Multiple peptide immunoreactivities in the nervous system of Aeschna cyanea (Insecta, Odonata). An immunohistochemical study using antisera to cholecystokinin octapeptide, somatoliberin, vasoactive intestinal peptide, motilin and proctolin

By use of the indirect immunoperoxidase method, the brain, the suboesophageal ganglion and the corpora cardiaca of the dragonfly Aeschna cyanea have been shown to be immunoreactive to proctolin antiserum and to several mammalian peptide antisera including unsulfated cholecystokinin octapeptide (CCK-8 NS) (Andriès et al. 1989), vasoactive intestinal peptide (VIP), human somatoliberin (hGRF) (Andriès et al. 1984) and motilin antisera. Immunohistochemical studies have been performed on material fixed in a solution of picricacid paraformaldehyde or in Bouin Hollande's sublimate solution. Antisera were applied on alternate sections or, according to the elution-restaining method of Tramu et al. (1978), one after another on the same section. Multiple peptide immunoreactivities appear expressed in the brain and the suboesophageal ganglion. Cells reactive to both hGRF and VIP antisera show also gastrin/CCK-like immunoreactivity and some of them are also detected by motilin antiserum. Besides, some cells immunopositive to CCK-8 NS and motilin antisera do not show hGRF or VIP immunoreactivity. At least, two pairs of protocerebral cells appear immunoreactive to both CCK-8 NS and proctolin antisera. Therefore, the present observations support our previously developed idea (Andriès et al. 1989) that the population of CCK-like cells is heterogenous.     

Multiple peptide immunoreactivities in the nervous system of Aeschna cyanea (Insecta,Odonata)

Summary By use of the indirect immunoperoxidase method, the brain, the suboesophageal ganglion and the corpora cardiaca of the dragonfly Aeschna cyanea have been shown to be immunoreactive to proctolin antiserum and to several mammalian peptide antisera including unsulfated cholecystokinin octapeptide (CCK-8 NS) (Andriès et al. 1989), vasoactive intestinal peptide (VIP), human somatoliberin (hGRF) (Andriès et al. 1984) and motilin antisera. Immunohistochemical studies have been performed on material fixed in a solution of picricacid paraformaldehyde or in Bouin Hollande's sublimate solution. Antisera were applied on alternate sections or, according to the elution-restaining method of Tramu et al. (1978), one after another on the same section. Multiple peptide immunoreactivities appear expressed in the brain and the suboesophageal ganglion. Cells reactive to both hGRF and VIP antisera show also gastrin/CCK-like immunoreactivity and some of them are also detected by motilin antiserum. Besides, some cells immunopositive to CCK-8 NS and motilin antisera do not show hGRF or VIP immunoreactivity. At last, two pairs of protocerebral cells appear immunoreactive to both CCK-8 NS and proctolin antisera. Therefore, the present observations support our previously developed idea (Andriès et al. 1989) that the population of CCK-like cells is heterogenous.     

Tryptophanyl-tRNA synthetase-like immunoreactivity in the central nervous system and midgut of the migratory locust. Comparisons with gastrin-cholecystokinin-like and octopamine-like immunoreactivity

A tryptophanyl-tRNA synthetase (TrpRS)-immunoreactivity is localized in various neurosecretory cells of all ganglia of the central nervous system of the Orthoptera Locusta migratoria, except in deutocerebrum, and in endocrine cells of the midgut. It has been observed that TrpRS-like material never co-localizes either with CCK-like or octopamine-like material. TrpRS immunoreactive perikarya and processes that ramify extensively throughout the neuropiles have been detected in the protocerebrum, optic lobes, tritocerebrum, suboesophageal, thoracic and abdominal ganglia. In the lateral protocerebrum, a particular TrpRS pathway different from the lateral gastrin cholecystokinin (CCK-8(s] pathway is revealed, certain of these processes terminating in the glandular part of the corpora cardiaca. In the metathoracic ganglion, have been observed numerous immunoreactive cell bodies and processes in the neuropiles. Some of them constitute a major pathway and which are distinct from octopamine (OA) cells but in close vicinity with the latter. In the midgut immunopositive TrpRS-like cells are dispersed among the regenerative and digestive cells of the epithelium; they are different from gastrin-cholecystokinin positive cells. The various TrpRS-like immunoreactivities identified in Locusta indicate that TrpRS-like material may occur in different tissues of organisms other than Vertebrates. These results suggest also that TrpRS-like enzyme could be involved in functions other than aminoacylation, as in Vertebrates.     

