Brain-derived neurotrophic factor-like neuropeptide is secreted as a neurohormone from specific brain neurons into the corpus allatum in the silkworm Bombyx mori

The aim of this study was to investigate the secretion of brain‐derived neurotrophic factor (BDNF)‐like neuropeptide in the silkworm, Bombyx mori , by using immunocytochemical techniques on the brain and retrocerebral complex of fifth instar larvae. In the brain, four pairs of median neurosecretory cell (MNC) bodies and six pairs of lateral neurosecretory cell (LNC) bodies had distinct immunoreactivities to this peptide, suggesting that this peptide is produced from two types of brain neuron. These reactivities were much stronger in the MNC than in the LNC. Labeled MNC projected their axons into the contralateral corpora allata, to which axons of labeled MNC were eventually innervated, through decussation in the median region, contralateral nerve corporis cardiaci I and nerve corpora allata I. Labeled LNC extended their axons into the ipsilateral corpora allata to be innervated through the ipsilateral nerve corporis cardiaci II and nerve corpora allata I. These results suggest that BDNF is secreted as a neurohormone from MNC and LNC of the brain into the corpora allata.     

New features of the brain-retrocerebral neuroendocrine complex of the locust Schistocerca vaga (scudder)

Summary The techniques of axonal iontophoresis and cobalt sulfide precipitation were used to elucidate the relationships of the brain's neurosecretory cell groups and the retrocerebral complex of the locust Schistocerca vaga. The axons of the nervi corporis cardiaci I (NCC I) arise (1) from the medial neurosecretory cells of the protocerebrum, showing only limited branching, looping or spiraling; and (2) from a cell group previously undescribed for this species, located in the tritocerebrum. The axons project into the neurohemal and the glandular portions of the corpora cardiaca and into the hypocerebral ganglion, but not into the corpora allata. Axons of the NCC II arise from the lateral neurosecretory cells of the protocerebrum and project into the center of the corpora allata via the nervi corporis allati I (NCA I), as well as into the neurohemal and glandular portions of the corpora cardiaca. Axons of the NCC III arise from another newly described cell group in the tritocerebrum and end in both the corpora cardiaca and corpora allata. Axons of the NCA II arise from cells in the subesophageal ganglion and also end in the corpora allata.Supported by NIH Predoctoral Fellowship No. 5 F 01 GM 43816-03, NSF Grant GB-23033 and NIH Grant CA-05045 to H. A. Bern and USPHS Grant 1 R 01 NS09404 to C.H.F. Rowell.I wish to express my gratitude to Professors H.A. Bern and C.H.F. Rowell for unending encouragement and advice. I am indebted to Dr. Mick O'Shea for instruction in the cobalt/axonal iontophoresis method, and to Ms. Bea Bacher for excellent technical assistance.     

Immunological evidence for an allatostatin-like neuropeptide in the central nervous system of Schistocerca gregaria,Locusta migratoria and Neobellieria bullata

Methanolic brain extracts of Locusta migratoria inhibit in vitro juvenile hormone biosynthesis in both the locust L. migratoria and the cockroach Diploptera punctata. A polyclonal antibody against allatostatin-5 (AST-5) (dipstatin-2) of this cockroach was used to immunolocalize allatostatin-5-like peptides in the central nervous system of the locusts Schistocerca gregaria and L. migratoria and of the fleshfly Neobellieria bullata. In both locust species, immunoreactivity was found in many cells and axons of the brain-retrocerebral complex, the thoracic and the abdominal ganglia. Strongly immunoreactive cells were stained in the pars lateralis of the brain with axons (NCC II and NCA I) extending to and arborizing in the corpus cardiacum and the corpora allata. Although many neurosecretory cells of the pars intercerebralis project into the corpus cardiacum, only 12 of them were immunoreactive and the nervi corporis cardiaci I (NCC I) and fibers in the nervi corporis allati II (NCA II) connecting the corpora allata to the suboesophageal ganglion remained unstained. S. gregaria and L. migratoria seem to have an allatostatin-like neuropeptide present in axons of the NCC II and the NCA I leading to the corpus cardiacum and the corpora allata. All these data suggest that in locusts allatostatin-like neuropeptides might be involved in controlling the production of juvenile hormone by the corpora allata and, perhaps, some aspects of the functioning of the corpus cardiacum as well. However, when tested in a L. migratoria in-vitro juvenile hormone-biosynthesis assay, allatostatin-5 did not yield an inhibitory or stimulatory effect. There is abundant AST-5 immunoreactivity in cell bodies of the fleshfly N. bullata, but none in the CA-CC complexes. Apparently, factors that are immunologically related to AST-5 do occur in locusts and fleshflies but, the active protion of the peptide required to inhibit JH biosynthesis in locusts is probably different from that of AST-5.     

