Localization of vasopressin-, vasoactive intestinal polypeptide-, peptide histidine isoleucine-and somatostatin-mRNA in rat suprachiasmatic nucleus

Summary Messenger RNAs (mRNA) coding for vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), somatostatin and vasopressin were localized in the suprachiasmatic nucleus (SCN) of the rat hypothalamus using in situ hybridization histochemistry. Specific mRNA coding for each of these peptides was distributed in areas coextensive with the immunohistochemical localization of the appropriate peptide. The autoradiographic signal produced with probes to VIP and PHI created dense concentrations of silver grains over neuronal perikarya in the ventrolateral SCN, and the coextensive distribution of both VIP-and PHI-mRNAs suggests that both peptides are synthesized within the same neurons. The distribution of somatostatin-mRNA was distinct from that of VIP and PHI. Labeled neurons are observed at the interface of the two SCN subdivisions and the distribution of these neurons is identical to those shown to contain somatostatin immunoreactivity. Vasopressin-mRNA is also differentially concentrated within neurons in the dorsomedial subdivision of the SCN in an area that is coextensive with vasopressin-immunoreactive perikarya. The discrete pattern of hybridization for each of these mRNAs indicates that each of these peptides are synthesized in SCN neurons and reaffirms the differential distribution of each of these chemically defined cell populations within cytoarchitecturally distinct subdivisions of the nucleus.     

Immunoelectronmicroscopic localization of vasopressin in the rat suprachiasmatic nucleus

Summary The classical areas for arginine-vasopressin (AVP) synthesis are the magnocellular supraoptic (SON) and paraventricular nuclei. More recently AVP was also demonstrated in neurons of the parvocellular suprachiasmatic nucleus (SCN) of the rat. This result was substantiated in the present study by means of immunoelectron microscopy, by subjecting sections to antivasopressin plasma. Conventional electron microscopy revealed dense-core vesicles in the SCN cell bodies and fibres (mean diameter 94.7±0.9 nm and 84.0±1.1 nm respectively). These vesicles were infrequent within the cell bodies and could not be accumulated by ethanol administration. Immunoelectron microscopy showed a positive reaction in the cell bodies and fibres within vesicles of 93.7±1.1 nm and 98.5±1.2 nm respectively. By comparison, the cell bodies and fibres of the SON showed immunoreactive granules of 143.0±1.8 and 147.3±1.8 nm respectively. The presence in the SCN of AVP in vesicles of different size than those in the SON suggests that synthesis of this substance is indeed occurring within the SCN cells.Supported by The Foundation for Medical Research FUNGOThe authors wish to thank Dr. L.A. Sternberger (Edgewood Arsenal, Md., U.S.A.) for the peroxidase-anti-peroxidase complex, Dr. J.G. Streefkerk (Free University, Amsterdam) and the members of our project group Neuroendocrinology for their suggestions, Mr. P.S. Wolters and Miss A. van der Veiden for their skilled assistance     

Light- and electron microscopic localization of vasopressin or a vasopressin-like substance in the neurons of the rat suprachiasmatic nucleus

Summary In the suprachiasmatic nucleus of the rat light microscopic immunostaining for vasopressin reveals a distribution pattern of the immunoreactive material different from that known for the supraoptic nucleus. Among non-stained neurons positive-reacting perikarya display a cap- or tiplike labeling. The area of the suprachiasmatic nucleus is marked by delicate vasopressin-positive fibers. At the ultrastructural level the reaction product, after incubation with anti-vasopressin, is localized in small elementary granules unevenly distributed over the cytoplasm. Groups of axons containing specifically labeled granules contact non-reacting fibers.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung Volkswagenwerk     

Distribution of vasoactive intestinal peptide-like and neurophysin-like immunoreactive neurons and acetylcholinesterase staining in the ring dove hypothalamus with emphasis on the question of an avian suprachiasmatic nucleus

Summary Two nuclei, termed here the medial hypothalamic nucleus and the lateral hypothalamic retinorecipient nucleus, are possible homologs of the mammalian suprachiasmatic nucleus. As the mammalian suprachiasmatic nucleus is characterized by a dense concentration of vasoactive intestinal peptide (VIP)-and neurophysin (NP)-immunoreactive neurons and an absence of acetylcholinesterase (AChE) staining, we decided to examine these factors in the ring dove hypothalamus. Neither the medial hypothalamic nucleus nor the lateral hypothalamic retinorecipient nucleus contained either VIP-or NP-like immunoreactive neurons. The lateral hypothalamic retinorecipient nucleus stained darkly for AChE. Although there was some overlap in the distribution of VIP-and NP-like immunoreactive neurons, a clustering of both types into a well defined nucleus was not observed. Therefore, an avian homolog to the mammalian suprachiasmatic nucleus must differ in its chemoarchitecture from that of mammalian species described to date.     

