Paraventricular Nucleus mRNA Research Articles

Myocardial infarction-induced disruption of blood-brain barrier in hypothalamic paraventricular nucleus is mediated by interleukin-17A signaling in rat

Increased inflammatory cytokines in the circulation act in the brain to promote neuroinflammation and sympathetic excitation in myocardial infarction-induced heart failure (HF). However, the mechanisms by which proinflammatory cytokines interrupt blood-brain barrier (BBB) to reach brain remain unclear. We recently reported that the levels of interleukin (IL)-17A, a key inflammatory mediator of immune responses, are significantly elevated in circulation and the brain in HF. IL-17A has been shown to disrupt the integrity of BBB in in vitro model and in vivo rodent model of experimental autoimmune encephalomyelitis. The present study sought to determine whether BBB in hypothalamic paraventricular nucleus (PVN), a key cardiovascular/autonomic region of the brain within the BBB, was damaged in HF and if so, whether IL-17A contributes to BBB disruption in this disease setting. Adult male Sprague Dawley rats underwent coronary artery ligation to induce HF or sham surgery (Sham). Some HF rats were pretreated with bilateral PVN microinjections of an IL-17 receptor A (IL-17RA) siRNA AAV virus or a scrambled siRNA control. The left ventricular function and infarction size were evaluated by echocardiography. Four weeks after coronary artery ligation, BBB permeability was assessed with fluorescein isothiocyanate (FITC, 10 kDa)-dextran extravasation and molecular measurements of its biomarkers. FITC was injected through left carotid artery at a rate of 300 μl/min (10 mg/ml, 3 ml/kg) and allowed to circulate for 30 min. We found that HF rats displayed noticeable FITC extravasation within the PVN area when compared with brain cortex after injections. FITC extravasation was not observed in the PVN and brain cortex in Sham rats. Compared with Sham rats, HF rats had significantly increased PVN mRNA expression of caveolin-1 (1.05± 0.13 vs 3.30 ± 0.48*, fold changes; *P<0.05), a molecular marker of transcytosis, and decreased PVN mRNA levels of occludin and zonula occludens-1 (1.05± 0.14 vs 0.57 ± 0.13*; 1.05± 0.10 vs 0.54 ± 0.03*, respectively), two important tight junction-associated molecules. However, the mRNA expression of caveolin-1, occludin or zonula occludens-1 did not display significant difference in the brain cortex between HF and Sham rats. Pretreatment with IL-17RA siRNA, substantially reduced FITC extravasation within the PVN and reversed mRNA expression of caveolin-1 (1.08 ± 0.20 vs. 0.51 ± 0.09*), occludin (1.07 ± 0.15 vs. 1.86 ± 0.18*) or zonula occludens-1 (1.02 ± 0.08 vs.1.84 ± 0.24*) in HF rats when compared with HF rats pretreated with a scrambled siRNA control. These data indicated that BBB permeability is increased in the PVN in myocardial infarction-induced HF. The activated IL-17A/IL-17RA signaling in BBB endothelium in the PVN in HF may play an important role in disrupting BBB, probably by a molecular mechanism to augment transcellular transport and reduce tight junction protein expression. Supported by NIH grants R01 HL-139521 & HL-155091 to SGW This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protection by -Biotics against Hypertension Programmed by Maternal High Fructose Diet: Rectification of Dysregulated Expression of Short-Chain Fatty Acid Receptors in the Hypothalamic Paraventricular Nucleus of Adult Offspring.

The role of short-chain fatty acids (SCFAs) in the brain on the developmental programming of hypertension is poorly understood. The present study explored dysregulated tissue levels of SCFAs and expression of SCFA-sensing receptors in the hypothalamic paraventricular nucleus (PVN), a key forebrain region engaged in neural regulation of blood pressure of offspring to maternal high fructose diet (HFD) exposure. We further investigated the engagement of SCFA-sensing receptors in PVN in the beneficial effects of -biotics (prebiotic, probiotic, synbiotic, and postbiotic) on programmed hypertension. Maternal HFD during gestation and lactation significantly reduced circulating butyrate, along with decreased tissue level of butyrate and increased expression of SCFA-sensing receptors, GPR41 and olfr78, and tissue oxidative stress and neuroinflammation in PVN of HFD offspring that were rectified by oral supplement with -biotics. Gene silencing of GPR41 or olfr78 mRNA in PVN also protected adult HFD offspring from programmed hypertension and alleviated the induced oxidative stress and inflammation in PVN. In addition, oral supplement with postbiotic butyrate restored tissue butyrate levels, rectified expressions of GPR41 and olfr78 in PVN, and protected against programmed hypertension in adult HFD offspring. These data suggest that alterations in tissue butyrate level, expression of GPR41 and olfr78, and activation of SCFA-sensing receptor-dependent tissue oxidative stress and neuroinflammation in PVN could be novel mechanisms that underlie hypertension programmed by maternal HFD exposure in adult offspring. Furthermore, oral -biotics supplementation may exert beneficial effects on hypertension of developmental origin by targeting dysfunctional SCFA-sensing receptors in PVN to exert antioxidant and anti-inflammatory actions in the brain.

