Rostral Ventrolateral Medulla Of Rats Research Articles

Oxidative stress in the rostral ventrolateral medulla modulates excitatory and inhibitory inputs in spontaneously hypertensive rats

The rostral ventrolateral medulla (RVLM) of the brainstem and the paraventricular nucleus (PVN) of the hypothalamus play crucial roles in central cardiovascular regulation. In hypertensive rats, an imbalance of excitatory and inhibitory inputs to the RVLM enhances central sympathetic outflow. Increased reactive oxygen species (ROS) in the RVLM also contribute to sympathoexcitation, leading to hypertension. The aim of the present study was to elucidate whether ROS in the RVLM modulate synaptic transmission via excitatory and inhibitory amino acids and influence the excitatory inputs to the RVLM from the PVN in spontaneously hypertensive rats (SHRs). We transfected adenovirus vectors encoding the manganese superoxide dismutase (AdMnSOD) gene to scavenge ROS in the RVLM both in Wistar-Kyoto rats and SHRs. The decreases in blood pressure and renal sympathetic nerve activity (RSNA) evoked by injecting kynurenic acid, a glutamate receptor blocker, into the RVLM were attenuated, and the increases in blood pressure and RSNA evoked by injecting bicuculline, a γ-amino butyric acid (GABA) receptor blocker, into the RVLM were enhanced in AdMnSOD-transfected SHRs compared with adenovirus vectors encoding the β-galactosidase (AdLacZ) gene-transfected SHRs. Furthermore, the increases in blood pressure and RSNA evoked by injecting bicuculline into the PVN were attenuated in AdMnSOD-transfected SHRs compared with AdLacZ-transfected SHRs. These findings suggest that ROS in the RVLM enhance glutamatergic excitatory inputs and attenuate GABAergic inhibitory inputs to the RVLM, thereby increasing sympathoexcitatory input to the RVLM from the PVN in SHRs.

Malnutrition affects the pressor response to microinjection of L-glutamate into the RVLM of awake rats

Post-weaning protein malnutrition is often related to the development of cardiovascular and metabolic diseases in humans, as well to changed content of neurotransmitters in the central nervous system under experimental conditions. The rostral ventrolateral medulla (RVLM) is a bulbar region that contains sympathetic premotor neurons; the excitatory amino acid L-glutamate seems to be the main neurotransmitter at this level. The aim of the present study was to evaluate the possible change in the L-glutamate sensitivity of the RVLM neurons of malnourished animals. Male Fischer rats were divided into two groups: control (n = 15) and malnourished (n = 19). Four days before the experiments, guide cannulas were implanted bilaterally in direction of the RVLM for microinjection of L-glutamate. Twenty-four hours before the experiments, the femoral artery was cannulated for cardiovascular recordings. The results showed that the baseline heart rate increased in malnourished compared to control animals (412.18 ± 16.03 bpm vs. 370.74 ± 9.59 bpm, respectively). Malnourished animals presented a dissimilar concentration-dependent pressor response curve to L-glutamate and an attenuated baroreflex gain. Our results suggest that post-weaning protein restriction affects glutamatergic neurotransmission of the baroreflex at the RVLM level.

Amelioration of central cardiovascular regulatory dysfunction by tropomyocin receptor kinase B in a mevinphos intoxication model of brain stem death

Little information exists on the mechanisms that precipitate brain stem death, the legal definition of death in many developed countries. We investigated the role of tropomyocin receptor kinase B (TrkB) and its downstream signalling pathways in the rostral ventrolateral medulla (RVLM) during experimental brain stem death. An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos bilaterally into the RVLM of Sprague-Dawley rats was used, in conjunction with cardiovascular, pharmacological and biochemical evaluations. A significant increase in TrkB protein, phosphorylation of TrkB at Tyr(516) (pTrkB(Y516) ), Shc at Tyr(317) (pShc(Y317) ) or ERK at Thr(202) /Tyr(204) , or Ras activity in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Microinjection bilaterally into RVLM of a specific TrkB inhibitor, K252a, antagonized those increases. Pretreatment with anti-pShc(Y317) antiserum, Src homology 3 binding peptide (Grb2/SOS inhibitor), farnesylthioacetic acid (Ras inhibitor), manumycin A (Ras inhibitor) or GW5074 (Raf-1 inhibitor) blunted the preferential augmentation of Ras activity or ERK phosphorylation in RVLM and blocked the up-regulated NOS I/protein kinase G (PKG) signalling, the pro-life cascade that sustains central cardiovascular regulation during experimental brain stem death. Activation of TrkB, followed by recruitment of Shc/Grb2/SOS adaptor proteins, leading to activation of Ras/Raf-1/ERK signalling pathway plays a crucial role in ameliorating central cardiovascular regulatory dysfunction via up-regulation of NOS I/PKG signalling cascade in the RVLM in brain stem death. These findings provide novel information for developing therapeutic strategies against this fatal eventuality.

