Rat Hypothalamic Paraventricular Nucleus Research Articles

Leptin acts in the rat hypothalamic paraventricular nucleus to induce gastric mucosal damage.

Leptin is produced and secreted by adipocytes to regulate body weight homeostasis. Leptin acts centrally to reduce weight by decreasing food intake and increasing energy expenditure. The paraventricular nucleus (PVN) is a central nervous system structure suggested as a site at which leptin acts to exert its central effects. Leptin microinjection (10(-6) M, 0.5 microl) into the PVN of urethan-anesthetized male Sprague-Dawley rats (150-300 g) resulted in significant gastric damage (mean score = 1.75, n = 16). Damage scores were significantly different than those observed after saline microinjection into the PVN (mean score = 0.00, n = 5, P < 0. 05), or leptin microinjection into non-PVN sites (mean score = 0.33, n = 6, P < 0.05). There were no changes in blood pressure (mean area under curve = 401.9 +/- 224.2 mmHg * s, n = 11, P > 0.05) or heart rate (mean area under curve = 40.9 +/- 25.9 beats, n = 10, P > 0.05) in response to leptin microinjection into PVN. These results suggest that leptin acts on a functionally specific population of PVN neurons involved in the control of gastrointestinal function.

Effect of inhibition of glucose and fat metabolism on galanin-R1 receptor mRNA levels in the rat hypothalamic paraventricular and supraoptic nuclei.

Using in situ hybridization histochemistry we have studied the effect of glucose and fat deprivation on galanin-R1 receptor (GAL-R1-R) mRNA levels in the rat paraventricular (PVN) and supraoptic (SON) nuclei after single and repeated i.p. administration of a glucose antimetabolite 2-deoxy-D-glucose (DG), as well as of a fatty acid antimetabolite, sodium mercaptoacetate (MA), treatments known to increase food intake. Both DG and MA injections caused an increase in levels of GAL-R1 mRNA transcripts in the PVN and SON. These results indicate that glucose and fat deprivation increase the sensitivity of PVN and SON neurons to galanin, and that regulation of receptor levels may be an important mechanism in galaninergic signalling.

Corticosterone Can Facilitate as Well as Inhibit Corticotropin-Releasing Hormone Gene Expression in the Rat Hypothalamic Paraventricular Nucleus

Corticosterone Can Facilitate as Well as Inhibit Corticotropin-Releasing Hormone Gene Expression in the Rat Hypothalamic Paraventricular Nucleus

Corticosterone can facilitate as well as inhibit corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus.

We have used in situ hybridization to investigate how basal levels of circulating corticosterone modulate CRH gene transcription in the neuroendocrine parvicellular part of the hypothalamic paraventricular nucleus (PVHmpd) during sustained hypovolemia. In the absence of the stressor, the accumulation rate of the CRH primary transcript exhibited a dose dependency on low maintained levels of plasma corticosterone similar to that previously reported for the mature messenger RNA (mRNA); levels declined as plasma corticosterone increased. In response to hypovolemia, the absence of corticosterone compromised CRH gene transcription mechanisms to mount the activated response seen in intact animals. However, adrenalectomized rats with low doses of corticosterone (insufficient to normalize thymus weights) showed an augmented mRNA response compared with that in intact animals. When replaced corticosterone normalized thymus weights, the magnitude of the mRNA response was reduced to that seen in intact animals. In contrast to CRH gene regulation, PVHmpd proenkephalin mRNA levels were unaffected by corticosterone concentrations. These results suggest that corticosterone affects CRH gene transcription in the PVHmpd using two mechanisms: first, inhibition, which probably uses type II glucocorticoid receptor-dependent mechanisms and contributes to classic negative feedback; and second, facilitation, which is seen at low plasma concentrations and maintains gene transcription in the presence of sustained stress, possibly using type I mechanisms. This suggests that one reason why adrenal insufficiency severely compromises survival of sustained stress is that CRH gene transcription cannot be maintained without previous exposure to low levels of plasma corticosterone.

