Paraventricular Nucleus Of Rats Research Articles

Expression of Mineralocorticoid and Glucocorticoid receptors in Pre‐autonomic Neurons of the Rat Paraventricular Nucleus

Plasma aldosterone levels that are inappropriately high for the sodium intake increases sympathetic nervous system (SNS) activity and the hypertension and increased SNS activity in experimental mineralocorticoid‐salt excess is abrogated by lesions of the paraventricular nucleus (PVN) or antagonists of the mineralocorticoid receptors (MR). It is uncertain whether physiological concentrations of aldosterone bind MR in the PVN; corticosterone and cortisol bind both MR and glucocorticoid (GR) receptors at physiological concentrations and MR and GR influence each other at transcriptional and functional levels. The retrograde tracer Fast Blue was injected into the IMLCC at the level of T4 and T10 of spinal cord after laminectomy to locate preautonomic neurons in the PVN. Immunofluorescent histochemistry was done in frozen modified STF‐fixed brains using specific anti‐bodies against the MR and GR to determine colocalization of these two receptors within pre‐autonomic neurons. Three different pseudo‐colors were assigned by Image‐Pro for better visualization. Expression of GR and MR varied in different parts of the PVN. Most colocalization was in mid‐PVN parvocellular neurons. Very few magnocellular pre‐autonomic neurons were found to express GR and/or MR. Adrenocortical steroids could have direct effects on sympathetic activity by binding MR and/or GR in pre‐autonomic neurons of the mid‐PVN. This limited area of expression may explain the inconsistent results of studies on the effect of PVN microinjections of MR and GR agonists and antagonists on sympathetic drive.Grant Funding Source: VA BLRDB‐ORD 1018X007080, NIH/HLBI RO1HL0272, and NIH/NCRR COBRE (Center for Psychiatric Neuroscience)

Central Mineralocorticoid Receptors and the Role of Angiotensin II and Glutamate in the Paraventricular Nucleus of Rats With Angiotensin II–Induced Hypertension

A chronic increase in circulating angiotensin II (Ang II) activates an aldosterone-mineralocorticoid receptor-ouabain neuromodulatory pathway in the brain that increases neuronal activation in hypothalamic nuclei, such as the paraventricular nucleus (PVN) and causes progressive hypertension. Several models of chronic sympathetic hyperactivity are associated with an increase in AT1 and glutamate receptor activation in the PVN. The current study evaluated whether increased angiotensin type 1 (AT1) and glutamate receptor-dependent signaling in the PVN contributes to the maintenance of blood pressure (BP) in Ang II-hypertensive Wistar rats, and the role of aldosterone-mineralocorticoid receptor pathway in this enhanced signaling. After subcutaneous infusion of Ang II for 2 weeks, in conscious rats BP and heart rate were recorded after (1) 10-minute bilateral infusions of candesartan and kynurenate in the PVN; (2) 1 hour intracerebroventricular infusion of eplerenone, and (3) candesartan and kynurenate after eplerenone. Candesartan or kynurenate in the PVN fully reversed the increase in BP from circulating Ang II. Kynurenate after candesartan or candesartan after kynurenate did not further lower BP. Intracerebroventricular infusion of eplerenone at 16 hours after its infusion fully reversed the increase in BP from circulating Ang II. After eplerenone, candesartan and kynurenate in the PVN did not further decrease BP. These findings suggest that increased mineralocorticoid receptor activation in the brain activates a slow neuromodulatory pathway that maintains enhanced AT1 and glutamate receptor-dependent signaling in the PVN, and thereby the hypertension from a chronic increase in circulating Ang II.

