Expression of Cre-dependent ChR2 in the preBötC

To manipulate specific molecularly defined preBötC neuronal populations (Fig. 1a), mice expressing Cre recombinase under the Dbx1, vesicular glutamate transporter 2 (Vglut2), or vesicular GABA/glycine transporter (Vgat) promoter were crossed with mice containing a loxP-flanked STOP cassette prior to a sequence for a ChR2-reporter fusion protein. Thus ChR2-reporter was selectively expressed in cells also expressing Cre recombinase. A subset of Cre-driver mice were crossed with Cre-dependent ZsGreen reporter mice to better represent the distribution of Cre+ cell bodies rather than the membrane bound ChR2-reporter (Fig. 1b–e). Since “Dbx1 neurons” within the preBötC express the Dbx1 gene transiently during embryonic development, a tamoxifen-inducible Cre (Dbx1CreERT2) was used and pregnant dams were given a tamoxifen pulse at E10.5 to induce Cre expression24,26. Similar to previous reports utilizing Dbx1CreERT225,30,31, reporter labeling was observed in transverse medullary slices within a well-defined region extending ventrolaterally from the hypoglossal (XII) motor nucleus encompassing the intermediate reticular formation (IRt), a region containing XII premotor neurons32, and the preBötC just ventromedial to ChAT+ nucleus ambiguus neurons (Fig. 1b). Although Dbx1CreERT2 preferentially labels neurons and glia in the preBötC and IRt and has been used in the majority of studies defining the role of Dbx1 neurons in respiratory rhythmogenesis (e.g., refs. 26,27,29,33,34,35), Dbx1CreERT2 will not label all Dbx1-derived preBötC neurons30. Therefore, we combined this approach with use of the Vglut2Cre line that specifically labels all glutamatergic preBötC neurons, including the vast majority Dbx1 neurons24,26. As expected, reporter fluorescence in Vglut2Cre and VgatCre mice was widely distributed within the medulla. However, strong reporter expression was observed in cell bodies within the preBötC of all animals from each experimental group (Fig. 1c–e), reflecting the heterogeneity of neuron subtypes within the preBötC.

Fig. 1 Anatomy of the heterogeneous preBötC. a Diagram showing estimated ratios of Dbx1 and non-Dbx1 excitatory and inhibitory neurons19, 24,25,26 with the corresponding promoters used to drive Cre expression within these preBötC subpopulations. b Schematic depicting key landmarks (NA nucleus amibiguus, IO inferior olive) and the pathway from preBötC rhythmogenesis to XII motor output and a representative 2.5× image of a transverse medullary preBötC section from an adult Dbx1-ZsGreen mouse. Blue arrows represent commissural connections between Dbx1 neurons24. c–e 20× z-projected images of Cre-dependent ZsGreen expression in preBötC neurons from VgatCre (c), Vglut2Cre (d), and Dbx1CreERT2 (e) mice. ChAT immunofluorescence demarks the NA dorsomedial to the preBötC Full size image

An intrinsic refractory period for preBötC neuron activity

To explore mechanisms regulating preBötC bursting, we recorded preBötC population activity in slices from Dbx1-ChR2 and Vglut2-ChR2 neonatal (P4–12) mice (Fig. 2a and Supplementary Fig. 1a). Excitatory neurons were stimulated contralateral to population recordings with 200 ms blue light pulses and the probability of light evoking a population burst was plotted against elapsed time following an endogenous preBötC burst (Fig. 2b). Confirming previous results29, we observed a refractory period of ~2 s following endogenous preBötC bursts during which stimulation of Dbx1 or Vglut2 neurons did not evoke a burst (Fig. 2a, b). Intracellular current-clamp recordings of Dbx1 neuron activity within the active preBötC network revealed that the magnitude of depolarization during inspiratory bursts was proportional to a membrane afterhyperpolarization (AHP) that followed36 (Fig. 2c), suggesting that intrinsic properties of preBötC neurons may play a significant role in the refractory period mechanism. To test this, rhythmically active preBötC neurons were synaptically isolated pharmacologically with antagonists of AMPA, N-methyl-d-aspartate, glycine, and GABA receptors (CNQX, CPP, strychnine, and gabazine, respectively) and current was injected to simulate a burst “drive potential”. Reminiscent of Dbx1 neuron activity within the network, isolated Dbx1 neurons were hyperpolarized following current injection, and the AHP depended on the intensity of the depolarizing current step (Fig. 2d). To examine how intrinsic AHP translates to a network effect, we quantified spiking activity during small (20 pA) consecutive current pulses following a larger (20–60 pA) depolarizing current step (Fig. 2e). The number of spikes evoked with each small current pulse was reduced 0.75–1.25 s following the initial larger depolarizing pulse and returned to near maximal levels by 2.25–2.75 s, corresponding to the duration of the refractory period for preBötC population activity (see Fig. 2b). The reduction in spiking activity was related to the intensity (Fig. 2e) and duration (Supplementary Fig. 2a) of the simulated drive potential. Synaptically isolated neurons were identified as Dbx1+ with depolarizing responses to light. Notably, spiking was also reduced following current injection in isolated Dbx1− neurons (Supplementary Fig. 2b), suggesting refractory mechanisms may be similar for Dbx1+ and Dbx1− preBötC neurons.