Magnocellular vasopressinergic neurons in explant cultures are rescued from cell death by ciliary neurotrophic factor and leukemia inhibiting factor

Selective death of magnocellular vasopressinergic neurons in the hypothalamus has been reported in cases of hereditary and idiopathic diabetes insipidus and after experimental lesions of the hypothalamo–neurohypophyseal pathway. To identify trophic factors that promote survival of these neurons, an in vitro model system was established in which organotypic cultures of the rat hypothalamic paraventricular nucleus were maintained in chemically-defined medium. We observe that the majority of magnocellular vasopressinergic neurons die in these cultures, while other cell populations such as corticotrophin-releasing factor producing parvicellular and oxytocin producing magnocellular cells retain a well preserved cytoarchitectonic organization. Degenerating vasopressinergic cells exhibit morphological signs of apoptosis and stained positively when analysed by the terminal deoxynucleotidyl transferase biotinylated dUTP nick end-labelling assay. Partial survival of vasopressinergic neurons occurred after co-culturing the paraventricular nucleus with neurohypophyseal explants, indicating that target-derived factors may be required for the survival of these neurons. Cell survival is dramatically increased by the administration of ciliary neurotrophic factor and leukemia inhibiting factor, but not by interleukin 6 or the members of the neurotrophin family. Reverse transcription–polymerase chain reaction followed by Southern analysis shows the presence of ciliary neurotrophic factor messenger RNA in the neurohypophysis. Thus, endogenous ciliary neurotrophic factor and leukemia inhibiting factor, produced by neurohypophyseal cells may function as a physiological survival factor for neurosecretory vasopressinergic neurons.

Activation by systemic angiotensin II of neurochemically identified neurons in rat hypothalamic paraventricular nucleus.

Using the immunohistochemical localization of the protein product of the immediate early gene, c-fos, to localize activated neurons in the paraventricular nucleus of the hypothalamus (PVN), we studied the chemical phenotypes of neurons activated by circulating angiotensin II (AII). We determined the proportions of activated PVN neurons that expressed AII type I receptor-like immunoreactivity (AT1-L) or the neurohormones vasopressin (VP) and oxytocin (OXY). In addition, we identified activated PVN neurons that putatively produce nitric oxide (NO) on the basis of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). Conscious rats received intravenous AII infusions at a rate sufficient to elevate mean arterial pressure by 40-60 mmHg for 90 min; control rats received infusions of vehicle. Brains were prepared for double immunohistochemistry [Fos-like immunoreactivity (FLI)/AT1-L, FLI/VP or FLI/OXY] or FLI/ NADPH-d histochemistry. Systemic AII infusions led to activation of 149+/-14 PVN neurons per section. In contrast, control animals showed activation of 21+/-6 PVN neurons per section. AII infusions elicited the activation of the following numbers of chemically identified PVN neurons per section: AT1-L, 24+/-5; VP, 26+/-5; OXY, 11+/-2; NADPH-d, 22+/-4. Control animals had few activated PVN neurons per section. For each of the chemically identified populations of PVN neurons, the following proportions were activated: AT1-L, 12.5%; VP, 15.2%; OXY, 7.2%; NADPH-d, 17.3%. The results suggest that PVN neurons producing the AT1 receptor, VP, OXY, and NO, participate in the mediation of the central responses to circulating AII.

An in situ hybridization histochemistry technique allowing simultaneous visualization by the use of confocal microscopy of three cellular mRNA species in individual neurons.

We present a specific and sensitive method for simultaneous detection of three mRNA species in individual neurons. The method relies on the use of riboprobes labeled with [35S]-UTP, digoxigenin-UTP, or biotin-UTP. The nonradioactive probes were sequentially revealed by incubation with anti-digoxigenin immunoglobulins or streptavidin conjugated to peroxidase, followed by the use of fluorochrome-labeled tyramides as peroxidase substrates. The radioactive probe was revealed by conventional autoradiography. There was no interaction among the different probes or the various detection systems. We demonstrate the use of this method by illustrating on laser scanning confocal microscopy the co-localization of the mRNAs coding for corticotropin-releasing factor (CRF), arginine vasopressin (AVP), or peptidylglycine alpha-amidating monooxygenase (PAM) in rat hypothalamic paraventricular nucleus (PVN) and its modulation by endogenous glucocorticoids. Our results suggest that this method could be used not only to study the regulation of the hypothalamo-pituitary-adrenal axis but also in various models in which mRNAs are present at low concentrations.