Gastrin‐releasing peptide acts via postsynaptic BB2 receptors to modulate inward rectifier K+ and TRPV1‐like conductances in rat paraventricular thalamic neurons

Gastrin-releasing peptide (GRP) is a bombesin-like peptide with a widespread distribution in mammalian CNS, where it has a role in food intake, circadian rhythm generation, fear memory, itch sensation and sexual behaviour. While it has been established that GRP predominantly excites neurons, details of the membrane mechanism involved in this action remain largely undefined. We used perforated patch clamp recording in acute brain slice preparations to investigate GRP-affected receptors and ionic conductances in neurons of the rat paraventricular thalamic nucleus (PVT). PVT is a component of the midline and intralaminar thalamus that participates in arousal, motivational drives and stress responses, and exhibits a prominence of GRP-like immunoreactive fibres. Exposure of PVT neurons to low nanomolar concentrations of GRP induced sustained TTX-resistant membrane depolarizations that could trigger rhythmic burst discharges or tonic firing. Membrane current analyses in voltage clamp revealed an underlying postsynaptic bombesin type 2 receptor-mediated inward current that resulted from the simultaneous suppression of a Ba(2+)-sensitive inward rectifier K(+) conductance and activation of a non-selective cation conductance with biophysical and pharmacological properties reminiscent of transient receptor potential vanilloid (TRPV) 1. A role for a TRPV1-like conductance was further implied by a significant suppressant influence of a TRPV1 antagonist on GRP-induced membrane depolarization and rhythmic burst or tonic firing. The results provide a detailed picture of the receptor and ionic conductances that are involved in GRP's excitatory action in midline thalamus.

Interleukin-10 inhibits angiotensin II-induced decrease in neuronal potassium current

Previously we demonstrated that viral-mediated increased expression of the anti-inflammatory cytokine interleukin-10 within the paraventricular nucleus of the hypothalamus significantly reduces blood pressure in normal rats made hypertensive by infusion of angiotensin II. However, the exact cellular locus of this interleukin-10 action within the paraventricular nucleus is unknown. In the present study we tested whether interleukin-10 exerts direct effects at its receptors located on hypothalamic neurons to offset the neuronal excitatory actions of angiotensin II via its type 1 receptors. The results indicated the presence of immunoreactive interleukin-10 receptors on neurons in normal rat paraventricular nucleus and that receptors for this cytokine were also expressed in neurons cultured from the hypothalamus. Patch-clamp electrophysiological recordings from these cultures revealed that extracellular application of interleukin-10 alone did not exert any alterations in neuronal membrane delayed rectifier or transient potassium currents. However, angiotensin II elicited a significant decrease in delayed rectifier potassium current, an effect that was abolished by interleukin-10 application. Since decreases in delayed rectifier potassium current contribute to increased neuronal excitability, this result is consistent with a direct inhibitory action of interleukin-10 on angiotensin-induced excitation of hypothalamic neurons. As such, these data are the first indication of a neuronal locus of action of interleukin-10 to temper the actions of angiotensin II in the hypothalamus.

Salusin-β in paraventricular nucleus increases blood pressure and sympathetic outflow via vasopressin in hypertensive rats

Salusin-β is a bioactive peptide with peripheral hypotensive, mitogenic and pro-atherosclerotic effects. The present study was designed to determine the roles of salusin-β in the paraventricular nucleus (PVN) and its relationship with arginine vasopressin (AVP) in hypertension and sympathetic activation. Renovascular hypertension was induced by two-kidney, one-clip (2K1C) in male Sprague-Dawley rats. Acute experiments were carried out 4 weeks after 2K1C or sham-operation under urethane and alpha-chloralose anaesthesia. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) were recorded. Microinjection of salusin-β into the PVN increased the RSNA, MAP, and HR in a dose-related manner, whereas anti-salusin-β IgG in the PVN decreased the RSNA and MAP, and abolished the effects of salusin-β in 2K1C rats. However, either salusin-β or anti-salusin-β IgG in the PVN failed to cause any significant effects in sham-operated rats. The number of salusin-β-like immunopositive neurons in the PVN was significantly increased in 2K1C rats. Salusin-β in the PVN increased the plasma AVP and norepinephrine levels in 2K1C rats, but not in sham-operated rats. Iv injection of dTyr(CH2)5(Me)AVP (an AVP V1-receptor antagonist, AAVP) decreased RSNA and MAP, and abolished the effects of salusin-β in the PVN in 2K1C rats. Microinjection of AAVP into the rostral ventrolateral medulla (RVLM), but not into the PVN, abolished the effects of salusin-β on RSNA and HR. Salusin-β in the PVN increases blood pressure, heart rate, and sympathetic outflow via both circulating AVP and AVP in the RVLM in hypertensive rats.