Relationship between distribution of Phox2b expressing neurons and capillary blood vessels in the parafacial region of the rostral ventrolateral medulla of rat

Relationship between distribution of Phox2b expressing neurons and capillary blood vessels in the parafacial region of the rostral ventrolateral medulla of rat

Transient middle cerebral artery occlusion and reperfusion alters inducible NOS expression within the ventrolateral medulla and modulates cardiovascular function during static exercise

A major cause of stroke is cerebral ischemia in regions supplied by the middle cerebral artery (MCA). In this study, we hypothesized that compromised cardiovascular function during static exercise may involve altered expression of inducible NOS (iNOS) protein within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM). We compared cardiovascular responses and iNOS protein expression within the left and right sides of both RVLM and CVLM in sham-operated rats and in rats with a 90 min left-sided MCA occlusion (MCAO) followed by 24h of reperfusion. Increases in blood pressure during a static muscle contraction were attenuated in MCAO rats compared with sham-operated rats. Also, iNOS expression within the left RVLM was augmented compared with the right RVLM in MCAO rats and compared with both RVLM quadrants in sham-operated rats. In contrast, compared with sham-operated rats and the right CVLM of MCAO rats, iNOS expression was attenuated in the left CVLM in left-sided MCAO rats. These data suggest that the attenuation of pressor responses during static exercise in MCAO rats involves overexpression of iNOS within the ipsilateral RVLM and attenuation in iNOS within the ipsilateral CVLM. Differential expression of iNOS within the medulla plays a role in mediating cardiovascular responses during static exercise following stroke.

Modulation of respiratory responses to chemoreflex activation by l-glutamate and ATP in the rostral ventrolateral medulla of awake rats

Presympathetic neurons in the different anteroposterior aspects of rostral ventrolateral medulla (RVLM) are colocalized with expiratory [Bötzinger complex (BötC)] and inspiratory [pre-Bötzinger complex (pre-BötC)] neurons of ventral respiratory column (VRC), suggesting that this region integrates the cardiovascular and respiratory chemoreflex responses. In the present study, we evaluated in different anteroposterior aspects of RVLM of awake rats the role of ionotropic glutamate and purinergic receptors on cardiorespiratory responses to chemoreflex activation. The bilateral ionotropic glutamate receptors antagonism with kynurenic acid (KYN) (8 nmol/50 nl) in the rostral aspect of RVLM (RVLM/BötC) enhanced the tachypneic (120 ± 9 vs. 180 ± 9 cpm; P < 0.01) and attenuated the pressor response (55 ± 2 vs. 15 ± 1 mmHg; P < 0.001) to chemoreflex activation (n = 7). On the other hand, bilateral microinjection of KYN into the caudal aspect of RVLM (RVLM/pre-BötC) caused a respiratory arrest in four awake rats used in the present study. Bilateral P2X receptors antagonism with PPADS (0.25 nmol/50 nl) in the RVLM/BötC reduced chemoreflex tachypneic response (127 ± 6 vs. 70 ± 5 cpm; P < 0.001; n = 6), but did not change the chemoreflex pressor response. In addition, PPADS into the RVLM/BötC attenuated the enhancement of the tachypneic response to chemoreflex activation elicited by previous microinjections of KYN into the same subregion (188 ± 2 vs. 157 ± 3 cpm; P < 0.05; n = 5). Our findings indicate that: 1) L-glutamate, but not ATP, in the RVLM/BötC is required for pressor response to peripheral chemoreflex and 2) both transmitters in the RVLM/BötC are required for the processing of the ventilatory response to peripheral chemoreflex activation in awake rats.

