Interleukin-1 beta depolarizes paraventricular nucleus parvocellular neurones

Interleukin-1 beta (IL-1 beta) is involved in hypothalamic regulation of corticotropin releasing hormone (CRH) secretion and consequent downstream modulation of the neuroimmune response. In this study, whole-cell patch clamp recordings of rat parvocellular neurones in a slice preparation of the paraventricular nucleus (PVN) of the hypothalamus were performed to examine the cellular effects of IL-1 beta. In response to 1 nm IL-1 beta, 65% of parvocellular neurones tested exhibited a clear depolarization, which was abolished in the presence of tetrodotoxin (TTX). This depolarization was partially dependent on nitric oxide formation, as demonstrated by attenuation of the response in the presence of N-omega-nitro-L-arginine methylester, a nitric oxide synthase inhibitor. The effects of IL-1 beta on responsive parvocellular neurones were associated with a decrease in the frequency of inhibitory post synaptic potentials (IPSPs). Bicuculline administration blocked the effects of IL-1 beta, suggesting that this cytokine modulates GABA-ergic output, resulting in a decrease in inhibitory input (IPSPs) and consequent depolarization. These data support the conclusion that IL-1 beta influences the excitability of parvocellular neurones in the PVN, as a secondary consequence of nitric oxide generation and modulation of GABAergic inhibitory input to these cells. They elucidate cellular correlates underlying the well-established neuroimmune roles of IL-1 beta in the paraventricular nucleus of the hypothalamus.     

Norepinephrine in the paraventricular nucleus stimulates corticosterone release

Hypothalamic cells containing corticotropin-releasing factor are believed to be densely innervated by noradrenergic terminals. However, the role of norepinephrine (NE) in the control of the hypothalamo-pituitary-adrenal axis has remained undefined, with both excitatory and inhibitory effects suggested by the literature. The present experiments tested the effects of direct hypothalamic infusion of NE on the release of corticosterone (CORT) in awake and freely moving rats. Norepinephrine infusion into the paraventricular nucleus (PVN) produced a dose-dependent increase in circulating levels of CORT. In a mapping study, this stimulatory effect of NE was found to be anatomically localized. The strongest rise in CORT levels (up to 12 micrograms%) was observed after injection into the PVN, where NE acted in a dose-dependent fashion. A somewhat smaller effect was also detected with NE in the dorsomedial nucleus, while no response occurred after injection just dorsal to the PVN, into the ventromedial or supraoptic nuclei, or into the lateral or posterior hypothalamus. Serotonin infusion into the PVN produced a small but statistically reliable increase in circulating CORT levels. However, dopamine injection into this nucleus had no observable effect. These results agree with recent studies suggesting an excitatory function of PVN NE in the pituitary-adrenal axis.     

Distributions of periventricular projections of the paraventricular nucleus to the median eminence and arcuate nucleus

Neuronal projections from the periventricular subnucleus of the hypothalamic paraventricular nucleus to the median eminence and the arcuate nucleus were investigated in the rat by the anterograde tract-tracer, Phaseolus vulgaris leucoagglutinin. The vast majority of labeled fibers coursed ventrally along the third ventricle and distributed in the external layer of the median eminence bilaterally, with ipsilateral predominance moving caudalwards. Periventricular fibers also terminated in the arcuate nucleus, but this innervation was exclusively ipsilateral.     

Norepinephrine applied in the paraventricular hypothalamic nucleus stimulates vasopressin release

The present experiments were designed to investigate the effect of norepinephrine (NE) applied directly in the area of the paraventricular nucleus (PVN) of the hypothalamus on arginine-vasopressin (AVP) release and blood pressure. A microinjection of 0.4 micrograms NE in the PNV produced a plasma AVP level of 26.3 +/- 5.3 pg/ml compared to 5.3 +/- 0.6 pg/ml in controls receiving dextrose (P less than 0.001). This rise was associated with blood pressure elevations varying between 10 and 13 mm Hg, lasting for about 5 min. Systemic injection of an antivasopressor AVP antagonist reversed or prevented the blood pressure rise induced by NE microinjection. The data suggest that locally applied NE in vasopressinergic neurons of the hypothalamus stimulates the release of AVP and induces an AVP-dependent rise in blood pressure.     

