Interdependent feedback regulation of breathing by the carotid bodies and the retrotrapezoid nucleus.

The retrotrapezoid nucleus (RTN) regulates breathing in a CO2 - and state-dependent manner. RTN neurons are glutamatergic and innervate principally the respiratory pattern generator; they regulate multiple aspects of breathing, including active expiration, and maintain breathing automaticity during non-REM sleep. RTN neurons encode arterial PCO2 /pH via cell-autonomous and paracrine mechanisms, and via input from other CO2 -responsive neurons. In short, RTN neurons are a pivotal structure for breathing automaticity and arterial PCO2 homeostasis. The carotid bodies stimulate the respiratory pattern generator directly and indirectly by activating RTN via a neuronal projection originating within the solitary tract nucleus. The indirect pathway operates under normo- or hypercapnic conditions; under respiratory alkalosis (e.g. hypoxia) RTN neurons are silent and the excitatory input from the carotid bodies is suppressed. Also, silencing RTN neurons optogenetically quickly triggers a compensatory increase in carotid body activity. Thus, in conscious mammals, breathing is subject to a dual and interdependent feedback regulation by chemoreceptors. Depending on the circumstance, the activity of the carotid bodies and that of RTN vary in the same or the opposite directions, producing additive or countervailing effects on breathing. These interactions are mediated either via changes in blood gases or by brainstem neuronal connections, but their ultimate effect is invariably to minimize arterial PCO2 fluctuations. We discuss the potential relevance of this dual chemoreceptor feedback to cardiorespiratory abnormalities present in diseases in which the carotid bodies are hyperactive at rest, e.g. essential hypertension, obstructive sleep apnoea and heart failure.

Is plasticity within the retrotrapezoid nucleus responsible for the recovery of the PCO2 set-point after carotid body denervation in rats?

KEY POINTS: Arterial PCO2 is kept constant via breathing adjustments elicited, at least partly, by central chemoreceptors (CCRs) and the carotid bodies (CBs). The CBs may be active in a normal oxygen environment because their removal reduces breathing. Thereafter, breathing slowly returns to normal. In the present study, we investigated whether an increase in the activity of CCRs accounts for this return. One week after CB excision, the hypoxic ventilatory reflex was greatly reduced as expected, whereas ventilation and blood gases at rest under normoxia were normal. Optogenetic inhibition of Phox2b-expressing neurons including the retrotrapezoid nucleus, a cluster of CCRs, reduced breathing proportionally to arterial pH. The hypopnoea was greater after CB excision but only in a normal or hypoxic environment. The difference could be simply explained by the loss of fast feedback from the CBs. We conclude that, in rats, CB denervation may not produce CCR plasticity. We also question whether the transient hypoventilation elicited by CB denervation means that these afferents are active under normoxia. ABSTRACT: Carotid body denervation (CBD) causes hypoventilation and increases the arterial PCO2 set-point; these effects eventually subside. The hypoventilation is attributed to reduced CB afferent activity and the PCO2 set-point recovery to CNS plasticity. In the present study, we investigated whether the retrotrapezoid nucleus (RTN), a group of non-catecholaminergic Phox2b-expressing central respiratory chemoreceptors (CCRs), is the site of such plasticity. We evaluated the contribution of the RTN to breathing frequency (FR ), tidal volume (VT ) and minute volume (VE ) by inhibiting this nucleus optogenetically for 10 s (archaerhodopsinT3.0) in unanaesthetized rats breathing various levels of O2 and/or CO2 . The measurements were made in seven rats before and 6-7 days after CBD and were repeated in seven sham-operated rats. Seven days post-CBD, blood gases and ventilation in 21% O2 were normal, whereas the hypoxic ventilatory reflex was still depressed (95.3%) and hypoxia no longer evoked sighs. Sham surgery had no effect. In normoxia or hypoxia, RTN inhibition produced a more sustained hypopnoea post-CBD than before; in hyperoxia, the responses were identical. Post-CBD, RTN inhibition reduced FR and VE in proportion to arterial pH or PCO2 (ΔVE : 3.3 ± 1.5% resting VE /0.01 pHa). In these rats, 20.7 ± 8.9% of RTN neurons expressed archaerhodopsinT3.0. Hypercapnia (3-6% FiCO2 ) increased FR and VT in CBD rats (n = 4). In conclusion, RTN regulates FR and VE in a pH-dependent manner after CBD, consistent with its postulated CCR function. RTN inhibition produces a more sustained hypopnoea after CBD than before, although this change may simply result from the loss of the fast feedback action of the CBs.

Proton detection and breathing regulation by the retrotrapezoid nucleus.

We discuss recent evidence which suggests that the principal central respiratory chemoreceptors are located within the retrotrapezoid nucleus (RTN) and that RTN neurons are directly sensitive to [H(+) ]. RTN neurons are glutamatergic. In vitro, their activation by [H(+) ] requires expression of a proton-activated G protein-coupled receptor (GPR4) and a proton-modulated potassium channel (TASK-2) whose transcripts are undetectable in astrocytes and the rest of the lower brainstem respiratory network. The pH response of RTN neurons is modulated by surrounding astrocytes but genetic deletion of RTN neurons or deletion of both GPR4 and TASK-2 virtually eliminates the central respiratory chemoreflex. Thus, although this reflex is regulated by innumerable brain pathways, it seems to operate predominantly by modulating the discharge rate of RTN neurons, and the activation of RTN neurons by hypercapnia may ultimately derive from their intrinsic pH sensitivity. RTN neurons increase lung ventilation by stimulating multiple aspects of breathing simultaneously. They stimulate breathing about equally during quiet wake and non-rapid eye movement (REM) sleep, and to a lesser degree during REM sleep. The activity of RTN neurons is regulated by inhibitory feedback and by excitatory inputs, notably from the carotid bodies. The latter input operates during normo- or hypercapnia but fails to activate RTN neurons under hypocapnic conditions. RTN inhibition probably limits the degree of hyperventilation produced by hypocapnic hypoxia. RTN neurons are also activated by inputs from serotonergic neurons and hypothalamic neurons. The absence of RTN neurons probably underlies the sleep apnoea and lack of chemoreflex that characterize congenital central hypoventilation syndrome.

