Phase dependent response of vasoactive intestinal polypeptide to light and darkness in the suprachiasmatic nucleus

Responsiveness of the vasoactive intestinal polypeptide (VIP) content to light and darkness in the rat suprachiasmatic nucleus (SCN) was examined by enzyme immunoassay of micropunched tissues. VIP content in the SCN has been shown to decrease monotonically in animals maintained in illumination. Decreases in VIP content in the SCN in response to both 6-h light and dark pulses depended on the phase of the circadian cycle when the pulses were applied. Light imposed at circadian time (CT) 18 or CT 22 was more effective in suppressing VIP levels than light exposure of the same intensity imposed at CT 0 or CT 6. Darkness interrupting continuous light was more effective at around CT 0 and less effective at around CT 12. These results suggest that VIP responsiveness to light and darkness in the SCN is regulated by the circadian clock in different ways and are correlated with phase-dependent phase shifts in the activity rhythm after light and dark pulses.     

Prolactin-releasing peptide is a potent mediator of stress responses in the brain through the hypothalamic paraventricular nucleus

The effects of i.c.v. administration of prolactin-releasing peptide on neurons in the paraventricular nucleus of rats and plasma corticosterone levels were examined by measuring changes in Fos-like immunoreactivity, c-fos mRNA using in situ hybridization histochemistry, and plasma corticosterone using a specific radioimmunoassay. Approximately 80% of corticotropin-releasing hormone immunoreactive cells exhibited Fos-like immunoreactivity in the parvocellular division of the paraventricular nucleus 90 min after i.c.v. administration of prolactin-releasing peptide. The greatest induction of the c-fos mRNA expression in the paraventricular nucleus was observed 30 min after administration of prolactin-releasing peptide, and occurred in a dose-related manner. Plasma corticosterone levels were also significantly increased 30 min after administration of prolactin-releasing peptide. Next, the effects of restraint stress, nociceptive stimulus and acute inflammatory stress on the expression of the prolactin-releasing peptide mRNA in the dorsomedial hypothalamic nucleus, nucleus of the solitary tract and ventrolateral medulla were examined using in situ hybridization histochemistry for prolactin-releasing peptide mRNA. Restraint stress and acute inflammatory stress upregulated the prolactin-releasing peptide mRNA expression in the nucleus of the solitary tract and ventrolateral medulla. Nociceptive stimulus upregulated the prolactin-releasing peptide mRNA expression in the ventrolateral medulla. Finally, we observed that pretreatment (i.c.v. administration) with an anti-prolactin-releasing peptide antibody significantly attenuated nociceptive stimulus-induced c-fos mRNA expression in the paraventricular nucleus. These results suggest that prolactin-releasing peptide is a potent and important mediator of the stress response in the brain through the hypothalamic paraventricular nucleus.     

Coexistence of glucocorticoid receptor-like immunoreactivity with neuropeptides in the hypothalamic paraventricular nucleus

Summary The paraventricular nucleus (PVN) of male albino rats was analyzed for the presence of glucocorticoid receptor-like immunoreactivity (GR-LI) in neuropeptide containing neurons. Using immunohistochemistry, coronal sections trough the entire PVN were double-stained with a mouse monoclonal antibody against GR and one of the following antisera: rabbit antiserum to corticotropin releasing factor (CRF), neurotensin (NT), enkephalin (ENK), cholecystokinin (CCK), thyrotropin releasing hormone (TRH), galanin (GAL), peptide histidine isoleucine (PHI), vasoactive intestinal polypeptide (VIP), somatostatin (SOM) or tyrosine hydroxylase (TH). For comparison the occurrence of GR-LI in NT-, SOM-, NPY- or TH-positive neurons of the arcuate nucleus was also studied. Our results indicate that GR-LI is present in the parvocellular part of the PVN but not in its magnocellular portion. Virtually every parvocellular neuron in the PVN containing one of the above mentioned peptides was also positive for GR, with the exception of SOM neurons, of which only about two thirds showed detectable levels of GR-LI. All TH-positive, presumably dopamine neurons in the PVN were GR-positive. In the arcuate nucleus all TH- and NPY-positive neurons as well as a large proportion of the SOM- and NT-immunoreactive neurons contained GR-LI. The results indicate that in the PVN, in addition to the CRF neurons, certain peptidergic neurons in the parvocellular part of the PVN, without any established role in the control of ACTH synthesis and release, may also be under glucocorticoid control. This seems to be the case also for most arcuate neurons.     