Oxidative stress in the cardiovascular center has a pivotal role in the sympathetic activation in hypertension

Activation of the sympathetic nervous system has an important role in the pathogenesis of hypertension. However, the precise mechanisms involved are not fully understood. Oxidative stress may be important in hypertension as well as in other cardiovascular disorders. We investigated the role of oxidative stress, particularly in the rostral ventrolateral medulla (RVLM), which is known as the cardiovascular center in the brainstem, in the activation of the sympathetic nervous system in hypertension. We observed that the reactive oxygen species (ROS) production increases in the RVLM in hypertensive rats, thereby enhancing the central sympathetic outflow, which leads to hypertension. Furthermore, the environmental factors of high salt intake and a high-calorie diet may also increase the ROS production in the RVLM, thereby activating the central sympathetic outflow and increasing the risk of hypertension. The activation of the nicotinamide adenine dinucleotide phosphate oxidase via the angiotensin type 1 (AT1) receptors is suggested to be the major source of ROS production, and an altered downstream signaling pathway is involved in the activation of the RVLM neurons, leading to enhanced central sympathetic outflow and hypertension. Thus, the brain AT1 receptors may be novel therapeutic targets, and, in fact, oral treatment with angiotensin receptor blockers has been found to inhibit the central AT1 receptors, despite the blood-brain barrier.

Proteomic investigation of a neural substrate intimately related to brain death

Our current understanding on brain death remains limited despite its clinical importance. This study evaluated whether the proteome expressed in the rat rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is uniquely different from other brain areas, using the cerebral cortex, which is defunct under persistent vegetative state for comparison. We found that a group of antioxidant proteins, including members of the peroxiredoxin (Prx) family (Prx-1, Prx-2, Prx-5, Prx-6), thioredoxin and mitochondrial manganese superoxide dismutase, exhibited significantly higher protein and mRNA expression levels in RVLM when compared to cerebral cortex. Tissue oxygen, ATP contents and ATP synthase subunits α and β in RVLM were also significantly elevated. On the other hand, protein and mRNA levels of members of the ubiquitin-proteasome system, including proteasome subunit α type-1, ubiquitin, uniquitin-conjugating enzyme E2 N, ubiquitin carboxyl-terminal hydrolase isozyme L1 and L3, were comparable in both brain regions. We conclude that a significantly elevated level of antioxidant proteins and mRNA in RVLM is consistent with the exhibition of higher tissue oxygen tension and metabolic energy production in this neural substrate, which together constitute a safeguard mechanism against brain death.

Chronic Superoxide Signaling in the Rostral Ventrolateral Medulla (RVLM) is Essential For Goldblatt Hypertension

Reactive oxygen species (ROS) in peripheral cardiovascular tissues are implicated in the pathogenesis of Goldblatt (two kidney one clip, 2K1C) hypertension. We identified an imbalance between ROS generation and oxidant scavenging in the RVLM of Goldblatt rats. In 2K1C rats, the RVLM exhibited increased mRNA expression of NAD(P)H oxidase subunits. With a role in sympathetic activity generation, we tested whether chronic superoxide scavenging in RVLM would affect Goldblatt hypertension. In Wistar rats (male, 150–180g) superoxide dismutase was over expressed (Ad‐CMV‐CuZnSOD) chronically using an adenoviral vector 3 weeks after renal artery clipping. In 2K1C rats, the elevated AP and heart rate returned to control values one week after transfection, was maintained for 3 weeks and associated with decreased low frequency spectra of systolic blood pressure variability. We confirmed both reduced ROS levels and the presence of CuZnSOD in RVLM neurons immunohistochemically. No changes in arterial pressure were observed in control rats receiving Ad‐CMV‐eGFP in the RVLM. Thus, chronic superoxide signaling in the RVLM of the Goldblatt model is essential for the hypertension. Supported by CAPES and British Heart Foundation.

