Paraventricular Nucleus Of Conscious Rats Research Articles

Regulation of arterial pressure by the paraventricular nucleus in conscious rats: interactions among glutamate, GABA, and nitric oxide

The paraventricular nucleus (PVN) of the hypothalamus is an important site for autonomic and neuroendocrine regulation. Experiments in anesthetized animals and in vitro indicate an interaction among gamma-aminobutyric acid (GABA), nitric oxide (NO), and glutamate in the PVN. The cardiovascular role of the PVN and interactions of these neurotransmitters in conscious animals have not been evaluated fully. In chronically instrumented conscious rats, mean arterial pressure (MAP) and heart rate (HR) responses to microinjections (100 nl) in the region of the PVN were tested. Bilateral blockade of ionotropic excitatory amino acid (EAA) receptors (kynurenic acid, Kyn) in the PVN produced small but significant decreases in MAP and HR. GABAA receptor blockade (bicuculline, Bic), and inhibition of NO synthase [(NOS), N-(G)-monomethyl-L-arginine, L-NMMA] each increased MAP and HR. The NO donor sodium nitroprusside (SNP) produced depressor responses that were attenuated by Bic. NOS inhibition potentiated both pressor responses to the selective EAA agonist, N-methyl-D-aspartic acid (NMDA), and depressor responses to Kyn. Increases in MAP and HR due to Bic were blunted by prior blockade of EAA receptors. Thus, pressor responses to GABA blockade require EAA receptors and GABA neurotransmission contributes to NO inhibition. Tonic excitatory effects of glutamate in the PVN are tonically attenuated by NO. These data demonstrate that, in the PVN of conscious rats, GABA, glutamate, and NO interact in a complex fashion to regulate arterial pressure and HR under normal conditions.

Switching control of sympathetic activity from forebrain to hindbrain in chronic dehydration

We investigated the mechanisms responsible for increased blood pressure and sympathetic nerve activity (SNA) caused by 2-3 days dehydration (DH) both in vivo and in situ preparations. In euhydrated (EH) rats, systemic application of the AT(1) receptor antagonist Losartan and subsequent pre-collicular transection (to remove the hypothalamus) significantly reduced thoracic (t)SNA. In contrast, in DH rats, Losartan, followed by pre-collicular and pontine transections, failed to reduce tSNA, whereas transection at the medulla-spinal cord junction massively reduced tSNA. In DH but not EH rats, selective inhibition of the commissural nucleus tractus solitarii (cNTS) significantly reduced tSNA. Comparable data were obtained in both in situ and in vivo (anaesthetized/conscious) rats and suggest that following chronic dehydration, the control of tSNA transfers from supra-brainstem structures (e.g. hypothalamus) to the medulla oblongata, particularly the cNTS. As microarray analysis revealed up-regulation of AP1 transcription factor JunD in the dehydrated cNTS, we tested the hypothesis that AP1 transcription factor activity is responsible for dehydration-induced functional plasticity. When AP1 activity was blocked in the cNTS using a viral vector expressing a dominant negative FosB, cNTS inactivation was ineffective. However, tSNA was decreased after pre-collicular transection, a response similar to that seen in EH rats. Thus, the dehydration-induced switch in control of tSNA from hypothalamus to cNTS seems to be mediated via activation of AP1 transcription factors in the cNTS. If AP1 activity is blocked in the cNTS during dehydration, sympathetic activity control reverts back to forebrain regions. This unique reciprocating neural structure-switching plasticity between brain centres emphasizes the multiple mechanisms available for the adaptive response to dehydration.

Central Endogenous Vasopressin Induced by Central Salt‐Loading Participates in Body Fluid Homeostasis through Modulatory Effects on Neurones of the Paraventricular Nucleus in Conscious Rats

