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A brainstem homeostatic system senses CO 2 /H + to regulate ventilation, blood gases and acid-base balance. Neurons of the retrotrapezoid nucleus (RTN) and medullary raphe are both implicated in this mechanism as respiratory chemosensors, but recent pharmacological work suggested that the CO 2 /H + sensitivity of RTN neurons is mediated indirectly, by raphe-derived serotonin acting on 5-HT7 receptors. To investigate this further, we characterized Htr7 transcript expression in phenotypically identified RTN neurons using multiplex single cell qRT-PCR and RNAscope. Although present in multiple neurons in the parafacial region of the ventrolateral medulla, Htr7 expression was undetectable in most RTN neurons (Nmb + /Phox2b + ) concentrated in the densely packed cell group ventrolateral to the facial nucleus. Where detected, Htr7 expression was modest and often associated with RTN neurons that extend dorsolaterally to partially encircle the facial nucleus. These dorsolateral Nmb + /Htr7 + neurons tended to express Nmb at high levels and the intrinsic RTN proton detectors Gpr4 and Kcnk5 at low levels. In mouse brainstem slices, CO 2 -stimulated firing in RTN neurons was mostly unaffected by a 5-HT7 receptor antagonist, SB269970 (n = 11/13). At the whole animal level, microinjection of SB269970 into the RTN of conscious mice blocked respiratory stimulation by co-injected LP-44, a 5-HT7 receptor agonist, but had no effect on CO 2 -stimulated breathing in those same mice. We conclude that Htr7 is expressed by a minor subset of RTN neurons with a molecular profile distinct from the established chemoreceptors and that 5-HT7 receptors have negligible effects on CO 2 -evoked firing activity in RTN neurons or on CO 2 -stimulated breathing in mice. KEY POINTS: Neurons of the retrotrapezoid nucleus (RTN) are intrinsic CO 2 /H + chemosensors and serve as an integrative excitatory hub for control of breathing. Serotonin can activate RTN neurons, in part via 5-HT7 receptors, and those effects have been implicated in conferring an indirect CO 2  sensitivity. Multiple single cell molecular approaches revealed low levels of 5-HT7 receptor transcript expression restricted to a limited population of RTN neurons. Pharmacological experiments showed that 5-HT7 receptors in RTN are not required for CO 2 /H + -stimulation of RTN neuronal activity or CO 2 -stimulated breathing. These data do not support a role for 5-HT7 receptors in respiratory chemosensitivity mediated by RTN neurons., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: Parkinson's disease (PD) is associated with respiratory dysfunction. In the 6-OHDA rat model of PD this is seen as a reduction in respiratory frequency and minute ventilation during normoxia and hypercapnia stimulus. Respiratory dysfunction is caused by neuronal death of medullary respiratory nuclei in the 6-OHDA model of PD. Oxidative stress can be considered a strong candidate for neurodegeneration via miR-34c downregulation and pro-apoptotic signalling in respiratory neurons, preceding the functional impairment observed in the 6-OHDA model of PD., Abstract: Parkinson's disease (PD) is a neurodegenerative disease caused by dopaminergic neuron death in the substantia nigra (SN). New evidence has revealed that this neurodegeneration is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction and disruption of the blood-brain barrier (BBB) in the SN. In addition to classic symptoms, PD patients also exhibit respiratory failure. Here, we investigated whether oxidative stress was associated with neurodegeneration in a respiratory group (RG) of 6-OHDA-treated rats, which act as a model of PD. We analysed how oxidative stress affected apoptotic signalling in the RG 30 days after 6-OHDA treatment, shortly before commencement of breathing impairment (40 days). After 30 days, a dihydroethidium assay showed increased oxidative stress in the RG, anti-apoptotic signalling, as shown by an increase in p-Akt and BcL-2 and a decrease in Bax in the caudal aspect of the nucleus of the solitary tract (cNTS), and a decrease in p-p38 and Bax levels in the retrotrapezoid nucleus (RTN); pro-apoptotic signalling was indicated by a decrease in p-Akt and BcL-2 and an increase in Bax in the rostral ventral respiratory group (rVRG) and pre-Botzinger complex (preBotC). miR-34c, a known oxidative stress protector, was downregulated in 6-OHDA animals in the RC. After 40 days of 6-OHDA, the NTS, rVRG, preBotC and RTN exhibited reduced NeuN immunoreactivity, no BBB disruption and an increase in thiobarbituric acid reactivity. We conclude that in