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We sought to investigate whether RVLM iNOS activity and oxidative profile may participate in the reduction of sympathetic responsiveness in swimming trained normotensive rats. Sedentary (S) and swimming trained (T) Wistar male rats chronically instrumented with an arterial catheter and guide cannula into the RVLM were submitted to continuous pressure and heart rate (HR) recordings and determination of autonomic control (power spectral analysis) before and after unilateral RVLM iNOS inhibition (aminoguanidine, 250 pmol/100 nL). Other S and T rats received local l-glutamate microinjection (5 nmol/100 nL). In separate S and T groups not submitted to brainstem cannulation, fresh bilateral RVLM punchs were collected for iNOS gene expression (qPCR); reduced glutathione and lipid peroxidation quantification (spectrophotometry); iron-reducing antioxidant (FRAP) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS˙+) scavenger assays. iNOS gene expression was confirmed in fixed RVLM slices (immunofluorescence). T rats exhibited resting bradycardia, lower sympathovagal balance, reduced RVLM iNOS gene/protein expression and higher antioxidant capacity. Decreased iNOS expression was positively correlated with reduced HR. Pressor and tachycardic response to l-Glutamate were smaller in T rats. Aminoguanidine microinjection reduced sympathetic activity in S rats but did not change it in T rats expressing reduced RVLM iNOS content. Our data indicate that iNOS, expressed in the RVLM of normotensive male rats, has tonic effects on sympathetic activity and that swimming training is an efficient tool to reduce iNOS expression and augment the antioxidant defense, thus reducing glutamatergic responsiveness and sympathetic drive to cardiovascular effectors.

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Optogenetic stimulation of the adrenergic C1 neurons produces cardiorespiratory activation, and selective depletion of these cells attenuates breathing responses induced by hypoxia. The preBötzinger complex (preBötC) is a group of neurons located in the intermediate aspect of the ventrolateral medulla, critical for respiratory rhythmogenesis, and is modulated by glutamate and catecholamines. Our hypothesis is that selective activation of C1 neurons leads to breathing responses by excitatory connections with the preBötC neurons. Anatomical connection between C1 cells and preBötC was evaluated using retrograde (Cholera Toxin b; preBötC) and anterograde (LVV-PRSx8-ChR2-eYFP; C1 region) tracers. LVV-PRSx8-ChR2-eYFP (viral vector that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuron-preferring promoter (PRSx8) was also injected into the C1 region of male Wistar rats for the functional experiments. Anatomical results demonstrated that preBötC neurons receive projections from C1 cells, and these projections express tyrosine hydroxylase and vesicular glutamate transporter 2. Functional connection between C1 cells and preBötC was evaluated by photostimulation of ChR2-transduced C1 neurons before and after unilateral injection of the ionotropic glutamate antagonist, kynurenic acid (kyn), or cocktail of adrenergic antagonists in the preBötC. Kyn injection into preBötC blocked the increase in DiaEMG frequency without changing the MAP increase elicited by photostimulation of C1 neurons, while the injection of adrenergic antagonists into the preBötC did not change DiaEMG frequency and MAP increase induced by photostimulation of C1 cells. Our results suggest that the increase in breathing produced by photostimulation of C1 neurons can be caused by a direct glutamatergic activation of preBötC neurons.

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Chronic intermittent hypoxia (CIH) increases basal sympathetic nervous system activity, augments chemoreflex-induced sympathoexcitation, and raises blood pressure. All effects are attenuated by systemic or intracerebroventricular administration of angiotensin II type 1 receptor (AT1R) antagonists. This study aimed to quantify the effects of CIH on AT1R- and AT2R-like immunoreactivity in the rostroventrolateral medulla (RVLM) and paraventricular nucleus of the hypothalamus (PVN), central regions that are important components of the extended chemoreflex pathway. Eighteen Sprague-Dawley rats were exposed to intermittent hypoxia (FIO2 = 0.10, 1 min at 4-min intervals) for 10 hr/day for 1, 5, 10, or 21 days. After exposure, rats were deeply anesthetized and transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were removed and sectioned coronally into 50 µm slices. Immunohistochemistry was used to quantify AT1R and AT2R in the RVLM and the PVN. In the RVLM, CIH significantly increased the AT1R-like immunoreactivity, but did not alter AT2R immunoreactivity, thereby augmenting the AT1R:AT2R ratio in this nucleus. In the PVN, CIH had no effect on immunoreactivity of either receptor subtype. The current findings provide mechanistic insight into increased basal sympathetic outflow, enhanced chemoreflex sensitivity, and blood pressure elevation observed in rodents exposed to CIH.

