Electrophysiological properties and morphology of cardiac and pulmonary motoneurons within the dorsal motor nucleus of the vagus of rats.

The dorsal motor nucleus of the vagus (DMV) contains parasympathetic motoneurons that project to the heart and lungs. These motoneurons control ventricular excitability/contractility and airways secretions/blood flow, respectively. However, their electrophysiological properties, morphology and synaptic input activity remain unknown. One important ionic current described in DMV motoneurons controlling their electrophysiological behaviour is the A-type mediated by voltage-dependent K+ (Kv) channels. Thus, we compared the electrophysiological properties, synaptic activity, morphology, A-type current density, and single cell expression of Kv subunits, that contribute to macroscopic A-type currents, between DMV motoneurons projecting to either the heart or lungs of adult male rats. Using retrograde labelling, we visualized distinct DMV motoneurons projecting to the heart or lungs in acutely prepared medullary slices. Subsequently, whole cell recordings, morphological reconstruction and single motoneuron qRT-PCR studies were performed. DMV pulmonary motoneurons were more depolarized, electrically excitable, presented higher membrane resistance, broader action potentials and received greater excitatory synaptic inputs compared to cardiac DMV motoneurons. These differences were in part due to highly branched dendritic complexity and lower magnitude of A-type K+ currents. By evaluating expression of channels that mediate A-type currents from single motoneurons, we demonstrated a lower level of Kv4.2 in pulmonary versus cardiac motoneurons, whereas Kv4.3 and Kv1.4 levels were similar. Thus, with the distinct electrical, morphological, and molecular properties of DMV cardiac and pulmonary motoneurons, we surmise that these cells offer a new vista of opportunities for genetic manipulation providing improvement of parasympathetic function in cardiorespiratory diseases such heart failure and asthma.

The sympathetic nervous system exacerbates carotid body sensitivity in hypertension.

AIMS: The carotid bodies (CBs) of spontaneously hypertensive (SH) rats exhibit hypertonicity and hyperreflexia contributing to heightened peripheral sympathetic outflow. We hypothesized that CB hyperexcitability is driven by its own sympathetic innervation. METHODS AND RESULTS: To test this, the chemoreflex was activated (NaCN 50-100 µL, 0.4 µg/µL) in SH and Wistar rats in situ before and after: (i) electrical stimulation (ES; 30 Hz, 2 ms, 10 V) of the superior cervical ganglion (SCG), which innervates the CB; (ii) unilateral resection of the SCG (SCGx); (iii) CB injections of an α1-adrenergic receptor agonist (phenylephrine, 50 µL, 1 mmol/L), and (iv) α1-adrenergic receptor antagonist prazosin (40 µL, 1 mmol/L) or tamsulosin (50 µL, 1 mmol/L). ES of the SCG enhanced CB-evoked sympathoexcitation by 40-50% (P < 0.05) with no difference between rat strains. Unilateral SCGx attenuated the CB-evoked sympathoexcitation in SH (62%; P < 0.01) but was without effect in Wistar rats; it also abolished the ongoing firing of chemoreceptive petrosal neurones of SH rats, which became hyperpolarized. In Wistar rats, CB injections of phenylephrine enhanced CB-evoked sympathoexcitation (33%; P < 0.05), which was prevented by prazosin (26%; P < 0.05) in SH rats. Tamsulosin alone reproduced the effects of prazosin in SH rats and prevented the sensitizing effect of the SCG following ES. Within the CB, α1A- and α1B-adrenoreceptors were co-localized on both glomus cells and blood vessels. In conscious SH rats instrumented for recording blood pressure (BP), the CB-evoked pressor response was attenuated after SCGx, and systolic BP fell by 16 ± 4.85 mmHg. CONCLUSIONS: The sympathetic innervation of the CB is tonically activated and sensitizes the CB of SH but not Wistar rats. Furthermore, sensitization of CB-evoked reflex sympathoexcitation appears to be mediated by α1-adrenoceptors located either on the vasculature and/or glomus cells. The SCG is novel target for controlling CB pathophysiology in hypertension.

Peripheral chemoreflex activation induces expiratory but not inspiratory excitation of C1 pre-sympathetic neurones of rats.