Tryptophanyl-tRNA synthetase-like immunoreactivity in the central nervous system and midgut of the migratory locust. Comparisons with gastrin-cholecystokinin-like and octopamine-like immunoreactivity

Summary A tryptophanyl-tRNA synthetase (TrpRS)-immunoreactivity is localized in various neurosecretory cells of all ganglia of the central nervous system of the Orthoptera Locusta migratoria, except in deutocerebrum, and in endocrine cells of the midgut. It has been observed that TrpRS-like material never co-localizes either with CCK-like or octopamine-like material.TrpRS immunoreactive perikarya and processes that ramify extensively throughout the neuropiles have been detected in the protocerebrum, optic lobes, tritocerebrum, suboesophageal, thoracic and abdominal ganglia. In the lateral protocerebrum, a particular TrpRS pathway different from the lateral gastrin cholecystokinin (CCK-8(s)) pathway is revealed, certain of these processes terminating in the glandular part of the corpora cardiaca. In the metathoracic ganglion, have been observed numerous immunoreactive cell bodies and processes in the neuropiles. Some of them constitute a major pathway and which are distinct from octopamine (OA) cells but in close vicinity with the latter. In the midgut immunopositive TrpRS-like cells are dispersed among the regenerative and digestive cells of the epithelium; they are different from gastrin-cholecystokinin positive cells.The various TrpRS-like immunoreactivities identified in Locusta indicate that TrpRS-like material may occur in different tissues of organisms other than Vertebrates. These results suggest also that TrpRS-like enzyme could be involved in functions other than aminoacylation, as in Vertebrates.     

Evidence for a crustacean hyperglycemic hormone-like molecule in the nervous system of the stick insect,Carausius morosus

Summary Histological sections of the brain, suboesophageal ganglion, and the corpora cardiaca/corpora allata complex were examined for the presence of crustacean hyperglycemic hormone-like immunoreactive material. With the use of an antiserum directed against the hyperglycemic hormone of Carcinus maenas, immunofluorescence was found in the median portion of the pars intercerebralis, and the corpora cardiaca. Extracts of corpora cardiaca were examined by radioimmunoassay for competitive binding to the antiserum; one pair of corpora cardiaca contains at least 7 pg crustacean hyperglycemic hormone-like material.     

NPY-like peptides occur in the nervous system and midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata

The distribution of the NPY-like substances in the nervous system and the midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata was determined by immunocytochemistry using an antiserum directed against synthetic porcine NPY. The peroxidase-antiperoxidase procedure revealed that NPY immunoreactive cell bodies and nerve fibers were observed in the brain, optic lobes, corpora cardiaca, suboesophageal ganglion and ventral nerve cord of the locust and in the brain, optic lobes and suboesophageal ganglion of the fleshfly. In the locust midgut, numerous endocrine cells and nerve fibers penetrating the outer musculature contained NPY-like immunoreactivity. The concentrations of NPY immunoreactive material in acetic acid extracts of locust brain, optic lobes, thoracic ganglia, ovaries and midguts was measured using a specific radioimmunoassay technique. The dilution curves of the crude tissue extracts were parallel to the standard curve. The highest amount of NPY-like immunoreactivity was found in the locust ovary and midgut. Reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay were used to characterize the NPY-like substances in the locust brain and midgut. HPLC-analysis revealed that NPY-immunoreactivity in the locust brain eluted as three separate peaks. The major peak corresponded to a peptide less hydrophobic than synthetic porcine NPY. RP-HPLC analysis of midgut extracts revealed the presence of an additional NPY-immunoreactive peak which had a retention time similar to the porcine NPY standard. The present data show the existence of a widespread network of NPY immunoreactive neurons in the nervous system of the locust and the fleshfly. Characterization of the immunoreactive substances indicates that peptides similar but not identical to porcine NPY are present in the central nervous system and midgut of insects.     

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.     

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.     

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

Summary 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, immunore-active 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.     