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Adrenaline is an important counter-regulatory hormone that helps restore glucose homeostasis during hypoglycaemia. However, the neurocircuitry that connects the brain glucose sensors and the adrenal sympathetic outflow to the chromaffin cells is poorly understood. We used electrical microstimulation of the perifornical hypothalamus (PeH) and the rostral ventrolateral medulla (RVLM) combined with adrenal sympathetic nerve activity (ASNA) recording to examine the relationship between the RVLM, the PeH and ASNA. In urethane-anaesthetised male Sprague-Dawley rats, intermittent single pulse electrical stimulation of the rostroventrolateral medulla (RVLM) elicited an evoked ASNA response that consisted of early (60±3ms) and late peaks (135±4ms) of preganglionic and postganglionic activity. In contrast, RVLM stimulation evoked responses in lumbar sympathetic nerve activity that were almost entirely postganglionic. PeH stimulation also produced an evoked excitatory response consisting of both preganglionic and postganglionic excitatory peaks in ASNA. Both peaks in ASNA following RVLM stimulation were reduced by intrathecal kynurenic acid (KYN) injection. In addition, the ASNA response to systemic neuroglucoprivation induced by 2-deoxy-d-glucose was abolished by bilateral microinjection of KYN into the RVLM. This suggests that a glutamatergic pathway from the perifornical hypothalamus (PeH) relays in the RVLM to activate the adrenal SPN and so modulate ASNA. The main findings of this study are that (i) adrenal premotor neurons in the RVLM may be, at least in part, glutamatergic and (ii) that the input to these neurons that is activated during neuroglucoprivation is also glutamatergic.

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BACKGROUND: It is well established that sympathetic nervous system is responsible for the onset, development and maintenance of neurogenic hypertension. The rostroventrolateral medulla (RVLM) and medullo-cervical pressor area (MCPA) are important central sympathoexcitatory regions whose role on neurogenic hypertension remains unknown. OBJECTIVE: To establish RVLM and MCPA roles in the long-term regulation of blood pressure by depressing their neuron activity through the over-expression of hKir2.1-potassium channel in conscious spontaneously hypertensive rats (SHR). METHODS: In SHR, a lentiviral vector LVV-hKir2.1 was microinjected into RVLM or MCPA areas. A sham group was injected with LVV-eGFP. Blood pressure (BP) and heart rate (HR) were continuously monitored for 75 days. Baroreflex and chemoreflex functions were evaluated. Baroreflex gain, chemoreflex sensitivity, BP and HR variability were calculated. RESULTS: LVV-hKir2.1 expression in RVLM, but not in MCPA, produced a significant time-dependent decrease in systolic, diastolic, mean-BP and LF of systolic BP at 60-days post-injection. No significant changes were seen in LVV-eGFP RVLM injected SHR. CONCLUSION: Data show that chronic expression of Kir2.1 in the RVLM of conscious SHR caused a marked and sustained decrease in BP without changes in the baro- and peripheral chemoreceptor reflex evoked responses. This decrease was mostly due to a reduction in sympathetic output revealed indirectly by a decrease in the power density of the SBP-LF band. Our data are amongst the firsts to demonstrate the role of the RVLM in maintaining BP levels in hypertension in conscious SHR. We suggest that a decrease in RVLM neuronal activity is an effective anti-hypertensive treatment strategy.

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Pseudorabies virus (PRV)-614 was injected into the tracheal wall of male MC4R-GFP transgenic mice, resulting in retrograde infections in spinal cord and autonomic premotor areas of the brain including the rostroventrolateral medulla (RVLM). This polysynaptic pathway to the airway may form the substrate underlying the impact of IML and RVLM on airway function. The neurochemical phenotype of MC4R-GFP-positive neurons was identified using fluorescence immunocytochemical labeling. PRV-614/MC4R-GFP dual labeled neurons were detected in spinal IML and the rostral ventrolateral medulla (RVLM). These data demonstrate the RVLM-IML pathway of synaptically connected neurons extending to the airway through melanocortinergic-sympathetic signaling.

