Effect of serotonin on differentiation of neurons producing vasoactive intestinal polypeptide in the rat suprachiasmatic nucleus

This study was aimed to evaluate the morphogenetic influence of serotonin on the differentiating vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus (SCN) in rats. The comparative morpho-functional analysis of VIP neurons was made in adult rats which developed under normal metabolism of serotonin or in its deficiency. The serotonin deficiency was induced in foetuses by injections of p-chlorophenilalanine to pregnant mothers. Control rats received the saline. According to our data, there was no difference in the VIP mRNA concentration and most probably in VIP synthesis in the SCN in adult rats following prenatal serotonin depletion compared to the control. However, the serotonin deficiency resulted in increase of the VIP concentration in cell bodies and of the VIP neurones number. The size of the VIP-neurones did not change in pharmacologically treated rats compared to the controls showing no functional hypertrophy of the neurones as a result of the activation of specific syntheses. From the above data, it follows that serotonin provides an imprinting influence differentiating the VIP neurones, contributing most probably to development of the VIP release mechanism.     

Immunohistochemical evidence of serotoninergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus

By applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered diffusely throughout the neuroplasma at various densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptic membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.     

Catecholaminergic innervation of the suprachiasmatic nucleus in the adult rat: ultrastructural relationships with neurons containing vasoactive intestinal peptide or vasopressin

Catecholaminergic fibers in the suprachiasmatic nucleus of adult rats were investigated by use of light- and electron-microscopic immunocytochemistry. The suprachiasmatic nucleus receives a modest density of tyrosine hydroxylase-containing axons, homogeneously distributed in the nucleus and forming varicosities throughout its entire rostro-caudal extension. Immunolabeling with antibodies against dopamine showed that this catecholamine input comprises a dopaminergic component. Many tyrosine hydroxylase-positive cells were localized at the immediate periphery of the suprachiasmatic nucleus. With electron-microscopic examination, dendrites of these neurons were found within the limits of the nucleus as well as at a border zone between the suprachiasmatic nucleus proper and the optic tract where they received unlabeled synapses, providing a morphological support for a possible role of dopaminergic neurons in the integration and/or transfer of light-related signals. More than 91% of catecholaminergic axonal varicosities were found to establish morphologically defined synapses with dendrites. To investigate whether these synapses might be shared with neurons of one or both of the two main peptidergic populations of the nucleus, namely vasoactive intestinal peptide- and vasopressin-containing neurons, we carried out doublelabeling experments combining immunoperoxidase and immunogold-silver labeling. Results showed only a few cases of direct association of the catecholaminergic terminals with these peptidergic categories. In both types of dually stained sections, catecholaminergic synapses were preferentially made with unlabeled dendrites. The homogeneous distribution of tyrosine hydroxylase-immunoreactive fibers in the suprachiasmatic nucleus could therefore reflect a lack of significant catecholaminergic innervation of both vasoactive intestinal peptide- and vasopressin-synthesizing neurons.     

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     

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.     

Involvement of cyclic AMP and calcium in exocrine protein secretion induced by vasoactive intestinal polypeptide in rat parotid cells

The effects of vasoactive intestinal polypeptide (VIP) on exocrine protein secretion were studied in enzymatically dispersed cell aggregates from rat parotid glands. VIP (10(-9) - 10(-7) M) stimulated secretion of alpha-amylase in a dose-dependent manner. The VIP-induced release of alpha-amylase was potentiated in the presence of a phosphodiesterase inhibitor. Basal levels of cyclic AMP of the dispersed cells were increased 6.7-fold after stimulation for 10 min by VIP (10(-7) M). The VIP-induced release of alpha-amylase was reduced by 40% when cells were incubated in a Ca2+-free medium in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Efflux of 45Ca2+ was significantly increased over basal levels by stimulation with VIP (10(-8) and 10(-7) M), but this increased efflux was approximately only half the increased efflux induced by carbachol (10(-5) M). VIP had no effect on the incorporation of [14C]leucine into protein by parotid cells, whereas incorporation was reduced to 30% of the control value by carbachol (10(-5) M). Thus, the VIP-ergic secretory response in the rat parotid gland is associated with a raised intracellular cyclic AMP level and the mobilisation of a different intracellular Ca2+ pool than that mobilised by carbachol. It is, therefore, closely analogous to the beta-adrenergic response.     