Fig. 2 Intrinsic refractory properties of preBötC neurons. a Diagram of in vitro brainstem slice preparation and example of the refractory period for evoked preBötC population bursts in a Dbx1-ChR2 and Vglut2-ChR2 slice. b Quantified probability of light-evoking a preBötC burst by stimulation excitatory neurons in Dbx1-ChR2 (n = 7) and Vglut2-ChR2 (n = 7) slices. (p > 0.05 at all time points, two-way ANOVA and Bonferroni’s multiple comparisons test, means ± s.e.m. c Overlaid activity of two Dbx1+ neurons in the spontaneously active preBötC network demonstrating the relationship between burst drive potential and AHP (arrow). Quantified data from n = 10 Dbx1+ neurons shown on the right (linear regression analysis, p < 0.0001; slope = −0.52 ± 0.058). d Representative example (10 overlaid sweeps) of a synaptically isolated Dbx1+ neuron showing hyperpolarization (arrow) and reduced spiking following a 600 ms 50 pA current step (upper). Membrane potential (average of 10 sweeps) before and after graded current step intensities (bottom). e Example showing transiently reduced number of spikes evoked by small (20 pA, 400 ms) current steps during the intrinsic refractory period following a 30 pA current step in a synaptically isolated Dbx1+ neuron (note the depolarization and spiking evoked by light); average normalized number of spikes evoked at different time points following current steps of varied intensity (n = 7 neurons from n = 7 slices; means ± s.e.m.) Full size image

Inhibition modulates the refractory period

Next, we examined how inhibitory preBötC neurons influence the activity of excitatory Dbx1 preBötC neurons and rhythmogenesis. Since the preBötC contains both glycinergic18,19 and GABAergic17 neurons, we recorded from rhythmic preBötC neurons in Dbx1-ChR2 slices during progressive bath application of strychnine (1 µM) and gabazine (1 µM) to block glycinergic and GABAergic fast synaptic inhibition, respectively (Fig. 3a). Strychnine and gabazine increased drive potential area (Fig. 3a) as well as the amplitude of preBötC population bursts (Fig. 3b). Thus both glycinergic and GABAergic mechanisms restrain Dbx1 neuron activity during synchronized population activity.