Separate neurons in the paraventricular nucleus project to the median eminence and to the medulla or spinal cord

Double labeling experiments with the retrogradely transported fluorescent tracers bisbenzimide and True blue indicate that separate populations of cells in the paraventricular nucleus of the hypothalamus project to the median eminence and to the dorsomedial medulla or spinal cord in the rat. The cell populations giving rise to each projection are, however, at least partially intermixed in different parts of the nucleus.     

Methamphetamine stimulates the release of neuropeptide Y and noradrenaline from the paraventricular nucleus in rats

Effects of methamphetamine (MAP) on the extracellular neuropeptide Y (NPY) and noradrenaline (NA) levels were examined in the vicinity of the paraventricular nucleus (PVN) of freely moving rats by means of push-pull perfusion. The NA and NPY levels increased significantly in 30–60 min and reached the maximum level in 90–120 min after intraperitoneal administration of MAP. The effects were dose-dependent. The maximum levels were 1.6-fold of the pretreatment level for NPY and 7-fold for NA, respectively, when 5.0 mg/kg b.w. of methamphetamine was administered. It is concluded that MAP stimulates the releases of paraventricular NPY and NA, but the effect is more strong for NA than for NPY.     

Enkephalin-immunoreactive neurons in the parvicellular subdivisions of the paraventricular nucleus project to the external zone of the median eminence.

The enkephalin-immunoreactive neurons that project to the external zone of the median eminence were identified on thin paraffin and thick vibratome sections using a combination of retrograde labeling with peripherally administered Fluoro-Gold and immunocytochemistry. The vast majority of the enkephalin-immunoreactive neurons that project to the external zone of the median eminence (ME) reside in the paraventricular nucleus (PVN) of the hypothalamus. Within the PVN, the majority of these hypophysiotropic neurons are located in the medial parvicellular subdivision, while a smaller number can be detected in the anterior and the periventricular subdivisions. Although many enkephalin-immunoreactive neurons are present in other hypophysiotropic areas of the hypothalamus, such as the medial preoptic area, the anterior periventricular area, and the arcuate nucleus, only a few of these can be retrogradely labeled from the ME. These results provide morphological evidence for the key role of paraventricular enkephalin-immunoreactive neurons in the regulation of neuroendocrine functions.     

Central carbachol stimulates vasopressin release into interstitial fluid adjacent to the paraventricular nucleus

We have used an in vivo double microdialysis probe technique in conscious rats to determine whether the application of carbachol to one paraventricular nucleus (PVN) can result in increased local release of vasopressin from that PVN. Experiments were carried out 24 h after placement of microdialysis probes lateral to each PVN. When both probes were perfused initially with 0.9% NaCl, vasopressin was detected in the outflow (dialysate) from both probes. When carbachol (100 μg/ml) was included in the perfusate of one probe for the first 10 min of a 30-min collection period, while the other probe continued to be perfused with saline alone, there was a seven-fold increase in the concentration of vasopressin in the dialysate from the carbachol-perfused probe; the vasopressin concentration in the dialysate from the contralateral probe increased only slightly. The plasma vasopressin concentration was also elevated. When one of the paired probes was perfused with carbachol (100 μg/ml) for 30 min, there were similar increases in the concentration of vasopressin in the dialysate from both probes and a sustained increase in the plasma vasopressin concentration. Thus, vasopressin is released into the interstitial fluid adjacent to the PVN under basal conditions, and this release can be substantially increased when vasopressin secretion to the periphery is stimulated.     

