Fig. 5: TGOT increases the frequency of preBötC inspiratory bursts, depolarizes preBötC^OT-R neurons that are inspiratory bursting and amplifies the glycinergic post-synaptic currents occurring in nA neurons during inspiratory bursts in rhythmic slices in vitro.

a–c, TGOT (0.5 µM) increases the frequency of preBötC inspiratory bursts in rhythmic preBötC slices from neonatal wild-type mice (n = 12 mice; ⎰preBötC, integrated extracellular recordings; V_m, whole-cell current-clamp recordings). Gray shaded areas in (a) are expanded in (b). Wilcoxon two-sided matched-pairs signed rank test (c). The violin plot is represented in gray, dashed line indicates the median and dotted lines represent the quartiles (c). d,e, TGOT (0.5 µM) increases the frequency of preBötC inspiratory bursts in preBötC ‘island’ preparations (n = 5 mice; d, representative trace; e, quantifications). Paired two-sided t-test. The violin plot is represented in gray, dashed line indicates the median and dotted lines represent the quartiles (e). f–h, PreBötC^OT-R neurons recorded in rhythmic preBötC slices from neonatal Oxtr-Cre; Ai14(Rosa26-LSL-tdTomato) mice display an inspiratory bursting pattern of activity (f), and TGOT (0.5 µM) depolarizes preBötC^OT-R neurons following the blockade of fast synaptic transmission (bicuculline (Bic), 10 µM; strychnine (Stry), 5 µM; CNQX, 20 µM; n = 7 mice) (g, representative traces; h, quantifications). Paired two-sided t-test. The violin plot is represented in gray, dashed line indicates the median and dotted lines represent the quartiles (h). i, Example of a recorded preBötC^OT-R neuron (dye⁺ and OT-R;tdTomato⁺, white arrows) located in the NK1-R-expressing preBötC region (n = 4 neurons recorded that were labeled and localized a posteriori). Scale bars: full image, 40 µm; inset, 20 µm. j, All respiratory-modulated nA neurons recorded (n = 10 wild-type mice) were hyperpolarized during the preBötC inspiratory bursts, causing inhibition of their spiking activity. k,l, nA neurons showed volleys of inhibitory currents during preBötC inspiratory bursts in the control condition (I_m, whole-cell voltage-clamp recordings; V_h, −40 mV holding voltage), which were amplified by TGOT (0.5 µM) application (n = 10 mice; k, representative traces; l, quantifications). During TGOT application, bicuculline (10 µM) application had no effect, while strychnine (5 µM) application either abolished all currents (n = 5 mice, top traces in k) or exposed the presence of volleys of excitatory currents during preBötC inspiratory bursts (n = 3 mice, bottom traces in k, further characterized in Extended Data Fig. 8d). Control vs TGOT, paired two-sided t-test. Δ changes following TGOT, bicuculline and strychnine applications, Friedman test with Dunn’s multiple comparison. Violin plots are represented in gray, the dashed lines indicate the median, the dotted lines represent the quartiles and the green line represents the limit above which the recorded currents are inhibitory and under which they are excitatory (l). m, Example of a recorded nA neuron (arrows indicate the dye⁺ recorded neuron that is Phox2b⁺ and ChAT⁺; n = 5 neurons recorded that were labeled and localized a posteriori). Scale bars: full image, 40 µm; inset, 20 µm. Detailed statistics are presented in Supplementary Table 1.

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