AIMS: Stimulation of peripheral chemoreceptors, as during hypoxia, increases breathing and respiratory-related sympathetic bursting. Activation of catecholaminergic C1 neurones induces sympathoexcitation, while its ablation reduces the chemoreflex sympathoexcitatory response. However, no study has determined the respiratory phase(s) in which the pre-sympathetic C1 neurones are recruited by peripheral chemoreceptor and whether C1 neurone activation affects all phases of respiratory modulation of sympathetic activity. We addressed these unknowns by testing the hypothesis that peripheral chemoreceptor activation excites pre-sympathetic C1 neurones during inspiration and expiration. METHODS: Using the in situ preparation of rat, we made intracellular recordings from baroreceptive pre-sympathetic C1 neurones during peripheral chemoreflex stimulation. We optogenetically activated C1 neurones selectively and compared any respiratory-phase-related increases in sympathetic activity with that which occurs following stimulation of the peripheral chemoreflex. RESULTS: Activation of peripheral chemoreceptors using cytotoxic hypoxia (potassium cyanide) increased the firing frequency of C1 neurones and both the frequency and amplitude of their excitatory post-synaptic currents during the phase of expiration only. In contrast, optogenetic stimulation of C1 neurones activates inspiratory neurones, which secondarily inhibit expiratory neurones, but produced comparable increases in sympathetic activity across all phases of respiration. CONCLUSION: Our data reveal that the peripheral chemoreceptor-mediated expiratory-related sympathoexcitation is mediated through excitation of expiratory neurones antecedent to C1 pre-sympathetic neurones; these may be found in the Kölliker-Fuse nucleus. Despite peripheral chemoreceptor excitation of inspiratory neurones, these do not trigger C1 neurone-mediated increases in sympathetic activity. These studies provide compelling novel insights into the functional organization of respiratory-sympathetic neural networks.

Intrinsic and synaptic mechanisms controlling the expiratory activity of excitatory lateral parafacial neurones of rats.

Active expiration is essential for increasing pulmonary ventilation during high chemical drive (hypercapnia). The lateral parafacial (pFL ) region, which contains expiratory neurones, drives abdominal muscles during active expiration in response to hypercapnia. However, the electrophysiological properties and synaptic mechanisms determining the activity of pFL expiratory neurones, as well as the specific conditions for their emergence, are not fully understood. Using whole cell electrophysiology and single cell quantitative RT-PCR techniques, we describe the intrinsic electrophysiological properties, the phenotype and the respiratory-related synaptic inputs to the pFL expiratory neurones, as well as the mechanisms for the expression of their expiratory activity under conditions of hypercapnia-induced active expiration, using in situ preparations of juvenile rats. We also evaluated whether these neurones possess intrinsic CO2 /[H+ ] sensitivity and burst generating properties. GABAergic and glycinergic inhibition during inspiration and expiration suppressed the activity of glutamatergic pFL expiratory neurones in normocapnia. In hypercapnia, these neurones escape glycinergic inhibition and generate burst discharges at the end of expiration. Evidence for the contribution of post-inhibitory rebound, CaV 3.2 isoform of T-type Ca2+ channels and intracellular [Ca2+ ] is presented. Neither intrinsic bursting properties, mediated by persistent Na+ current, nor CO2 /[H+ ] sensitivity or expression of CO2 /[H+ ] sensitive ion channels/receptors (TASK or GPR4) were observed. On the other hand, hyperpolarisation-activated cyclic nucleotide-gated and twik-related K+ leak channels were recorded. Post-synaptic disinhibition and the intrinsic electrophysiological properties of glutamatergic neurones play important roles in the generation of the expiratory oscillations in the pFL region during hypercapnia in rats. KEY POINTS: Hypercapnia induces active expiration in rats and the recruitment of a specific population of expiratory neurones in the lateral parafacial (pFL ) region. Post-synaptic GABAergic and glycinergic inhibition both suppress the activity of glutamatergic pFL neurones during inspiratory and expiratory phases in normocapnia. Hypercapnia reduces glycinergic inhibition during expiration leading to burst generation by pFL neurones; evidence for a contribution of post-inhibitory rebound, voltage-gated Ca2+ channels and intracellular [Ca2+ ] is presented. pFL glutamatergic expiratory neurones are neither intrinsic burster neurones, nor CO2 /[H+ ] sensors, and do not express CO2 /[H+ ] sensitive ion channels or receptors. Post-synaptic disinhibition and the intrinsic electrophysiological properties of glutamatergic neurones both play important roles in the generation of the expiratory oscillations in the pFL region during hypercapnia in rats.

