Peripheral Chemoreceptors Tune Inspiratory Drive via Tonic Expiratory Neuron Hubs in the Medullary Ventral Respiratory Column Network

Models of brain stem ventral respiratory column (VRC) circuits typically emphasize populations of neurons, each active during a particular phase of the respiratory cycle. We have proposed that “tonic” pericolumnar expiratory (t-E) neurons tune breathing during baroreceptor-evoked reductions and central chemoreceptor-evoked enhancements of inspiratory (I) drive. The aims of this study were to further characterize the coordinated activity of t-E neurons and test the hypothesis that peripheral chemoreceptors also modulate drive via inhibition of t-E neurons and disinhibition of their inspiratory neuron targets. Spike trains of 828 VRC neurons were acquired by multielectrode arrays along with phrenic nerve signals from 22 decerebrate, vagotomized, neuromuscularly blocked, artificially ventilated adult cats. Forty-eight of 191 t-E neurons fired synchronously with another t-E neuron as indicated by cross-correlogram central peaks; 32 of the 39 synchronous pairs were elements of groups with mutual pairwise correlations. Gravitational clustering identified fluctuations in t-E neuron synchrony. A network model supported the prediction that inhibitory populations with spike synchrony reduce target neuron firing probabilities, resulting in offset or central correlogram troughs. In five animals, stimulation of carotid chemoreceptors evoked changes in the firing rates of 179 of 240 neurons. Thirty-two neuron pairs had correlogram troughs consistent with convergent and divergent t-E inhibition of I cells and disinhibitory enhancement of drive. Four of 10 t-E neurons that responded to sequential stimulation of peripheral and central chemoreceptors triggered 25 cross-correlograms with offset features. The results support the hypothesis that multiple afferent systems dynamically tune inspiratory drive in part via coordinated t-E neurons. understanding the connectivity and functions of oscillating networks in the brain is a major goal in neuroscience (e.g., Akam and Kullmann 2014). Brain stem circuit mechanisms that control the drive to breathe are of particular interest not only because of their vital functions in maintaining ventilation and cardiovascular coupling (Nicholls and Paton 2009). The brain stem respiratory network can concurrently express multiple rhythms, in addition to the basic breathing rhythm, with frequencies spanning several orders of magnitude (Dick et al. 2005; Funk and Parkis 2002; Morris et al. 2010). These circuits for breathing have remarkable adaptive and emergent properties (Devinney et al. 2013; Lindsey et al. 1992, 2012; Ramirez et al. 2012) and participate in the generation of numerous other behaviors (Bartlett and Leiter 2012), including vocal communication (McLean et al. 2013), coughing and swallowing, and their coordinated expression as a metabehavioral response to aspiration (Pitts et al. 2013; Shannon et al. 2000). Breathing may also bind orofacial sensations (Kleinfeld et al. 2014) and be involved in hippocampal processes for learning and memory (Lockmann and Belchior 2014). Core circuits for generating the respiratory motor pattern are found in the ventral respiratory column (VRC; Richter and Smith 2014) and are embedded in a larger pontomedullary respiratory network (Nuding et al. 2009a; Segers et al. 2008). Models of the VRC typically emphasize interactions among populations of neurons, each active only during a particular phase of the respiratory cycle or at a phase transition (Lindsey et al. 2012). However, pericolumnar “tonic” neurons with or without respiratory-modulated discharge patterns have also been identified and proposed to serve as intermediaries in the regulation of breathing by sensory (e.g., baroreceptor, chemoreceptor) systems and by internal state-dependent drives (Orem 1989). We have proposed that the VRC includes an excitatory inspiratory (I) neuron chain tuned by feedforward and recurrent inhibition from other I neurons and by the inspiratory-phase inhibitory actions of tonically firing expiratory (t-E) neurons, i.e., cells that are active predominantly during the expiratory (E) phase but also discharge during the I phase under eupneic-like normocapnic conditions (O\u27Connor et al. 2012; Segers et al. 1987). Several observations support the hypothesis that t-E neurons serve as a downstream network “hub” (van den Heuvel and Sporns 2013) where signals from multiple afferent systems functionally converge for modulation of inspiratory drive. First, baroreceptor-evoked reductions in inspiratory drive are associated with increased firing rates in t-E neurons that have inhibitory functional connections with inspiratory premotor or motor neurons (Lindsey et al. 1998). Second, central chemoreceptor-evoked enhancement of inspiratory drive occurs during suppressed inspiratory-phase activity in t-E neurons (Ott et al. 2012). Third, such disinhibitory amplification of inspiratory drive also occurs during a bout of coughing (Segers et al. 2012). In the course of these prior studies, we identified cross-correlation feature sets indicative of distributed functional inhibitory connectivity of t-E neurons within the column (Ott et al. 2012; Segers et al. 2012) and noted that some pairs of t-E neurons exhibited short-timescale spike synchrony. One aim of the present work was to confirm and extend that result. Such coordinated firing would be consistent with the hub concept and cooperative behavior among t-E neurons that could influence the efficacy or duration of their collective actions on common targets. Peripheral chemoreceptors of the carotid body rapidly sense changes in arterial O2 and CO2-pH and also modulate the drive to breathe (Kumar and Prabhakar 2012), although the functional connections through which they act upon VRC circuits remain incompletely understood (Nuding et al. 2009b; Spyer and Gourine 2009). Having previously identified differential peripheral chemoreceptor modulation of inspiratory neurons in the pre-Bötzinger region and more caudal medullary domains (Morris et al. 1996, 2001), we had the second objective of testing the hypothesis that carotid chemoreceptors enhance inspiratory drive via downstream disinhibitory actions of t-E neurons upon caudal columnar inspiratory neurons. Numerous disorders of breathing and cardio-respiratory coupling are associated with dysfunctional chemoreceptor drive mechanisms (Dempsey and Smith 2014; Garcia et al. 2013; Perez and Keens 2013; Plataki et al. 2013). Brain mechanisms that mediate the separate and joint actions of central chemoreceptors, sensors of brain CO2 and pH, and the peripheral chemoreceptors are topics of active research and debate (Duffin and Mateika 2013; Nuding et al. 2009b; Teppema and Smith 2013; Wilson and Day 2013). Thus a related third aim was to determine whether t-E neurons are dually modulated by both central and peripheral chemoreceptor influences. We employed electrode arrays with individual submicrometer electrode depth adjustments to monitor VRC neurons at multiple sites simultaneously. Acquired data sets were screened for short-timescale correlations indicative of paucisynaptic functional connectivity and altered firing rates during chemoreceptor perturbation