Differential delayed responses of arginine vasotocin and its receptors in septo-hypothalamic brain structures and anterior pituitary that sustain hypothalamic–pituitary-adrenal (HPA) axis functions during acute stress

Recently, we proposed that corticotropin releasing hormone (CRH) neurons in the nucleus of hippocampal commissure (NHpC), located in the septum, function as a part of the traditional hypothalamic–pituitary-adrenal (HPA) axis in avian species. CRH and its receptor, CRHR1, are regulated differently in the NHpC compared to the paraventricular nucleus (PVN) following feed deprivation (FD). Therefore, we followed up our work by examining arginine vasotocin (AVT), the other major ACTH secretagogue, and its receptors, V1aR and V1bR, gene expression during FD stress in the NHpC, PVN, and ventral mediobasal hypothalamus/median eminence (MBHv/ME). The objectives were to 1) identify AVT perikarya, fibers and its two major receptors, V1aR and V1bR, in the NHpC, PVN, and MBHv/ME using immunohistochemistry, 2) determine the effect of stress on AVT, V1aR and V1bR mRNA expression in the same three brain structures, NHpC, PVN, and MBHv/ME; and, 3) ascertain the expression pattern of V1aR and V1bR mRNA in the anterior pituitary and measure plasma stress hormone, corticosterone (CORT), concentration following FD stress. Male chicks (Cobb 500), 14 days of age, were divided into six groups (10 birds/treatment) and subjected to different times of FD stress: (Control, 1 h, 2 h, 3 h, 4 h, and 8 h). For each bird, blood, brain, and anterior pituitary were sampled and frozen immediately. The NHpC, PVN, and MBHv/ME were micro-dissected for RT-PCR. Data were analyzed using one-way ANOVA followed by Tukey Kramer HSD test using a significance level of p < 0.05. Perikarya of AVT neurons were identified in the PVN but not in the NHpC nor MBHv/ME, and only V1aR-immunoreactivity (ir) was observed in the three structures, however, gene expression data for AVT and its two receptors were obtained in all structures. Both AVT and V1aR mRNA are expressed and increased significantly in the PVN following FD stress (p < 0.01). For the first time, V1bR mRNA was documented in the avian brain and specifically shown upregulated in the NHpC and PVN (p < 0.01) following stress. Additionally, delayed significant gene expression of AVT and its receptors in the PVN showed a positive feedback relationship responsible for maintaining CORT release. In contrast, a significant downregulation of AVT mRNA and upregulation of V1aR mRNA occurred in the NHpC (p < 0.01) during FD showing a negative feedback relationship between AVT and its receptors, V1aR and V1bR. Within the MBHv/ME and anterior pituitary, a gradual increase of AVT mRNA in PVN as well as MBHv/ME was associated with significant upregulation of V1bR (p < 0. 01) and downregulation of V1aR (p < 0.01) in both MBHv/ME and anterior pituitary indicating AVT regulates its receptors differentially to sustain CORT release and control overstimulation of the anterior pituitary during a stress response.

Increased activity of the orexin system in the paraventricular nucleus contributes to salt-sensitive hypertension.

The orexin system is involved in arginine vasopressin (AVP) regulation, and its overactivation has been implicated in hypertension. However, its role in salt-sensitive hypertension (SSHTN) is unknown. Here, we tested the hypothesis that hyperactivity of the orexin system in the paraventricular nucleus (PVN) contributes to SSHTN via enhancing AVP signaling. Eight-week-old male Dahl salt-sensitive (Dahl S) and age- and sex-matched Sprague-Dawley (SD) rats were placed on a high-salt (HS; 8% NaCl) or normal-salt (NS; 0.4% NaCl) diet for 4 wk. HS intake did not alter mean arterial pressure (MAP), PVN mRNA levels of orexin receptor 1 (OX1R), or OX2R but slightly increased PVN AVP mRNA expression in SD rats. HS diet induced significant increases in MAP and PVN mRNA levels of OX1R, OX2R, and AVP in Dahl S rats. Intracerebroventricular infusion of orexin A (0.2 nmol) dramatically increased AVP mRNA levels and immunoreactivity in the PVN of SD rats. Incubation of cultured hypothalamus neurons from newborn SD rats with orexin A increased AVP mRNA expression, which was attenuated by OX1R blockade. In addition, increased cerebrospinal fluid Na+ concentration through intracerebroventricular infusion of NaCl solution (4 µmol) increased PVN OX1R and AVP mRNA levels and immunoreactivity in SD rats. Furthermore, bilateral PVN microinjection of the OX1R antagonist SB-408124 resulted in a greater reduction in MAP in HS intake (-16 ± 5 mmHg) compared with NS-fed (-4 ± 4 mmHg) anesthetized Dahl S rats. These results suggest that elevated PVN OX1R activation may contribute to SSHTN by enhancing AVP signaling.NEW & NOTEWORTHY To our best knowledge, this study is the first to investigate the involvement of the orexin system in salt-sensitive hypertension. Our results suggest that the orexin system may contribute to the Dahl model of salt-sensitive hypertension by enhancing vasopressin signaling in the hypothalamic paraventricular nucleus.