Heightened respiratory-parasympathetic coupling to airways in the spontaneously hypertensive rat.

KEY POINTS: Carotid body (CB) chemoreceptors are hyperactive in hypertension, and their acute activation produces bronchoconstriction. We show that the respiratory-modulated bronchiolar tone, pulmonary parasympathetic efferent activity, and the firing frequency and synaptic excitation of bronchoconstrictor motoneurones in the nucleus ambiguus were all enhanced in spontaneous hypertensive (SH) rats. In SH rats, CB denervation reduced the respiratory-related parasympathetic-mediated bronchoconstrictor tone to levels seen in normotensive rats. Chemoreflex evoked bronchoconstrictor tone was heightened in SH versus normotensive rats. The intrinsic electrophysiological properties and morphology of bronchoconstrictor motoneurones were similar across rat strains. The heightened respiratory modulation of parasympathetic-mediated bronchoconstrictor tone to the airways in SH rats is caused by afferent drive from the CBs. ABSTRACT: Much research has described heightened sympathetic activity in hypertension and diminished parasympathetic tone, especially to the heart. The carotid body (CB) chemoreceptors exhibit hyperreflexia and are hyperactive, providing excitatory drive to sympathetic networks in hypertension. Given that acute CB activation produces reflex evoked bronchoconstriction via activation of parasympathetic vagal efferents, we hypothesised that the parasympathetic bronchoconstrictor activity is enhanced in spontaneously hypertensive (SH) rats and that this is dependent on CB inputs. In situ preparations of Wistar and SH rats were used in which bronchiolar tone, the pulmonary branch of the vagus (pVN) and phrenic nerves were recorded simultaneously; whole cell patch clamp recordings of bronchoconstrictor vagal motoneurones were also made from the nucleus ambiguus. Bronchiolar tone, pVN and bronchoconstrictor motoneurones were respiratory modulated and this modulation was enhanced in SH rats. These differences were all eliminated after CB denervation. Stimulation of the CBs increased the phrenic frequency that caused a summation of the respiratory-related increases in pVN, resulting in the development of bronchoconstrictor tone. This tone was exaggerated in SH rats. The enhanced respiratory-parasympathetic coupling to airways in SH rats was not due to differences in the intrinsic electrophysiological properties of bronchoconstrictor motoneurones but reflected heightened pre-inspiratory- and inspiratory-related synaptic drive. In summary, in SH rats the phasic respiratory modulation of parasympathetic tone to the airways is elevated and the greater development of this bronchoconstrictor tone is caused by the heightened afferent drive originating from the CBs. Thus, targeting the CBs may prove effective for increasing lower airway patency.

Chronic intermittent hypoxia increases excitability and synaptic excitation of protrudor and retractor hypoglossal motoneurones.

KEY POINTS: Dysfunctions in the hypoglossal control of tongue extrinsic muscles are implicated in obstructive sleep apnoea (OSA) syndrome. Chronic intermittent hypoxia (CIH), an important feature of OSA syndrome, produces deleterious effects on the motor control of oropharyngeal resistance, but whether the hypoglossal motoneurones innervating the tongue extrinsic muscles are affected by CIH is unknown. We show that CIH enhanced the respiratory-related activity of rat hypoglossal nerve innervating the protrudor and retractor tongue extrinsic muscles. Intracellular recordings revealed increases in respiratory-related firing frequency and synaptic excitation of inspiratory protrudor and retractor hypoglossal motoneurones after CIH. CIH also increased their intrinsic excitability, depolarised resting membrane potential and reduced K+ -dominated leak conductance. CIH affected the breathing-related synaptic control and intrinsic electrophysiological properties of protrudor and retractor hypoglossal motoneurones to optimise the neural control of oropharyngeal function. ABSTRACT: Inspiratory-related tongue movements and oropharyngeal motor actions are controlled mainly by the protrudor and retractor extrinsic tongue muscles, which are innervated by the hypoglossal motoneurones. Chronic intermittent hypoxia (CIH), an important feature of obstructive sleep apnoea syndrome, produces detrimental effects on the contractile function of the tongue extrinsic muscles and the medullary inspiratory network of rodents. However, the impact of the CIH on the electrophysiological properties of protrudor and retractor hypoglossal motoneurones has not been described before. Using nerves and intracellular recordings in in situ preparation of rats (5 weeks old), we tested the hypothesis that CIH (FiO2 of 0.06, SaO2 74%, during 30-40 s, every 9 min, 8 h/day for 10 days) increases the intrinsic excitability of protrudor and retractor motoneurones from the hypoglossal motor nucleus of rats. Recordings of hypoglossal nerve, before its bifurcation to innervate the tongue protrudor and retractor muscles, revealed that CIH enhances its pre-inspiratory, simultaneously with the presence of active expiration, and inspiratory activities. These changes were mediated by increases in the respiratory-related firing frequency and synaptic excitation of inspiratory protrudor and retractor hypoglossal motoneurones. Besides, CIH increases their intrinsic excitability and depolarises resting membrane potential by reducing a K+ -dominated leak conductance. In conclusion, CIH enhances the respiratory-related neural control of oropharyngeal function of rats by increasing the synaptic excitation, intrinsic excitability, and reducing leak conductance in both protrudor and retractor hypoglossal motoneurones. We propose that these network and cellular changes are important to optimise the oropharyngeal resistance in conditions related to intermittent hypoxia.

