreactivity in the snail, Helix aspersa, has been demonstrated by immunocytochemistry using 2 regionspecific antisera. One, designated NPF3, was raised against a synthetic N-terminal fragment of Helix aspersa NPF; the other, designated PP221, was raised against the C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) but cross-reacts fully with the analogous C-terminal region of Helix aspersa NPF. The distribution of NPF immunoreactivity has also been compared with that of FMRFamide using alternate serial sections of Helix aspersa ganglia. Results showed that NPF immunoreactivity was abundant and widespread in the central and peripheral nervous systems and the pattern of immunostaining obtained using both region-specific antisera was similar. Likewise, immunocytochemistry of neural tissues of a congeneric species, Helix pomatia, and 2 prosobranch gastropods, Buccinum undatum and Littorina littorea, produced similar staining patterns with both antisera. However, in the cephalopod mollusc, Loligo vulgaris, and the cestode, Moniezia expansa, positive immunostaining was only obtained with the C-terminal PP antiserum. Immunostaining of alternate serial sections of Helix aspersa ganglia with NPF3, and an antiserum raised to FMRFamide, showed that while a few neurones were immunoreactive with one antiserum only, in the majority, both immunoreactivities were co-localised. NPF thus appears to be an important neuropeptide of widespread distribution in Helix aspersa and the differential immunocytochemical staining obtained using the 2 region-specific antisera would suggest a high degree of primary structural conservation within the gastropod molluscs, but lack of conservation of the N-terminal region of the peptide in other invertebrate groups.     

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     

Segregation of FMRF amide-immunoreactive efferent fibers from NPY-immunoreactive amacrine cells in goldfish retina

Summary Immunocytochemical studies were conducted on goldfish to determine whether a retinal efferent fiber system, immunoreactive to the tetrapeptide Phe-Met-Arg-Phe-NH₂ (FMRFamide), might contain instead a substance similar to one of the 36-amino acid pancreatic polypeptides, the C-terminus of which is similar to FMRFamide.Our results demonstrate the presence of two separate peptidergic systems, one containing FMRFamide-like, and the other pancreatic polypeptide-like peptides. Antisera to FMRFamide reveal the efferent fibers, whose axons exit the optic nerve and terminate in layer 1 of the inner plexiform layer, as previously described. Antisera to porcine neuropeptide Y, and to avian and bovine pancreatic polypeptides label a sparse population of putative amacrine cell bodies and a dense fiber plexus in layers 1, 3, and 5 of the inner plexiform layer. Based on intensity of staining, this amacrine cell peptide appears to be most similar to neuropeptide-Y.Radioimmunoassay and immunocytochemical staining of retinas in which the efferent fiber peptide was depleted by optic nerve crush confirm in large part the observation that the two peptide systems are distinct. However, there is some cross-recognition of the FMRFamide-like tissue antigen by pancreatic polypeptide antibodies.Double-label studies with antisera to tyrosine hydroxylase and neuropeptide-Y indicate that the pancreatic polypeptide antigen is not co-localized with catecholamines.     

An FMRFamide antiserum differentiates between populations of antigens in the brain and retrocerebral complex of the locust,Schistocerca gregaria

Summary The distribution of FMRFamide-irmunoreactive cell bodies in the brain and retrocerebral complex of the locust, Schistocerca gregaria, is described. Most of the immunoreactive cell bodies are found in the pars intercerebralis and in the optic lobes. Many, but not all, of the cell bodies also react with an antiserum raised against bovine pancreatic polypeptide, but this antiserum also reveals another population of cells that stain selectively with this antiserum. In addition to the cell bodies, numerous immunoreactive processes are revealed by both antisera in neuropilar regions of the brain. The results of blocking experiments suggest that a differential distribution of three locust antigens can be determined from the examination of alternate serial sections stained with the two antisera used.     