Fig. 3 Inhibition restrains excitatory preBötC neuron activity and the refractory period. a Example Dbx1+ neuron activity under baseline conditions and following progressive blockade of glycinergic and gabaergic synaptic inhibition with strychnine (1 µM) and gabazine (1 µM), respectively. Average normalized inspiratory drive potential area (n = 7) of Dbx1+ neurons during preBötC bursts in strychnine and gabazine (*p < 0.05, ***p<0.001; means ± s.e.m.; one-way repeated measures ANOVA and Bonferroni’s multiple comparisons test). b Example of preBötC population activity from a Dbx1-ChR2 slice during inhibition block and failure to light-evoke bursts during the refractory period, and normalized burst amplitude of integrated preBötC population activity (n = 11 slices; *p < 0.05, **p<0.01, ****p<0.0001; means ± s.e.m.; Friedman test and Dunn’s multiple comparisons test). c Quantified probability of evoking a burst (0.5 s bins) relative to time post endogenous burst (s) from Dbx1-ChR2 slices during inhibition block (~100–150 trials in each condition from each slice; n = 4 slices; means ± s.e.m.). d Data from a representative Dbx1-ChR2 stimulation experiment comparing the phase shift (stimulus cycle duration/average cycle duration) elicited compared to the time of the light stimulus relative to the preceding endogenous burst. Note that in strychnine (yellow) and strychnine+gabazine (orange) failed bursts are more common for a longer duration following endogenous bursts (358 stimulations). Average data shown in Supplementary Fig 1. e Cumulative probability of a spontaneous burst (0.05 probability bins) relative to time post endogenous burst (s) from Dbx1-ChR2 slices during inhibition block (~100–150 trials in each condition from each slice; n = 4 slices; means ± s.e.m.). Vertical dashed lines correspond to the end of the refractory period and an increasing probability of spontaneous bursting Full size image

To determine how inhibition influences the refractory period for preBötC population bursting, we delivered light pulses randomly during the respiratory cycle while recording contralateral preBötC population activity under baseline conditions and after blocking glycinergic and then GABAergic, synaptic transmission (Fig. 3b). The probability of light-evoking a population burst was plotted against elapsed time following an endogenous preBötC burst (Fig. 3c). Under baseline conditions, a burst was evoked ~50% of the time 1.08 s and ~100% of the time 2.75 s following an endogenous burst. When glycinergic inhibition was blocked, a burst was evoked ~50% of the time at 2.25 s and ~100% of the time at 3.75 s. After blocking glycinergic and GABAergic inhibition, there was a ~50% chance of evoking a burst 3.80 s and a ~100% chance 5.75 s following an endogenous preBötC burst. The change in cycle duration (phase shift) relative to the time of light onset was also assessed (Fig. 3d, Supplementary Fig. 1b), revealing similar results. Thus both glycinergic and GABAergic inhibition reduce the refractory period for preBötC bursting. Further, we found that longer refractory periods were associated with slower preBötC rhythms since preBötC burst frequency was reduced during blockade of inhibition (Fig. 3e). Note, that in all conditions (baseline, strychnine, and strychnine+gabazine), the end of the refractory period corresponded with an increased probability of an endogenous burst occurring.

Similar mechanisms also apply to sighs, large amplitude biphasic bursts generated by reconfiguration of the preBötC network37,38 that are followed by a “post-sigh apnea”39. We found that Dbx1 neuron drive potential, AHP, and the refractory period were increased during sigh bursts and relative to normal “eupneic” bursts (Supplementary Fig. 3). Thus we posit that the post sigh apnea results, at least in part, from an exaggerated refractory period due to increased preBötC activity during sighs.

Phase-dependent activity of preBötC inhibitory neurons

Next, we explored the activity of inhibitory preBötC neurons in Vgat-ChR2 slices (Fig. 4a). Individual neurons were identified as Vgat+ with a depolarizing response to blue light, whereas Vgat− neurons typically hyperpolarized, followed by postinhibitory rebound40 coincident with a preBötC population burst at the end of light stimulation (Fig. 4b). Inhibitory preBötC neurons rhythmically active during the inspiratory phase were abundant18. Fourteen out of the 37 (37%) recorded inspiratory neurons were Vgat+ (Fig. 4b), although none had pre-inspiratory activity (Supplementary Fig. 4). PreBötC neurons active during the expiratory phase were much less common41. However, all recorded expiratory neurons (10/10) were Vgat+ (Fig. 4c). Expiratory neurons became inspiratory following blockade of synaptic inhibition (Fig. 4c), indicating that they receive concurrent excitation and inhibition, with inhibition dominating under control conditions42,43,44. Indeed, some inhibitory neurons were silenced during small preBötC bursts but excited during larger bursts or sighs45 (Supplementary Fig. 5).

