Figure 5.

Excitatory role for SST⁺ glutamatergic preBötC neurons and a SST⁺ neuron-mediated inhibitory pathway within preBötC. (A–D) Cre dependent ChR2 expression targeted to preBötC SST neurons. A, Representative confocal mosaic micrograph of sagittal brainstem section of SST-Cre mouse showing the extent of eYFP⁺, SST⁺ and NK1R⁺ neurons 4 weeks after injection of AAV2/1-Ef1α-DIO-ChR2-eYFP into preBötC. Scale bar, 500 μm. VII, facial nucleus; BötC, Bötzinger Complex; NA, nucleus ambiguus; LRN, lateral reticular nucleus; LSO, lateral superior olive. B, Rostrocaudal distribution of eYFP (green), marking location of neurons expressing ChR2 (n = 4) and SST⁺ (red) neurons relative to caudal boundary of facial nucleus. C, High-magnification single channel and overlay confocal micrographs of square in circled area demarking the preBötC in A show small fusiform NK1R⁺ neurons (red, white arrows) were immunoreactive for eYFP (green). Large NK1R⁺ neurons (red, purple arrows) were not colocalized with eYFP immunoreactivity. Section also labeled for NeuN (blue) immunoreactivity. Scale bar, 50 μm. D, High-magnification micrograph showing colocalization (yellow) of ChR2-eYFP (green) and preBötC SST⁺ neurons (red) in ChR2-transfected SST-Cre mice. Section also labeled for NeuN (blue) immunoreactivity. Scale bar, 50 μm. (E–H) Excitatory effect elicited by bilateral preBötC SPP in ChR2-transfected SST-Cre mice. E, Left: Augmented inspiratory bursts induced by bilateral preBötC SPP in early inspiration. Scale bar, 1 s. Right: Bilateral preBötC SPP in early inspiration effects on normalized (norm) ∫GG_EMG prestimulation (Pre), during stimulation (Stim) and poststimulation (Post) (*, P<0.05; n=4). F, Ectopic inspiratory burst elicited by bilateral preBötC SPP during midexpiration in ChR2-transfected SST-Cre mice. Scale bar, 1 s. G, Phase response curve shows bilateral preBötC SPP in ChR2-transfected SST-Cre mice led to phase advances. *, comparisons versus control phase shift (Ctrl), P<0.05. H, Each of three bilateral preBötC SPPs in ChR2-transfected SST-Cre mice induced a breath during an inflation-induced apnea. Dotted line represents CPAP (8 cm H₂O). Scale bar, 5 s. (I–J) The remaining inhibitory effect in virus transfected SST-Cre mice was mediated by GABA and/or glycine. I, Left: Bilateral preBötC LPP in ChR2 transfected SST-Cre mice did not induce apnea but slowed down breathing frequency. Scale bar, 2 s. Right: T_I (top) and T_E (bottom) measured before (Pre) and during bilateral preBötC LPP (Stim) (*, P<0.05; n=4). J, Left: Bilateral preBötC LPP no longer produced prolongation in T_E after blocking inhibition within preBötC in ChR2 transfected SST-Cre mice. Scale bar, 5 s. Right: T_I (top) and T_E (bottom) measured Pre and during Stim in B+S. Error bars represent mean±SD.