Interleukin-1beta release in the supraoptic nucleus area during osmotic stimulation requires neural function

Interleukin (IL)-1beta is present throughout the magnocellular neuroendocrine system and co-depletes with oxytocin and vasopressin from the neural lobe during salt-loading. To examine whether IL-1beta is released from the dendrites/soma of magnocellular neurones during osmotic stimulation, microdialysis adjacent to the supraoptic nucleus (SON) in conscious rats was combined with immunocapillary electrophoresis and laser-induced fluorescence detection to quantify cytokine in 5-min dialysates collected before (0-180 min; basal), and after (180-240 min), hypertonic saline injected s.c. (1.5 m NaCl). Osmotic release of IL-1beta was compared after inhibiting local voltage-gated channels for Na+ (tetrodotoxin) and Ca2+ (cadmium and nickel) or by reducing intracellular Ca2+ stores (thapsigargin). Immunohistochemistry combined with microdialysis was used to localise cytokine sources (IL-1beta+) and microglia (OX-42+). Under conditions of microdialysis, the basal release of IL-1beta+ in the SON area was measurable and stable (pg/ml; mean +/- SEM) from 0-60 min (2.2 +/- 0.06), 60-120 min (2.32 +/- 0.05) and 120-180 min (2.33 +/- 0.06), likely originating locally from activated microglia (OX42+; IL-1beta+; ameboid, hypertrophied) and magnocellular neurones expressing IL-1beta. In response to osmotic stimulation, IL-1beta increased progressively in dialysates of the SON area by a mechanism dependent on intracellular Ca2+ stores sensitive to thapsigargin and, similar to dendritic secretion of oxytocin and vasopressin, required local voltage-gated Na+ and Ca2+ channels for activation by osmoregulatory pathways from the forebrain. During osmotic stimulation, neurally dependent release of IL-1beta in the SON area likely upregulates osmosensitive cation currents on magnocellular neurones (observed in vitro by others), to facilitate dendritic release of neurohypophysial hormones.     

Chronic infusion of norepinephrine and clonidine into the hypothalamic paraventricular nucleus

Previous experiments have shown that acute injection of NE and CLON into the PVN initiates a short-term feeding response in satiated rats. This study examined, in brain-cannulated rats, the impact of remote, chronic injections of NE, CLON, or saline on daily food intake and body weight gain. Over a period of 14 days, NE was infused into the PVN, either continuously at a rate of 12 nm/microliter/hr, or discretely at a rate of 6 nm/microliter/sec. In addition, the alpha 2-adrenergic agonist CLON was infused into the PVN discretely at a rate of 3 nm/0.5 microliter/30 sec. Relative to saline infusion, chronic (continuous or discrete) stimulation of the PVN with either of these drugs was effective in potentiating daily food intake by 12-19% and in increasing body weight gain, from approximately 1.5 g/day to 3.3 g/day. This evidence indicates that medial hypothalamic NE, especially within the PVN, is sufficiently robust to alter long-term feeding patterns and body weight regulation.     

Correlations of norepinephrine release in the paraventricular nucleus with plasma corticosterone and leptin after systemic lipopolysaccharide: blockade by soluble IL-1 receptor

The purpose of the study was to investigate the effects of systemic lipopolysaccharide (LPS) on norepinephrine (NE) release in the paraventricular nucleus (PVN) and on plasma concentrations of corticosterone and leptin. Soluble IL-1 receptor (sIL-1R) was used to determine the role of interleukin-1 (IL-1) in these effects. Adult male rats were implanted with a push-pull cannula in the PVN and a jugular catheter to facilitate blood sampling. On the day of the experiment, after the collection of a pretreatment blood and perfusate sample, rats were injected (i.p.) with the vehicle for LPS (saline), 2.5 or 10 microg/kg BW LPS. Other groups of animals were treated i.p. with 25 microg of sIL-1R, or a combination of 10 microg/kg BW of LPS and 25 microg of sIL-1R, 5 min before and 90 min after LPS. Blood and perfusate samples were collected at 30-min intervals for 6 h. NE concentrations in the perfusate were measured using HPLC-EC and corticosterone and leptin levels in the plasma were measured using radioimmunoassay. NE release in the PVN was dose dependent and increased significantly within 90 min in response to the high dose of LPS and reached maximum levels around 180 min before declining gradually to pretreatment levels at 330 min. The corticosterone profile in LPS-treated animals was similar to the NE release profile in the PVN. In contrast, the LPS-induced increase in leptin levels reached a maximum at 210 min and remained elevated even at the end of the observation period. Treatment with sIL-1R completely blocked the LPS-induced effects. It is concluded that LPS stimulates NE release in the PVN and increases plasma concentrations of corticosterone and leptin and that these effects are mediated at least in part by IL-1.     