Proctolin-like immunoreactivity in the central and peripheral nervous systems of the locust, Locusta migratoria

Proctolin-like immunoreactivity (PLI) was widely distributed in the locust, Locusta migratoria, within the central, peripheral and stomatogastric nervous systems, as well as the digestive system and retrocerebral complex. Proctolin-like immunoreactivity was observed in cells and processes of the brain and all ganglia of the ventral nerve cord. Of interest, PLI was found in the lateral neurosecretory cells, which send axons within the paired nervi corporis cardiaci II (NCC II) to the corpus cardiacum (CC). The CC contained extensive processes displaying PLI, which continued on within the paired nervi corporis allata (NCA) to the paired corpora allata (CA) where the axons entered and branched therein. The frontal and hypocerebral ganglia of the stomatogastric nervous system contained PLI within processes, resulting in a brightly staining neuropile. Each region of the gut contained PLI in axons and processes of varying patterns and densities. The paired ingluvial ganglia contained PLI, including an extensively stained neuropile and immunoreactive axons projecting through the nerves to the foregut. The hindgut contained PLI within longitudinal tracts, with lateral projections originating from the 8th abdominal ganglion via the proctodeal nerve. The midgut contained PLI in a regular latticework pattern with many varicosities and blebs. No difference in PLI in cells and processes of the central nervous system (CNS) was found between males and females.     

Neuroendocrine organs of the lemon-butterfly, Papilio demoleus L. II. The corpora cardiaca-allata complex of the adult.

The retrocerebral endocrine organs of the adult lemon-butterfly; Papilio demoleus have been described. The organs are subaortic lying closely behind the brain. While the nervi corporis cardiaci I (NCCI) originate from the protocerebrum of the brain, the NCCII seem to take their origin in the tritocerebrum in common with another nerve named earlier as the tegumentary nerve. The corpora cardiaca (CC) and corpora allata (CA) are closely approximated to each other obliterating the nervi corporis allati (NCA) which are conspicuous in the larva of the same species. An intercardiacal bridge (ICB) connects the CC of the two sides and acts as a possible centre of distribution for the neurosecretory material (NSM) to the gut. Histological evidence suggests that the NSM inside the CC remains intraaxonal without being primarily unloaded in the organs for storage. The intrinsic secretory cell of the CC are intimately associated with the neurosecretory fibres from the brain and bear fairly thick axons. No NSM could be detected in the CA of this insect.     

Control of C-16 juvenile hormone biosynthesis in active corpora allata of female African locusts

Summary

Corpora allata from 8-day-old female Locusta migratoria, during the phase of yolk deposition, exhibit high rates of C-16 juvenile hormone (JH) biosynthesis. The effect of different potential factors which may be involved in the regulation of corpora allata activity is reported. The biosynthetic activity of corpora allata was determined by radiochemical assay.

In maturing females, no changes in corpora allata activity are detected during one daily cycle. Starvation reduces JH biosynthesis only 3 days after the beginning of the food deprivation. Suppression of the median neurosecretory material by electrocoagulation of the internal cardiaca tract (TCC-I) does not disturb JH biosynthesis whereas the transection of the allata I nerve fibres (NCA-I) or the electrocoagulation of the lateral neurosecretory pericarya results in a rapid decline of JH biosynthesis. These data indicate that the median and lateral allatotropins are different, and that only the lateral neurosecretory material exerts an allatostimulating action on corpora allata at the time of vitellogenesis. The corpora allata response to the median allatotropin changes during oocyte growth. C-16 JH and/or 20-hydroxyecdysone treatments in vitro (addition in the culture medium) and in vivo (injection in female) do not influence JH production in our experimental conditions.     