Fig. 4 PreBötC inhibitory neuron populations have distinct effects on inspiratory burst frequency. a Diagram of Vgat-ChR2 brainstem slice configuration used to identify inhibitory preBötC neuron activity relative to preBötC burst phase. b Representative activity of a Vgat− inspiratory neuron showing post-inhibitory rebound following light stimulation (left) and a Vgat+ neuron with inspiratory activity (right). 14/37 (37%) recorded inspiratory neurons were inhibitory. c Example Vgat+ expiratory neuron at baseline and during blockade of synaptic inhibition with strychnine (1 µM) and gabazine (1 µM) (n = 3). Note that expiratory activity transitions to inspiratory activity following blockade of synaptic inhibition. 10/10 (100%) recorded expiratory neurons were inhibitory. d Example recording of preBötC population activity from a Vgat-ChR2 slice during phase-specific light stimulation. Average data shown in Fig. 5c Full size image

Phasic inhibition elicits rapid preBötC bursting

To examine phase-dependent effects of inhibitory neurons on the ongoing preBötC rhythm, we unilaterally or bilaterally stimulated Vgat+ neurons specifically during inspiration or expiration with light pulses threshold-triggered by preBötC population bursts. While blocking synaptic inhibition slowed the rhythm (see Fig. 3b, e), increasing inhibition optogenetically during inspiration increased preBötC burst frequency (Figs. 4d and 5b, c). Bilateral light stimulation elicited stable and robust high frequency bursting (314 ± 46% increase to 1.37 ± 0.16 Hz), whereas bursting was more irregular and slower on average during unilateral stimulation (93 ± 16% increase to 0.90 ± 0.08 Hz). Importantly, frequency was increased beyond the maximum possible frequency predicted by the refractory period (1 burst/~2 s or 0.5 Hz). During bilateral stimulation of Vgat+ neurons, increases in burst frequency were laser power-dependent, suggesting frequency can be tuned by the amount of inhibitory neuron activation (Supplementary Fig. 6a). These effects were blocked in strychnine and gabazine (Supplementary Fig. 6b). Unlike phasic stimulation during inspiration, activation of inhibitory neurons during the expiratory phase slowed preBötC bursting by −37 ± 7% during bilateral stimulation and −26 ± 4% during unilateral stimulation (Figs. 4d and 5c). Thus inhibition has phase-dependent effects on respiratory frequency23, and reducing the refractory period with inhibition allows a rapid preBötC rhythm.

Fig. 5 Phasic inhibition, but not excitation, drives high preBötC burst frequencies. a Representative inspiratory neuron and preBötC population recording during bilateral light stimulation specifically during the expiratory phase in a Dbx1-ChR2 rhythmic brainstem slice. b Representative inspiratory neuron and preBötC population activity during threshold-triggered bilateral light stimulation specifically during the inspiratory phase in a Vgat-ChR2 slice. c Average changes in frequency in Vgat-ChR2 slices during unilateral and bilateral preBötC stimulation during inspiration (n = 8) and expiration (n = 6) compared to Dbx1-ChR2 stimulation during inspiration (n = 5) and expiration (n = 5; one-way ANOVA and Bonferonni’s multiple comparisons test; *p < 0.001). d Intracellular recording of a Vgat− neuron and preBötC population activity during brief light pulses (200 ms) in a Vgat-ChR2 slice. Note that rebound spiking is reduced during the refractory period. Quantified probability of evoking a population burst via postinhibitory rebound relative to the time of light stimulation following an endogenous burst (0.5 s bins; n = 7 slices, ~100–150 trials/slice; mean ± s.e.m.) compared to the probability of evoked bursts during Dbx1 stimulation (data shown in Fig. 2b), demonstrating similar refractory periods Full size image

Next, we compared these results with the effects of phasic stimulation of excitatory neurons. Dbx1 neuron stimulation during inspiration had very little effect on frequency, with decreases of −10 ± 3% elicited during bilateral stimulation and −10 ± 3% during unilateral stimulation (Fig. 5c). Targeting light stimulation specifically during the expiratory phase only moderately increased burst frequency (Fig. 5a, c). Bilateral stimulation increased frequency by 32 ± 15% to 0.41 ± 0.05 Hz and unilateral stimulation by 23 ± 11% to 0.37 ± 0.03 Hz, levels similar to that predicted by the refractory period. Changes in burst frequency were similar for bilateral and unilateral stimulation of excitatory neurons, and phase-specific stimulations in Vglut2-ChR2 slices yielded similar results (Supplementary Fig. 6c).