An identified dorsal unpaired median neurone and bilaterally projecting neurones exhibiting bovine pancreactic polypeptide-like/FMRFamide-like immunoreactivity in abdominal ganglia of the migratory locust

Summary Three antisera were used to study the distribution and anatomy of bovine pancreatic polypeptide (BPP)-like/FMRFamide-like immunoreactive neurones within the unfused abdominal ganglia of the migratory locust, Locusta migratoria. All the antisera used stained two or more clusters of perikarya, localized anteriorly and posteriorly near the midline within each unfused abdominal ganglion. Double labelling experiments with intracellular dye injection, or differential backfilling, combined with subsequent immunostaining were carried out to identify these neurones. Two of the antisera (antisera 1 and 2, both raised against FMRFamide) stained three groups of midline neurones, located anterior dorsal, anterior ventral and posterior dorsal within the ganglion. Neurones of the former of these two clusters projected via the anterior median nerve to a neurohaemal organ. The posterior cluster of midline cells comprised immunopositive perikarya all but one of which also projected via the anterior median nerve to innervate the neurohaemal organ. Double labelling with Lucifer yellow and antisera 1 and 2 showed that the remaining neurone was the previously identified doral unpaired median (DUM)heart1 neurone. The third antiserum (AK141), also raised against FMRFamide, stained neurones within an anterior dorsal cluster, and in a posterior cluster. Double labelling with differential Co²⁺/Ni²⁺-backfilling and the antiserum 3 (AK141) demonstrated that the large neurones of both clusters belonged to the population of bilaterally projecting neurones (BPNs), including the DUMheart1 neurone. Since the antisera cross-react with BPP and fail to label neurones when preadsorped with BPP or FMRFamide, we conclude that the labelled neurones contain polypeptides of the FMRFamide/BPP-family.     

Postembryonic development of Arg-Phe-amide-like and cholecystokinin-like immunoreactive neurons in the blowfly optic lobe

Summary The adult optic lobes of the blowfly Calliphora erythrocephala were found to be innervated by more than 2000 neurons immunoreactive to antisera raised against the neuropeptides FMRFamide, its fragment RFamide, and gastrin/cholecystokinin (CCK). All of the CCK-like immunoreactive (CCK-IR) neurons also reacted with antisera to RFamide, FMRFamide and pancreatic polypeptide. A few RFamide/FMRFamide-like immunoreactive (RF-IR) neurons did not react with CCK antisera; they reacted instead with antisera to Leu-enkephalin and Met-enkephalin-Arg⁶-Phe⁷. The RF-IR neurons are, thus, heterogeneous with respect to their contents of immunoreactive peptides. Two of the RF-IR neuron types innervating the adult optic lobes could be traced in their entirety only after following their postembryonic development, because of the complexity of the trajectories of the immunoreactive neuronal process in the adult insect. The majority of the cell bodies of the RF-IR and CCK-IR neurons lie within the optic lobes and are derived from imaginal neuroblasts of the inner and outer optic anlagen. Six of the peptidergic neurons are, however, metamorphosing larval neurons with their cell bodies in the central part of the protocerebrum. The full extent of immunoreactivitiy is not attained in some of the neurons until the late pupal or early adult stage. The larval optic center was also found to be innervated by neurons immuno-reactive with both RFamide and CCK antisera. The cell bodies of these RF-IR/CCK-IR neurons are located near the developing lamina (one on each side). In the 24 h pupa, the cell bodies of these neurons are still immunoreactive, but thereafter they cannot be immunolabeled apparently due to cell death or a change in transmitter phenotype.     

Immunocytochemical localization of peptidergic cells in the neuro-endocrine system of the Colorado potato beetle,Leptinotarsa decemlineata,with antisera against vasopressin,vasotocin and oxytocin

Summary Antisera against vasopressin, vasotocin, oxytocin, neurophysin-1 and neurophysin-2 were used to investigate immunocytochemically the presence of neurons containing substances antigenically related to these peptides in the nervous system of the Colorado potato beetle. Ten different antisera were used, four against vasopressin, three against oxytocin and one against vasotocin, neurophysin-1, and neurophysin-2. Immunoreactivity was shown by all antisera except those against the neurophysins. The vasopressin antisera all gave different results. One antiserum revealed only a single neuron pair, whereas others revealed in addition one or two other different cell groups. The oxytocin antisera likewise revealed different neurons. The fixation procedure influenced the outcome of the immunocytochemical reaction. Immunoreactivity as revealed by vasopressin, vasotocin and oxytocin antisera is often co-localized in the same neurons; solid phase adsorptions showed that this is due to cross-reactivity of the antisera. Some of the immunoreactive neurons are identical to those recently described to contain a bovine pancreatic polypeptide/FMRFamide-like peptide. This co-localization is probably not due to a cross-reaction. These findings indicate the presence of several vasopressin-like and oxytocin-like substances which in the Colorado potato beetle all have a different degree of immunocytochemical resemblance to vasopressin and oxytocin.     