Immunohistochemical Localization of Prothoracicotropic Hormone-Producing Neurosecretory Cells in the Brain of Bombyx mori

A monoclonal antibody that recognized the Bombyx prothoracicotropic hormone (PTTH) was produced by immunizing mice with a synthetic pentadecapeptide corresponding to the amino-terminal portion of Bombyx PTTH. The antibody recognized both intact and reduced forms of PTTH. Immunohistochemistry with this antibody has demonstrated that PTTH is produced by two pairs of dorso-lateral neurosecretory cells of the brain and transported to the corpora allata by axons running through the contralateral hemisphere of the brain. Immunoreactive axon terminals in the corpora allata were localized between the glandular cells, suggesting that PTTH is released at the inner part of this organ.     

Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata

Summary A monoclonal antibody against allatostatin I was used to demonstrate the allatostatin-immunoreactive pathways between the brain and the corpus cardiacum-corpus allatum complex in the adult cockroach Diploptera punctata. The antibody was two to three orders of magnitude more sensitive to allatostatin I than to the other four known members of the allatostatin family. Whole and sectioned brains in which immunoreactivity was localized with horseradish peroxidase-H₂O₂-diaminobenzidine reaction showed strongly immunoreactive cells in the pars lateralis of the brain with axons leading to and arborizing in the corpus cardiacum and the corpus allatum. Although many neurosecretory cells of the pars intercerebralis project to the corpora allata only, four strongly immunoreactive cells were evident here (two pairs on either side), and these did not project to the corpus cardiacum and corpus allatum but rather terminated within the protocerebrum in areas in which lateral cells also formed arborizations. Immunoreactivity was found in many other cells in the brain, especially in the tritocerebrum.     

ACTIVATION OF THE CORPORA ALLATA IN RELATION TO OVARIAN MATURATION IN THE SEASONAL FORMS OF THE BUTTERFLY, POLYGONIA C-AUREUM L.

In Polygonia c-aureum , there are two seasonal forms, viz. , the summer form and the autumn form. Previous experiments (E ndo , 1970) showed that the corpora allata of the summer form accelerate the maturation of ovarian follicles soon after imaginal ecdysis. On the other hand, the corpora allata of the autumn form do not stimulate ovarian maturation during a period of 2 to 3 weeks after the emergence.
In the summer form (S) as well as in the autumn form (A), the corpora allata remained in an inactive state for about 15 days after emergence when they had been isolated microsurgically from the brain and the corpora cardiaca during the larval period. Further, when separation of the brain into the right and left hemispheres or ablation of the medial neurosecretory group cells of the pars intercerebralis had been carried out on S-pupae of 28 hr after pupation or of earlier ages, they developed into autumn form in respect to wing pattern and their corpora allata did not stimulate ovarian maturation until 2 to 3 weeks after emergence. On the other hand, when the above operations had been performed 34 hr after the pupation or of later ages, they developed into summer form in respect to wing pattern and ovarian maturation was evident soon after the emergence. In these cases, stimulation of the corpora allata is closely connected with the development of seasonal-forms of wing pattern.
From these experiments, it is clear that the medial neurosecretory group cells of the pars intercerebralis in S-insects stimulate the corpora allata about 30 hr after pupation by way of the nervi corporis cardiaci and the activated corpora allata promote ovarian maturation throughout adult life. In A-insects, on the other hand, the medial neurosecretory group cells of the brain are inactive and fail to activate the corpora allata which in turn have no influence on ovarian maturation.     

Extracerebral caphalic neuroendocine complex of the blattids,Periplaneta americana (L.) and Neostylopyga rhombifolia (Stoll): An in situ study

