Photostimulation of Retrotrapezoid Nucleus Phox2b-Expressing NeuronsIn VivoProduces Long-Lasting Activation of Breathing in Rats

Abstract

The retrotrapezoid "nucleus" (RTN), located in the rostral ventrolateral medullary reticular formation, contains a bilateral cluster of ϳ1000 glutamatergic noncatecholaminergic Phox2b-expressing propriobulbar neurons that are activated by CO 2 in vivo and by acidification in vitro. These cells are thought to function as central respiratory chemoreceptors, but this theory still lacks a crucial piece of evidence, namely that stimulating these particular neurons selectively in vivo increases breathing. The present study performed in anesthetized rats seeks to test whether this expectation is correct. We injected into the left RTN a lentivirus that expresses the lightactivated cationic channel ChR2 (channelrhodopsin-2) (H134R mutation; fused to the fluorescent protein mCherry) under the control of the Phox2-responsive promoter PRSx8. Transgene expression was restricted to 423 Ϯ 38 Phox2b-expressing neurons per rat consisting of noncatecholaminergic and C1 adrenergic neurons (3:2 ratio). Photostimulation delivered to the RTN region in vivo via a fiberoptic activated the CO 2 -sensitive neurons vigorously, produced a long-lasting (t 1/2 ϭ 11 s) increase in phrenic nerve activity, and caused a small and short-lasting cardiovascular stimulation. Selective lesions of the C1 cells eliminated the cardiovascular response but left the respiratory stimulation intact. In rats with C1 cell lesions, the mCherry-labeled axon terminals originating from the transfected noncatecholaminergic neurons were present exclusively in the lower brainstem regions that contain the respiratory pattern generator. These results provide strong evidence that the Phox2b-expressing noncatecholaminergic neurons of the RTN region function as central respiratory chemoreceptors.

Nalcn Is a "Leak" Sodium Channel That Regulates Excitability of Brainstem Chemosensory Neurons and Breathing

“…Although structurally related to the Na V family of voltage-dependent Na ϩ -selective channels, Nalcn is TTX resistant and forms a voltage-independent, nonselective cation channel that generates a "leak" inward current, carried primarily by Na ϩ ions under physiological conditions (Lu et al, 2007(Lu et al, , 2009Ren, 2011;Cochet-Bissuel et al, 2014); Nalcn current can be further enhanced by various neuromodulators (Lu et al, 2009;Ren, 2011;Cochet-Bissuel et al, 2014). Notably, global genetic deletion of Nalcn leads to disrupted respiration and early postnatal death in mice (Lu et al, 2007), and patients with Nalcn mutations present with respiratory dysfunction, among other disorders (Al-Sayed et al, 2013;Köroglu et al, 2013;Cochet-Bissuel et al, 2014;Chong et al, 2015;Gal et al, 2016). A contribution of Nalcn to endogenous currents in respiratory neurons has not been demonstrated.…”

Defective Respiratory Rhythmogenesis and Loss of Central Chemosensitivity in Phox2b Mutants Targeting Retrotrapezoid Nucleus Neurons

“…First identified as a site projecting extensively to the more caudal respiratory columns (Connelly et al, 1989;Smith et al, 1989), it has been attributed a role in central chemoreception, i.e., the activation of breathing by increased pCO 2 through the detection of changes in pH Mulkey et al, 2004;Abbott et al, 2009). RTN neurons are molecularly defined by expression of Vglut2, neurokinin1 receptor (NK1R), and the Phox2b transcription factor and lack of tyrosine hydroxylase (TH) (Stornetta et al, 2006;Onimaru et al, 2008).…”

Optogenetics in neuroscience: what we gain from studies in mammals

“…The retrotrapezoid nucleus (RTN) is thought to be involved in sensitization to CO 2 , and photostimulation of the RTN increases activity of the phrenic nerve [39] . Moreover, active expiration occurs after photoactivation [68] .…”