RU-486 blocks stress-induced enhancement of proenkephalin gene expression in the paraventricular nucleus of rat hypothalamus

The purpose of this study was to investigate the glucocorticoid (GC) mediated regulation of corticotropin-releasing hormone (CRH) and proenkephalin (PE) gene expressions in the paraventricular nucleus (PVN) of the hypothalamus during physical stress induced by a single intraperitoneal (i.p.) injection of hypertonic saline (9% NaCl). Previous intracerebroventricular (i.c.v.) administration of the type II glucocorticoid receptor (GR) antagonist RU-486 (20 ng/rat), increased the basal CRH mRNA levels in the PVN but had no effect on PE gene expression. Stress induced by injection of hypertonic saline increased both CRH and PE mRNA levels in PVN. Administration of RU-486 completely blocked the stress-induced increase of PE mRNA levels, but failed to alter the CRH mRNA levels in the PNV. These data suggests that, under these experimental conditions, endogenous GC are necessary for a normal PE response to hypertonic saline stress.

Nitric oxide in paraventricular nuclei of rat hypothalamus under extreme conditions

The activity of nitroxide synthetase (NOS) was studied histochemically and levels of oxytocin and vasopressin immunocytochemically in rat hypothalamus paraventricular nuclei in on-earth experiments simulating space flight conditions with landing. Colonization of oxytocin and NOS was found in large-cell neurons of paraventricular nuclei. After 15 days under conditions of simulated microgravitation followed by 1-day double gravitation and a short-term (1 day) macrogravitation, activities of NOS and content of neuropeptides increased in large-cell neurons of paraventricular nuclei of experimental animals.

Sexually dimorphic MK801-induced c-fos in the rat hypothalamic paraventricular nucleus

MK801 induces Fos-like immunoreactivity (FLI) in the paraventricular nucleus (PVN) in a sex, age, hormone and dosage dependent manner. MK801 (1.0 mg/kg) elicited greater behavioural disruption, but less FLI in the PVN of cycling and lactating female rats, compared to like-treated males. Results from gonadectomized rats with or without acute steroid replacement resembled those seen in intact rats but sex differences were extinguished. At a lower dose (0.1 mg/kg), behavioural effects were diminished but females exhibited more behavioural disruption. At this dose however, more FLI was seen in the PVN of females compared to males, but only male-lactating female differences were significant. Taken together, the data suggest that the action of MK801 on the PVN is influenced by gonadal steroids.

Distributions of FOS and NADPH-D positive neurons of rat hypothalamic paraventricular nucleus after intraperitoneal administration of IL-1β

Distributions of FOS and NADPH-D positive neurons of rat hypothalamic paraventricular nucleus after intraperitoneal administration of IL-1β

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on corticotropin-releasing hormone ( CRH) gene expression in the rat hypothalamic paraventricular nucleus

Pituitary adenylate cyclase-activating peptide (PACAP) is a 38-amino-acid polypeptide, first isolated from hypothalamus, which directly stimulates in vitro the production of cAMP as well as the release of several pituitary hormones, such as growth hormone and luteinizing hormone. In vivo, PACAP has been shown to stimulate ACTH release. The presence of PACAP receptors in several brain areas, including the hypothalamus, suggests that this peptide might play a role as a neurotransmitter/neuromodulator and might be involved in the regulation of hypophysiotropic neurohormones. In order to study the role of PACAP on corticotropin-releasing hormone (CRH) neuron, we have investigated the effects of intracerebroventricular (i.c.v.) and intravenous (i.v.) injections of PACAP and the potent PACAP antagonist PACAP(6–38) on CRH gene expression in the hypothalamic paraventricular nucleus (PVN) in the male rat. The levels of CRH mRNA were evaluated by quantitative in situ hybridization. The i.c.v. injection of PACAP (4 μg/kg b.wt.) produced a 22% increase in the hybridization signal, an effect which was completely prevented by the concomitant injection of the PACAP antagonist (4 μg/kg b.wt.). On the other hand, the administration of the PACAP antagonist induced by itself a 40% decrease in the amounts of CRH mRNA. The i.v. injection of the same peptides (100 μg/kg. b.wt.) produced very similar results. These data strongly suggest that PACAP is involved in the positive regulation of CRH gene expression via specific central receptors and then can play a role as a neurotransmitter/neuromodulator. The effect observed after i.v. injection of PACAP also suggests that the circulating levels of PACAP can play a role in the modulation of CRH gene expression. PACAP might then be involved in the regulation of the HPA axis by a double mechanism: stimulation of CRH gene expression at the central level and direct effect on pituitary corticotrophs.

