Pathway-specific patterns of the co-existence of substance P,calcitonin gene-related peptide,cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig

Summary The co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labelling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (1) Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. (2) Neurons with immunoreactivity to SP, CGPR and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. (3) Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. (4) Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin.These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.     

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.This work was supported by INTAS (grant 01-2117), CRDF (grant RB1-2321-MO-02), and the Russian Foundation for Basic Research (grants 05-04-48724 and 03-04-48179).     

Action and immunoreactivity of neuropeptides in the buccal neuromuscular system of a prosobranch mollusc,Rapana thomasiana

Summary In a prosobranch mollusc, Rapana thomasiana, the catch-relaxing peptide H-Ala-Met-Pro-Met-Leu-Arg-Leu-NH₂ (CARP) was found to depress the contraction of the radula protractor and retractor elicited by electrical stimulations. The action of CARP was in contrast to that of other neuropeptides, H-Phe-Met-Arg-Phe-NH₂ (FMRFamide) and H-Phe-Leu-Arg-Phe-NH₂ (FLRFamide), which enhanced the contraction of the radula protractor and retractor, respectively. By immunohistochemical examinations, FMRFamide-like immunoreactive neurons were found on the rostral side of the right buccal ganglion and the caudal side of the left ganglion, where some CARP-like immunoreactive neurons were also distributed, indicating a possible coexistence of FMRFamide and CARP. FMRFamide- and CARP-like immunoreactivities were also detected in the neuropile of buccal ganglia, radula nerves arising from the ganglia, and nerve fibers in the radula muscles. The present results suggest that FMRFamide- and CARP-like peptides are involved in the regulation of the contraction of the radula muscles.     

Neuropeptide distribution in the cervico-thoracic paravertebral ganglia of the cat with particular reference to calcitonin gene-related peptide immunoreactivity

Summary Paraffin sections of cervical and upper thoracic paravertebral ganglia of the cat were investigated by immunohistochemistry using antisera directed against calcitonin gene-related peptide (CGRP). The relationships of CGRP-immunoreactive structures to those exhibiting immunoreactivity to antisera against other regulatory peptides and dopamine--hydroxylase (DBH), respectively, were studied in consecutive sections. Singly scattered CGRP-immunoreactive neuronal perikarya were observed in the superior and middle cervical ganglia as well as in the stellate ganglion. These neurons also displayed immunoreactivity to vasoactive intestinal polypeptide (VIP), and some additionally exhibited faint substance-P immunoreactivity. DBH- and neuropeptide Y-immunoreactive ganglion cells were not identical with CGRP-immunoreactive neuronal cell bodies.According to the immunoreactive properties of varicosities, which abut on CGRP/VIP-immunoreactive perikarya, three types of CGRP/VIP-immunoreactive ganglion cells could be distinguished: (1) CGRP/VIP-immunoreactive neurons being surrounded by somatostatin-immunoreactive nerve fibers, (2) neurons being approached by both DBH- and met-enkephalin-immunoreactive varicosities, and (3) neurons receiving both DBH- and neurotensin-immunoreactive fibers. The stellate and upper thoracic ganglia harbored clusters of intensely VIP-immunoreactive somata, which lacked CGRP-immunoreactivity. Fine somatostatin-immunoreactive and coarse CGRP-immunoreactive fibers were distributed within these clusters, whereas patches of neurotensin-immunoreactive fibers were complementarily arranged. At all segmental levels investigated, a few postganglionic neurons were approached by both CGRP-immunoreactive and substance P-immunoreactive varicosities, but lacked a VIP-immunoreactive innervation. Therefore, CGRP/substance P-immunoreactive fiber baskets appeared rather to be of extraganglionic origin than to emerge from intraganglionic CGRP/VIP/SP neurons. CGRP-immunoreactive cell bodies or fibers were absent in clusters of small paraganglionic cells, but some of the solitary paraganglionic cells displayed CGRP-immunoreactivity. Our findings establish the presence of CGRP-immunoreactivity in a population of sympathetic neurons in the cat. A highly differentiated, segment-dependent organizational pattern of neuropeptides in cervico-thoracic paravertebral ganglia was demonstrated.Supported by Deutsche Forschungsgemeinschaft grant He 919/6-2     

