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.

The C1 neurons reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM, IVLM). They use glutamate as a fast transmitter and synthesize catecholamines plus various neuropeptides. These neurons regulate the hypothalamic pituitary axis via direct projections to the paraventricular nucleus and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. The presympathetic C1 cells, located in the RVLM, are probably organized in a roughly viscerotopic manner and most of them regulate the circulation. C1 cells are variously activated by hypoglycemia, infection or inflammation, hypoxia, nociception, and hypotension and contribute to most glucoprivic responses. C1 cells also stimulate breathing and activate brain stem noradrenergic neurons including the locus coeruleus. Based on the various effects attributed to the C1 cells, their axonal projections and what is currently known of their synaptic inputs, subsets of C1 cells appear to be differentially recruited by pain, hypoxia, infection/inflammation, hemorrhage, and hypoglycemia to produce a repertoire of stereotyped autonomic, metabolic, and neuroendocrine responses that help the organism survive physical injury and its associated cohort of acute infection, hypoxia, hypotension, and blood loss. C1 cells may also contribute to glucose and cardiovascular homeostasis in the absence of such physical stresses, and C1 cell hyperactivity may contribute to the increase in sympathetic nerve activity associated with diseases such as hypertension. C1 neurons; blood pressure; brain stem BEST KNOWN for their contribution to the control of arterial pressure (AP), the C1 neurons have also been implicated in many other physiological processes ranging from neuroendocrine responses to infection and inflammation, glucose homeostasis, reproduction, breathing, thermoregulation, hypothalamo-pituitary axis (HPA)-mediated stress responses, and food consumption. The purpose of this review is to summarize the most salient information concerning the C1 cells, to point out some of the remaining gaps in our current knowledge, and to suggest a few unifying physiological principles that could account for these seemingly disparate observations. Based on the various effects attributed to the C1 cells and what is currently known of their synaptic inputs, we propose that these neurons are, figuratively speaking, the body's "emergency medical technicians." By this we imply that these neurons produce stereotyped autonomic, metabolic, and neuroendocrine responses designed to help the organism survive major acute physical stresses such as accidental, pathological, or dive-related hypoxia or physical injury and its associated cohort of acute infection, blood loss, and hypotension. These emergency responses include, in the short term and depending on the stress, vasoconstriction, cardioinhibition, or acceleration, breathing stimulation, antidiuresis, changes in metabolism, and gastrointestinal (GI) functions designed to conserve pe...

SummaryThe retrotrapezoid nucleus (RTN) contains non-cholinergic non-catecholaminergic glutamatergic neurons that express the transcription factor Phox2b (chemically coded or "cc" RTN neurons). These cells regulate breathing and may be central chemoreceptors. Here we explore their ultrastructure and their acid-sensitivity using two novel BAC eGFP-Phox2b transgenic mice (B/G, GENSAT JX99) in which respectively 36% and 100% of the cc RTN neurons express the transgene in complete or partial anatomical isolation from other populations of eGFP neurons.All but one eGFP-labeled RTN neuron recorded in these mice were acid-activated in slices. These cells contained VGLUT2 mRNA and 50% contained preprogalanin mRNA (determined by singlecell PCR in the B/G mouse). Two neuronal subgroups were revealed which differed in discharge rate at pH 7.3 (Type I ~2; Type II ~4 Hz) and the degree of alkalization that silenced the cells (Type I: 7.4 -7.6; Type II: 7.8 -8.0). Medial to the RTN, C1 neurons recorded in a tyrosinehydroxylase-GFP mouse were pH-insensitive between pH 6.9 -7.5.Ultrastructural studies demonstrated that eGFP-labeled RTN neurons were surrounded by numerous capillaries and were often in direct contact with glial cells, pericytes and the basement membrane of capillaries. Terminals contacting large proximal eGFP-dendrites formed mainly symmetric, likely inhibitory, synapses. Terminals on more distal eGFP-dendrites formed preferentially asymmetric, presumably excitatory, synapses.In sum, C1 cells are pH-insensitive whereas cc RTN neurons are uniformly acid-sensitive. The RTN neurons receive inhibitory and excitatory synaptic inputs and may have unfettered biochemical interactions with glial cells and the local microvasculature.