A cardiaca-allatal commissural plexus (CACP) lies between and partly overlapping the postcommissural lobes of the corpora cardiaca (CC), the nervi corpori allati I (NCA I) and the corpora allata (CA). CACP, which is often continuous posteriorly with a complicated postallatal plexus (PAP), comprises a variable number of connectives with neurosecretory processes linking the cardiaca-commissural organ or dorsal cardiac commissure (containig tritocerebral fibres) to the NCA I. the allatal commissure and the CA. Neurosecretory processes are exchanged between the two halves of the cephalic neuroendocrine complex (CNC) both intracerebrally at different locales, possibly to ensure functional synchrony of CNC components. NCA I and CACP are drawn out with their stroma to varying extents over the CA. Histophysiological evidence suggests that part of the stainable secretion stored in, and or in axonal transit through CA may be released through CA surface; NCA I, the nervi cardiostomatogastrici, CACP, perhaps also NCA II may function as neurohaemal areas. A “directed” neurosecretory pathway could be distinguished from PAP to the foregut and the fat body. The degree of spatial intimacy detected between neurosecretory and stomatogastric components of CNC suggests that the two systems may function in an integrated fashion. The recurrent-oesophageal nerve complex serves not only for a direct transport of neurosecretion, but also as one of the sites of its release.     

Effects of electrocoagulation of cerebral neurosecretory cells and implantation of corpora allata on oöcyte development in Locusta migratoria

The implantation of active corpora allata into intact Locusta females during growth accelerates pre-vitellogenic oöcyte growth and vitellogenesis. Localised stimulation of yolk deposition follows the implantation of active corpora allata between the ovarioles demonstrating a gonadotrophic rôle for the corpus allatum hormone. Electrocoagulation of the median neurosecretory cells of the brain prevents vitellogenesis whilst pre-vitellogenic oöcyte growth occurs normally. Implantation of active corpora allata into females with ablated cerebral neurosecretory cells promotes vitellogenesis in a proportion of test animals although mature oöcytes are never produced.It is suggested that the rôle of the median neurosecretory cells during egg development in Locusta is primarily concerned with the activation and maintenance of activity of the corpora allata. The corpus allatum hormone acts both metabolically and gonadotrophically.     

Identification of neuronal pathways between the stomatogastric nervous system and the retrocerebral complex of the cockroach Periplaneta americana (L.)

Summary The neuronal pathways connecting the stomatogastric nervous system with the retrocerebral complex of the cockroach, Periplaneta americana, were investigated by means of axonal cobalt chloride iontophoresis. Somata in the hypocerebral ganglion and in the nervus recurrens sending their axons to different parts of the stomatogastric nervous system were traced. Some axons in the oesophageal nerve arise from large perikarya in the anterior part of the pars intercerebralis and pass via the NCCI to the corpora cardiaca and the oesophageal nerve. They form a profuse dendritic tree in the protocerebrum. Fibers of the NCC I and NCC II as well as the NCA I and NCA II enter the stomatogastric nervous system via the hypocerebral ganglion.     

Dynamics and metamorphosis of an identifiable peptidergic neuron in an insect

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion. 1994 John Wiley & Sons, Inc.     

Studies on the neurosecretory system of Ranatra elongata fabricius (Hemiptera: Nepidae) with reference to the distal fate of NCC I and II

With the help of PF and PAVB bulk-stained preparations and sections the neurosecretory system of Ranatra elongata has been described. Two medial, each of 9-10 cells, and two lateral, each of 3-4 cells, groups of neurosecretory cells have been observed in the protocerebrum. Only the A-cells have been found to be positive to PAVB histo- and cyto-chemical technique. Axons of the A-cells after traversing the proto- and deuto-cerebrum emerge from the tritocerebrum as the NCC I. The NCC I after bypassing the corpora cardiaca penetrate the aorta wall. There is no physical continuity between the corpora cardiaca and the NCC I and the two are separable. The NSM from the A-cells, transported by their nerve fibers, has been observed in the aorta wall. On the basis of large accumulation on NSM in the aorta wall the latter has been considered as the storage-and-release organ for the A-material. Corpora cardiaca are found to be devoid of A-material. Axons from the B-cells, after emergence from the tritocerebrum as the NCC II, have been observed to penetrate the corpora cardiaca. On the basis of ample amounts of B-material the glands have been considered as the storage-and-release organ for the B-material only. Observations are compared with results on related species and it is concluded that two independent organ complexes constitute the neurosecretory systemt of R. elongata. The A-cells, their pathways, the NCC I, and the aorta wall comprise the first; and the B-cells, their pathways, the NCC II, and the corpora cardiaca the second. The former is concerned with the elaboration, transportion, storage-and-release of the A-material and the latter with the B-material. Finally arguments are presented to include the aorta wall in the neurosecretory system.     