Altered functionality of the corticotrophin-releasing hormone receptor-2 in the hypothalamic paraventricular nucleus of hyperphagic maternally separated rats

Early-life stress induces endocrine and metabolic alterations that increase food intake and overweight in adulthood. The stress response activates the corticotropin-releasing hormone (CRH) and urocortins' (Ucns) system in the hypothalamic paraventricular nucleus (PVN). These peptides induce anorexic effects through CRH-R2 receptor activation; however, chronic stressed animals develop hyperphagia despite of high PVN CRH expression.We analyzed this paradoxical behavior in adult rats subjected to maternal separation (MS) for 180min/daily during post-natal days 2–14, evaluating their body weight gain, food intake, serum corticosterone and vasopressin concentrations, PVN mRNA expression of CRH-R1, CRH-R2, CRH, Ucn2, Ucn3, vasopressin and CRH-R2 protein levels.MS adults increased their feeding, weight gain as well as circulating corticosterone and vasopressin levels, evincing chronic hyperactivity of the stress system. MS induced higher PVN CRH, Ucn2 and CRH-R2 mRNA expression and protein levels of CRH-R2 showed a tendency to decrease in the cellular membrane fraction. An intra-PVN injection of the CRH-R2 antagonist antisauvagine-30 in control adults increased receptor's mRNA expression, mimicking the observed PVN receptor's up-regulation of early-life MS adults.An injection of Ucn-2 directly into the PVN reduced food intake and increased PVN pCREB/CREB ratio in control animals; in contrast, Ucn-2 was unable to reduce food intake and enhance phosphorylated-CREB levels in PVN of MS rats.In conclusion, the chronic hyperactivity of the stress axis and PVN CRH-R2 resistance to Ucn2 effects, supported impaired receptor functionality in MS animals, probably due to its chronic stimulation by CRH or Ucn2, induced by early-life stress.

Abstract P327: Increased Brain iNOS Contributes to Hypertension in Dahl Salt Sensitive Rats

The hypothalamic paraventricular nucleus (PVN) is one of the key central nuclei to play an important role in regulating arterial blood pressure (ABP) of salt-sensitive hypertension (SSH). However, the detailed molecular mechanism(s) whereby the PVN increases ABP are not well understood. Here, we tested the hypothesis that high salt (HS) loading increases expression of iNOS in the PVN which contributes to SSH. Six-week-old male Dahl salt sensitive (Dahl S) rats and age matched Sprague Dawley (SD) rats were fed either a HS (4% NaCI) or a normal salt (NS, 0.4% NaCl) diet (n=4~7/group). Mean arterial pressure (MAP) was measured via tail cuff method. Five weeks following diet treatment, HS diet induced hypertension in Dahl S rats (HS: 153±9; vs. NS: 122±2 mmHg, P&lt;0.05), but not in SD rats (HS: 107±3; vs. NS: 107±2 mmHg). Rats were then euthanized and PVN tissues were punched out for real time PCR. The HS diet induced dramatic increases in mRNA levels of iNOS (25-fold), and Fra1 (3.6-fold), a chronic neuronal activation marker, in Dahl S rat but not in SD rats. Next, we investigated the effect of intracerebroventricular (ICV) administration of hypertonic saline on PVN iNOS and Fra1 expression in SD rats. Anesthetized adult male SD rats received ICV infusion of isotonic NaCI (0.15 M, 2μl, as control) or hypertonic NaCI (2M, 2μl) (n=7~8/group). Three hours following ICV infusion, rats were euthanized and PVN mRNA levels of iNOS and Fra1 were assayed. ICV hypertonic saline increased mRNA levels of iNOS (9.5-fold) and Fra1 (4.1-fold). We further tested whether these increases in iNOS and Fra1 expression occurred in neurons. Incubation of hypertonic saline (10 mM NaCI) for 3 hours increased iNOS (6-fold) and Fra1 (2.8-fold) mRNA levels in neuronal cultures from the hypothalamus containing the PVN. Finally, we tested whether increased iNOS activity contributes to ABP elevation in Dahl SSH. In anaesthetized Dahl S rats, bilateral PVN microinjection of the iNOS inhibitor, aminoguanidine (250 pmol) significantly decreased MAP in HS treated animals compared to rats with a NS diet (HS: -13±3; vs. NS: -2±2 mmHg, P&lt;0.05) (n=5/group). These observations suggest that HS intake increases iNOS expression in PVN neurons, which may contribute to the central neural mechanism of Dahl SSH.

High Salt Challenge Augments Expression of Proinflammatory Cytokines and Contributes to Elevated Neuronal Activity in the Hypothalamic Paraventricular Nucleus