the 6-OHDA model of PD, oxidative stress contributes to neurodegeneration in medullary respiratory neurons., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Key Points: A strong association between disordered breathing patterns, elevated sympathetic activity, and enhanced central chemoreflex drive has been shown in experimental and human heart failure (HF). The aim of this study was to determine the contribution of catecholaminergic rostral ventrolateral medulla catecholaminergic neurones (RVLM-C1) to both haemodynamic and respiratory alterations in HF. Apnoea/hypopnoea incidence (AHI), breathing variability, respiratory-cardiovascular coupling, cardiac autonomic control and cardiac function were analysed in HF rats with or without selective ablation of RVLM-C1 neurones. Partial lesion (∼65%) of RVLM-C1 neurones reduces AHI, respiratory variability, and respiratory-cardiovascular coupling in HF rats. In addition, the deleterious effects of central chemoreflex activation on cardiac autonomic balance and cardiac function in HF rats was abolished by ablation of RVLM-C1 neurones. Our findings suggest that RVLM-C1 neurones play a pivotal role in breathing irregularities in volume overload HF, and mediate the sympathetic responses induced by acute central chemoreflex activation., Abstract: Rostral ventrolateral medulla catecholaminergic neurones (RVLM-C1) modulate sympathetic outflow and breathing under normal conditions. Heart failure (HF) is characterized by chronic RVLM-C1 activation, increased sympathetic activity and irregular breathing patterns. Despite studies showing a relationship between RVLM-C1 and sympathetic activity in HF, no studies have addressed a potential contribution of RVLM-C1 neurones to irregular breathing in this context. Thus, the aim of this study was to determine the contribution of RVLM-C1 neurones to irregular breathing patterns in HF. Sprague-Dawley rats underwent surgery to induce volume overload HF. Anti-dopamine β-hydroxylase-saporin toxin (DβH-SAP) was used to selectively lesion RVLM-C1 neurones. At 8 weeks post-HF induction, breathing pattern, blood pressures (BP), respiratory-cardiovascular coupling (RCC), central chemoreflex function, cardiac autonomic control and cardiac function were studied. Reduction (∼65%) of RVLM-C1 neurones resulted in attenuation of irregular breathing, decreased apnoea-hypopnoea incidence (11.1 ± 2.9 vs. 6.5 ± 2.5 events h -1 ; HF+Veh vs. HF+DβH-SAP; P < 0.05) and improved cardiac autonomic control in HF rats. Pathological RCC was observed in HF rats (peak coherence >0.5 between breathing and cardiovascular signals) and was attenuated by DβH-SAP treatment (coherence: 0.74 ± 0.12 vs. 0.54 ± 0.10, HF+Veh vs. HF+DβH-SAP rats; P < 0.05). Central chemoreflex activation had deleterious effects on cardiac function and cardiac autonomic control in HF rats that were abolished by lesion of RVLM-C1 neurones. Our findings reveal that RVLM-C1 neurones play a major role in irregular breathing patterns observed in volume overload HF and highlight their contribution to cardiac dysautonomia and deterioration of cardiac function during chemoreflex activation., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Key Points: The embryonic PHOX2B-progenitor domain generates neuronal and glial cells which together are involved in chemosensory control of breathing and sleep homeostasis. Ablating PHOX2B-derived astrocytes significantly contributes to secondary hypoxic respiratory depression as well as abnormalities in sleep homeostasis. PHOX2B-derived astrocyte ablation results in axonal pathologies in the retrotrapezoid nucleus., Abstract: We identify in mice a population of ∼800 retrotrapezoid nucleus (RTN) astrocytes derived from PHOX2B-positive, OLIG3-negative progenitor cells, that interact with PHOX2B-expressing RTN chemosensory neurons. PHOX2B-derived astrocyte ablation during early life results in adult-onset O 2 chemoreflex deficiency. These animals also display changes in sleep homeostasis, including fragmented sleep and disturbances in delta power after sleep deprivation, all without observable changes in anxiety or social behaviours. Ultrastructural evaluation of the RTN demonstrates that PHOX2B-derived astrocyte ablation results in features characteristic of degenerative neuro-axonal dystrophy, including abnormally dilated axon terminals and increased amounts of synapses containing autophagic vacuoles/phagosomes. We conclude that PHOX2B-derived astrocytes are necessary for maintaining a functional O 2 chemosensory reflex in the adult, modulate sleep homeostasis, and are key regulators of synaptic integrity in the RTN region, which