Upregulation of AT1R and iNOS in the Rostral Ventrolateral Medulla (RVLM) Is Essential for the Sympathetic Hyperactivity and Hypertension in the 2K-1C Wistar Rat Model

We hypothesized that upregulation of angiotensin type 1 receptor (AT(1)R) and inducible nitric oxide (NO) synthase (iNOS) within the rostral ventrolateral medulla (RVLM) could contribute to two-kidney, one-clip (2K-1C) hypertension. The experiments were performed in male Wistar rats, 6 weeks after the renal surgery. The animals were divided into control (SHAM, n = 18) and hypertensive groups (2K-1C, n = 18). Bilateral tissue punches were taken from sections containing the RVLM to perform iNOS gene expression analyses by the real-time PCR technique, and AT(1)R and iNOS protein expression analyses by western blotting. In addition, we injected losartan (1 nmol), an AT(1)R antagonist, and aminoguanidine (250 pmol), an iNOS inhibitor, bilaterally into the RVLM to analyze the mean arterial pressure (MAP) and renal sympathetic nerve activity (rSNA). iNOS mRNA expression levels were greater (P < 0.05) in the 2K-1C group compared to the SHAM group. Furthermore, the AT(1)R and iNOS protein expression were significantly increased in the RVLM of 2K-1C rats compared to SHAM rats. Injection of losartan into the RVLM reduced the MAP (11%) and rSNA (18%) only in the 2K-1C rats, whereas injection of aminoguanidine in the same region decreased the MAP (31%) and rSNA (34%) in hypertensive rats. The present study suggests that upregulation of AT(1)R and iNOS in the RVLM is important in the maintenance of high blood pressure and renal sympathetic activation in 2K-1C hypertension.

The RNA Binding Complex Translin‐Trax Mediates Pro‐Excitatory Activity in Neurons

In animal models of neurogenic hypertension the Rostral Ventrolateral Medulla (RVLM), of the brain, displays increased excitatory activity that is linked to elevated sympathetic tone and blood pressure. Analysis of gene expression in the RVLM of normotensive Wistar Kyoto (WKY) and Spontaneous Hypertensive Rat (SHR) revealed reduction of the protein Translin (TSN) in SHR. TSN and its partner Trax (TRX) form a complex that is thought to mediate dendritic trafficking of mRNA. To examine the functional effect of this complex, we dialyzed purified TSN‐TRX into cultured brain neurons from either WKY or SHR and measured its effect on the rate of action potential firing (APF). Basal APF rate was 1.4 Hz in WKY neurons and after 20 min control treated neurons showed a similar rate of 1.3 Hz. In contrast, TSN‐TRX treatment mediated a significant increase in APF rate to 1.9 Hz. Tsn alone had no effect in WKY neurons and SHR neurons showed no response to TSN‐TRX. Pretreatment with the RNA translation inhibitor, cycloheximide, abolished the excitatory effect of Tsn‐TRX in WKY neurons. Our results suggest that the excitatory effect of TSN‐TRX in WKY neurons is translation‐dependent. We hypothesize that homeostatic feedback mechanisms in SHR neurons lead to downregulation of TSN levels and the resistance of these cells to the excitatory effects of the TSN‐TRX complex. This work was supported by funding from NHLBI, NIDA of the NIH and NARSAD.

Telmisartan inhibits sympathetic nerve activity through the reduction of oxidative stress and release of GABA in RVLM of hypertensive rats

BackgroundWe have demonstrated that angiotensin II‐dependent reactive oxygen species (ROS) and ƒÁ‐amino butyric acid (GABA) in the rostral ventrolateral medulla (RVLM) play a major role in regulating sympathetic nerve activity (SNA) in hypertensive rats. The aim of this study was to determine whether oral administration of angiotensin II type 1 receptor (AT1R) blocker, telmisartan, decreases SNA via the reduction of ROS and release of GABA in the RVLM of hypertensive rats.Methods and ResultsWe divided stroke‐prone spontaneously hypertensive rats (SHRSP), as a hypertensive model with enhanced SNA, into 2 groups, telmisartan‐group (TLM) and hydralazine‐group (HYD). Telemetered mean blood pressure (MBP) was reduced in both groups to a similar level. SNA and heart rate (HR) were significantly decreased in TLM, whereas increased in HYD. ROS in the RVLM were significantly lower in TLM than in HYD. The release of GABA measure by microdialysis was significantly higher in TLM than in HYD (15.1±0.7 vs 8.2±1.1 pmol/sample, n=5 for each, P&lt;0.01). Microinjection of angiotensin II into the RVLM increased MBP and HR, and decreased GABA in the RVLM in HYD to a greater extent than in TLM.ConclusionTelmisartan decreases AT1R‐dependent ROS and increases GABA in the RVLM, thereby decreases SNA as well as blood pressure in SHRSP.

