Pacemakers handshake synchronization mechanism of mammalian respiratory rhythmogenesis

Abstract

Inspiratory and expiratory rhythms in mammals are thought to be generated by pacemaker-like neurons in 2 discrete brainstem regions: pre-Bötzinger complex (preBötC) and parafacial respiratory group (pFRG). How these putative pacemakers or pacemaker networks may interact to set the overall respiratory rhythm in synchrony remains unclear. Here, we show that a pacemakers 2-way "handshake" process comprising pFRG excitation of the preBötC, followed by reverse inhibition and postinhibitory rebound (PIR) excitation of the pFRG and postinspiratory feedback inhibition of the preBötC, can provide a phase-locked mechanism that sequentially resets and, hence, synchronizes the inspiratory and expiratory rhythms in neonates. The order of this handshake sequence and its progression vary depending on the relative excitabilities of the preBötC vs. the pFRG and resultant modulations of the PIR in various excited and depressed states, leading to complex inspiratory and expiratory phase-resetting behaviors in neonates and adults. This parsimonious model of pacemakers synchronization and mutual entrainment replicates key experimental data in vitro and in vivo that delineate the developmental changes in respiratory rhythm from neonates to maturity, elucidating their underlying mechanisms and suggesting hypotheses for further experimental testing. Such a pacemakers handshake process with conjugate excitation-inhibition and PIR provides a reinforcing and evolutionarily advantageous fail-safe mechanism for respiratory rhythmogenesis in mammals.

Fig. 1.

Pacemakers handshake model of respiratory rhythm generation in neonates. (A) Pacemaker-like neurons in the pFRG and preBötC constitute the putative ERG and IRG (expiratory and inspiratory rhythm generators). EPG and IPG are expiratory and inspiratory pattern generators. PreBötC inhibits pFRG pacemaker (presumably via some inhibitory interneuron, data not shown) during the inspiratory phase and induces a PIR during the post-I phase. A post-I feedback provides a putative “inspiratory off-switch” through direct or indirect synaptic inhibition of the preBötC. (B and C) Model simulations of pFRG and preBötC membrane potential trajectories with alternating pre-I, inspiratory, and post-I bursts evidencing a forward handshake sequence. PIR is robustly induced with inhibitory synapse reversal potentials of −94 mV in B and −53mV in C. Int. preBötC, integrated preBötC activity. (D) Corresponding intrinsic pFRG membrane potential trajectories without preBötC inhibition. Duration of an uninterrupted pFRG burst (562 ms) is comparable with those of the PIR-induced post-I bursts in B (757 ms) and C (542 ms).

Fig. 2.

Criticality of pFRG and preBötC phase resetting by brief pFRG stimulation. The model accurately simulates the experimentally observed inspiratory–expiratory phase resetting elicited by pFRG stimulation (7, 8, 40). (A) pFRG stimulation (amplitude, 10 pA; duration, 40 ms) within 60% of the preBötC cycle (T_s ≤ 60% of T_c) resets the expiratory rhythm and prolongs the stimulated expiration. (B) Stimulation at T_s = 67% evokes a premature preBötC burst that terminates the expiratory phase and resets the inspiratory phase. (C) Summary plots showing the time-critical nature of preBötC phase resetting predicted by the model. T_SE, duration of the stimulated expiration. T_PSE, duration of the poststimulus expiration.

Fig. 3.

Model simulation of opioid-induced fractional quantal slowing of breathing. (A) preBötC bursts are skipped whenever the random excitatory transmission to the depressed preBötC fails to reach the firing threshold, as indicated by the oblique arrows and shaded vertical bars. Note that the durations of the pre-I and post-I bursts for nonskipped cycles vary randomly from cycle to cycle but always in opposite directions, i.e., a shorter pre-I is followed by a longer post-I. (B) Quanta of preBötC cycle duration for varying skipped cycles (n = 0, 1, 2, 3). Note that the quantal increments of cycle duration (T₁, T₂, … with T_n ≈ n·T₁ and T₁ < T_ctrl) are fractional instead of integer multiples of the normal cycle duration T_ctrl. (C) Histogram of skipping 0–3 preBötC bursts in the simulation compared with similar experimental data derived from figure 2 in ref. . (D) Skipped cycle duration expressed as a percentage of normal cycle duration (T₁/T_ctrl ≈70% in simulation, compared with the experimental value of ≈74% from figure 2 in ref. 12).

Fig. 4.

pFRG initiation and preBötC veto/promotion of handshake. (A) Durations of the pre-I burst when the preBötC is silent (E_{Leak-preBötC} = −61mV as in Figs. 1–3) and when it is spontaneously bursting (E_{Leak-preBötC} = −59mV). (B) Model prediction of pFRG-preBötC phase locking with full handshake sequence present and the lack of phase locking during quantal breathing (preBötC-depressed) or ectopic breathing (preBötC-stimulated). The thick lines indicate the predicted relations when the preBötC is not depressed. Normally, the preBötC's spontaneous burst frequency is lower than the pFRG's (f_preBötC < f_pFRG); hence the inspiratory rhythm is always entrained by the expiratory rhythm to the same frequency (f_I/f_E = 1). During quantal breathing the pre-I input is occasionally vetoed by the preBötC for lack of excitability and hence f_I/f_E < 1. When the preBötC network is excited such that f_preBötC > f_pFRG (while the pFRG remains fairly excitable to entrain the preBötC), the inspiratory rhythm has two components: a weak, free-running ectopic rhythm (identity line) produced by the asynchronous preBötC network and a “normal” rhythm that remains phased-locked to the expiratory rhythm (horizontal line). These model predictions are in agreement with experimental observations reported in ref. .

Fig. 5.

Simulation of no, reverse, and half handshakes when the preBötC is more excitable than the pFRG. Simulation settings are: E_{Leak-preBötC} = −57.0 mV, g_I = 0.5 nS. (A) No handshake. When the pFRG is sufficiently depressed (E_Leak-pFRG ≤ −64.0 mV) the preBötC becomes the only active pacemaker that drives the respiratory rhythm. (B) Reverse handshake. When the pFRG excitability is partially restored (E_Leak-pFRG = −63.2 mV) the driving preBötC burst may induce PIR of the pFRG which, in turn, triggers a second preBötC burst in the absence of post-I inhibition of the preBötC. This peculiar phenomenon occurs only within a narrow band of depressed pFRG excitability relative to preBötC and hence, is rarely discernible experimentally (although a wider band may be possible in full-blown pFRG and preBötC population networks). (C) Half handshake. At even less depressed pFRG excitability levels (E_Leak-pFRG = −62.3 mV) the pFRG-triggered preBötC burst may induce a delayed PIR of the pFRG which, in turn, triggers a new preBötC burst, and so on.