Effects of Stresscopin on Rat Hypothalamic Paraventricular Nucleus Neurons In Vitro

The effects of stresscopin (SCP) on rat paraventricular nucleus (PVN) neurons were examined using whole-cell patch-clamp recordings and single-cell reverse-transcription multiplex polymerase chain reaction (SC-RT-mPCR) techniques. Under current-clamp conditions, bath application of SCP (100 nM) induced inhibition in 35.2% (37/105) of putative magnocellular neurons and 24.7% (20/81) of putative parvocellular neurons, and excitation in 5.7% (6/105) of putative magnocellular neurons and 18.5% (15/81) of putative parvocellular neurons. SCP-induced inhibition persisted in the presence of a mixture of TTX, a voltage-gated Na+ channel blocker, CNQX, an AMPA/kainate receptor antagonist and bicuculline, a GABAA receptor antagonist, whereas SCP-induced excitation of PVN neurons was reversed by the mixture. The SCP-induced inhibition of PVN neurons was abolished by bath application of antisauvagine-30, a selective CRF receptor 2 (CRF-R2) antagonist. Under voltage-clamp conditions, SCP evoked outward currents at the holding potential (−60 mV), which reversed near the potassium equilibrium potential. The SCP-evoked membrane currents were completely blocked by bath application of tertiapin-Q, a selective blocker of G protein-activated inwardly rectifying potassium (GIRK) channels. SC-RT-mPCR analysis indicated that all the SCP-sensitive PVN neurons (57 SCP-inhibited neurons, 21 SCP-excited neurons) expressed CRF-R1 and CRF-R2 mRNAs. Among SCP-hyperpolarized PVN neurons, oxytocin (OT) mRNA was detected in 91.8% of putative magnocellular neurons and 45.0% of putative parvocellular neurons. OT mRNA was also detected in 26.6% of SCP-depolarized parvocellular neurons, but not in SCP-depolarized magnocellular neurons. These results indicate that SCP inhibits a subpopulation of PVN neurons, especially OTergic magnocellular neurons, by enhancing the activity of GIRK channels via CRF-R2.

Inner capillary diameter of hypothalamic paraventricular nucleus of female rat increases during lactation

BackgroundThe role of the endothelial cell (EC) in blood flow regulation within the central nervous system has been little studied. Here, we explored EC participation in morphological changes of the anterior hypothalamic paraventricular nucleus (PVN) microvasculature of female rats at two reproductive stages with different metabolic demand (virginity and lactation). We measured the inner capillary diameter (ICD) of 800 capillaries from either the magnocellular or parvocellular regions. The space occupied by neural (somas, dendrites and axons) and glial, but excluding vascular elements of the neurovascular compartment was also measured in 100-μm2 sample fields of both PVN subdivisions.ResultsThe PVN of both groups of animals showed ICDs that ranged from 3 to 10 microns. The virgin group presented mostly capillaries with small ICD, whereas the lactating females exhibited a significant increment in the percentage of capillaries with larger ICD. The space occupied by the neural and glial elements of the neurovascular compartment did not show changes with lactation.ConclusionsOur findings suggest that during lactation the microvasculature of the PVN of female rats undergoes dynamic, transitory changes in blood flow as represented by an increment in the ICD through a self-cytoplasmic volume modification reflected by EC changes. A model of this process is proposed.

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.

