Distribution and functional significance of Met-enkephalin-Arg6-Phe7-and Met-enkephalin-Arg6-Gly7-Leu8-like peptides in the blowflyCalliphora vomitoria

Summary Neuronal pathways in the retrocerebral complex and thoracico-abdominal ganglionic mass of the blowflyCalliphora vomitoria have been identified immunocytochemically with antisera against the extended-enkephalins, Met-enkephalin-Arg⁶-Phe⁷ (Met-7) and Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8). Neurons of the hypocerebral ganglion, immunoreactive to Met-8, have axons in the crop duct nerve and terminals in muscles of the crop and its duct. Certain neurons of the hypocerebral ganglion are also immunoreactive to Met-7, and axons from these cells innervate the heart. Met-8 immunoreactive nerve terminals invest the cells of the corpus allatum. The source of this material is believed to ve a single pair of lateral neurosecretory cells in the brain. There is no Met-7 immunoreactive material in the corpus allatum. In the corpus cardiacum neither Met-7 nor Met-8 immunoreactivity is present in the cells. However, in the neuropil of the gland certain fibres, with their origins elsewhere, do contain Met-8 immunoreactivity. The most prominent neurons in the thoracic ganglion are the Met-7 immunoreactive ventral thoracic neurosecretory cells, axons from which project to neurohaemal areas in the dorsal neural sheath and also, via the ventral connective, to the brain. Co-localisation studies show that the perikarya of these cells are immunoreactive to antisera raised against several vertebrate-type peptides, such as Met-7, gastrin/cholecystokinin and pancreatic polypeptide. However, their axons and terminals show varying amounts of the peptides, suggesting differential transport and utilisation. Only a few cells in the thoracic ganglion are immunoreactive to Met-8 antisera. These lie close to the nerve bundles suppling the legs. In the abdominal ganglion, Met-8 immunoreactive neurons project to the muscles of the hindgut. This study suggests that the extended enkephalin-like peptides ofCalliphora may have a variety of different roles: as neurotransmitter or neuromodulator substances; in the direct innervation of effector organs; and as neurohormones.     

Distribution and functional significance of Met-enkephalin-Arg6-Phe7- and Met-enkephalin-Arg6-Gly7-Leu8-like peptides in the blowfly Calliphora vomitoria

Summary Neuronal pathways immunoreactive to antisera against the extended-enkephalins, Met-enkephalin-Arg⁶-Phe⁷ (Met-7) and Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8), have been identified in the brain of the blowfly Calliphora vomitoria. Co-localisation with other enkephalins in certain neurons suggests that a precursor similar to preproenkephalin A exists in insects and that differential enzymatic processing occurs as in vertebrates. Co-localisations of the extended-enkephalin-like peptides with other vertebrate-type peptides, including cholecystokinin and pancreatic polypeptide, also occur. The enkephalinergic pathways are specific, comprising a few groups of highly characteristic neurons and areas of neuropil. Of special interest is the finding that parts of the antennal chemosensory and the optic lobe visual systems contain Met-8 immunoreactive neurons. Within the median neurosecretory cell groups, some of the giant neurons show immunoreactivity to Met-8 and others to both Met-8 and Met-7. Fibres from these cells project to the corpus cardiacum and also to the suboesophageal ganglion, where arborisations occur in the tritocerebral neuropil. Co-localisation studies of these cells have shown that at certain terminals, one particular type of peptide is the dominant neuroregulator, whilst at other terminals, within the same cell, a different co-synthesised peptide predominates. Several groups of lateral neurosecretory cells show clearly defined enkephalinergic pathways, most of which have connections with the central body. The complex patterns of immunoreactivity seen in terminals in the different parts of the central body, suggest an important role for the enkephalin-like peptides in the integration of multimodal sensory inputs. The physiological functions of the extended-enkephalin-like peptides in the brain of Calliphora is still unknown, but the anatomical evidence suggests they may have a role similar to that in mammals, where they are thought to control aspects of feeding behaviour.     