Threshold-like pattern of neuronal activation in the hypothalamus during treadmill running: establishment of a minimum running stress (MRS) rat model

Despite the indication that the hypothalamo-pituitary-adrenal (HPA) axis is activated during treadmill running, there have not been any studies focusing on the relationship between exercise intensity and region-specific neural activities in hypothalamus. To address this, rats were subjected to 30 min of running, either at middle (supra-LT, 25 m min(-1)) or low speeds (sub-LT, 15 m min(-1)), and c-Fos-(+) cells were counted and compared with control rats. Significant increases in blood glucose and lactate levels, and plasma ACTH and osmolality levels were observed during supra-LT running. Only supra-LT running significantly increased c-Fos induction in various hypothalamic regions, namely, the medial preoptic area (MPO), periventricular nucleus (Pe), suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), parvocellular division of the paraventricular nucleus (pPVN), anterior hypothalamic area (AH), arcuate nucleus (ARC) and posterior hypothalamic nucleus (PH). However, sub-LT caused no effect on c-Fos accumulation. This indicates that the hypothalamus responds uniquely to running in a threshold-like pattern distinct from the speed-dependent pattern previously reported for the medulla oblongata [Ohiwa et al., 2006a,b]. In addition, these results showed a physiologic basis for mild exercise useful for establishing our minimum running stress (MRS) rat model, or the running conditions that minimize the activation of the HPA axis.     

束缚应激大鼠下丘脑内一氧化氮合酶与c-fos蛋白的共存

为了探讨下丘脑内一氧化氮与应激之间的关系，本实验应用NADPH-d组化法和C-fos免疫组化技术相结合的方法，对束缚应激大鼠下丘脑内一氧化氮合酶（NOS）和c-fos蛋白的分布以及两者的共存关系进行了观察。结果表明，大鼠在束缚应激4h后，（1）室旁核大细胞部和视上核可见密集深染的NOS阳性大细胞；（2）室旁核小细胞部、背内侧核、穹窿周核、环状核、腹内侧核腹外侧部、结节内侧核、外侧区、室周区、乳头体前核和内侧核的外侧区等核团出现疏密不等和深浅不一的NOS阳性细胞；（3）C-fos蛋白以室旁核表达最为强烈，视前区、背内侧核、弓状核和外侧区亦有较强的表达；（4）在外侧区、室旁核小细胞部及其附近的室周区、背内侧核及乳头体前核腹侧部约有10％～15％的中、小型NOS阳性细胞同时表达C-fos蛋白，室旁核大细胞部则仅有较少的NOS阳性大细胞表达C－fos蛋白，在结节区、结节内侧核和视上核视交叉后部则偶见双标细胞。结果提示上述下丘脑核团内的部分一氧化氮能神经元与束缚应激反应有关。     

Regional expression of c-fos antigen in the basal forebrain following intraventricular infusions of angiotensin and its modulation by drinking either water or saline.

The expression of c-fos protein was examined in the basal forebrains of male rats 60 min following intracerebroventricular infusions of 250 pmol angiotensin II. Levels of corticosterone and vasopressin were also measured at the same time point. In animals not allowed access to water after infusion, angiotensin II induced intense c-fos expression in a band of neurons extending throughout the anterior region of the third ventricle region, including the organum vasculosum of the lamina terminalis, the median preoptic nucleus (nucleus medianus) and the subfornical organ. There were also high levels of expression in the hypothalamic supraoptic nucleus and the paraventricular nucleus, particularly its lateral (magnocellular) region, though other, parvicellular areas were also affected. No other area of the hypothalamus was altered. There was increased c-fos expression in the central nucleus of the amygdala and the bed nucleus of the stria terminalis. Allowing rats to drink during the 60-min survival period modified this pattern of response. c-fos was markedly reduced in the supraoptic nucleus and the paraventricular nucleus but not in the other areas examined, including the anterior region of the third ventricle and the amygdala. When water was withheld for 15 min, but then allowed, rats drank the same total volume but c-fos expression was no longer inhibited in either the supraoptic nucleus or paraventricular nucleus. When rats were given 0.9% saline to drink, they ingested about three times as much as water, but angiotensin II-induced c-fos expression was similar to that in rats denied access to water. The pattern was similar following access to 1.8% saline, though levels in the organum vasculosum of the lamina terminalis were reduced. There was a marked correlation between the number of c-fos-positive neurons in the supraoptic nucleus or paraventricular nucleus and plasma levels of corticosterone 60 min after infusion, but not with arginine-vasopressin levels. These experiments show that angiotensin II induces highly localized expression of c-fos in areas known to be concerned with the dipsogenic and endocrine actions of this peptide, and that this pattern is selectively altered by allowing the animal to drink solutions of different tonicity. Immediate-early gene expression is a novel and valuable method of determining the neural response to peptides at the cellular level.     

Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain

The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.     

Vasoactive intestinal polypeptide/peptide histidine isoleucine immunoreactive neuron systems in the basal hypothalamus of the rat with special reference to the portal vasculature: an immunohistochemical and in situ hybridization study

Using immunohistochemistry and in situ hybridization, we have analysed the distribution of vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in hypothalamus of male and female Sprague-Dawley rats under normal and experimental conditions. In most cases there was a good overlap between the distribution of VIP- and PHI-immunoreactive structures. At the median eminence level precapillary arterioles along its lateral aspect were surrounded by dense networks of VIP/PHI-positive fibers, suggesting that these peptides, via their vasodilatory property, may be involved in control of blood flow through portal vessels. Furthermore, a thick VIP/PHI-containing nerve bundle of variable size was observed on the surface of the median eminence in coronal, horizontal and sagittal sections. Also this bundle could be of importance for portal circulation, but VIP/PHI released may reach the anterior pituitary level and play a role in, for example, control of prolactin release. Although different lesions were performed, the origin of the VIP/PHI nerves around lateral blood arterioles and of the bundle is still unclear, but is in all probability peripheral. Within the median eminence of untreated rats only few positive nerve endings were seen in the external layer, but after 48 h hypophysectomy a large number of PHI-immunoreactive fibers could be observed. With regard to cell bodies the suprachiasmatic nucleus contained VIP/PHI-immunoreactive neurons even in untreated rats. After colchicine administration fluorescent cells were in addition seen in several other hypothalamic nuclei, including the parvocellular paraventricular nucleus. After hypophysectomy, with in situ hybridization, VIP mRNA could be demonstrated in magno- and parvocellular neurons of the paraventricular nucleus, whereas in control rats VIP mRNA was undetectable. These results demonstrate that VIP/PHI are present in at least three systems of direct neuroendocrine importance: (1) in nerves related to the blood vessels in the median eminence and presumably involved in control of blood flow through the portal system; (2) in parvocellular paraventricular neurons, presumably related to stress-induced prolactin release; and (3) in magnocellular neurons after certain experimental manipulations.     

Rhythmic expression of Fos-related proteins within the rat suprachiasmatic nucleus during constant retinal illumination.

Within the retinorecipient or ventrolateral subfield of suprachiasmatic nuclei (SCN) in rodents, expression of the protein product of the c-fos proto-oncogene, Fos, is regulated by light. In the present study, the expression of Fos and Fos-related proteins within the SCN was examined immunocytochemically for evidence of rhythmic variation in rats sacrificed at different circadian times during exposure to constant retinal illumination (LL). In all animals, nuclear Fos immunoreactivity was mainly confined to an area of the SCN that was coextensive with neuropeptide Y-immunopositive fibers distinguishing the ventrolateral subfield of the nucleus. Moreover, Fos-immunostaining within the ventrolateral SCN of rats exposed to LL fluctuated over the course of the circadian cycle, such that the density of immunopositive cells within this subfield was 2 times greater during the subjective night than during the subjective day. Since Fos expression within the SCN oscillates in the absence of photoperiodic time cues and since the peak of this oscillation coincides with the circadian times when light modulates the periodicity of the SCN pacemaker, these data provide further evidence that expression of the c-fos gene may be a molecular signal in the circadian timekeeping mechanism in the SCN and its regulation by photic stimuli.     

双侧眼球摘除对大鼠视交叉上核的影响──免疫组化及光电镜体视学研究

采用双侧眼球摘除大鼠模型，术后存活不同时间，用免疫组化法探讨视交叉上核内ＶＩＰ和ＡＶＰ免疫反应强度和面积的变化；对变化明显的２１ｄ组进一步用光镜和电镜体现学方法进行形态学研究。＜１＞免疫组化研究：ＶＩＤ免疫染色，２１ｄ组免疫反应强度和面积均比对照组降低（Ｐ＜０．０５），２ｄ和７ｄ组免疫反应强度和面积与对照组相比无显著差异。ＡＶＰ免疫染色，三个时间实验组免疫反应强度和面积均与对照组无明显差异。＜２＞体视学研究：实验组ＶＩＰ样神经元胞体体积、胞核体积、核仁平均直径均明显缩小，粗面内质网和高尔基复合体表面积密度明显下降。以上所见提示摘除眼球２ｌｄ时，视交叉上核内直接接受光信息的ＶＩＰ样神经元的免疫活性下降是因阻断光传入所造成的。光传入的阻断和视神经纤维的溃变使视交叉上核中ＶＩＰ样神经元的结构呈现功能性改变，推测这些变化将引起该神经元肽类物质含量的下降。     

