IMMUNOHISTOCHEMICAL LOCALIZATION OF DELTA SLEEP-INDUCING PEPTIDE-LIKE IMMUNOREACTIVITY IN THE CENTRAL NERVOUS SYSTEM AND PITUITARY OF THE FROG RANA RIDIBUNDA Laurent Yon, M. Feuilloley, Y. Charnay, H. Vaudry To cite this version: Laurent Yon, M. Feuilloley, Y. Charnay, H. Vaudry. IMMUNOHISTOCHEMICAL LOCALIZATION OF DELTA SLEEP-INDUCING PEPTIDE-LIKE IMMUNOREACTIVITY IN THE CENTRAL NERVOUS SYSTEM AND PITUITARY OF THE FROG RANA RIDIBUNDA. Neuroscience, Else- vier - International Brain Research Organization, 1992, 47, pp.221-240. 10.1016/0306-4522(92)90135- O. hal-01706416 HAL Id: hal-01706416 https://hal-normandie-univ.archives-ouvertes.fr/hal-01706416 Submitted on 18 Jul 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. IMMUNOHISTOCHEMICAL LOCALIZATION OF DELTA SLEEP-INDUCING PEPTIDE-LIKE IMMUNOREACTIVITY IN THE CENTRAL NERVOUS SYSTEM AND PITUITARY OF THE FROG RANA RIDIBUNDA L. YoN,* M. Ffil.Jn.LOLEY,*Y. ÛIARNAyt and H. VAUDRY*t •European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, University of Rouen, 76134 Mont-Saint-Aignan, France tDivision of Morphological Psychopathology, University Department of Psychiatry, 1225 Chêne-Bourg, Geneva, Switzerland Allltract-The purpose of the present study was to investigate the distribution of delta sleep-inducing peptide in the brain and pituitary of the frog Rana ridibunda and to determine the possible effect of this nonapeptide on adrenocorticotropic hormone and corticosteroid secretion. Delta sleep-inducing peptide­ like immunoreactivefibres were observedthroughout the brain of the frog. Thesefibres generally exhibited the characteristics of glial cell processes. Scarce delta sleep-inducing peptide-positive fibres were seen in the olfactory bulb and in the periventricular areas of the telencephalon. In the diencephalon, numerous delta sleep-inducing peptide-containing processes were noted in the preoptic nucleus, the infundibular nuclei and the median eminence. A few cerebrospinal lluid-contacting cells were visuali7.Cd in the ventral nucleus of the infundibulum. Delta sleep-inducing peptide-positive fibres were also observed in the mesencephalon, radiating through the different layers of the tectum. In the cerebellum, ail Purkinje cells exhibited delta sleep-inducing peptide-like immunoreactivity. More caudally,numerous delta sleep-induc­ ing peptide-positivefibres were noted in the vestibular nucleus of the rhombencephalon.A densenetwork of delta sleep-inducing peptide-containingfibres was seenin the pars nervosaof the pituitary. In the distal lobe, a population of endocrine cells located in the anteroventral region contained delta sleep-inducing peptide-immunoreactive material. Labellingof consecutivesections of the pituitary by delta sleep-inducing peptide and adrenocorticotropic hormone antiserum revealed that a delta sleep-inducing peptide-related peptide is expressed in corticotroph cells. The possible role of delta sleep-inducing peptide in the control of adrenocorticotropichormone and corticosteroidrelease was studied invitro, using theperifusion system technique. Administration of graded doses of delta sleep-inducing peptide (from 10-1 to 10-6 M) to perifused frog anterior pituitary cells did not affect the spontaneous release of adrenocorticotropic hormone. In addition, prolonged infusion of delta sleep-inducing peptide (I0-6 M) did not alter the stimulatory effect of corticotropin-releasing factor (10-7 M) on adrenocorticotropic hormone secretion. Similarly, exposureof frog interrenal slicesto delta sleep-inducing peptidedid not induceany modification of spontaneous or adrenocorticotropic hormone-evoked secretion of corticosterone and aldosterone. Our results provide the first evidence for the presence of a delta sleep-inducing peptide-related peptide in lower vertebrates. The occurrenceof delta sleep-inducing peptide-likeimmunoreactivity in specificareas of the brain suggests that the peptidemay act as a neuromodulator.Although delta sleep-inducing peptide was detected in pituitary corticotrophs, we did not observe any effect of exogenous delta sleep-inducing peptide on adrenocorticotropic hormone or corticosteroid secretion in vitro. The nonapeptide delta sleep-inducing peptide(DSIP) rence of DSIP-immunoreactive material was also was originallycharacterized from the cerebral venous demonstrated in peripheral organs such as the pitu­ blood of the rabbit for its hypnogenic activity.43 itary,7 ·8·11·13·14·17·46 stomach,22 intestine9 and adrenal Immunocytochemical and biochemical studies have medullary gland.7·17 Howcver, the exact nature of the demonstrated that DSIP is widely distributed in the immunoreactive material is still discussed.Chromato­ CNS of man,47 cat,13 rabbit11 and rat.14·18 The occur- graphie analysis of tissue extracts and body fluids suggests the existence of a family of DSIP-related peptides. 