Lactate does not activate the carotid body of Wistar rat.

The carotid body's glomus cells are the primary sensors of hypoxia in mammals. Previous studies suggested that the glomus cells' hypoxia sensitivity is mediated by lactate in mice. This molecule increases the intracellular [Ca2+] and induces exocytosis in glomus cells, activating the carotid sinus nerve (the axons of chemoreceptive petrosal neurons). On the other hand, how lactate affects the activity of carotid body of rats is still unknown. We hypothesized that lactate activates the carotid body of rats. In Wistar rats, we measured the changes in the electrical properties of isolated glomus cells and petrosal chemoreceptive neurons in in situ preparations in response to different concentrations of lactate. Superfusion of both physiological and supraphysiological concentrations of lactate did not affect the membrane conductance and potential of glomus cells. Moreover, lactate injected into the carotid body did not activate the anatomically and physiologically identified chemoreceptive petrosal neurons. We conclude that the carotid body of Wistar rats is not sensitive to lactate.

Active expiratory oscillator regulates nasofacial and oral motor activities in rats.

NEW FINDINGS: What is the central question of this study? Does the parafacial respiratory group (pFRG), which mediates active expiration, recruit nasofacial and oral motoneurons to coordinate motor activities that engage muscles controlling airways in rats during active expiration. What is the main finding and its importance? Hypercapnia/acidosis or pFRG activation evoked active expiration and stimulated the motoneurons and nerves responsible for the control of nasofacial and oral airways patency simultaneously. Bilateral pFRG inhibition abolished active expiration and the simultaneous nasofacial and oral motor activities induced by hypercapnia/acidosis. The pFRG is more than a rhythmic oscillator for expiratory pump muscles: it also coordinates nasofacial and oral motor commands that engage muscles controlling airways. ABSTRACT: Active expiration is mediated by an expiratory oscillator located in the parafacial respiratory group (pFRG). Active expiration requires more than contracting expiratory muscles as multiple cranial nerves are recruited to stabilize the naso- and oropharyngeal airways. We tested the hypothesis that activation of the pFRG recruits facial and trigeminal motoneurons to coordinate nasofacial and oral motor activities that engage muscles controlling airways in rats during active expiration. Using a combination of electrophysiological and pharmacological approaches, we identified brainstem circuits that phase-lock active expiration, nasofacial and oral motor outputs in an in situ preparation of rat. We found that either high chemical drive (hypercapnia/acidosis) or unilateral excitation (glutamate microinjection) of the pFRG evoked active expiration and stimulated motoneurons (facial and trigeminal) and motor nerves responsible for the control of nasofacial (buccal and zygomatic branches of the facial nerve) and oral (mylohyoid nerve) motor outputs simultaneously. Bilateral pharmacological inhibition (GABAergic and glycinergic receptor activation) of the pFRG abolished active expiration and the simultaneous nasofacial and oral motor activities induced by hypercapnia/acidosis. We conclude that the pFRG provides the excitatory drive to phase-lock rhythmic nasofacial and oral motor circuits during active expiration in rats. Therefore, the pFRG is more than a rhythmic oscillator for expiratory pump muscles: it also coordinates nasofacial and oral motor commands that engage muscles controlling airways in rats during active expiration.