Assessment of the Role of Oxytocin Receptors in Phenylpropanolamine-Induced Anorexia in Rats

M cMAHON, L. R. AND PAUL J. WELLMAN. Assessment of the role of oxytocin receptors in phenylpropanolamine induced anorexia in rats. PHARMACOL BIOCHEM BEHAV 57(4) 767–770, 1997.—The anorexic effects of phenylpropanolamine (PPA) have been attributed to activation by PPA of α1-adrenoceptors within rat hypothalamic paraventricular nucleus (PVN). The PVN, however, is a nexus for a number of ascending and descending fibers systems that release transmitters and modulators known to inhibit appetite. The focus of the present study was to assess the possibility that oxytocin activity might play a role in the anorexic action of PPA. The present study therefore examined the effects of systemic administration of the oxytocin antagonist L-366,948 on PPA-induced anorexia. Adult male rats ( n = 10 per group) were pretreated (IP) with either 0, 1, or 2 mg/kg L-366,948 15 min prior to treatment injections (IP) of either 0, 5, 10 and 15 mg/kg PPA. Food and water intakes were recorded for a 30 min period (1600 h) starting 30 min after the treatment injection. Rats pretreated with vehicle and then treated with PPA exhibited a dose-dependent suppression of feeding with a maximal effect evident at 15 mg/kg PPA. Pretreatment with 1 or 2 mg/kg L-366,948 alone did not alter feeding nor did these doses alter the anorexia induced by PPA. These results suggest that direct or indirect oxytocin activity is not a factor in the anorexic action of PPA, a finding that further strengthens the notion that PPA inhibits food intake via activation of α1-adrenoceptors.

Endogenous neurotensin regulates hypothalamic-pituitary-adrenal axis activity and peptidergic neurons in the rat hypothalamic paraventricular nucleus.

Adrenocorticotropin (ACTH) secretion depends primarily on hypophysiotrophic factors released from neurons of the paraventricular nucleus of the hypothalamus. However, the neurochemical factors controlling these neurons, in particular neuropeptides, have had little investigation. In this study, we have investigated the role of neurotensin in the regulation of the different components of the hypothalamo-pituitary-adrenal (HPA) axis under basal and stress conditions in rats. For this purpose, animals were implanted with bilateral cannulae filled with crystals of the neurotensin antagonist, SR 48692, and which were located above the paraventricular nucleus. Five days after surgery, the effects of SR 48692 implants were studied on basal and stress-induced secretion of ACTH and corticosterone. Such treatment did not modify plasma levels of ACTH and corticosterone in basal conditions but reduced ACTH but not corticosterone levels after tail cut procedure. After an exposure to a novel environment for 30 min, both ACTH and corticosterone plasma levels were reduced in the SR 48692-treated group. In situ hybridization studies revealed that chronic administration of SR 48692 induced a significant reduction of CRF mRNA levels in the parvocellular division of the paraventricular nucleus of the hypothalamus. In addition, a 2-fold increase in basal levels of plasma vasopressin associated with an increase in vasopressin mRNA levels in the magnocellular neurons of the paraventricular nucleus was also detected. Finally, the basal plasma levels of oxytocin were not affected by the same treatment. Taken together, these findings strongly suggest that endogenous neurotensin in the paraventricular nucleus plays a tonic stimulatory role on HPA axis activity and an inhibitory effect on vasopressin secretion.