Immunocytochemical localization of peptidergic cells in the neuro-endocrine system of the Colorado potato beetle, Leptinotarsa decemlineata, with antisera against vasopressin, vasotocin and oxytocin

Antisera against vasopressin, vasotocin, oxytocin, neurophysin-1 and neurophysin-2 were used to investigate immunocytochemically the presence of neurons containing substances antigenically related to these peptides in the nervous system of the Colorado potato beetle. Ten different antisera were used, four against vasopressin, three against oxytocin and one against vasotocin, neurophysin-1, and neurophysin-2. Immunoreactivity was shown by all antisera except those against the neurophysins. The vasopressin antisera all gave different results. One antiserum revealed only a single neuron pair, whereas others revealed in addition one or two other different cell groups. The oxytocin antisera likewise revealed different neurons. The fixation procedure influenced the outcome of the immunocytochemical reaction. Immunoreactivity as revealed by vasopressin, vasotocin and oxytocin antisera is often co-localized in the same neurons; solid phase adsorptions showed that this is due to cross-reactivity of the antisera. Some of the immunoreactive neurons are identical to those recently described to contain a bovine pancreatic polypeptide/FMRFamide-like peptide. This co-localization is probably not due to a cross-reaction. These findings indicate the presence of several vasopressin-like and oxytocin-like substances which in the Colorado potato beetle all have a different degree of immunocytochemical resemblance to vasopressin and oxytocin.     

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.     

FMRFamide-like immunoreactivity in the metathoracic ganglion of the locust (Schistocerca gregaria)

Summary The distribution of FMRFamide-like immunoreactivity in the metathoracic ganglion of the locust, Schistocerca gregaria, has been investigated in serial semithin transverse sections with the use of the peroxidase-antiperoxidase (PAP) technique. The topographical distribution of approximately 120 immunopositive neurons was established. Antiserum against bovine pancreatic polypeptide (BPP) stains the same ganglionic cells as FMRFamide-antiserum, yet this staining is largely blocked after preabsorption to FMRFamide. A comparison of these results with those from other studies suggests that there may be more than one type of endogenous RFamide-like peptide.     

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.     

Distribution of SchistoFLRFamide-like immunoreactivity in the adult ventral nervous system of the locust,Schistocerca gregaria

SchistoFLRFamide (PDVDHVFLRF-NH₂) is one of the major endogenous neuropeptides of the FMRF-amide family found in the nervous system of the locust,Schistocerca gregaria. To gain insights into the potential physiological roles of this neuropeptide we have examined the distribution of SchistoFLRFamide-like immunoreactivity in the ventral nervous system of adult locusts by use of a newly developed N-terminally specific antibody. SchistoFLRFamide-like immunoreactivity in the ventral nerve cord is found in a subgroup of the neurones that are immunoreactive to an antiserum raised against bovine pancreatic polypeptide (BPP). In the suboesophageal ganglion three groups of cells stain, including one pair of large posterior ventral cells. These cells are the same size, in the same location in the ganglion and have the same branching pattern as a pair of BPP immunoreactive cells known to innervate the heart and retrocerebral glandular complex of the locust. In the thoracic and abdominal ganglia two and three sets of cells, respectively, stain with both the SchistoFLRFamide and BPP antisera. In the abdominal ganglia the immunoreactive cells project via the median nerves to the intensely immunoreactive neurohaemal organs.     

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

Summary 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 immuno-histochemistry 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 FMRFamide-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.     