Adenoviral and Adeno-Associated Viral Vectors-Mediated Neuronal Gene Transfer to Cardiovascular Control Regions of the Rat Brain

Viral vectors have been utilized extensively to introduce genetic material into the central nervous system. In order to investigate gene functions in cardiovascular control regions of rat brain, we applied WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) enhanced-adenoviral (Ad) and adeno-assoicated virus (AAV) type 2 vectors to mediate neuronal gene delivery to the paraventricular nucleus of the hypothalamus, the nucleus tractus solitarius and the rostral ventrolateral medulla, three important cardiovascular control regions known to express renin-angiotensin system (RAS) genes. Ad or AAV2 harboring an enhanced green fluorescent protein (EGFP) reporter gene or the angiotensin type 2 receptor gene were microinjected into these brain regions in adult rats. Our results demonstrated that both AAV2 and Ad vectors elicited long-term neuronal transduction in these regions. Interestingly, we found that the WPRE caused expression of GFP driven by the synapsin1 promoter in pure glial cultures or co-cultures of neurons and glia derived from rat hypothalamus and brainstem. However, in rat paraventricular nucleus WPRE did not cause expression of GFP in glia. This demonstrates the potential use of these vectors in studies of physiological functions of certain genes in the cardiovascular control regions of the brain.

Effect of Barodenervation on Cardiovascular Responses Elicited from the Hypothalamic Arcuate Nucleus of the Rat

We have previously reported that chemical stimulation of the hypothalamic arcuate nucleus (ARCN) in the rat elicited increases as well as decreases in blood pressure (BP) and sympathetic nerve activity (SNA). The type of response elicited from the ARCN (i.e., increase or decrease in BP and SNA) depended on the level of baroreceptor activity which, in turn, was determined by baseline BP in rats with intact baroreceptors. Based on this information, it was hypothesized that baroreceptor unloading may play a role in the type of response elicited from the ARCN. Therefore, the effect of barodenervation on the ARCN-induced cardiovascular and sympathetic responses and the neurotransmitters in the hypothalamic paraventricular nucleus (PVN) mediating the excitatory responses elicited from the ARCN were investigated in urethane-anesthetized adult male Wistar rats. Bilateral barodenervation converted decreases in mean arterial pressure (MAP) and greater splanchnic nerve activity (GSNA) elicited by chemical stimulation of the ARCN with microinjections of N-methyl-D-aspartic acid to increases in MAP and GSNA and exaggerated the increases in heart rate (HR). Combined microinjections of NBQX and D-AP7 (ionotropic glutamate receptor antagonists) into the PVN in barodenervated rats converted increases in MAP and GSNA elicited by the ARCN stimulation to decreases in MAP and GSNA and attenuated increases in HR. Microinjections of SHU9119 (a melanocortin 3/4 receptor antagonist) into the PVN in barodenervated rats attenuated increases in MAP, GSNA and HR elicited by the ARCN stimulation. ARCN neurons projecting to the PVN were immunoreactive for proopiomelanocortin, alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH). It was concluded that increases in MAP and GSNA and exaggeration of tachycardia elicited by the ARCN stimulation in barodenervated rats may be mediated via release of alpha-MSH and/or ACTH and glutamate from the ARCN neurons projecting to the PVN.

Detraining Differentially Preserved Beneficial Effects of Exercise on Hypertension: Effects on Blood Pressure, Cardiac Function, Brain Inflammatory Cytokines and Oxidative Stress

AimsThis study sought to investigate the effects of physical detraining on blood pressure (BP) and cardiac morphology and function in hypertension, and on pro- and anti-inflammatory cytokines (PICs and AIC) and oxidative stress within the brain of hypertensive rats.Methods and ResultsHypertension was induced in male Sprague-Dawley rats by delivering AngiotensinII for 42 days using implanted osmotic minipumps. Rats were randomized into sedentary, trained, and detrained groups. Trained rats underwent moderate-intensity exercise (ExT) for 42 days, whereas, detrained groups underwent 28 days of exercise followed by 14 days of detraining. BP and cardiac function were evaluated by radio-telemetry and echocardiography, respectively. At the end, the paraventricular nucleus (PVN) was analyzed by Real-time RT-PCR and Western blot. ExT in AngII-infused rats caused delayed progression of hypertension, reduced cardiac hypertrophy, and improved diastolic function. These results were associated with significantly reduced PICs, increased AIC (interleukin (IL)-10), and attenuated oxidative stress in the PVN. Detraining did not abolish the exercise-induced attenuation in MAP in hypertensive rats; however, detraining failed to completely preserve exercise-mediated improvement in cardiac hypertrophy and function. Additionally, detraining did not reverse exercise-induced improvement in PICs in the PVN of hypertensive rats; however, the improvements in IL-10 were abolished.ConclusionThese results indicate that although 2 weeks of detraining is not long enough to completely abolish the beneficial effects of regular exercise, continuing cessation of exercise may lead to detrimental effects.