Ultrastructural distribution of alpha-bungarotoxin binding sites in the suprachiasmatic nucleus of the rat hypothalamus

Summary The distribution of (¹²⁵I) alpha bungarotoxin (-BTX) binding sites in the suprachiasmatic nucleus (SCN) of the adult female rat was examined by electron-microscopic autoradiography. The ultrastructural distribution of silver grains was analysed by line source, direct point count, and 50% probability circle methods. Real grain distribution was significantly different from that of randomly generated hypothetical grains. Line source analysis demonstrated two populations of sources: one associated with membranes, and one inside neuronal structures. Probability circle analysis of shared grains indicated that membrane-bound-radioactive sources were mainly asssociated with axo-dendritic appositions. Only a small proportion of labeled neuronal interfaces exhibited synaptic differentiations in the plane of section. However, the compartment containing synaptic terminals was the most enriched when comparing real to hypothetical grains. Probability circle analysis of exclusive grains demonstrated that sources that were not associated with neuronal plasma membranes were likely to be within nerve cell bodies and dendrites. It is concluded that the majority of specifically labeled -BTX binding sites in the SCN is membrane bound, and may be associated with axodendritic synaptic transmission. The presence of a significant proportion of the label in the soma and dendrites of suprachiasmatic neurons 24 h after ventricular infusion suggests that some of the labeled binding sites (junctional or nonjunctional) may be internalized within these two compartments.     

Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate

Receptors for porcine vasoactive intestinal peptide have been characterized in isolated epithelial cells of rat ventral prostate. The interaction of ¹²⁵I-labelled VIP with cells was rapid, reversible, specific, saturable and dependent on temperature. Degradation of peptide and receptors was minimized at 15°C. At apparent equilibrium, the binding of ¹²⁵I-labelled peptide was competitively inhibited by native VIP in the 1·10⁻¹⁰−10⁻⁷ M range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a K_d = 4.0 nM and a low binding capacity (0.12 pmol VIP/mg cell protein), and a low-affinity class with a K_d = 17.8 nM and a high binding capacity (1.6 pmol VIP/mg cell protein). Chicken VIP and porcine secretin exhibited a 7-fold higher and a 7-fold lower affinity than porcine VIP for binding sites, respectively. Glucagon, Leu-enkephalin, Met-enkephalin and somatostatin were ineffective. The presence of high-affinity receptors for VIP together with previous reports on the occurrence of VIP-containing neurones innervating the male genitourinary tract strongly suggest that this peptide may be important in the physiological regulation of the functions of prostatic epithelium.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Ovarian innervation develops before initiation of folliculogenesis in the rat

Summary Sympathetic neurotransmitters have been shown to be present in the ovary of the rat during early postnatal development and to affect steroidogenesis before the ovary becomes responsive to gonadotropins, and before the first primordial follicles are formed. This study was undertaken to determine if development of the ovarian innervation is an event that antedates the initiation of folliculogenesis in the rat, Rattus norvegicus. Serial sections of postnatal ovaries revealed a negligible frequency of follicles 24 h after birth (about 1 primordial follicle per ovary). Twelve hours later there were about 500 follicles per ovary, a number that more than doubled to about 1300 during the subsequent 12 h, indicating that an explosive period of follicular differentiation occurs between the end of postnatal days 1 and 2. Electron microscopy demonstrated that before birth the ovaries are already innervated by fibers containing clear and dense-core vesicles. Immunohistochemistry performed on either fetal (day 19) or newborn (less than 15h after birth) ovaries showed the presence of catecholaminergic nerves, identified by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. While some of these fibers innervate blood vessels, others are associated with primordial ovarian cells, thereby suggesting their participation in non-vascular functions. Since prefollicular ovaries are insensitive to gonadotropins, the results suggest that the developing ovary becomes subjected to direct neurogenic influences before it acquires responsiveness to gonadotropins.     

