Immunocytochemical mapping of neuronal pathways from brain to corpora cardiaca/corpora allata in the cockroach Diploptera punctata with antisera against Met-enkephalin-Arg6-Gly7-Leu8

Summary Neuronal circuits in the brain and retrocerebral complex of the cockroach Diploptera punctata have been mapped immunocytochemically with antisera directed against the extended enkephalin, Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8). The pathways link median and lateral neurosecretory cells with the corpus cardiacum/corpus allatum complex. In females, nerve fibres penetrate the corpora allata and varicosities or terminals, immunoreactive to Met-8, surround the glandular cells. Males differ in having almost no Met-8 immunoreactivity in the corpora allata. The corpora cardiaca of both males and females are richly supplied with Met-8 immunoreactive material, in particular in the cap regions immediately adjacent to the corpora allata. A similarity in the amino-acid sequences of Met-8 and the C-terminus of the recently characterised allatostatins of D. punctata suggests that the pathways identified with the Met-8 antisera may be the same as those by which the allatostatins are transported from the brain to the corpus allatum. In comparative studies on the blowfly Calliphora vomitoria, similar neuronal pathways have been identified except that no sexual dimophism with respect to amounts of immunoreactive material within the corpus allatum has been observed. These results suggest a possible homology in the neuropeptide regulation of the gland.     

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

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

Regulation of Cyclic AMP Accumulation by Peptide Hormone Receptors in Immunocytochemically: Defined Astroglial Cells

Primary cultures of neonatal murine brain have been reported to express multiple receptors that regulate adenylate cyclase activity. Since for the most part these results were obtained with mixed cell cultures, it has been difficult to define receptor profiles for specific cell types. With this concern in mind a series of studies has been initiated designed to identify specific receptors present on highly purified, immunocytochemically defined astroglia derived from the cerebral cortices of neonatal rats. In this study the capacity of a variety of peptide hormones to regulate cyclic AMP metabolism in these cells was examined. Fibroblasts derived from the meninges represent a predictable source of contamination in primary CNS culture. Thus, to assign more clearly specific receptors to the astroglial cell population, receptor-mediated regulation of cyclic AMP accumulation was also examined in fibroblasts. Cyclic AMP accumulation in astroglia was stimulated by catecholamines (acting at beta 1-adrenergic receptors), prostaglandin E1, vasoactive intestinal polypeptide, alpha-melanocyte-stimulating hormone, and adrenocorticotropin. Bombesin, luteinizing hormone-releasing hormone, neurotensin, thyrotropin-releasing hormone, somatostatin, secretin, and vasopressin did not significantly increase cyclic AMP levels in these cultures. Catecholamines, acting at alpha 2-adrenergic receptors, and somatostatin inhibited agonist-stimulated cyclic AMP accumulation. In meningeal cell cultures catecholamines (acting at beta 2- and alpha 2-adrenergic receptors) and prostaglandin E1 regulated cyclic AMP levels. However, vasoactive intestinal peptide did not stimulate and somatostatin did not inhibit cyclic AMP accumulation in these cells.     

‘Neurosecretion’ by a classic cholinergic innervation apparatus

Summary Nerve terminals forming typical synapses with adrenal chromaffin tissues have been examined in the goldfish, frog (Rana pipiens), hamster and rat. Presumptive secretory inclusions present in the terminals are of two distinct types. Electron-lucent synaptic vesicles 30–50 nm in diameter are densely clustered adjacent to membrane thickenings and presumably discharge their contents into the synaptic clefts. Secretory granules (i.e. large dense-cored vesicles) 60–100 nm in diameter are more abundant in other parts of the terminals. Sites of granule exocytosis have been observed in each of the animals investigated. They are usually encountered within apparently undifferentiated areas of plasmalemma and only rarely occur within synaptic thickenings. Granule exocytosis from within synaptic terminals and chromaffin gland cells is most readily observed in specimens exposed, prior to fixation, to saline solutions containing both tannic acid, and 4-aminopyridine and/or elevated levels of K⁺. These findings show that the pattern of secretory discharge, involving both synaptic and non-synaptic release, which is widespread in invertebrate central nervous systems, is also characteristic of vertebrate, peripheral cholinergic terminals.     

Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin-releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon (Columba livia). Alterations following lactation and exposure to cold

Summary The distribution of VIP- and TRH-immunoreactivity in neurons and processes within the hypothalamus of the pigeon was investigated with light-microscopic immunocytochemical techniques. Most of the VIP-containing neurons are concentrated in the middle and caudal parts of the hypothalamus, with the greatest concentration of perikarya occurring in the medial and lateral part of the ventromedial hypothalamic nucleus and the infundibular nucleus. These cells give rise to axons that seem to extend into the median eminence. An extensive network of VIP-immunoreactive fibers and varicosities occupy the external layer of the median eminence. The majority of TRH-containing neurons is found in the anterior hypothalamus with the greatest concentration of cells in the magnocellular preoptic, medial preoptic, suprachiasmatic and paraventricular nuclei. TRH-immunoreactive fibers and varicosities form a dense arborization in the external layer of the median eminence. Lactation seems to induce substantial changes in VIP as well as in TRH-immunostaining in the median eminence and other hypothalamic regions as compared to control, sexually active animals. Furthermore, TRH-immunoreactivity decreased in the median eminence following 60-min exposure to cold. These results suggest that VIP- and TRH-containing pathways in the pigeon hypothalamus are involved in the mediation of neuroendocrine responses.     