Morphine and anandamide coupling to nitric oxide stimulates GnRH and CRF release from rat median eminence: neurovascular regulation

Nitric oxide (NO) is involved in neurohormonal secretion from median eminence neuroendocrine nerve terminals. We report that stimulation of NO release from median eminence fragments including vascular tissues occurs by μ₃ receptor activation by morphine, or by cannabinoid type 1 receptor activation by anandamide. The released levels of NO are lower after anandamide than after morphine stimulation. These processes can be blocked by L-NAME, a specific nitric oxide synthase inhibitor, by naloxone for the morphine-stimulated NO release, or SR 141716A, a specific CB1 receptor inhibitor, for the anandamide-stimulated NO release. Furthermore, morphine and anandamide, by this NO dependent process, influences neurohormonal release from median eminence nerve terminals within 10 min. Via this NO dependent process, morphine stimulates both GnRH and CRF release, whereas anandamide selectively stimulates GnRH release. These observations together with previous data suggest that morphine and the anandamide-stimulated NO originates from the vascular endothelium of the portal plexus. These results indicate that endothelial cells of the median eminence may be involved in the release of neurohormones.     

Neurons in the paraventricular nucleus projecting to the median eminence: a study of their afferent connections from peripheral baroreceptors, and from the A1-catecholaminergic area in the ventrolateral medulla

In male rats anesthetized with urethane-chloralose, extracellular recordings were made from tuberoinfundibular (TI) neurons in the paraventricular nucleus (PVN) identified by antidromic stimulation of the median eminence. Activation of baroreceptors induced by intravenous administration of phenylephrine inhibited approximately two-thirds of the 32 TI-neurons tested. Electrical stimulation of the A1-catecholaminergic area in the ventrolateral medulla produced an excitation in one half of the 28 neurons. Microinjection of L-glutamate (40 nl of 0.5 M solution of pH 7.4-7.6) to the same A1-area evoked an excitation in 7 out of 8 TI-neurons tested. The results show that some TI-neurons in the PVN receive inhibitory synaptic inputs from the baroreceptors and excitatory inputs from neurons in the A1-catecholaminergic area.     

Stress increases oxytocin release within the hypothalamic paraventricular nucleus

Evidence indicates that the hypothalamic paraventricular nucleus (PVN) and oxytocin (OT) neurons in particular play a role in the physiological response to stress. Microdialysis (MD) experiments were performed to determine whether OT is released into the PVN during shaker stress. Male rats were prepared with venous catheters and PVN guide cannulae. OT and vasopressin (VP) release into PVN and peripheral blood were measured under basal conditions and during and after shaker stress (10 min at 110 cycles/min). Stress produced a specific increase in PVN and plasma OT. Dialysate OT levels were 0.3±0.1, 2.8±1.2 and 1.3±0.6 pg/sample (control, stress and recovery, respectively). Plasma OT was significantly increased during stress (3.7±1.2 vs. 11.7±2.3 pg/ml, basal vs. stress, respectively). When MD probes were located outside the PVN, there was no increase in OT release, demonstrating site specificity. Stress produced no change in VP levels, either in dialysate or plasma. These results show that OT, but not VP, is released into the PVN and peripheral blood in response to shaker stress. The data raise the possibility that local release of OT into the PVN plays a role in the neuroendocrine stress cascade.     

