Distinct rhythm generators for inspiration and expiration in the juvenile rat

Abstract

Inspiration and active expiration are commonly viewed as antagonistic phases of a unitary oscillator that generates respiratory rhythm. This view conflicts with observations we report here in juvenile rats, where by administration of fentanyl, a selective mu-opiate agonist, and induction of lung reflexes, we separately manipulated the frequency of inspirations and expirations. Moreover, completely transecting the brainstem at the caudal end of the facial nucleus abolished active expirations, while rhythmic inspirations continued. We hypothesize that inspiration and expiration are generated by coupled, anatomically separate rhythm generators, one generating active expiration located close to the facial nucleus in the region of the retrotrapezoid nucleus/parafacial respiratory group, the other generating inspiration located more caudally in the preBötzinger Complex.

Figure 1

Transition from control to quantal pattern of breathing in an 11-day-old vagotomized rat after fentanyl Top three traces, recordings of tidal volume (V_T), genioglossus muscle electromyographic (EMG_GG) and abdominal muscle electromyographic (EMG_ABD) activity. Bottom, timing bars indicating occurrence of inspiratory airflow (inspiration) and expiratory motor activity (EMG_ABD). A, control recording had all normal (I-E) cycles (i.e. cycles with inspiration and expiration). B, onset of quantal breathing as an occasional sequence of inspiration–expiration–expiration, i.e. E-only cycle (cycle without inspiration), is observed at 9 min after an initial dose of fentanyl (20 μg kg⁻¹s.c.). C, at 4 min after a supplemental dose of fentanyl (10 μg kg⁻¹), there are more E-only cycles. Note split EMG_ABD bursts in I-E cycles, and single EMG_ABD burst in E-only cycles.

Figure 2

Quantal pattern of breathing in an 8-day-old vagotomized rat given fentanyl A, intervals between successive EMG_ABD bursts (circles) and inspirations (squares) for 220 consecutive respiratory cycles: 120 I-E cycles (mean duration of I-E cycles ) and 100 E-only cycles (mean duration of E-only cycles ). B, recording of epoch shown in A (∼(150 s, 190 s); green squares represent normal, i.e. I-E cycles (1, 3, 6, 8, 9), and red circles represent E-only cycles (2, 4, 5, 7). Traces from top to bottom: cycle number, EMG_GG, EMG_ABD, their integrals (∫EMG_GG and ∫EMG_ABD), V_T and respiratory flow. Vertical dotted lines mark the onset of each EMG_ABD burst. E-only cycles differed from I-E cycles; see C, D and text. C, ∫EMG_GG and ∫EMG_ABD on an expanded time scale to show pattern of bursting in an I-E cycle (cycle 1); note that ∫EMG_GG has two distinct components (I-EMG_GG and E-EMG_GG) and can have no delay between them or be separate, e.g. Fig. 3, paired vertical lines. EMG_ABD activity has a two-burst structure. D, comparison of an I-E cycle (cycle 1) and an E-only cycle (cycle 2, red).

Figure 3

Response to continuous positive airway pressure (CPAP) in an 11-day-old rat with intact vagi and transected pons given fentanyl Top traces, V_T (continuous line) with superimposed resting lung volume (dashed line), EMG_GG and EMG_ABD. Paired vertical lines above the V_T trace indicate I-E cycles where termination of an I-EMG_GG burst and onset of subsequent E-EMG_GG burst were separate; this is consistent with distinct sources of these bursts. Bottom, timing bars (see Fig. 1). Initially, inspiratory frequency/expiratory frequency (f_I/f_E) = 0.7, i.e. on average 3 of 10 cycles were E-only. With CPAP = 3 cmH₂O, f_I/f_E= 0, as I-E cycles were completely suppressed. Note that during CPAP the amplitude and duration of the expiratory efforts increased, and V_T generated by ‘passive’ inflations matched that of active inspirations.

