Vasoactive intestinal polypeptide neuron changes in the senile rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is thought to be the main neuronal oscillator underlying circadian rhythmicity of different biological phenomena such as sleep-wakefulness and body temperature. Although numerous studies in old rats showed that circadian organization is clearly disturbed in senescence, no decrease in total SCN cell number has been observed. However, in an earlier study we found a significant decrease of approximately 30% in the number of immunocytochemically-stained vasopressin (VP) neurons in the SCN of the old rat. The aim of the present study was to examine whether another group of SCN neurons, i.e., the vasoactive intestinal polypeptide (VIP) cells, shows age-related changes parallel with disturbances found in sleep/wake parameters. Immunocytochemical staining with antiVIP followed by morphometric analysis revealed a 36% decrease in the number of immunoreactive VIP neurons in the SCN of old rats as compared to young ones. The average size of the remaining VIP cells increased in aged rats. The rapid-eye-movement (REM)-sleep time was negatively correlated with the immunoreactive VIP cell number in the old animals. VP and VIP alterations in the SCN may constitute an anatomical substrate for the circadian disturbances observed in senescence.     

Vasoactive intestinal polypeptide afferents to the bed nucleus of the stria terminalis in the rat: an immunohistochemical and biochemical study

Vasoactive intestinal polypeptide (VIP) has a markedly heterogeneous distribution in the rat bed nucleus of the stria terminalis. The dorsal bed nucleus contains the highest concentration of VIP in the rat brain, with the exception of the suprachiasmatic nucleus, 4-fold higher than the VIP concentration in the frontal cortex. These biochemical findings agree well with the immunohistochemical analysis of this area. The bed nucleus is also a heterogeneous nucleus with respect to the afferent VIP pathways which innervate it. A combination of immunohistochemical and biochemical techniques was used to examine VIP innervation of the bed nucleus after knife cuts designed to interrupt ascending brainstem, stria terminalis and ventral amygdalofugal inputs to the bed nucleus. The results obtained suggest that (1) ascending pathways arising in the mesencephalon at the level of the dorsal raphe nucleus send VIP fibers to the dorsal but not the ventral bed nucleus, (2) afferent VIP fibers which travel to the bed nucleus via the stria terminalis contribute a diffuse VIP innervation to both the dorsal and ventral bed nucleus and (3) a newly described ventral amygdalofugal VIP pathway to the bed nucleus contributes a major input to the dorsal, but not to the ventral bed nucleus. These three pathways probably account for the entire extrinsic VIP input to the bed nucleus. The finding that the bed nucleus is heterogeneous both with respect to VIP content and afferent VIP inputs serves to clarify previous, apparently discrepant, reports that both the stria terminalis and ascending pathways constitute the major VIP input to the bed nucleus of the stria terminalis.     

Distribution of Met-enkephalyl-Arg-Gly-Leu in rat larynx: partial coexistence with vasoactive intestinal polypeptide, peptide histidine isoleucine and neuropeptide Y

Using light microscopic (LM) enzyme-immunohistochemistry on deparaffinized adjacent sections Met-enkephalyl-Arg-Gly-Leu (ME-RGL) immunoreactivity was found to partially coexist with immunoreactive neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in intrinsic laryngeal neurons of the rat. Further ME-RGL-immunoreactive (ir) fibres were found around glands in the subepithelium, in connective tissue of striated muscle and in the perichondrium, as well as around arterial and venous blood vessels. They frequently contacted mast cells and macrophages. The presence of ME-RGL indicates pro-enkephalin-related origin of this novel laryngeal opioid system. From the specific target relations and close interrelations of fibres staining for opioids with those staining for the other peptides--which are known to be more or less characteristic of the sympathetic (NPY), parasympathetic (VIP, PHI) and sensory (calcitonin gene-related peptide; CGRP) subdivisions of the peripheral nervous system--we deduce that opioid/non-opioid interactions might co-control various laryngeal functions, e.g. glandular secretion, blood flow, immune and inflammatory responses and/or might be of relevance in trophic mechanisms.     

Vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivity colocalize with vasopressin-like immunoreactivity in the canine hypothalamo-neurohypophysial neuronal system

The distribution of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine amide (PHI) was investigated in the canine hypothalamus by immunocytochemistry. VIP- and PHI-like immunoreactive neurons were detected in the magnocellular supraoptic and paraventricular nucleus. These magnocellular VIP- and PHI-producing neurons coexist with vasopressin-like immunoreactivity and send axons to the median eminence and neurohypophysis. These findings may serve as an anatomical basis for studying the function of VIP and PHI on pituitary hormone secretion.     

Subcellular distribution of the immunoreactive peptide histidine isoleucine in the rat brain and release from synaptosomes in vitro

Subcellular fractionation studies of rat brain homogenate revealed that peptide histidine isoleucine-like immunoreactivity (PHI-LI) was enriched in synaptosomal preparations. PHI-LI was released by high potassium concentrations from synaptosomal pellets incubated in vitro. These results suggest a possible physiological role of PHI in the central nervous system as a neurotransmitter or modulator of synaptic function.     

Effect of the glucocorticoid milieu on the immunostaining of peptide histidine isoleucine (PHI) in the rat hypothalamus

Effects of adrenalectomy (ADX) or dexamethasone (DEX) treatment on the immunostaining of hypothalamic peptide histidine isoleucine (PHI) were examined in male rats. After colchicine treatment, PHI-containing cell bodies were observed in the suprachiasmatic nucleus (SCN) and the parvocellular division of the paraventricular nucleus (PVN). ADX increased and DEX dose-dependently decreased the number of PHI-immunopositive neurons in the PVN. The number of SCN-PHI neurons was not affected by any treatment employed in this study. These results suggest that PVN-PHI neurons are under the effects of the glucocorticoid milieu, and that the neurons may be involved in the glucocorticoid regulation of adrenocorticotropin and prolactin secretion.     

Basic study of peptide histidine isoleucine (PHI) immunoreactivity in the rat respiratory tract

Peptide histidine isoleucine (PHI) as well as vasoactive intestinal peptide (VIP) is considered to be a neurotransmitter of non-adrenergic and non-cholinergic inhibitory nerves in the respiratory tract. We tried to measure PHI immunoreactivity in rat respiratory tracts to evaluate the movement of PHI. A high titer of PHI antiserum which did not react with VIP was obtained from a rabbit immunized with rat PHI, and rat PHI radioimmunoassay was established with this antiserum. Tracheas, extrapulmonary bronchi and lungs were dissected from rats sacrificed by microwave irradiation. They were homogenized with 0.5 N acetic acid and centrifuged at 35,000 X g. Lyophilized supernates were used as samples for the radioimmunoassay. PHI immunoreactivity per g wet weight tissue was 5.12 +/- 1.35 (mean +/- SD) pmol in the tracheas, 1.66 +/- 1.15 pmol in the extrapulmonary bronchi and 0.12 +/- 0.06 pmol in the lungs. PHI immunoreactivity occurred more in the central airways than in the peripheral ones. VIP immunoreactivity in rat respiratory tracts, assayed simultaneously by radioimmunoassay using commercialized VIP antiserum, was as strong as the of PHI, suggesting that PHI and VIP may be produced from the same precursor at the ratio of 1:1 in rat respiratory tracts.     

A peptide histidine isoleucine/peptide histidine methionine-like peptide in the rabbit retina: colocalization with vasoactive intestinal peptide, synaptic relationships and activation of adenylate cyclase activity

