Interactions between respiratory oscillators in adult rats

Robert TR Huckstepp; Lauren E Henderson; Kathryn P Cardoza; Jack L Feldman

doi:10.7554/eLife.14203

. 2016 Jun 14;5:e14203. doi: 10.7554/eLife.14203

Interactions between respiratory oscillators in adult rats

Robert TR Huckstepp ¹, Lauren E Henderson ¹, Kathryn P Cardoza ¹, Jack L Feldman ^1,^*

Editor: Jan-Marino Ramirez²

PMCID: PMC4907693 PMID: 27300271

Abstract

Breathing in mammals is hypothesized to result from the interaction of two distinct oscillators: the preBötzinger Complex (preBötC) driving inspiration and the lateral parafacial region (pF_L) driving active expiration. To understand the interactions between these oscillators, we independently altered their excitability in spontaneously breathing vagotomized urethane-anesthetized adult rats. Hyperpolarizing preBötC neurons decreased inspiratory activity and initiated active expiration, ultimately progressing to apnea, i.e., cessation of both inspiration and active expiration. Depolarizing pF_L neurons produced active expiration at rest, but not when inspiratory activity was suppressed by hyperpolarizing preBötC neurons. We conclude that in anesthetized adult rats active expiration is driven by the pF_L but requires an additional form of network excitation, i.e., ongoing rhythmic preBötC activity sufficient to drive inspiratory motor output or increased chemosensory drive. The organization of this coupled oscillator system, which is essential for life, may have implications for other neural networks that contain multiple rhythm/pattern generators.

DOI: http://dx.doi.org/10.7554/eLife.14203.001

Research Organism: Rat

eLife digest

Mammals breathe air into and out of their lungs to absorb oxygen into the body and to remove carbon dioxide. The rhythm of breathing is most likely controlled by two groups of neurons in a part of the brain called the brain stem. One group called the preBötzinger Complex drives breathing in (inspiration), and normally, breathing out (expiration) occurs when the muscles responsible for inspiration relax. The other group of neurons – known as the lateral parafacial region – controls extra muscles that allow us to increase our breathing when we need to, such as during exercise.

Huckstepp et al. set out to determine how these two groups of neurons interact with one another in anesthetized rats to produce a reliable and efficient pattern of breathing. The experiments provide further evidence that inspiration is mainly driven by the preBötzinger Complex. Whilst activity from the lateral parafacial region is needed to cause the rats to breathe out more forcefully than normal, a second low level of activity from another source is also required. This source could either be the preBötzinger Complex, or some unknown neurons that change their activity in response to the levels of oxygen and carbon dioxide in the blood or fluid of the brain.

Further investigation is required to identify how these interactions go awry in diseases that affect breathing, such as sleep apneas.

DOI: http://dx.doi.org/10.7554/eLife.14203.002

Introduction

Coupled oscillator neural networks driving behavior are widespread, e.g., for swimming (Grillner, 2003), and locomotion (Goulding, 2009; Talpalar et al., 2013). Amongst complex and vital behaviors in mammals, breathing, an exceptionally reliable and continuous behavior throughout postnatal life, is one that we may be closest to understanding (Feldman and Kam, 2015). Not only do we know the location of the neural microcircuits that generate respiratory rhythm, but we also have direct, accurate and reliable behavioral measures of the output, i.e., breathing. We hypothesize that the respiratory rhythm central pattern generator (CPG) in mammals is comprised of two oscillators (Feldman et al., 2013): inspiratory rhythm originates in the preBötzinger Complex (preBötC) in the ventrolateral medulla (Smith et al., 1991) and active expiratory rhythm originates in the rostral medulla ventrolaterally adjacent to the facial nucleus (parafacial lateral region; pF_L) (Pagliardini et al., 2011; Huckstepp et al., 2015).

Two parafacial regions that potentially overlap and whose anatomical descriptions are incomplete and ambiguous, the retrotrapezoid nucleus (RTN) and parafacial respiratory group/embryonic parafacial (pFRG/e-PF), contain overlapping subpopulations neurons that express the neurokinin-1 receptor (NK1R), the homeobox gene Phox2b, and the glutamate transporter 2 (VGlut2) (Nattie and Li, 2002; Mulkey et al., 2004; Onimaru et al., 2008; Thoby-Brisson et al., 2009; Ramanantsoa et al., 2011). Previously, we used unbiased descriptors to partition the ventral and lateral parafacial regions, designating them as parafacial lateral (pF_L) and parafacial ventral (pF_V). These parafacial regions are chemosensitive (Mulkey et al., 2004; Onimaru et al., 2008; Marina et al., 2010; Onimaru et al., 2012), but can be functionally separated by: i) their contribution to active expiration, with the pF_V providing drive to expiration (Huckstepp et al., 2015; Silva et al., 2016) and the pF_L containing a presumptive expiratory rhythm generator (Pagliardini et al., 2011; Huckstepp et al., 2015), and; ii) the presence of neurons with respiratory rhythmic behavior in pF_L (or presumptively equivalent areas) (Onimaru and Homma, 2003; Thoby-Brisson et al., 2009; Pagliardini et al., 2011).

In mammals at rest, during wakefulness and sleep, when active breathing movements are primarily inspiratory, generation of the underlying rhythm appears driven by the preBötC. As metabolic demand increases, e.g., during exercise, the pF_L appears to turn on to produce active expiration. Thus, while breathing is a unified act of inspiratory and expiratory airflow, we postulate this behavior results from the coordinated interaction of two anatomically and functionally distinct oscillators (Mellen et al., 2003; Janczewski and Feldman, 2006; Pagliardini et al., 2011; Huckstepp et al., 2015). To understand the generation and control of respiration we need to determine how these two oscillators interact. For example, in adult rats the preBötC can generate inspiratory rhythm while the pF_L is quiescent (Pagliardini et al., 2011; Huckstepp et al., 2015). Is the converse true?

To investigate their independent and interactive functions, we used a pharmacogenetic approach to selectively inhibit the preBötC and/or activate the normally quiescent pF_L. We bilaterally transfected the preBötC with the G_i/o-coupled allatostatin receptors (AlstR), which when activated by allatostatin (Alst) silences transfected preBötC neurons (Tan et al., 2008). In the same rats, we bilaterally transfected the pF_L with the G_q-coupled HM₃D DREADD receptor (HM₃DR) that when activated by clozapine-N-oxide (CNO) depolarizes (Armbruster et al., 2007) transfected pF_L neurons. We slowed respiration in a controlled manner by titrating the dose of Alst applied to the AlstR-transfected preBötC, allowing us to examine the dynamics of this presumptive coupled oscillator system as we shifted the balance of activity from the preBötC to the pF_L. Depressing preBötC activity resulted in quantal slowing of breathing, similar to slowing of breathing following opiate depression of preBötC activity in vitro and in juvenile rats (Mellen et al., 2003; Janczewski and Feldman, 2006). As preBötC activity waned, burstlets appeared on inspiratory muscle electromyograms (EMGs) and in airflow, consistent with our postulate that burstlets, not bursts, in the preBötC are rhythmogenic (Kam et al., 2013). In complementary experiments, we applied CNO to the HM₃DR transfected pF_L to activate this normally quiescent oscillator. We confirmed that pF_L activation initiates active expiration (Pagliardini et al., 2011; Huckstepp et al., 2015). By combining these protocols, i.e., silencing the preBötC while simultaneously driving the pF_L, we removed some confounding factors from these initial experiments. Importantly, we found that active expiration could not be induced when preBötC inspiratory driven motor activity was suppressed and chemosensory drive was absent, indicating that in adult rats active expiration is driven by the pF_L but requires an additional source of network excitation such as ongoing preBötC activity or chemosensory drive. The organization of this coupled oscillator system may have implications for other neural networks that contain multiple central pattern generators.

Results

Viral targeting of the preBötC and pF_L

We made two pairs of viral injections in each adult rat, i.e., bilateral injections into the preBötC (Figure 1A,B) and into the pF_L (Figure 1C,D). In histological sections the preBötC is defined as the neurokinin-1 receptor (NK1R) dense area ventral to the semi-compact nucleus ambiguous (Figure 1A,B) and the pF_L is defined as the area ventral to the lateral edge of the facial nucleus, juxtaposed to the spinal trigeminal tract (Figure 1C,D) (Huckstepp et al., 2015). In representative 40 µm sections: from preBötC injection sites, 161 ± 42, representing 82 ± 6%, neurons expressed GFP (n=3); from pF_L injection sites, 112 ± 31, representing 81 ± 3%, neurons expressed mCitrine (n=3). Transfection sites ranged from ~350–600 µm in diameter. As the responses to Alst and CNO did not differ between the largest and smallest injections sites, the effects of receptor activation were due to silencing or driving of the preBötC and pF_L respectively and not to spread of virus to neighboring regions. We found no labeling of neurons in regions of the medulla other than within the injection sites (data not shown). In particular, we found no fluorescent reporters, i.e., eGFP or mCitrine, in the Bötzinger complex (BötC; Figure 1E,F).

Silencing the preBötC initially slowed breathing frequency (f) and induced active expiration, but eventually resulted in apnea

Hyperpolarizing preBötC AlstR-transfected neurons reduced f, ultimately progressing to apnea (Tan et al., 2008). In anesthetized adult rats at rest transfected with AlstR in the preBötC (n=8; Figure 2), Alst injected bilaterally into the preBötC initially decreased f (42 iqr 20 to 22 iqr 11 s; p=0.008), increased T_I (0.4 iqr 0.1 to 0.5 iqr 0.2 s; p=0.04) and T_E (1.0 iqr 0.7 to 3.2 iqr 1.0 s; p=0.008), decreased V_T (4.6 iqr 0.7 to 2.4 iqr 0.8 mL; p=0.008) and ∫Dia_EMG (25 iqr 13 to 15 iqr 4 arbitrary units (a.u.); p=0.8), and did not alter ∫GG_EMG (8.6 iqr 9.8 to 11.1 iqr 13.7 a.u.; p=0.008). When rats became mildly hypercapnic (ETCO₂ increased from 39.7 iqr 5.4 to 43.4 iqr 10.6 mm Hg; p=0.008), they exhibited expiratory-modulated ∫Abd_EMG (0.3 iqr 0.2 to 9.7 iqr 9.3 a.u., e,g., Figure 2Aii, Bii), the signature of active expiration (Pagliardini et al., 2011). After inspiratory activity ceased (10.4 iqr 2.7 mins post Alst injection) and rats became severely hypercapnic (ETCO₂ 57.8 iqr 22.3 mm Hg), ∫Abd_EMG briefly became tonic (17 iqr 5 s.; Figure 2Aiii, Biii) before becoming silent, i.e., no activity.