Lack of effect of vasoactive intestinal peptide antagonists on blood flow in the rat thyroid

We used three putative vasoactive intestinal peptide (VIP) antagonists: 1) [4Cl-D-Phe⁶,Leu¹⁷]VIP, 2) [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, and 3) VIP(10–28) to assess the involvement of endogenous VIP in the regulation of thyroid hormone secretion and thyroid blood flow (BF). We measured thyroid BF in ketamine-pentobarbital-anesthetized rats using the microsphere technique. Increases in thyroid BF induced by VIP administration (30 pmol-1.5 nmol/100 g b.wt.) were not affected by any of the three compounds tested at doses 10–100 times higher than that of VIP. These compounds (3–15 nmol/100 g b.wt.) also failed to affect basal thyroid BF or hormone secretion. Increases in pancreatic and salivary gland BFs induced by VIP (30 pmol/100 g b.wt.) were also not affected by [4Cl-D-Phe⁶,Leu¹⁷]VIP or [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂ (3 nmol/100 g b.wt.). These results indicate that the three compounds tested are not effective inhibitors of VIP receptors in the thyroid vasculature and, therefore, they cannot be used in the investigation of the functional significance of endogenous VIP in the regulation of thyroid BF.     

Immunohistochemical localization of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat

Summary The indirect peroxidase-antiperoxidase immunohistochemical technique was used to investigate the possible presence of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat. Considerable numbers of VIP-immunoreactive fibers were seen in the pineal gland. A moderate amount of VIP-immunoreactive fibers was present in the median eminence, the posterior lobe of the pituitary and the area postrema, but only few fibers were found in the organum vasculosum laminae terminalis. No immunoreactivity was observed in the subfornical organ or the subcommissural organ. The circumventricular organs investigated were completely free of VIP-immunoreactive perikarya. In the circumventricular organs, VIP-immunoreactive fibers were visible between the parenchymal cells and in the perivascular spaces. The presence of coarse VIP-immunoreactive terminals in apposition to the portal vessels in the external layer of the median eminence indicates that VIP may be secreted directly into the pituitary portal circulation, thus influencing the anterior pituitary cells. The presence of large VIP-immunoreactive boutons in the posterior lobe of the pituitary suggests a secretion of VIP directly into the systemic circulation. In the pineal gland, a dense innervation by VIP-immunoreactive fibers was found in the peripheral superficial part of organ, with fibers penetrating into its central portion where they mainly terminate near in vicinity of the capillaries. In the area postrema, VIP-immunoreactive material was mainly found at the ventral border of the organ. In addition to the secretion of VIP into the bloodstream via the circumventricular organs, this study provides evidence that VIP exerts specific influence on the cellular elements of these organs.     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15°C, the response occurred in the 1·10⁻¹⁰−10⁻⁷ M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1·10⁻⁶ M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.     

Rearrangement of serotonin-immunoreactive fibers in the denervated rat suprachiasmatic nucleus after transplantation of fetal raphe tissue

Summary Pieces of fetal midbrain raphe tissue were transplanted into the third ventricle or the ventral hypothalamic region near the suprachiasmatic nucleus (SCN) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. The ability of grafted serotonin neurons to reinnervate the SCN in the host rats was studied by means of immunohistochemistry 1 and 3 months after transplantation. In both the intraventricular and intraparenchymal transplant experiments, reinnervation by outgrowing serotonin fibers was observed in the hypothalamus of host rats at 1 and 3 months after surgery. At both survival periods, there was no abundant arborization of serotonin fibers in the SCN, while the preoptic and periventricular areas of the host rats displayed a pattern of serotonergic innervation resembling that in normal (untreated) rats. It is suggested that within the SCN the regenerating serotonin fibers may be exposed to an inhibitory environment.     