Figure 4

Rhythmic lung inflations increased f_E and rhythmic lung deflations increased f_I in a 9-day-old rat with intact vagi given fentanyl in which all cycles were E-only Traces, EMG_ABD, EMG_GG and tracheal pressure (TP). A and B, Breuer-Hering inflation/deflation reflexes before administration of fentanyl. A, inflation reflex triggered by brief lung inflations (upward deflections of TP trace) leads to apnoea and briefly inhibits EMG_ABD activity. B, brief lung deflation triggers an extra inspiration, and sustained lung deflation increases f_I from 92 to 208 min⁻¹. C and D, after administration of fentanyl in a dose that eliminated inspiratory activity, i.e, all cycles were E-only. C, brief lung inflations applied shortly after the beginning of each EMG_ABD burst increased f_E from 11 to 29 bursts min⁻¹ and shortened EMG_ABD bursts from 2.2 to 0.4 s. D, brief lung deflations triggered inspirations (indicated by further downward deflections of TP trace) and terminated ongoing EMG_ABD bursts, e.g. D-1. Note that f_E remained fairly constant despite shortening of EMG_ABD bursts. D-1, the first deflation in D on an expanded time scale. Note that EMG_ABD burst was not terminated until the onset of inspiration.

Figure 5

Response to lung deflation in an 11-day-old rat with intact vagi and transected pons given fentanyl Traces, tracheal pressure (TP), respiratory flow, EMG_ABD, EMG_GG. Bottom, timing bars (see Fig. 1). Arrows mark onset of EMG_ABD bursts. Negative pressure of −1.5 cmH₂O did not change f_E but did increase f_I, increasing f_I/f_E from 0.7 to 1.0. Negative pressure of −3 cmH₂O recruited additional inspirations (triangles/dotted lines). f_I increased leading to f_I/f_E= 2.5. The frequency of I activity locked to EMG_ABD bursts was similar to that of preceding I-E cycles, and was clearly different from the rhythm of inspirations ‘in between’ EMG_ABD bursts (triangles). Note that during lung deflation, EMG_ABD bursts became shorter.

Figure 6

Naloxone increased f_I/f_E in an 11-day-old rat with intact vagi given fentanyl A, intervals between successive EMG_ABD bursts (black circles), and between inspirations preceded (blue squares) or not preceded (red squares) by an EMG_ABD burst. A small dose of naloxone (0.03 mg kg⁻¹s.c.) was injected (arrow) and antagonized fentanyl-induced changes in breathing apparent after ∼300 s (double arrow). Note that naloxone had little effect on f_E, but increased f_I to the point where some inspirations (red squares) occurred in between E-bursts. B, quantal breathing after fentanyl but before naloxone. C, ∼300 s after injection of naloxone, ectopic inspiratory bursts were induced.

Figure 7

Transection rostral to the facial nucleus did not affect breathing pattern, whereas transection at the caudal end of the facial nucleus eliminated EMG_ABD bursts (but not inspirations) in vagotomized rats A, breathing after transection at the level just rostral to the VII nucleus (dotted line, ‘rostral-t’ in C) in rats that spontaneously recovered from quantal pattern of breathing. B, breathing after transection marked as ‘caudal-t’ in C. Only this most caudal transection completely eliminated EMG_ABD bursts. Tonic EMG_ABD activity, inhibited during inspirations, could still be observed. A–1 and B–2, expanded time-scale of flow and V_T during the first cycles of traces in A and B. C, sagittal representation of medulla and pons showing the levels of the most rostral (‘rostral-t’) and caudal (‘caudal-t’) transections. Intermediate transections were also made (see text). C-1 and C-2, medullary sections (40 μm thick) of remaining medulla cut at the same angle as ‘caudal-t’ transection. The first section (C-1), showing the caudal end of the VII nucleus, was modestly damaged, but the next section (C-2), showing rostral end of BötC, was intact in every rat (n = 5). V, motor trigeminal nucleus; VII, facial nucleus; LRN, lateral reticular nucleus; preBötC, preBötzinger Complex; BötC, Bötzinger Complex; pFRG, parafacial respiratory group; RTN, retrotrapezoid nucleus; SO, superior olivary complex; Ac, ambiguus nucleus compact.