Previous studies have demonstrated that brain proinflammatory cytokines (PIC) play an important role in neurogenic hypertension. However, little is known about the role of brain inflammation in salt sensitive hypertension (SSHTN). In this study, we tested the hypothesis that the PIC expression is increased in the hypothalamic paraventricular nucleus (PVN) and contributes to increased neuronal activity in SSHTN. To test this hypothesis, 8‐week‐old Dahl salt sensitive (DS) rats were divided into two groups (n=3~ 5/group) and fed with either a high salt (HS, 4% NaCI) or normal salt (NS, 0.4% NaCl) diet. Four weeks following diet treatment, rats were euthanized, the PVN was punched out, and real time PCR were performed to assay genes of interest. HS treatment resulted in respective 8 fold, 4 fold, and 7 fold increases in TNFα, IL1β and IL6 mRNA levels in the PVN. In addition, Fra1, a chronic neuronal activation marker, was also increased by 4 fold. Additionally, in order to examine the action of acute hypertonic NaCI on the expression of PIC and neuronal activation marker, adult male SD rats were divided into two groups (n=3~4 per group) and received intracerbroventricular infusion of either hypertonic (2M, 2μl), or isotonic (0.15 M, 2μl) NaCI under anesthetized condition. Three hours following the infusion, rats were euthanized and PVN mRNA levels of PIC and Fra1 assayed. Results indicate that hypertonic saline infusion induced significant increases in mRNA levels of PIC including TNFα (6‐fold), IL1β (13‐fold) and IL6 (21‐fold), as well as neuronal activation marker Fra1 (8‐fold). Finally, we tested whether or not the increase in PIC expression occurred in neurons. Incubation of hypertonic saline (10 mM NaCI plus full medium) for 3 hours resulted in a 12‐fold increase in IL6 and 2.8 fold increase in Fra1 mRNA levels in neuronal cultures from the hypothalamus containing the PVN of neonatal SD rats. These observations suggest that HS intake induced inflammation in the PVN may contribute to increased sympathetic nerve activity and therefore to SSHTN developmentSupport or Funding InformationFunding support: AHA 11SDG7420029, Zhiying Shan; R15‐HL122952, Qinghui Chen

Activation of corticotropin-releasing factor neurons and microglia in paraventricular nucleus precipitates visceral hypersensitivity induced by colorectal distension in rats

Visceral hypersensitivity is a major contributor to irritable bowel syndrome and other disorders with visceral pain. Substantial evidence has established that glial activation and neuro-glial interaction play a key role in the establishment and maintenance of visceral hypersensitivity. We recently demonstrated that activation of spinal microglial toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor κB (NF-κB) signaling facilitated the development of visceral hypersensitivity in a rat model developed by neonatal and adult colorectal distensions (CRDs). Hypothalamic paraventricular nucleus (PVN) plays a pivotal role in the pathogenesis of chronic pain. In this study, we examined the mechanism by which microglia and neurons in PVN establish and maintain visceral hypersensitivity and the involvement of TLR4 signaling. Visceral hypersensitivity was precipitated by adult colorectal distension (CRD) only in rats that experienced neonatal CRDs. Visceral hypersensitivity was associated with an increase in the expression of c-fos, corticotropin-releasing factor (CRF) protein and mRNA in PVN, which could be prevented by intra-PVN infusion of lidocaine or small interfering RNA targeting the CRF gene. These results suggest PVN CRF neurons modulate visceral hypersensitivity. Adult CRD induced an increase in the expression of Iba-1 (a microglial marker), TLR4 protein, and its downstream effectors MyD88, NF-κB, as well as proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) only in rats that experienced neonatal CRDs. Intra-PVN infusion of minocycline, a nonselective microglial inhibitor, attenuated the hyperactivity of TLR4 signaling cascade, microglial activation, and visceral hypersensitivity. Taken together, these data suggest that neonatal CRDs induce a glial activation in PVN. Adult CRD potentiates the glial and CRF neuronal activity, and precipitates visceral hypersensitivity and pain. TLR4 signaling and proinflammatory cytokines TNF-α and IL-1β may participate in neuro-glial interaction during the pathogenesis of visceral hypersensitivity.

Activation of angiotensin II type‐1 receptors stimulates microglial production of pro‐inflammatory cytokines in hypothalamic paraventricular nucleus of heart failure rats

Activated microglia produce pro‐inflammatory cytokines, which contribute to sympathetic activity in heart failure (HF). Microglia express angiotensin (ANG) II type‐1 receptors (AT1R), an important component of the renin‐angiotensin system. However, the relationships between the renin‐angiotensin system, activation of microglia and increased pro‐inflammatory cytokines in key cardiovascular regulatory centers of the brain has not been fully elucidated. We hypothesized that activation of AT1R mediate microglial release of pro‐inflammatory cytokines in the hypothalamic paraventricular nucleus (PVN) in HF. Rats underwent coronary ligation to induce HF or sham surgery. HF rats received a 4 week intracerebroventricular infusion of vehicle, the microglial inhibitor minocycline or the AT1R antagonist losartan. Compared with sham, vehicle‐treated HF rats had increased (P&lt;0.05) microglial marker CD68 and Emr1 mRNA, and augmented tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), cyclooxygenase‐2 (COX‐2) and AT1R mRNA in PVN. Minocycline‐treated HF rats had less CD68 and Emr1 mRNA and reduced TNF‐α, IL‐1β and COX‐2 but not AT1R mRNA in PVN. Losartan‐treated HF rats also had less Emr1 mRNA and reduced TNF‐α, IL‐1β and COX‐2 in PVN, as well as reduced AT1R mRNA. These results suggest that activation of AT1R contributes to microglial production of pro‐inflammatory cytokines in the PVN of HF rats.