Antisera against peptide histidine isoleucine and peptide histidine methionine were found to label a subpopulation of amacrine and displaced amacrine cells in the rabbit retina with processes ramifying in sublaminas 1, 3 and 5 of the inner plexiform layer. Preadsorption controls demonstrated that this immunoreactivity was specific for a peptide histidine isoleucine- or peptide histidine methionine-like (peptide histidine isoleucine/peptide histidine methionine-like) peptide, and was not caused by cross-reactivity of the peptide histidine isoleucine or peptide histidine methionine antibodies with vasoactive intestinal peptide vasoactive intestinal peptide. In double-label studies, vasoactive intestinal peptide and peptide histidine isoleucine/peptide histidine methionine-like immunoreactivity were colocalized in the same population of retinal neurons. Electron microscopic analysis revealed that the peptide histidine isoleucine/peptide histidine methionine-labelled cells interacted with processes of bipolar cells, amacrine cells and ganglion cells. Peptide histidine methionine and peptide histidine isoleucine were slightly less potent than vasoactive intestinal peptide in stimulating adenylate cyclase activity in the rabbit retina, while the related peptides secretin, glucagon, and the C-terminal vasoactive intestinal peptide fragment, vasoactive intestinal peptide (10-28), showed little or no stimulatory activity. Stimulation of adenylate cyclase by high concentrations of vasoactive intestinal peptide and peptide histidine methionine were non-additive. These results suggest that a peptide histidine isoleucine/peptide histidine methionine-like peptide may function as a neuroactive peptide in the mammalian retina, and that this peptide appears to be cosynthesized and colocalized with vasoactive intestinal peptide and to mimic the activity of vasoactive intestinal peptide through interaction with vasoactive intestinal peptide receptor-adenylate cyclase complexes.     

Distribution, ontogeny and projections of cholecystokinin-8, vasoactive intestinal polypeptide and gamma-aminobutyrate-containing neuron systems in the rat spinal cord: an immunohistochemical analysis

The distribution, ontogeny and fiber projections of cholecystokinin-8, vasoactive intestinal polypeptide and gamma-aminobutyrate-containing neuronal systems in the rat spinal cord were investigated by means of immunocytochemistry. Immunoreactive fibers to cholecystokinin-8, vasoactive intestinal polypeptide and glutamate decarboxylase (gamma-aminobutyrate-synthesizing enzyme, used as a marker of gamma-aminobutyrate) were widely distributed in the spinal cord, being particularly concentrated in the superficial dorsal horn, suggesting a close relationship to the pain transmission system. Cholecystokinin-8-containing neurons were mostly distributed in the dorsal laminae and glutamate decarboxylase-containing neurons were distributed in both the dorsal and ventral horns. Vasoactive intestinal polypeptide-containing neurons were detected in the lateral spinal nucleus and the lamina X. Cholecystokinin-8 and vasoactive intestinal polypeptide immunoreactive structures first appeared on gestational day 17-18. Although no substantial change in immunoreactive structures was observed during the fetal period, they increased markedly after birth. On the other hand, glutamate decarboxylase-positive structures appeared at gestational day 16 and those in the grey matter reached a maximum content at birth; both groups were present in adult animals. Transection of the upper cervical cord resulted in accumulations of cholecystokinin-8 and glutamate decarboxylase rostral to the lesion, revealing the presence of supraspinal projections of cholecystokinin-8 and glutamate decarboxylase to the spinal cord. The same experimental procedure demonstrated the existence of vasoactive intestinal polypeptide-mediating neuronal projections to the supraspinal level, as the accumulating fibers occurred in the area caudal to the lesion.     

Interaction between vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in stimulating the secretion of prolactin from rat anterior pituitary cells in vitro

Interaction between vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in regulating the secretion of prolactin (PRL) from the pituitary was investigated in the rat in vitro using two different methods: (1) short incubation of anterior pituitary cells and (2) superfusion of the pituitary cell column. PRL levels in the incubation medium were raised by addition of either VIP or PHI in concentrations of 10(-9) M to 10(-7) M in a dose-related manner. When both peptides were simultaneously added, additive stimulating effect on PRL release was obtained below the VIP concentration of 10(-7) M, in which no additive effect of PHI was revealed. PRL release from superfused rat pituitary cells was stimulated by 5-min pulses of VIP (10(-7) M), PHI (10(-7) M) and TRH (10(-8) M). Infusion of VIP for 200 min in the concentration of 0.3 x 10(-7) M resulted in an increase in basal release of PRL and blunted PRL release induced by not only VIP but also PHI stimulation, whereas PRL release induced by TRH was not affected. Infusion of PHI (0.3 x 10(-7) M, 0.7 x 10(-7) M and 10(-7) M) for 200 min also dose-relatedly suppressed PRL release induced by not only PHI but also VIP without any change in PRL release induced by TRH. These findings suggest that VIP and PHI may act through a common binding site on the pituitary lactotroph in the rat.     

