Melina P. da Silva

Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats

The respiratory pattern generator modulates the sympathetic outflow, the strength of which is enhanced by challenges produced by hypoxia. This coupling is due to the respiratory-modulated presympathetic neurons in the rostral ventrolateral medulla (RVLM), but the underlining electrophysiological mechanisms remain unclear. For a better understanding of the neural substrates responsible for generation of this respiratory-sympathetic coupling, we combined immunofluorescence, single cell qRT-pCR, and electrophysiological recordings of the RVLM presympathetic neurons in in situ preparations from normal rats and rats submitted to a metabolic challenge produced by chronic intermittent hypoxia (CIH). Our results show that the spinally projected cathecholaminergic C1 and non-C1 respiratory-modulated RVLM presympathetic neurons constitute a heterogeneous neuronal population regarding the intrinsic electrophysiological properties, respiratory synaptic inputs, and expression of ionic currents, albeit all neurons presented persistent sodium current-dependent intrinsic pacemaker properties after synaptic blockade. A specific subpopulation of non-C1 respiratory-modulated RVLM presympathetic neurons presented enhanced excitatory synaptic inputs from the respiratory network after CIH. This phenomenon may contribute to the increased sympathetic activity observed in CIH rats. We conclude that the different respiratory-modulated RVLM presympathetic neurons contribute to the central generation of respiratory-sympathetic coupling as part of a complex neuronal network, which in response to the challenges produced by CIH contribute to respiratory-related increase in the sympathetic activity.

Purinergic receptors in the carotid body as a new drug target for controlling hypertension

In view of the high proportion of individuals with resistance to antihypertensive medication and/or poor compliance or tolerance of this medication, new drugs to treat hypertension are urgently needed. Here we show that peripheral chemoreceptors generate aberrant signaling that contributes to high blood pressure in hypertension. We discovered that purinergic receptor P2X3 (P2rx3, also known as P2x3) mRNA expression is upregulated substantially in chemoreceptive petrosal sensory neurons in rats with hypertension. These neurons generate both tonic drive and hyperreflexia in hypertensive (but not normotensive) rats, and both phenomena are normalized by the blockade of P2X3 receptors. Antagonism of P2X3 receptors also reduces arterial pressure and basal sympathetic activity and normalizes carotid body hyperreflexia in conscious rats with hypertension; no effect was observed in rats without hypertension. We verified P2X3 receptor expression in human carotid bodies and observed hyperactivity of carotid bodies in individuals with hypertension. These data support the identification of the P2X3 receptor as a potential new target for the control of human hypertension.

Gaseous modulators in the control of the hypothalamic neurohypophyseal system.

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are gaseous molecules produced by the brain. Within the hypothalamus, gaseous molecules have been highlighted as autocrine and paracrine factors regulating endocrine function. Therefore, in the present review, we briefly discuss the main findings linking NO, CO, and H2S to the control of body fluid homeostasis at the hypothalamic level, with particular emphasis on the regulation of neurohypophyseal system output.

Respiratory Network Enhances the Sympathoinhibitory Component of Baroreflex of Rats Submitted to Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (CIH) produces respiratory-related sympathetic overactivity and hypertension in rats. In this study, we tested the hypothesis that the enhanced central respiratory modulation of sympathetic activity after CIH also decreases the sympathoinhibitory component of baroreflex of rats, which may contribute to the development of hypertension. Wistar rats were exposed to CIH or normoxia (control group) for 10 days. Phrenic nerve, thoracic sympathetic nerve, and neurons in the rostral ventrolateral medulla and caudal ventrolateral medulla were recorded in in situ preparations of rats. Baroreflex regulation of thoracic sympathetic nerve, rostral ventrolateral medulla, and caudal ventrolateral medulla neurons activities were evaluated in different phases of respiration in response to either aortic depressor nerve stimulation or pressure stimuli. CIH rats presented higher respiratory-related thoracic sympathetic nerve and rostral ventrolateral medulla presympathetic neurons activities at the end of expiration in relation to control rats, which are indexes of respiratory-related sympathetic overactivity. Baroreflex-evoked thoracic sympathetic nerve inhibition during expiration, but not during inspiration, was enhanced in CIH when compared with control rats. In addition, CIH selectively enhanced the expiratory-related baroreceptor inputs, probably through caudal ventrolateral medulla neurons, to the respiratory-modulated bulbospinal rostral ventrolateral medulla presympathetic neurons. These findings support the concept that the onset of hypertension, mediated by sympathetic overactivity, after 10 days of CIH is not secondary to a reduction in sympathoinhibitory component of baroreflex. Instead, it was observed an increase in the gain of sympathoinhibitory component in in situ preparations of rats, suggesting that changes in the respiratory-related sympathetic network after CIH also play a key role in preventing greater increase in arterial pressure.

Pacemaking Property of RVLM Presympathetic Neurons.

Despite several studies describing the electrophysiological properties of RVLM presympathetic neurons, there is no consensus in the literature about their pacemaking property, mainly due to different experimental approaches used for recordings of neuronal intrinsic properties. In this review we are presenting a historical retrospective about the pioneering studies and their controversies on the intrinsic electrophysiological property of auto-depolarization of these cells in conjunction with recent studies from our laboratory documenting that RVLM presympathetic neurons present pacemaking capacity. We also discuss whether increased sympathetic activity observed in animal models of neurogenic hypertension (CIH and SHR) are dependent on changes in the intrinsic electrophysiological properties of these cells or due to changes in modulatory inputs from neurons of the respiratory network. We also highlight the key role of INaP as the major current contributing to the pacemaking property of RVLM presympathetic neurons.

