Postsynaptic mechanisms of CO₂ responses in parafacial respiratory neurons of newborn rats

Abstract

Key points• The central chemoreceptors for respiratory control in the medulla sense changes in CO 2 concentration and regulate respiratory activity.• Neurons that express a transcription factor, Phox2b, in the parafacial region of the rostral and ventrolateral medulla are excited by hypercapnic stimulation and are proposed to play an important role in central chemoreception.• In this study, we show evidence that Phox2b-expressing parafacial neurons in neonatal rats were sensitive to hypercapnia via direct action on the postsynaptic membrane without contribution of putative presynaptic or other calcium-dependent components.• Since these parafacial neurons are also a part of the respiratory rhythm generator in neonates, they are essential for postnatal survival, which is probably due to their contribution to central chemoreception as well as respiratory rhythm generation. AbstractThe parafacial respiratory group (pFRG) in the rostral ventrolateral medulla of the newborn rat is predominantly composed of pre-inspiratory (Pre-I) neurons and is involved in respiratory rhythm generation. The subgroup located close to the ventral surface (at least partially overlapping the retrotrapezoid nucleus, RTN) expresses the Phox2b transcription factor and responds to hypercapnic stimulation with strong depolarization, which suggests it has a role in central chemoreception. Although a CO 2 response of pFRG/RTN neurons has been confirmed in the presence of tetrodotoxin (TTX), it is unknown whether the depolarization involved in this response is induced by a direct postsynaptic response of pFRG/RTN neurons or by any presynaptic components mediated by Ca 2+ -dependent mechanisms. In this study, we examined the effects of ATP or substance P receptor antagonists on hypercapnic responses of rostral pFRG/RTN neurons. We tested effects of Cd 2+ and low Ca 2+ -high Mg 2+ in the presence of TTX. The experiments were performed in in vitro brainstem-spinal cord preparations from newborn rats in which Pre-I neurons reflect the discharge pattern of the pFRG. We found that ATP receptor and substance P receptor antagonists do not block membrane potential responses to hypercapnic stimulation (2% → 8%) of pFRG/RTN neurons in the rostral parafacial region. Moreover, rostral pFRG/RTN neurons were depolarized by hypercapnia under conditions where the contribution of presynaptic components was inhibited in the presence of TTX and Cd 2+ or in a low Ca 2+ -high Mg 2+ solution containing TTX and Cd 2+ . All cases (except some cases in a low Ca 2+ -high Mg 2+ solution) of membrane depolarization by hypercapnic stimulation were accompanied with an increase in input resistance. These neurons were predominantly Phox2b immunoreactive. suggest that the response of pFRG/RTN neurons to hypercapnia is induced by direct action on the postsynaptic membrane via closing of K + channels.

Regulation of Breathing and Autonomic Outflows by Chemoreceptors

“…In keeping with this interpretation, RTN neurons are activated by CO 2 and acidification in coronal slices of neonate rodent brain (Fig. 5G2) even when synaptic activity is reduced by addition of blockers of glutamatergic, glycinergic, and GABAergic ionotropic transmission plus PPADS, a blocker of ATP receptors (231, 300, 355) or by incubation in low calcium/high magnesium solution (328). Finally, these cells also retain their CO 2 sensitivity after acute isolation (446) (Fig.…”

“…Numerous electrophysiological studies subsequently showed that acidification or increases in PCO 2 (10%–15%) alters the discharge rate and/or membrane potential of brainstem neurons recorded in such preparations as the whole neonatal brainstem and brain slices, usually from neonatal rodents, or in culture (76,77,167,203,300,328,447,448,458). A substantial proportion of neurons (15%–45%) selected at random within a given region are mildly activated/depolarized and a similar proportion are inhibited/hyperpolarized by acidification in the range of 0.2 to 0.5 pH or more (15% CO 2 ), the balance being unaffected.…”

Neural Control of Breathing and CO2 Homeostasis

“…Because effects of astrocytic opto-depolarization were blocked by MRS 2179, a P2Y1 purinergic receptor antagonist, it was proposed that astrocytes, not neurons, are the cellular proton/CO 2 sensors responsible for respiratory chemoreception, with ATP providing an obligate excitatory signal from astrocytes to neurons via P2Y1 receptors (Gourine et al, 2010). However, later work revealed that RTN neurons are intrinsically chemosensitive (Wang et al, 2013b), and that various P2 receptor antagonists, including MRS 2179, reduced the activation of RTN neurons by CO 2 modestly, at best (Mulkey et al, 2006; Onimaru et al, 2012; Wenker et al, 2012). …”

Neuromodulation by Extracellular ATP and P2X Receptors in the CNS

“…Moreover, recent work has shown that the purinergic component of the respiratory drive is at most 30%, a fraction that relies on hemichannel mediated ATP release, and that the actions of endogenous ATP on RTN neurons were not mediated by P2Y1 receptors, leaving open the intriguing possibility P2X receptors play a critical role (Wenker et al, 2012). Furthermore, in newborn rats, RTN chemoreception depends only on the intrinsic chemosensitivity of neurons (Onimaru et al, 2012). In summary, the available data indicate that a component of central chemoreception is mediated by astrocyte ATP that likely acts on P2X receptors in the RTN, demonstrating how ATP neuromodulation has profound effects on RTN neuron function with clear behavioral outcomes in the form of respiratory responses to hypercapnia.…”