Gestational Hypoxia Induces Sex-Differential Methylation of Crhr1 Linked to Anxiety-like Behavior

Stress during gestation increases vulnerability to disease and changes behavior in offspring. We previously reported that hypoxia and restraint during pregnancy sensitized the hypothalamic-pituitary-adrenal (HPA) axis and induced anxiety-like behavior in the adult offspring. Here, we report that gestational intermittent hypoxia (GIH) elicited a sex-dependent anxiety-like behavior in male P90 offspring and activation of corticotropin-releasing hormone (CRH) and CRH type-1 receptor (CRHR1) mRNA in the hypothalamic paraventricular nucleus (PVN) and in male E19 hypothalamus. These linked to demethylation at several specific sites of CpG island of Crhr1 promoter in P90 PVN and E19 embryo hypothalamus in GIH groups. Crhr1 DNA demethylation is more crucial in CpG island 1 than island 2 for activation of CRHR1 mRNA. DNMT3b is required for the Crhr1 DNA methylation than DNMT1 and DNMT3a in increased CRHR1 mRNA. We first address a novel hypothesis that GIH-induced male-sex-dependent demethylation at CpG sites of Crhr1 DNA in promoter triggers elevation of CRHR1 mRNA in PVN and anxiety-like behavior in adult offspring.

Activation of Brain Peroxisome Proliferator‐Activated Receptor‐γ Inhibits Lipopolysaccharide‐Induced Central Inflammation and Sympathetic Excitation

Inflammation in the brain cardiovascular regulatory regions has recently emerged as an important factor contributing to augmented sympathetic drive in hypertension and heart failure. Activation of peroxisome proliferator‐activated receptor (PPAR)‐γ, a nuclear transcription factor, has been shown to inhibit inflammation in brain and peripheral tissues. We hypothesized that activation of brain PPAR‐γ would inhibit hypothalamic inflammation and sympathetic excitation in response to a lipopolysaccharide (LPS) challenge. In anesthetized rats, intracerebroventricular (ICV) injection of LPS (5 μg) increased (p&lt;0.05, vs baseline) renal sympathetic nerve activity (RSNA, 44.2 ± 7.2 %), mean blood pressure (MBP, 13.9 ± 2.5 mmHg ) and heart rate (HR, 97.2 ± 21.6 bpm). LPS decreased (p&lt;0.05, vs vehicle) PPAR‐γ mRNA (−28.7 %) in hypothalamic paraventricular nucleus (PVN), and increased PVN mRNA for tumor necrosis factor‐α (311.7 %), interleukin‐1β (374.1 %), and cyclooxygenase‐2 (341.1 %), as well as cerebrospinal fluid levels of prostaglandin E2 (171.2 %). ICV pretreatment with the PPAR‐γ agonist pioglitazone (10 μg) significantly attenuated all of these LPS‐induced responses. These data demonstrate that brain PPAR‐γ activation counteracts central inflammatory mechanisms leading to sympathetic excitation and so may be an appropriate target for therapeutic intervention in heart failure and hypertension.

EP3receptors mediate PGE2-induced hypothalamic paraventricular nucleus excitation and sympathetic activation

Prostaglandin E(2) (PGE(2)), an important mediator of the inflammatory response, acts centrally to elicit sympathetic excitation. PGE(2) acts on at least four E-class prostanoid (EP) receptors known as EP(1), EP(2), EP(3), and EP(4). Since PGE(2) production within the brain is ubiquitous, the different functions of PGE(2) depend on the expression of these prostanoid receptors in specific brain areas. The type(s) and location(s) of the EP receptors that mediate sympathetic responses to central PGE(2) remain unknown. We examined this question using PGE(2), the relatively selective EP receptor agonists misoprostol and sulprostone, and the available selective antagonists for EP(1), EP(3), and EP(4). In urethane-anesthetized rats, intracerebroventricular (ICV) administration of PGE(2), sulprostone or misoprostol increased renal sympathetic nerve activity, blood pressure, and heart rate. These responses were significantly reduced by ICV pretreatment with the EP(3) receptor antagonist; the EP(1) and EP(4) receptor antagonists had little or no effect. ICV PGE(2) or misoprostol increased the discharge of neurons in the hypothalamic paraventricular nucleus (PVN). ICV misoprostol increased the c-Fos immunoreactivity of PVN neurons, an effect that was substantially reduced by the EP(3) receptor antagonist. Real-time PCR detected EP(3) receptor mRNA in PVN, and immunohistochemical studies revealed sparsely distributed EP(3) receptors localized in GABAergic terminals and on a few PVN neurons. Direct bilateral PVN microinjections of PGE(2) or sulprostone elicited sympathoexcitatory responses that were significantly reduced by the EP(3) receptor antagonist. These data suggest that EP(3) receptors mediate the central excitatory effects of PGE(2) on PVN neurons and sympathetic discharge.