Figure 2. — (A) Effect of Alst application to left preBötC (unilateral, first red arrow and dashed line) then right preBötC (bilateral, second red arrow and dashed line); gray arrow and dashed line mark onset of mechanical ventilation. (B) Expanded traces from A, indicated by shaded epochs (i-iii): (Bi) Activity at rest. (**Bii**) and (**Biii**) Activity following bilateral Alst injection. (C) Comparison between ventilation in rats at rest (Rst) and following Alst: (Ci) before Alst had taken full effect (≈Bii). (**Cii**) After Alst had taken full effect (≈Biii). Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest parameter, i.e., f, T_I, T_E, V_T, ∫GG_EMG, ∫Dia_EMG, or ∫Abd_EMG, value regardless of whether it belonged to control or Alst group. f – frequency, T_I – inspiratory period T_E, – expiratory period, V_T – tidal volume, ∫GG_EMG – integrated genioglossus electromyogram, ∫Dia_EMG – integrated diaphragm electromyogram, ∫Abd_EMG – integrated abdominal electromyogram.

**DOI:** http://dx.doi.org/10.7554/eLife.14203.004

The decrease in f was not continuous (n=8; Figures 2Bii, 3A–C), but instead quantal (Mellen et al., 2003). In a representative example (Figure 3C), kernel density plot estimations determined the optimal bandwidth, i.e., bin size, to be 0.44 s and revealed a multimodal distribution with 3 peaks: control respiratory periods were ~2.1 s, and after Alst injections into the preBötC, respiratory periods increased by quantal multiples of this baseline, i.e., 4.2 and 6.3 s (Figure 3Ci). During longer periods, low amplitude activity, which was not present during eupnea, appeared at times when normal breaths were expected (n=8; Figure 3A,B red arrows). This low amplitude Dia_EMG and airflow activity are postulated to represent the inspiratory motor outflow manifestation of preBötC burstlets (Kam et al., 2013), i.e., low levels of rhythmogenic neural population activity in the preBötC that under normal conditions occur in the absence of motor output, being transmitted to motoneuron pools (see Figure 7A in Kam et al., 2013). Ultimately, inspiratory activity ceased, i.e., no phasic ∫Dia_EMG or ∫GG_EMG, and expiratory-related ∫Abd_EMG, i.e., active expiration, terminated, resulting in apnea (n=8; Figure 2; p=0.008 for all variables).

We tested for nonspecific effects of Alst injections in non-transfected rats (n=8; Figure 4). Injection of Alst into the preBötC did not alter f (47 iqr 12 to 54 iqr 12 s; p=0.1), T_I (0.3 iqr 0.0 to 0.3 iqr 0.0 s; p=0.1), T_E (1.0 iqr 0.3 to 0.8 iqr 0.3 s; p=0.3), V_T (4.7 iqr 1.3 to 4.7 iqr 1.3 mL; p=0.3), ∫Dia_EMG (34 iqr 13 to 34 iqr 14 a.u.; p=0.5), ∫GG_EMG (14 iqr 11 to 13 iqr 10 a.u.; p=0.8), nor induce expiratory-related ∫Abd_EMG (0.3 iqr 0.1 to 0.3 iqr 0.1 a.u.; p=0.3). As there were no effects of Alst in the absence of AlstRs, the cessation of inspiratory activity and termination of expiratory-related ∫Abd_EMG following injections of Alst into the preBötC of transfected rats resulted from inactivation of AlstR-transfected preBötC neurons.

Activation of the pF_L leads to active expiration

As we previously found nonspecific effects of CNO at a concentration of 100 µM (Huckstepp et al., 2015), we tested for nonspecific effects of 90 µM CNO in non-transfected rats (n=8; Figure 5). 90 µM CNO did not alter f (54 iqr 11 to 53 iqr 9 s; p=0.6), T_I (0.3 iqr 0.0 to 0.3 iqr 0.0 s; p=0.2), T_E (0.8 iqr 0.3 to 0.8 iqr 0.2 s; p=0.6), V_T (4.6 iqr 1.5 to 4.7 iqr 1.6 mL; p=0.5), Dia_EMG (33 iqr 13 to 34 iqr 14 a.u.; p=1.0), GG_EMG (13 iqr 12 to 13 iqr 13 a.u.; p=0.1), nor induce expiratory-related ∫Abd_EMG (0.3 iqr 0.1 to 0.3 iqr 0.1 a.u.; p=0.3). There were no effects of 90 µM CNO in the absence of HM₃DRs.

Figure 5. — (A) Effect of CNO applied to ventral surface (pink shaded area). (B) Expanded traces from A, indicated by shaded epochs (**i-–ii**): (Bi) Activity at rest. (**Bii**) Activity in presence of CNO. (C) Comparison between ventilation in rats at rest and in presence of CNO. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, ∫GG_EMG, ∫Dia_EMG, or ∫Abd_EMG regardless of whether it belonged to control or CNO group. Abbreviations defined in Figure 2.

**DOI:** http://dx.doi.org/10.7554/eLife.14203.007

We predicted that depolarizing pF_L neurons would elicit active expiration, similar to the effect of their disinhibition or optogenetic photoactivation (Pagliardini et al., 2011). In anesthetized adult rats at rest transfected with HM₃DRs in the pF_L (n=8; Figure 6), CNO (90 µM) increased f (37 iqr 12 to 42 iqr 7 s; p=0.04), did not alter T_I (0.3 iqr 0.1 to 0.3 iqr 0.1 s; p=0.8) decreased T_E (1.3 iqr 0.6 to 1.1 iqr 0.3 s; p=0.02), increased V_T (3.9 iqr 1.3 to 4.2 iqr 1.3 mL; p=0.02), ∫Dia_EMG (25 iqr 12 to 27 iqr 12 a.u.; p=0.02), and ∫GG_EMG (7.6 iqr 6.6 to 8.4 iqr 5.6 a.u.; p=0.04), and induced expiratory-related ∫Abd_EMG (0.3 iqr 0.2 to 3.0 iqr 4.0 a.u.; p=0.02). Thus depolarizing pF_L neurons altered respiration in a similar manner to optogenetic photoactivation (Pagliardini et al., 2011) for all measured variables common to both studies. We conclude that depolarization of HM₃DR-transfected pF_L neurons resulted in active expiration.

Figure 6. — (A) Effect of CNO applied to ventral surface (pink shaded area). (B) Expanded traces from A, indicated by grey shaded epochs (**i-ii**): (Bi) Activity at rest. (**Bii**) Activity in presence of CNO. (C) Comparison between ventilation in rats at rest and in presence of CNO. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for that parameter, i.e., f, T_I, T_E, V_T, ∫GG_EMG, ∫Dia_EMG, or ∫Abd_EMG regardless of whether it belonged to control or CNO group. Abbreviations defined in Figure 2.

**DOI:** http://dx.doi.org/10.7554/eLife.14203.008

Silencing the preBötC during active expiration leads to apnea

The preBötC can generate inspiratory rhythm while the pF_L is quiescent (Pagliardini et al., 2011). Can the pF_L generate a respiratory rhythm when the preBötC is quiescent? If active expiration is independent of inspiratory activity, then active expiration driven by activation of HM₃DRs in pF_L should persist after cessation of inspiratory activity resulting from injection of Alst in the preBötC. In anesthetized adult rats with an active expiratory breathing pattern induced by CNO, injection of Alst into the preBötC led to a significant decrease in respiratory activity (n=8; p=0.008 for all variables; Figure 7), ultimately resulting in apnea, with no rhythmicity in V_T, ∫Dia_EMG, ∫GG_EMG, and ∫Abd_EMG. After rhythmic inspiratory activity ceased, ∫Abd_EMG continued tonically for a short duration (15 iqr 12 s; Figure 7A,Bii) before disappearing, which was not different from the duration of tonic ∫Abd_EMG following the onset of apnea in the absence of CNO (Figure 2Biii; p=0.7). When inspiratory activity stopped, rats were severely hypercapnic (ETCO₂ 64.6 iqr 14.5 mm Hg; Figure 7Bii), this was not different from the hypercapnia at the onset of apnea in the absence of CNO (ETCO₂ 57.8 iqr 22.3 mm Hg; p=0.8). Rats were mechanically ventilated to restore CO₂ and O₂ levels to within the normal range (Figure 7). To assess the state of the respiratory CPG the mechanical ventilator was intermittently turned off. Shortly after allatostatin injections into the preBötC, no respiratory activity was seen following cessation of mechanical ventilation, and rats were placed back on the ventilator. As the time from allatostatin injection in the preBötC increased, removal from the ventilator led to brief periods of spontaneous breathing. When spontaneous breathing occurred, the return of inspiratory activity always preceded the return of expiratory abdominal activity; active expiration did not return until V_T reached 3.2 iqr 1.7 mL, ∫Dia_EMG reached 13.1 iqr 8.2 a.u., and ∫GG_EMG reached 5.2 iqr 4.2 a.u. (n=8; Figure 8). That is, at low levels of reinitiated inspiratory activity there was no active expiration, which appeared only after inspiratory activity reached a higher value. If rats were able to spontaneously breathe without ventilation, they were allowed to do so. However, most early periods of spontaneous breathing deteriorated, and when rats became apneic again they were re-ventilated; in 5 of 6 rats spontaneous breathing occurred during ventilation, and in the remaining rat, spontaneous breathing following removal from the ventilator was sustained. When spontaneous breathing occurred during ventilation the return of inspiratory activity always preceded the return of expiratory abdominal activity. Therefore, following silencing of the preBötC with Alst in the presence of CNO to drive the pF_L, at no time during the re-initiation of spontaneous breathing, either with or without mechanical ventilation, did active expiration occur in the absence of inspiratory activity and chemosensory drive.

Figure 7. — (A) Integrated traces from a single experiment showing effect of Alst injection to left preBötC (unilateral, first red arrow and dashed line) then right preBötC (bilateral, second red arrow and dashed line), in presence of CNO (pink shaded area); gray arrow and dashed line mark onset of mechanical ventilation. (B) Expanded traces from A indicated by shaded epochs: (Bi) In presence of CNO only. (**Bii**) Following Alst in presence of CNO. (C) Comparison between ventilation in rats in presence of CNO and following Alst in presence of CNO. Lines connect data from individual experiments, box and whisker plots show combined data. Data are normalized to highest value for that parameter, i.e., ∫GG_EMG, ∫Dia_EMG, or ∫Abd_EMG regardless of whether it belonged to CNO or CNO with Alst group. Abbreviations defined in Figure 2.

**DOI:** http://dx.doi.org/10.7554/eLife.14203.009

Figure 8. — (A) Integrated traces from one experiment showing effect of removing rat from ventilator (dark grey shaded area), in presence of CNO (pink shaded area), following induction of apnea by microinjection of Alst into the preBötC of preBötC-AlstR transfected rats. Intervening period between the two traces, whilst ventilation was ongoing and continuous, has been removed (double break), so traces can be expanded. (B) Expanded traces from A (indicated by black dashed lines), showing how measurements were taken for inspiratory parameters when active expiration returned. (C) Inspiratory parameters when active expiration returned. Dots represent individual experiments, box and whisker plots show combined data. (Ci) Tidal volume (V_T) (**Cii**) ∫Dia_EMG, (**Ciii**) ∫GG_EMG. (D) Integrated traces from one experiment showing the return of inspiratory and expiratory activity during mechanical ventilation, in presence of CNO (pink shaded area), following induction of apnea by microinjection of Alst into the preBötC of preBötC-AlstR transfected rats. Abbreviations defined in Figure 2.