Peripherally secreted arginine vasopressin (AVP) plays a role in controlling body fluid homeostasis, and central endogenous AVP acts as a neurotransmitter or neuromodulator. The limbic system, which appears to exert an inhibitory effect on the endocrine hypothalamus, is also innervated by fibres that contain AVP. We examined whether central endogenous AVP is also involved in the control of body fluid homeostasis. To explore this possibility, we examined neuronal activity in the paraventricular nucleus of the hypothalamus (PVN), periventricular parts of the PVN and limbic brain areas, as well as AVP mRNA expression in the PVN and the peripheral secretion of AVP after central salt-loading in rats that had been pretreated i.c.v. with the AVP V(1) receptor antagonist OPC-21268. Neuronal activity in the PVN evaluated in terms of Fos-like immunoreactivity (FLI), especially in the parvocellular subdivisions, was suppressed. On the other hand, FLI was enhanced in the lateral septum, the bed nucleus of the stria terminalis and the anterior hypothalamic area. Similarly, AVP mRNA expression was enhanced in the magnocellular subnucleus of the PVN, despite the lack of a significant difference in the peripheral AVP level between OPC-21268- and vehicle-pretreated groups. We recorded renal sympathetic nerve activity (RSNA) as sympathetic nerve outflow during central salt-loading. The suppression of RSNA was significantly attenuated by i.c.v. pretreatment with OPC-21268. These results suggest that the suppression of RSNA during central salt-loading might be the result of a decrease in neuronal activity in the parvocellular subdivisions of the PVN via the inhibitory action of central endogenous AVP. The parvocellular and magnocellular neurones in the PVN might show different responses to central salt-loading to maintain body fluid homeostasis as a result of the modulatory role of central endogenous AVP.

Modulatory effects of GABA in the Paraventricular Nucleus of conscious rats subjected to exercise training

The paraventricular nucleus (PVN) of the hypothalamus is an important site for autonomic and neuroendocrine regulation, where gamma‐aminobutyric acid (GABA) system play an important role. The central mechanisms underlying modulatory effects of exercise training, have yet to be characterized. The aim of this study was analyze the cardiovascular effects of microinjection of bicuculine (BIC), muscimol (Musc), into the PVN of conscious rats submitted to swimming training (ST). After the ST protocol, adult male Wistar rats (250 to 300 g) were anesthetized for implantation of guide cannulas to PVN. After 5 days, a femoral artery and a femoral vein were catheterized for recording mean arterial pressure (MAP) and drugs injection. 24 hours later, unilateral microinjection of BIC (n=8) in the control rats (C) produced an increase in MAP (23±3 mmHg) and HR (93±12 bpm). After ST (n=9) the cardiovascular effects of BIC were attenuated for MAP and did not change for HR (DMAP= 15±2 mmHg; DHR= 88±19 bpm). A bilateral microinjection of Musc in C rats induced a decrease in MAP and HR (DMAP= −14±1 mmHg; DHR=−95±18 bpm). After ST Musc promoted a higher decrease in MAP and no alterations in HR (DMAP= −28±4 mmHg, DHR=−54±12 bpm). Our data showed that ET seems to enhance GABA tonic inhibition in the PVN and/or a blunted effect in the excitatory system to regulate cardiovascular function, which are in line with a decreased sympathetic activity.

Exposure to a hot environment can activate rostral ventrolateral medulla‐projecting neurones in the hypothalamic paraventricular nucleus in conscious rats

A major integrative site within the brain for autonomic function is the hypothalamic paraventricular nucleus (PVN). Several studies have suggested that the PVN may be involved in the responses regulating body temperature. Hyperthermia elicits redirection of blood flow from the viscera to the periphery and involves changes in sympathetic nerve activity mediated by the central nervous system. The hypothalamic PVN includes neurones that project to the rostral ventrolateral medulla (RVLM), an important autonomic region involved in the tonic regulation of sympathetic nerve activity. This pathway could contribute to the cardiovascular changes induced by hyperthermia. The PVN has a high concentration of nitrergic neurones and it is known that nitric oxide within the brain mediates heat dissipation. Thus the aims of this study were to determine whether RVLM-projecting neurones in the PVN are activated by heat and whether those neurones are also nitrergic. The results show that, compared with control conditions, exposure of conscious rats to a hot environment of 39 degrees C significantly increased the number of neurones containing a Fos-positive nucleus (a marker of activation) and significantly increased the number of activated RVLM-projecting neurones in the PVN. Also, although heating significantly increased the number of activated nitrergic PVN neurones, triple-labelled neurones (i.e. activated, nitrergic and RVLM projecting) in the PVN were rarely observed. The results suggest that RVLM-projecting neurones in the PVN may play a role in responses to heat exposure but these are not nitrergic.

