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Comparative Study
, 61 (4), 835-41

Optogenetic Stimulation of c1 and Retrotrapezoid Nucleus Neurons Causes Sleep State-Dependent Cardiorespiratory Stimulation and Arousal in Rats

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Comparative Study

Optogenetic Stimulation of c1 and Retrotrapezoid Nucleus Neurons Causes Sleep State-Dependent Cardiorespiratory Stimulation and Arousal in Rats

Stephen B G Abbott et al. Hypertension.

Abstract

C1 catecholaminergic neurons and neurons of the retrotrapezoid nucleus are integrative nodes within the brain stem network regulating cardiorespiratory reflexes elicited by hypoxia and hypercapnia, stimuli that also produce arousal from sleep. In the present study, Channelrhodopsin-2 was selectively introduced into these neurons with a lentiviral vector to determine whether their selective activation also produces arousal in sleeping rats. Sleep stages were identified from electroencephalographic and neck muscle electromyographic recordings. Breathing was measured using unrestrained whole body plethysmography and blood pressure by telemetry. During nonrapid eye movement sleep, unilateral photostimulation of the C1 region caused arousal in 83.0±14.7% of trials and immediate and intense cardiorespiratory activation. Arousal during photostimulation was also observed during rapid eye movement sleep (41.9±5.6% of trials), but less reliably than during nonrapid eye movement sleep. The cardiorespiratory responses elicited by photostimulation were dramatically smaller during rapid eye movement sleep than nonrapid eye movement sleep or wakefulness. Systemic α1-adrenoreceptor blockade reduced the cardiorespiratory effects of photostimulation but had no effect on the arousal caused by photostimulation during nonrapid eye movement sleep. Postmortem histology showed that neurons expressing Channelrhodopsin 2-mCherry were predominantly catecholaminergic (81%). These results show that selective activation of C1 and retrotrapezoid nucleus neurons produces state-dependent arousal and cardiorespiratory stimulation. These neurons, which are powerfully activated by chemoreceptor stimulation, may contribute to the sleep disruption associated with obstructive sleep apnea.

Conflict of interest statement

Conflict of interest: None

Figures

Figure 1
Figure 1. Photostimulation of ChR2+ C1/RTN neurons during non-rapid eye movement sleep (NREMS) causes arousal
A, Photostimulation trial during NREMS in a ChR2+ rat. Open arrowheads indicate first signs of cortical desynchronization. Inset. Expanded view of the initial respiratory effects and cortical desynchronization in A. Flow- barometric plethysmographic recording of breathing, EEG- electroencephalograph, EMG-neck electromyograph. Y-axis: Flow- 50 ml/s, EEG and EMG- 0.5 mV. B, The probability of arousal during photostimulation in NREMS in ChR2+ and control rats (N= 17 ChR2+ and 8 control rats, **P<0.01, ***P<0.0001). C, Cumulative frequency of arousals during photostimulation in NREMS (1 s bins, N= 12 ChR2+ and 8 control rats).
Figure 2
Figure 2. Photostimulation of ChR2+ C1/RTN neurons during rapid eye movement sleep (REMS) causes arousal
A, As in Figure 1A except during REMS. B, The probability of an arousal during photostimulation in REMS in ChR2+ and control rats (N= 13 ChR2+ and 8 control rats, **P<0.01). C, Cumulative frequency of arousals during photostimulation in REMS (1 s bins, N=12 ChR2+ and 8 control rats).
Figure 3
Figure 3. The respiratory effect of ChR2+ C1/RTN neuron photostimulation is sleep state dependent
A, Photostimulation trials (20 Hz) spanning 3 arousal states (1: NREMS to wake, 2: REMS, 3: REMS, 4: REMS to wake, 5: wake). Note that the ventilatory response is attenuated during REMS trials. Y-axis: Flow- 50 ml/s, EEG and EMG- 0.5 mV. B, Changes in minute volume (MV) during photostimulation in waking, NREMS, and REMS in ChR2+ and control rats (*P<0.05, **P<0.01, ***P<0.001, repeated measures two-way ANOVA comparing sleep state & stimulus frequency in ChR2+ rats. ### P<0.001, two-way ANOVA comparing the presence of ChR2 & sleep state at a stimulus frequency of 20 Hz.
Figure 4
Figure 4. Arousal during ChR2+ C1/RTN neuron photostimulation is correlated with changes in ventilation during NREMS, but not REMS
A–B, X-Y scatter plots of the change in MV and the probability of arousal during 2, 10 and 20 Hz photostimulation trials in ChR2+ rats during NREMS (A) and REMS (B). (N=12 for NREMS and 10 for REMS). Correlation determined using Pearson’s correlation calculation.
Figure 5
Figure 5. The cardiovascular effect of ChR2+ C1/RTN neuron photostimulation is sleep state dependent
A, Effects of 3 s photostimulation trials (20Hz, 5 ms) on arterial pressure (AP) and heart rate (HR) during each arousal state in ChR2+ rats. Y-axis: Flow- 50 ml/s, EEG and EMG- 0.5 mV. B, Normalized time course changes in MAP and HR during a 3 s stimulus trains during each arousal state in ChR2+ rats (N=6).
Figure 6
Figure 6. Alpha1-adrenoreceptor antagonism does not prevent arousal, but attenuates the cardiorespiratory activation during photostimulation
A, Effects of photostimulation before (left) and after (right) administration of prazosin (1mg/kg, I.P.). Y-axis: Flow- 35 ml/s, EEG and EMG- 0.25 mV. B–D, Average cardiovascular (B), respiratory (C) and arousal response to photostimulation before (open columns) and after prazosin treatment (closed columns) (N=6, * P<0.05, ** P<0.01, *** P<0.001, paired Student’s t-test).
Figure 7
Figure 7. ChR2-mCherry is preferentially expressed by C1 and RTN neurons
A, Rostro-caudal distribution of neurons expressing ChR2-mCherry, tyrosine hydroxylase (TH) or both in the rostral ventrolateral medulla (N=22 ChR2+ rats). B, Location of the fiber placement in all experiments (N= 22 ChR2+ and 8 control rats). Scale bar: 0.5 mm.

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