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.

Molkov YI, Zoccal DB, Moraes DJ, Paton JF, Machado BH, Rybak IA. Intermittent hypoxia-induced sensitization of central chemoreceptors contributes to sympathetic nerve activity during late expiration in rats. J Neurophysiol 105: 3080 -3091, 2011. First published April 6, 2011 doi:10.1152/jn.00070.2011.-Hypertension elicited by chronic intermittent hypoxia (CIH) is associated with elevated activity of the thoracic sympathetic nerve (tSN) that exhibits an enhanced respiratory modulation reflecting a strengthened interaction between respiratory and sympathetic networks within the brain stem. Expiration is a passive process except for special metabolic conditions such as hypercapnia, when it becomes active through phasic excitation of abdominal motor nerves (AbN) in late expiration. An increase in CO 2 evokes late-expiratory (late-E) discharges phase-locked to phrenic bursts with the frequency increasing quantally as hypercapnia increases. In rats exposed to CIH, the late-E discharges synchronized in AbN and tSN emerge in normocapnia. To elucidate the possible neural mechanisms underlying these phenomena, we extended our computational model of the brain stem respiratory network by incorporating a population of presympathetic neurons in the rostral ventrolateral medulla that received inputs from the pons, medullary respiratory compartments, and retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG). Our simulations proposed that CIH conditioning increases the CO 2 sensitivity of RTN/pFRG neurons, causing a reduction in both the CO 2 threshold for emerging the late-E activity in AbN and tSN and the hypocapnic threshold for apnea. Using the in situ rat preparation, we have confirmed that CIHconditioned rats under normal conditions exhibit synchronized late-E discharges in AbN and tSN similar to those observed in control rats during hypercapnia. Moreover, the hypocapnic threshold for apnea was significantly lowered in CIH-conditioned rats relative to that in control rats. We conclude that CIH may sensitize central chemoreception and that this significantly contributes to the neural impetus for generation of sympathetic activity and hypertension.

Key pointsr Hypoxia activates peripheral chemoreceptors producing an increase in breathing and arterial pressure.r In conditions of sustained hypoxia, an increase in ventilation and arterial blood pressure is observed that persists after the return to normoxia.r We show in rats that sustained hypoxia for 24 h produces glutamate-dependent changes in the activity of expiratory and sympathetic neurones of the rostral ventrolateral medulla, which are essential for the control of respiratory and sympathetic activities.r These neuronal changes induced by sustained hypoxia are critical for the emergence of coupled active expiration and augmented sympathetic activity.r These findings contribute to a better understanding of cardiorespiratory adjustments associated with sustained hypoxia in individuals experiencing high altitudes.Abstract Individuals experiencing sustained hypoxia (SH) exhibit adjustments in the respiratory and autonomic functions by neural mechanisms not yet elucidated. In the present study we evaluated the central mechanisms underpinning the SH-induced changes in the respiratory pattern and their impact on the sympathetic outflow. Using a decerebrated arterially perfused in situ preparation, we verified that juvenile rats exposed to SH (10% O 2 ) for 24 h presented an active expiratory pattern, with increased abdominal, hypoglossal and vagal activities during late-expiration (late-E). SH also enhanced the activity of augmenting-expiratory neurones and depressed the activity of post-inspiratory neurones of the Bötzinger complex (BötC) by mechanisms not related to changes in their intrinsic electrophysiological properties. SH rats exhibited high thoracic sympathetic activity and arterial pressure levels associated with an augmented firing frequency of pre-sympathetic neurones of the rostral ventrolateral medulla (RVLM) during the late-E phase. The antagonism of ionotropic glutamatergic receptors in the BötC/RVLM abolished the late-E bursts in expiratory and sympathetic outputs of SH rats, indicating that glutamatergic inputs to the BötC/RVLM are essential for the changes in the expiratory and sympathetic coupling observed in SH rats. We also observed that the usually silent late-E neurones of the retrotrapezoid nucleus/parafacial respiratory group became active in SH rats, suggesting that this neuronal population may provide the excitatory drive essential to the emergence of active expiration and sympathetic overactivity. We conclude that short-term SH induces the activation of medullary expiratory neurones, which affects the pattern of expiratory motor activity and its coupling with sympathetic activity.