Intact Catecholamine Inputs to the Forebrain are Required for Appropriate Regulation of Corticotrophin‐Releasing Hormone and Vasopressin Gene Expression by Corticosterone in the Rat Paraventricular Nucleus

Corticotrophin-releasing hormone (CRH) neuroendocrine neurones in the paraventricular nucleus of the hypothalamus (PVH) drive adrenocorticotrophic hormone (ACTH) and thereby glucocorticoid release from pituitary corticotrophs and the adrenal cortex, respectively. Glucocorticoids suppress the ability of neuroendocrine corticotrophin-releasing hormone (CRH) neurones to synthesise and release ACTH secretogogues. Despite the importance of glucocorticoids as regulatory signals to CRH neurones in the extended time domain, how and where they act in this capacity is still not fully understood. Ascending catecholamine projections encode important cardiovascular, metabolic and other visceral information to the rat PVH and surrounding hypothalamus. These afferents have previously been implicated as targets for glucocorticoid action, including a role in the feedback regulation of PVH neuroendocrine neurones. To determine the contribution of these neurones to the long-term actions of corticosterone on CRH and vasopressin (AVP) gene expression in the PVH, we used an immunocytotoxin (a conjugate of the cytotoxin saporin and an antibody against dopamine-β-hydroxylase) that specifically ablates adrenergic and noradrenergic neurones. Lesions were administered to intact animals and to adrenalectomised animals with either no corticosterone or corticosterone replacement that provided levels above those required to normalise Crh expression. The ability of elevated levels of corticosterone to suppress Crh expression was abolished in animals lacking catecholaminergic innervation of the PVH. No effect was seen in the absence of corticosterone or in animals with intact adrenals. Furthermore, Avp expression, which is increased in CRH neurones following adrenalectomy, was suppressed in adrenalectomised catecholaminergic-lesioned animals. Interactions between corticosterone and catecholaminergic projections to the hypothalamus therefore make significant contributions to the regulation of Crh and Avp expression. However, the importance of catecholamine inputs is only apparent when circulating corticosterone concentrations are maintained either below or above those required to maintain the activity of the hypothalamic-pituitary-adrenal axis that is seen in intact animals.

Hydrogen Sulfide in Paraventricular Nucleus Enhances Sympathetic Activity and Cardiac Sympathetic Afferent Reflex in Chronic Heart Failure Rats

BackgroundIntracerebroventricular infusion of NaHS, a hydrogen sulfide (H2S) donor, increased mean arterial pressure (MAP). This study was designed to determine the roles of H2S in the paraventricular nucleus (PVN) in modulating sympathetic activity and cardiac sympathetic afferent reflex (CSAR) in chronic heart failure (CHF).Methodology/Principal FindingsCHF was induced by left descending coronary artery ligation in rats. Renal sympathetic nerve activity (RSNA) and MAP were recorded under anesthesia. CSAR was evaluated by the RSNA and MAP responses to epicardial application of capsaicin. PVN microinjection of low doses of a H2S donor, GYY4137 (0.01 and 0.1 nmol), had no significant effects on RSNA, MAP and CSAR. High doses of GYY4137 (1, 2 and 4 nmol) increased baseline RSNA, MAP and heart rate (HR), and enhanced CSAR. The effects were greater in CHF rats than sham-operated rats. A cystathionine-β-synthase (CBS) inhibitor, hydroxylamine (HA) in PVN had no significant effect on the RSNA, MAP and CSAR. CBS activity and H2S level in the PVN were decreased in CHF rats. No significant difference in CBS level in PVN was found between sham-operated rats and CHF rats. Stimulation of cardiac sympathetic afferents with capsaicin decreased CBS activity and H2S level in the PVN in both sham-operated rats and CHF rats.ConclusionsExogenous H2S in PVN increases RSNA, MAP and HR, and enhances CSAR. The effects are greater in CHF rats than those in sham-operated rats. Endogenous H2S in PVN is not responsible for the sympathetic activation and enhanced CSAR in CHF rats.