Mapping of enkephalin-related peptides in the nervous system of the blowfly,Calliphora vomitoria,and their co-localization with cholecystokinin (CCK)- and pancreatic polypeptide (PP)-like peptides

Summary The distribution of enkephalin-like immunoreactive material has been studied in the CNS of C. vomitoria. The presence of both Met- and Leu-enkephalin-related peptides is suggested by differential immunostaining with a variety of antisera. Comparisons made between certain of the enkephalin-immunoreactive perikarya, nerve fibres and terminals with cells in corresponding positions as evidenced in previously published neuroanatomical studies of the dipteran brain have suggested specific enkephalinergic pathways. As examples, one Met-enkephalin-immunoreactive neuron appears to link the lobula with the dorsal protocerebrum, and a group of Leu-enkephalin cells in the pars intercerebralis appear to have arborisations in both the central body (fan-shaped body) and the tritocerebral neuropil around the oesophageal foramen. Neuronal pathways of this type indicate that the enkephalin-like peptides of the fly brain are functioning as neurotransmitters and/or neuromodulators. In the thoracic ganglia, symmetrically arranged cells, immunoreactive to both Met- and Leu-enkephalin antisera, are positioned ventrally in pairs on either side of the mid-line in a sagittal plane. Very little immunoreactive material is observed in the neuropil, however, and the source of the accumulation of Leu-enkephalin-immunoreactivity in the dorsal neural sheath is not certain. It is suggested that this material, in contrast to that present in areas of the brain, acts as a neurohormone and that it may have a physiological role following its release into the haemolymph. The enkephalin-like immunoreactive material of certain neurons identified within the brain and thoracic ganglion shows a complex pattern of co-existence with pancreatic polypeptide- and gastrin/cholecystokinin-like peptides.     

Immunocytochemical mapping of gastrin/CCK-like peptides in the neuroendocrine system of the blowfly Calliphora vomitoria (Diptera)

Summary The distribution of gastrin/CCK-like immunoreactive material has been studied in the retrocerebral complex of Calliphora. The material reacts with antisera specific for the common COOH terminus of gastrin and CCK but not with N-terminal antisera. The three thoracic ganglia and the fused abdominal ganglia each contain a specific number of symmetrically arranged immunoreactive cells both dorsally and ventrally in pairs on either side of the midline in a sagittal plane. The neuropil of these ganglia also contains a considerable amount of immunoreactive fibres and droplets. Reconstructed axonal pathways suggest that some of the nerve fibres have their origins within the brain and/or the suboesophageal ganglion. Immunoreactive material may also be seen apparently leaving the thoracic ganglion posteriorly via the abdominal nerves, and there is strong evidence of a neurohaemal organ within the dorsal sheath in the region of the metathoracic and abdominal ganglia. There appears to be a direct correlation between the content of peptidergic material of cells and fibres and the age and diet of the flies. The corpus cardiacum contains COOH-terminal specific gastrin/CCK-like material within the intrinsic cells and in the neuropil. It is present also in the cardiac-recurrent nerve entering the corpus cardiacum anteriorly and in the nerves leaving the gland dorsoposteriorly, the aortic or cardiac nerves. It is not observed, however, in the nerves leaving the corpus cardiacum ventroposteriorly, the so-called oesophageal, gastric or crop-duct nerves. The corpus allatum and the hypocerebral ganglion do not contain immunoreactive material of this type. Gastrin/CCK-like and secretin-like immunoreactive materials appear to co-exist in the cells of the corpus cardiacum and co-existence of gastrin/CCK-like and pancreatic polypeptide like substances occurs within certain cells of the thoracic ganglion.     