Since postinhibitory rebound can trigger preBötC bursts40 (see Fig. 4b), we sought to determine whether this mechanism could overcome the refractory period to drive rapid preBötC bursting. Using Vgat-ChR2 slices, we optically stimulated preBötC inhibitory neurons with light pulses randomly during the respiratory cycle and calculated the probability of evoking a population burst via postinhibitory rebound (Fig. 5d) and the phase shift (Supplementary Fig. 1a). Rebound bursts were rarely generated during the first ~2 s following an endogenous preBötC burst, indicating a refractory period for postinhibitory rebound very similar to that determined for Dbx1 stimulation (Fig. 5d). Thus unless the refractory period is reduced by limiting the activity of excitatory neurons during inspiration, rebound mechanisms per se cannot overcome the refractory period to drive rapid bursting.

PreBötC inhibition is partially mediated by vagal afferents

To explore how excitatory and inhibitory mechanisms contribute to the control of breathing frequency in the intact respiratory network where frequencies are typically much faster (Supplementary Fig. 7), we recorded inspiratory activity from the hypoglossal nerve in anesthetized, spontaneously breathing, optogenetic adult mice with open access to the ventral medullary surface (Fig. 6a). Optogenetic stimulations of preBötC subpopulations were performed before and after transection of the vagal nerves, resulting in a constant slow breathing rate46. Vagal afferents respond to lung stretch and have a phasic inhibitory influence on breathing to prevent over inflation of the lungs (i.e., Breuer–Hering reflex)47. To confirm that transection of the vagal nerves reduces inhibition locally within the preBötC, we compared the effects of vagotomy with bilateral nanoinjection of strychnine (250 µM) and gabazine (250 µM) into the preBötC (Fig. 6b–e). Similar to Janczewski et al.20, but see Marchenko et al.21, both vagotomy and inhibition blockade slowed, but did not stop, breathing and increased XII amplitude. Changes in frequency and XII amplitude were negatively correlated (linear regression, p < 0.0001). Slowed frequency was primarily due to an increase in expiratory time (Te) (154 ± 20%); however, inspiratory time (Ti) was also increased (55 ± 5%). Importantly, nanoinjection of strychnine and gabazine into the preBötC had a larger effect on XII frequency and amplitude than vagotomy (Fig. 6e). Thus, consistent with our results in vitro (see Figs. 3 and 4b), not all preBötC inhibition is mediated by sensory feedback. Application of continuous positive airway pressure to induce lung stretch confirmed that the Breuer–Hering reflex was eliminated following blockade of preBötC inhibition20 (Supplementary Fig. 8). Therefore, activity of vagal afferents ultimately inhibits breathing via inhibitory mechanisms within the preBötC.

Fig. 6 Effects of preBötC sensory feedback inhibition on hypoglossal motor output in vivo. a Schematic of the surgical approach to access the ventral brainstem for bilateral preBötC nanoinjections, photostimulation, and vagotomy. b Representative trace of integrated hypoglossal (XII) nerve activity during bilateral injection of strychnine and gabazine (150 nl, 250 µM each) with frequency and amplitude overlaid (10 s bins). c Example transverse hemisection showing injection site marked by flourospheres localized to the preBötC in a Dbx1-ChR2 mouse. d Representative trace of XII activity during bilateral vagotomy with frequency and amplitude overlaid (10 s bins). e Quantified changes in frequency and amplitude ~5 min following vagotomy (n = 47) and blockade of preBötC fast synaptic inhibition (n = 6; unpaired two-tailed t-tests with Welch’s correction; **p < 0.01, ****p < 0.0001). f Bright field image (left) and Dbx1-ChR2 fluorescence (middle) of the ventral medulla showing the location of preBötC photostimulation relative to the basilar artery (BA), caudal cerebellar artery (CCA), and intersection of the vertebral arteries (VA). Example extracellular recording demonstrating pre-inspiratory activity relative to XII nerve activity at photostimulation sites (right). g Quantified coordinates of injected fluorospheres (bottom) demonstrate that photostimulation sites were consistent across experimental groups (Dbx1, n = 14; Vglut2, n = 14; Vgat, n = 5; Cre-, n = 10; two-way ANOVA and Bonferonni’s post hoc test; n.s., not significant, p > 0.05) Full size image