Inhibition of Cyclooxygenase-2 Reduces Hypothalamic Excitation in Rats with Adriamycin-Induced Heart Failure

BackgroundThe paraventricular nucleus (PVN) of the hypothalamus plays an important role in the progression of heart failure (HF). We investigated whether cyclooxygenase-2 (COX-2) inhibition in the PVN attenuates the activities of sympathetic nervous system (SNS) and renin-angiotensin system (RAS) in rats with adriamycin-induced heart failure.Methodology/Principal FindingHeart failure was induced by intraperitoneal injection of adriamycin over a period of 2 weeks (cumulative dose of 15 mg/kg). On day 19, rats received intragastric administration daily with either COX-2 inhibitor celecoxib (CLB) or normal saline. Treatment with CLB reduced mortality and attenuated both myocardial atrophy and pulmonary congestion in HF rats. Compared with the HF rats, ventricle to body weight (VW/BW) and lung to body weight (LW/BW) ratios, heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular peak systolic pressure (LVPSP) and maximum rate of change in left ventricular pressure (LV±dp/dtmax) were improved in HF+CLB rats. Angiotensin II (ANG II), norepinephrine (NE), COX-2 and glutamate (Glu) in the PVN were increased in HF rats. HF rats had higher levels of ANG II and NE in plasma, higher level of ANG II in myocardium, and lower levels of ANP in plasma and myocardium. Treatment with CLB attenuated these HF-induced changes. HF rats had more COX-2-positive neurons and more corticotropin releasing hormone (CRH) positive neurons in the PVN than did control rats. Treatment with CLB decreased COX-2-positive neurons and CRH positive neurons in the PVN of HF rats.ConclusionsThese results suggest that PVN COX-2 may be an intermediary step for PVN neuronal activation and excitatory neurotransmitter release, which further contributes to sympathoexcitation and RAS activation in adriamycin-induced heart failure. Treatment with COX-2 inhibitor attenuates sympathoexcitation and RAS activation in adriamycin-induced heart failure.

Changes in GABAergic inputs in the paraventricular nucleus maintain sympathetic vasomotor tone in chronic heart failure

The paraventricular nucleus (PVN) of the hypothalamus is an important region of the brain involved in the regulation of sympathetic vasomotor tone. Accumulating evidence supports the idea that a change in hypothalamic γ-aminobutyric acid (GABA)-ergic inhibitory and glutamatergic excitatory inputs contribute to the exacerbated sympathetic drive in chronic heart failure (HF). The purpose of this study was to determine whether a possible imbalance between glutamatergic and GABAergic inputs to the PVN contributes to increased sympathetic outflow in HF in two different sympathetic territories. Renal (RSNA) and splanchnic sympathetic nerve activity (SSNA), mean arterial blood pressure (MAP) and heart rate were recorded from urethane-anesthetized HF or sham rats. The NMDA-glutamate and GABA-A receptor densities within the PVN were quantified in HF and sham rats by autoradiography. Bilateral microinjection of kynurenic acid (4nmol) into the PVN decreased MAP and RSNA and SSNA in HF but not in sham rats. Furthermore, in response to GABA-A blockade in the PVN by bicuculline (400 pmol), hypertension and SSNA were reduced in HF compared to sham. The quantification of ionotropic NMDA receptors and GABA-A receptors in the PVN showed a significant reduction of GABA-A in HF rats; however, the NMDA density in the PVN did not differ between groups. Thus, this study provides evidence that the sympathoexcitation is maintained by an imbalance between GABAergic and glutamatergic inputs in the PVN in HF. The reduced GABAergic input results in relatively augmented glutamatergic actions in the PVN of HF rats.