The release of a pheromonotropic neuropeptide, PBAN, in the turnip moth Agrotis segetum, exhibits a circadian rhythm

In the female turnip moth, Agrotis segetum, a pheromone biosynthesis activating neuropeptide (PBAN) stimulates sex pheromone biosynthesis which exhibits a daily rhythm. Here we show data supporting a circadian rhythm in PBAN release from the corpora cardiaca, which we propose regulates the endogenous rhythm in sex pheromone biosynthesis. This conclusion is drawn as the observed daily rhythm in PBAN-like immunoreactivity in the hemolymph is persistent in constant darkness and is phase-shifted by an advanced light:dark cycle. PBAN-like immunoreactivity was found in the brain, the optic lobe, the suboesophageal ganglion and in the retrocerebral complex. In each hemisphere ca. 10 immunopositive neurons were observed in the pars intercerebralis and a pair of stained somata in the dorso-lateral protocerebrum. A cluster of cells containing PBAN-like immunoreactive material was found in the tritocerebrum and three clusters of such cells were found in the SOG. Their processes reach the corpora cardiaca via nervi corporis cardiaci and the dorsal surface of the corpora allata via the nervi corporis allati.     

Gastrin/CCK-like immunoreactivity in the nervous system of coelenterates

Using immunocytochemistry, gastrin/CCK-like immunoreactivity is found in sensory nerve cells in the ectoderm of the mouth region of hydra and in nerve cells in the endoderm of all body regions of the sea anemone tealia. These results are corroborated by radioimmunoassay: One hydra contains at least 5 fmole and one tealia at least 2 nmole gastrin/CCK-like immunoreactivity. Reactivities towards gastrin and CCK antisera with different specificities suggest that the coelenterate gastrin/CCK-like peptide contains the C-terminal amino-acid sequence common to mammalian gastrin and CCK. In addition the radioimmunochemical data indicate that the coelenterate peptide also contains an amino-acid sequence that resembles the sequence 20-30 of porcine CCK-33, but that no other sequences of gastrin are present. Thus, it is probably more CCK-like than gastrin-like.     

Gastrin/CCK-like immunoreactivity in the nervous system of coelenterates

Summary Using immunocytochemistry, gastrin/CCK-like immunoreactivity is found in sensory nerve cells in the ectoderm of the mouth region of hydra and in nerve cells in the endoderm of all body regions of the sea anemone tealia. These results are corroborated by radioimmunoassay: One hydra contains at least 5 fmole and one tealia at least 2 nmole gastrin/CCK-like immunoreactivity. Reactivities towards gastrin and CCK antisera with different specificities suggest that the coelenterate gastrin/CCK-like peptide contains the C-terminal amino-acid sequence common to mammalian gastrin and CCK. In addition the radioimmunochemical data indicate that the coelenterate peptide also contains an amino-acid sequence that resembles the sequence 20–30 of porcine CCK-33, but that no other sequences of gastrin are present. Thus, it is probably more CCK-like than gastrin-like.     

An active principle from the corpora cardiaca,corpora allata and suboesophageal ganglion of the locust,inducing egg diapause in Bombyx mori

The activity of the substance(s) which are contained in the cephalic endocrine organs of the locust which induce egg diapause in Bombyx mori was examined by implantation and injection of saline extracts of these organs. Extracts from the median and lateral neurosecretory parts of the locust brain were not effective in inducing egg diapause. Extracts of the corpora cardiaca, corpora allata, and suboesophageal ganglion of the locust induced diapause eggs in Bombyx pharate adults from which the suboesophageal ganglion had been removed. The first two extracts could induce egg diapause even in isolated abdomens of pharate adults of Bombyx. In the locust corpora cardiaca, the activity was present only in the glandular lobe and not in the nervous region. This activity decreased when the nervi corporis cardiaci I and II and of nervi corporis allati I were cut. Allatectomy also brought about a decrease in the activity in the glandular lobe which could not be restored by the injection of juvenile hormone. The activity in the corpora allata was enhanced slightly by the disconnection though not significantly.From these results, it is assumed that the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust contain and active principle(s) capable of inducing egg diapause in Bombyx mori. The nervous connections between the brain, corpora cardiaca, and corpora allata are essential for the accumulation of the active substance(s) in the glandular lobes of the corpora cardiaca.     

Distribution of locustamyotropin-like immunoreactivity in the nervous system of Locusta migratoria.