Comitogenic effects of vasoactive intestinal polypeptide on rat hepatocytes

The primary mitogens such as epidermal growth factor and transforming growth factor-α are known to stimulate DNA synthesis in primary cultures of adult rat hepatocytes. Vasoactive intestinal polypeptide (VIP) was found to amplify DNA synthesis induced by the primary mitogens and thus acted as a comitogen. The comitogenic effect of VIP was specific for the culture medium, suggesting that minor components in the medium were required for hepatocytes to fully respond to VIP. Glutamic acid is probably one of these minor components, although other components present in the nutrient-rich medium were also necessary for the full comitogenic effect. Other comitogens such as insulin, vasopressin, and angiotensin II interacted additively with low concentrations of VIP. The comitogenic effect of VIP was also found in hepatocytes cultured from regenerating rat liver after a partial hepatectomy. In the regenerating hepatocyte cultures, VIP can act as a mitogen even in the absence of the primary mitogen EGF. VIP mRNA was found in several organs including brain, intestine, and liver, and its expression was slightly induced in liver 24 h after a partial hepatectomy. These results suggest that VIP can act as a hepatic comitogen and may play a role in liver cell proliferation. © 1996 Wiley-Liss, Inc.     

Age-dependent effects of conditioning on cholinergic and vasopressin systems in the rat suprachiasmatic nucleus

Active shock avoidance was used to explore the impact of behavioural stimulation on the neurochemistry of the suprachiasmatic nucleus. We have found previously that the expression of muscarinic acetylcholine receptors in the suprachiasmatic nucleus of young rats was significantly enhanced 24 hours after fear conditioning. Here, we investigated whether this observation is age-dependent. We used 26 month-old Wistar rats with a deteriorated circadian system, and compared them with young rats (4 months of age) with an intact circadian system. Vasopressin, representing a major output system of the suprachiasmatic nucleus, was studied in addition to muscarinic receptors. Young rats showed a significant increase in immunostaining for muscarinic acetylcholine receptors 24 h after training, corroborating earlier observations. Aged rats did not show such an increase. In contrast, aged rats did show an increase in vasopressin immunoreactivity 24 h after fear conditioning, both at the level of content and cell number, while young rats did not reveal a significant rise. Thus, it seems that these two neurochemical systems in the suprachiasmatic nucleus are regulated independently. The results further demonstrate that the circadian pacemaker is influenced by fear conditioning in an age-dependent manner.     

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.     

Effects of vasoactive intestinal peptide genotype on circadian gene expression in the suprachiasmatic nucleus and peripheral organs

The neuropeptide vasoactive intestinal polypeptide (VIP) has emerged as a key candidate molecule mediating the synchronization of rhythms in clock gene expression within the suprachiasmatic nucleus (SCN). In addition, neurons expressing VIP are anatomically well positioned to mediate communication between the SCN and peripheral oscillators. In this study, we examined the temporal expression profile of 3 key circadian genes: Per1, Per2 , and Bmal1 in the SCN, the adrenal glands and the liver of mice deficient for the Vip gene (VIP KO), and their wild-type counterparts. We performed these measurements in mice held in a light/dark cycle as well as in constant darkness and found that rhythms in gene expression were greatly attenuated in the VIP-deficient SCN. In the periphery, the impact of the loss of VIP varied with the tissue and gene measured. In the adrenals, rhythms in Per1 were lost in VIP-deficient mice, while in the liver, the most dramatic impact was on the phase of the diurnal expression rhythms. Finally, we examined the effects of the loss of VIP on ex vivo explants of the same central and peripheral oscillators using the PER2::LUC reporter system. The VIP-deficient mice exhibited low amplitude rhythms in the SCN as well as altered phase relationships between the SCN and the peripheral oscillators. Together, these data suggest that VIP is critical for robust rhythms in clock gene expression in the SCN and some peripheral organs and that the absence of this peptide alters both the amplitude of circadian rhythms as well as the phase relationships between the rhythms in the SCN and periphery.     

Interaction of vasoactive intestinal peptide with rat small intestinal epithelial cells after intestinal resection

Specific binding of vasoactive intestinal peptide (VIP) and VIP-stimulated c y c l i c AMP accumulation were studied in small intestinal epithelial cells (both of crypt and villous levels) 3, 7 and 14 d after a 60% resection of the small intestine . The affinity, but not the binding capacity, of VIP receptors decreased during the adaptive hyperplastic response. Basal cyclic AMP levels were similar in cells of both control and resected rats. Resection induced a decrease of potency, but not of efficiency, of VIP on the stimulation of cyclic AMP accumulation.     

Molecular variants of vasoactive intestinal polypeptide in dog, rat and hog

Molecular forms of vasoactive intestinal polypeptide (VIP) have been examined in the gut and brain of dog, rat and hog. Fractionation of acid extracts on CM-Sephadex revealed three components cross-reacting in a radioimmunoassay using an amino-terminal specific antiserum. One of the components was compatible with standard porcine octacosapeptide VIP, the other two eluted earlier and are so likely to be less positively charged peptides. However, after gel filtration on Sephadex G50, the same peaks of activity eluted in a similar position to porcine VIP indicating similar molecular size. There were marked species differences in the distribution of the different molecular forms. For example, in both muscle and mucosal layers of the rat intestine 50–90% of total immunoreactive VIP was attributable to the molecular variants, while in hog colon the variants were found predominantly in the mucosa and accounted for about 50% of total immunoreactivity. In contrast a form of VIP compatible with the authentic peptide accounted for over 75% of activity in the brain of all three species. The biological activity of the VIP variants is not known but clearly caution needs to be exercised in interpreting the physiological significance of studies on the action, release and metabolism of VIP.     