Evidence that some preganglionic sympathetic neurons in the rat contain vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunoreactivities.

Physiological studies have established that preganglionic sympathetic nerve fibers innervating the rat superior cervical ganglion release a second transmitter, in addition to acetylcholine. Based on pharmacological and histochemical investigations, possible candidates for this non-cholinergic neurotransmitter include vasoactive intestinal peptide and peptide histidine isoleucine amide. For example, previous immunohistochemical studies have demonstrated that antisera raised against both of these peptides stain neural processes in the rat preganglionic cervical sympathetic trunk and in the superior cervical ganglion. In the present study, it was found that, when the cervical sympathetic trunk was ligated, vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities built up on both sides of the ligature. In addition, examination of the thoracic spinal cord in colchicine-treated animals revealed vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivies in neuronal cell bodies in the intermediolateral cell column and in the region of the lateral funiculus adjacent to it. In a second group of animals in which retrograde tracing techniques were used, these two regions of the spinal cord were shown to contain most of the cell bodies of the preganglionic neurons that project to the superior cervical ganglion. Smaller numbers of retrogradely labeled neurons were found dorsal to the central canal and in the nucleus intercalatus. When either vasoactive intestinal peptide- or peptide histidine isoleucine amide-like immunostaining and retrograde labeling were examined in the same animals, double-labeled neurons were found in the intermediolateral cell column and in the lateral funiculus. These data demonstrate that vasoactive intestinal peptide- and peptide histidine amide-like immunoreactivities are present in certain of the preganglionic neurons that project to the superior cervical ganglion, supporting the hypothesis that vasoactive intestinal peptide and peptide histidine isoleucine amide are released in the ganglion when these preganglionic neurons are activated.     

Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection

The existence of a direct projection from retinal ganglion cells to vasoactive intestinal peptide (VIP)-like immunoreactive neuronal elements in the rat suprachiasmatic nucleus (SCN) was revealed by combining analysis of degenerating axons following enucleation and electron microscopic immunocytochemistry. Degenerating axons appeared to make synaptic contact with VIP-like immunoreactive dendrite and neuronal perikarya in the ventral part of the SCN. The possibility of neuronal input from retinal ganglion cells to axons of VIP-like immunoreactive neurons was also suspected since axo-axonic synapses were detected between degenerating axons and axons with VIP-like immunoreactivity. Thus, VIP-like immunoreactive neurons in the SCN receive several neuronal inputs, including those from the retina, and may play a significant role in circadian entrainment.     

The distribution of histidine decarboxylase mRNA in the rat brain: an in situ hybridization study using synthetic oligonucleotide probes

L-Histidine decarboxylase catalyzes the formation of histamine from the amino acid L-histidine. We have studied the distribution of neurons expressing mRNA for histidine decarboxylase in adult rat brain using in situ hybridization with synthetic oligonucleotide probes. The expression of mRNA for histidine decarboxylase was detected in the hypothalamic tuberomammillary nucleus that has been shown to contain histidine decarboxylase-like and histamine-like immunoreactivity, but not in any other brain area. This method may prove useful in studying the physiological role of central histaminergic neurons.     

