Expression of Phox2b by Brainstem Neurons Involved in Chemosensory Integration in the Adult Rat

Abstract

Central congenital hypoventilation syndrome is caused by mutations of the gene that encodes the transcription factor Phox2b. The syndrome is characterized by a severe form of sleep apnea attributed to greatly compromised central and peripheral chemoreflexes. In this study, we analyze whether Phox2b expression in the brainstem respiratory network is preferentially associated with neurons involved in chemosensory integration in rats. At the very rostral end of the ventral respiratory column (VRC), Phox2b was present in many VGlut2 (vesicular glutamate transporter 2) mRNA-containing neurons. These neurons were functionally identified as the respiratory chemoreceptors of the retrotrapezoid nucleus (RTN). More caudally in the VRC, many fewer neurons expressed Phox2b. These cells were not part of the central respiratory pattern generator (CPG), because they were typically cholinergic visceral motor neurons or catecholaminergic neurons (presumed C1 neurons). Phox2b was not detected in serotonergic neurons, in the A5, A6, and A7 noradrenergic cell groups nor within the main cardiorespiratory centers of the dorsolateral pons. Phox2b was expressed by many solitary tract nucleus (NTS) neurons including those that relay peripheral chemoreceptor information to the RTN. These and previous observations by others suggest that Phox2b is expressed by an uninterrupted chain of neurons involved in the integration of peripheral and central chemoreception (carotid bodies, chemoreceptor afferents, chemoresponsive NTS neurons projecting to VRC, RTN chemoreceptors). The presence of Phox2b in this circuit and its apparent absence from the respiratory CPG could explain why Phox2b mutations disrupt breathing automaticity during sleep without causing major impairment of respiration during waking.

Widespread depolarization during expiration: A source of respiratory drive?

“…The RTN, a central chemoreceptor site responsible for the 'drive' to breathe [43], is important in maintaining involuntary breathing during anesthesia [44] and may be essential for involuntary breathing during sleep [45]. Chemoreceptors in the RTN receive inhibitory input from stretch receptors in the lungs, most likely SARs, during lung inflation [28].…”

Breathing withPhox2b

“…PHOX2B AND THE RETROTRAPEZOID NUCLEUS Two independent lines of research have now established a link between Phox2b and the RTN, one of the two main contenders for the role of CO 2 /pH sensor in the CNS ( §2). First, neurons of the adult rat RTN, defined physiologically by their responsiveness to CO 2 in vivo (Mulkey et al 2004;reviewed in Guyenet 2008), and phenotyped as Vglut2 þ (but negative for glutamate decarboxylase and tyrosine hydroxylase) and located at the ventral medullary surface under the facial nucleus and extending approximately 500 mm caudal to it, were all found to express Phox2b (Stornetta et al 2006). Second, inspection of the hindbrain of Phox2b 27Ala/þ newborn mice that have no response to hypercapnia ( §2) showed that there was an 85 per cent depletion of the Phox2b þ /Vglut2 þ RTN neurons (Dubreuil et al 2008).…”

“…Moreover, these cells receive direct projections from a subpopulation of Phox2b þ nTS neurons that themselves relay PaO 2 responsiveness by virtue of an input from the CB, via the petrosal ganglion (Stornetta et al 2006), which are all dependent on Phox2b. Thus, the circuitry for monitoring blood gases, which provides a major drive to breathe, might consist of an uninterrupted Phox2b-dependent four-neuron circuit.…”

Measurement of purine release with microelectrode biosensors

“…The rostral area appears to correspond to the retrotrapezoid nucleus. Guyenet and colleagues have adduced considerable evidence for the existence of chemosensitive neurons in this nucleus that appear important in the regulation of breathing, at least in the neonate [45,46]. Neurons of the medullary raphe have also been shown to be chemosensitive [47,48] and associated with blood vessels in a manner highly suggestive of a chemosensory function [48].…”