is necessary for the chemosensory control of breathing. These data also highlight how defects in embryonic development may manifest as neurodegenerative pathology in an adult., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Key Points: Cholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to the retrotrapezoid nucleus (RTN) are considered to be important for sleep-wake state-dependent control of breathing. The RTN also receives cholinergic input from the postinspiratory complex. Stimulation of the PPTg increases respiratory output under control conditions but not when muscarinic receptors in the RTN are blocked. The data obtained in the present study support the possibility that arousal-dependent modulation of breathing involves recruitment of cholinergic projections from the PPTg to the RTN., Abstract: The pedunculopontine tegmental nucleus (PPTg) in the mesopontine region has important physiological functions, including breathing control. The PPTg contains a variety of cell types, including cholinergic neurons that project to the rostral aspect of the ventrolateral medulla. In addition, cholinergic signalling in the retrotrapezoid nucleus (RTN), a region that contains neurons that regulate breathing in response to changes in CO 2 /H + , has been shown to activate chemosensitive neurons and increase inspiratory activity. The present study aimed to identify the source of cholinergic input to the RTN and determine whether cholinergic signalling in this region influences baseline breathing or the ventilatory response to CO 2 in conscious male Wistar rats. Retrograde tracer Fluoro-Gold injected into the RTN labelled a subset of cholinergic PPTg neurons that presumably project directly to the chemosensitive region of the RTN. In unrestrained awake rats, unilateral injection of the glutamate (10 mm/100 nL) in the PPTg decreased tidal volume (V T ) but otherwise increased respiratory rate (f R ) and net respiratory output as indicated by an increase in ventilation (V E ). All respiratory responses elicited by PPTg stimulation were blunted by prior injection of methyl-atropine (5 mm/50-75 nL) into the RTN. These results show that stimulation of the PPTg can increase respiratory activity in part by cholinergic activation of chemosensitive elements of the RTN. Based on previous evidence that cholinergic PPTg projections may simultaneously activate expiratory output from the pFRG, we speculate that cholinergic signalling at the level of RTN region could also be involved in breathing regulation., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Key Points: It is recognized that seizures commonly cause apnoea and oxygen desaturation, but there is still a lack in the literature about the respiratory impairments observed ictally and in the post-ictal period. Respiratory disorders may involve changes in serotonergic transmission at the level of the retrotrapezoid nucleus (RTN). In this study, we evaluated breathing activity and the role of serotonergic transmission in the RTN with a rat model of tonic-clonic seizures, the Wistar audiogenic rat (WAR). We conclude that the respiratory impairment in the WAR could be correlated to an overall decrease in the number of neurons located in the respiratory column., Abstract: Respiratory disorders may involve changes in serotonergic neurotransmission at the level of the chemosensitive neurons located in the retrotrapezoid nucleus (RTN). Here, we investigated the central respiratory chemoreflex and the role of serotonergic neurotransmission in the RTN with a rat model of tonic-clonic seizures, the Wistar audiogenic rat (WAR). We found that naive or kindled WARs have reduced resting ventilation and ventilatory response to hypercapnia (7% CO 2 ). The number of chemically coded (Phox2b + /TH - ) RTN neurons, as well as the serotonergic innervation to the RTN, was reduced in WARs. We detected that the ventilatory response to serotonin (1 mm, 50 nl) within the RTN region was significantly reduced in WARs. Our results uniquely demonstrated a respiratory impairment in a genetic model of tonic-clonic seizures, the WAR strain. More importantly, we demonstrated an overall decrease in the number of neurons located in the ventral respiratory column (VRC), as well as a reduction in serotonergic neurons in the midline medulla. This is an important step forward to demonstrate marked changes in neuronal activity and breathing impairment in the WAR strain, a genetic model of epilepsy., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Key Points: ACh is an important modulator of breathing, including at the level of the retrotrapezoid nucleus (RTN), where