**DOI:** http://dx.doi.org/10.7554/eLife.14203.010

Technical consideration

Our experimental design required long lasting activation of receptors at 4 separate loci, i.e., preBötC bilaterally and pF_L bilaterally. Optogenetics can have millisecond resolution, making them a valuable tool for studying breathing on a breath by breath basis, e.g., (Pagliardini et al., 2011). However opsins can rapidly (>seconds) desensitize and their activation requires placement of optic fibers to illuminate each transfected area, a particularly challenging problem for four regions in the brainstem. Therefore, we chose a pharmacogenetic approach to excite the pF_L by activation of HM₃DRs and inhibit the preBötC by activation of AlstRs. This way we were able to switch neurons in these regions on or off over a period of minutes by application of CNO to the ventral surface of the medulla, and careful titration of the dose and infusion rate of Alst injected into the preBötC.

We note AAV2/5 can retrogradely transfect of some types of afferent neurons, e.g., injections of AAV2/5 into the entorhinal cortex retrogradely labels a subset of dentate gyrus neurons, but not the dense afferent projections from other nuclei (Aschauer et al., 2013). We assert that our results are unlikely to be confounded by retrograde transfection of distant neurons: i) Other than the injection site, we found no labeling of medullary neurons following transfection of the pF_L with AAV2/5 viruses that coexpress mCitrine with a DREADD receptor, in this (Figure 1E,F) or our previous (Huckstepp et al., 2015) study; ii) CNO was applied directly to the ventral medullary surface, and the effective concentration for receptor activation would be limited to the first few hundred microns beneath the ventral surface, and; iii) we find essentially the same effects of activation of neurons in the pF_L as optogenetic photoactivation of the same neurons transfected with lentivirus (Pagliardini et al., 2011), which is not retrogradely transported.

Here we discuss the output of the preBötC and pF_L, which ultimately form the final output of the respiratory network. Though we do not refer to other nuclei, we do not rule out their contribution to the control of respiration; for example following silencing of the preBötC, the increase in chemosensory drive to the respiratory oscillators, may come from increased drive from other respiratory-related nuclei, such as the pontine nuclei, i.e., locus coreleus, parabrachial nucleus, and the Kolliker Fuse nucleus, the ventral respiratory group or other medullary nuclei, i.e., RTN/pF_V, medullary raphe, nucleus tractus solitarii, or even astrocytes. In addition, due to the necessity to perform injections into the medulla, we were unable to record directly from the preBötC or pF_L. Therefore, the activity of the preBötC and pF_L, was assessed by motor output recorded from respiratory muscles, which may not always reflect the activity of these oscillators, as they may have subthreshold activity, e.g., (Kam et al., 2013).

Discussion

Many rhythmic behaviors are driven by neural networks that (presumably) contain coupled oscillators (Grillner, 2003; Goulding, 2009). Here, we examined the coupled oscillator microcircuit that controls breathing, a motor behavior that is presently unique insofar as the locations of its oscillators, i.e., preBötC and pF_L, are known (Smith et al., 1991; Pagliardini et al., 2011; Feldman et al., 2013; Feldman and Kam, 2015; Huckstepp et al., 2015). In rats, while the function of the preBötC as the critical site for generation of inspiratory rhythm is consistent across all relevant developmental stages, from the third trimester in utero through adulthood, and state, e.g., sleep-wake, rest-exercise (Smith et al., 1991; Janczewski and Feldman, 2006; Tan et al., 2008; Kam et al., 2013), the presumptive function of the pF_L and its coupling with the preBötC appears to change developmentally and with state (Figure 9) (Onimaru and Homma, 2003; Iizuka and Fregosi, 2007; Oku et al., 2007; Onimaru et al., 2008; Thoby-Brisson et al., 2009; Pagliardini et al., 2011; Huckstepp et al., 2015). To understand the context in which we interpret our data, we first summarize this disparate literature with reference to Figure 9.

Figure 9. — Functional connections of undetermined connectivity are indicated as broken lines. As pF_L neurons are excitatory (Onimaru et al., 2008; Thoby-Brisson et al., 2009) and lack inhibitory markers (Ellenberger, 1999; Tanaka et al., 2003), inhibitory connections from pF_L to preBötC are indirect (see Figure 7 in Huckstepp et al., 2015). (A) Embryonic stage (all data *in vitro*). (Ai) pF_L (red circle) oscillates at embryonic day 14.5 (E14.5). (**Aii**) preBötC (blue circle) oscillates at embryonic day 15.5 (E15.5) and couples to the pF_L, where it excites and inhibits different groups of pF_L neurons. (**Aiii**) preBötC and pF_L can oscillate independently of each other following a transverse section caudal to facial nucleus. (**Aiv**) pF_L can oscillate in the absence of preBötC following bath application of a glutamatergic antagonist (CNQX). (Av) preBötC can oscillate in the absence of the pF_L following bath application of a sodium channel blocker (riluzole) (Thoby-Brisson et al., 2009). (B) Postnatal stage. (Bi) In late fetal (Thoby-Brisson et al., 2005; Bouvier et al., 2008) and postnatal rats (Smith et al., 1991), the preBötC can oscillate in the absence of the pF_L in transverse slices, and (**Bii**) the pF_L can oscillate independently following suppression of preBötC rhythm by bath application of opioid agonists (Takeda et al., 2001; Janczewski et al., 2002). Biii) Immediately following birth, respiratory rhythm is driven by pF_L (Onimaru and Homma, 2003; Oku et al., 2007). (**Biv**) Shortly after birth (>1 day), the breathing CPG becomes driven by the preBötC (Oku et al., 2007). (C) Juvenile stage. (Ci) Expiration and inspiration alternate and are reciprocally coupled. (**Cii**) PreBötC and pF_L are differentially affected by fentanyl, which shifts breathing to an expiratory-dominant pattern. (Cii + iii) preBötC and pF_L can be independently suppressed by activation of Breuer-Hering deflation reflex (BHDR; **Cii**) or inflation reflex (BHIR; **Ciii**) (Janczewski and Feldman, 2006). (D) Adult Stage: (Di) breathing is inspiratory driven by preBötC while pF_L activity is normally suppressed at rest (also see Pagliardini et al., 2011 and Huckstepp et al., 2015); (**Dii**) activation of HM₄DR transfected pF_L neurons by CNO (see Figure 6) or optogenetic activation (Pagliardini et al., 2011), or suppression of AlstR transfected preBötC neurons with Alst (see Figure 2, 7) can induce active expiration; (**Dii**) as preBötC neurons project to the pF_V but do not appear to project to the pF_L (Tan et al., 2010), excitatory drive from the preBötC to the pF_L is most likely through an intermediate excitatory relay, such as the pF_V. (**Diii**) Depression of inspiration by Alst, in presence or absence of CNO, leads to tonic expiratory activity during hypercapnia (see Figures 2, 7) or (**Div**) apnea during hypoxia (see Figures 2, 7). (Dv) As breathing returns, abdominal activity remains absent until inspiratory activity is near normal levels (see Figure 8), implicating an indirect involvement of preBötC excitatory neurons in expiration either through its excitatory projections throughout breathing CPG (Tan et al., 2010), including pF_V that contributes to expiratory activity (Huckstepp et al., 2015), or through mechanosensory feedback that can provides expiratory drive (Remmers, 1973; Davies and Roumy, 1986; Janczewski and Feldman, 2006).

**DOI:** http://dx.doi.org/10.7554/eLife.14203.011

From third trimester in utero to adulthood, NK1R-expressing neurons lying under the lateral edge of the facial nucleus and extending laterally out to the spinal trigeminal tract have respiratory-rhythmic burst activity (Onimaru et al., 2008; Thoby-Brisson et al., 2009; Pagliardini et al., 2011; Huckstepp et al., 2015). In E14.5 mice, these neurons are designated as comprising the embryonic parafacial nucleus (e-pF) (Thoby-Brisson et al., 2009); in postnatal rats, these neurons are designated as comprising the parafacial respiratory group (pFRG) (Onimaru and Homma, 2003), and; in the adult rat, these neurons are designated as comprising the pF_L (Pagliardini et al., 2011; Huckstepp et al., 2015). While 3 distinctly different groups of NK1R neurons expressing respiratory-rhythmic bursting could exist in sequence within this homologous anatomical location at distinctly different developmental time points, the most parsimonious interpretation is of one population of neurons studied at different developmental time points; we postulate that this is the case. For the balance of the DISCUSSION, we will refer to this location as the pF_L.

NK1R-expressing neurons in the ventral respiratory column (including the parafacial region) are almost exclusively glutamatergic (Guyenet et al., 2002). In embryonic and postnatal rodents, the pF_L is comprised of glutamatergic NK1R-expressing neurons (Onimaru et al., 2008; Thoby-Brisson et al., 2009). In the adult rodent, the presumptive pF_L expiratory oscillator is comprised (mostly) of NK1R-expressing neurons (Figure 1D, see also Huckstepp et al., 2015). Since the pF_L and pF_V do not appear to contain any inhibitory neurons (Ellenberger, 1999; Stornetta and Guyenet, 1999; Tanaka et al., 2003; Fortuna et al., 2008; Abbott et al., 2009), we conclude that any resultant inhibitory action associated with their activity occurs through an intermediate relay of inhibitory neurons, perhaps located in the preBötC (Morgado-Valle et al., 2010) or BötC (Schreihofer et al., 1999).

In E14.5 mice, the pF_L is rhythmic before the preBötC appears to form (Figure 9Ai; see also Thoby-Brisson et al., 2009). At E15.5, the preBötC forms (Thoby-Brisson et al., 2005) and it becomes rhythmically coupled to the pF_L (Thoby-Brisson et al., 2009). At this time: i) the preBötC and pF_L can oscillate independently following a transverse section caudal to facial nucleus (Figure 9Aiii); ii) the pF_L can oscillate in the absence of preBötC rhythm following bath application of the glutamatergic antagonist CNQX (Figure 9Aiv), and; iii) the preBötC can oscillate in the absence of the pF_L following administration of the sodium channel blocker riluzole (Figure 9Av) (Thoby-Brisson et al., 2009). Thus embryonically, the preBötC and pF_L are independent, coupled oscillators, and rhythmic output of the network is a convolution of the faster rhythm of the pF_L and the slower rhythm of the preBötC (Thoby-Brisson et al., 2009); the pF_L (Thoby-Brisson et al., 2009) provides excitatory drive to the preBötC, while bursting in the preBötC, comprised of inhibitory (Rahman et al., 2015) and excitatory neuronal activity (Gray et al., 2010), inhibits and excites pF_L neurons (Thoby-Brisson et al., 2009). Whether the projections between the preBötC and pF_L are direct or indirect (Figure 9Aii) is yet to be determined (Thoby-Brisson et al., 2009).