Neuronal types in the spinal dorsal gray of the turtle Chrysemys d'orbigny: a Golgi study

At thoracic and lumbar levels the spinal dorsal gray of young specimens of the turtle Chrysemys d'orbigny consists of a cell-free neuropil and an aggregation of perikarya termed here the lateral column of the dorsal horn (LCDH). Nerve cell clusters also occur in the dorsal commissure. The main neuropil area can be divided into a thin superficial layer containing some myelinated fibers (neuropil area Ib) and a compact core composed of unmyelinated axon terminals, dendritic branches, and thin glial processes (neuropil area II). A looser neuropil area is located at the horn base (neuropil area III). The so-called marginal zone of de Lange represents a fourth synaptic field termed here neuropil area Ia. The LCDH consists of neurons of different size and shape. Two peculiar nerve cell types have been recognized in the dorsal horn: giant and bitufted neurons. The former exhibits a large dendritic arbor, which after passing through neuropil areas II and Ib projects into neuropil area Ia and the adjacent white matter. Most frequently Golgi-stained giant neurons have perikarya and dendritic domains on the same side (ipsilateral giant neurons). There are also heterolateral giant neurons whose dendritic branches invade the opposite horn. Bitufted neurons are characterized by the presence of two main dendritic shafts connecting neuropil area II of both dorsal horns. At neuropil levels the major dendritic branches ramify profusely giving rise to short tortuous terminal processes. Perikarya of bitufted neurons occur in the dorsal commissure. The LCDH also contains many small and medium-sized neurons. These are oriented in two main directions: parallel or radial with respect to the dorsal horn surface. The population of horizontally oriented neurons comprises two subtypes termed here alpha and beta. Radially oriented neurons are pleomorphic, defying precise, unequivocal classification.     

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     

Immunocytochemical identification of structures containing putative red pigment-concentrating hormone in two species of decapod crustaceans

Summary By use of an antiserum raised against the Nterminal sequence pGlu-Leu-Asn-Phe..., common to red pigment-concentrating hormone (RPCH) of Pandalus borealis and three structurally similar insect neuropeptides, putative RPCH-immunopositive structures were revealed in the eyestalks of Carcinus maenas and Orconectes limosus and in the brain and thoracic ganglion (TG) of C. maenas. In the eyestalks, complete neurosecretory pathways were demonstrated, consisting of perikarya, axons and terminals in the neurohemal organ, the sinus gland (SG). In C. maenas approximately 20 small RPCH cells are present as a distinct group adjacent to the medulla terminalis ganglionic X-organ (MTGXO, XO). They are morphologically different from the larger XO perikarya, which contain the crustacean hyperglycemic hormone (CHH). The occurrence of both neuropeptides in distinct neurosecretory pathways was ascertained by immunologic double staining (PAP/gold) or by analysis of consecutive sections. In addition, a group of two to four larger RPCH cells is located in the proximal part of the MT. In O. limosus, RPCH cells are found in the XO. Cells corresponding to the proximal MT cells of C. maenas were not found. In both species, a few more weakly staining immunopositive perikarya were observed in clusters of cell somata of the optic ganglia. It is uncertain whether these are connected to the SG.In the brain of C. maenas, several smaller and three larger perikarya were consistently observed in the dorsal lateral cell somata adjacent to the olfactory lobes. In the optic nerve, two axons that project into the eyestalk were stained. Some axons were also observed in the ventral median neuropil of the brain. In the TG, RPCH cells were found in small numbers in median positions, i.e., in clusters of somata between the ganglia of the appendages.HPLC analysis of the red pigment-concentrating activity from the SG of C. maenas revealed that the retention time of the neuropeptide is similar but not identical to that of Pandalus borealis RPCH.     

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH₂ and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu³-Glu⁴- in place of -Asp³-Asp⁴-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.     