evidence suggests that ACh is essential for the maintenance of breathing. Despite this potentially important physiological role, little is known about the mechanisms responsible for the cholinergic control of RTN function. In the present study, we show at the cellular level that ACh increases RTN chemoreceptor activity by a CO2/H(+) independent mechanism involving M1/M3 receptor-mediated inositol 1,4,5-trisphosphate/Ca(+2) signalling and downstream inhibition of KCNQ channels. These results dispel the theory that ACh is required for RTN chemoreception by showing that ACh, similar to serotonin and other modulators, controls the activity of RTN chemoreceptors without interfering with the mechanisms by which these cells sense H(+). By identifying the mechanisms by which wake-on neurotransmitters such as ACh modulate RTN chemoreception, the results of the present study provide a framework for understanding the molecular basis of the sleep-wake state-dependent control of breathing., Abstract: ACh has long been considered important for the CO2/H(+)-dependent drive to breathe produced by chemosensitive neurons in the retrotrapezoid nucleus (RTN). However, despite this potentially important physiological role, almost nothing is known about the mechanisms responsible for the cholinergic control of RTN function. In the present study, we used slice-patch electrophysiology and pharmacological tools to characterize the effects of ACh on baseline activity and CO2/H(+)-sensitivity of RTN chemoreceptors, as well as to dissect the signalling pathway by which ACh activates these neurons. We found that ACh activates RTN chemoreceptors in a dose-dependent manner (EC50 = 1.2 μm). The firing response of RTN chemoreceptors to ACh was mimicked by a muscarinic receptor agonist (oxotremorine; 1 μm), and blunted by M1- (pirezenpine; 2 μm) and M3- (diphenyl-acetoxy-N-methyl-piperidine; 100 nm) receptor blockers, but not by a nicotinic-receptor blocker (mecamylamine; 10 μm). Furthermore, pirenzepine, diphenyl-acetoxy-N-methyl-piperidine and mecamylamine had no measurable effect on the CO2/H(+)-sensitivity of RTN chemoreceptors. The effects of ACh on RTN chemoreceptor activity were also blunted by inhibition of inositol 1,4,5-trisphosphate receptors with 2-aminoethoxydiphenyl borate (100 μm), depletion of intracellular Ca(2+) stores with thapsigargin (10 μm), inhibition of casein kinase 2 (4,5,6,7-tetrabromobenzotriazole; 10 μm) and blockade of KCNQ channels (XE991; 10 μm). These results show that ACh activates RTN chemoreceptors by a CO2/H(+) independent mechanism involving M1/M3 receptor-mediated inositol 1,4,5-trisphosphate/Ca(+2) signalling and downstream inhibition of KCNQ channels. Identifying the components of the signalling pathway coupling muscarinic receptor activation to changes in chemoreceptor activity may provide new potential therapeutic targets for the treatment of respiratory control disorders., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Central chemoreception is the mechanism by which CO₂/H(+) -sensitive neurons (i.e. chemoreceptors) regulate breathing in response to changes in tissue CO₂/H(+) . Neurons in the retrotrapezoid nucleus (RTN) directly regulate breathing in response to changes in tissue CO₂/H(+) and function as a key locus of respiratory control by integrating information from several respiratory centres, including the medullary raphe. Therefore, chemosensitive RTN neurons appear to be critically important for maintaining breathing, thus understanding molecular mechanisms that regulate RTN chemoreceptor function may identify therapeutic targets for the treatment of respiratory control disorders. We have recently shown that KCNQ (Kv7) channels in the RTN are essential determinants of spontaneous activity ex vivo, and downstream effectors for serotonergic modulation of breathing. Considering that loss of function mutations in KCNQ channels can cause certain types of epilepsy including those associated with sudden unexplained death in epilepsy (SUDEP), we propose that dysfunctions of KCNQ channels may be one cause for epilepsy and respiratory problems associated with SUDEP. In this review, we will summarize the role of KCNQ channels in the regulation of RTN chemoreceptor function, and suggest that these channels represent useful therapeutic targets for the treatment of respiratory control disorders., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