SOD1 gene transfer into paraventricular nucleus attenuates hypertension and sympathetic activity in spontaneously hypertensive rats

Excessive sympathetic activity contributes to the initiation and progression of hypertension. Reactive oxygen species in the paraventricular nucleus (PVN) is involved in sympathetic overdrive and hypertension. The present study was designed to investigate whether superoxide dismutase 1 (SOD1) overexpression in the PVN attenuated sympathetic activation and hypertension. Adenoviral vectors containing human SOD1 or null adenoviral vectors were microinjected into the PVN of Wistar rats and spontaneously hypertensive rats (SHR). Significant depressor effects were observed from weeks 1 to 4 after SOD1 gene transfer in SHR. Acute experiments were carried out at the end of the 3rd week. In the PVN, superoxide anion and angiotensin II levels were increased while SOD1 activity and protein expression were decreased in SHR, which were attenuated by SOD1 overexpression in the PVN. However, SOD1 overexpression had no significant effect on the SOD2 activity in the PVN. The blood pressure response to ganglionic blockade, cardiac sympathetic nerve activity, and cardiac sympathetic afferent reflex (CSAR) were enhanced, and the plasma norepinephrine level was increased in SHR, which were prevented by SOD1 gene transfer in the PVN. Furthermore, SOD1 overexpression decreased the ratio of left ventricular weight to body weight, cross-sectional areas of myocardial cells, media thickness, and the media/lumen ratio of small arteries in the heart in SHR. These results indicate that SOD1 overexpression in the PVN reduces arterial blood pressure, attenuates excessive sympathetic activity and CSAR, and improves myocardial and vascular remodeling in SHR.

Abstract 420: Role of Ang II and Glutamate in the PVN of Rats with Ang II-induced Hypertension

A chronic increase in circulating Ang II increases neuronal activation in hypothalamic nuclei such as the paraventricular nucleus (PVN) and causes progressive hypertension. These effects can be largely prevented by central infusion of blockers of an aldosterone-ouabain neuromodulatory pathway that enhances the activity of angiotensinergic sympathoexcitatory pathways. A number of models of chronic sympathetic hyperactivity are associated with an increase in glutamate receptor and AT 1 -receptor activation in the PVN. The present study evaluated whether these mechanisms in the PVN also contribute to the elevated BP from a chronic increase in plasma Ang II by chronic subcutaneous (sc) infusion of Ang II for 2 weeks. One week following bilateral stereotaxic cannulation of the PVN, bilateral infusion (300 nL/min/side) of AT 1 and glutamate receptor blockers, candesartan and kynurenate were performed, and BP and HR were recorded in conscious Wistar rats. Data are mean±SE; (n=4-5/group). *p<0.05 vs. baseline Kynurenate or candesartan in the PVN both fully reversed the increase in BP from sc infusion of Ang II for 2 weeks. Infusion of candesartan after kynurenate or kynurenate after candesartan did not further lower BP. These findings suggest that enhanced AT 1 -receptor and glutamate receptor activation in the PVN both contribute to the elevated BP in Wistar rats with chronically elevated plasma Ang II. The effects of increased AT 1 -receptor activation in the PVN appear to be mediated by local glutamate release.

Social interaction attenuates stress responses following chronic stress in maternally separated rats

Early life stress has been implicated as a risk factor for functional gastrointestinal (GI) disorders. Hypothalamic oxytocin (OXT) is well known to regulate social interactions and affiliative behaviors. We have shown that maternal separation (MS) induces GI dysmotility and impair hypothalamic OXT expression in response to chronic homotypic stress (CHS). We studied whether social interaction can improve GI dysmotility and OXT expression in MS rats. Male neonatal SD rats were exposed to MS for 180min from postnatal day (PND)-2 to PND-14. After weaning, 3MS rats were housed together (pure MS). In another group, 1MS rat was housed with 2 control rats (mixed MS). Anxiety-like behaviors were evaluated in elevated plus maze (EPM). Solid gastric emptying (GE) and colonic transit (CT) were measured following CHS loading. Expression of corticotropin releasing factor (CRF) and OXT in the paraventricular nucleus (PVN) were evaluated by real time RT-PCR and immunohistochemistry. Pure MS rats demonstrated increased anxiety-like behaviors, which were significantly reduced in mixed MS rats. Delayed GE (31.5±2.8%, n=6) and accelerated CT [Geometric center (GC) =8.9±0.8, n=6] observed in pure MS rats were restored in mixed MS rats (GE=67.8±3.8%, GC=6.7±1.2, n=6, P<0.05) following CHS. OXT mRNA expression was upregulated, while CRF mRNA expression was downregulated in mixed MS rats, compared to pure MS rats. The number of OXT-immunoreactive cells was significantly increased following CHS at the PVN in mixed MS rats. Our study may contribute to the treatment strategies for GI motility disorders associated with early life stress.