In postnatal rats, respiratory rhythm appears similar to the embryo; the glutamatergic pF_L provides excitatory drive to the preBötC (Onimaru and Homma, 2003; Onimaru et al., 2012), while bursting of the preBötC, comprised of inhibitory (Morgado-Valle et al., 2010) and excitatory neurons (Gray et al., 2010), inhibits and excites different subsets of pF_L neurons (Takeda et al., 2001; Onimaru et al., 2007). At this time: i) the preBötC can oscillate in transverse slices, i.e., in the absence of the pF_L (Figure 9Bi) (Smith et al., 1991) and; ii) the pF_L can oscillate following suppression of preBötC rhythm by bath application of opioid agonists (Figure 9Bii) (Takeda et al., 2001; Janczewski et al., 2002). Thus postnatally, the preBötC and pF_L are also independent, coupled oscillators. Immediately following birth, respiratory rhythm appears driven by the pF_L (Figure 9Biii) (Onimaru and Homma, 2003; Oku et al., 2007), perhaps to protect against the opioid surge in the fetal brain during birth (Janczewski et al., 2002) that would act to suppress preBötC activity (Takeda et al., 2001). Shortly after birth (~P2-P4), the breathing central pattern generator (CPG) matures, and becomes driven by the preBötC (Oku et al., 2007; Mckay et al., 2009; Kennedy, 2015) (Figure 9Biv), and remains so throughout life.

In juvenile rats, the preBötC and pF_L are reciprocally coupled (9Ci) but are still differentially affected by fentanyl, which shifts breathing to an expiratory-dominant pattern (9Cii) (Janczewski and Feldman, 2006). Also, the preBötC and pF_L can be independently suppressed by the Breuer-Hering deflation reflex (BHDR; 9Cii) or inflation reflex (BHIR; 9Ciii) (Janczewski and Feldman, 2006).

Here, we used a novel pharmacogenetic strategy to investigate how the preBötC and pF_L interact to produce the appropriate breathing pattern in adult spontaneously breathing vagotomized rats (Figure 9D). The preBötC is the dominant oscillator, active at rest to drive inspiratory movements (Figure 9Di, see also Pagliardini et al., 2011 and Huckstepp et al., 2015). The pF_L is the subsidiary conditional oscillator, quiescent at rest (Figure 9Di); in the presence of concurrent, or reduced, preBötC activity (Figure 2), and appropriate inputs, such as an increase in expiratory drive through activation of exogenous receptors (Figure 6, see also Pagliardini et al., 2011) or altered blood gases (Huckstepp et al., 2015), the pF_L drives active expiration (Figure 9Dii). In the absence of preBötC activity, chemosensory drive increases network excitability and drives active expiration, though abdominal activity is tonic due to a lack of phasic inhibition from the preBötC (Figure 9Diii). However, the pF_L appears incapable of independently driving respiratory movements in the adult rat when preBötC activity is silenced by exogenous receptors (Figures 2, 7, 9Div) or when preBötC activity is low and blood gases are normal (Figures 8, 9Dv). As preBötC neurons project to the pF_V but do not appear to project to the pF_L (Tan et al., 2010), excitatory drive from the preBötC to the pF_L is most likely through an intermediate excitatory relay, such as the pF_V (Figure 9Dii).

Silencing the preBötC leads to quantal slowing of breathing and transmission of burstlets to motoneuron pools

Hyperpolarizing preBötC neurons initially decreased f, V_T, and ∫Dia_EMG (Figures 2, 3, see also Tan et al., 2008). Breathing slowed in a quantal manner (Mellen et al., 2003) with missed inspiratory bursts, rather than through a gradual increase in period (Figures 2Bii, 3A–C). Concurrently, as drive within the preBötC diminished and chemosensory drive increased, presumptive burstlets in the preBötC (Kam et al., 2013) appeared to be transmitted to motor pools, leading to low level inspiratory motor activity, i.e., ∫Dia_EMG and ∫GG_EMG, resulting in minimal inspiratory airflow (Figure 3A–B). Here, lowered drive in the preBötC from activation of the allatostatin receptor creates burstlets in the preBötC; under normal conditions these burstlet signals are not strong enough to drive motor activity. However, increased excitability in the premotor and motor network caused by increased chemosensory drive (from decreased ventilation) causes these normally subthreshold events to become suprathreshold, and thus these burstlets are transmitted to the motor output. During quantal slowing of breathing, inspiratory activity on the Dia_EMG and GG_EMG was interspersed with Abd_EMG activity (Figures 2A-B, 7A–B). Interestingly, even when expiratory activity was at its highest, low level preBötC activity, as seen as burstlets on the Dia_EMG and GG_EMG was still able to inhibit Abd_EMG activity (Figure 7Bii). These observations are consistent with our hypothesis that burstlets originate in the preBötC and, under atypical conditions can be transmitted to motoneuron pools to be seen as small events in muscle EMGs (Kam et al., 2013).

Silencing the preBötC leads first to active expiration then to apnea

Following hyperpolarization of preBötC neurons by activation of AlstRs, active expiration appeared (Figures 2, 3, 9Dii). Hypoxia and hypercapnia can themselves induce active expiration (Iizuka and Fregosi, 2007; Huckstepp et al., 2015), whether changes in blood gases are sensed by pF_L neurons, or by neurons driving the pF_L, remains to be determined. In either case, active expiration could have been due to increased hypoxic and/or hypercapnic drive to the pF_L as inspiratory movements waned (Figures 2A–B, 7A–B, 9Dii). Alternatively, active expiration could be due to disinhibition of a conditional expiratory oscillator resulting from a loss of (presumptive) inhibitory preBötC drive (Kuwana et al., 2006; Morgado-Valle et al., 2010) to the pF_L (Figure 9Dii). Subsequently, when inspiratory motor outflow disappeared and hypercapnia and hypoxia inexorably increased, active expiration ceased (Figures 2, 9Div, see also Tan et al., 2008), perhaps due to the inhibitory effect of severe hypoxia on expiratory motor output (Sears et al., 1982; Fregosi et al., 1987). At normal blood gas levels, active expiration may be seen in the absence of inspiratory motor activity resulting from lung inflation, e.g., in juvenile rat (Janczewski and Feldman, 2006) (Figure 9C). Nonetheless, from the data presented here in the adult rat, we conclude a necessary role of preBötC excitatory drive in generating active expiration in normal breathing (discussed further below).

Activating the pF_L can induce active expiration but not during to apnea

Depolarizing pF_L neurons by activation of HM₃DRs (Figure 6) changes breathing in a manner similar to their disinhibition or optogenetic photostimulation (Pagliardini et al., 2011). In the presence of inspiratory motor output, depolarizing pF_L neurons led to substantial active expiration, but when inspiration ceased so did active expiration (Figures 7, 9Div). Tupal et al observed the absence of expiratory rhythm in perinatal mice lacking Dbx1 neurons and concluded that Dbx1-derived parafacial neurons are an essential component of an expiratory oscillator (Tupal et al., 2014). Our data suggests an alternative interpretation, that suppressing preBötC neuron activity, either acutely as done here or perhaps genetically via Dbx1 deletion, is sufficient to prevent active expiration, without any need to invoke explicit perturbations of the pF_L.

Loss of inhibitory output from the preBötC leads to a loss of phasic expiratory activity

As rats went from eupnea to apnea following suppression of preBötC activity, and after initiation of active expiration, ∫Abd_EMG transitioned from phasic to tonic: ∫Abd_EMG oscillated with a normal antiphase relationship to inspiration when preBötC drive and hypercapnia were moderate, but became tonic during apnea accompanied by severe hypercapnia (Figures 2A–B, 7A–B, 9Diii). Similarly, in anesthetized cats as ventilation moves in the opposite direction from hypocapnic apnea to eupnea with a resultant increase in CO₂, expiratory motor activity transitions from tonic to phasic (Sears et al., 1982). We suggest, like Sears et al, that near the transition from eupnea to apnea, expiratory activity is phasic due to periodic inhibition of tonic activity during inspiration, and becomes tonic once the phasic inspiratory inhibition is lost (Figures 2Bii–iii, 7Bii, 9Diii). We suggest the loss of phasic inhibition is the result of silencing of inhibitory preBötC inspiratory neurons by Alst (Kuwana et al., 2006; Morgado-Valle et al., 2010), implying a significant role for inhibitory preBötC neurons in shaping expiratory output.

Respiratory network activity is a requirement for active expiration

Interestingly, tonic ∫ABD_EMG activity was not seen during the transition from apnea to eupnea (Figures 8Aii, 9Dv) in contrast to its presence during the reverse transition from eupnea to apnea (Figures 7Bii, 9Diii). Following apnea during ventilation to maintain blood gases, when pF_L neurons were excited by activation of HM₃DRs there were three phases to the re-initiation of breathing. Initially, soon after silencing the preBötC, no inspiratory or expiratory activity was seen, even in the absence of mechanical ventilation. Next, presumably as the effect of Alst on preBötC neurons was waning, no inspiratory or expiratory activity was seen during ventilation, but spontaneous breathing was present upon removal from the ventilator. Here, spontaneous breathing was likely due to increased chemosensory drive to the preBötC overcoming the waning hyperpolarizing effect of the Alst on preBötC neurons. Ultimately, as spontaneous breathing continued and chemosensory drive diminished, rats would once again become apneic and require mechanical ventilation (Figure 8A–C). During this phase, upon removal from the ventilator, ∫Abd_EMG did not return until inspiratory motor, and presumably preBötC, activity reached a threshold level (Figure 8A–C). Finally, spontaneous breathing occurred during ventilation, once the effect of Alst on preBötC neurons had worn off (Figure 8D). During this phase, inspiratory activity on the GG_EMG and Dia_EMG always returned before Abd_EMG (Figure 8D). At no time during mechanical ventilation, or during the periods where rats were briefly removed from the ventilator, did active expiration occur in the absence of inspiration. As blood gases were normal (Figure 8), the loss of expiratory activity is unlikely to be due to excessive hypoxia or hypercapnia. Thus there appears to be a minimum level of respiratory network excitability required for active expiration. In the absence of preBötC activity, this network excitability may be provided by chemosensory drive, accounting for the ∫Abd_EMG activity seen shortly before the onset of apnea. However following recovery from apnea the increase in network excitability is provided by the preBötC either directly through its extensive excitatory projections throughout the respiratory network (Tan et al., 2010), e.g., the pF_V that can modulate expiratory activity (Marina et al., 2010; Huckstepp et al., 2015), or indirectly when V_T became large enough to induce mechanosensory feedback (that can provide expiratory drive, e.g., Remmers, 1973; Davies and Roumy, 1986; Janczewski and Feldman, 2006).