The effect of lactation on induced Fos-like immunoreactivity in the rat hypothalamic paraventricular nucleus

Lactating rats display a period of blunted hypothalamo-pituitary-adrenal (HPA) response to a variety of stressors. This hyporesponsiveness is reported to be dependent upon continuous mother–pup interactions. In this study, computer-assisted densitometric methods were used to measure levels of induced Fos-like immunoreactivity (FLI) in the hypothalamic paraventricular nucleus (PVN) of lactating and non-lactating rats. Adrenalectomy (ADX) induces elevated levels of FLI in the PVN of non-lactating rats. We have observed that, between post-partum day (pd) 4 and pd 21, the level of ADX-induced FLI in the PVN of lactating rats follows a U-shaped distribution; that the persistence of this phenomenon is dependent upon continued mother–pup interaction and that sustained mother–pup interaction beyond the end of the normal suckling period (pd 21) does not extend the period of refractoriness. We have further determined that both the non-specific neural activator Metrazole, and the glutamate agonist N-methyl- d, l-aspartate (NMA), induced smaller increases in FLI in the PVN of lactating rats compared to non-lactating cohorts, and that the suppressing effect of lactation on Metrazole-induced FLI does not extend to all brain regions. These results suggest that mechanisms responsible for the onset and maintenance of the so-called lactational stress-hyporesponsive period (LSHRP) include altered function of glutamatergic pathways. © 1997 Elsevier Science B.V. All rights reserved.

Antidiuretic action of tachykinin NK-3 receptor in the rat paraventricular nucleus

Studies were performed on the central antidiuretic actions via the tachykinin NK-3 receptor in the rat hypothalamic paraventricular nucleus (PVN). Microinjections of the selective tachykinin NK-3 receptor agonist senktide (2–200 pmol) into the PVN resulted in prolonged inhibition of urine output in water-loaded rats, its effect being dose-dependent. The antidiuretic action of senktide was blocked by pretreatment with the vasopressin V2 receptor antagonist OPC-31260 (1 mg/kg, i.v.), but not by microinjection of the angiotensin II AT-1 receptor antagonist losartan (1 nmol) into the PVN. NK-3 receptor mRNA was strongly detected in the magnocellular part of the PVN and the supraoptic nucleus (SON) of the hypothalamus as detected by in situ hybridization histochemistry. Moreover, [ 3H]senktide binding sites were also detected in the PVN and the SON by receptor autoradiography. These findings suggest that NK-3 receptors in the PVN may be involved in water regulation by stimulation of vasopressin secretion from the posterior pituitary gland, and that vasopressin caused water reabsorbtion via the kidney V2 receptor.

Physiological Mapping of Local Inhibitory Inputs to the Hypothalamic Paraventricular Nucleus

Local inhibitory synaptic inputs to neurons of the rat hypothalamic paraventricular nucleus (PVN) were studied by using glutamate microstimulation and conventional intracellular and whole-cell patch-clamp recording in coronal, horizontal, and parasagittal slices of rat hypothalamus. PVN cells were classified as magnocellular or parvocellular neurons on the basis of electrophysiological and post hoc immunohistochemical analyses; GABA-producing neurons were localized with in situ hybridization. Glutamate microstimulation of different sites around the PVN evoked volleys of postsynaptic potentials in 43% of the PVN neurons tested. Some responses to stimulation at each site were blocked by bicuculline, suggesting that they were mediated by the activation of presynaptic GABA neurons. In the coronal plane, presynaptic inhibitory sites were located lateral to the PVN and ventral to the fornix, corresponding to the lateral hypothalamic area and the posterior bed nucleus of the stria terminalis (BNST). In the horizontal plane, presynaptic inhibitory sites were found rostral, lateral, and caudal to the nucleus, corresponding to parts of the anterior hypothalamic area, the posterior BNST, the medial preoptic area, and the dorsomedial hypothalamus. In the parasagittal plane, presynaptic inhibitory neurons were revealed at sites rostral and caudal to the nucleus, corresponding to the medial preoptic area and the dorsomedial hypothalamus, and in a site dorsal to the optic chiasm that included the suprachiasmatic nucleus. These presynaptic sites each contained GABA-producing neurons based on in situ hybridization with a glutamic acid decarboxylase riboprobe and together formed a three-dimensional ring around the PVN. Unexpectedly, both magnocellular and parvocellular neurons received inhibitory synaptic inputs from common sites.