Centrally acting adrenomedullin in the long-term potentiation of sympathetic vasoconstrictor activity induced by intermittent hypoxia in rats.

NEW FINDINGS: What is the central question of this study? Adrenomedullin in the rostral ventrolateral medulla (RVLM) increases sympathetic activity; given that adrenomedullin is released during hypoxia, what are the effects of its agonism and antagonism in the RVLM after chronic intermitent hypoxia (CIH) exposure? What is the main finding and its importance? CIH exposure sensitizes adrenomedullin-dependent mechanisms in the RVLM, supporting its role as a sympathoexcitatory neuromodulator. A novel mechanism was identified for the generation of sympathetic overdrive and hypertension associated with hypoxia, providing potential guidance on new therapeutic approaches for controlling sympathetic hyperactivity in diseases such as sleep apnoea and neurogenic hypertension. ABSTRACT: Adrenomedullin in the rostral ventrolateral medulla (RVLM) has been shown to increase sympathetic activity whereas the antagonism of its receptors inhibited this autonomic activity lowering blood pressure in conditions of hypertension. Given that hypoxia is a stimulant for releasing adrenomedullin, we hypothesized that the presence of this peptide in the RVLM associated with chronic intermittent hypoxia (CIH) would cause sympathetic overdrive. Juvenile male rats (50-55 g) submitted to CIH (6% oxygen every 9 min, 8 h day-1 for 10 days) were studied in an arterially perfused in situ preparation where sympathetic activity was recorded. In control rats (n = 6), exogenously applied adrenomedullin in the RVLM raised baseline sympathetic activity when combined with episodic activation of peripheral chemoreceptors (KCN 0.05%, 5 times every 5 min). This sympathoexcitatory response was markedly amplified in rats previously exposed to CIH (n = 6). The antagonism of adrenomedullin receptors in the RVLM caused a significant reduction in sympathetic activity in the CIH group (n = 7), but not in controls (n = 8). The transient reflex-evoked sympathoexcitatory response to peripheral chemoreceptor stimulation was not affected by either adrenomedullin or adrenomedullin receptor antagonism in the RVLM of control and CIH rats. Our findings indicate that CIH sensitizes the sympathoexcitatory networks within the RVLM to adrenomedullin, supporting its role as an excitatory neuromodulator when intermittent hypoxia is present. These data reveal novel state-dependent mechanistic insights into the generation of sympathetic overdrive and provide potential guidance on possible unique approaches for controlling sympathetic discharge in diseases such as sleep apnoea and neurogenic hypertension.

The Logic of Carotid Body Connectivity to the Brain.

The carotid body has emerged as a therapeutic target for cardio-respiratory-metabolic diseases. With the expansive functions of the chemoreflex, we sought mechanisms to explain differential control of individual responses. We purport a remarkable correlation between phenotype of a chemosensory unit (glomus cell-sensory afferent) with a distinct component of the reflex response. This logic could permit differential modulation of distinct chemoreflex responses, a strategy ideal for therapeutic exploitation.

Enhancement of excitatory transmission in NTS neurons projecting to ventral medulla of rats exposed to sustained hypoxia is blunted by minocycline.

KEY POINTS: Rats subjected to sustained hypoxia (SH) present increases in arterial pressure (AP) and in glutamatergic transmission in the nucleus tractus solitarius (NTS) neurons sending projections to ventrolateral medulla (VLM). Treatment with minocycline, a microglial inhibitor, attenuated the increase in AP in response to SH. The increase in the amplitude of glutamatergic postsynaptic currents in the NTS-VLM neurons, induced by postsynaptic mechanisms, was blunted by minocycline treatment. The number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS of minocycline-treated rats. The data show that microglial recruitment/proliferation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the observed increase in AP. ABSTRACT: Short-term sustained hypoxia (SH) produces significant autonomic and respiratory adjustments and triggers activation of microglia, the resident immune cells in the brain. SH also enhances glutamatergic neurotransmission in the NTS. Here we evaluated the role of microglial activation induced by SH on the cardiovascular changes and mainly on glutamatergic neurotransmission in NTS neurons sending projections to the ventrolateral medulla (NTS-VLM), using a microglia inhibitor (minocycline). Direct measurement of arterial pressure (AP) in freely moving rats showed that SH (24 h, fraction of inspired oxygen ( FI,O2 ) 0.1) in vehicle and minocycline (30 mg/kg i.p. for 3 days)-treated groups produced a significant increase in AP in relation to control groups under normoxic conditions, but this increase was significantly lower in minocycline-treated rats. Whole-cell patch-clamp recordings revealed that the active properties of the membrane were comparable among the groups. Nevertheless, the amplitudes of glutamatergic postsynaptic currents, evoked by tractus solitarius stimulation, were increased in NTS-VLM neurons of SH rats. Changes in asynchronous glutamatergic currents indicated that the observed increase in amplitude was due to postsynaptic mechanisms. These changes were blunted in the SH group previously treated with minocycline. Using immunofluorescence, we found that the number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS neurons of minocycline-treated rats. Our data support the concept that microglial activation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the increase in AP observed in this experimental model.