Summary

We have developed a new strategy using complementary pharmacogenetics for studying coupled oscillator systems. By independently altering the excitability of two anatomically and functionally separate respiratory oscillators, we have uncovered a fundamental interaction and further delineated their role within the breathing CPG. We conclude that though respiration results from the interaction of two distinct oscillators, the preBötC (inspiration) and the pF_L (expiration) are not organized as symmetrical half centers. Instead in the adult rat, the preBötC is the dominant oscillator and the pF_L is the subsidiary conditional oscillator that is normally suppressed at rest; whereas the preBötC can drive breathing alone, the pF_L is unable to drive breathing (including active expiration) in the absence of an additional form of network excitation, i.e., ongoing rhythmic preBötC activity sufficient to drive inspiratory motor output or increased chemosensory drive when changes in blood gases are below the threshold where hypoxia is sufficient to inhibit abdominal muscle recruitment. This hierarchy is established by the sensitivity of the system to each oscillator, even when preBötC drive is low, it is able to drive inspiratory bursts and inhibit expiration, whereas even when pF_L drive is high, it requires a certain amount of network excitability from other sources to drive expiratory activity. This asymmetrical organization may be relevant to other neural networks that contain hierarchically organized coupled oscillators for pattern generation, such as may underlie the asymmetrical acts of flexion and extension in locomotion (Grillner, 2003; Talpalar et al., 2013). The interactions of the preBötC and pF_L change with development and maturation (Figure 9), and with state, represent another layer of complexity in understanding the neural control of breathing.

Materials and methods

Viral design and handling

Two different viruses were used: AAV-2/5 hSyn-HA-hM₃D(Gq)-IRES-mCitrine (6 × 10¹² vp/ml; HM₃DR; UNC Gene Therapy Vector Core, Chapel Hill, NC), and; AAV-DJ synapsin-allatostatin receptor-GFP (Huckstepp et al., 2015) (5.8 × 10¹³ vp/ml; AlstR; SALK institute GT³ Core, La Jolla, CA). The viruses were stored at -80°C. For injection, viruses were held at 4°C and loaded into pipettes.

Viral transfection (Figure 1)

All protocols were approved by the UCLA Chancellor’s Animal Research Committee. Male Sprague-Dawley rats (350–450 g) were anesthetized by intraperitoneal ketamine (100 mg/kg; Clipper Distribution Co, St Joseph, MO), xylazine (10 mg/kg; Lloyd Inc, Shenandoah, IA), and atropine (1 mg/kg; Westward Pharmaceutical Co, Eatontown, NJ), supplemented with isofluorane (0.5–2%; Piramal Healthcare Ltd, India) as required. Rats were placed prone in a stereotaxic apparatus (Kopf Instruments, St, Tujunga, CA), with the head positioned with Bregma 5 mm below Lambda, on a heating pad to maintain body temperature at 37 ± 0.5°C. The dorsal medullary surface was exposed and pipettes placed stereotaxically into the preBötC or pF_L. Coordinates were (lateral, rostral, ventral from obex in mm): preBötC; (2.0, 0.9, 2.8), and pF_L; (2.5, 1.8, 3.5). Viral solutions were pressure injected (100–150 nL) with a Picospritzer II (General Valve Corp, Fairfield, NJ) controlled by a pulse generator. Pipettes were left in place for 3–5 min to prevent solution backflow up the pipette track. We injected AlstR-expressing AAV in preBötC and HM₃DR-expressing AAV in pF_L. Postoperatively, rats received buprenorphine (0.1 mg/kg; Reckitt Benckiser, UK) intraperitoneally and meloxicam (2 mg/kg; Norbrook Inc, UK) subcutaneously, and antibiotics (10 days; TMS: Hi-Tech Pharmacal, Amityville, NY) and meloxicam (4 days; 0.05 mg/mL) in their drinking water. Rats were allowed 3–6 weeks for recovery and viral expression, with food and water ad libitum.

Ventral approach

Anesthesia was induced with isofluorane and maintained with urethane (1.2–1.7 g/kg; Sigma, St Louis, MO) in sterile saline via a femoral catheter. Rats were placed supine in a stereotaxic apparatus on a heating pad to maintain body temperature at 37 ± 0.5°C. The trachea was cannulated, and respiratory flow was monitored via a flow head (GM Instruments, UK). A capnograph (Type 340: Harvard Apparatus, Holliston, MA) was connected to the tracheal tube to monitor expired CO₂, as a proxy of blood gases homeostasis. Paired electromyographic (EMG) wires (Cooner Wire Co, Chatsworth, CA) were inserted into genioglossal (GG), diaphragmatic (Dia), and oblique abdominal muscles (Abd). Anterior neck muscles were removed, a basiooccipital craniotomy exposed the ventral medullary surface, and the dura was resected. A bilateral vagotomy was performed to remove confounding factors such as feedback from lung stretch receptors that can drive abdominal activity (Janczewski and Feldman, 2006), after which exposed tissue around the neck and mylohyoid muscle were covered with dental putty (Reprosil; Dentsply Caulk, Milford, DE) to prevent drying. As micturition is inhibited under anesthesia, rats bladders were expressed preceding and during the experiment to remove any risk of autonomic dysreflexia from bladder distension; to maintain fluid balance rats were given an IP injection of saline every time the bladder was expressed. Rats were left for 30 min for breathing to stabilize. At rest, spontaneous breathing consisted of alternating active inspiration and passive expiration. From a ventral approach allatostatin (Alst; 10 μM; ~100-200 nL; Antagene Inc, Sunnyvale, CA) in sterile saline was injected bilaterally into the preBötC to hyperpolarize neurons transfected by AlstRs. Coordinates were (lateral from the basilar artery, caudal from the rostral hypoglossal nerve rootlet, dorsal from the ventral surface in mm): preBötC; (2.0, 0.6, 0.7). Small adjustments were made to avoid puncturing blood vessels. Rats were ventilated for the duration of ensuing apnea. After breathing stabilized, clozapine-N-oxide (CNO; 90 µM; Santa Cruz Biotechnology, Dallas, TX) in sterile saline was applied to the ventral medullary surface to depolarize pF_L neurons transfected with HM₃DRs. Once breathing stabilized, rats received a second set of bilateral injections of Alst. Though rats were ventilated with room air for the duration of ensuing apnea, they were intermittently removed from the ventilator to assess spontaneous breathing, and drives to inspiration and expiration. Ventilation depths and speed were chosen to match end-tidal CO₂ to that when the rat was spontaneously breathing room air. Once rats were spontaneously breathing and no longer required ventilation, CNO was removed from the ventral surface of the medulla and the medulla was washed in PBS. All 8 rats underwent the entire procedure, i.e., there were 8 biological replicates, and were only exposed to each condition once, i.e., there were no technical replicates. In age-matched rats not transfected with AlstR- or HM₃DR-expressing AAVs, we injected Alst into the preBötC or applied CNO to the medullary surface to see if these protocols produced non-specific effects All 8 rats underwent the entire procedure, i.e., there were 8 biological replicates, and were only exposed to each condition once, i.e., there were no technical replicates.

Localization of transfected neurons

Rats were sacrificed by overdose of urethane and transcardially perfused with saline followed by cold (4°C) paraformaldehyde (PFA; 4%). The medulla was harvested and postfixed in 4% PFA overnight at 4°C, then cryoprotected in sucrose (30%) in standard PBS (1–3 days at 4°C). PBS contained (mM): NaCl 137, KCl 2.7, Na₂HPO₄ 10, KH₂PO₄ 1.8, pH 7.4. Brainstems were transversely sectioned at 40 μm. Free-floating sections were incubated overnight in PBS containing 0.1% Triton X-100 (PBT) and 1° antibodies (1:500): mouse anti-NeuN (EMD Millipore, Billerica, MA), rabbit anti-neurokinin 1 receptor (NK1R: EMD Millipore), and chicken anti-green fluorescent protein (GFP: Aves lab, Tigard, OR). The tissue was washed in PBS, 6 times for 5 min and then incubated separately for 2–4 hr in a PBT containing 2° antibodies (1:250): Donkey anti-mouse AlexFluor 647, donkey anti-rabbit rhodamine red, donkey anti-chicken AlexFluor 488 (Jackson ImmunoResearch Laboratories Inc, West Grove, PA). The tissue was washed in PBS, 6 times for 5 min. Slices were mounted onto polylysine-coated slides, dehydrated overnight at 22°C, and coverslipped using Cytoseal 60 (Electron Microscopy Sciences, Hatfield, PA). Slides were analyzed using a fluorescent microscope with AxioVision acquisition software (AxioCam2, Zeiss, Germany).

Data analysis and statistics

Sample sizes were calculated using Gpower 3 v3.1.9.2 (http://www.ats.ucla.edu/stat/gpower/); using a ‘means: Wilcoxon signed-rank test (matched pairs)’ test, with a desired power of 90%, at a 5% significance level, and an effect size of 1.15 (calculated from the initial effect of Alst on respiratory frequency). Data were only included from animals where the preBötC and pF_L were successfully targeted bilaterally, and no data were excluded from these animals. All statistical analysis was performed in Igor Pro (WaveMetrics, Lake Oswego, OR).

EMG signals and airflow measurements were collected using preamplifiers (P5; Grass technologies, Rockland, MA) connected to a Powerlab AD board (ADInstruments, Australia) in a computer running LabChart software (ADInstruments), and were sampled at 400 Hz/channel. High pass filtered (>0.1 Hz) flow head measurements were used to calculate: tidal volume (V_T, peak amplitude of the integrated airflow signal during inspiration, converted to mL by comparison to calibration with a 3 mL syringe), inspiratory duration (T_I, beginning of inspiration until peak V_T), expiratory duration (T_E, peak V_T to the beginning of the next inspiration), and f (1/[T_I+T_E]). EMG data, expressed in arbitrary units (A.U.), were integrated (τ=0.05 s; ∫Dia_EMG, ∫GG_EMG, and ∫Abd_EMG) and peak amplitude of each signal was computed for each cycle. To obtain control values, all parameters except end-tidal CO₂ (ETCO₂), were averaged over 20 consecutive cycles preceding each experimental manipulation ( ${\bar{X}}_{c o n t r o l}$ ). After Alst, measurements were taken at 2 time points: i) 20 cycles were averaged where only partial effects were seen, and; ii) 20 points were averaged following apnea. After CNO, 20 cycles were averaged after breathing had stabilized. After Alst in the presence of CNO, 20 points were averaged following apnea. In the presence or absence of CNO, capnograph peaks were averaged for 10 cycles preceding Alst, and for 5–10 cycles preceding apnea. Following removal from ventilation, the amplitude of the inspiratory bursts and ∫Dia_EMG and ∫GG_EMG activity immediately preceding the first ∫Abd_EMG burst (Figure 8, blue lines dashed lines) were recorded to calculate inspiratory parameters at which active expiration returned.