Combination of inflammatory cytokines increases nitrite and nitrate levels in the paraventricular nucleus of conscious rats

Inflammatory cytokines stimulate glial cells in vitro to produce nitric oxide (NO) from inducible NO synthase (iNOS). Whether the stimulation with cytokines produces NO derived from iNOS has not hitherto been demonstrated in the vivo brain. Nitrite and nitrate (NOx −) levels in the rat paraventricular nucleus (PVN) were measured before and after intraparenchymal microinjection of cytokines with a microdialysis technique. The cytokines, tumor necrosis factor (TNF)-α (10 ng), interleukin (IL)-1β (2 ng), and interferon (IFN)-γ (2 ng) were microinjected. None of the cytokines alone had any effect on the NOx − levels for 8 h. But a combination of TNF-α and IFN-γ gradually increased NOx − levels beginning at 140 min after the microinjection, and NOx − levels reached 1.8 times the basal level at 380 min. A combination of TNF-α and IL-1β increased NOx − beginning at 340 min, reaching 1.7 times the basal level at 440 min, whereas a combination of IL-1β and IFN-γ had no effect. Microinjection of a mixture of all three cytokines increased NOx − levels beginning at 120 min, reaching 3.3 times the basal level at 400 min. Aminoguanidine, which is a selective inhibitor of iNOS, reduced NOx − levels induced by the mixed cytokine treatment. Semi-quantitative RT–PCR for iNOS mRNA was done. The intensity of the iNOS mRNA band for the cytokine-treated PVN was stronger than that for the vehicle-treated PVN. These results suggest that the increased NOx − after the treatment with mixed cytokines were dependent on iNOS activity. This is the first report to indicate that only cytokines induce NOS in vivo in the brain.

Renal effects of angiotensin II receptor subtype 1 and 2-selective ligands injected into the paraventricular nucleus of conscious rats

We determined the effects of losartan and CGP42112A (selective ligands of the AT 1 and AT 2 angiotensin receptors, respectively) and salarasin (a relatively nonselective angiotensin receptor antagonist) on urinary volume and urinary sodium and potassium excretion induced by administration of angiotensin II (ANG II) into the paraventricular nucleus (PVN) of conscious rats. Both the AT 1 and AT 2 ligands and salarasin administered in the presence of ANG II elicited a concentration-dependent inhibition of urine excretion, but losartan inhibited only 75% of this response. The IC 50 for salarasin, CGP42112A, and losartan was 0.01, 0.05, and 6 nM, respectively. Previous treatment with saralasin, CGP42112A and losartan competitively antagonized the natriuretic responses to PVN administration of ANG II, and the IC 50 values were 0.09, 0.48, and 10 nM, respectively. The maximum response to losartan was 65% of that obtained with saralasin. Pretreatment with saralasin, losartan, and CGP42112A injected into the PVN caused shifts to the right of the concentration–response curves, but the losartan concentrations were disproportionately greater compared with salarasin or CGP42112A. The IC 50 values were 0.06, 0.5, and 7.0 for salarasin, CGP42112A, and losartan, respectively. These results suggest that both AT 1 and AT 2 receptor subtypes in the PVN are involved in ANG II-related urine, sodium, and potassium excretion, and that the inhibitory responses to AT 2 blockade are predominant.

Differential Regulation of Corticotropin-Releasing Hormone and Vasopressin Gene Transcription in the Hypothalamus by Norepinephrine

All stress-related inputs are conveyed to the hypothalamus via several brain areas and integrated in the parvocellular division of the paraventricular nucleus (PVN) where corticotropin-releasing hormone (CRH) is synthesized. Arginine vasopressin (AVP) is present in both magnocellular and parvocellular divisions of the PVN, and the latter population of AVP is colocalized with CRH. CRH and AVP are co-secreted in the face of certain stressful stimuli, and synthesis of both peptides is suppressed by glucocorticoid. CRH and AVP stimulate corticotropin (ACTH) secretion synergistically, but the physiological relevance of the dual corticotroph regulation is not understood. Norepinephrine (NE) is a well known neurotransmitter that regulates CRH neurons in the PVN. We explored the mode of action of NE on CRH and AVP gene transcription in the PVN to examine the effect of the neurotransmitter on multiple genes that are responsible for a common physiological function. After NE injection into the PVN of conscious rats, CRH heteronuclear (hn) RNA increased rapidly and markedly in the parvocellular division of the PVN. AVP hnRNA did not change significantly in either the parvocellular or magnocellular division of the PVN after NE injection. The present results show that the transcription of CRH and AVP genes is differentially regulated by NE, indicating the complexity of neurotransmitter regulation of multiple releasing hormone genes in a discrete hypothalamic neuronal population.