Injection and photostimulation sites were marked with flourospheres (Fig. 6c, f). We found the optimal location to target the preBötC was 1.35 mm (48%) lateral from the basilar artery, 1.29 mm (47%) caudal to the caudal cerebellar artery, and 0.30 mm (12%) rostral to the intersection of the vertebral arteries. Extracellular recordings in this area revealed inspiratory population activity that occurred prior to XII bursts (Fig. 6f). Imaging of fluorospheres injected at the stimulation site at the end of each experiment confirmed that there were no differences in the stimulation sites between experimental groups (Fig. 6g).

Limitations on excitatory preBötC mechanisms in vivo

To test the ability of excitatory mechanisms to generate rapid breathing in vivo, we recorded hypoglossal activity during a continuous 10 s photostimulation of overlapping glutamatergic Dbx1-ChR2 and Vglut2-ChR2 subpopulations (Fig. 7). Light stimulation in vagus-intact Dbx1-ChR2 mice increased XII nerve burst amplitude (31 ± 3%) but, surprisingly, had almost no effect on breathing frequency (5 ± 2%). In contrast, stimulation in Vglut2-ChR2 mice elicited a moderate increase in both breathing frequency (23 ± 3%) and XII amplitude (24 ± 3%) (Fig. 7a, c). During slow breathing following loss of vagal sensory feedback, continuous light stimulation of the preBötC moderately increased breathing frequency in both Dbx1-ChR2 (24 ± 5%) and Vglut2-ChR2 (21 ± 5%) mice (Fig. 7b, d). Effects on XII amplitude during preBötC photostimulation were abolished in vagotomized mice, indicating that vagotomy minimizes dynamic amplitude responses28.

Fig. 7 Excitatory preBötC neurons have a limited ability to generate rapid breathing in vivo. a, b Representative hypoglossal (XII) nerve activity during a continuous 10 s stimulation of the preBötC in Dbx1-ChR2, Vglut2-ChR2, and Cre- control mice with the vagus intact (a) and following vagotomy (b). c, d Average XII nerve burst frequency and amplitude (1 s bins) during continuous light stimulation with the vagus intact (c) (Dbx1, n = 11; Vglut2, n = 10; Cre−, n = 10) and following vagotomy (d) (Dbx1, n = 10; Vglut2, n = 10; Cre−, n = 8; means ± s.e.m.). e, f Average changes in inspiratory and expiratory time (Ti and Te, respectively) during continuous stimulation in vagus-intact (e) and vagotomized (f) mice demonstrating that changes in breathing frequency are primarily the result of reduced Te (mean ± s.e.m.; two-way ANOVA and Bonferonni’s multiple comparisons test; **p < 0.01, ***p < 0.001, ****p < 0.0001) Full size image

Under both vagus-intact and vagotomized conditions, changes in breathing frequency were reflected in reduced Te (Fig. 7e,f). Results were similar across increasing laser powers (Supplementary Fig. 9). Consistent with our results in vitro (see Fig. 5c), unilateral and bilateral stimulations of excitatory neurons had similar, moderate effects on breathing frequency (Supplementary Fig. 9a), suggesting that there is a ceiling effect for increased breathing frequency elicited by activation of excitatory preBötC neural populations.

