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, 118 (12), 1491-501

Selective Optogenetic Stimulation of the Retrotrapezoid Nucleus in Sleeping Rats Activates Breathing Without Changing Blood Pressure or Causing Arousal or Sighs

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Selective Optogenetic Stimulation of the Retrotrapezoid Nucleus in Sleeping Rats Activates Breathing Without Changing Blood Pressure or Causing Arousal or Sighs

Peter G R Burke et al. J Appl Physiol (1985).

Abstract

Combined optogenetic activation of the retrotrapezoid nucleus (RTN; a CO2/proton-activated brainstem nucleus) with nearby catecholaminergic neurons (C1 and A5), or selective C1 neuron stimulation, increases blood pressure (BP) and breathing, causes arousal from non-rapid eye movement (non-REM) sleep, and triggers sighs. Here we wished to determine which of these physiological responses are elicited when RTN neurons are selectively activated. The left rostral RTN and nearby A5 neurons were transduced with channelrhodopsin-2 (ChR2(+)) using a lentiviral vector. Very few C1 cells were transduced. BP, breathing, EEG, and neck EMG were monitored. During non-REM sleep, photostimulation of ChR2(+) neurons (20s, 2-20 Hz) instantly increased V̇e without changing BP (13 rats). V̇e and BP were unaffected by light in nine control (ChR2(-)) rats. Photostimulation produced no sighs and caused arousal (EEG desynchronization) more frequently in ChR2(+) than ChR2(-) rats (62 ± 5% of trials vs. 25 ± 2%; P < 0.0001). Six ChR2(+) rats then received spinal injections of a saporin-based toxin that spared RTN neurons but destroyed surrounding catecholaminergic neurons. Photostimulation of the ChR2(+) neurons produced the same ventilatory stimulation before and after lesion, but arousal was no longer elicited. Overall (all ChR2(+) rats combined), ΔV̇e correlated with the number of ChR2(+) RTN neurons whereas arousal probability correlated with the number of ChR2(+) catecholaminergic neurons. In conclusion, RTN neurons activate breathing powerfully and, unlike the C1 cells, have minimal effects on BP and have a weak arousal capability at best. A5 neuron stimulation produces little effect on breathing and BP but does appear to facilitate arousal.

Keywords: A5 noradrenergic neurons; arousal; hypercapnia; non-REM sleep; retrotrapezoid nucleus.