For each rat we obtained ${\bar{X}}_{c o n t r o l}$ , and the average of 20 cycles during the stimulus ( ${\bar{X}}_{s t i m u l u s}$ ). ${\bar{X}}_{c o n t r o l}$ values and their associated ${\bar{X}}_{s t i m u l u s}$ values for each parameter in each rat were combined into a single data set. To facilitate graphical comparisons data were normalized to the highest value in the data set regardless of whether it belonged to ${\bar{X}}_{c o n t r o l}$ or ${\bar{X}}_{s t i m u l u s}$ group (C in Figure 2, 4–7). Therefore the highest value in the data set, whether it be ${\bar{X}}_{c o n t r o l}$ or ${\bar{X}}_{s t i m u l u s}$ , was 1.0; except for measurements of V_T, ∫Dia_EMG, and ∫GG_EMG following ventilation, which are displayed as absolute values. Recorded data were not normally distributed, and were therefore analyzed using non-parametric statistical test, and reported as median and interquartile range (IQR). Statistical tests performed in Igor Pro (WaveMetrics), are 2-sided Wilcoxon signed-rank tests with a significance level of p≤0.05. Data are displayed as box and whisker plots for comparison of groups, and as line graphs for individual experiments. Kernel density estimations (Parzen, 1962; Epanechnikov, 1969), were used to determine the distribution of respiratory periods. After calculating the optimal bandwidth, i.e., bin size (Park and Marron, 1990; Sheather and Jones, 1991), the data was smoothed (Cao et al., 1994) and plotted. The modality of kernel density plots, were used to assess baseline respiratory periods and whether breathing slowed by quantal integers of that baseline. Bandwidth selection, data smoothing, and kernel density plots were performed in Microsoft excel (Microsoft Corporation, Redmond, WA) using an add-in written by the royal society of chemistry (http://www.rsc.org/Membership/Networking/InterestGroups/Analytical/AMC/Software/kerneldensities.asp).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grant:

National Institutes of Health NIH HL 70029 to Jack L Feldman.

Additional information

Competing interests

The authors declare that no competing interests exist.

Author contributions

RTRH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

LEH, Acquisition of data, Drafting or revising the article.

KPC, Acquisition of data, Drafting or revising the article.

JLF, Conception and design, Analysis and interpretation of data, Drafting or revising the article.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols (IACUC protocol #1994-159-73) were approved by the UCLA Chancellor's Animal Research Committee. Recovery surgery was performed under Ketamine/xylazine anesthesia and accompanied bybuprenorphine and meloxicam analgesia, non-recovery surgery was performed under urethane anesthesia, and every effort was made to minimize suffering.

References

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eLife. 2016 Jun 14;5:e14203. doi: 10.7554/eLife.14203.012

Decision letter

Editor: Jan-Marino Ramirez¹

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Interactions between respiratory oscillators in adult rats" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

We recognize that this study describes the novel observation that active expiration turns tonic if the preBötzinger complex is experimentally silenced. The reviewers congratulate you and your co-authors for the cutting-edge approaches that allowed you to independently lesion the preBötzinger complex and pFRG region. Based on these manipulations, you and your co- authors conclude that active expiration (and the oscillator in the pFRG) requires rhythmic inhibition from the preBötC to generate expiration in the adult. Since this is not the case in juvenile animals, you proposed that there must be a developmental change in the role of the pFRG in breathing.

The decision to reject is based on the reviewers' major concern that these experiments do not suffice to justify your major conclusion. In all cases silencing the preBötzinger complex is associated with an apnea while the animal is not ventilated. Under these conditions it cannot be avoided that the animal will become hypoxic. Thus, it is not clear whether the cessation of inspiration and expiration is due to the cessation of the preBötzinger complex or the hypoxic conditions associated with the apnea. It is well-known that hypoxic conditions will lead to the cessation of inspiration and expiration. It is also well known that adults are more sensitive to hypoxia than juveniles/neonates.

Thus, the reviewers demand an additional set of new experiments. To justify your conclusion it is critical to silence the preBötC while ventilating the animal, show stable oxygenation in the absence of preBötC activity, and then demonstrate whether under these physiologically stable conditions active expiration can be induced, persists or disappears when inspiration ceases neuronally. These experiments will not be feasible within the allowed two months, hence we will not be able to consider your paper. For further details, please see the individual reviews.

Reviewer #1:

This study is an extension of a discovery that was made ten years ago (J.Physiol. 2006) indicating that two rhythm generators underlie the control of breathing: the preBötC for Inspiration, and the pFRG (first discovered by Onimaru) responsible for expiration ("active expiration"). The study does not contribute any new insights to this well-known concept, since this concept has been demonstrated not only in juveniles (the original 2006 study), but also in adults (J. Neuroscience, same lead author).

The only new discovery is based on the observation that active expiration turns tonic if the preBötzinger complex is lesioned/silenced. This observation leads the authors to conclude that active expiration (and the oscillator in the pFRG) requires rhythmic inhibition from the preBötC to generate expiration in the adult. The authors conclude, that there must be a developmental change in the role of the pFRG in breathing.

However, based on the experiments submitted here, this conclusion is not justified.

In all cases when apnea is induced by lesioning/silencing the preBötzinger complex the animal is not ventilated. This means, the animal will be exposed to severe hypoxia, which could (and will) indirectly induce the apnea and cessation of inspiration and expiration. This is a well-known phenomenon, and not at all surprising.

Thus, in order to arrive at the conclusion that the authors propose in this study, they need to add new experiments i.e. block or silence the preBötC while ventilating the animal show stable oxygenation and induce active expiration and then see whether under these physiologically stable conditions active expiration persists or disappears when inspiration ceases. Given that this is the major conclusion on which this paper rests, this experiment is absolutely essential.

Another major issue is the interpretation of the quantal slowing. First, it would be interesting to relate this to expiration, as has been done in prior studies. Secondly, the interpretation/speculation that the quantal slowing is explained by the presence of burstlets is not based on any experiment. These burstlets as far as I understand the original publication are "subthreshold" oscillations within the respiratory rhythm generator – without burst generation. If this is the case, how would the authors explain the transmission of these subthreshold oscillations to the motor output?

Reviewer #2:

I congratulate the investigators on a well-conducted study and a well-written manuscript that provides a significant advancement in understanding of mechanisms of respiratory rhythm generation. I am not requesting any specific changes in the manuscript prior to publication. However, I have a few suggestions for the authors to consider for additions to the discussion.

First, rostral pontine nuclei provide an important contribution to the control of breathing. What are the authors' speculations regarding this contribution to the two oscillators shown herein?

Second, what are the authors' thoughts on interactions between these two respiratory oscillators during normal physiological conditions particularly under conditions of NREM and REM sleep?

Third, this study shows the effects of acute, brief alterations in activity of the two oscillators. What happens when either one of the oscillators is altered on a long term or chronic basis? Studies by J. Wenninger et al., K. Krause et al., and S. Neumueller et al. seem to indicate that there is plasticity within the respiratory rhythm generation system.

Reviewer #3:

Authors investigated the interaction between two coupled oscillators in the lower brainstem of anesthetized adult rats. This is an important issue in neuroscience. The experimental design is straightforward and results are clear. However, I found some problems in the interpretation of results.

General comments:

The most important findings seemed to be that active expiration could not be induced when preBötC inspiratory driven activity was suppressed, based on results in Figures 7 and 8. Indeed, Figure 7 (and also Figure 2) shows that cessation of rhythmic inspiratory activity after Alst injection in preBötC was followed by disappearance of Abd activity. Consequently, authors concluded that in adult rats active expiration is driven by the pF_L but requires ongoing preBötC activity. My concern is that these results may be due to transient unphysiological condition like extremely high CO₂ (and low O₂) of blood gases. To avoid such unphysiological situation, therefore, experiments should be done under following conditions to verify their hypothesis; using ventilator, CO₂ level should be kept in suitable values to induce Abd activity in the presence (or absence) of CNO, then injection of Alst to preBötC should be performed to stop Dia activity and to examine Abd activity.

In Figure 3, they showed quantal inhibition of Dia activity. This result is very important, although they did not discuss much about this result. If possible, authors should also show Abd activity (or GG activity) together with Dia activity during quantal slowing. Analysis of such data may be useful to discuss interaction of two oscillators. The quantal inhibition of respiratory rhythm is known in newborn (and maybe juvenile) animal. Can this phenomenon in the present study be explained in similar mechanisms of interaction between two coupled oscillators like newborn rodent? It would be also helpful for further understanding of coupled oscillator to discuss whether there are any other examples of such quantal inhibition in adult animal.

Related to above questions, activity of two oscillators was assessed by motor neuron outputs in the present study. Central activity of oscillators may not always appear in motor output; i.e. motor output may be not equivalent to central activity to determine CPG property.

Specific comments:

Discussion section, "Shortly after birth (>1 day), the breathing central pattern generator (CPG) is driven by the preBötC (Oku et al., 2007) (Figure 9Biv), and…”: This part is questionable. This optical recording paper seemed to show no direct evidence for postnatal change of the CPG and there is no electrophysiological study to support this idea. More accurate explanation is needed.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "Interactions between respiratory oscillators in adult rats" for further consideration at eLife. Your revised article has been favorably evaluated by a Senior editor and two reviewers, one of whom is a member of our Board of Reviewing Editors.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Reviewer #1:

This is a revision of a previously submitted manuscript. The authors have done a great job in addressing the reviewers’ comments. I have no further comments. But I think it is a solid contribution to the field, and the findings are novel and of general interest. The finding that the pFRG oscillator requires input from the preBötC is surprising which makes this paper very interesting.

Reviewer #2:

I know there were concerns previously that the absence of expiratory activity during prolonged silencing of the preBötC could be due to the effect of hypoxia on expiratory muscle activity. However the discussion in subsection “Respiratory network activity is a requirement for active expiration” seems to counter this concern. That is as the effect of hyperpolarizing effect of Alst on preBötC neurons progressively diminished, the rats were ventilated but upon removal from the ventilator, Abd activity did not return until presumably preBötC activity reached a threshold level. Dia and GG activity always returned before Abd activity. When the rats were ventilated to maintain normal blood gases, there was no Abd activity when the rats were briefly removed from the ventilator; thus, it does not seem that hypoxia could be causing the depressed Abd activity. Accordingly, I accept their point that a minimal amount of preBötC activity seems to be necessary drive the expiratory oscillator.