Galanin-, neuropeptide Y- and enkephalin-like immunoreactivities in catecholamine-storing paraganglia of the fetal guinea pig and newborn pig

Summary The occurrence and distribution of several neuropeptides and transmitter enzymes have been investigated by means of indirect immunofluorescence histochemistry in preaortal and carotid body-like paraganglia of the fetal guinea pig and the newborn pig. Preaortal paraganglia from the celiac and inferior mesenteric ganglion regions in fetal guinea pigs showed cell bodies immunoreactive (IR) for tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), neuropeptide Y (NPY), galanin (GAL) and metenkephalin (ENK). Almost all cells were IR for TH and DBH, whereas NPY-like immunoreactivity (-LI), GAL-LI and ENK-LI occurred less frequently. Direct double-labeling revealed the coexistence of NPY/GAL, NPY/ENK and GAL/ENK in paraganglion cells from the celiac and inferior mesenteric region. Nerve fibers and terminals were IR for ENK; fibers IR for calcitonin-gene-related peptide (CGRP) were present in the inferior mesenteric ganglion region. Preaortal paraganglia cells from the newborn pig showed TH-LI, DBH-LI, GAL-LI and ENK-LI, the distribution pattern being similar to that seen in the guinea pig; however, NPY-LI was absent. Carotid-body-like paraganglia from the newborn pig showed cell bodies IR to TH, GAL and ENK. Few cells were seen with DBH-LI. A rich supply of nerve fibers with CGRP-LI was present; some fibers exhibited ENK-LI and CCK-LI. In the adjacent superior cervical ganglion, ganglion cell bodies showed immunoreactivity to TH, DBH and NPY. A small number of cells were positive for GAL, CGRP and vasoactive intestinal polypeptide (VIP). Physiological activation of the paraganglia, leading to release or increase in catecholamines, may also change the content of the neuropeptides present in the paraganglia.     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.     

Localization of FMRFamide- and ACEP-1-like immunoreactivities in the nervous system and heart of a pulmonate mollusc,Achatina fulica

Immunohistochemical localization of two neuropeptides possibly involved in the regulation of cardiac activity in a pulmonate mollusc, Achatina fulica Férussac, was studied. On the ventral surface of the right cerebral ganglion, more than 50 neurons with diameters of 30–50 m showed immunoreactivity to the antiserum of the neuropeptide FMRFamide. Many were also immunoreactive to an antiserum raised against Achatina cardio-excitatory peptide-1 (ACEP-1). Although FMRFamidelike immunoreactive neurons occurred in all components of the subesophageal ganglia, identifiable ACEP-1-like immunoreactive neurons were located only in the visceral ganglion and the right parietal ganglion. In the heart, FMRFamide- and ACEP-1-like immunoreactive fibers were restricted to the atrium and the aortic end of the ventricle, consistent with morphological observations of cardiac innervation. The present results suggest that FMRFamide-and ACEP-1-like peptides are involved in regulating the heart beat of this snail.     

Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain

Locustatachykinin I is one of four closely related myotropic neuropeptides isolated from brain and corpora-cardiaca complexes of the locust Locusta migratoria. Antiserum was raised against locustatachykinin I for use in immunocytochemistry. It was found that the antiserum recognizes also locustatachykinin II and hence probably also the other two locustatachykinins due to their similarities in primary structure. Locustatachykinin-like immunoreactive (LomTK-LI) neurons were mapped in the brain of the locust, L. migratoria. A total of approximately 800 Lom TK-LI neurons were found with cell bodies distributed in the proto-, deutoand tritocerebrum, in the optic lobes and in the frontal ganglion. Processes of these neurons innervate most of the synaptic neuropils of the brain and optic lobes, as well as the frontal ganglion and hypocerebral ganglion. The widespread distribution of LomTK-LI neurons in the locust brain indicates an important role of the locustatachykinins in signal transfer or regulation thereof. As a comparison neurons were mapped with an antiserum against the cockroach myotropic peptide leucokinin I. This antiserum, which probably recognizes the native peptide locustakinin, labels a population of about 140 neurons distinct from the LomTK-LI neurons (no colocalized immunoreactivity). These neurons have cell bodics that are distributed in the proto- and tritocerebrum and in the optic lobe. The processes of the leucokinin-like immunoreactive (LK-LI) neurons do not invade as large areas in neuropil as the Lom TK-LI neurons do and some neuropils, e.g. the mushroom bodies, totally lack innervation by LK-LI fibers. In some regions, however, the processes of the Lom TK-LI and LK-LI neurons are superimposed: most notably in the central body and optic lobes. A functinal relation between the two types of neuropeptide in the locust brain can, however, not be inferred from the present findings.     

Distribution of immunoreactive neuropeptides in the pancreas of the bullfrog,Rana catesbeiana,demonstrated by immunofluorescence

Indirect double immunofluorescence labelling for eight neuropeptides in the pancreas of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of certain neuropeptides in the exocrine and endocrine pancreas. Immunoreactivity of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and galanin (GAL) was localized in nerve fibers distributed between the acini and around the duct system and vasculature of the exocrine pancreas. In these regions, CGRP-immunoreactive fibers were more numerous than those containing the other five peptides. Almost all SP fibers showed coexistence of SP with CGRP, and about one third of fibers also showed coexistence of SP with VIP, NPY, FMRF, and GAL. In the endocrine pancreas, SP, CGRP, VIP, and GAL were recognized in the nerve fibers around and within the islets of Langerhans, and VIP and GAL fibers were more numerous than SP and CGRP fibers. All CGRP fibers, and about half of the VIP and GAL fibers were immunoreactive for SP. NPY- and FMRF-immunoreactive cells were found at the periphery of the islets. These findings suggest that the exocrine and endocrine pancreatic functions of the bullfrog are under the control of peptidergic innervation.     

PBAN/pyrokinin peptides in the central nervous system of the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

The pyrokinin/pheromone-biosynthesis-activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including the stimulation of pheromone biosynthesis in female moths, muscle contraction, induction of embryonic diapause, melanization, acceleration of puparium formation, and termination of pupal diapause. We have used immunocytochemical techniques to demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of the fire ant, Solenopsis invicta. Polyclonal antisera against the C-terminal end of PBAN have revealed the location of the peptide-producing cell bodies and axons in the central nervous system. Immunoreactive material is detectable in at least three groups of neurons in the subesophageal ganglion and corpora cardiaca of all adult sexual forms. The ventral nerve cord of adults consists of two segmented thoracic ganglia and four segmented abdominal ganglia. Two immunoreactive pairs of neurons are present in the thoracic ganglia, and three neuron pairs in each of the first three abdominal ganglia. The terminal abdominal ganglion has no immunoreactive neurons. PBAN immunoreactive material found in abdominal neurons appears to be projected to perisympathetic organs connected to the abdominal ganglia. These results indicate that the fire ant nervous system contains pyrokinin/PBAN-like peptides, and that these peptides are released into the hemolymph. In support of our immunocytochemical results, significant pheromonotropic activity is found in fire ant brain-subesophageal ganglion extracts from all adult fire ant forms (queens, female and male alates, and workers) when extracts are injected into decapitated females of Helicoverpa zea. This is the first demonstration of the presence of pyrokinin/PBAN-like peptides and pheromonotropic activity in an ant species. This research was supported in part by a US-Israel Binational Science Foundation Grant (no. 2003367).     

