Functional Connectivity in Raphé-Pontomedullary Circuits Supports Active Suppression of Breathing During Hypocapnic Apnea

Hyperventilation is a common feature of disordered breathing. Apnea ensues if CO2 drive is sufficiently reduced. We tested the hypothesis that medullary raphé, ventral respiratory column (VRC), and pontine neurons have functional connectivity and persistent or evoked activities appropriate for roles in the suppression of drive and rhythm during hyperventilation and apnea. Phrenic nerve activity, arterial blood pressure, end-tidal CO2, and other parameters were monitored in 10 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated cats. Multielectrode arrays recorded spiking activity of 649 neurons. Loss and return of rhythmic activity during passive hyperventilation to apnea were identified with the S-transform. Diverse fluctuating activity patterns were recorded in the raphé-pontomedullary respiratory network during the transition to hypocapnic apnea. The firing rates of 160 neurons increased during apnea; the rates of 241 others decreased or stopped. VRC inspiratory neurons were usually the last to cease firing or lose rhythmic activity during the transition to apnea. Mayer wave-related oscillations (0.04–0.1 Hz) in firing rate were also disrupted during apnea. Four-hundred neurons (62%) were elements of pairs with at least one hyperventilation-responsive neuron and a correlational signature of interaction identified by cross-correlation or gravitational clustering. Our results support a model with distinct groups of chemoresponsive raphé neurons contributing to hypocapnic apnea through parallel processes that incorporate disfacilitation and active inhibition of inspiratory motor drive by expiratory neurons. During apnea, carotid chemoreceptors can evoke rhythm reemergence and an inspiratory shift in the balance of reciprocal inhibition via suppression of ongoing tonic expiratory neuron activity. breathing is a remarkably robust behavior that is activated at birth and continues until death, yet the brain stem neural network controlling it is even more remarkable in its malleability. For example, talking, swallowing, and coughing are motor acts that alter the breathing pattern and, rather than simply inhibiting breathing, the neural substrate for these motor acts transiently appropriates and reconfigures the respiratory pattern generator (Bolser et al. 2011, 2013; Shannon et al. 2004). However, what about conditions when breathing stops? Hyperventilation is a component of dangerous underwater breath-holding behaviors (Boyd et al. 2015; Craig 1961) and a common feature of disordered breathing (for discussion, see Abdala et al. 2014; Dempsey 2005; Laffey and Kavanagh 2002). If the drive from CO2 is sufficiently reduced, hypocapnic apnea, a transient cessation of breathing, ensues with its attendant and potentially adverse consequences (Bitter et al. 2011; Harper et al. 2013; Javaheri and Dempsey 2013; Leung et al. 2012; Sankri-Tarbichi et al. 2009; Sankri-Tarbichi 2012). During the transition from eupneic-like breathing to hyperventilatory apnea, phrenic motoneurons (Prabhakar et al. 1986) and phasic respiratory-modulated brain stem neurons either cease to discharge or assume a tonic pattern of activity (Bainton and Kirkwood 1979; Batsel 1967; Cohen 1968; Haber et al. 1957; Nesland and Plum 1965; Orem and Vidruk 1998; St. John 1998; Sun et al. 2001, 2005). With one exception (Cohen 1968), these studies recorded neurons in the ventral respiratory column (VRC; Smith et al. 2013), one at a time. This approach precludes assessment of local connectivity within the VRC and of distributed interactions with pontine and raphé neurons of the respiratory network (Nuding et al. 2009a; Segers et al. 2008). The circuit mechanisms for hypocapnic apnea remain poorly understood. Both reduced excitatory chemoreceptor drive and active inhibitory processes may contribute to the suspended state of the respiratory central pattern generator. Peripheral chemoreceptors of the carotid body monitor changes in arterial O2 and CO2-pH (Kumar and Prabhakar 2012), and central chemoreceptors, distributed among various brain stem sites, sense brain CO2-pH (Nattie and Li 2012). Mechanisms of their joint and separate influences on pattern-generating circuits are subjects of active research (Duffin and Mateika 2013a,b; Phillipson et al. 1981; Teppema and Smith 2013a,b; Wilson and Day 2013a,b). Central chemoreceptors and their follower neurons, collectively termed chemoresponsive, may be either functionally excited or inhibited by an increase in PaCO2 (e.g., Bochorishvili et al. 2012; Dean et al. 1989; Guyenet et al. 2010; Marina et al. 2010; Nuding et al. 2009b; Ott et al. 2011, 2012; Richerson et al. 2001). Medullary raphé neurons have diverse responses to hypercapnia and acidosis: firing rates of serotonergic neurons increase (Brust et al. 2014; Iceman et al. 2013; Severson et al. 2003; Veasey et al. 1995; Wang et al. 1998, 2001), whereas GABAergic raphé neurons are functionally inhibited (Iceman et al. 2014). These results are consistent with the hypothesis that distinct populations of chemoresponsive raphé neurons produce an additive “push-pull” enhancement of breathing via excitation and disinhibition, respectively (Richerson et al. 2001), a notion similar to that proposed for baroreceptor-evoked modulation of breathing via raphé-mediated excitation and disinhibition of ventral respiratory column expiratory neurons (Lindsey et al. 1998). The distinct chemoresponsive profiles of different raphé neuron populations led us to conjecture that cells effectively excited during hypercapnia would exhibit decreased firing rates during hypocapnia and vice versa for neurons inhibited during hypercapnia. This possibility and gaps in our knowledge of network interactions motivated us to test the hypothesis that medullary raphé, VRC, and pontine neurons have functional connectivity as well as persistent and evoked activities appropriate for roles in the suppression of respiratory drive and rhythm during hyperventilation and hypocapnic apnea. Sears et al. (1982) demonstrated reciprocal tonic activation of inspiratory and expiratory motor neurons during hypocapnic apnea. A PaCO2 drive below the apneic threshold may promote expiratory activity and functionally suppress inspiration. Hypoxia associated with apnea evokes increased peripheral chemoreceptor activity, enhances or elicits tonic inspiratory motor neuron activities, and can reestablish respiratory rhythmogenesis. Fluctuations in this peripheral chemoreceptor-mediated “inspiratory shift,” operating through unknown circuit mechanisms, may contribute to periodic breathing in heart failure and central sleep apnea (Lovering et al. 2012). Our multielectrode arrays allow concurrent single-unit recordings from multiple brain stem nuclei that generate and modulate breathing. This approach is well suited for testing our hypothesis and assessment of the activity patterns of many neurons under the same conditions. Thus we recorded changes in firing rates during different chemoreceptor-evoked perturbations of breathing and evaluated spike trains for correlation features indicative of functional connectivity. Preliminary accounts of this work have been presented (Lindsey et al. 2014; Nuding et al. 2005, 2013)