Immunocytochemical localization of prolactin-like antigenic determinants in the neuroendocrine system of the honeybee (Apis mellifica)

In the brain of the adult worker bee (Apis mellifica) prolactin-like (PRL) immunoreactive cells were localized in the lateral neurosecretory cell region and the subesophageal ganglion by means of the PAP procedure. These cells emit nerve fibers which pass through the neuropile of the brain to the corpora cardiaca where a great number of immunoreactive axon terminals is present. Test with antisera against rat pituitary prolactin and human luteinizing hormone were negative. These results indicate that hPRL material is produced in neurosecretory cells of the bee brain and transferred via axons to the corpora cardiaca for storage and subsequent release into haemolymph.     

Neuropepride Regulators of Insect Corpora Allata

SYNOPSIS. Neuropeptides of the insect brain that regulate juvenilehormone synthesis by the corpora allata include allatotropins,stimulatory modulators, and allatostatins, inhibitory modulators.A radiochemical assay for juvenile hormone synthesis by corporaallata in vitro was utilized in the high pressure liquid chromatographicisolation of brain neuropeptides leading to the determinationof their primary structure. Identified are an allatotropin andan allatostatin from a Lepidopteran, Manduca sexta, and a familyof five allatostatins from a Dictyopteran, Diploptera punctata.These neuropeptides are all unique, effective at low concentration(10^–10 to 10^–8 M), act quickly (within hrs) andappear to be effective only within the same order of insectsas that from which the peptides were isolated. The physiologicalstate of the corpora allata conditions the effectiveness ofthe allatostatins of D. punctata. These neuropeptide regulatorsof corpora allatal function may have multiple regulatory roles.This is indicated for D. punctata allatostatin I by specificreceptors in brain and fat body as well as in corpora allatalmembrane preparations, and also by immunocytochemical localizationof allatostatin I in medial nerve cells that terminate withinthe brain as well as in the lateral neurosecretory cells thatterminate on corpus allatum cells.     

Cockroach allatostatin-immunoreactive neurons and effects of cockroach allatostatin in earwigs

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to demonstrate the presence of allatostatin-immunoreactive cells and fiber tracts in the neuroendocrine system of the earwig Euborellia annulipes. The corpora cardiaca cells were not immunoreactive, nor were the neurosecretory endings of fiber tracts from the brain to the corpora cardiaca. No immunoreactive material was detected in the corpus allatum, although the corpus allatum contained neurosecretory endings, and some cells of the brain, including medial and lateral protocerebral cells, showed immunoreactivity. In addition, the recurrent and esophageal nerves were allatostatin-positive. The last abdominal ganglion contained immunoreactive somata, and immunoreactive axons of the proctodeal nerve innervated the rectum, anterior intestine, and posterior midgut. We did not detect reactive endocrine cells in the midgut. Allatostatin I at concentrations of 10^–5 and 10^–7 M did not inhibit juvenile hormone biosynthesis by E. annulipes corpora allata in vitro. This was true for glands of low activity from 2-day females and brooding females, as well as for relatively high activity glands from 10-day females. In contrast, 10^–7 M allatostatin I significantly and reversibly decreased hindgut motility. Motility was decreased in hindguts of high endogenous motility from 2-day females and in those of relatively low activity from brooding females. These results support the notion that a primary function of allatostatin might be to reduce gut motility. Arch. Insect Biochem. Physiol. 38:155–165, 1998. © 1998 Wiley-Liss, Inc.     