H igh sympathetic activity is associated with hypertension 1-6 and many other diseases including heart failure, 7,8 insulin resistance, 9 and obesity. 10,11 Sympathetic nerve activity increases progressively as blood pressure rises from normal to moderate to severe hypertension in humans. 12 Interestingly, in both animal models of hypertension 13 and in patients with hypertension 3,6 elevated sympathetic activity precedes hypertension. The mechanisms driving excessive sympathetic activity in hypertension remain unresolved. Likely mechanisms contributing include elevated reflex afferent drive from both peripheral chemoreceptors 14,15 and kidney, 16,17 saltangiotensin II, 18-21 inflammation, [22][23][24] and respiration. 13,[25][26][27][28][29] Respiratory modulation of sympathetic activity or respiratory-sympathetic coupling has been observed in many sympathetic postganglionic outflows. 13,21,[28][29][30][31][32][33] Clinical studies have purported that by controlling breathing depth and rate (using RespeRate) blood pressure can be lowered 34 in patients with hypertension. 35 Recently, we found that in the spontaneously hypertensive (SH) rats, a genetic animal model of hypertension, 13 as well in rats submitted to chronic intermittent hypoxia, 27,29,36 there is augmented coupling of the central respiratory network to sympathetic circuits. This is expressed as elevations in sympathetic bursts in the late inspiratory (or beginning of postinspiratory; post-I) phases in the thoracic sympathetic nerve relative to age/sex-matched normotensive rats and is present before rats develop hypertension. 13 Simms et al 13 focused only on phrenic (central inspiratory) related modulation of sympathetic nerve activity and did not consider changes in expiratory neuronal activity at either the motor or neuronal level, so the central neural mechanisms for enhancing this coupling are unknown. In other models of hypertension (angiotensin II-salt hypertension), rostral ventrolateral medulla (RVLM) presympathetic neurons also show enhanced respiratory-related activity, 28 suggesting that this fundamental mechanism exists in both genetic and environmental induced hypertensive animal models. The unknown issue is whether enhanced respiratory-sympathetic coupling reflects either enhanced excitability of RVLM premotor sympathetic Abstract-A major aspect of hypertension is excessive sympathetic activity but the reasons for this remain elusive.Sympathetic tone is increased in the spontaneously hypertensive (SH) rat reflecting, in part, enhanced respiratorysympathetic coupling. We aimed to identify which respiratory cells might have altered properties. Using the working heart-brain stem preparation, we monitored simultaneously sympathetic and respiratory nerve activity in combination with intracellular recordings of physiologically characterized medullary presympathetic or respiratory neurons. In SH rats, respiratory modulation of both inspiratory and postinspiratory phases of sympathetic activity was larger relative to Wistar rats. An additiona...

Central mechanisms of coupling between respiratory and sympathetic systems are essential for the entrainment between the enhanced respiratory drive and sympathoexcitation in response to hypoxia. However, the brainstem nuclei and neuronal network involved in these respiratory-sympathetic interactions remain unclear. Here, we evaluated whether the increase in expiratory activity and expiratory-modulated sympathoexcitation produced by the peripheral chemoreflex activation involves the retrotrapezoid nucleus/parafacial respiratory region (RTN/pFRG). Using decerebrated arterially perfused in situ rat preparations (60-80 g), we recorded the activities of thoracic sympathetic (tSN), phrenic (PN), and abdominal nerves (AbN) as well as the extracellular activity of RTN/pFRG expiratory neurons, and reflex responses to chemoreflex activation were evaluated before and after inactivation of the RTN/pFRG region with muscimol (1 mM). In the RTN/pFRG, we identified late-expiratory (late-E) neurons (n = 5) that were silent at resting but fired coincidently with the emergence of late-E bursts in AbN after peripheral chemoreceptor activation. Bilateral muscimol microinjections into the RTN/pFRG region (n = 6) significantly reduced basal PN frequency, mean AbN activity, and the amplitude of respiratory modulation of tSN (P < 0.05). With respect to peripheral chemoreflex responses, muscimol microinjections in the RTN/pFRG enhanced the PN inspiratory response, abolished the evoked late-E activity of AbN, but did not alter either the magnitude or pattern of the tSN reflex response. These findings indicate that the RTN/pFRG region is critically involved in the processing of the active expiratory response but not of the expiratory-modulated sympathetic response to peripheral chemoreflex activation of rat in situ preparations.

