Looking for inspiration: new perspectives on respiratory rhythm

Abstract

Recent experiments in vivo and in vitro have advanced our understanding of the sites and mechanisms involved in mammalian respiratory rhythm generation. Here we evaluate and interpret the new evidence for two separate brainstem respiratory oscillators and for the essential role of emergent network properties in rhythm generation. Lesion studies suggest that respiratory cell death might explain morbidity and mortality associated with neurodegenerative disorders and ageing.

Figure 1. Ventral view of en bloc brainstem showing voltage-dependent respiratory neuronal activity

Frames of averaged (∼100 respiratory cycles) optical signals from bath-applied voltage-dependent dye (Di-2-ANEPEQ) reveal two discrete regions of respiratory-modulated neurons in the ventrolateral medulla. Averaging was synchronized to the onset of rhythmic inspiratory burst activity in the C4 ventral nerve root (black trace below each image; burst duration marked by blue bar). The red marker below each frame shows the time of each frame relative to C4 output. One cluster of activity appears at the top (rostral) of each frame ∼400 ms before inspiratory onset, whereas a second more caudal cluster appears ∼100 ms before. The rostral cluster corresponds to the retrotrapezoid nucleus/parafacial respiratory group and the caudal cluster corresponds to the preBötzinger Complex (BOX 2). Reproduced, with permission, from REF. 21.

Figure 2. Opiate agonists induce quantal slowing of inspirations without affecting frequency of active expirations

a | Sequential plots of inspiratory period in vitro before and after treatment with the μ-opiate agonist DAMGO (d-Ala(2),NMePhe(4),Gly-ol(5)enkephalin, red bar). The top trace shows continuous slowing of rhythm in the slice; the bottom trace shows quantal slowing of rhythm en bloc. b | Simultaneous recordings of inspiratory burst activity in control and DAMGO-treated XII nerve and preBötzinger Complex (preBötC) inspiratory neurons (top two pairs of traces) and pre-inspiratory (pre-I) neurons in the retrotrapezoid nucleus/parafacial respiratory group (bottom pair). Arrows in DAMGO traces indicate subthreshold events in preBötC neurons during skipped bursts, which occur at the approximate time expected for inspiratory bursts at control frequency, or unperturbed bursting in pre-I neurons. c | In vivo recordings in juvenile rats following fentanyl injection. Traces on the left show typical recording with normal cycles (1, 3, 6) interspersed with cycles without inspiratory activity (2,4,5). ∫EMG_ABD, integrated abdominal muscle electromyogram (EMG); ∫EMG_GG, integrated genioglossus muscle EMG; flow, air flow (up or down for inspiratory or expiratory air flow, respectively); V_T, tidal volume. Traces on the right show superimposition of ∫EMG_GG and ∫EMG_ABD in a normal cycle (black) compared with an inspiratory skipped cycle (red). Note the marked differences, especially in the ∫EMG_ABD burst. Panels a and b reproduced, with permission, from REF. 19 © (2003) Cell Press. Panel c reproduced, with permission, from REF. 22.

Figure 3. Summary of our view of the gross organ ization of respiratory rhythmogenesis in the brainstem of mammals

We propose that there are two oscillators that are differentially affected by various inputs, such as opiates and lung inflation and deflation. The more rostral oscillator is located in the region of the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) and appears to drive active expiratory activity. It might not be rhythmic in mammals at rest, when there is little or no active expiration. The more caudal oscillator is located in the preBötzinger Complex (preBötC) and appears to drive inspiratory activity. Substance P-saporin (SP-SAP) lesion of preBötC neurokinin 1 receptor (NK1R) neurons disrupts breathing. Transections between the two oscillators disrupt expiratory motor outflow, while inspiratory activity continues unabated. Lung inflation enhances the activity of the expiratory oscillator and depresses the inspiratory oscillator, which serves, ultimately, to reduce lung volume, whereas lung deflation has the opposite effect on the inspiratory and expiratory oscillators. We further speculate that the mechanism of rhythmogenesis in the preBötC involves a group pacemaker. However, this model does not resolve the question of why lesions of the preBötC NK1R neurons in adult rats should disrupt rhythm and render breathing ineffective.

Figure 4. Lesions of the preBötzinger Complex significantly disturb breathing pattern in an unrestrained, unanaesthetized adult rat

SP-SAP was injected bilaterally into the preBötzinger Complex (preBötC) and by (or shortly after) day 10 post-injection had killed >80% of preBötC neurokinin 1 receptor (NK1R) neurons. Under control conditions, breathing is continuous throughout the sleep–wake cycle, as determined from electroencephalogram (EEG) and neck electromyogram (EMG) signals (not shown). However, on day 8 after SP-SAP administration breathing pattern ceases to be normal during REM (rapid eye movement) sleep, when apnoea develops and ventilation is restored only on waking, a cycle that then repeats. DIA_EMG, integrated diaphragmatic EMG; I_amp, inspiratory amplitude; NREM, non-REM sleep; W, wakefulness. Reproduced, with permission, from REF. 56.

Figure 5. Group-pacemaker hypothesis of respiratory rhythm generation

The membrane potential of a rhythm-generating neuron is shown (V_M, top trace) with network activity, represented by XII motor output (XII, bottom). Images to the right of the traces depict neuronal activity at different stages of the cycle. 1 is the refractory state that follows inspiration, in which activity-dependent outward currents depress membrane potential, and excitatory synapses in the network are inactive. During epoch 2, the most excitable neurons recover from post-burst hyperpolarization and begin to spike at a low rate. By 3 these highly excitable cells begin to synaptically activate other neurons, leading to aggregation of network activity itself due to recurrent synaptic excitation — a positive-feedback process. The inspiratory burst (4) ensues once a critical number of cells in the network are activated by recurrent excitation. In this final step, synaptic inputs recruit burst-generating intrinsic currents such as Ca²⁺-activated nonspecific cationic current (I_CAN) and persistent Na⁺ current (I_NaP), which give rise to large inspiratory burst potentials with high-frequency spike activity. Inspiratory bursts terminate owing to intrinsic properties of cells that can reverse the positive feedback process, including Ca²⁺-dependent K⁺ currents and electrogenic pumps, which are recruited by cationic influx during inspiration.