Suppression by glucocorticoid of the immunoreactivity of corticotropin-releasing factor and vasopressin in the paraventricular nucleus of rat hypothalamus

The effect of glucocorticoid on the production of corticotropin-releasing factor (CRF) and vasopressin in the paraventricular nucleus of the hypothalamus (PVH) was examined immunocytochemically. Intraperitoneal administration of dexamethasone sulfate in a dose of 0.1 mg/day suppressed the immunoreactivity of CRF and vasopressin in the medial parvocellular divisions of the PVH of the rat subsequent to bilateral adrenalectomy. In the magnocellular divisions, suppression of vasopressin-immunoreactivity was not observed. These results suggest that the vasopressin in the medial parvocellular divisions plays a distinct role from that in the magnocellular divisions, the former having functional significance in the hypothalamo-hypophysio-adrenal axis.     

Immunoreactivity of hypothalamo-neurohypophysial neurons which secrete corticotropin-releasing hormone (CRH) and vasopressin (Vp): immunocytochemical evidence for a correlation with their functional state in colchicine-treated rats

Summary The specific immunoreactivity of neurons containing corticotropin-releasing hormone (CRH) or vasopressin (Vp) was studied both centrally, in the parvocellular division of the paraventricular nucleus, and distally, in the external median eminence. Control rats were compared with adrenalectomized rats and with animals supplemented with corticosterone or dexamethasone, either without additional treatment, or 24, and 48 h after an intraventricular injection of colchicine. In all groups of animals, colchicine induced a progressive and parallel decrease in both CRH and Vp immunoreactivity within the axons of the external median eminence. A semi-quantitative estimation of this axonal immunostaining showed that the decrease was clearly correlated with the axons' releasing activity according to the different functional states of the adrenocorticotropic system. Increased rates of hormonal release induced by adrenalectomy could be seen in the accelerated depletion of axonal immunoreactivity whereas corticosteroid supplementation had the opposite effect. Correspondingly, the progressive intensification of the CRH and Vp immunoreactivity within the perikarya following colchicine treatment was further markedly enhanced in adrenalectomized rats and diminished after corticosteroid supplementation. Taken together, these data suggest that in these neurons, perikaryal hormone synthesis may be closely related to the releasing activity of the axon terminals. They further point to appropriate colchicine treatment as useful tool for evaluating the functional state of CRH and Vp neurons of the parvocellular paraventricular nucleus under various experimental conditions.     

Presence of estrogen receptor immunoreactivity in the oxytocin-containing magnocellular neurons projecting to the neurohypophysis in the guinea-pig.

The immunoperoxidase technique was used on adjacent sections of guinea-pig brain to compare precisely the distribution of estrogen receptor-immunoreactive cells and progesterone receptor-immunoreactive cells in the supraoptic nucleus and the paraventricular nucleus. Only estrogen receptor-immunoreactive neurons were found in the supraoptic nucleus. A large number of estrogen receptor-positive cells were observed in the periventricular magnocellular groups throughout the rostrocaudal extent of the paraventricular nucleus, whereas only a few progesterone receptor-immunoreactive cells were scattered in the anterior portion of this region. We used a combination of axonal tracing with double immunocytochemical detection to determine whether estradiol acts directly on the oxytocin-immunoreactive neurons which project to the neurohypophysis. Oxytocin-immunoreactive cells were found in the supraoptic nucleus, ventrally to the optic pathways, in subchiasmatic and retrochiasmatic areas, and in the anterior hypothalamic area. These cells were also retrogradely labeled by Granular Blue when this tracer was injected intravenously. In the paraventricular nucleus, the Granular Blue/oxytocin-positive cells were observed in the periventricular magnocellular groups whereas Granular Blue labeled neurons were found in both parvocellular and magnocellular components. We found that almost all the oxytocin-immunoreactive cells revealed estrogen receptor immunoreactivity. In conclusion, the comparative study of distribution of estrogen receptors and progesterone receptors in the guinea-pig supraoptic and paraventricular nuclei indicates that, in the supraoptic nucleus, only estrogen receptors are present and that, in the paraventricular nucleus, they are far more numerous than progesterone receptors. The present findings demonstrate that the magnocellular cells which contain estrogen receptors are oxytocinergic. In addition, these cells are retrogradely labeled pointing to a neurohypophysial projection. It is likely that estradiol controls the hypothalamo-neurohypophysial oxytocin system by direct action on the magnocellular neurons.     