Crustacean cardioactive peptide in the nervous system of the locust,Locusta migratoria: an immunocytochemical study on the ventral nerve cord and peripheral innervation

Summary Crustacean cardioactive peptide-immunoreactive neurons occur in the entire central nervous system of Locusta migratoria. The present paper focuses on mapping studies in the ventral nerve cord and on peripheral projection sites. Two types of contralaterally projecting neurons occur in all neuromers from the subesophageal to the seventh abdominal ganglia. One type forms terminals at the surface of the thoracic nerves 6 and 1, the distal perisympathetic organs, the lateral heart nerves, and on ventral and dorsal diaphragm muscles. Two large neurons in the anterior part and several neurons of a different type in the posterior part of the terminal ganglion project into the last tergal nerves. In the abdominal neuromers 1–7, two types of ipsilaterally projecting neurons occur, one of which gives rise to neurosecretory terminals in the distal perisympathetic organs, in peripheral areas of the transverse, stigmata and lateral heart nerves. Four subesophageal neurons have putative terminals in the neurilemma of the nervus corporis allati II, and in the corpora allata and cardiaca. In addition, several immunoreactive putative interneurons and other neurons were mapped in the ventral nerve cord. A new in situ whole-mount technique was essential for elucidation of the peripheral pathways and targets of the identified neurons, which suggest a role of the peptide in the control of heartbeat, abdominal ventilatory and visceral muscle activity.Abbreviations AG abdominal ganglia - AM alary muscle - AMN alary muscle nerve - CA corpus allatum - CC corpus cardiacum - dPSO distal perisympathetic organ - LHN lateral heart nerve - LT CCAP-immunoreactive lateral tract - NCA nervus corporis allati - NCC nervus corporis cardiaci - NM neuromer - PMN paramedian nerve - PSO perisympathetic organ - SOG subesophageal ganglion - VDM ventral diaphragm muscles - VNC ventral nerve cord     

Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta

Summary Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta were individually reconstructed. Serotonin immunoreactivity was detected in 19–20 bilaterally symmetrical pairs of interneurons in the midbrain and 10 pairs in the suboesophageal ganglion. These neurons were also immunoreactive with antisera against DOPA decarboxylase. All major neuropil regions except the protocerebral bridge are innervated by these neurons. In addition, efferent cells are serotonin-immunoreactive in the frontal ganglion (5 neurons) and the suboesophageal ganglion (2 pairs of neurons). The latter cells probably give rise to an extensive network of immunoreactive terminals on the surface of the suboesophageal ganglion and suboesophageal nerves. Most of the serotonin-immunoreactive neurons show a gradient in the intensity of immunoreactive staining, suggesting low levels of serotonin in cell bodies and dendritic arbors and highest concentrations in axonal terminals. Serotonin-immunoreactive cells often occur in pairs with similar morphological features. With one exception, all serotonin-immunoreactive neurons have bilateral projections with at least some arborizations in identical neuropil areas in both hemispheres. The morphology of several neurons suggests that they are part of neuronal feedback circuits. The similarity in the arborization patterns of serotonin-immunoreactive neurons raises the possibility that their outgrowing neurites experienced similar forces during embryonic development. The morphological similarities further suggest that serotonin-immunoreactive interneurons in the midbrain and suboesophageal ganglion share physiological characteristics.Abbreviations CNS central nervous system - DDC DOPA decarboxylase - LAL lateral accessory lobe - SLI serotonin-like immunoreactivity - SOG suboesophageal ganglion - VLP ventro-lateral protocerebrum     

Morphology and central synaptic connections of the efferent neurons innervating the crayfish hindgut

Summary The distribution, morphology and synaptic connections of the hindgut efferent neurons in the last (sixth) abdominal ganglion of the crayfish, Orconectes limosus, have been investigated using light and electron microscopy in conjunction with retrograde cobalt/nickel and HRP labeling through the intestinal nerve. The hindgut efferent neurons occur singly and in clusters, and are unipolar. Their axonal projections are uniform and consist of a thick primary neurite with typical lateral projections and limited arborization of varicose fibers in the ganglionic neuropil. They also send lower order axon processes to the ganglionic neural sheath, where they arborize profusely, forming a network of varicose fibers. The majority of the efferent neurons project to the anterior part of the hindgut. HRP-labeled axon profiles are found in both pre- and postsynaptic position in the neuropil of the ganglion. HRP-labeled axon profiles also establish pre- and postsynaptic contacts in the intestinal nerve root. All hindgut efferent terminals contain similar synaptic vesicle populations: ovoid agranular vesicles (50–60 nm) and a few large granular vesicles (100–200 nm). It is suggested that the hindgut efferent neurons in the last abdominal ganglion are involved in: (1) innervation of the hindgut; (2) central integrative processes; (3) en route synaptic modification of efferent and afferent signals in the intestinal nerve; (4) neurohumoral modulation of peripheral physiological processes.Fellow of the Alexander von Humboldt Stiftung     