On the other hand, I am concerned by the conclusion as stated in the Abstract that "in anesthetized adult rats, active expiration is driven by the pF_L but requires an additional form of network excitation, i.e., ongoing rhythmic preBötC activity sufficient to drive inspiratory motor output or increased chemosensory drive". If increased chemosensory drive can drive the pF_L oscillator, then why doesn't the eventual severe hypercapnia during apnea produced by silencing the preBötC activate expiratory muscle activity? Am I missing something?

eLife. 2016 Jun 14;5:e14203. doi: 10.7554/eLife.14203.013

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Please accept the resubmission our manuscript for eLife. In light of the reviewers comments we have performed additional analysis of the data to support our findings.

We have provided an addition panel to Figure 8 from rats we ventilated in the presence of CNO (Pink shading) where end-tidal CO₂ was maintained at normal resting levels (~45 mmHg). This panel shows that active expiration could occur during ventilation, and that no expiratory activity was seen until inspiratory activity returned during ventilation.

Furthermore, upon revising the manuscript we realized that certain statements made within the text could have been made clearer; for example that in the absence of preBӧtC activity, active expiration can occur in the presence of increased chemosensory drive. We recognize that as authors we must accept the blame when readers, including reviewers, miss key points. We have rewritten the manuscript to make this and other points even more explicit.

Reviewer #1:

We appreciate the reviewer’s statement that our proposal that there are separate inspiratory and expiratory oscillators is a “well-known” concept, but there is much work to be done to show how these oscillators interact, and there are still those not convinced of this. We are unaware of any paper (ours or anyone else’s) that looks at active expiration after silencing the preBötC, or how inspiratory and expiratory oscillators interact to pattern breathing in the adult rat. Thus this study does indeed contribute several key new insights to this “well-known” concept.

This reviewer’s surmise of our conclusion is not what we intended to convey; our fault for not making our conclusion sufficiently transparent. We did not say that active expiration requires inhibitory input; rather we said that the phasic pattern of expiration requires inhibition from the preBötC. This is based on the observation that when the preBötC stops firing (including preBötC inhibitory neurons) then the expiratory rhythm transforms from phasic to tonic; this is an idea first proposed by Sears and colleagues. We stated in the Discussion section “We suggest, like Sears et al., that near the transition from eupnea to apnea, expiratory activity is phasic due to periodic inhibition of tonic activity during inspiration, and becomes tonic once the phasic inspiratory inhibition is lost (Figures 2Bii-iii, 7Bii, 9Diii). We suggest the loss of phasic inhibition is the result of silencing of inhibitory preBötC inspiratory neurons by Alst (Kuwana et al., 2006; Morgado-Valle et al., 2010), implying a significant role for inhibitory preBötC neurons in shaping expiratory output.”

Moreover, we actually postulated that silencing of inhibitory neurons in the preBӧtC may actually increase expiratory activity, through disinhibition of the pF_L. As we stated “Alternatively, active expiration could be due to disinhibition of a conditional expiratory oscillator resulting from a loss of (presumptive) inhibitory preBötC drive (Kuwana et al., 2006; Morgado-Valle et al., 2010) to the pF_L (Figure 9Dii).”

However, based on the experiments submitted here, this conclusion is not justified.

We explicitly acknowledged the inhibitory effect of hypoxia, and its role in the cessation of on expiratory activity. As we stated in the Discussion section “when inspiratory motor outflow disappeared and hypercapnia and hypoxia inexorably increased, active expiration ceased (Figures 2, 9Div, see also Tan et al., 2008), perhaps due to the inhibitory effect of severe hypoxia on expiratory motor output (Sears et al., 1982; Fregosi et al., 1987)”.

In fact, it is most likely the hypercapnia and hypoxia during hypopnea that allows tonic activity of abdominal muscles to continue after inspiratory activity ceases. As seen in Figure 2, where there is expiratory activity without activation of HM₃DRs in the pF_L, and stated in the Discussion section “In either case, active expiration could have been due to increased hypoxic and/or hypercapnic drive to the pF_L as inspiratory movements waned”

Thus, in order to arrive at the conclusion that the authors propose in this study, they need to add new experiments. I.e. block or silence the preBötC while ventilating the animal show stable oxygenation and induce active expiration and then see whether under these physiologically stable conditions active expiration persists or disappears when inspiration ceases. Given that this is the major conclusion on which this paper rests, this experiment is absolutely essential.

Even though we acknowledge the role of hypoxia on the cessation of expiratory activity, a careful examination of the data and a conservative interpretation leads to the parsimonious conclusion that hypoxia per se could NOT have participated in the absence of expiration activity during ventilation, or the delay in expiratory activity following removal from the ventilator. Rats were ventilated on average for ~18 min following the onset apnea after which removal from the ventilator led to the onset of inspiratory activity (Figure 8A,B). Thus, rats were normoxic for ~18 min during which time the pF_L was being excited by the activation of HM₃DRs and the preBötC was inhibited by activation of AlstRs and presumably silent; during this time, no abdominal activity was seen. That expiratory activity returned within 30 secs of being removed from the ventilator (Figure 8A, B) means that the HM₃DRs were still driving the pF_L, and the pF_L was still capable of driving abdominal activity. The lack of abdominal activity during ventilation was not the result of mechanical actions of the ventilator, as expiratory activity was apparent during ventilation towards the end of the experiment (see Figure 8D). Therefore, the most parsimonious explanation for the lack of abdominal activity during ventilation when inspiratory motor activity is absent is that expiratory drive from the pF_L alone is insufficient to drive expiration and requires an additional source of network excitation, such as inspiratory activity or chemosensory drive.

We have added a new panel in Figure 8, panel D, to show that expiration returns after inspiratory activity, and that abdominal activity can still occur during ventilation. We have also expanded the Results, Discussion and Summary to make this clearer.

Results section”: “Rats were mechanically ventilated to restore CO₂ and O₂ levels to within the normal range (Figure 7). […] Therefore, following silencing of the preBötC with Alst in the presence of CNO to drive the pF_L at no time during the re-initiation of spontaneous breathing, either with or without mechanical ventilation, did active expiration occur in the absence of inspiratory activity and chemosensory drive.”

Discussion section: “Following apnea during ventilation to maintain blood gases, when pF_Lneurons were excited by activation of HM₃DRs there were three phases to the re-initiation of breathing. […] As blood gases were normal (Figure 8), the loss of expiratory activity is unlikely to be due to excessive hypoxia or hypercapnia.”

Summary: “the pF_L is unable to drive breathing (including active expiration) in the absence of an additional form of network excitation, i.e., preBötC activity or chemosensory drive. This hierarchy is established by the sensitivity of the system to each oscillator, even when preBӧtC drive is low, it is able to drive inspiratory bursts and inhibit expiration, whereas even when pF_L drive is high, it requires a certain amount of network excitability from other sources to drive expiratory activity.”

It was in fact essential to the conclusions of this paper that rats were allowed to become moderately hypercapnic and moderately hypoxic during the hypopnea leading up to the apnea, as this allowed us to see that in the absence of inspiratory activity, abdominal activity could persist if network excitability was increased by chemosensory drive, if only for a short period until it was inhibited by severe hypoxia. Furthermore, following apnea rats were immediately placed on the ventilator to remove hypoxic inhibition of expiratory activity, which showed that in conditions with no preBötC activity and no hypercapnic drive in normoxic conditions there was no expiratory activity. The experimental design allowed us to assess expiratory activity under the following conditions.

pF_L activity	preBӧtC activity	Hypercapnia	Hypoxia	Abdominal activity
No	Yes	No	No	Absent
Yes (driven by CO₂)	Low	Moderate	Moderate	Phasic
Yes (driven by CO₂)	Absent	Moderate	Moderate	Tonic
No (inhibited by hypoxia)	Absent	High	High	Absent
No	Absent	No	No	Absent
Yes (driven by HM₃DR)	Yes	No	No	Phasic
Yes (driven by HM₃DR and CO₂)	Low	Moderate	Moderate	Phasic
Yes (driven by HM₃DR and CO₂)	Absent	Moderate	Moderate	Tonic
Yes (driven by HM₃DR, but inhibited by hypoxia)	Absent	High	High	Absent
Yes (driven by HM₃DR)	Absent	No	No	Absent

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We have also expanded the Methods section to make our protocol clearer. “Though rats were ventilated with room air for the duration of ensuing apnea, they were intermittently removed from the ventilator to assess spontaneous breathing, and drives to inspiration and expiration. […] Once rats were spontaneously breathing and no longer required ventilation, CNO was removed from the ventral surface of the medulla and the medulla was washed in PBS.”

In our original burstlet paper (Kam et al. 2013), burstlets are by definition subthreshold events. However, as made clear in Kam et al. 2013 paper (Figure 7 and the associated text), burstlets can be transmitted to motor output if specific conditions are met; namely lowered excitability in preBötC with increased drive to respiratory motor pools. We believe that these conditions are met in our experiments: lowered drive in the preBötC resulting from activation of the allatostatin receptor depresses bursting while burstlets may continue in the preBötC. Under normal conditions burstlets are not strong enough to drive activity in motor pools. However, increased excitability in the motor pools caused by increased chemosensory drive (from decreased ventilation) causes these normally subthreshold events to become suprathreshold and thus these burstlets are transmitted to the motor pool. As we stated in the Results section, “This low amplitude Dia_EMG and airflow activity are postulated to represent the inspiratory motor outflow, i.e., EMG, manifestation of preBötC burstlets (Kam et al., 2013), i.e., low levels of rhythmogenic neural population activity in the preBötC that under normal conditions occur in the absence of motor output, being transmitted to motoneuron pools (see Figure 7A in Kam et al., 2013).”

We have also expanded the Discussion to make this clearer. “Here, lowered drive in the preBötC from activation of the allatostatin receptor creates burstlets in the preBötC; under normal conditions these burstlet signals are not strong enough to drive motor activity. […] Interestingly, even when expiratory activity was at its highest, low level preBötC activity, as seen as burstlets on the Dia_EMG and GG_EMG was still able to inhibit Abd_EMG activity (Figure 7Bii).”

Reviewer #2:

First, rostral pontine nuclei provide an important contribution to the control of breathing. What are the authors' speculations regarding this contribution to the two oscillators shown herein?

Second, what are the authors' thoughts on interactions between these two respiratory oscillators during normal physiological conditions particularly under conditions of NREM and REM sleep?

We are delighted that this reviewer enjoyed our manuscript. Although no specific changes were requested, we did consider the reviewer’s suggestions in the revised manuscript.

Though the contribution of respiratory-related areas to network output is beyond the remits of this study, we do not in any way rule it out. In fact some of the chemosensory drive seen during moderate hypercapnia and hypoxia likely comes from sources outside of the preBötC and pF_L. So as not to break up the flow of the discussion we have added a section to the technical considerations section to discuss this possibility. Results section “Here we discuss the output of the preBötC and pF_L, which ultimately form the final output of the respiratory network. […]Therefore, the activity of the preBötC and pF_L, was assessed by motor output recorded from respiratory muscles, which may not always reflect the activity of these oscillators, as they may have subthreshold activity, e.g., Kam et al., 2013.”

