Contiguous brain regions associated with a given behavior are increasingly being divided into subregions associated with distinct aspects of that behavior. Using recently developed neuronal hyperpolarizing technologies, we functionally dissect the parafacial region in the medulla, which contains key elements of the central pattern generator for breathing that are important in central CO 2 -chemoreception and for gating active expiration. By transfecting different populations of neighboring neurons with allatostatin or HM 4 D G i/o -coupled receptors, we analyzed the effect of their hyperpolarization on respiration in spontaneously breathing vagotomized urethaneanesthetized rats. We identify two functionally separate parafacial nuclei: ventral (pF V ) and lateral (pF L ). Disinhibition of the pF L with bicuculline and strychnine led to active expiration. Hyperpolarizing pF L neurons had no effect on breathing at rest, or changes in inspiratory activity induced by hypoxia and hypercapnia; however, hyperpolarizing pF L neurons attenuated active expiration when it was induced by hypercapnia, hypoxia, or disinhibition of the pF L . In contrast, hyperpolarizing pF V neurons affected breathing at rest by decreasing inspiratory-related activity, attenuating the hypoxia-and hypercapnia-induced increase in inspiratory activity, and when present, reducing expiratory-related abdominal activity. Together with previous observations, we conclude that the pF V provides a generic excitatory drive to breathe, even at rest, whereas the pF L is a conditional oscillator quiet at rest that, when activated, e.g., during exercise, drives active expiration.

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 (pFL) 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 pFL 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 pFL 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

Recently, based on functional differences, we subdivided neurons juxtaposed to the facial nucleus into two distinct populations, the parafacial ventral and lateral regions, i.e., pFV and pFL. Little is known about the composition of these regions, i.e., are they homogenous or heterogeneous populations? Here, we manipulated their excitability in spontaneously breathing vagotomized urethane anesthetized adult rats to further characterize their role in breathing. In the pFL, disinhibition or excitation decreased breathing frequency (f) with a concomitant increase of tidal volume (VT), and induced active expiration; in contrast, reducing excitation had no effect. This result is congruent with pFL neurons constituting a conditional expiratory oscillator comprised of a functionally homogeneous set of excitatory neurons that are tonically suppressed at rest. In the pFV, disinhibition increased f with a presumptive reflexive decrease in VT; excitation increased f, VT and sigh rate; reducing excitation decreased VT with a presumptive reflexive increase in f. Therefore, the pFV, has multiple functional roles that require further parcellation. Interestingly, while hyperpolarization of the pFV reduces ongoing expiratory activity, no perturbation of pFV excitability induced active expiration. Thus, while the pFV can affect ongoing expiratory activity, presumably generated by the pFL, it does not appear capable of directly inducing active expiration. We conclude that the pFL contains neurons that can initiate, modulate, and sustain active expiration, whereas the pFV contains subpopulations of neurons that differentially affect various aspects of breathing pattern, including but not limited to modulation of ongoing expiratory activity.

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