Cardiovascular responses produced by microinjection of serotonin-receptor agonists into the paraventricular nucleus in conscious rats.

Activation of serotonin (5-hydroxytryptamine, 5-HT) receptors in the brain produces cardiovascular responses by altering autonomic outflow. The paraventricular nucleus (PVN) contains a modest density of 5-HT receptors and has connections to autonomic centers. Experiments were designed to determine whether cardiovascular responses were produced by the administration of 5-HT2- and 5-HT1A-receptor agonists into the PVN of conscious rats. The microinjection of the 5-HT2-receptor agonist DOI [(+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] into the PVN produced dose-dependent (1-10 nmol) increases in heart rate and blood pressure; the peak responses were +39 +/- 10 beats/min and +6 +/- 2 mm Hg, respectively. Both responses were blocked by the concomitant administration of the selective 5-HT2-receptor antagonist LY53857 into the PVN. By contrast, the microinjection of the selective 5-HT1A-receptor agonist R(+)-8-OH-DPAT [R(+)8-hydroxy-2-(di-n-propylamino) tetralin HBr; 1-10 nmol] into the PVN did not affect blood pressure or heart rate. These data suggest that 5-HT neurons projecting from the raphe nuclei to or near the PVN can participate in the central control of the cardiovascular system by way of 5-HT2 receptors. Apparently 5-HT neurons terminating in the PVN can increase blood pressure and heart rate and produce sympathoadrenal activation, metabolic and hormonal responses consistent with those observed in several different stress paradigms.

Serotonin stimulates corticotropin-releasing factor gene expression in the hypothalamic paraventricular nucleus of conscious rats

To examine the direct effects of serotonin (5-HT) on the release and synthesis of corticotropin-releasing factor (CRF) in the hypothalamic paraventricular nucleus (PVN), 5-HT was microinjected just onto the bilateral PVN of conscious rats. Plasma adrenocorticotropic hormone (ACTH) levels peaked at 30 min and returned to the basal levels in 90 min. Northern blot analysis revealed that the CRF messenger RNA (mRNA) level in the PVN as well as the proopiomelanocortin mRNA level in the anterior pituitary significantly increased 120 min after the 5-HT injections (50–250 nmol/side). Pretreatment with intracerebroventricular (i.c.v.) injection of pindobind 5-HT 1A (5 nmol) or LY-278584 (500 nmol) completely abolished the 5-HT-induced ACTH response, whereas LY-53857 (100 nmol) was without effect. These results suggest that 5-HT stimulates CRF release, which has interactions with 5-HT 1A and 5-HT 3 receptors on CRF neurons in the PVN, and activates CRF synthesis in conscious rats.

Effect of abdominal vagotomy on interleukin-1 induced nitric oxide release in the paraventricular nucleus in conscious rats

The anti-psoriatic efficacy of orally administered methotrexate loaded chitin nanogel (MCNG) was evaluated (two doses- 2.715 mg/kg and 5.143 mg/kg) and compared against orally administered methotrexate tablet MTX (5.143 mg/kg). MCNG at both dose levels of 2.715 mg/kg and 5.143 mg/kg exhibited significant anti-psoriatic activity which is very much comparable with MTX, caused normalization of histological features and inflammatory score associated with induced psoriasis. Biodistribution studies revealed the presence of drug in serum and in vital organs at all the three cases with highest amount in MCNG at 5.143 mg/kg dose, followed by MTX tablet and are lowest in MCNG at 2.715 mg/kg dose. MCNG at the highest dose of 5.143 mg/kg caused liver, lung and kidney toxicities on sub acute toxicity studies and MTX tablet was found to be toxic on liver and lung on sub chronic toxicity studies. MCNG 2.715 mg/kg was found to be safe on both sub acute and sub chronic administrations, suggesting that it can provide sufficient serum and tissue level of methotrexate necessary to clear psoriatic lesions, without inducing systemic toxicity and expected to be a better alternative for orally administered conventional methotrexate tablet for patients who need systemic medications for psoriasis.