Locustamyotropin-like immunoreactivity was visualized in the nervous system of Locusta migratoria by means of the peroxidase antiperoxidase method. Highly specific antibodies to the carboxy-terminus of the locustamyotropins were obtained by elution through an affinity column to which Lom-MT II was covalently bound. Specific cells in the nervous system of Locusta migratoria contain substances immunoreactive to anti-locustamyotropin. In total, about 100 cells immunoreactive to the Lom-MT-II antiserum were detected in the head ganglia, in the abdominal neuromeres of the metathoracic ganglion, and in the five free abdominal ganglia. In the brain, immunoreactive cell groups were situated in the inner and outer edge of the tritocerebrum. Prominent axon bundles tightly surround the tractus I to the corpora cardiaca. The corpora allata were innervated by the nervus corporis allati I coming from the corpora cardiaca and by fibers in the nervus corporis allati II originating from cell bodies in the suboesophageal ganglion. Immunoreactive cell bodies in the suboesophageal and abdominal ganglia are distributed along the anterior posterior midline axis, both dorsally and ventrally. The processes of the cell bodies in the abdominal ganglia leave the ganglia and were traced in the respective median nerves into the neurohaemal organs. Since the Lom-MT-II antiserum cross-reacts with all peptides of the locustamyotropin family that have a carboxy-terminus in common, these cells may contain one or several locustamyotropins. The Lom-MT antiserum also recognizes pheromone biosynthesis activating neurohormone, as was revealed by the intensive labeling of suboesophageal cell bodies in Bombyx mori.     

Serotonin immunoreactive neurons in the central nervous system of an insect (Periplaneta americana)

Serotonin-like immunoreactivity was mapped in the central nervous system (CNS) of the cockroach, Periplaneta americana. Immunoreactive staining occurred in every ganglion of the CNS. The largest numbers of immunoreactive somata were detected in the optic lobes and the brain, and lowest numbers in the first and second thoracic ganglia. Dense stained fibers, ramifications, and varicosities were found in all ganglia, and numerous axon like processes occurred in all interganglionic connectives. Immunoreactive processes were not, however, detected in most of the peripherally projecting nerve roots. Processes were found only in roots of the suboesophageal ganglion and the tritocerebral lobes of the brain. A comparison of the map for serotonin immunoreactivity with one generated for the pentapeptide transmitter proctolin suggests that the two systems overlap only in the suboesophageal ganglion and the tritocerebrum. The amine and peptide may co-occur in neurons in these regions. The serotonin immunoreactive system appeared significantly different from the octopaminergic system of the ventral nerve cord. Seventy-two potentially identifiable immunoreactive cells were located in the cockroach CNS. Some of these may be suitable for physiological study of the functional role of serotonin.     

Quantification of periviscerokinin‐1 in the nervous system of the American cockroach,Periplaneta americana: An insect neuropeptide with unusual distribution

This study was undertaken to reveal the quantitative distribution of the insect neuropeptide periviscerokinin‐1 (Pea‐PVK‐1) in the central nervous system of Periplaneta americana and to demonstrate that neurons stained in a previous immunohistochemical study contain authentic Pea‐PVK‐1. For this, we combined ELISA, HPLC, and MALDI‐TOF mass spectrometry. The high specificity of the used antiserum enabled the quantification of Pea‐PVK‐1 in unseparated tissue extracts. No cross‐reactivities with other insect neuropeptides were detected in ELISA. Only two immunoreactive fractions, coeluting with synthetic Pea‐PVK‐1 in its oxidized and nonoxidized form, were found in HPLC‐separated extracts of the brain, suboesophageal ganglion, metathoracic ganglion, second abdominal ganglion with or without perisympathetic organ, and terminal ganglion. By using MALDI‐TOF mass spectrometry, we were able to confirm the existence of authentic Pea‐PVK‐1 in these fractions. The abdominal perisympathetic organs contained 6.3 pmol Pea‐PVK‐1 per animal; another 1.3 pmol were found in the abdominal ganglia. More than 90% of the total 8.2 pmol in the central nervous system was found in the abdominal ganglia and their perisympathetic organs. The corpora cardiaca and corpora allata did not contain immunoreactive material, suggesting that Pea‐PVK‐1 is not released by the cephalic neurohaemal system. The quantitative distribution of Pea‐PVK‐1 differs considerably from that of other known insect neuropeptides. Arch. Insect Biochem. Physiol. 40:203–211, 1999. © 1999 Wiley‐Liss, Inc.     