Extracellular signal-regulated kinase 1/2 plays a pro-life role in experimental brain stem death via MAPK signal-interacting kinase at rostral ventrolateral medulla

BackgroundAs the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this fatal phenomenon. The present study assessed the hypothesis that extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinases (MAPKs) that is important for cell survival and is activated specifically by MAPK kinase 1/2 (MEK1/2), plays a pro-life role in RVLM during brain stem death. We further delineated the participation of MAPK signal-interacting kinase (MNK), a novel substrate of ERK in this process.MethodsAn experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague-Dawley rats was used, in conjunction with cardiovascular, pharmacological and biochemical evaluations.ResultsResults from ELISA showed that whereas the total ERK1/2 was not affected, augmented phosphorylation of ERK1/2 at Thr202 and Tyr204 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Furthermore, pretreatment by microinjection into the bilateral RVLM of a specific ERK2 inhibitor, ERK activation inhibitor peptide II (1 nmol); a specific MEK1/2 inhibitor, U0126 (5 pmol); or a specific MNK1/2 inhibitor, CGP57380 (5 pmol) exacerbated the hypotension and blunted the augmented life-and-death signals exhibited during the pro-life phase. Those pretreatments also blocked the upregulated nitric oxide synthase I (NOS I)/protein kinase G (PKG) signaling, the pro-life cascade that sustains central cardiovascular regulatory functions during experimental brain stem death.ConclusionsOur results demonstrated that activation of MEK1/2, ERK1/2 and MNK1/2 in RVLM plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during brain stem death via upregulation of NOS I/PKG signaling cascade in RVLM.

Downregulated Kv4.3 expression in the RVLM as a potential mechanism for sympathoexcitation in rats with chronic heart failure

Elevated central angiotensin II (ANG II) plays a critical role in the sympathoexcitation of chronic heart failure (CHF) by stimulating upregulated ANG II type 1 receptors (AT(1)R) in the rostral ventrolateral medulla (RVLM). However, the link between enhanced ANG II signaling and alterations in the electrophysiological characteristics of neurons in the RVLM remains unclear. In the present experiments, we screened for potentially altered genes in the medulla of rats with CHF that are directly related to neuronal membrane conductance using the Rat Genome 230 2.0 Array GeneChip. We found that CHF rats exhibited a 2.1-fold reduction in Kv4.3 gene expression, one of the main voltage-gated K(+) channels, in the medulla. Real-time RT-PCR and Western blot analysis confirmed the downregulation of Kv4.3 in the RVLM of CHF rats. In intact animals, we found that microinjection of the voltage-gated potassium channel blocker, 4-aminopyridine, into the RVLM evoked a sympathoexcitation and hypertension in both normal and CHF rats. CHF rats exhibited smaller responses to 4-aminopyridine than did normal rats. Finally, we used a neuronal cell line (CATH.a neurons) to explore the effect of ANG II on Kv4.3 expression and function. We found that ANG II treatment significantly downregulated mRNA and protein expression of Kv4.3 and decreased the A-type K(+) current. Employing this cell line, we also found that the ANG II-induced inhibition of Kv4.3 mRNA expression was attenuated by the superoxide scavenger Tempol and the p38 MAPK inhibitor SB-203580. The effects of ANG II were abolished by the AT(1)R antagonist losartan. We conclude that the sympathoexcitation observed in the CHF state may be due, in part, to an ANG II-induced downregulation of Kv4.3 expression and subsequent decrease in K(+) current, thereby increasing the excitability of neurons in the RVLM. The ANG II-induced inhibition of Kv4.3 mRNA expression was mediated by ANG II-AT(1)R-ROS-p38 MAPK signaling.

Anteroposterior distribution of AT 1 angiotensin receptors in caudal brainstem cardiovascular regulatory centers of the rat