Thyroxine-induced expression of pyroglutamyl peptidase II and inhibition of TSH release precedes suppression of TRH mRNA and requires type 2 deiodinase

Suppression of TSH release from the hypothyroid thyrotrophs is one of the most rapid effects of 3,3',5'-triiodothyronine (T(3)) or thyroxine (T(4)). It is initiated within an hour, precedes the decrease in TSHβ mRNA inhibition and is blocked by inhibitors of mRNA or protein synthesis. TSH elevation in primary hypothyroidism requires both the loss of feedback inhibition by thyroid hormone in the thyrotrophs and the positive effects of TRH. Another event in this feedback regulation may be the thyroid hormone-mediated induction of the TRH-inactivating pyroglutamyl peptidase II (PPII) in the hypothalamic tanycytes. This study compared the chronology of the acute effects of T(3) or T(4) on TSH suppression, TRH mRNA in the hypothalamic paraventricular nucleus (PVN), and the induction of tanycyte PPII. In wild-type mice, T(3) or T(4) caused a 50% decrease in serum TSH in hypothyroid mice by 5 h. There was no change in TRH mRNA in PVN over this interval, but there was a significant increase in PPII mRNA in the tanycytes. In mice with genetic inactivation of the type 2 iodothyronine deiodinase, T(3) decreased serum TSH and increased PPII mRNA levels, while T(4)-treatment was ineffective. We conclude that the rapid suppression of TSH in the hypothyroid mouse by T(3) occurs prior to a decrease in TRH mRNA though TRH inactivation may be occurring in the median eminence through the rapid induction of tanycyte PPII. The effect of T(4), but not T(3), requires the type 2 iodothyronine deiodinase.

Abstract 383: Depletion of Perivascular Macrophages of the Blood-Brain Barrier Reduces Sympathetic Drive in Rats after Myocardial Infarction

Pro-inflammatory cytokines induce cyclooxygenase-2 (COX-2) activity and synthesis of prostaglandin E2 (PGE 2 ) in perivascular macrophages of hypothalamic paraventricular nucleus (PVN). Cytokine-induced PGE 2 synthesis in the brain contributes to increased sympathetic nerve activity following myocardial infarction (MI). Perivascular macrophages can be selectively depleted by clodronate liposomes. Depletion of perivascular macrophages of the blood-brain barrier will reduce COX-2 expression and cytokine-induced sympathetic nerve activity in rats after MI. Clodronate liposomes (CLOD), control liposomes (LIPO) or vehicle (VEH, artificial CSF) were injected (1 μl/min over 40 minutes) into lateral ventricle in rats within 24 hours of MI, or normal rats. One week after injection, perivascular macrophages were completely destroyed and COX-2 immunoreactivity was eliminated in PVN of all CLOD-treated rats but not in LIPO or VEH-treated rats. Activated microglia were unaffected by CLOD. Compared with sham-operated rats (n=10), VEH-treated MI rats (n=11) had increased COX-2 (0.09±0.01* vs 0.05±0.01, normalized to GAPDH, *P&lt;0.05), interleukin-1β (IL-1β, 0.48±0.06* vs 0.21±0.03) and tumor necrosis factor - α (TNF-α, 0.50±0.09* vs 0.22±0.08) mRNA in PVN, IL-1β (23±2* vs 15±1, pg/ml), TNF-α (5±1* vs 2±0, pg/ml), norepinephrine (1146±143* vs 450±66, pg/ml) levels in plasma, PGE 2 (1152±109* vs 480±84, pg/ml) in CSF and Fra-like-positive neurons (40±3* vs 21±2) in PVN. CLOD treatment of MI rats (n=11) normalized COX-2 mRNA in PVN and reduced CSF PGE 2 (by 30%#, #P&lt;0.05 vs VEH), plasma norepinephrine (by 34%#) and Fra-like positive PVN neurons (by 29%#), but had no effects on IL-1β and TNF-αin PVN or plasma. In normal rats, CLOD treatment (n=9) reduced (P&lt;0.05, vs VEH) sympatho-excitatory responses (renal sympathetic nerve activity, blood pressure and heart rate) induced by intracarotid artery injection of TNF-α (0.5 μg/kg). No effects were found in LIPO-treated MI or normal rats. Pro-inflammatory cytokines stimulate sympathetic excitation after MI by inducing COX-2 activity and PGE 2 production in perivascular macrophages of the blood-brain barrier.

Mitogen-Activated Protein Kinases Mediate Upregulation of Hypothalamic Angiotensin II Type 1 Receptors in Heart Failure Rats

In heart failure (HF), angiotensin II type 1 receptor (AT(1)-R) expression is upregulated in brain regions regulating sympathetic drive, blood pressure, and body fluid homeostasis. However, the mechanism by which brain AT(1)-R are upregulated in HF remains unknown. The present study examined the hypothesis that the angiotensin II (Ang II)-triggered mitogen-activated protein kinases (MAPKs) p44/42, p38, and c-Jun N-terminal kinase contribute to upregulation of the AT(1)-R in the hypothalamus of rats with HF. AT(1)-R protein, AT(1)-R mRNA, and AT(1)-R immunoreactivity increased in the paraventricular nucleus of hypothalamus and the subfornical organ of rats with ischemia-induced HF compared with sham-operated controls. Phosphorylated p44/42 MAPK, c-Jun N-terminal kinase, and p38 MAPK also increased in paraventricular nucleus and subfornical organ. A 4-week ICV infusion of the AT(1)-R antagonist losartan decreased AT(1)-R protein and phosphorylation of p44/42 MAPK, c-Jun N-terminal kinase, and p38 MAPK in the HF rats. A 4-week ICV infusion of the p44/42 MAPK inhibitor PD98059 or the c-Jun N-terminal kinase inhibitor SP600125 significantly decreased AT(1)-R protein and AT(1)-R immunoreactivity in the paraventricular nucleus and subfornical organ, but the p38 MAPK inhibitor SB203580 did not. Treatment with ICV losartan, PD98059, and SP600125 had no effect on AT(1)-R expression by Western blot in sham-operated rats. In untreated HF rats 4 weeks after coronary ligation, a 3-hour ICV infusion of PD98059, SP600125, or losartan reduced AT(1)-R mRNA in paraventricular nucleus and subfornical organ. These data indicate that MAPK plays an important role in the upregulation of AT(1)-R in the rat forebrain in HF and suggest that Ang II upregulates its own receptor by this mechanism.