Localization of allatostatin-immunoreactive material in the central nervous system,stomatogastric nervous system,and gut of the cockroach Blattella germanica

Immunoreactivity against peptides of the allatostatin family having a typical YXFGL-NH₂ C-terminus has been localized in different areas of the central nervous system, stomatogastric nervous system and gut of the cockroach Blattella germanica. In the protocerebrum, the most characteristic immunoreactive perikarya are situated in the lateral and median neurosecretory cell groups. Immunoreactive median neurosecretory cells send their axons around the circumesophageal connectives to form arborizations in the anterior neuropil of the tritocerebrum. A group of cells in the lateral aspect of the tritocerebrum project to the antennal lobes in the deutocerebrum, where immunoreactive arborizations can be seen in the periphery of individual glomeruli. Nerve terminals were shown in the corpora allata. These terminals come from perikarya situated in the lateral neurosecretory cells in the pars lateralis and in the subesophageal ganglion. Immunoreactive axons from median neurosecretory cells and from cells positioned in the anteriormost part of the tritocerebrum enter together in the stomatogastric nervous system and innervate foregut and midgut, especially the crop and the valve between the crop and the midgut. The hindgut is innervated by neurons whose perikarya are located in the last abdominal ganglion. Besides immunoreactivity in neurons, allatostatin-immunoreactive material is present in endocrine cells distributed within the whole midgut epithelium. Possible functions for these peptides according to their localization are discussed. Arch. Insect Biochem. Physiol. 37:269–282, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical localization of gastrin-cholecystokinin-like material in the central nervous system of the migratory locust

Brain, corpora cardiaca (CC)-corpora allata (CA) complex, suboesophageal ganglion, thoracic and abdominal ganglia of adults, larvae and embryos of Locusta migratoria have been immunohistochemically screened for gastrin cholecystokinin (CCK-8(s]-like material. In adult, numerous immunoreactive neurons and nerve fibres are located, with a marked symmetry, in various parts of the brain and throughout the ventral nerve cord. In the median part of the brain, cell bodies belonging neither to cellular type A1 nor A2 (following Victoria blue-paraldehyde fuchsin staining) are immunopositive; their processes terminate in the upper protocerebral neuropile. In lateral parts of the brain, external cell bodies send axons into CC and some up to CA, other internal have processes which terminate in the neuropile of the brain. Two of these latter cells react also with methionine-enkephalin antiserum. In the ventral nerve cord, in addition to numerous perikarya, immunoreactive arborizations terminate in the neuropile or in close association with the sheath, at the dorsal part of all ganglia. This CCK-8(s) distribution pattern is observed only at the two last larval instars, but is precociously detected in the abdominal nerve cord of embryos, one day before hatching.     

Three-dimensional architecture of identified cerebral neurosecretory cells in an insect

The organization of identified neurosecretory cell groups in the larval brain of the tobacco hornworm, Manduca sexta, was investigated immunocytologically. Computer-assisted three-dimensional reconstruction was used to examine the architecture of the neurosecretory cell groups. The group III lateral neurosecretory cells (L-NSC-III) which produce the prothoracicotropic hormone are located dorsolaterally in the protocerebrum and extend axons medially that decussate to the contralateral lobe prior to exiting the brain through the nervi corporis cardiaci I + II. The group IIa2 medial neurosecretory cells (M-NSC IIa2) are located anteriorly in the medial dorsal protocerebrum. The axons of these cells also exit the brain via the contralateral nervi corporis cardiaci I + II. However, their axons traverse a different pathway through the brain from that of the L-NSC III axons. Each of the cell groups possesses elaborate dendrites with terminal varicosities. The dendrites can be classified into specific fields based upon their location and projection pattern within the brain. The dendrites for these two neurosecretory cell groups overlap in specific regions of the protocerebral neuropil. After the axons of these neurosecretory cells exit the brain through the retrocerebral nerve, they innervate the corpus allatum where they arborize to form neurohemal terminals in strikingly different patterns. The L-NSC III penetrate throughout the glandular structure and the M-NSC IIa2 terminals are restricted to the external sheath. A third group of cerebral neurosecretory cells, the ventromedial neurons (VM) which stain with the monoclonal antibody to prothoracicotropic hormone in Manduca, are located anteriorly in the medial region of the brain. The axons of these cells do not exit the brain to the retrocerebral complex, but rather pass through the circumesophageal connectives and ventral nerve cord. These neurons appear to be the same VM neurons that produce eclosion hormone. One dendritic field of the L-NSC III terminates in close apposition to the VM neurons. The distinct morphologies of these neurosecretory cell groups in relation to other cell groups and the distribution of neuropeptides within the neurons suggest that insect neurosecretory cells, like their vertebrate counterparts, may have multiple regulatory roles.