Angiotensin II acts on Ang II type 1 (AT 1) receptors in areas of the caudal brainstem involved in cardiovascular regulation. In particular, activation of AT 1 receptors in the rostral ventrolateral medulla (RVLM) has been suggested to contribute to hypertension. However, the characteristics of AT 1 receptors in the RVLM of rat, the species in which the most experimental work has been done, are not well documented. This study evaluated AT 1 receptor binding along a 2.7-mm length of rat medulla, which included the full extent of the RVLM and the caudal ventrolateral medulla (CVLM). Sections of medulla from female rats cut on a cryostat were incubated with five concentrations of 125I-sarcosine 1, isoleucine 8 angiotensin II to assess the density ( B max) and dissociation constant ( K D) of the receptors for the radioligand. The dorsomedial medulla (DMM) displayed a high density of AT 1 binding (1207 ± 100 fmol/g), which peaked at 0.4 mm rostral to the calamus scriptorius (approximately 14 mm caudal to Bregma). The RVLM and CVLM displayed significantly lower ( p < 0.01) densities of AT 1 binding, 278 ± 38 and 379 ± 64 fmol/g, respectively. However, the dissociation constants were significantly lower (i.e., higher affinity) in RVLM and CVLM (164 ± 38 and 178 ± 27 pM, respectively,) than in DMM (328 ± 12 pM, p < 0.01 and p < 0.05, respectively). These results provide an anatomical and pharmacological framework for future studies on the role in cardiovascular regulation of AT 1 receptors in the caudal brainstem.

P7.8 Role of cAMP dependent effectors and voltage gated calcium channels in the rat rostral ventrolateral medulla and in the spinal cord in the tonic and reflex control of arterial pressure in hypertension and normotension

It is clear that a large number of neurotransmitters and neuromodulators act at G-protein coupled receptors (GPCR) influencing the activity of presympathetic neurons in the rostral ventrolateral medulla (RVLM). The role of some of these GPCR play in the RVLM on the tonic and reflex control of arterial pressure has been investigated. For example, activation of muscarinic receptors increases arterial pressure and enhances the baroreceptor reflex whilst attenuating and inhibiting the chemoreceptor and somatosympathetic reflexes respectively [1] whereas activation of somatostatin receptors evokes profound hypotension and attenuates the somatosympathetic and chemoreceptor reflexes but does not alter sympathetic baroreflex function [2].

Sex differences in the subcellular distribution of angiotensin type 1 receptors and NADPH oxidase subunits in the dendrites of C1 neurons in the rat rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM), a region critical for the tonic and reflex control of arterial pressure, contains a group of adrenergic (C1) neurons that project to the spinal cord and directly modulate pre-ganglionic sympathetic neurons. Epidemiological data suggest that there are gender differences in the regulation of blood pressure. One factor that could be involved is angiotensin II signaling and the associated production of reactive oxygen species (ROS) by NADPH oxidase, which is emerging as an important molecular substrate for central autonomic regulation and dysregulation. In this study dual electron microscopic immunolabeling was used to examine the subcellular distribution of the angiotensin type 1 (AT1) receptor and two NADPH oxidase subunits (p47 and p22) in C1 dendritic processes, in tissue from male, proestrus (high estrogen) and diestrus (low estrogen) female rats. Female dendrites displayed significantly more AT1 labeling and significantly less p47 labeling than males. While elevations in AT1 labeling primarily resulted from higher levels of receptor on the plasma membrane, p47 labeling was reduced both on the plasma membrane and in the cytoplasm. Across the estrous cycle, proestrus females displayed significantly higher levels of AT1 labeling than diestrus females, which resulted exclusively from plasma membrane density differences. In contrast, p47 labeling did not change across the estrous cycle, indicating that ROS production might reflect AT1 receptor membrane density. No significant differences in p22 labeling were observed. These findings demonstrate that both sex and hormonal levels can selectively affect the expression and subcellular distribution of components of the angiotensin II signaling pathway within C1 RVLM neurons. Such effects could reflect differences in the capacity for ROS production, potentially influencing short term excitability and long term gene expression in a cell group which is critically involved in blood pressure regulation, potentially contributing to gender differences in the risk of cardiovascular disease.

Apelin Gene Transfer Into the Rostral Ventrolateral Medulla Induces Chronic Blood Pressure Elevation in Normotensive Rats