Effect of vasoactive intestinal peptide on serotonin release in the suprachiasmatic area of the rat. Modulation by oestradiol

The effect of vasoactive intestinal peptide (VIP) on spontaneous and induced release of newly synthesized 5-hydroxytryptamine (5-HT) was studied in the suprachiasmatic area (SCA) using a superfusion system. To test the possible modualtion by E₂ on the interaction VIP-5-HT, the experiments were conducted on male, ovariectomized (OVX) and ovariectomized oestradiol implanted rats (OVX-E₂). VIP (10^?7 M) infused for 15 min caused an increase of 5-HT release from SCA of male and OVX. The positive effect of VIP on 5-HT release results partially from an inhibition of the reuptake of 5-HT: in male and OVX SCA, VIP inhibited the ³H-5-HT uptake by 40 to 50%. The infusion of VIP before a pulse of K⁺ (10-20-30-56 mM) leads to a potentialisation of the evoked release suggesting that VIP sensitized the presynaptic membrane to the process linking depolarization and release. When SCA taken from OVX-E₂ were exposed to VIP, 5-HT uptake and consequently 5-HT release were unchanged. The present results suggest that the metabolism of 5-HT in the SCA is influenced by VIP and that this regulation may be modulated by E₂. This interaction between E₂, VIP and 5-HT at the SCA level may be involved in the regulation of phasic LH and prolactin surge.     

Nocturnal expression of phosphorylated-ERK1/2 in gastrin-releasing peptide neurons of the rat suprachiasmatic nucleus

Extracellular regulated kinase (ERK) signalling is believed to play roles in various aspects of circadian clock mechanisms. In this study, we show in rat that the nuclear versus cytoplasmic intracellular distribution of the phosphorylated forms of ERK1/2 (P-ERK1/2) in the central clock, namely the suprachiasmatic nucleus (SCN), is proportionally constant across the light/dark cycle while the spatial distribution and neurochemical phenotype of cells expressing these activated forms are time-regulated according to a daily rhythm and light-regulated. P-ERK1/2 was exclusively found in neuronal elements. At daytime, it was detected throughout the dorsoventral extent of the SCN, partly within neurons synthesizing either arginine-vasopressin or vasoactive intestinal peptide (VIP). At night time, it was segregated in the ventrolateral aspect of the nucleus, within a cluster of cells 45% of which were gastrin-releasing peptide (GRP) neurons with or without co-localization with VIP. After a light pulse at night, expression of P-ERK1/2 increased in GRP neurons but also appeared in a population of neurons that stained for VIP only. These data show that the GRP neurons are closely associated with ERK1/2 activation at night and point to the importance of ERK1/2 signalling not only in intra-SCN transmission of photic information but also in maintenance of neuronal rhythms in the SCN.     

Catecholaminergic innervation of GRF-containing neurons in the rat hypothalamus revealed by electron-microscopic cytochemistry

Summary The Catecholaminergic innervation of neurons containing growth hormone-releasing factor (GRF) was examined by use of a method which combined either 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of ³H-noradrenaline with immunocytochemistry for GRF in the same tissue sections at the electron-microscopic level. In the ventrolateral part of the arcuate nucleus of the rat hypothalamus a large number of immunonegative axon terminals were found to make synaptic contact with GRF-like immunoreactive (GRF-LI) cell bodies and processes. ³H-noradrenaline autoradiography or 5-OHDA-labeling combined with GRF immunocytochemistry revealed that axon terminals labeled with ³H-noradrenaline or 5-OHDA make synaptic contact with the GRF-LI nerve cell bodies and processes. These findings indicate that catecholamine-containing neurons innervate GRF neurons to regulate GRF secretion via synapses in the rat arcuate nucleus.     

Post-coital ultrastructural changes in neurons of the suprachiasmatic nucleus of the rabbit

Summary The effects of coitus on the ultrastructure of neurons in the suprachiasmatic nucleus of the rabbit were studied. Changes first became apparent 1/2 h after coitum in neurons located near capillaries. More pronounced ultrastructural changes were observed in large neurons removed at 1 h post-coitus. These changes, characterized by well developed Golgi systems and rough endoplasmic reticulum, the presence of large dense-core vesicles and a significant increase in both neuronal and nuclear size, were also evident in neurons observed at 2 to 10 h post coitum. Similar ultrastructural features were not observed in the neurons of the control animals. The post-coital ultrastructural changes observed within these neurons suggest high synthetic activity which may concern the production sites of the neurohormone LH-RF. Two populations of dense-core vesicles were observed: a) those with a mean diameter of 849 Å, and b) those with a mean diameter of 1542 Å. The small dense-core vesicle is probably monoamine in nature; the larger vesicle may contain the neurohormone LH-RF. A third vesicle type with a mean diameter of 1836 Å and characterized by a granular content of low electron density was also observed. That this vesicle represents the immature form of the large (1542 Å) dense-core vesicle is suggested; however, morphological evidence supporting this hypothesis is inconclusive. There is also no evidence for the storage of secreted materials within the soma of these neurons. Immediate transport toward the median eminence is suggested.This investigation was supported by grants to the two senior authors from the Medical Research Council of Canada.     