Suprachiasmatic nucleus neurons immunoreactive for vasoactive intestinal polypeptide have synaptic contacts with axons immunoreactive for neuropeptide Y: an immunoelectron microscopic study in the rat

An electron microscopic study showed by using a dual immunolabeling technique that in the suprachiasmatic nucleus of the rat, axon terminals immunoreactive for neuropeptide Y (NPY) made synaptic contacts upon neurons immunoreactive for vasoactive intestinal polypeptide (VIP). Diaminobenzidine (DAB)-labeled NPY axon terminals made synaptic contacts on silver-gold-labeled VIP perikarya and dendritic processes. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles labeled with DAB chromogen. At the synaptic portion, a symmetrical thickening of the pre- and post-synaptic membranes was evident.     

Age-related changes in rates of basal secretion of immunoreactive vasoactive intestinal polypeptide and dopamine into pituitary stalk blood from the hypothalamus in anesthetized male rats

Age-related changes in prolactin (PRL) in systemic blood plasma, and in secretions of hypothalamic vasoactive intestinal polypeptide (VIP), an important candidate for PRL-releasing factor, and dopamine, a PRL-inhibiting factor, into the pituitary stalk blood were investigated. The experiments were performed on male urethane-chloralose-anesthetized Wistar rats of three different ages, i.e., (1) adult rats 5-8 months old, (2) middle-aged rats 12-15 months old, and (3) aged rats 24-26 months old. The concentration of immunoreactive PRL (iPRL) in systemic blood plasma of the aged rats was significantly higher than that of the adult rats (p less than 0.01). The secretion rate of hypothalamic immunoreactive VIP (iVIP) into the pituitary stalk blood was unchanged during aging, while that of dopamine was markedly increased in the aged rats in comparison with the value in both adult and middle-aged rats (p less than 0.01). These results indicate that the basal secretion of hypothalamic VIP is well maintained, while that of hypothalamic dopamine is augmented in aged rats with hyperprolactinemia. It can be assumed that the increase in the pituitary PRL secretion is a primary event during aging in rats, and that a high circulating level of PRL may facilitate the hypothalamic dopamine secretion through the activation of a negative feedback system of the hormone.     

Occurrence of secretogranin II in the prolactin-immunoreactive neurons of the rat lateral hypothalamus: an in situ hybridization and immunocytochemical study

The occurrence of secretogranin II in a neuron population of the rat lateral hypothalamus specifically detected by an anti-serum to ovine prolactin was examined. As this population was previously reported to synthesize dynorphin, the distribution of neurons recognized by ovine prolactin-, dynorphin B- and secretogranin II anti-sera was investigated on adjacent sections of hypothalami. The prolactin immunoreactive neurons were the only cells in the lateral hypothalamus to be stained by secretogranin II anti-serum. Moreover, coupling immunocytochemical detection and in situ hybridization with an oligonucleotide probe complementary to secretogranin II mRNA showed that these neurons expressed the secretogranin II gene. These new findings should help to study the physiological role of the prolactin immunoreactive neurons of the lateral hypothalamus.     

Endocrinology: Vasoactive intestinal polypeptide and peptide histidine methionine. Presence in human follicular fluid and effects on DNA synthesis and steroid secretion in cultured human granulosa/lutein cells

Vasoactive intestinal polypeptide (VIP) and peptide histidinemethionine (PHM) originate from the same precursor molecule,preproVIP. In the present study we examined the concentrationsof VIP and PHM in human follicular fluid and their effects oncultured human granulosa/lutein cells. Follicular fluid andcells were obtained from patients undergoing in-vitro fertilizationfor tubal infertility. The concentrations of VIP and PHM inpre-ovulatory human follicular fluid were measured radioimmunochemically.Granulosa/lutein cells isolated from follicular fluid were culturedunder serum-free conditions with VIP and PHM in varying concentrations(0.1, 10, 1000 nmol/l). [³H]Thymidine incorporation in the cellsand oestradiol as well as progesterone concentrations in theculture medium were measured. The mean (± SEM) concentrationsof VIP and PHM were 6.8 ± 0.1 and 7.7 ± 0.8 pmol/l,respectively. VIP at a concentration of 10 nmol/l caused a significantincrease in (3H) thymidine incorporation, and at 1000 nmol/1a significant increase in oestradiol secretion was observed.VIP had no effect on progesterone secretion. PHM at the concentrationstested did not influence any of the activities. We concludethat VIP and PHM are present in human preovulatory follicularfluid and that VIP stimulates DNA synthesis and oestradiol secretionin cultured human granulosa/lutein cells. This indicates thatVIP and perhaps PHM participate in the local nervous regulationof human ovarian function     

Her-2/neu expression in salivary duct carcinoma: an immunohistochemical and chromogenic in situ hybridization study.