Vagal feedback differentially modulates excitatory neurons

To further investigate the differential frequency response observed during continuous stimulation of Dbx1 and Vglut2 neurons, we selectively stimulated the preBötC during the inspiratory or expiratory phase with light pulses threshold-triggered by XII nerve bursts (Fig. 8). With the vagus intact, selective stimulation during inspiration in both Dbx1-ChR2 and Vglut2-ChR2 mice elicited a similar increase in XII amplitude (Dbx1: 22 ± 5%, Vglut2: 19 ± 3) and decrease in breathing frequency (Dbx1: −13 ± 1%, Vglut2: −11 ± 1%) primarily via prolonged Te (Dbx1: 24 ± 1%, Vglut2:16 ± 4%) (Fig. 8a, c). Thus, with vagal feedback intact, increasing the activity of excitatory preBötC neurons during an inspiratory burst delays the subsequent burst and slows breathing. In contrast, during expiratory stimulation, breathing frequency was increased (Dbx1: 15 ± 2%, Vglut2: 28 ± 4%) via a reduction in Te (Dbx1: −24 ± 2%, Vglut2: −42 ± 3%), and the effect was greater in Vglut2-ChR2, compared to Dbx1-ChR2 mice (Fig. 8a, c). It is notable that, for Dbx1 stimulation, the amount frequency was reduced during inspiratory stimulation was nearly equivalent to the amount it was increased during expiratory stimulation, likely leading to the minimal frequency effect of continuous stimulation.

Fig. 8 Differential frequency control of overlapping excitatory preBötC populations is specific to expiratory stimulation in vagus-intact, but not in vagotomized, mice. a, b Representative XII nerve activity during respiratory phase-specific inspiratory or expiratory preBötC stimulation in Dbx1-ChR2 and Vglut2-ChR2 mice with the vagus intact (a) and following vagotomy (b). c, d Average changes in frequency, Ti, Te, and XII burst amplitude during inspiratory and expiratory stimulation in vagus-intact (c) (Dbx1, n = 5; Vglut2, n = 6) and vagotomized (d) (Dbx1, n = 4; Vglut2, n = 4) mice (mean ± s.e.m.; two-way ANOVA and Bonferonni’s multiple comparisons test; *p < 0.05, ***p < 0.001). Note that Vglut2 stimulation only elicits a larger frequency effect than Dbx1 stimulation when sensory feedback inhibition is intact (vagus intact) and when stimulation is specific to the expiratory phase Full size image

During slow breathing following vagotomy46, phase-specific stimulation of either excitatory population during inspiration no longer had an effect on XII frequency or amplitude, suggesting that excitatory neurons are nearly maximally active during preBötC bursts in vagotomized mice (Fig. 8b, d). Similar to results obtained in vagus-intact mice, breathing frequency was increased during expiratory stimulation (Dbx1: 45 ± 6%, Vglut2: 45 ± 11%) due to decreased Te (Dbx1: −37 ± 2%, Vglut2: −39 ± 4%). However, vagotomy eliminated the differential effect of Dbx1-ChR2 and Vglut2-ChR2 stimulation (Fig. 8b, d), suggesting vagal feedback differentially modulates the ability of preBötC excitatory populations to increase breathing frequency specifically during the expiratory phase. Despite increasing frequency by ~40% to 1.0 ± 0.2 Hz, stimulation of either excitatory preBötC subpopulation could not drive breathing to pre-vagotomy frequencies (2.1 ± 0.5 Hz, p < 0.0001), further indicating that excitatory mechanisms alone have a limited ability to drive rapid breathing.

Vagal feedback limits the refractory period

We next sought to characterize the refractory period for breathing in vivo. Unlike in vitro stimulations where bursts were clearly evoked or not, stimulations in vivo typically resulted in a shift in the onset of the subsequent inspiratory burst. Therefore, the change in the cycle duration containing the stimulus relative to previous, unperturbed cycles (i.e., “phase shift”) was used to assess the refractory period in vivo, which was defined as the time between the end of inspiration and the stimulus phase that elicited the maximum phase shift.

In vagus-intact Dbx1-ChR2 mice, we observed a refractory period of ~200 ms (Fig. 9a). However, in Vglut2-ChR2 mice, a phase advance was elicited when stimulation occurred within the first 200 ms following the end of inspiration (Fig. 9a, c). Indeed, the phase shift elicited by Vglut2-ChR2 stimulation was significantly greater than the Dbx1 phase shift during the first 200 ms following inspiration (p < 0.05), while near the end of the respiratory cycle, Vglut2-ChR2 and Dbx1-ChR2 stimulation had equivalent effects (p > 0.05). Following vagotomy, the refractory period was increased and the differential effect of Dbx1-ChR2 and Vglut2-ChR2 stimulation was lost (Fig. 9b, d), such that stimulation did not result in a phase advance for a period of 500–600 ms following the end of inspiration in both Dbx1-ChR2 and Vglut2-ChR2 mice. Near the end of the refractory period, failed bursts were occasionally apparent (arrows in Fig. 9b); and if stimulations occurred later in the respiratory cycle, a phase advance was elicited. Thus loss of sensory feedback inhibition exaggerates the refractory period, thereby slowing breathing frequency.