We used optogenetics to determine the global respiratory effects produced by selectively stimulating raphe obscurus (RO) serotonergic neurons in anesthetized mice and to test whether these neurons detect changes in the partial pressure of CO 2 , and hence function as central respiratory chemoreceptors. Channelrhodopsin-2 (ChR2) was selectively (ϳ97%) incorporated into ϳ50% of RO serotonergic neurons by injecting AAV2 DIO ChR2-mCherry (adeno-associated viral vector double-floxed inverse open reading frame of ChR2-mCherry) into the RO of ePet-Cre mice. The transfected neurons heavily innervated lower brainstem and spinal cord regions involved in autonomic and somatic motor control plus breathing but eschewed sensory related regions. Pulsed laser photostimulation of ChR2-transfected serotonergic neurons increased respiratory frequency (fR) and diaphragmatic EMG (dEMG) amplitude in relation to the duration and frequency of the light pulses (half saturation, 1 ms; 5-10 Hz). dEMG amplitude and fR increased slowly (half saturation after 10 -15 s) and relaxed monoexponentially (tau, 13-15 s). The breathing stimulation was reduced ϳ55% by methysergide (broad spectrum serotonin antagonist) and potentiated (ϳ16%) at elevated levels of inspired CO 2 (8%). RO serotonergic neurons, identified by their entrainment to short light pulses (threshold, 0.1-1 ms) were silent (nine cells) or had a low and regular level of activity (2.1 Ϯ 0.4 Hz; 11 cells) that was not synchronized with respiration. These and nine surrounding neurons with similar characteristics were unaffected by adding up to 10% CO 2 to the breathing mixture. In conclusion, RO serotonergic neurons activate breathing frequency and amplitude and potentiate the central respiratory chemoreflex but do not appear to have a central respiratory chemoreceptor function.

Activation of rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons e.g. by hypoxia is thought to increase sympathetic outflow thereby raising blood pressure (BP). Here we test whether these neurons also regulate breathing and cardiovascular variables other than BP. Selective expression of ChR2-mCherry by RVLM-CA neurons was achieved by injecting Cre-dependent vector AAV2-EF1α-DIO-ChR2-mCherry unilaterally into RVLM of dopamine-beta-hydroxylaseCre/0 (DβHCre/0) mice. Photostimulation of RVLM-CA neurons increased breathing in anesthetized and conscious mice. In conscious mice, photostimulation primarily increased breathing frequency and this effect was fully occluded by hypoxia (10% O2). In contrast, the effects of photostimulation were largely unaffected by hypercapnia (3 and 6% CO2). The associated cardiovascular effects were complex (slight bradycardia and hypotension) and, using selective autonomic blockers, could be explained by co-activation of the sympathetic and cardiovagal outflows. ChR2-positive RVLM-CA neurons expressed VGLUT2 and their projections were mapped. Their complex cardiorespiratory effects are presumably mediated by their extensive projections to supraspinal sites such as the ventrolateral medulla, the dorsal vagal complex, the dorsolateral pons, and selected hypothalamic nuclei (dorsomedial, lateral, paraventricular nuclei). In sum, selective optogenetic activation of RVLM-CA neurons in conscious mice revealed two important novel functions of these neurons, namely breathing stimulation and cardiovagal outflow control, effects that are attenuated or absent under anesthesia and are presumably mediated by the numerous supraspinal projections of these neurons. The results also suggest that RVLM-CA neurons may underlie some of the acute respiratory response elicited by carotid body stimulation but contribute little to the central respiratory chemoreflex.

