Selective optogenetic stimulation of the retrotrapezoid nucleus in 2 sleeping rats activates breathing without changing blood pressure or 3 causing arousal or sighs . 4

27 Combined optogenetic activation of the retrotrapezoid nucleus (RTN, a CO2/proton28 activated brainstem nucleus) with nearby catecholaminergic neurons (C1 and A5), or selective 29 C1 neuron stimulation, increases blood pressure (BP) and breathing, causes arousal from non30 REM sleep and triggers sighs. Here we wished to determine which of these physiological 31 responses are elicited when RTN neurons are selectively activated. The left rostral RTN and 32 nearby A5 neurons were transduced with channelrhodopsin-2 (ChR2) using a lentiviral vector. 33 Very few C1 cells were transduced. BP, breathing, EEG and neck EMG were monitored. During 34 non-REM sleep, photostimulation of ChR2 neurons (20s, 2-20Hz) instantly increased VE 35 without changing BP (13 rats). VE and BP were unaffected by light in nine control (ChR2) rats. 36 Photostimulation produced no sighs and caused arousal (EEG desynchronization) more 37 frequently in ChR2 than ChR2 rats (62 ± 5% of trials vs. 25 ± 2%; p < 0.0001). Six ChR2 rats 38 then received spinal injections of a saporin-based toxin that spared RTN neurons but destroyed 39 surrounding catecholaminergic neurons. Photostimulation of the ChR2 neurons produced the 40 same ventilatory stimulation before and after lesion but arousal was no longer elicited. Overall 41 (all ChR2 rats combined), ΔVE correlated with the number of ChR2 RTN neurons whereas 42 arousal probability correlated with the number of ChR2 catecholaminergic neurons. 43 In conclusion, RTN neurons activate breathing powerfully and, unlike the C1 cells, have 44 minimal effects on BP and have a weak arousal capability at best. A5 neuron stimulation 45 produces little effect on breathing and BP, but does appear to facilitate arousal. 46 47 by 10.2.32.246 on O cber 7, 2016 http://jaysiology.org/ D ow nladed fom Introduction 48 Hypoxia and hypercapnia, alone or in combination, activate breathing and, if severe 49 enough, produce arousal (17, 60, 62, 63). The carotid bodies are largely responsible for the 50 hypoxic ventilatory stimulation and for hypoxia-induced arousal because both responses are 51 eliminated by ablating these organelles (17). How and where CNS hypercarbia elicits the 52 hypercapnic ventilatory reflex (HCVR) is still debated and the CNS pathways responsible for 53 arousal to hypoxia or hypercapnia are even more conjectural. The prevailing view is that both 54 the HCVR and CO2-induced arousal result from the combined action of CO2 on myriads of CNS 55 pH-sensitive neurons, including serotonergic, noradrenergic and orexinergic neurons (13, 21, 29, 56 41, 49, 59, 67). 57 The retrotrapezoid nucleus (RTN) is a key structure for the HCVR. RTN neurons are 58 glutamatergic and seem to operate both as a central respiratory chemoreceptor and chemoreflex 59 integrator, i.e., these neurons detect acid, directly and via surrounding astrocytes, and receive 60 convergent inputs from other neurons that regulate the HCVR, e.g., CNS orexinergic and 61 aminergic neurons and the carotid bodies (4, 5, 9, 36, 65). In the present study we examine 62 whether RTN could also play a role in CO2-induced arousal and blood pressure control. 63 At present, the most selective way to manipulate RTN neurons in adult rodents in vivo is 64 to use PRSx8-promoter-containing lentiviral vectors that express their actuator (opsins, 65 allatostatin receptor etc.) selectively in Phox2b-positive neurons (1, 4, 5, 44, 53). The main 66 disadvantage of these vectors is that they transduce both RTN and surrounding 67 catecholaminergic neurons (C1 and A5 neurons). Simultaneous activation of these two classes of 68 neurons produces massive breathing stimulation and other effects including BP increase, arousal 69 and sighs (1, 47). All these effects, including breathing stimulation, can be evoked, albeit to 70 by 10.2.32.246 on O cber 7, 2016 http://jaysiology.org/ D ow nladed fom different degrees, by activating the C1 neurons selectively (22). The best documented function of 71 the C1 neurons is their contribution to the baroreflex (36, 38) but many of them also receive 72 powerful excitatory inputs from the carotid bodies (77). Increased sympathetic vasomotor tone, 73 arousal and sighs are characteristic effects of hypoxia (10, 63), raising the possibility that the C1 74 neurons mediate these responses with some selectivity (22). However, the C1 neurons belong to 75 the reticular core of the medulla oblongata and activation of many other types of neurons within 76 this region could conceivably also cause arousal and raise BP, the RTN included. 77 The C1 and A5 catecholaminergic neurons that are coextensive with RTN are bulbospinal 78 and can be selectively destroyed by spinal injections of the ribosome-inactivating toxin anti79 dopamine β-hydroxylase-saporin (5, 24, 64, 71). We took advantage of this toxin to identify 80 which physiological effects are elicited by selective activation of RTN neurons. We transduced 81 both RTN and nearby catecholaminergic neurons with channelrhodopsin-2 (ChR2) using a 82 previously described PRSx8-contining LVV (5, 9, 47). We first examined the effects produced 83 by photostimulation of ChR2-expressing neurons and then repeated the experiments after 84 lesioning spinally-projecting catecholaminergic neurons (5). The responses that persisted after 85 such lesions were therefore attributed to the selective activation of ChR2-transduced RTN 86 neurons. The specific questions addressed in this study are whether these residual responses 87 include hyperpnea, BP increase and arousal from non-REM sleep or sighs. 88