Fig. 9 Vagal sensory feedback limits the refractory period for excitatory preBötC populations. a, b Example integrated XII nerve activity during brief (100–200 ms) light stimulation at different time points within the respiratory cycle in vagus-intact (a) and vagotomized (b) Dbx1-ChR2, Vglut2-ChR2, and Vgat-ChR2 mice. Note the difference in the refractory period for partially overlapping Vglut and Dbx1 populations in vagus-intact mice. Arrows indicate failed bursts near the end of the refractory period. Traces are scaled to the expected duration of the respiratory cycle (expected phase). c, d Quantified phase shift elicited during stimulations across the respiratory cycle (stimulus phase 0 = beginning of inspiration) in vagus-intact (c) (Dbx1, n = 10; Vglut2, n = 10; Vgat, n = 5; Cre−, n = 9) and vagotomized mice (d) (Dbx1, n = 9; Vglut2, n = 7; Vgat, n = 3; Cre−, n = 9; mean ± s.e.m.). Data from Cre− control mice shown in black. Refractory periods for Dbx1-ChR2 (blue) and Vglut2-ChR2 (green) shown on equivalent time scales relative to the average respiratory cycle duration in vagus-intact and vagotomized conditions. Note the exaggerated refractory period in vagotomized mice and the minimal effect of Vgat-ChR2 stimulation during the refractory period Full size image

Inhibitory neurons promote rapid dynamic breathing

Next, we examined the phase-dependent effects of stimulating inhibitory preBötC neurons in vivo by randomly delivering brief light pulses to the preBötC of Vgat-ChR2 mice (Fig. 9). In both vagus-intact and vagotomized conditions, a large phase advance was elicited by light pulses occurring during inspiration, consistent with our results in vitro. Light pulses during the expiratory phase elicited a phase delay, with longer delays near the end of Te. However, in vagotomized mice, light pulses had little effect during the time following inspiration that corresponded to the refractory period (Note the flat area of the curve in Fig. 9d), suggesting that activity of inhibitory neurons during the refractory period has minimal effects on breathing (also see Fig. 5d). Interestingly, in contrast to in vitro preparations, stimulation of inhibitory neurons during expiration did not evoke postinhibitory rebound in vivo23, calling into question models of respiratory rhythm generation implicating postinhibitory rebound as a driver of rapid breathing based on a very slow non-physiological rhythms generated in vitro40.

Finally, we optogenetically stimulated inhibitory preBötC neurons threshold-triggered by integrated XII nerve activity. If the preBötC was stimulated unilaterally, breathing frequency was increased initially but then became irregular in both vagus-intact and vagotomized mice (Fig. 10a, b), consistent with our observations in vitro. Since the irregularity could reflect discoordination between the left and right preBötC due to far fewer commissural connections between inhibitory versus excitatory neurons48, we stimulated the preBötC bilaterally during inspiration. Bilateral inspiratory stimulation elicited a stable increase in breathing frequency that was larger in vagotomized (403.3 ± 74.2%) versus vagus-intact (140.9 ± 15.4%) mice (Fig. 10c). Increased frequency was due to decreases in both Ti and Te. Importantly, unlike stimulation of excitatory preBötC neurons (see Fig. 8), inspiratory stimulation in vagotomized Vgat-ChR2 mice increased breathing frequency to 4.6 ± 1.0 Hz, far beyond spontaneous frequencies with the vagus intact (1.9 ± 0.3 Hz) and beyond the maximum frequency predicted by the Dbx1 refractory period (~2.5 Hz in vagus-intact and ~1.1 Hz in vagotomized mice). Unilateral stimulation of inhibitory neurons during expiration decreased breathing frequency, whereas bilateral expiratory stimulation caused apnea23 (Fig. 10a, b). Collectively, these data reveal a critical role for inhibition in the generation of high-frequency breathing in vitro and in vivo.