915 Effect of abdominal vagotomy on interleukin-1β induced nitric oxide release in the paraventricular nucleus in conscious rats

915 Effect of abdominal vagotomy on interleukin-1β induced nitric oxide release in the paraventricular nucleus in conscious rats

2418 Effect of abdominal vagotomy on interleukin-1β induced noradrenaline release in the paraventricular nucleus in conscious rats

Seasonal breeding mammals exhibit a reversible annual cycle of fertility, and thus represent valuable models for investigating the organization of luteinizing hormone-releasing hormone (LHRH) neurons which mediate the neuroendocrine control of reproduction. Electron microscopic immunocytochemistry was used to examine the ultrastructure of LHRH neurons and their projections in a seasonal breeder, the golden hamster, housed under photoperiodic conditions which are reproductively stimulatory. LHRH perikarya in the diagonal band, medial septum, and preoptic area were bipolar or unipolar cells which possessed nuclei with prominent indentations and multiple nucleoli. LHRH reaction product within these cells was associated with neurosecretory granules and the rough endoplasmic reticulum (RER), particularly those portions of RER adjacent to the outer nuclear envelope. LHRH cell bodies and dendrites in the hamster received an extremely limited amount of synaptic input; of a total of twenty-five cells analyzed, we found only five instances of morphologically identified synapses onto immunoreactive dendrites. Occasionally close somatic appositions were seen between LHRH cells and non-immunoreactive neurons, and in one instance between two LHRH somas. In the organum vasculosum of the lamina terminalis (OVLT), LHRH fibers appeared as isolated strings of varicosities. In contrast, within the median eminence immunoreactive axonal profiles were frequently bundled together in fascicles although synaptic specializations were not observed between individual axons. The existence of contacts between LHRH axons in the median eminence, coupled with the extreme paucity of synaptic inputs onto LHRH neurons in this species, suggests that the median eminence may be a site where neural or hormonal signals could influence the coordinated release of LHRH from cells of dispersed origin.

Catecholaminergic inhibition by hypercortisolemia in the paraventricular nucleus of conscious rats.

Administration of glucocorticoids decreases the release of corticotropin-releasing hormone and in vitro turnover of norepinephrine (NE) in the paraventricular nucleus (PVN) of the hypothalamus, and immobilization (IMMO) markedly increases NE release and stimulates corticotropin-releasing hormone neurons in the PVN. This study assessed whether hypercortisolemia affects in vivo indexes of catecholaminergic activation in the PVN. Microdialysis was used to simultaneously measure PVN microdialysate concentrations of NE, the neuronal NE metabolite dihydroxyphenylglycol, the extraneuronal NE metabolite methoxyhydroxyphenylglycol, and the dopamine metabolite dihydroxyphenylacetic acid before, during, and after 2 h of IMMO. Catecholamine synthesis was examined based on elevations of 3,4-dihydroxyphenylalanine levels after local perfusion with NSD-1015, an inhibitor of L-aromatic acid decarboxylase. Cortisol (CORT; 25 mg/kg.day) or vehicle (VEH; saline) was infused sc for 7 days via an osmotic minipump. CORT-treated rats had lower basal NE, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, and dihydroxyphenylacetic acid levels and significantly smaller levels of all these compounds during IMMO than VEH-treated rats. CORT-treated rats also had less NSD-1015-induced accumulation of microdialysate 3,4-dihydroxyphenylalanine at baseline and during IMMO than VEH-treated rats. Basal and IMMO-induced plasma ACTH and corticosterone responses were reduced in CORT-treated rats. The results indicate that chronic hypercortisolemia decreases basal levels and stress-induced increments in indexes of release, metabolism, turnover, and synthesis of catecholamines in the PVN and suggest that glucocorticoids restrain the limit of hypothalamo-pituitary-adrenocortical axis activation during stress by attenuating catecholamine synthesis and release in the PVN.

Effect of acetylcholine on corticotropin-releasing factor gene expression in the hypothalamic paraventricular nucleus of conscious rats.