Salivary duct carcinoma (SDC) shares significant morphologic and immunophenotypic overlap with ductal carcinoma of the breast, including HER-2/neu expression. Previous studies have detected HER-2/neu at the protein level in SDCs; however, no study, to date, has assayed whether this expression is related to gene amplification detected by chromogenic in situ hybridization (CISH). Formalin-fixed, paraffin-embedded tissue sections from 12 previously diagnosed SDCs were evaluated by immunohistochemistry (IHC) and CISH for HER-2/neu status. Result concordance was seen in all 12 cases. A total of 4 SDCs were positive by IHC; all 4 cases showed amplification with CISH. The remaining 8 cases were negative by IHC and showed no gene amplification with CISH. SDCs in this study show HER-2/neu overexpression on both the protein and gene levels in approximately 30% of cases. These findings suggest a role may exist for Herceptin (trastuzumab) based therapy in some SDC patients.     

Morphological changes of c-Fos-like immunoreactivity in rat cerebral cortex after cerebral ischemia and reperfusion with special reference to vasoactive intestinal polypeptide

We investigated morphological changes in neurons with c-Fos-like immunoreactivity (c-Fos-LI) after cerebral ischemia by light and electron microscopic immunocytochemistry. Strong c-Fos-LI was observed in layers II–VI of the cerebral cortex with an especially abundant distribution in the nuclei of layers II, IV, and VI ipsilateral to the lesioned side. Reperfusion after ischemia had a greater effect on the expression of c-Fos-LI than did permanent ischemia. Vasoactive intestinal peptide (VIP)-positive neurons were seen scattered in layers II–V of the cerebral cortex. Some VIP-positive neurons showed c-Fos-LI after ischemia. Electron microscopy revealed c-Fos-LI in euchromatin in the nuclei of c-Fos-positive cells. Dilatation of the cisternae of the rough endoplasmic reticulum and the presence of numerous secondary lysosomes were found in neurons on the lesioned side after 12h of reperfusion. Some VIP-containing neurons revealed c-Fos-LI with reperfusion after ischemia by a double immunostaining method on the same tissue section. These findings suggest that ischemia potentiates c-fos expression in VIP- or other transmitter- or modulator-containing neurons, thereby protecting from neuronal cell death.     

Light and electron microscope study on developing intestinal mucosa in rat fetuses with special reference to the obliteration of the intestinal lumen

The development of the small intestine in rat fetuses was studied by light and electron microscopy with special reference to morphological events during the stage of obliteration. Prior to this stage, the epithelium lining the intestinal lumen was simple, tall and columnar and consisted of undifferentiated cells mutually joined by the adluminal junctional complex. At the stage of obliteration, the epithelium appeared stratified and the intestinal lumen was identified as a narrow irregular slit. In addition to the junctional complex surrounding the intestinal lumen, secondary junctional complexes were scattered in the more basal parts of the epithelium. The intra-epithelial cavity was formed within the macula occludens of the secondary junctional complex and became larger by division of the surrounding cells. Since the small intestinal epithelium was essentially simple columnar, mitosis occurred facing the lumen. At the stage of obliteration when the number of epithelial cells was rapidly increasing, mitosis occurred facing not only the main intestinal lumen but also the intra-epithelial cavity. As the intra-epithelial cavities fused with the main intestinal lumen, mesenchyme invaded to form villi and the epithelium returned to a simple columnar form. Consequently, the intestinal lumen was enlarged and villi were formed. At the same time, absorptive cells with a brush border appeared in the villous epithelium and immature goblet cells were occasionally seen.