Distribution of five peptides, three general neuroendocrine markers, and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: an immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I-II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I-III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I-II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Regulatory peptides in the pancreas of two species of elasmobranchs and in the Brockmann bodies of four teleost species

Summary Immunohistochemistry was used to localize regulatory peptides in endocrine cells and nerve fibres in the pancreas of two species of elasmobranchs (starry ray,Raja radiata and spiny dogfish,Squalus acanthias), and in the Brockmann bodies of four teleost species (goldfish,Carassius auratus, brown troutSalmo trutta, rainbow trout,Oncorhynchus mykiss and cod,Gadus morhua). In the elasmobranchs, the classical pancreatic hormones somatostatin, glucagon and insulin were present in endocrine cells of the islets. In addition, endocrine cells were labelled with antisera to enkephalins, FMRF-amide, gastrin/cholecystokinin-(CCK)/caerulein, neurotensin, neuropeptide Y (NPY), and peptide YY (PYY). Nerve fibres were demonstrated with antisera against bombesin, galanin and vasoactive intestinal polypeptide (VIP). These nerve fibres innervated the walls of blood vessels, in the exocrine as well as the endocrine tissue. In the four teleost species immunoreactivity to somatostatin, insulin and glucagon was intense in the Brockmann bodies. Cells were labelled with antisera to enkephalin, neurotensin, FMRFamide, gastrin/CCK/ caerulein, NPY, PYY and VIP. Only a few nerve fibres were found with antisera against dopamine--hydroxylase (DBH, cod), enkephalin (met-enkephalin-Arg-Phe, cod), bombesin (cod), gastrin/CCK/caerulein (cod) and VIP. Galanin-like-immunoreactive fibres were numerous in the Brockmann bodies of all teleosts examined. Immunoreactivity to calcitonin gene-related peptide (CGRP), substance P, tyrosine hydroxylase (TH), and phenyl-N-methyl transferase (PNMT) could not be found in any of the species studied.     

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

Summary the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.     

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 m); (ii) large neurons (80–100 m); (iii) medium-sized neurons (40–70 m); (iv) small neurons (12–30 m). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.     

A large proportion of afferent neurons innervating the uterine cervix of the cat contain VIP and other neuropeptides

Summary Axonal tracing techniques were used in combination with immunohistochemistry to examine the distribution of neuropeptides in afferent pathways from the uterine cervix of the cat. Primary afferent neurons innervating the uterine cervix were identified by axonal transport of the dye, fast blue, injected into the cervix. Fifteen to twenty-five days after the injection, dorsal root ganglia (L1–S3) were removed and incubated for 48–72 h in culture medium containing colchicine to increase the levels of peptides. Calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), leucine-enkephalin (LENK), somatostatin, substance P and vasoactive intenstinal polypeptide (VIP) were identified by use of indirect immunohistochemical techniques. Eighty-four percent of uterine cervix afferent neurons were identified in the sacral dorsal root ganglia (S1–S3), and 16% in the middle lumbar dorsal root ganglia (L3–L4). In sacral dorsal root ganglia, VIP was present in the highest percentage of dye-labeled cells (71%), CGRP in 42%, and substance P in 18% of the cells. CCK and LENK were present in 13% of the cells. In lumbar dorsal root ganglia, CGRP (51%) was most prominent peptide followed by VIP (34%), substance P (28%), LENK (17%) and CCK (13%). Somatostatin was present in the ganglia but did not occur in dye-labeled neurons. In conclusion, the uterine cervix of the cat receives a prominent VIP-and CGRP-containing afferent innervation. The percentage of neurons containing VIP is three to five times higher than the percentage of these neurons in afferent pathways to other pelvic organs. These observations coupled with the results of physiological studies suggest that VIP is an important transmitter in afferent pathways from the cervix.     

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.     