Hyperexcitability and plasticity induced by sustained hypoxia on rectus abdominis motoneurons.

KEY POINTS: Acute hypoxia induces active expiration in rectus abdominis (RA) muscles in conscious freely moving rats, although its overall contribution is smaller than in internal oblique (IO) muscles. Tonically active and silent RA motoneurons were identified in in vitro preparations of rat spinal cords. Sustained hypoxia (SH) increased the synaptic strength and induced morphological changes in tonically active RA motoneurons. Expiratory RA motoneurons were recorded in the in situ preparation and SH enhanced both the excitability and the synaptic transmission in those firing during the stage 2 expiration. The present study contributes to a better understanding of the mechanisms involved in SH recruitment of RA motoneurons to induce active expiration in rats. ABSTRACT: Rectus abdominis (RA) motoneurons translate the complex respiratory brainstem inputs into effective muscle contractions. Despite their fundamental role in respiration, their functional and morphological properties are not fully understood. In the present study, we investigated for the first time the contribution of RA muscle to active expiration and characterized RA motoneurons regarding their electrical, molecular and morphological profiles in control rats and in rats submitted to sustained hypoxia (SH), which induces chronic recruitment of abdominal muscles. Electromyographic experiments in conscious freely moving control rats and SH rats showed that RA contributes to active expiration induced by acute hypoxia, although its contribution is smaller than in internal oblique muscles. in vitro whole-cell patch clamp recordings from RA motoneurons revealed two populations of cells: tonically active and silent. SH induced hyperexcitability in the tonically active cells by changing their action potential properties, and EPSCs. Three-dimensional morphological reconstructions of these cells showed that SH increased the dendritic complexity, stimulated the appearance of dendrite spines, and increased the somatic area and volume. Physiologically identified RA motoneurons, firing in two distinct phases of expiration, were recorded in the brainstem-spinal cord in situ preparation of rats. SH increased the firing frequency and EPSCs of neurons firing during stage 2 expiration. Taken together, our results show that RA motoneurons reconfigure their biophysical properties, morphology and synaptic strength to produce an appropriate expiratory drive in response to SH in rats.

Locus Coeruleus as a vigilance centre for active inspiration and expiration in rats.

At rest, inspiration is an active process while expiration is passive. However, high chemical drive (hypercapnia or hypoxia) activates central and peripheral chemoreceptors triggering reflex increases in inspiration and active expiration. The Locus Coeruleus contains noradrenergic neurons (A6 neurons) that increase their firing frequency when exposed to hypercapnia and hypoxia. Using recently developed neuronal hyperpolarising technology in conscious rats, we tested the hypothesis that A6 neurons are a part of a vigilance centre for controlling breathing under high chemical drive and that this includes recruitment of active inspiration and expiration in readiness for flight or fight. Pharmacogenetic inhibition of A6 neurons was without effect on resting and on peripheral chemoreceptors-evoked inspiratory, expiratory and ventilatory responses. On the other hand, the number of sighs evoked by systemic hypoxia was reduced. In the absence of peripheral chemoreceptors, inhibition of A6 neurons during hypercapnia did not affect sighing, but reduced both the magnitude and incidence of active expiration, and the frequency and amplitude of inspiration. These changes reduced pulmonary ventilation. Our data indicated that A6 neurons exert a CO2-dependent modulation of expiratory drive. The data also demonstrate that A6 neurons contribute to the CO2-evoked increases in the inspiratory motor output and hypoxia-evoked sighing.

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.

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.

Corrigendum: Pacemaking Property of RVLM Presympathetic Neurons.

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

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.

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.

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.

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.