Differences in the suprachiasmatic nucleus and lower subparaventricular zone of diurnal and nocturnal rodents

Diurnal and nocturnal species are profoundly different in terms of the temporal organization of daily rhythms in physiology and behavior. The neural bases for these divergent patterns are at present unknown. Here we examine functional differences in the suprachiasmatic nucleus (SCN) and one of its primary targets in a diurnal rodent, the unstriped Nile grass rat (Arvicanthis niloticus) and in a nocturnal one, the laboratory rat (Rattus norvegicus). Grass rats and laboratory rats were housed in a 12:12 light:dark cycle, and killed at six time points. cFos-immunoreactive rhythms in the SCN of grass rats and laboratory rats were similar to those reported previously, with peaks early in the light phase and troughs in the dark phase. However, cFos-immunoreactivity in the lower subparaventricular zone (LSPV) of grass rats rose sharply 5 h into the dark phase, and remained high through the first hour after light onset, whereas in laboratory rats it peaked 1 h after light onset and was low at all other sampling times. Daily cFos rhythms in both the SCN and the LSPV persisted in grass rats, but not in laboratory rats, after extended periods in constant darkness. In grass rats, the endogenous cFos rhythm in the LSPV, but not the SCN, was present both in calbindin-positive and in calbindin-negative cells. Cells that expressed cFos at night in the region of the LSPV in grass rats were clearly outside of the boundaries of the SCN as delineated by Nissl stain and immunoreactivity for vasopressin and vasoactive intestinal peptide. The LSPV of the grass rat, a region that receives substantial input from the SCN, displays a daily rhythm in cFos expression that differs from that of laboratory rats with respect to its rising phase, the duration of the peak and its dependence on a light/dark cycle. These characteristics may reflect the existence of mechanisms in the LSPV that enable it to modulate efferent SCN signals differently in diurnal and nocturnal species.     

Immunohistochemical evidence for the presence of neuropeptides in the hypothalamic suprachiasmatic nucleus of ground squirrels

The cytoarchitecture and immunocytochemical distribution of neuropeptides (corticotropin-releasing factor, CRF; neuropeptide Y, NPY; oxytocin, OXY; vasopressin, VP; and vasoactive intestinal polypeptide, VIP) were studied in the hypothalamic suprachiasmatic nuclei (SCN) in male and female ground squirrels of two species (Spermophilus tridecemlineatus and S. richardsonii). Immunoreactive (IR) perikarya were found in sections incubated with VP or VIP antisera. VP-IR cell bodies were seen in the dorsal and medial parts of the nucleus in colchicine-treated animals. IR fibers were distributed throughout the SCN. In the ventral part of the nucleus, VIP-IR cells were seen in untreated animals and were more pronounced in colchicine-treated animals. VIP-IR fibers and terminals form a dense plexus throughout the nucleus. Furthermore, NPY-IR terminals and fibers with multiple varicosities, but no IR perikarya, were present in the suprachiasmatic nuclei. Within the borders of the SCN, no cell bodies or fibers were stained with CRF or OXY antisera in any animal.     

后叶加压素在下丘脑的分布和释放途径及失水对正中隆起中后叶加压素的影响

我们用免疫细胞化学方法(ICC)对大白鼠下丘脑中加压素(VP)免疫反应阳性的神经元作了较详细研究。观察到VP阳性神经元存在于下丘脑室旁核(PVN)各亚核、视上核(SON)、交叉上核(SCN)、室周核(PN)及一些附属核团,包括环状核(CN)、交叉后核(RCN)、下丘脑外侧核(HLN)、穹窿周围核(PFN)。且发现一特殊的VP阳性胞体聚集区—很可能是多巴胺神经元聚集区A_(14)中的细胞,位于第三脑室侧壁中1/3段两侧,腹内侧核背侧。本文首次观察到在PVN和SON之间有VP阳性的神经纤维相联系。ICC和免疫电镜研究进一步证明在正中隆起外带存在VP阳性纤维,含大颗粒囊泡的VP末梢紧邻门脉毛细血管。将HRP注入第四脑室用HRP逆行追踪与ICC方法相结合,在PVN中观察到双标细胞,与直接用ICC法所见VP阳性轴突伸入第三脑室腔的结果一致。说明PVN中存在接触脑脊液神经元。干渴动物正中隆起的内、外带中的VP免疫反应明显减弱。     