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     

Autoradiographic localization of vasoactive intestinal polypeptide receptors in the rat mesenteric vascular tree

By the use of combined in vitro radioreceptor binding and autoradiographic techniques, we analyzed the pharmacological properties and the anatomical localization of the vasoactive intestinal polypeptide (VIP) receptor in rat superior mesenteric artery and in medium and small mesenteric artery branches. 125I-VIP was bound by sections of rat superior mesenteric artery in a manner consistent with the labeling of specific VIP receptors, with Kd and Bmax values of 0.23 nM and 0.71 pmol/mg protein respectively. Inhibition of 125I-VIP binding with VIP and related peptides gives the following rank order of potency: VIP greater than peptide histidine methionine greater than secretin. Light microscope autoradiography reveals specific VIP binding sites within the medial layer of superior mesenteric artery and its branches. Medium and small sized vessels are richer in 125I-VIP binding sites than the larger ones.     

Autoradiographic distribution of vasoactive intestinal polypeptide receptors in rabbit and rat small intestine

Vasoactive intestinal peptide (VIP) is found in the enteric nervous system of all layers of the small intestine. In the gastrointestinal tract, VIP receptors coupled to adenylate cyclase are present on epithelial, smooth muscle and possibly mononuclear cells. This study analyzes the distribution of VIP binding using in vitro autoradiographic techniques. VIP binding was present in high density in the mucosal layer of rabbit duodenum, jejunum and ileum. Low VIP binding was noted over the smooth muscle layers or the lymphoid follicles. Similar results were obtained in rat small intestine. The density of VIP binding was greatest in duodenal mucosa but was present in lower density in jejunal and ileal mucosa. Again, low VIP binding was noted in the smooth muscle layers or lymphoid follicles. Thus, autoradiographic maps of small intestine indicate that VIP receptors are found primarily in the small intestinal mucosa.     

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.     

Gastric and intestinal release of vasoactive intestinal polypeptide in the milk-fed lamb

Vasoactive intestinal polypeptide (VIP) has been proposed as the neurotransmitter of the atropine-resistant relaxation of gastric structures in the lamb. To examine this proposal VIP concentrations in plasma from arterial, gastric venous and intestinal venous blood were measured in healthy conscious lambs before, during and after teasing with, and sucking of milk. Basal arterial plasma VIP concentrations were undetectable (less than 3 pmol/l) and remained so during and after feeding. Before feeding VIP was detected in only 2 of 12 gastric venous plasma samples (5 and 13 pmol/l). During teasing with food there were increments in VIP of 19 +/- 4 pmol/l and during feeding of 27 +/- 5 pmol/l. VIP concentration in gastric venous plasma rapidly returned to fasting levels after cessation of sucking. In contrast VIP in the intestinal venous plasma did not rise during teasing or upon commencement of sucking but a peak increment of 34 +/- 6 pmol/l occurred at 5 min after cessation of feeding. The results are consistent with the hypotheses that VIP is released in anticipation of and during sucking from inhibitory neurones involved in relaxation of gastric structures and that intestinal release of VIP is a consequence of entry of digesta into the small intestine.     

Synaptology of the rat suprachiasmatic nucleus

Within the suprachiasmatic nucleus (SCN) of the rat the fine structure of the synapses and some features of their topological arrangement were studied. Five types of synapses could be distinguished with certainty: A. Two types of Gray-type-I (GTI) or asymmetrical synapses (approximately 33%). The presynaptic elements contain strikingly different types of mitochondria. Size of clear vesicles: approximately 450 A. Synapses with subjunctional bodies often occur, among these also "crest synapses". Localization: dendritic shafts and spines, rarely somata. B. Three types of Gray-type-2 (GTII) or symmetrical synapses (approximately 66%):1) Axo-dendritic and -somatic (=AD) synapses. Size of clear vesicles: approximately 500 A. 2) Invaginated axo-dendritic and -somatic (=IAD) synapses with club-like postsynaptic protrusions within the presynaptic elements (PreE1). Size of clear vesicles is very variable: approximately 400-1,000 A. 3) Dendro-dendritic, -somatic and somato-dendritic (=DD) synapses occurring at least partly in reciprocal arrangements. They represent an intrinsic system. Shape of clear vesicles: often oval; sucrose treatment partly produces flattening. Dense core-vesicles (dcv) are found in all GTII- and most of the GTI-synapses after three-dimensional reconstruction. All types of synapses (mostly GTII-synapses) can be enclosed by multilamellar astroglial formations. The synapses often occur in complex synaptic arrangements. Dendrites and somata of females show significantly more multivesiculated bodies than those of males. Further pecularities of presynaptic (PreELs) and postsynaptic elements (PostELs) within the SCN are described and discussed.