Effects of Central Sodium On Epithelial Sodium Channels (ENaC) Expression In Rat Brain

Mineralocorticoid receptors (MR) and ENaC mediate pressor responses to increases of CSF [Na+]. We evaluated MR‐dependent effects of central sodium on ENaC subunits (α, β ,γ) expression as well as its endogenous regulator, Sgk1. Male Wistar rats were infused icv for 2 weeks with regular or Na+‐rich artificial CSF (aCSF) with or without spironolactone. Brain areas studied included ependyma of the anteroventral third ventricle, supraoptic nucleus (SON), paraventricular nucleus (PVN) and subfornical organ (SFO). In ependyma, Na+‐rich aCSF increased expression of the α and β subunits, primarily in the basolateral membrane. Spironolactone did not prevent these changes. In SON, Na+ increased expression of mRNA for α and γ subunits without affecting α protein levels, and increased Sgk1 mRNA levels although a decrease in phosphorylated Sgk1 protein (p‐Sgk1) was noted. Spironolactone prevented the increases in mRNA encoding ENaC but not Sgk1. In PVN, Na+ decreased the α mRNA but not protein levels, and significantly decreased p‐Sgk1 protein with no change in Sgk1 mRNA. In SFO, Na+ only significantly increased γ mRNA. Spironolactone did not prevent the changes in ENaC mRNA in PVN or SFO. Regulation of ENaC and Sgk1 expression in the brain in response to Na+ appears quite region specific. Spironolactone prevents only some of the increases in ENaC subunit expression implying that, in addition to aldosterone, other regulators are involved.

Analysis of the anxiolytic-like effect of TRH and the response of amygdalar TRHergic neurons in anxiety

Thyrotropin-releasing hormone (TRH) was first described for its neuroendocrine role in controlling the hypothalamus-pituitary-thyroid axis (HPT). Anatomical and pharmacological data evidence its participation as a neuromodulator in the central nervous system. Administration of TRH induces various behavioural effects including arousal, locomotion, analepsy, and in certain paradigms, it reduces fear behaviours. In this work we studied the possible involvement of TRHergic neurons in anxiety tests. We first tested whether an ICV injection of TRH had behavioural effects on anxiety in the defensive burying test (DBT). Corticosterone serum levels were quantified to evaluate the stress response and, the activity of the HPT axis to distinguish the endocrine response of TRH injection. Compared to a saline injection, TRH reduced cumulative burying, and decreased serum corticosterone levels, supporting anxiolytic-like effects of TRH administration. The response of TRH neurons was evaluated in brain regions involved in the stress circuitry of animals submitted to the DBT and to the elevated plus maze (EPM), tests that allow to correlate biochemical parameters with anxiety-like behaviour. In the DBT, the response of Wistar rats was compared with that of the stress-hypersensitive Wistar Kyoto (WKY) strain. Behavioural parameters were analysed in recorded videos. Animals were sacrificed 30 or 60min after test completion. In various limbic areas, the relative mRNA levels of TRH, its receptors TRH-R1 and -R2, and its inactivating ectoenzyme pyroglutamyl peptidase II (PPII), were determined by RT-PCR, TRH tissue content by radioimmunoassay (RIA). The extent of the stress response was evaluated by measuring the expression profile of CRH, CRH-R1 and GR mRNA in the paraventricular nucleus (PVN) of the hypothalamus and in amygdala, corticosterone levels in serum. As these tests demand increased physical activity, the response of the HPT axis was also evaluated. Both tasks increased the levels of serum corticosterone. WKY rats showed higher anxiety-like behaviour in the DBT than Wistar, as well as increased PVN mRNA levels of CRH and GR. TRH mRNA levels increased in the PVN and TSH values remained unchanged in both strains although TRH content decreased in the medial basal hypothalamus of Wistar rats only. TRH content was measured in several limbic regions but only amygdala showed specific task-related changes after DBT exposure of both strains: increased TRH content. Expression of TRH mRNA decreased in the amygdala of Wistar, suggesting inhibition of TRHergic neuronal activity in this region. The participation of amygdalar TRH neurons in anxiety was confirmed in the EPM where TRH expression and release correlated with the number of entries, and the % of time spent in open arms, supporting an anxiolytic role of these TRH-neurons. These results contribute to the understanding of the involvement of TRH during emotionally charged situations and shed light on the participation of particular circuits in related behaviours.