Identification of neuroendocrine cells producing a diuretic hormone in the tobacco hornworm moth,Manduca sexta

Summary Separate antisera were raised to the N- and C-terminal half of the diuretic hormone from Manduca sexta. Antisera against the two halves of this peptide recognized the same cells in M. sexta, and preabsorption of the antisera with the peptides used as antigens abolished the immunoreactivity, confirming their specificity. The antisera reacted with two median neurosecretory cells on each side of the protocerebral groove in larvae, and with a group of about 80 small median neurosecretory cells in the adult, as well as their axons to, and their axon terminals in, the corpora cardiaca. During the early pupal stages, small cells, which are possibly derived from a common neuroblast, differentiate into immunoreactive neurosecretory cells, which explains the large increase in cell numbers in the adult. In the sleepy sulphur butterfly, Eurema nicippe, homologous median neurosecretory cells in the adult were immunoreactive with both antisera.     

Lobula plate and ocellar interneurons converge onto a cluster of descending neurons leading to neck and leg motor neuropil in Calliphora erythrocephala

Summary In the fly, Calliphora erythrocephala, a cluster of three Y-shaped descending neurons (DNOVS 1–3) receives ocellar interneuron and vertical cell (VS4–9) terminals. Synaptic connections to one of them (DNOVS 1) are described. In addition, three types of small lobula plate vertical cell (sVS) and one type of contralateral horizontal neuron (Hc) terminate at DNOVS 1, as do two forms of ascending neurons derived from thoracic ganglia. A contralateral neuron, with terminals in the opposite lobula plate, arises at the DNOVS cluster and is thought to provide heterolateral interaction between the VS4–9 output of one side to the VS4–9 dendrites of the other. DNOVS 2 and 3 extend through pro-, meso-, and metathoracic ganglia, branching ipsilaterally within their tract and into the inner margin of leg motor neuropil of each ganglion. DNOVS 1 terminates as a stubby ending in the dorsal prothoracic ganglion onto the main dendritic trunks of neck muscle motor neurons. Convergence of VS and ocellar interneurons to DNOVS 1 comprises a second pathway from the visual system to the neck motor, the other being carried by motor neurons arising in the brain. Their significance for saccadic head movement and the stabilization of the retinal image is discussed.     

FMRFamide-like immunoreactivity in the isolated,in vivo cultured ventral ganglion of the fly,Sarcophaga bullata (Parker) (Diptera: Sarcophagidae)

The influence of peripheral connectivity on the survival and differentiation of Phe-Met-Arg-Phe-amide-like immunoreactive (FLI) neurons in the ventral ganglion (VG) of the fly Sarcophaga bullata (Diptera: Sarcophagidae) was examined. Isolated larval VG were cultured in vivo for 13 days. The ganglia had undergone metamorphosis and resembled in situ metamorphosed VG in morphology and in the number and location of FLI neurons. The 3 pairs of large thoracic FLI neurons survived and became translocated to the midventral position extending immunoreactive axons into the dorsal neuropil. The 5 pairs of small FLI neurons also appeared de novo in the abdominal ganglion. However, the dorsal neural sheath of the cultured VG was devoid of FMRFamide-like immunoreactivity that was so characteristic of adult VG, which suggests the importance of peripheral connectivity for the metamorphic modification of FLI neurons.     

Constancies in the neuronal architecture of the suboesophageal ganglion at metamorphosis in the beetleTenebrio molitor L.