Regulation of corticotropin-releasing factor type 1 (CRF1) receptor messenger ribonucleic acid in the paraventricular nucleus of rat hypothalamus by exogenous CRF

Regulation of corticotropin-releasing factor type 1 (CRF1) receptor messenger ribonucleic acid in the paraventricular nucleus of rat hypothalamus by exogenous CRF

Regulation of corticotropin-releasing factor type 1 (CRF1) receptor messenger ribonucleic acid in the paraventricular nucleus of rat hypothalamus by exogenous CRF.

The present study sought to examine the effects of intracerebroventricular (icv) administration of corticotropin-releasing factor (CRF) on the expression of CRF1 receptor messenger RNA (mRNA) within the hypothalamus as determined by quantitative in situ hybridization histochemistry. Adult male Sprague-Dawley rats were stereotaxically implanted with guide cannulae directed towards the right lateral ventricle. After 8-10 days of recovery, either 10 microliters CRF (5 micrograms) or vehicle solution was injected into the lateral ventricle over a 2-min period. The rats were then deeply anesthetized at 15, 60, and 180 min after icv injection, transcardially perfused, and their brains cut into 30-micron coronal sections. Brain sections were then processed using standard radioactive in situ hybridization histochemistry revealing the expression of the CRF1 receptor mRNA. Low to moderate basal levels of CRF1 receptor transcript were observed in several regions of the forebrain. However, the hybridization signal for the mRNA encoding the CRF1 receptor was barely detectable in the paraventricular nucleus of the hypothalamus (PVN) of vehicle-injected rats. In contrast, 180 min after icv administration of CRF, a significant increase in CRF1 receptor transcript was measured specifically in the PVN, despite having virtually any hybridization signal before 180 min. This increase in the level of receptor transcription by CRF was restricted to the type 1 receptor subtype because the hybridization signal for the CRF2 alpha receptor mRNA was unaffected in the brain regions in which it was located. Moreover, we confirmed previous findings of a CRF-induced neuronal activation of parvocellular neurosecretory cells of the PVN, as assessed by c-fos mRNA expression. This neuronal activation induced by exogenous CRF was also associated with a rapid and strong induction of CRF heteronuclear RNA selectively in the rat PVN, a phenomenon abolished by a pretreatment with a CRF receptor antagonist. These results provide evidence that elevated levels of central CRF may trigger CRF1 receptor transcription selectively in the PVN. This positive feedback of CRF on its own receptor may represent a functional adaptation of the hypothalamic-pituitary-adrenal axis in response to stress.

GABA and glutamate mediate rapid neurotransmission from suprachiasmatic nucleus to hypothalamic paraventricular nucleus in rat.

1. Intracellular sharp electrode and whole-cell patch-clamp recording from characterized paraventricular nucleus (PVN) neurones in rat hypothalamic slices were used to study the synaptic mechanism and associated neurotransmitters that mediate their response to suprachiasmatic nucleus (SCN) stimulation. 2. Electrical stimulation restricted to SCN evoked short-latency inhibitory postsynaptic potentials (IPSPs) or combinations of IPSPs and excitatory postsynaptic potentials (EPSPs) in all (n = 59) PVN neurones tested. Type I neurones (n = 18) were magnocellular and a majority (13/18) demonstrated monosynaptic IPSPs that reversed polarity at the chloride equilibrium potential and were sensitive to bicuculline. 3. Type II (n = 10) and III parvocellular (n = 13), and unclassifiable neurones (n = 18) displayed combinations of IPSPs and EPSPs following similar stimuli applied to SCN. IPSP blockade with bicuculline uncovered SCN-evoked monosynaptic dual-component EPSPs that were sensitive to N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists. In addition, chemical microstimulation within SCN was associated with transient increases in spontaneous EPSPs recorded from these PVN neurones. 4. These data imply that the amino acids GABA and glutamate are important mediators of fast monosynaptic transmission from SCN to defined neurones in PVN, and are candidates for conveying circadian rhythmicity to PVN regulation of neuroendocrine and autonomic processes.