Peptide immunocytochemistry of neurons projecting to the retrocerebral complex in the blow fly, <Emphasis Type="Italic">Protophormia terraenovae</Emphasis>

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to characterize neurons with somata in the pars intercerebralis (PI), pars lateralis (PL), and subesophageal ganglion (SEG), designated as PI neurons, PL neurons, and SEG neurons, respectively, all of which project to the retrocerebral complex in the blow fly, Protophormia terraenovae. Immunocytochemistry combined with backfills through the cardiac-recurrent nerve revealed that at least two pairs of PI and SEG neurons for each were FMRFamide-immunoreactive. Immunoreactivity against [Arg7]-corazonin, beta-pigment-dispersing hormone (beta-PDH), cholecystokinin8, or FMRFamide was observed in PL neurons. Immunoreactive colocalization of [Arg7]-corazonin with beta-PDH, [Arg7]-corazonin with cholecystokinin8, or beta-PDH with FMRFamide was found in two to three somata in the PL of a hemisphere. Based on their anatomical and immunocytochemical characteristics, PI neurons were classified into two types, PL neurons into six types, and SEG neurons into two types. Fibers in the retrocerebral complex showed [Arg7]-corazonin, beta-PDH, cholecystokinin8, and FMRFamide immunoreactivity. Cholecystokinin8 immunoreactivity was also detected in intrinsic cells of the corpus cardiacum. The corpus allatum was densely innervated by FMRFamide-immunoreactive varicose fibers. These results suggest that PI, PL, and SEG neurons release [Arg7]-corazonin, beta-PDH, cholecystokinin8, or FMRFamide-like peptides from the corpus cardiacum or corpus allatum into the hemolymph, and that some PL neurons may simultaneously release several neuropeptides.     

Mercuric chloride-induced Gastrin/Cholecystokinin 8 immunoreactivity in the central nervous system of the terrestrial slug Semperula maculata: an immunohistochemical study

We measured the immunoreactivity of the neuropeptide gastrin cholecystokinin 8 (gastrin/CCK 8) in neurons of the terrestrial slug Semperula maculata following acute treatment with mercuric chloride (HgCl₂). The distribution of gastrin/CCK 8 was analyzed in neurons of different regions, specifically from cerebral ganglia (procerebrum (pro-c), mesocerebrum (meso-c) and metacerebrum (meta-c). In the control group, neurons of pedal, pleural, parietal and visceral ganglia showed positive immunoreactivity using vertebrate antiserum against gastrin/CCK 8. Gastrin/CCK 8 immunoreactivity was also seen in the fibers and neuropil region of all ganglia. In the cerebral ganglion, 10, 12 and 8 % of the neurons from pro-c, meso-c and meta-c, respectively, were stained with the antibody. The immunostaining was increased in neurons (giant, large, medium and small) after HgCl₂ treatment. The treatment greatly increased the mucin content within the neurons. Exposure to HgCl₂ enhanced gastrin immunoreactivity in the neurons and this increased with time. Results are discussed in the context of neuropathology in cerebral ganglia associated with the feeding behavior of Semperula maculata.     

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.     

Distribution of neuropeptides in the porcine stellate ganglion

The localization and distribution of neuropeptides including neuropeptide Y (NPY), [Met⁵]enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P and somatostatin (SOM) were analyzed in the stellate ganglion of the pig by use of the indirect immunofluorescence technique. NPY, MEAGL, SOM, VIP and CGRP immunoreactivities were found to exist in subpopulations of neuronal cell bodies of the stellate ganglion. A population of the small intensely fluorescent (SIF) cells showed MEAGL immunoreactivity. In addition, the presence of NPY-, MEAGL-, CGRP-, SP-, SOM- and VIP-immunoreactive nerve fibers and axonal varicosities were observed in the stellate ganglion. The localization and pattern of distribution of these peptides in the porcine stellate ganglion were compared with studies carried out on stellate ganglia of other mammalian species.