To examine the physiological role of cholinergic input in the regulation of CRF neurons in the paraventricular nucleus (PVN) of the hypothalamus, acetylcholine (ACh) was microinjected bilaterally into the dorsolateral border of the PVN of conscious rats. Changes in the levels of POMC messenger RNA (mRNA) in the anterior pituitary, CRF mRNA in hypothalamic tissue containing the PVN, and plasma ACTH were assessed. Plasma ACTH concentrations increased in a dose-dependent manner after ACh injection (1-100 pmol/side), reaching a peak 30 min after ACh injection and returning to baseline within 120 min. The POMC mRNA level in the anterior pituitary and the hypothalamic CRF mRNA level increased in a dose-dependent manner 120 min after ACh (0.1-10 pmol/side) injection. Intracerebroventricular pretreatment with atropine completely abolished the ACh-induced increase in plasma ACTH concentrations, whereas pretreatment with hexamethonium was without significant effect. The intracerebroventricular injection of ACh also increased plasma ACTH concentrations in a dose-dependent manner in conscious rats, but not in pentobarbital-anesthetized animals. Thus, cholinergic hypothalamic input stimulates CRF gene expression in the PVN and CRF secretion into the portal circulation under physiological conditions. The use of conscious animals is essential in elucidating the physiological roles of neurotransmitters and other modulators regulating CRF neurons.

Catecholaminergic inhibition by hypercortisolemia in the paraventricular nucleus of conscious rats

Administration of glucocorticoids decreases the release of corticotropin-releasing hormone and in vitro turnover of norepinephrine (NE) in the paraventricular nucleus (PVN) of the hypothalamus, and immobilization (IMMO) markedly increases NE release and stimulates corticotropin-releasing hormone neurons in the PVN. This study assessed whether hypercortisolemia affects in vivo indexes of catecholaminergic activation in the PVN. Microdialysis was used to simultaneously measure PVN microdialysate concentrations of NE, the neuronal NE metabolite dihydroxyphenylglycol, the extraneuronal NE metabolite methoxyhydroxyphenylglycol, and the dopamine metabolite dihydroxyphenylacetic acid before, during, and after 2 h of IMMO. Catecholamine synthesis was examined based on elevations of 3,4-dihydroxyphenylalanine levels after local perfusion with NSD-1015, an inhibitor of L-aromatic acid decarboxylase. Cortisol (CORT; 25 mg/kg.day) or vehicle (VEH; saline) was infused sc for 7 days via an osmotic minipump. CORT-treated rats had lower basal NE, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, and dihydroxyphenylacetic acid levels and significantly smaller levels of all these compounds during IMMO than VEH-treated rats. CORT-treated rats also had less NSD-1015-induced accumulation of microdialysate 3,4-dihydroxyphenylalanine at baseline and during IMMO than VEH-treated rats. Basal and IMMO-induced plasma ACTH and corticosterone responses were reduced in CORT-treated rats. The results indicate that chronic hypercortisolemia decreases basal levels and stress-induced increments in indexes of release, metabolism, turnover, and synthesis of catecholamines in the PVN and suggest that glucocorticoids restrain the limit of hypothalamo-pituitary-adrenocortical axis activation during stress by attenuating catecholamine synthesis and release in the PVN.

Effect of acetylcholine on corticotropin-releasing factor gene expression in the hypothalamic paraventricular nucleus of conscious rats

To examine the physiological role of cholinergic input in the regulation of CRF neurons in the paraventricular nucleus (PVN) of the hypothalamus, acetylcholine (ACh) was microinjected bilaterally into the dorsolateral border of the PVN of conscious rats. Changes in the levels of POMC messenger RNA (mRNA) in the anterior pituitary, CRF mRNA in hypothalamic tissue containing the PVN, and plasma ACTH were assessed. Plasma ACTH concentrations increased in a dose-dependent manner after ACh injection (1-100 pmol/side), reaching a peak 30 min after ACh injection and returning to baseline within 120 min. The POMC mRNA level in the anterior pituitary and the hypothalamic CRF mRNA level increased in a dose-dependent manner 120 min after ACh (0.1-10 pmol/side) injection. Intracerebroventricular pretreatment with atropine completely abolished the ACh-induced increase in plasma ACTH concentrations, whereas pretreatment with hexamethonium was without significant effect. The intracerebroventricular injection of ACh also increased plasma ACTH concentrations in a dose-dependent manner in conscious rats, but not in pentobarbital-anesthetized animals. Thus, cholinergic hypothalamic input stimulates CRF gene expression in the PVN and CRF secretion into the portal circulation under physiological conditions. The use of conscious animals is essential in elucidating the physiological roles of neurotransmitters and other modulators regulating CRF neurons.