The peripheral apelin system plays a significant role in cardiovascular homeostasis and in the pathophysiology of cardiovascular diseases. However, the central effect of this neurohormonal system in neural control of cardiovascular function remains poorly understood. Thus, this study was undertaken to evaluate the effect of apelin in the rostral ventrolateral medulla (RVLM) on blood pressure, cardiac function, and sympathetic nerve activity. Apelin mRNA and protein levels were detected with real-time RT-PCR and Western blots, respectively. Expression of apelin was significantly enhanced in the RVLM of spontaneously hypertensive rat (SHR) compared with normotensive Wistar-Kyoto (WKY) rats. To study the functional consequence of upregulated apelin expression, apelin was overexpressed by bilateral microinjection of the AAV2-apelin viral vector into the RVLM of WKY rats. Immunofluorescence staining and Western blots demonstrated that microinjection of AAV2-apelin into the RVLM resulted in a significant increase in apelin expression, which was associated with a chronic elevation in blood pressure and cardiac hypertrophy. In addition, direct microinjection of exogenous apelin-13 (200 pmol in 50 nL) into the RVLM caused a 20 mm Hg elevation in blood pressure and a 24% increase in sympathetic nerve activity. The present study is the first to show that apelin expression is enhanced in the RVLM of SHR versus WKY rats and that overexpression of this gene in the RVLM results in chronic blood pressure elevation and cardiac hypertrophy in normotensive rats. Thus, the apelin system in the RVLM may play a very important role in central blood pressure regulation and in the pathogenesis of hypertension.

5α‐reductase mRNA is increased in the rostral ventrolateral medulla (RVLM) of term pregnant rats

In term pregnant rats, baseline GABAergic inhibition of the RVLM is increased, while baseline GABAergic inhibition of the hypothalamic paraventricular nucleus (PVN) is decreased. The major metabolite of progesterone, 3α‐hydroxy‐dihydroprogesterone (3α‐OH‐DHP), is a potent endogenous positive modulator of GABAA receptors and CNS levels of this neurosteroid are increased in pregnancy. Local regulation of the enzymes responsible for formation of 3α‐OH‐DHP, 5α‐reductase (5α‐RDA) and 3α‐hydroxysteroid dehydrogenase (3α‐HSD), is one factor which determines brain region specific actions of 3α‐OH‐DHP. Expression of mRNA for 5α‐RDA and 3α‐HSD (reference gene = MAPK) was determined in tissue punches of the RVLM and PVN from nonpregnant (NP) and term pregnant (P) rats. In the PVN, pregnancy did not change relative expression of 5α‐RDA [NP(7)= 1.0 ±0.23; P(7)= 0.64± 0.15] or 3α‐HSD [NP(5)= 1.0 ±0.38; P(6)= 0.84± 0.23]. In the RVLM, expression of 3α‐HSD [NP(5)= 1.0 ±0.54; P(6)= 0.67± 0.44] was not different between NP and P rats. However, there was a strong trend for increased expression of 5α‐RDA in P (5) [2.44± 0.89] compared to NP(5) [1.0 ±0.28] rats. Although preliminary, these data suggest that increased expression of 5α‐RDA in the RVLM of pregnant rats, could result in increased formation of the major metabolite of progesterone and potential positive modulation of GABAA receptors. (NIH HL 36245)

Paraventricular nucleus (PVN) neurons projecting to the rostral ventrolateral medulla (RVLM) contain both oxytocin and glutamate

The PVN is crucial to the CNS regulation of sympathetic outflow. Glutamate is the prevalent excitatory neurotransmitter in the CNS, including the PVN. Oxytocin (OT) is specifically produced by PVN and supraoptic nucleus neurons, and microinjection of OT into the RVLM induces a pressor response. The current study is aimed at determining whether glutamate PVN neurons projecting to the RVLM contain OT as a co‐transmitter. Real time RT‐PCR analyses of PVN tissues from 4‐6 week‐old Sprague‐Dawley (SD) rats revealed the presence of OT and phosphate‐activated glutaminase (PAG; specific marker of glutamate neurons) mRNAs. Microinjection of AAV2‐PAG‐eGFP (200 nL; 1.2 x 1012 gc/mL) bilaterally into PVN of 5 week‐old SD rats produced glutamate neuron‐specific expression of GFP within two weeks. Isolation and ex‐vivo culture of PVN cells from these rats produced GFP labeled neurons, and single‐cell PCR analysis of these revealed that 8 out of 13 (61.53%) were OT‐positive. To identify the phenotype of the GFP expressing glutamate neurons that project to RVLM, fluorescent retrobeads were microinjected bilaterally into the RVLM of SD rats that had received injections of AAV2‐PAG‐eGFP into the PVN. Immunocytochemistry indicated that GFP‐ and retrobead‐labeled PVN neurons contained immunoreactive OT. Thus we conclude that RVLM‐projecting PVN neurons contain both OT and glutamate as major excitatory neurotransmitters.