11β-Hydroxysteroid Dehydrogenase Type 2 Activity in Hypothalamic Paraventricular Nucleus Modulates Sympathetic Excitation

Aldosterone stimulates the sympathetic nervous system by binding to a select population of brain mineralocorticoid receptors (MR). These MR have an equal affinity for corticosterone that is present in substantially higher concentrations, but are held in reserve for aldosterone by activity of the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2), which converts corticosterone to an inactive metabolite. Thus, colocalization of MR and 11beta-HSD-2 activity may help identify brain regions that mediate the effects of aldosterone. The present studies tested the hypothesis that 11beta-HSD-2 activity regulates MR-mediated responses in the paraventricular nucleus (PVN) of the hypothalamus, a forebrain region implicated in sympathetic regulation. Real-time-polymerase chain reaction revealed the presence of 11beta-HSD-2 mRNA in PVN. In anesthetized adult male Sprague-Dawley rats, microinjection of the 11beta-HSD-2 inhibitor carbenoxolone (CBX) into PVN increased mean arterial pressure, heart rate, and renal sympathetic nerve activity. Intracerebroventricular injections of CBX excited PVN neurons and increased mean arterial pressure, heart rate, and renal sympathetic nerve activity. The ability of CBX to increase sympathetic activity by inhibiting 11beta-HSD-2, thereby permitting corticosterone to activate MR, was confirmed by the following: Intracerebroventricular glycyrrhizic acid, another 11beta-HSD-2 inhibitor, mimicked the sympathoexcitatory effects of CBX; the sympathoexcitatory effects of CBX were blocked by spironolactone, a MR antagonist. Neither CBX nor glycyrrhizic acid elicited a response in adrenalectomized rats. These findings suggest that MR in PVN contribute to sympathetic regulation and may be activated by aldosterone or corticosterone (or cortisol in humans) depending on the state of 11beta-HSD-2 activity.

Hypothalamic paraventricular nucleus, but not vasopressin, participates in chronic hyperactivity of the HPA axis in diabetic rats

Diabetes mellitus (DM), as chronic stress activates the hypothalamo-pituitary-adrenocortical axis. We examined whether arginine vasopressin (AVP) and the hypothalamic paraventricular nucleus (PVN) participate in DM-induced chronic stress symptoms. AVP-deficient Brattleboro or PVN-lesioned Wistar rats were used with heterozygous or sham-operated controls. The rats were studied 2 wk after a single injection of streptozotocin. The appearance of DM (enhanced water consumption and blood glucose elevation) and the chronic stress-like somatic changes (body weight decrease, thymus involution, adrenal gland hypertrophy) were not influenced by the lack of AVP. By contrast, PVN lesion significantly attenuated DM-induced thymus involution and adrenal gland hypertrophy as well as the increase in water consumption. The corticotropin-releasing hormone mRNA in PVN was diminished by DM and elevated by the lack of AVP without interaction. DM elevated the proopiomelanocortin (POMC) mRNA in the anterior lobe of the pituitary. The lack of AVP had no effect, whereas lesioning the PVN significantly diminished the elevation. The elevated basal corticosterone plasma levels detectable in DM were influenced neither by the lack of AVP nor by lesioning the PVN. Thus the lack of AVP had no influence on DM-induced chronic stress symptoms, but lesioning the PVN attenuated part of them. However, the lack of elevation in POMC mRNA after PVN lesion, together with the maintained corticosterone elevation, suggests that direct adrenal gland activation occurs in untreated DM.

Stress integration after acute and chronic predator stress: differential activation of central stress circuitry and sensitization of the hypothalamo-pituitary-adrenocortical axis.

Predator exposure is a naturalistic stressor of high ethological relevance. In the current study, our group examined central and peripheral integration of stress responses in rats after acute or repeated exposure to a natural predator (cat). Acute cat exposure rapidly induced hypothalamo-pituitary-adrenocortical (HPA) axis activation and paraventricular nucleus (PVN) CRH mRNA production. Repeated daily cat exposure (7 and 14 d) also up-regulated PVN mRNA CRH expression, but did not result in frank adrenocortical hyperactivity. Unlike other chronic homotypic stress regimens, repeated cat exposure facilitated corticosterone secretion after the 6th or 13th day of exposure. Notably, ACTH secretion and central amygdaloid nucleus CRH mRNA expression were enhanced in animals that were preexposed to the holding chamber relative to chamber-naive rats, suggesting that contextual cues can sensitize subsequent responses to a fearful stimulus. Analysis of c-fos activation was then used to identify brain circuits activated by acute predator stress. Cat exposure elicited a pattern of central c-fos activation that differed substantially from that after either restraint or hypoxia. Predator-specific c-fos mRNA induction was observed in several brain regions comprising the hypothetical brain defense circuit (bed nucleus of the stria terminalis, medial region of the ventromedial nucleus, and dorsal premammillary nucleus). Surprisingly, acute cat exposure did not induce c-fos expression in the PVN. In summary, the data indicate that 1) predation stress invokes a unique stress circuitry that promotes homotypic sensitization of the HPA axis, and 2) familiarization of animals to testing environments can prime central stress pathways to respond robustly to novel threats.