In adult rats, there is no expiratory activity seen during sleep under normal physiological conditions, so we expect the interaction between the oscillators to be the same during sleep as they are during quiet wakefulness. To make this clear we have slightly expanded the line in the Introduction section to read: “In mammals at rest, during wakefulness and sleep, when active breathing movements are primarily inspiratory, generation of the underlying rhythm appears driven by the preBötC”.

The effect of chronic lesioning of the respiratory oscillators and network compensation for these changes is complicated. The Neumueller et al. 2011 paper, shows that destruction of the preBӧtC can be compensated for if the lesion occurs incrementally over time, however the Tupal et al. 2014 paper shows that loss of DBX1 neurons from birth cannot be compensated for. To properly address this subject would take considerable discussion in the manuscript, thus to keep our discussion concise we have not covered this subject in this paper.

Reviewer #3:

General comments:

This is partly correct, we do conclude that preBӧtC is a requirement for rats who are normoxic and normocapnic, hence the return of inspiratory activity always precedes the return of expiratory activity following apnea and ventilation. However, we conclude that expiratory activity can occur in the absence of inspiration if chemosensory drive is high, hence the appearance of tonic abdominal activity following the cessation of inspiratory activity. We concluded that expiratory activity requires a certain level of network activity from another source, either inspiratory activity or chemosensory drive. As we stated “Thus there appears to be a minimum level of respiratory network excitability required for active expiration. […] However following recovery from apnea the increase in network excitability is provided by the preBötC either directly through its extensive excitatory projections throughout the respiratory network (Tan et al., 2010), e.g., the pF_V that can modulate to expiratory activity (Marina et al., 2010; Huckstepp et al., 2015), or indirectly when V_T became large enough to induce mechanosensory feedback (that can provide expiratory drive (Remmers, 1973; Davies and Roumy, 1986; Janczewski and Feldman, 2006))”

We have also expanded the Abstract, Introduction, Discussion and Summary to make this clearer.

Introduction: “We conclude that in anesthetized adult rats active expiration is driven by the pF_L but requires an additional form of network excitation, i.e., ongoing rhythmic preBötC activity sufficient to drive inspiratory motor output or increased chemosensory drive.”

Introduction: “Importantly, we found that active expiration could not be induced when preBötC inspiratory driven motor activity was suppressed and chemosensory drive was absent, indicating that in adult rats active expiration is driven by the pF_L but requires an additional source of network excitation such as ongoing preBötC activity or chemosensory drive.”

Discussion section: “The pF_L is the subsidiary conditional oscillator, quiescent at rest (Figure 9Di); in the presence of concurrent, or reduced, preBötC activity (Figure 2), and appropriate inputs, such as an increase in expiratory drive through activation of exogenous receptors (Figure 6, see also Pagliardini et al., 2011) or altered blood gases (Huckstepp et al., 2015), the pF_L drives active expiration (Figure 9Dii). In the absence of preBötC activity, chemosensory drive increases network excitability and drives active expiration, though abdominal activity is tonic due to a lack of phasic inhibition from the preBötC (Figure 9Diii).”

My concern is that these results may be due to transient unphysiological condition like extremely high CO₂ (and low O₂) of blood gases. To avoid such unphysiological situation, therefore, experiments should be done under following conditions to verify their hypothesis; using ventilator, CO₂ level should be kept in suitable values to induce Abd activity in the presence (or absence) of CNO, then injection of Alst to preBötC should be performed to stop Dia activity and to examine Abd activity.

For CO₂ levels to be kept in suitable values to induce Abd activity in the absence of CNO, the rat would have to be chronically hypercapnic. In that situation, we would expect abdominal activity to continue, as chemosensory drive can drive abdominal activity in the absence of inspiratory activity, see previous answer. As discussed above, our experimental design allowed us to test the interactions of the preBötC and pF_L under an array of conditions. See table above.

The effect of quantal slowing on ABD and GG in conjunction with Dia can be seen in Figure 2B and 7Bii, and so was not included in Figure 3, which shows burstlet activity and how quantal slowing was calculated. We have added the following sentence to expand on this in the Discussion, “During quantal slowing of breathing, inspiratory activity on the Dia_EMG and GG_EMG was interspersed with Abd_EMG activity (Figures 2A-B, 7A-B). Interestingly, even when expiratory activity was at its highest, low level preBötC activity, as seen as burstlets on the Dia_EMG and GG_EMG was still able to inhibit Abd_EMG activity (Figure 7Bii).”

We agree that motor output may not always reflect oscillator activity, as described in our burstlet paper (Kam et al., 2013). We added the following statement to the technical considerations section to make this clear. “In addition, due to the necessity to perform injections into the medulla, we were unable to record directly from the preBötC or pF_L. Therefore, the activity of the preBötC and pF_L, was assessed by motor output recorded from respiratory muscles, which may not always reflect the activity of these oscillators, as they may have subthreshold activity, e.g., Kam et al., 2013.”

Specific comments:

We have added two new references, which provide evidence of the maturation of respiratory system from an ePF/pFRG lead network to a preBӧtC lead network.

Discussion section: “Shortly after birth (~P2-P4), the breathing central pattern generator (CPG) matures, and becomes driven by the preBötC (Oku et al., 2007; Mckay et al., 2009; Kennedy, 2015) (Figure 9Biv), and remains so throughout life.”

[Editors' note: the author responses to the re-review follow.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Reviewer #1:

This is a revision of a previously submitted manuscript. The authors have done a great job in addressing the reviewers comments. I have no further comments. But I think it is a solid contribution to the field, and the findings are novel and of general interest. The finding that the pFRG oscillator requires input from the preBötC is surprising which makes this paper very interesting.

We thank the reviewer for his/her kind comments.

Reviewer #2:

We are pleased that our revisions to the manuscript have made this point clearer.

We previously showed that both hypercapnia and hypoxia, when moderate and brief, lead to expiratory-related abdominal activity (Huckstepp et al., J Neurosci, 2016). In the paper under review, we conclude that the moderate hypercapnia and hypoxia following cessation of inspiratory activity is responsible for the tonic abdominal muscle activity at the beginning of apnea. As stated in the Discussion section “In either case, active expiration could have been due to increased hypoxic and/or hypercapnic drive to the pF_L as inspiratory movements waned”. This underlies our statement in the Summary that additional network excitation from increased chemosensory drive can lead to abdominal activity in the absence of preBötC activity.

We acknowledge that severe hypoxia profoundly inhibits abdominal muscle recruitment (Fregosi et al., J Appl Physiol, 1987), suggesting that the cessation of abdominal activity during apnea is due to severe hypoxia resulting from the prolongation of the apnea. We state: “when inspiratory motor outflow disappeared and hypercapnia and hypoxia inexorably increased, active expiration ceased (Figures 2, 9Div, see also Tan et al., 2008), perhaps due to the inhibitory effect of severe hypoxia on expiratory motor output (Sears et al., 1982; Fregosi et al., 1987)”.

Nonetheless, we maintain that a detailed examination of the data leads to the parsimonious conclusion that hypoxia does NOT underlie the absence of expiratory activity during ventilation (that assures proper gas exchange and avoids hypoxia and hypercapnia), or the delay in the onset of expiratory activity following removal from the ventilator (see the reviewer’s previous comment and section “Respiratory network activity is a requirement for active expiration for discussion”).

In our Summary and Abstract we state that an additional form of network excitation is required for active expiration in anesthetized adult rats, as even when driving the pF_L by activation of the HM₃D receptor, abdominal activity does not occur in the absence of another source of network excitation. We conclude that the additional network excitation necessary for recruitment of abdominal muscles can come from: 1) ongoing rhythmic preBötC activity sufficient to drive inspiratory motor output, as abdominal activity is not seen in the absence of inspiratory motor output when chemosensory drive is low or absent, or 2) increased chemosensory drive, as tonic abdominal activity is seen immediately after the cessation of inspiratory activity, when drive from the preBötC is low or absent but chemosensory drive is moderate.

We have redacted the Summary to make this point clearer: “the pF_Lis unable to drive breathing (including active expiration) in the absence of additional network excitation, i.e., ongoing rhythmic preBötC activity sufficient to drive inspiratory motor output or increased chemosensory drive when changes in blood gases are below the threshold where hypoxia is sufficient to inhibit abdominal muscle recruitment.”

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PERMALINK

Interactions between respiratory oscillators in adult rats

Robert TR Huckstepp

Lauren E Henderson

Kathryn P Cardoza

Jack L Feldman

Roles

Abstract

eLife digest

Introduction

Results

Viral targeting of the preBötC and pFL

Figure 1. Transfection of neurons in preBötC and pFL.

Silencing the preBötC initially slowed breathing frequency (f) and induced active expiration, but eventually resulted in apnea

Figure 2. Hyperpolarizing preBötC neurons reduced ventilation and induced active expiration, but eventually resulted in apnea.

Figure 3. Hyperpolarizing preBötC neurons leads to quantal slowing of breathing and burstlet-like activity in ∫DiaEMG.

Figure 4. Alst in absence of AlstRs does not affect breathing.

Activation of the pFL leads to active expiration

Figure 5. CNO in absence of HM3DRs does not affect respiration.

Figure 6. Depolarizing pFL neurons elicits active expiration.

Silencing the preBötC during active expiration leads to apnea

Figure 7. Hyperpolarizing preBötC neurons leads to apnea, and loss of active expiration even with activation of pFL.

Figure 8. Following apnea, in presence of CNO, active expiration only returns after inspiratory activity returns.

Technical consideration

Discussion

Figure 9. Developmental and state-dependent changes in coupling between pFL and preBötC.

Silencing the preBötC leads to quantal slowing of breathing and transmission of burstlets to motoneuron pools

Silencing the preBötC leads first to active expiration then to apnea

Activating the pFL can induce active expiration but not during to apnea

Loss of inhibitory output from the preBötC leads to a loss of phasic expiratory activity

Respiratory network activity is a requirement for active expiration

Summary

Materials and methods

Viral design and handling

Viral transfection (Figure 1)

Ventral approach

Localization of transfected neurons

Data analysis and statistics

Funding Statement

Funding Information

Additional information

Competing interests

Author contributions

Ethics

References

Decision letter

Roles

Author response

ACTIONS

PERMALINK

RESOURCES

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Viral targeting of the preBötC and pF_L

Figure 1. Transfection of neurons in preBötC and pF_L.

Figure 3. Hyperpolarizing preBötC neurons leads to quantal slowing of breathing and burstlet-like activity in ∫Dia_EMG.

Activation of the pF_L leads to active expiration

Figure 5. CNO in absence of HM₃DRs does not affect respiration.

Figure 6. Depolarizing pF_L neurons elicits active expiration.

Figure 7. Hyperpolarizing preBötC neurons leads to apnea, and loss of active expiration even with activation of pF_L.

Figure 9. Developmental and state-dependent changes in coupling between pF_L and preBötC.

Activating the pF_L can induce active expiration but not during to apnea