In this review, we examine why blood pressure (BP) and sympathetic nerve activity (SNA) increase during a rise in central nervous system (CNS) P(CO(2)) (central chemoreceptor stimulation). CNS acidification modifies SNA by two classes of mechanisms. The first one depends on the activation of the central respiratory controller (CRG) and causes the much-emphasized respiratory modulation of the SNA. The CRG probably modulates SNA at several brain stem or spinal locations, but the most important site of interaction seems to be the caudal ventrolateral medulla (CVLM), where unidentified components of the CRG periodically gate the baroreflex. CNS P(CO(2)) also influences sympathetic tone in a CRG-independent manner, and we propose that this process operates differently according to the level of CNS P(CO(2)). In normocapnia and indeed even below the ventilatory recruitment threshold, CNS P(CO(2)) exerts a tonic concentration-dependent excitatory effect on SNA that is plausibly mediated by specialized brain stem chemoreceptors such as the retrotrapezoid nucleus. Abnormally high levels of P(CO(2)) cause an aversive interoceptive awareness in awake individuals and trigger arousal from sleep. These alerting responses presumably activate wake-promoting and/or stress-related pathways such as the orexinergic, noradrenergic, and serotonergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have brainwide projections that contribute to the CO(2)-induced rise in breathing and SNA by facilitating neuronal activity at innumerable CNS locations. In the case of SNA, these sites include the nucleus of the solitary tract, the ventrolateral medulla, and the preganglionic neurons.

Summary The C1 neurons are a nodal point for blood pressure control and other autonomic responses. Here we test whether these rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons use glutamate as a transmitter in the dorsal motor nucleus of the vagus (DMV). After injecting Cre-dependent AAV2 DIO-Ef1α-channelrhodopsin2(ChR2)-mCherry (AAV2) into the RVLM of dopamine-beta-hydroxylase Cre transgenic mice (DβHCre/0), mCherry was detected exclusively in RVLM-CA neurons. Within the DMV >95% mCherry-immunoreactive (-ir) axonal varicosities were tyrosine hydroxylase-ir and the same proportion were vesicular glutamate transporter 2 (VGLUT2)-ir. VGLUT2-mCherry co-localization was virtually absent when AAV2 was injected into the RVLM of DβHCre/0;VGLUT2flox/flox mice, into the caudal VLM (A1 noradrenergic neuron-rich region) of DβHCre/0 mice or into the raphe of ePetCre/0 mice. Following injection of AAV2 into RVLM of TH-Cre rats, phenylethanolamine N-methyl transferase (PNMT) and VGLUT2 immunoreactivities were highly co-localized in DMV within EYFP-positive or EYFP-negative axonal varicosities. Ultrastructurally, mCherry terminals from RVLM-CA neurons in DβHCre/0 mice made predominantly asymmetric synapses with ChAT-ir DMV neurons. Photostimulation of ChR2-positive axons in DβHCre/0 mouse brain slices produced EPSCs in 71% of tested DMV preganglionic neurons (PGNs) but no IPSCs. Photostimulation (20 Hz) activated PGNs up to 8 spikes/sec (current clamp). EPSCs were eliminated by tetrodotoxin, reinstated by 4-aminopyridine and blocked by ionotropic glutamate receptor blockers. In conclusion, VGLUT2 is expressed by RVLM-CA (C1) neurons in rats and mice regardless of the presence of AAV2, the C1 neurons activate DMV parasympathetic preganglionic neurons monosynaptically and this connection uses glutamate as an ionotropic transmitter.

SUMMARYBreathing automaticity and CO 2 regulation are inseparable neural processes. The retrotrapezoid nucleus (RTN), a group of glutamatergic neurons that express the transcription factor Phox2b, may be a crucial nodal point through which breathing automaticity is regulated to maintain CO 2 constant. This review updates the analysis presented in prior publications. Additional evidence that RTN neurons have central respiratory chemoreceptor properties is presented but this is only one of many factors that determine their activity. The RTN is also regulated by powerful inputs from the carotid bodies and, at least in the adult, by many other synaptic inputs. We also analyze how RTN neurons may control the activity of the downstream central respiratory pattern generator. Specifically, we review the evidence which suggests that RTN neurons a) innervate the entire ventral respiratory column, and b) control both inspiration and expiration. Finally, we argue that the RTN neurons are the adult form of the parafacial respiratory group in neonate rats.