Synaptic Depression Influences Inspiratory-Expiratory Phase Transition in Dbx1 Interneurons of the preBötzinger Complex in Neonatal Mice

Abstract

The brainstem preBötzinger complex (preBötC) generates the rhythm underlying inspiratory breathing movements and its core interneurons are derived from Dbx1-expressing precursors. Recurrent synaptic excitation is required to initiate inspiratory bursts, but whether excitatory synaptic mechanisms also contribute to inspiratory-expiratory phase transition is unknown. Here, we examined the role of short-term synaptic depression using a rhythmically active neonatal mouse brainstem slice preparation. We show that afferent axonal projections to Dbx1 preBötC neurons undergo activity-dependent depression and we identify a refractory period (∼2 s) after endogenous inspiratory bursts that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1 preBötC neurons. We demonstrate that the duration of the refractory period-but neither the cycle period nor the magnitude of endogenous inspiratory bursts-is sensitive to changes in extracellular Ca(2+). Further, we show that postsynaptic factors are unlikely to explain the refractory period or its modulation by Ca(2+). Our findings are consistent with the hypothesis that short-term synaptic depression in Dbx1 preBötC neurons influences the inspiratory-expiratory phase transition during respiratory rhythmogenesis.

Significance statement: Theories of breathing's neural origins have heretofore focused on intrinsically bursting "pacemaker" cells operating in conjunction with synaptic inhibition for phase transition and cycle timing. However, contemporary studies falsify an obligatory role for pacemaker-like neurons and synaptic inhibition, giving credence to burst-generating mechanisms based on recurrent excitation among glutamatergic interneurons of the respiratory kernel. Here, we investigated the role of short-term synaptic depression in inspiratory-expiratory phase transition. Until now, this role remained an untested prediction of mathematical models. The present data emphasize that synaptic properties of excitatory interneurons of the respiratory rhythmogenic kernel, derived from Dbx1-expressing precursors, may provide the core logic underlying the rhythm for breathing.

Keywords: breathing; central pattern generator; oscillation; respiration.

Figure 1.

Rhythmically active slices expose Dbx1 preBötC neurons. A, Rostral slice surface from a Dbx1^CreERT2;Rosa26^tdTomato mouse pup showing hypoglossal motor nucleus (XII), semicompact division of the nucleus ambiguus (scNA), and the principal loop of the inferior olive (IO_loop) sites colocalized with the preBötC (left). Dotted box marks the preBötC. B, Whole-cell recordings in Dbx1^CreERT2;Rosa26^tdTomato (top) and Dbx1^CreERT2;Rosa26^{thChR2-tdTomato} (bottom) mouse slices. Shown are tdTomato (left), Dodt contrast microscopy (middle), and Alexa Fluor 488 introduced via patch pipette (right). C, Inspiratory bursts in the Dbx1 preBötC neuron from B (top) with XII motor output.

Figure 2.

Activity-dependent synaptic depression in Dbx1 preBötC neurons. A, Evoked EPSPs in response to 5 Hz electrical stimulation of midline-crossing axons (top); group data from eight Dbx1 preBötC neurons (time synced) are also shown (bottom). Red circles show EPSP amplitudes; black bars show mean ± SD. B, Relative frequency of failures to evoke an EPSP as a function of pulse number.

Figure 3.

Light-evoked inspiratory-like bursts in Dbx1^CreERT2;Rosa26^{hChR2-tdTomato} mouse slices. A, Pharmacology of evoked bursts. Time calibration applies to A. B, Laser pulses delivered at increasing intervals after endogenous inspiratory bursts. Voltage calibration applies to A and B. C, Burst amplitude and area plotted versus the time interval between the endogenous inspiratory burst and stimulus onset. Endogenous control bursts are plotted at the 0 s tick. Time calibration for B and C is the abscissa.

Figure 4.

sEPSPs, dendritic AMPA pulses, and extracellular Ca²⁺ modulation of the refractory period. A, sEPSPs before (magenta) and after (cyan) an endogenous inspiratory burst and the cumulative probability histogram for sEPSP amplitude. Calibrations apply to A. B, Postsynaptic responses to repetitive dendritic AMPA pulses separated by 4, 3, 2, 1, or 0.5 s (left). The amplitude of the second (black) or fifth (gray) postsynaptic response, normalized to the first response, is plotted for each time interval (right). Bars show means. Voltage calibration applies to B; time calibration can be inferred by AMPA pulse interval timing. C, Minimum refractory period after an endogenous inspiratory burst (e.g., Fig. 3B) plotted for different [Ca²⁺]_o (Ca). Voltage and time calibrations apply to Ca. Refractory period (Cb), respiratory cycle period (Cc), endogenous inspiratory burst amplitude and area (Cd), and input resistance (Ce) are plotted for each [Ca²⁺]_o. Symbols show individual experiments; solid lines represent sample means.