FMRFamide- and pancreatic polypeptide-like immunoreactivity of endocrine cells in the midgut of a mosquito

Immunocytochemical surveys of midguts from female mosquitoes, Aedes aegypti, reveal that half of the estimated 500 endocrine cells in a midgut contain a substance recognized by antisera to bovine pancreatic polypeptide and a molluscan peptide, FMRFamide (phenylalanine-methionine-arginine-phenylalanine-amide). With light microscopy the cells resemble an endocrine type because of their basal position in the epithelium, conical shape, and, in some instances, apical extensions to the lumen. At the ultrastructural level, the immunoreactive substance is contained specifically within the secretory granules of such cells. Immunoreactive cells are distributed exclusively in the midgut region where blood is stored, and ingestion of vertebrate blood reduces the number of such cells and the intensity of reaction in others. These two facts suggest that a blood meal stimulates release of the immunoreactive substance from the cells. Since the immunocytochemical localization is supplemented by a demonstrated secretory response, the cells are considered to be peptidergic endocrine cells.     

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.     

Immunohistochemical investigations of neuropeptides in the brain,corpora cardiaca,and corpora allata of an adult lepidopteran insect,Manduca sexta (L)

In the brain of adult specimens of the tobacco hornworm moth, Manduca sexta (L), cells immunoreactive for several kinds of neuropeptides were localized by means of the PAP procedure, by use of antisera raised against mammalian hormones or hormonal peptides. In contrast, no such neurosecretory cells were found in the corpora cardiaca and corpora allata (CC/CA); in the CC/CA, however, immunoreactive nerve fibres were observed, reaching these organs from the brain. The neurosecretory cells found in the brain were immunoreactive with at least one of the following mammalian antisera, namely those raised against the insulin B-chain, somatostatin, glucagon C-terminal, glucagon N-terminal, pancreatic polypeptide (PP), secretin, vasoactive intestinal polypeptide (VIP), glucose-dependent insulinotropic peptide (GIP), gastrin C-terminus, enkephalin, alpha- and beta-endorphin, Substance P, and calcitonin. No cells were immunoreactive with antisera specific for detecting neurons containing the insulin A-chain, nerve growth factor, epidermal growth factor, insulin connecting peptide (C-peptide), polypeptide YY (PYY), gastrin mid-portion (sequence 6-13), cholecystokinin (CCK) mid-portion (sequences 9-20 and 9-25), neurotensin C-terminus, bombesin, motilin, ACTH, or serotonin. All the neuropeptide-immunoreactive cells observed emitted nerve fibers passing through the brain to the CC and in some cases also to the CA. In CC these immunoreactive nerve fibers tended to accumulate near the aorta. It was speculated that neuropeptides are released into the circulating haemolymph and act as neurohormones.     

Neuropeptides regulate the cardiac activity of a prosobranch mollusc,Rapana thomasiana

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

Production of a specific antiserum by synthetic C-terminal fragment of glucagon

Seven rabbits were immunized with a synthetic C-terminal glucagon fragment [15--29] conjugated with bovine serum albumin by means of glutaraldehyde. Antisera for glucagon were produced in all the animals after six injections of the conjugate. One of them revealed a higher titer antiserum (G42), which did not cross react with gut glucagon-like immunoreactive material, secretin, insulin, gastric inhibitory polypeptide or vasoactive intestinal peptide. From the results of inhibition of 125 I-glucagon in binding with the antiserum by various glucagon-related fragments the immunogenic determinant of the antiserum was proved to be in the C-terminal residue of the glucagon molecule, although peptide [17--29] or [21--29] reacted weakly with the antiserum. The plasma glucagon levels measured by antiserum G 42 during an arginine test in five normal subjects were superposed on those obtained by other antiserum (G21), specific for pancreatic glucagon. Furthermore, a comparable standard curve for glucagon was obtained using antiserum G42, when a labelled p-hydroxyphenylacetylated glucagon fragment [15--29] was employed as a tracer. The present study clearly demonstrated that the C-terminal glucagon fragment could yield a specific antiserum for pancreatic glucagon, supporting the proposal that the C-terminal fragment of glucagon is responsible for such specific antisera. Furthermore, it is concluded that immunoassay for glucagon could be performed using the labelled glucagon fragment as a tracer.     

Immunocytochemical characterization of the accessory medulla in the cockroach Leucophaea maderae

Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.