The rostral ventrolateral medulla oblongata (RVLM) contains two functionally distinct types of neurons that control and orchestrate cardiovascular and respiratory responses to hypoxia and hypercapnia. One group is composed of the central chemoreceptor neurons of the retrotrapezoid nucleus, which provides a CO₂/H(+) -dependent drive to breathe and serves as an integration centre and a point of convergence of chemosensory information from other central and peripheral sites, including the carotid bodies. The second cluster of RVLM cells forms a population of neurons belonging to the C1 catecholaminergic group that controls sympathetic vasomotor tone in resting conditions and in conditions of hypoxia and hypercapnia. Recent evidence suggests that ATP-mediated purinergic signalling at the level of the RVLM co-ordinates cardiovascular and respiratory responses triggered by hypoxia and hypercapnia by activating retrotrapezoid nucleus and C1 neurons, respectively. The role of ATP-mediated signalling in the RVLM mechanisms of cardiovascular and respiratory activities is the main subject of this short review., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Several brain regions are thought to function as important sites of chemoreception including the nucleus of the solitary tract (NTS), medullary raphe and retrotrapezoid nucleus (RTN). In the RTN, mechanisms of chemoreception involve direct H(+)-mediated activation of chemosensitive neurons and indirect modulation of chemosensitive neurons by purinergic signalling. Evidence suggests that RTN astrocytes are the source of CO2-evoked ATP release. However, it is not clear whether purinergic signalling also influences CO2/H(+) responsiveness of other putative chemoreceptors. The goals of this study are to determine if CO2/H(+)-sensitive neurons in the NTS and medullary raphe respond to ATP, and whether purinergic signalling in these regions influences CO2 responsiveness in vitro and in vivo. In brain slices, cell-attached recordings of membrane potential show that CO2/H(+)-sensitive NTS neurons are activated by focal ATP application; however, purinergic P2-receptor blockade did not affect their CO2/H(+) responsiveness. CO2/H(+)-sensitive raphe neurons were unaffected by ATP or P2-receptor blockade. In vivo, ATP injection into the NTS increased cardiorespiratory activity; however, injection of a P2-receptor blocker into this region had no effect on baseline breathing or CO2/H(+) responsiveness. Injections of ATP or a P2-receptor blocker into the medullary raphe had no effect on cardiorespiratory activity or the chemoreflex. As a positive control we confirmed that ATP injection into the RTN increased breathing and blood pressure by a P2-receptor-dependent mechanism. These results suggest that purinergic signalling is a unique feature of RTN chemoreception.

Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO(2)H+. A brainstem region called the retrotrapezoid nucleus (RTN) contains a population of CO2/H+-sensitive neurons that appears to function as an important chemoreceptor. Evidence also indicates that CO2-evoked ATP release from RTN astrocytes modulates activity of CO2/H+-sensitive neurons; however, the extent to which purinergic signalling contributes to chemoreception by RTN neurons is not clear and the mechanism(s) underlying CO2/H+-evoked ATP release is not fully elucidated. The goals of this study are to determine the extent to which ATP contributes to RTN chemoreception both in vivo and in vitro, and whether purinergic drive to chemoreceptors relies on extracellular Ca(2+) or gap junction hemichannels. We also examine the possible contribution of P2Y1 receptors expressed in the RTN to the purinergic drive to breathe.We show that purinergic signalling contributes, in part, to the CO(2)/H+ sensitivity of RTN neurons. In vivo, phrenic nerve recordings of respiratory activity in adult rats show that bilateral injections of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS, a P2 receptor blocker) decreased the ventilatory response to CO2 by 30%. In vitro, loose-patch recordings from RTN neurons show that P2 receptor blockers decreased responsiveness to both 10% and 15% CO2 also by 30%. In the slice, the contribution of purinergic signalling to RTN chemoreception did not increase with temperature (22–35◦C) and was retained in low extracellular Ca2+ medium. Conversely, the gap junction blockers carbenoxolone and cobalt decreased neuronal CO2/H+ sensitivity by an amount similar to P2 receptor antagonists. Inhibition of the P2Y1 receptor in the RTN had no effect on CO2 responsivness in vitro or in vivo; thus, the identity of P2 receptors underlying the purinergic component of RTN chemoreception remains unknown. These results support the possibility that CO2/H+-evoked ATP release is mediated by a mechanism involving gap junction hemichannels.