The cycling and distribution of PER-like antigen in relation to neurons recognized by the antisera to PTTH and EH in Thermobia domestica

The cephalic nervous system of the firebrat contains antigens recognized by antisera to the clock protein period (PER), the prothoracicotropic hormone (PTTH) and the eclosion hormone (EH). The content of the 115 kDa PER-like antigen visualized on the western blots fluctuates in diurnal rhythm with a maximum in the night. The oscillations entrained in a 12:12 h light/dark (LD) cycle persist in the darkness and disappear in continuous light. They are detected by immunostaining in 14 pairs of the protocerebral neurons and are extreme in four suboesophageal neurons and two cells in each corpus cardiacum that contain PER only during the night phase. No circadian fluctuations occur in three lightly stained perikarya of the optic lobe. Five cell bodies located in each brain hemisphere between the deuto-and the tritocerebrum retain weak immunoreactivity under constant illumination. In all cells, the staining is confined to the cytoplasm and never occurs in the cell nuclei. The cells containing PER-like material do not react with the anti-PTTH and anti-EH antisera, which recognize antigens of about 50 and 20 kDa, respectively. The anti-PTTH antiserum stains in each brain hemisphere seven neurons in the protocerebrum, eight in the optic lobe, and 3–5 in the posterior region of the deutocerebrum. The antiserum to EH reacts in each hemisphere with just two cells located medially to the mushroom bodies. No cycling of the PTTH-like and EH-like antigens was detected.     

Endocrine cells with gastrin/cholecystokinin-like immunoreactivity in the pancreas of the spiny dogfish,Squalus acanthias

Endocrine cells containing gastrin/cholecystokinin (CCK)-like immunoreactivity were localized to the islet tissue in the pancreas of the spiny dogfish. Most of these cells were located in the 'intestinal' lobe of the pancreas; only occasional cells were observed in the 'splenic' lobe. The gastrin/CCK-like immunoreactive cells were often co-localized with the 'classical' pancreas hormones (insulin, glucagon and somatostatin). Radioimmunoassay of water extracts with a C-terminally directed antiserum revealed high levels of immunoreactive material in the intestinal part (48.6 +/- 19.9 pmol/g) and lower levels (4.5 +/- 0.6 pmol/g) in the splenic part. Acetic acid extracts of the intestinal lobe contained low levels (6.8 +/- 3.3 pmol/g) of gastrin/CCK-like immunoreactivity, whereas corresponding extracts of the splenic part showed no immunoreactivity. When the extracts were subjected to DEAE ion-exchange chromatography the gastrin/CCK-like peptides eluted as a major peak. After Sephadex gel filtration, pooled immunoreactive material from the main DEAE chromatographic peak eluted at a position close to that of CCK4. Further characterization by ion-exchange and reversed-phase HPLC showed that, in general, the immunoreactive material behaved like the shorter forms of the gastrin/CCK family (CCK4/G5 and CCK8/Cae 3-10).     

Immunoreactive material resembling vertebrate neuropeptides and neurophysins in the brain,suboesophageal ganglion,corpus cardiacum and corpus allatum of the dictyopteran Periplaneta americana L.

Summary The peroxidase-antiperoxidase technique (Vandesande and Dierickx 1976) with antibodies raised against several vertebrate neuropeptides and neurophysins, was applied] to 4-m Paraplast-embedded serial sections of in situ fixed brains and adjacent suboesophageal ganglion (SOG), corpora cardiaca (CC) and corpora allata (CA) of the blattarian insect Periplaneta americana L. Substances immunologically related to bovine neurophysin I (NPI) and II (NPII), synthetic arginine vasopressin (AVP) and synthetic oxytocin (OT) were found to be differentially distributed in the central nervous system. The differences among all four antigens demonstrated became clearly evident by immunohistochemical double-staining procedures (Vandesande 1983); no overlapping was observed. The same double-staining technique revealed that these vertebrate-type substances occur in other neurosecretory cells and axons than those containing CRF- and ACTH-like material as reported earlier (Verhaert et al. 1984).