Neuropeptide Y, epinephrine and norepinephrine in the paraventricular nucleus: Stimulation of feeding and the release of corticosterone, vasopressin and glucose

The paraventricular nucleus (PVN) is known to have an important function in mediating a variety of behavioral and endocrine responses. In the present study, the responsiveness of the PVN to the effects of the coexisting neurotransmitters, neuropeptide Y (NPY), epinephrine (EPI) and norepinephrine (NE), was examined. Albino rats were each chronically implanted with a swivel brain-cannula that permits chemicals to be infused without disturbing the animals' ongoing behavior. When infused into the PVN, each of these neurotransmitters elicited a reliable feeding response during the first hour after injection. The response to EPI was significantly stronger than that of NE and NPY, while the latency to eat after injection was considerably longer for NPY as compared to the catecholamines. In tests with food absent, each of these substances also increased blood levels of corticosterone (EPI greater than NE = NPY) and vasopressin (NPY greater than EPI greater than NE) and revealed a significant positive correlation between circulating levels of these two hormones. In addition, EPI and NE, in contrast to NPY, caused a simultaneous rise in blood glucose, producing levels that were positively correlated with the hormones. No relationship, however, was detected between these endocrine changes and the rats' feeding-stimulatory responses. Together with other evidence, these results suggest that adrenergic as well as noradrenergic innervation to the PVN has a key role in the behavioral and endocrine systems of this nucleus and, moreover, that NPY generally mimics the effects of these catecholamines in the PVN.     

Capsaicin increases GFAP and glutamine synthetase immunoreactivity in rat arcuate nucleus and median eminence

Antibodies to glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) were used to determine the effect of s.c. capsaicin (after 75 min) on astroglial cells in the rat arcuate nucleus-median eminence (ARC-ME). Compared to vehicle, capsaicin significantly increased GFAP and GS immunoreactivity in the ARC-ME. Co-localization of GFAP and GS was observed in the ARC-ME complex. Since GS is primarily responsible for glutamate-glutamine metabolism, the increase in total immunostaining for GFAP-and GS- staining suggests a functional adjustment to cope with some of the capsaicin-induced effects. Together with the involvement of nitric oxide synthase in the ARC-ME response to capsaicin, these observations indicate activity-dependent plasticity of the neuron-glia network in response to this stressful/noxious stimulus.     

Microinjection of thyrotropin-releasing hormone in the paraventricular nucleus of the hypothalamus stimulates gastric contractility

Changes in gastric contractility following microinjection of thyrotropin-releasing hormone (TRH) into the paraventricular nucleus of the hypothalamus (PVN) were examined in fasted, urethane-anesthetized rats. Gastric contractility was measured with extraluminal force transducers and analysed by computer. Unilateral and bilateral PVN microinjections of TRH (0.5 and 1.0 μg) significantly increased the force index of gastric contractions from 0 to 60 min postinjection, when compared with animals microinjected with 0.1 μg TRH, 0.1% BSA or TRH (0.5 and 1.0 μg TRH) in sites adjacent to the PVN. The gastric force index was also significantly elevated from 61 to 120 min postinjection in rats receiving bilateral PVN microinjections of TRH (0.5 and 1.0 μg). Peak gastric responses occurred within 10–20 min postinjection and represented an approximately eight-fold increase over basal values. In the remaining groups, the force index was not significantly altered from preinjection values. The excitatory action of TRH (1.0 μg) on gastric contractility was completely abolished by subdiaphragmatic vagotomy. These results suggest that TRH acts within the PVN to stimulate gastric contractility via vagal-dependent pathways.     

Substance P-like immunoreactive neurons in the arcuate nucleus project to the median eminence in rat

Substance P-containing nerve fibers in the median eminence of rat arise in cells located in the arcuate nucleus. Two days following surgical lesioning of the median eminence immunoreactive substance P accumulated in neuronal perikarya in the middle part (rostrocaudally) of the arcuate nucleus, mainly in its ventromedial portion. Substance P-immunostained cells appeared nowhere else in the hypothalamus following surgical lesion of the median eminence while they were found in several hypothalamic and extrahypothalamic cell groups after colchicine treatment.