State-dependent control of breathing by the retrotrapezoid nucleus.

KEY POINTS: This study explores the state dependence of the hypercapnic ventilatory reflex (HCVR). We simulated an instantaneous increase or decrease of central chemoreceptor activity by activating or inhibiting the retrotrapezoid nucleus (RTN) by optogenetics in conscious rats. During quiet wake or non-REM sleep, hypercapnia increased both breathing frequency (fR ) and tidal volume (VT ) whereas, in REM sleep, hypercapnia increased VT exclusively. Optogenetic inhibition of RTN reduced VT in all sleep-wake states, but reduced fR only during quiet wake and non-REM sleep. RTN stimulation always increased VT but raised fR only in quiet wake and non-REM sleep. Phasic RTN stimulation produced active expiration and reduced early expiratory airflow (i.e. increased upper airway resistance) only during wake. We conclude that the HCVR is highly state-dependent. The HCVR is reduced during REM sleep because fR is no longer under chemoreceptor control and thus could explain why central sleep apnoea is less frequent in REM sleep. ABSTRACT: Breathing has different characteristics during quiet wake, non-REM or REM sleep, including variable dependence on PCO2. We investigated whether the retrotrapezoid nucleus (RTN), a proton-sensitive structure that mediates a large portion of the hypercapnic ventilatory reflex, regulates breathing differently during sleep vs. wake. Electroencephalogram, neck electromyogram, blood pressure, respiratory frequency (fR ) and tidal volume (VT ) were recorded in 28 conscious adult male Sprague-Dawley rats. Optogenetic stimulation of RTN with channelrhodopsin-2, or inhibition with archaerhodopsin, simulated an instantaneous increase or decrease of central chemoreceptor activity. Both opsins were delivered with PRSX8-promoter-containing lentiviral vectors. RTN and catecholaminergic neurons were transduced. During quiet wake or non-REM sleep, hypercapnia (3 or 6% FI,CO2 ) increased both fR and VT whereas, in REM sleep, hypercapnia increased VT exclusively. RTN inhibition always reduced VT but reduced fR only during quiet wake and non-REM sleep. RTN stimulation always increased VT but raised fR only in quiet wake and non-REM sleep. Blood pressure was unaffected by either stimulation or inhibition. Except in REM sleep, phasic RTN stimulation entrained and shortened the breathing cycle by selectively shortening the post-inspiratory phase. Phasic stimulation also produced active expiration and reduced early expiratory airflow but only during wake. VT is always regulated by RTN and CO2 but fR is regulated by CO2 and RTN only when the brainstem pattern generator is in autorhythmic mode (anaesthesia, non-REM sleep, quiet wake). The reduced contribution of RTN to breathing during REM sleep could explain why certain central apnoeas are less frequent during this sleep stage.

Hypoxia silences retrotrapezoid nucleus respiratory chemoreceptors via alkalosis.

In conscious mammals, hypoxia or hypercapnia stimulates breathing while theoretically exerting opposite effects on central respiratory chemoreceptors (CRCs). We tested this theory by examining how hypoxia and hypercapnia change the activity of the retrotrapezoid nucleus (RTN), a putative CRC and chemoreflex integrator. Archaerhodopsin-(Arch)-transduced RTN neurons were reversibly silenced by light in anesthetized rats. We bilaterally transduced RTN and nearby C1 neurons with Arch (PRSx8-ArchT-EYFP-LVV) and measured the cardiorespiratory consequences of Arch activation (10 s) in conscious rats during normoxia, hypoxia, or hyperoxia. RTN photoinhibition reduced breathing equally during non-REM sleep and quiet wake. Compared with normoxia, the breathing frequency reduction (Δf(R)) was larger in hyperoxia (65% FiO2), smaller in 15% FiO2, and absent in 12% FiO2. Tidal volume changes (ΔV(T)) followed the same trend. The effect of hypoxia on Δf(R) was not arousal-dependent but was reversed by reacidifying the blood (acetazolamide; 3% FiCO2). Δf(R) was highly correlated with arterial pH up to arterial pH (pHa) 7.5 with no frequency inhibition occurring above pHa 7.53. Blood pressure was minimally reduced suggesting that C1 neurons were very modestly inhibited. In conclusion, RTN neurons regulate eupneic breathing about equally during both sleep and wake. RTN neurons are the first putative CRCs demonstrably silenced by hypocapnic hypoxia in conscious mammals. RTN neurons are silent above pHa 7.5 and increasingly active below this value. During hyperoxia, RTN activation maintains breathing despite the inactivity of the carotid bodies. Finally, during hypocapnic hypoxia, carotid body stimulation increases breathing frequency via pathways that bypass RTN.