Bombesin increases dopamine function in rat brain areas

Bombesin is a tetradecapeptide heterogenously distributed in the mammalian brain. Bombesin (45 micrograms) given intracisternally (IC) to unanesthetized rats increased the accumulation of dihydroxyphenylalanine (DOPA) in striatum, olfactory tubercles and hypothalamus after DOPA-decarboxylase inhibition, thus indicating an increased dopamine synthesis. A dose-dependent increase in dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), the principal dopamine metabolites, was seen in several brain areas 1 hr after IC injection of bombesin (0-60 micrograms). In striatum and olfactory tubercles HVA increased more than DOPAC with a maximal increase after 30-45 micrograms. In a time-course experiment a biphasic change of dopamine metabolites was observed in the olfactory tubercles with an actual decrease in metabolite levels 4 hr after 60 micrograms IC bombesin injection. Co-administration of bombesin and naloxone (8 mg/kg IP) or ethanol (2.25 g/kg IP) did not affect the increase in dopamine metabolites seen after bombesin alone. The action of IC administered bombesin on dopamine function was most pronounced in hypothalamus indicating a neuroendocrine regulatory of the peptide.     

Topographical relationships between catecholamine-and neuropeptide-containing fibers in the median eminence of the newt,Triturus alpestris

Summary Dopaminergic and peptidergic nerve fibers were simultaneously demonstrated with a double-labeling technique at the ultrastructural level. The first antibody, raised against tyrosine hydroxylase, was applied during the preembedding phase and visualized with the peroxidase method. The second antibody, raised against one of the peptides met-enkephalin, somatostatin or gonadotropin-releasing hormone (GnRH), was applied to the ultrathin sections and visualized with gold-labeled goat anti-rabbit IgG. The fibers of both categories were present in the zona externa of the median eminence, frequently contacting the basal lamina of the portal vessels. In addition, topographical relationships between different types of nerve fibers were observed in the perivascular areas, although there were no morphological signs of synaptic specializations. Using serial sections, it could be established that one GnRH-fiber contacted both a dopaminergic fiber and a fiber immunoreactive for met-enkephalin. The observations support earlier physiological data concerning the regulation of the hypothalamo-hypophyseal axis, with special emphasis on the release of neurohormones in the median eminence of the newt.     

Determination of cholecystokinin tetrapeptide and cholecystokinin octapeptide sulfate in different rat brain regions by high-pressure liquid chromatography with electrochemical detection

A method for the determination of cholecystokinins in biological material, based on high-pressure liquid chromatography with direct electrochemical detection (HPLC-EC), is described. Using this method, the levels of cholecystokinin tetrapeptide and octapeptide sulfate in rat brain cortex, hippocampus, striatum, and brain stem were measured and found to be comparable to those reported using radioimmunoassay methods. We show that HPLC-EC is sensitive enough to accurately determine neuropeptides in brain tissue without prior derivatization and is therefore, due to its simplicity, an attractive alternative to existing methods.     

3D-QSAR based optimization of insect neuropeptide allatostatin analogs

Allatostatins (AST) are neuropeptides originally described as inhibitors of juvenile hormone (JH) synthesis in insects. Consequently, they have been considered as potential lead compounds for the discovery of new insect growth regulators (IGRs). In the present work, receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) was studied with 48 AST analogs, and a general approach for novel potent bioactive AST analogs is proposed. Hence, six novel AST analogs were designed and synthesized. Bioassays indicated that the majority novel analogs exhibited potent JH inhibitory activity, especially analog A6 (IC₅₀: 3.79?nmol/L), which can be used as lead compound to develop new IGRs.     

The role of activin in neuropeptide induction and pain sensation

Signals from target tissues play critical roles in the functional differentiation of neuronal cells, and in their subsequent adaptations to peripheral changes in the adult. Sensory neurons in the dorsal root ganglia (DRG) provide an excellent model system for the study of signals that regulate the development of neuronal diversity. DRG have been well characterized and contain both neurons that convey information from muscles about limb position, as well as other neurons that provide sensations from skin about pain information. Sensory neurons involved in pain sensation can be distinguished physiologically and antigenically, and one hallmark characteristic is that these neurons contain neuropeptides important for their functions. The transforming growth factor (TGF) beta family member activin A has recently been implicated in neural development and response to injury. During sensory neuron development, peripheral target tissues containing activin or activin itself can regulate pain neuropeptide expression. Long after development has ceased, skin target tissues retain the capacity to signal neurons about changes or injury, to functionally refine synapses. This review focuses on the role of activin as a target-derived differentiative factor in neural development that has additional roles in response to cutaneous injuries in the adult.