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.

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.

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

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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).

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Author details

1. Robert TR Huckstepp

   Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States

   Contribution

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

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-4410-3397

2. Lauren E Henderson

   Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States

   Contribution

   LEH, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Kathryn P Cardoza

   Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States

   Contribution

   KPC, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Jack L Feldman

   Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States

   Contribution

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

   For correspondence

   feldman@g.ucla.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-3692-9412

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