Pressor response to microinjection of clonidine into the hypothalamic paraventricular nucleus in conscious rats

We have reported that intracerebroventricular (i.c.v.) injection of clonidine causes pressor response in conscious rats. To determine the effective brain site, cardiovascular responses induced by unilateral microinjection of clonidine into various hypothalamic nuclei of conscious rats were studied. Microinjection of clonidine (5–20 μg/0.5 μl) into the paraventricular nucleus (PVN) of conscious rats dose-dependently produced a long-lasting pressor response with a decrease in heart rate, which mimicked the response to i.c.v. injection of clonidine. However, clonidine (10 μg) injection into various hypothalamic nuclei (anterior, posterior, ventromedial and dorsomedial nucleus) caused a small or no pressor response. In anesthetized rats, clonidine injected into the PVN induced a long-lasting depressor response concomitant with bradycardia. PVN pretreatment with theα 2-adrenoceptor antagonist, yohimbine (1 and 10 μg), dose-dependently inhibited the pressor response to PVN injected clonidine, but theα 1-adrenoceptor antagonist, prazosin (1 μg), had no significant effect. Central (i.c.v.) pretreatment with the vasopressin (AVP) V 1-receptor antagonist, [d(CH 2) 5Tyr(Me)]-AVP (0.5 and 2.0 μg), dose-dependently inhibited the pressor response to PVN injection of clonidine (10 μg), while systemic (i.v.) and local (intra-PVN injection) pretreatments with V 1-receptor antagonist (2.0 μg) had no effect. These results suggest that the pressor response to microinjection of clonidine into the PVN of conscious rats is mediated by endogenous brain AVP, which is released by activation ofα 2-adrenoceptors. It is also suggested that the PVN is a possible brain site for the pressor response to i.c.v. injected clonidine.

Noradrenergic activation in the paraventricular nucleus during acute and chronic immobilization stress in rats: an in vivo microdialysis study

In vivo microdialysis was used to study the effects of single (2 h) or repeated (2 h for 7 consecutive days) immobilization (IMMO) stress on extracellular fluid concentrations of norepinephrine (NE) and the deaminated metabolites of NE and dopamine, dihydroxyphenylglycol (DHPG) and dihydroxyphhenylacetic acid (DOPAC) in the paraventricular nucleus of conscious rats. During IMMO, NE, DHPG, and DOPAC levels increased markedly, with similar peak values and time courses in the repeatedly stressed and previously unstressed groups. NE levels during a 2-h baseline period were lower in the repeatedly stressed group than in the unstressed group (99 ± 9 pg/ml vs. 167 ± 13 pg/ml, P< 0.05), whereas DHPG (1,697 ± 263 pg/ml vs. 1,424 ± 194 pg/ml) and DOPAC (5,989 ± 863 pg/ml vs. 4,428 ± 1150 pg/ml) levels tended to be higher, so that the NE/DHPG ratio at baseline was significantly lower in the repeatedly stressed group ( P < 0.05). The results indicate that IMMO stress enhances NE release, reuptake, metabolism, and synthesis in the PVN. Repeated exposure to IMMO may decrease the microdialysate NE/DHPG ratio by inhibiting exocytotic release or enhancing neuronal reuptake of NE. In either case, the results suggest that repeated exposure to stress alters the release and disposition of NE in the PVN of conscious animals.

The Effect on Vasopressin Release of Microinjection of Cholinergic Agonists into the Paraventricular Nucleus of Conscious Rats

Abstract We have studied in conscious unrestrained rats under basal conditions the effect of activation of muscarinic and nicotinic receptors in the paraventricular nucleus on vasopressin secretion and mean arterial blood pressure. The microinjection of oxotremorine (0.2, 2 or 20 ng), a specific muscarinic agonist, produced a substantial, dose-dependent, transient increase in the plasma vasopressin concentration. There was also a rise in mean arterial blood pressure and a bradycardia that followed the same time-course as the change in plasma vasopressin levels. The microinjection of nicotine (0.1, 1 or 10 HQ) into the paraventricular nucleus had only questionable effects on vasopressin release and mean arterial blood pressure; heart rate was unaffected. These findings suggest that muscarinic receptors may be of primary importance in the paraventricular nucleus in the Cholinergic stimulation of vasopressin release.