Moraes DJ, Zoccal DB, Machado BH. Sympathoexcitation during chemoreflex active expiration is mediated by L-glutamate in the RVLM/Bötzinger complex of rats. J Neurophysiol 108: 610 -623, 2012. First published April 25, 2012 doi:10.1152/jn.00057.2012The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/Bötzinger/pre-Bötzinger complexes (RVLM/ BötC/pre-BötC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro-and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/BötC/preBötC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/BötC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 Ϯ 0.02 vs. 1 Ϯ 0.08 Hz; P Ͻ 0.05; n ϭ 18) and hypoglossal (HN) inspiratory response (41 Ϯ 2 vs. 82 Ϯ 2%; P Ͻ 0.05; n ϭ 8), but decreased postinspiratory (35 Ϯ 3 vs. 12 Ϯ 2%; P Ͻ 0.05) and late-expiratory (24 Ϯ 4 vs. 2 Ϯ1%; P Ͻ 0.05; n ϭ 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 Ϯ 6 vs. 40 Ϯ 2%; P Ͻ 0.05; n ϭ 5) and expiratory component of sympathoexcitatory (77 Ϯ 8 vs. 26 Ϯ 5%; P Ͻ 0.05; n ϭ 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/BötC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-BötC; n ϭ 16) abolished inspiratory responses [PN (n ϭ 16) and HN (n ϭ 6)], and no changes in magnitude of sympathoexcitatory (n ϭ 16) and expiratory (AbN and cVN; n ϭ 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/BötC and RVLM/preBötC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/BötC is essential to the coupling of active expiration and sympathoexcitatory response.

Humans ascending to high altitudes are submitted to sustained hypoxia (SH), activating peripheral chemoreflex with several autonomic and respiratory responses. Here we analyzed the effect of short-term SH (24 h, FIO 2 10%) on the processing of cardiovascular and respiratory reflexes using an in situ preparation of rats. SH increased both the sympatho-inhibitory and bradycardiac components of baroreflex and the sympathetic and respiratory responses of peripheral chemoreflex. Electrophysiological properties and synaptic transmission in the nucleus tractus solitarius (NTS) neurons, the first synaptic station of afferents of baroreflexes and chemoreflexes, were evaluated using brainstem slices and whole-cell patch-clamp. The second-order NTS neurons were identified by previous application of fluorescent tracer onto carotid body for chemoreceptor afferents or onto aortic depressor nerve for baroreceptor afferents. SH increased the intrinsic excitability of NTS neurons. Delayed excitation, caused by A-type potassium current (IK A ), was observed in most of NTS neurons from control rats. The IK A amplitude was higher in identified second-order NTS neurons from control than in SH rats. SH also blunted the astrocytic inhibition of IK A in NTS neurons and increased the synaptic transmission in response to afferent fibers stimulation. The frequency of spontaneous excitatory currents was also increased in neurons from SH rats, indicating that SH increased the neurotransmission by presynaptic mechanisms. Therefore, short-term SH changed the glia-neuron interaction, increasing the excitability and excitatory transmission of NTS neurons, which may contribute to the observed increase in the reflex sensitivity of baroreflex and chemoreflex in in situ preparation.

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