Hepatic metabolism of vasoactive intestinal polypeptide (VIP) in the rat

Vasoactive intestinal polypeptide (VIP) is released into the portal circulation by a meal stimulus, but is rapidly cleared from plasma. Although it is known to bind to receptors on liver cells, the role of the liver in the clearance of VIP is not clearly defined. We therefore studied the disappearance of VIP in recirculating and in single pass isolated perfused rat liver (IPRL) preparations. Disappearance of added VIP was rapid in recirculating IPRL experiments with a half life of ca. 30 min. In single-pass steady-state studies in which livers were perfused at 16 ml/min for 30 min, clearance of VIP was complete (16 ml/min) at concentrations of 500 fmol/ml, but clearance fell to 3 and 1 ml/min at perfusate concentrations of 8 and 40 pmol/ml respectively. Further experiments to evaluate whether VIP was disappearing in perfusate itself demonstrated substantial metabolism of VIP in perfusate which had previously been circulated through a liver for 90 min. The products of metabolism were identical to those found in the IPRL. We conclude that VIP is rapidly cleared as it passes through the isolated perfused rat liver model with a significant proportion of clearance attributable to release of a peptidase from the liver into the perfusate.     

Organotypic suprachiasmatic nuclei cultures of adult voles reflect locomotor behavior: differences in number of vasopressin cells

This study is the first to demonstrate organotypic culturing of adult suprachiasmatic nuclei (SCN). This approach was used to obtain organotypic SCN cultures from adult vole brain with a previously determined state of behavioral circadian rhythmicity. We examined vasopressin (AVP) immunoreactivity in these organotypic slice cultures. AVP is one of the major neuropeptides produced by the SCN, the main mammalian circadian pacemaker. AVP immunoreactivity in the SCN of adult common voles in vivo has been shown to correlate with the variability in expression of circadian wheel-running behavior. Here, cultures prepared from circadian rhythmic and nonrhythmic voles were processed immunocytochemically for AVP. Whereas in all cultures AVP could be observed, AVP immunoreactivity differed considerably between vole SCN cultures. SCN cultures from rhythmic voles contained significantly lower numbers of AVP immunoreactive (AVPir) cells per surface area than cultures from nonrhythmic voles. The correlation between timing of behavior and AVP immunoreactivity in vitro is similar to the correlation found earlier in vivo. Apparently, such correlation depends on intrinsic AVP regulation mechanisms of SCN tissue, and not on neural or hormonal input from the environment, as present in intact brain.     

Hyperalgesia in response to traumatic occlusion and GFAP expression in rat parabranchial nucleus: modulation with fluorocitrate

We have examined, by immunocytochemical methods and nociceptive behavior assessment in rats, whether astrocytes in the parabrachial nucleus (PBN) are involved in the regulation of traumatic occlusion. The expression of glial fibrillary acidic protein (GFAP) in PBN of ipsilateral and contralateral sides was up-regulated 4 h after occlusal changes in molars, reached peak levels at 24 h, and was then gradually down-regulated. PBN astrocytes activated by traumatic occlusion were found to have enlarged cell bodies and thickened processes within 8 h. An inhibitor of glia metabolism (FCA, fluorocitrate) reduced astrocyte activation and significantly attenuated the development of pain hypersensitivity in this model. The results suggested that the GFAP-immunoreactive astrocytes in PBN within the bridge of Varolius were activated by traumatic occlusion, and that they were involved in the transmission and modulation of nociceptive information in the central nervous system. However, although astrocytes in PBN are thus probably involved in causing post-occlusal hyperalgesia, we have not been able to exclude that astrocytes at other locations also contribute to this effect. Jinwu Chen and Jun Zhang contributed equally to this study. This study was supported by the National Nature Science Foundation of China (nos. 30400503 and 30572066).