Figures

Fig. 1.
Fig. 1.
Cardiorespiratory correlates of spontaneous arousal in Sprague-Dawley rats. A: spontaneous arousal from non-rapid eye movement (REM) sleep. Non-REM sleep was characterized by large amplitude, slow-wave EEG (delta frequencies: 0.5–4.5 Hz), a regular breathing rate (respiratory frequency; fR, breaths/min) and tidal volume (Vt; ml/100 g), steady blood pressure (BP; mmHg, via telemetry), and heart rate (HR; beats/min). Arousal followed a stereotypical sequence beginning with EEG desynchronization (loss of delta power; arrow #1), followed by tachycardia (arrow #2) and, typically, a sigh (arrow #3) followed by an apnea. B: spontaneous arousal from REM sleep. REM sleep was characterized by a highly synchronized EEG in the theta band (6–8 Hz), more rapid and irregular breathing rate, bradycardia, with increased BP and HR variability. Arousal from REM sleep followed the same sequence: EEG desynchronizarion, tachycardia, and sigh. Regular breathing and steady BP resumed shortly thereafter during quiet wake or with the recommencement of non-REM sleep. Air flow represents the raw whole body plethysmography signal (Flow). The rat had been habituated to the environment through repeated sojourns in the plethysmography chamber; au, arbitrary units.
Fig. 2.
Fig. 2.
Optogenetic stimulation of channelrhodopsin-2 (ChR2)-transduced neurons in intact rats. A: photostimulation (20 Hz, 20 s; gray bar) during non-REM sleep. The stimulus produced an immediate increase in breathing frequency (fR) and tidal volume, followed (∼3 s later) by EEG desynchronization (box in EEG trace is expanded at bottom) and, 4 s later, by tachycardia and a slight drop in BP. No sigh occurred with the arousal or the stimulus. B: photostimulation during REM sleep (20 Hz; 20 s; same rat in A) had no effect besides a slight increase in Vt. The box in the EEG trace is expanded at bottom to illustrate that the theta rhythm was unaffected by the stimulus.
Fig. 3.
Fig. 3.
Optogenetic stimulation of ChR2-transduced neurons in non-REM sleep stimulates breathing and produces arousal. A–C: breathing parameters during non-REM sleep before (baseline) and during photostimulation at 2, 10, or 20 Hz for 20 s [n = 6; 2-way repeated-measures (RM) ANOVA with Bonferroni's multiple comparisons]. Group data from the same 6 rats that later received intraspinal anti-dopamine β-hydroxylase-saporin (anti-DβH-saporin). V̇e, minute ventilation. D: cumulative arousal probability elicited by 20-s photostimulation (grey bar) at 2, 10, and 20 Hz in ChR2+ animals (n = 13) or 20-Hz photostimulation in control animals (n = 9). Control subjects (ChR2) were fully instrumented rats in which no neuron was transduced and the photostimulus was delivered. E: cumulative arousal probability in experimental ChR2+ (n = 13) and ChR2 control rats (n = 9). The 20-s photostimulus was delivered during periods of established non-REM sleep at time 0 and the cumulative arousal probability was measured at 40 s (see D); significance by two-way ANOVA with Bonferroni's multiple comparisons. F: cumulative probability of a sigh during the same 40-s window in experimental ChR2+ (n = 13) and control ChR2 rats (n = 9); not significant (ns) by two-way ANOVA. In A–F: **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 4.
Fig. 4.
Optogenetic stimulation of ChR2-transduced neurons during non-REM sleep after lesion of bulbospinal catecholaminergic neurons. Photostimulation (20 Hz, 20 s; gray bar) in non-REM sleep 2 wk after administration of the saporin conjugate into the thoracic spinal cord (same animal shown in Fig. 2A). This stimulus produced a similar large increase in breathing but had no effect on EEG, HR, or BP.
Fig. 5.
Fig. 5.
Optogenetic stimulation of ChR2-transduced neurons during non-REM sleep before and after lesion of bulbospinal catecholaminergic neurons: arousal, sighs, and respiratory stimulation. A: arousal probability elicited by a 20-Hz/20-s photostimulus (gray bar) measured 40 s after the onset of the stimulus in 6 rats before (intact ChR2+) and 2 wk after administration of the catecholaminergic neuron-specific toxin (lesion ChR2+) into the thoracic spinal cord. Control rats (ChR2; n = 9) were fully instrumented rats in which no neuron was transduced. B: arousal probability in control rats or in the experimental rats before (intact) and after lesion; one-way ANOVA with Tukey's multiple comparisons test. C: probability of sigh during the photostimulation period in control rats or experimental rats before (intact) and after lesion; one-way ANOVA with Tukey's multiple comparisons test. D–F: breathing parameters during non-REM sleep at rest and during photostimulation (20 s/20 Hz) in 6 rats determined before (intact) and after lesion of bulbospinal catecholaminergic neurons; two-way RM ANOVA with Bonferrroni's multiple comparisons test. G–I: change (Δ) in breathing parameters evoked by photostimulation in 6 rats before (intact) and after catecholaminergic neuron lesion. Breathing stimulation was identical before and after the lesion (paired Students t-test). In A–I: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 6.
Fig. 6.
Anatomical distribution of ChR2-transduced neurons before and after lesion of bulbospinal catecholaminergic neurons. A: rostrocaudal distribution of tyrosine hydroxylase (TH)-immunoreactive neurons in 8 intact and 6 lesioned rats. Counts were made on the left side of the brain in a 1-in-3 series of 30-μm-thick transverse sections. The section closest to the caudal end of the facial motor nucleus was assigned the level 11.6 mm caudal to bregma and served as reference point to align the brain sections from all the rats. The rostral cluster (bregma 11-8.5) consists of A5 noradrenergic neurons and is massively destroyed by the toxin. The caudal cluster (C1 neurons) consists of adrenergic/glutamatergic neurons that are more modestly damaged. Its rostral portion (bregma 11.5–13.5 mm) contains the bulk of the spinally projecting C1 cells. B: rostrocaudal distribution of ChR2-transduced neurons (mCherry-immunoreactive) in 8 intact rats. Transduced catecholaminergic neurons (TH+) and noncatecholaminergic neurons [putative retrotrapezoid nucleus (RTN) chemoreceptors] are separately represented. C: rostrocaudal distribution of ChR2-transduced neurons (mCherry-immunoreactive) in 6 lesioned rats. Transduced catecholaminergic neurons (TH+) and noncatecholaminergic neurons (putative RTN chemoreceptors) are represented separately. All transduced neurons were on the left side.
Fig. 7.
Fig. 7.
Correlations between the number and phenotype of ChR2-transduced neurons and the physiological response elicited by photostimulation. A: x–y plots of the increase in minute ventilation (ΔV̇e) vs. the number of ChR2+ RTN neurons (left), ChR2+ catecholaminergic neurons (middle), or total ChR2+ neurons (right). Photostimulation was 20 s/20 Hz in non-REM sleep. The degree of hyperpnea was correlated only with the number of ChR2+ RTN neurons. Pearson's r and significance (P) are indicated. B: plots of arousal probability elicited by 20-s/20-Hz photostimulation vs. the number of ChR2+ RTN neurons, ChR2+ catecholaminergic neurons, or all ChR2+ neurons. Arousal probability was only correlated with the number of ChR2+ catecholaminergic neurons.
Fig. 8.
Fig. 8.
Summary and working model. A: current finding: selective RTN stimulation increases breathing without producing arousal from non-REM sleep, sighs, or increasing BP, whereas stimulation of the noradrenergic A5 neurons produces arousal with little apparent effects on breathing or mean BP. B: working model for the contribution of A5, C1, and RTN neurons to arousal, breathing, and autonomic activation by hypoxia and hypercapnia: hypoxia activates C1 and A5 cells by a pathway stimulated by the O2-sensing carotid body (CB) afferents and relayed via caudal commissural commissural nucleus of the solitary tract (NTS) neurons. Systemic hypoxia may also activate C1 cells via intrinsic and local glial mechanisms (54, 76, 80). C1 cell stimulation reproduces most of the effects of hypoxia: arousal from non-REM sleep, sighs, and increases in breathing and BP (22). C1 cells innervate and excite major wake-promoting systems, the orexinergic and noradrenergic neurons, including the locus coeruleus (A6), A5, A2, and A1 (3, 15, 42). Sighs may be triggered upon arousal by norepinephrine release at the pre-Bötzinger complex via β-adrenergic signaling (79); sighs are probably linked to hypoxia via the same wake-promoting/vigilance systems. RTN regulates breathing selectively; CO2-induced arousal from non-REM sleep is not produced by RTN stimulation but is likely caused by 5HT and other wake-promoting systems that do not trigger sighs (not represented here; see Ref. 21). A5 neurons also innervate sympathetic preganglionic neurons (SPNs; not represented), which may cause some blood flow redistribution bu t little change in mean BP. CPG, respiratory central pattern generator.

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