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The parasympathetic control of heart rate arises from premotor cardiac vagal neurons (CVNs) located in the nucleus ambiguus (NA). Previous microinjection studies in NA show that dopamine evokes a decrease in heart rate, but the underlying mechanisms responsible for these responses were not identified. This study tested whether dopamine modulates inhibitory GABAergic and glycinergic and/or excitatory glutamatergic neurotransmission to CVNs. Retrogradely labeled CVNs were identified in an in vitro rat brainstem slice preparation and synaptic events were recorded using whole cell voltage clamp techniques. Bath application of dopamine (100 µM) had no effect on excitatory synaptic events, but reversibly inhibited the frequency (but not amplitude) of GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs. Similarly, dopamine (10 µM and 100 µM) inhibited glycinergic IPSC frequency by ~50% and 70% respectively. The reduction in inhibitory neurotransmission to CVNs by dopamine was prevented by the sodium channel blocker TTX (1µM) indicating that the dopamine mediated effects were action potential dependent. Dopamine evoked responses were mimicked by the D2-like receptor agonist, Quinpirole but not D1-like receptor agonist, SKF 38393. In addition, the dopamine mediated depression of inhibitory synaptic responses were prevented by the D2-like receptor antagonist sulpiride, but not by D1-like or adrenergic or serotonergic receptor antagonists, suggesting that these responses were D2-like receptor mediated and not D1-like or adrenergic or 5-HT receptor mediated. These data suggest that dopamine acts via dis-inhibition, and diminishes inhibitory GABAergic and glycinergic neurotransmission to CVNs, which would be predicted to increase parasympathetic activity to the heart and evoke a bradycardia.

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The mammalian diving response is a powerful autonomic adjustment to underwater submersion greatly affecting heart rate, arterial blood pressure, and ventilation. The bradycardia is mediated by the parasympathetic nervous system, arterial blood pressure is mediated via the sympathetic system and still other circuits mediate the respiratory changes. In the present study we investigate the cardiorespiratory responses and the brainstem neurons activated by voluntary diving of trained rats, and, compare them to control and swimming animals which did not dive. We show that the bradycardia and increase in arterial blood pressure induced by diving were significantly different than that induced by swimming. Neuronal activation was calculated after immunohistochemical processing of brainstem sections for Fos protein. Labeled neurons were counted in the caudal pressor area, the medullary dorsal horn, subnuclei of the nucleus tractus solitarii (NTS), the nucleus raphe pallidus (RPa), the rostroventrolateral medulla, the A5 area, the nucleus locus coeruleus, the Kölliker-Fuse area, and the external lateral and superior lateral subnuclei of the parabrachial nucleus. All these areas showed significant increases in Fos labeling when data from voluntary diving rats were compared to control rats and all but the commissural subnucleus of the NTS, A5 area, and RPa were significantly different from swimming rats. These data provide a substrate for more precise experiments to determine the role of these nuclei in the reflex circuits driving the diving response.

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The autonomic nervous system plays a key role in maintaining homeostasis under normal and pathological conditions. The sympathetic tone, particularly for the cardiovascular system, is generated by sympathetic discharges originating in specific areas of the brainstem. Aerobic exercise training promotes several cardiovascular adjustments that are influenced by the central areas involved in the output of the autonomic nervous system. In this review, we emphasize the studies that investigate aerobic exercise training protocols to identify the cardiovascular adaptations that may be the result of central nervous system plasticity due to chronic exercise. The focus of our study is on some groups of neurons involved in sympathetic regulation. They include the nucleus tractus solitarii, caudal ventrolateral medulla and the rostral ventrolateral medulla that maintain and regulate the cardiac and vascular autonomic tonus. We also discuss studies that demonstrate the involvement of supramedullary areas in exercise training modulation, with emphasis on the paraventricular nucleus of the hypothalamus, an important area of integration for autonomic and neuroendocrine responses. The results of these studies suggest that the beneficial effects of physical activity may be due, at least in part, to reductions in sympathetic nervous system activity. Conversely, with the recent association of physical inactivity with chronic disease, these data may also suggest that increases in sympathetic nervous system activity contribute to the increased incidence of cardiovascular diseases associated with a sedentary lifestyle.

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