Cervical stimulation activates A1 and locus coeruleus neurons that project to the paraventricular nucleus of the hypothalamus.

In female rats, stimulation of the uterine cervix during mating induces two daily surges of prolactin. Inhibition of hypothalamic dopamine release and stimulation of oxytocin neurons in the paraventricular nucleus (PVN) are required for prolactin secretion. We aim to better understand how stimulation of the uterine cervix is translated into two daily prolactin surges. We hypothesize that noradrenergic neurons in the A1, A2, and locus coeruleus (LC) are responsible for conveying the peripheral stimulus to the PVN. In order to determine whether projections from these neurons to the PVN are activated by cervical stimulation (CS), we injected a retrograde tracer, Fluoro-Gold (FG), into the PVN of ovariectomized rats. Fourteen days after injection, animals were submitted to artificial CS or handling and perfused with a fixative solution. Brains were removed and sectioned from the A1, A2, and LC for c-Fos, tyrosine hydroxylase (TH), and FG triple-labeling using immunohistochemistry. CS increased the percentage of TH/FG+ double-labeled neurons expressing c-Fos in the A1 and LC. CS also increased the percentage of TH+ neurons expressing c-Fos within the A1 and A2, independent of their projections to the PVN. Our data reinforce the significant contributions of the A1 and A2 to carry sensory information during mating, and provide evidence of a functional pathway in which CS activates A1 and LC neurons projecting to the PVN, which is potentially involved in the translation of CS into two daily prolactin surges.

Central Administration of Aminoprocalcitonin Inhibits Food Intake and Stimulates the Hypothalamic‐Pituitary‐Adrenal Axis in Rats via the Corticotrophin‐Releasing Factor System

Aminoprocalcitonin (N-PCT), a neuroendocrine peptide derived from procalcitonin, reduces food intake and body weight when administered centrally in rats. We have recently shown that N-PCT is expressed in brain areas known to be involved in energy homeostasis, including the paraventricular nucleus (PVN) of the hypothalamus, which contains a prominent population of corticotrophin-releasing factor (CRF)-synthesising neurones. CRF plays a pivotal role in the regulation of the hypothalamic-pituitary adrenal (HPA) axis and food intake. However, little is known about functional interactions of N-PCT and CRF. In the present study, we found endogenous N-PCT protein in the rat PVN. We also showed N-PCT immunoreactivity in PVN co-localised with NeuN, a neuronal marker, or glial fibrillary acidic protein, an astrocyte marker. Double staining immunohistochemistry revealed that N-PCT co-localised with CRF in parvocellular neurones of the PVN. Intracerebroventricular N-PCT administration increased CRF mRNA and content in the hypothalamus, suggesting that N-PCT stimulates the HPA axis and suppresses food intake and body weight via CRF-dependent pathways. In keeping with this, i.c.v. co-injection of D-Phe-CRF(12-41), a CRF receptor antagonist, significantly attenuated N-PCT-induced reduction in food intake and body weight in a dose-dependent manner. Furthermore, i.c.v. administration of N-PCT increased plasma adrenocorticotrophic hormone and corticosterone concentrations and induced the expression of Fos protein, a marker of neuronal activity, in parvocellular CRF neurones. These data collectively support the hypothesis that N-PCT inhibits food intake and body weight and stimulates the HPA axis via CRF-mediated pathways.