Role of ventral medullary catecholaminergic neurons for respiratory modulation of sympathetic outflow in rats

Sympathetic activity displays rhythmic oscillations generated by brainstem inspiratory and expiratory neurons. Amplification of these rhythmic respiratory-related oscillations is observed in rats under enhanced central respiratory drive or during development of neurogenic hypertension. Herein, we evaluated the involvement of ventral medullary sympatho-excitatory catecholaminergic C1 neurons, using inhibitory Drosophila allatostatin receptors, for the enhanced expiratory-related oscillations in sympathetic activity in rats submitted to chronic intermittent hypoxia (CIH) and following activation of both peripheral (hypoxia) and central chemoreceptors (hypercapnia). Pharmacogenetic inhibition of C1 neurons bilaterally resulted in reductions of their firing frequency and amplitude of inspiratory-related sympathetic activity in rats in normocapnia, hypercapnia or after CIH. In contrast, hypercapnia or hypoxia-induced enhanced expiratory-related sympathetic oscillations were unaffected by C1 neuronal inhibition. Inhibition of C1 neurons also resulted in a significant fall in arterial pressure and heart rate that was similar in magnitude between normotensive and CIH hypertensive rats, but basal arterial pressure in CIH rats remained higher compared to controls. C1 neurons play a key role in regulating inspiratory modulation of sympathetic activity and arterial pressure in both normotensive and CIH hypertensive rats, but they are not involved in the enhanced late-expiratory-related sympathetic activity triggered by activation of peripheral or central chemoreceptors.

Nitric Oxide Modulates HCN Channels in Magnocellular Neurons of the Supraoptic Nucleus of Rats by an S-Nitrosylation-Dependent Mechanism

The control of the excitability in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus has been attributed mainly to synaptic inputs from circunventricular organs. However, nitric oxide (NO), a gaseous messenger produced in this nucleus during isotonic and short-term hypertonic conditions, is an example of a modulator that can act directly on MNCs to modulate their firing rate. NO inhibits the electrical excitability of MNCs, leading to a decrease in the release of vasopressin and oxytocin. Although the effects of NO on MNCs are well established, the mechanism by which this gas produces its effect is, so far, unknown. Because NO acts independently of synaptic inputs, we hypothesized that ion channels present in MNCs are the targets of NO. To investigate this hypothesis, we used the patch-clamp technique in vitro and in situ to measure currents carried by hyperpolarization-activated and nucleotide-gated cation (HCN) channels and establish their role in determining the electrical excitability of MNCs in rats. Our results show that blockade of HCN channels by ZD7288 decreases MNC firing rate with significant consequences on the release of OT and VP, measured by radioimmunoassay. NO induced a significant reduction in HCN currents by binding to cysteine residues and forming S-nitrosothiol complexes. These findings shed new light on the mechanisms that control the electrical excitability of MNCs via the nitrergic system and strengthen the importance of HCN channels in the control of hydroelectrolyte homeostasis. SIGNIFICANCE STATEMENT Cells in our organism live in a liquid environment whose composition and osmolality are maintained within tight limits. Magnocellular neurons (MNCs) of the supra optic nucleus can sense osmolality and control the synthesis and secretion of vasopressin (VP) and oxytocin (OT) by the neurohypophysis. OT and VP act on the kidneys controlling the excretion of water and sodium to maintain homeostasis. Here we combined electrophysiology, molecular biology, and radioimmunoassay to show that the electrical activity of MNCs can be controlled by nitric oxide (NO), a gaseous messenger. NO reacts with cysteine residues (S-nitrosylation) on hyperpolarization-activated and nucleotide-gated cation channels decreasing the firing rate of MNCs and the consequent secretion of VP and OT.

Purinergic plasticity within petrosal neurons in hypertension

The carotid bodies are peripheral chemoreceptors and contribute to the homeostatic maintenance of arterial levels of O2, CO2, and [H+]. They have attracted much clinical interest recently because of the realization that aberrant signaling in these organs is associated with several pathologies including hypertension. Herein, we describe data suggesting that sympathetic overactivity in neurogenic hypertension is, at least in part, dependent on carotid body tonicity and hyperreflexia that is related to changes in the electrophysiological properties of chemoreceptive petrosal neurons. We present results showing critical roles for both ATP levels in the carotid bodies and expression of P2X3 receptors in petrosal chemoreceptive, but not baroreceptive, terminals in the etiology of carotid body tonicity and hyperreflexia. We discuss mechanisms that may underlie the changes in electrophysiological properties and P2X3 receptor expression in chemoreceptive petrosal neurons, as well as factors affecting ATP release by cells within the carotid bodies. Our findings support the notion of targeting the carotid bodies to reduce sympathetic outflow and arterial pressure, emphasizing the potential clinical importance of modulating purinergic transmission to treat pathologies associated with carotid body dysfunction but, importantly, sparing physiological chemoreflex function.

Corrigendum: Pacemaking Property of RVLM Presympathetic Neurons.

[This corrects the article on p. 424 in vol. 7, PMID: 27713705.].