Summary Topological organization of identified neurons has been characterized for the larval, pupal and imaginal suboeosphageal neuropil of the meal-worm beetleTenebrio molitor. Neuronal fate mapping allows identification of individually persisting neurons in the metamorphosing suboesophageal ganglion ofTenebrio. Analysis was performed on interneurons characterized by serotonin and CCAP (crustacean cardioactive peptide) immunohistochemistry, on motoneurons that innervate the dorsal and ventral longitudinal muscles, and on suboesophageal descending neurons. All these different populations of neurons show topologically invariant features throughout metamorphosis. Motoneurons, interneurons, and descending suboesophageal neurons of the imaginal suboeosphageal ganglion embody individually persisting larval interneurons. Impacts for a functional interpretation of the neuronal architecture of the suboesophageal ganglion are discussed.     

Aminergic neurons in the brain of blowflies and Drosophila: dopamine- and tyrosine hydroxylase-immunoreactive neurons and their relationship with putative histaminergic neurons

Summary The distribution and morphology of neurons reacting with antisera against dopamine (DA), tyrosine hydroxylase (TH) and histamine (HA) were analyzed in the blowflies Calliphora erythrocephala and Phormia terraenovae. TH-immunoreactive (THIR) and HA-immunoreactive (HAIR) neurons were also mapped in the fruitfly Drosophila melanogaster. The antisera against DA and TH specifically labeled the same neurons in the blowflies. About 300 neurons displayed DA immunoreactivity (DAIR) and THIR in the brain and subesophageal ganglion of the blowflies. Most of these neurons were located in bilateral clusters; some were distributed as bilateral pairs, and two ventral unpaired median (VUM) neurons were seen in the subesophageal ganglion. Immunoreactive processes were found in all compartments of the mushroom bodies except the calyces, in all divisions of the central body complex, in the medulla, lobula and lobula plate of the optic lobe, and in non-glomerular neuropil of protocerebrum, tritocerebrum and the subesophageal ganglion. No DA or TH immunoreactivity was seen in the antennal lobes. In Drosophila, neurons homologous to the blowfly neurons were detected with the TH antiserum. In Phormia and Drosophila, 18 HA-immunoreactive neurons were located in the protocerebrum and 2 in the subesophageal ganglion. The HAIR neurons arborized extensively, but except for processes in the lobula, all HAIR processes were seen in non-glomerular neuropil. The deuto- and tritocerebrum was devoid of HAIR processes. Double labeling experiments demonstrated that TH and HA immunoreactivity was not colocalized in any neuron. In some regions there wasm however, substantial superposition between the two systems. The morphology of the extensively arborizing aminergic neurons described suggests that they have modulatory functions in the brain and subesophageal ganglion.     

Putative neurohemal areas in the peripheral nervous system of an insect,Gryllus bimaculatus,revealed by immunocytochemistry

The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin immunoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.     

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.     

Neurons with histaminelike immunoreactivity in the segmental and stomatogastric nervous systems of the crayfishPacifastacus leniusculus and the lobsterHomarus americanus

Summary We used a polyclonal antiserum against histamine to map histaminelike immunoreactivity (HLI) in whole mounts of the segmental ganglia and stomatogastric ganglion of crayfish and lobster. Carbodiimide fixation permitted both HRP-conjugated and FITC-conjugated secondary antibodies to be used effectively to visualize HLI in these whole mounts. Two interneurons that send axons through the inferior ventricular nerve (ivn) and the stomatogastric nerve to the stomatogastric ganglion had strong HLI, both in crayfish and in lobster. These ivn interneurons were known from other evidence to be histaminergic. The neuropil of the stomatogastric ganglion in both crayfish and lobster contained brightly labeled terminals of axons that entered the ganglion from the stomatogastric nerve. No neuronal cell bodies in this ganglion had HLI. Each segmental ganglion contained at least one pair of neurons with HLI. Some neurons in the subesophageal ganglion and in each thoracic ganglion labeled very brightly. Axons of projection interneurons with strong HLI occurred in the dorsal lateral tracts of each segmental ganglion, and sent branches to the lateral neuropils and tract neuropils of each ganglion. All the labeled neurons were interneurons; no HLI was observed in peripheral nerves.