Injection of the neurotoxin saporin-substance P (SSP-SAP) into the retrotrapezoid nucleus (RTN) attenuates the central chemoreflex in rats. Here we ask whether these deficits are caused by the destruction of a specific type of interneuron that expresses the transcription factor Phox2b and is non-catecholaminergic (Phox2b(+)TH(-)). We show that RTN contains around 2100 Phox2b(+)TH(-) cells. Injections of SSP-SAP into RTN destroyed Phox2b(+)TH(-) neurons but spared facial motoneurons, catecholaminergic and serotonergic neurons and the ventral respiratory column caudal to the facial motor nucleus. Two weeks after SSP-SAP, the apnoeic threshold measured under anaesthesia was unchanged when fewer than 57% of the Phox2b(+)TH(-) neurons were destroyed. However, destruction of 70 +/- 3.5% of these cells was associated with a dramatic rise of the apnoeic threshold (from 5.6 to 7.9% end-expiratory P(CO(2))). In anaesthetized rats with unilateral lesions of around 70% of the Phox2b(+)TH(-) neurons, acute inhibition of the contralateral intact RTN with muscimol instantly eliminated phrenic nerve discharge (PND) but normal PND could usually be elicited by strong peripheral chemoreceptor stimulation (8/12 rats). Muscimol had no effect in rats with an intact contralateral RTN. In conclusion, the destruction of the Phox2b(+)TH(-) neurons is a plausible cause of the respiratory deficits caused by injection of SSP-SAP into RTN. Two weeks after toxin injection, 70% of these cells must be killed to cause a severe attenuation of the central chemoreflex under anaesthesia. The loss of an even greater percentage of these cells would presumably be required to produce significant breathing deficits in the awake state.

The retrotrapezoid nucleus (RTN) contains CO(2)-activated interneurons with properties consistent with central respiratory chemoreceptors. These neurons are glutamatergic and express the transcription factor Phox2b. Here we tested whether RTN neurons receive an input from slowly adapting pulmonary stretch receptors (SARs) in halothane-anaesthetized ventilated rats. In vagotomized rats, RTN neurons were inhibited to a variable extent by stimulating myelinated vagal afferents using the lowest intensity needed to inhibit the phrenic nerve discharge (PND). In rats with intact vagus nerves, RTN neurons were inhibited, also to a variable extent, by increasing positive end-expiratory pressure (PEEP; 2-6 cmH(2)O). The cells most sensitive to PEEP were inhibited during each lung inflation at rest and were instantly activated by stopping ventilation. Muscimol (GABA-A agonist) injection in or next to the solitary tract at area postrema level desynchronized PND from ventilation, eliminated the lung inflation-synchronous inhibition of RTN neurons and their steady inhibition by PEEP but did not change their CO(2) sensitivity. Muscimol injection into the rostral ventral respiratory group eliminated PND but did not change RTN neuron response to either lung inflation, PEEP increases, vagal stimulation or CO(2). Generalized glutamate receptor blockade with intracerebroventricular (i.c.v.) kynurenate eliminated PND and the response of RTN neurons to lung inflation but did not change their CO(2) sensitivity. PEEP-sensitive RTN neurons expressed Phox2b. In conclusion, RTN chemoreceptors receive an inhibitory input from myelinated lung stretch receptors, presumably SARs. The lung input to RTN may be di-synaptic with inhibitory pump cells as sole interneurons.

The present study explores how elevations in brain P(CO(2)) increase the sympathetic nerve discharge (SND). SND, phrenic nerve discharge (PND) and putative sympathoexcitatory vasomotor neurons of the rostral ventrolateral medulla (RVLM) were recorded in anaesthetized sino-aortic denervated and vagotomized rats. Hypercapnia (end-expiratory CO(2) from 5% to 10%) increased SND (97 +/- 6%) and the activity of RVLM neurons (67 +/- 4%). Injection of kynurenic acid (Kyn, ionotropic glutamate receptor antagonist) into RVLM or the retrotrapezoid nucleus (RTN) eliminated or reduced PND, respectively, but did not change the effect of CO(2) on SND. Bilateral injection of Kyn or muscimol into the rostral ventral respiratory group (rVRG-pre-Bötzinger region, also called CVLM) eliminated PND while increasing the stimulatory effect of CO(2) on SND. Muscimol injection into commissural part of the solitary tract nucleus (commNTS) had no effect on PND or SND activation by CO(2). As expected, injection of Kyn into RVLM or muscimol into commNTS virtually blocked the effect of carotid body stimulation on SND in rats with intact carotid sinus nerves. In conclusion, CO(2) increases SND by activating RVLM sympathoexcitatory neurons. The relevant central chemoreceptors are probably located within or close to RVLM and not in the NTS or in the rVRG-pre-Bötzinger/CVLM region. RVLM sympathoexcitatory neurons may be intrinsically pH-sensitive and/or receive excitatory synaptic inputs from RTN chemoreceptors. Activation of the central respiratory network reduces the overall sympathetic response to CO(2), presumably by activating barosensitive CVLM neurons and inhibiting RTN chemoreceptors.