Analysis of peptide histidine-isoleucine/vasoactive intestinal polypeptide-immunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor- and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus

The distribution of peptide histidine-isoleucine (PHI) and vasoactive intestinal polypeptide (VIP), two peptides derived from the same precursor molecule, was analysed with immunohistochemistry in the central nervous system of the rat, and to a limited extent in some other species including sheep, monkey and man. Special attention was focused on possible cross-reactivity between PHI antisera and corticotropin releasing factor in parvocellular neurons in the hypothalamic paraventricular nucleus projecting to the external layer of the median eminence. (1) Characterization of the PHI and VIP antisera revealed that they recognized different sequences of the peptide molecules. One of the PHI antisera (PHI-N), although mainly N-terminally directed, also probably contained an antibody population directed against the C-terminal amino acid in PHI which is an amidated isoleucine. Rat and human corticotropin releasing factor but not ovine also have an amidated isoleucine in C-terminal position. (2) PHI- and VIP-like immunoreactivity were found with parallel and overlapping distribution in all areas investigated in the rat central nervous system. In many cases coexistence of the two immunoreactivities could be directly demonstrated. PHI neurons were found in some areas so far not know to contain PHI/VIP neurons, including the dorsal septum, the septofimbrial nucleus, the stria terminalis and lamina V of the spinal cord. (3) Using an antiserum directed against the amino acid sequence 111-122 of the VIP/PHI precursor, immunoreactive cell bodies were seen in some areas containing VIP and PHI neurons. PHI- and VIP-like immunoreactivity were expressed in parallel in increasing amounts in the superficial laminae of the dorsal horn after transection of the sciatic nerve [G. P. McGregor et al. (1984) Neuroscience 13, 207-216; S. A. S. Shehab and M. E. Atkinson (1984) J. Anat. 139, 725; S. A. S. Shehab and M. E. Atkinson (1986) Expl Brain Res. 62, 422-430]. (5) The PHI-N antiserum stains large numbers of immunoreactive cells in the parvocellular part of the paraventricular nucleus and these cells are mostly identical with corticotropin releasing factor-positive neurons. Absorption experiments suggested that this PHI-N-like immunoreactivity to a large extent represented cross-reactivity with rat CRF and that earlier demonstration of many PHI-positive neurons in the paraventricular nucleus probably represents an artefact as proposed by F. Berkenbosch et al. (Neuroendocrinology 44, 338-346). However, some cells did, in fact, contain VIP- as well as PHI-like immunoreactivity as was shown with antisera not cross-reacting with corticotropin releasing factor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Right Atrial Stretch Induces Renal Nerve Inhibition and c-fos Expression in Parvocellular Neurones of the Paraventricular Nucleus in Rats

The paraventricular nucleus of the hypothalamus plays a pivotal role in the regulation of plasma volume. Part of the response to an increase in volume load is an inhibition of renal sympathetic nerve activity. The present experiments were designed to determine which subnuclei of the paraventricular nucleus are involved in this sympatho-inhibitory response. Experiments were performed on anaesthetised rats. Activated neurones were recognised by the expression of the early gene c-fos, identified by immunohistochemical labelling of its protein product Fos. Plasma volume loading with 4 % Ficoll 70, using an infusion-withdrawal procedure (2 ml over 1 min) repeated 15 times over 1 h revealed a total of 775 +/- 101 (n = 6) Fos-positive neurones scattered throughout both the magnocellular and parvocellular subnuclei. In comparison, sustained hypertension resulted in 452 +/- 56 (n = 3) Fos-positive neurones similarly distributed, whereas a normotensive control group (n = 3) displayed 115 +/- 18 Fos-positive neurones. Because of this lack of a specific effect we used a more selective stimulation of right atrial receptors via a balloon placed at the junction of the superior vena cava and the right atrium so it did not impede venous return. Inflation of the balloon inhibited renal sympathetic nerve activity (36 +/- 5 %, n = 7) and repetitive inflation over 1 h resulted in c-fos activation of a small number of neurones (54 +/- 14) located only in the parvocellular subnuclei. Whether these are inhibitory interneurones acting within the paraventricular nucleus, or spinally projecting neurones which inhibit or excite renal sympathetic activity by an action in the spinal cord remains to be determined.