25 tTo whom correspondence should be addressed. Pharmacological studies have shown that besides Abbreviations: ACTH,adrenocorticotropic hormone; ANF, its sleep-inducing activity,23.25.43 DSIP exhibits anal­ atrial natriuretic factor; CRF, corticotropin-releasing gesic55 and immunodepressor properties,54 and factor; DSIP, delta sleep-inducing peptide; GH, growth reduces blood pressure,2 6 motor activity21 and hormone; HEPES, N-2 hydroxyethylpiperazine-N-2- 56 ethanesulphonic acid; LH, luteinizing hormone; LH­ body temperature. Recent studies also suggest that RH, luteinizing hormone-releasing hormone; ix-MSH, DSIP is involved in the control of release of ix-melanocyte-stimulating hormone; MCH, melanin­ anterior pituitary hormones. In particular, DSIP concentrating hormone; NPY, neuropeptide Y; PBS, induces a marked decrease of corticotropin-releasing phosphate-buffered saline; RIA, radioimmunoassay; factor (CRF)-evoked adrenocorticotropic hormone SRIF, somatotropin-release inhibiting factor; TRH, 1 24 39 thyrotropin-releasing hormone. (ACTH) secretion in rodents and humans.8• 0· • Concurrently, DSIP stimulates the release of both sections (3-6 µm thick) were eut in a cryostat (Frigocut 29 42 luteinizing hormone (LH) • and growth hormone 2700, Reichert Jung) and mounted on glass slides coated (GH).28•31 DSIP has also been reported to act at the with gelatin. The slides were processed for indirect immu­ nofluorescence Jabelling as previously described. 15 Briefly. hypothalamic level to inhibit the release of somato­ tissue sections were incubated overnight at 4' C with a statin. 30 Taken together, these data support the con­ polyclonal antiserum or a monoclonal antibody to DSIP cept that DSIP exerts neurohormonal and/or (ascite fluid) diluted 1/100 in 0.1 M PBS containing 0.3% neuromodulatory functions in mammals. Triton X-100 and 1% human serum albumin. The sections were rinsed three times in PBS for 30 min and then Studies conceming the localization, characteriz­ incubated for I h at room temperature with fluorescein ation and biological effects of DSIP have only been isothiocyanate-conjugated goat anti-rat y-globulin (Nordic performed in mammals. The distribution of many Immunology) diluted 1/60. Finally, the sections were rinsed other neuropeptides, such as atrial natriuretic factor for 1 h in PBS and mounted with PBS-glycerol ( L' 1 ). (ANF),38 neuropeptide Y (NPY)16 ix-melanocyte­ The preparations were examined under a Leitz Orthoplan 6 microscope equipped with a Vario-Orthomat photographie stimulating hormone (ix-MSH) and their roles in the system. regulation of the hypothalamo-hypophyseal-adrenal Nomenclature of brain areas was based on the atlas or axis have been investigated in both mammalian and Wada.52 The anatomical structures were identified on non-mammalian vertebrates. These studies have sections stained with Cresyl Violet. The possible co-existence of DSIP- and ACTH-like im­ often revealed marked species differences and thus munoreactivity in the same cells of the pituitary was inves­ provide crucial information conceming the functions tigated using 3-µm consecutive sections. One section was of regulatory peptides during evolution. incubated with the R7 DSIP antiserum and the adjacent The aim of the present work was to investigate the section with the ACTH antiserum diluted 1/1500. For each distribution of DSIP-like immunoreactivity in the pair of sections, the first one was reversed so that the incubation was performed exactly on symmetrical sections. brain and pituitary of the frog Ranaridibunda, and to determine the possible role of DSIP on the hypotha­ Controls lamo-pituitary-adrenal axis. To study the specificity of the immunoreaction, the following controls were performed: (i) substitution of the different primary antisera with PBS; (ii) replacement of EXPERIMENTAL PROCEDURES polyclonal DSIP antibodies with non-immune rat serum diluted 1/100 and replacement of monoclonal antibodies by Animais normal ascite fluid; and (iii) pre-incubation of the DSIP Adult male frogs (Rana ridibunda)weighjng 30--40 g, were antibodies (diluted 1/100) with synthetic DSIP (1 o ·-6 M). obtainedfrom a commercial source(Couétard,