Retrotrapezoid Nucleus Chemoreceptors Research Articles

Loss-of-function of chemoreceptor neurons in the retrotrapezoid nucleus: What have we learned from it?

Central respiratory chemoreceptors are cells in the brain that regulate breathing in relation to arterial pH and PCO2. Neurons located at the retrotrapezoid nucleus (RTN) have been hypothesized to be central chemoreceptors and/or to be part of the neural network that drives the central respiratory chemoreflex. The inhibition or ablation of RTN chemoreceptor neurons has offered important insights into the role of these cells on central respiratory chemoreception and the neural control of breathing over almost 60 years since the original identification of acid-sensitive properties of this ventral medullary site. Here, we discuss the current definition of chemoreceptor neurons in the RTN and describe how this definition has evolved over time. We then summarize the results of studies that use loss-of-function approaches to evaluate the effects of disrupting the function of RTN neurons on respiration. These studies offer evidence that RTN neurons are indispensable for the central respiratory chemoreflex in mammals and exert a tonic drive to breathe at rest. Moreover, RTN has an interdependent relationship with oxygen sensing mechanisms for the maintenance of the neural drive to breathe and blood gas homeostasis. Collectively, RTN neurons are a genetically-defined group of putative central respiratory chemoreceptors that generate CO2-dependent drive that supports eupneic breathing and stimulates the hypercapnic ventilatory reflex.

Superoxide dismutase 2 deficiency is associated with enhanced central chemoreception in mice: Implications for breathing regulation

AimsIn mammals, central chemoreception plays a crucial role in the regulation of breathing function in both health and disease conditions. Recently, a correlation between high levels of superoxide anion (O2.-) in the Retrotrapezoid nucleus (RTN), a main brain chemoreceptor area, and enhanced central chemoreception has been found in rodents. Interestingly, deficiency in superoxide dismutase 2 (SOD2) expression, a pivotal antioxidant enzyme, has been linked to the development/progression of several diseases. Despite, the contribution of SOD2 on O2.- regulation on central chemoreceptor function is unknown. Accordingly, we sought to determine the impact of partial deletion of SOD2 expression on i) O2.−accumulation in the RTN, ii) central ventilatory chemoreflex function, and iii) disordered-breathing. Finally, we study cellular localization of SOD2 in the RTN of healthy mice. MethodsCentral chemoreflex drive and breathing function were assessed in freely moving heterozygous SOD2 knockout mice (SOD2+/− mice) and age-matched control wild type (WT) mice by whole-body plethysmography. O2.- levels were determined in RTN brainstem sections and brain isolated mitochondria, while SOD2 protein expression and tissue localization were determined by immunoblot, RNAseq and immunofluorescent staining, respectively. ResultsOur results showed that SOD2+/− mice displayed reductions in SOD2 levels and high O2.- formation and mitochondrial dysfunction within the RTN compared to WT. Additionally, SOD2+/− mice displayed a heightened ventilatory response to hypercapnia and exhibited overt signs of altered breathing patterns. Both, RNAseq analysis and immunofluorescence co-localization studies showed that SOD2 expression was confined to RTN astrocytes but not to RTN chemoreceptor neurons. Finally, we found that SOD2+/− mice displayed alterations in RTN astrocyte morphology compared to RTN astrocytes from WT mice. Innovation & conclusionThese findings provide first evidence of the role of SOD2 in the regulation of O2.- levels in the RTN and its potential contribution on the regulation of central chemoreflex function. Our results suggest that reductions in the expression of SOD2 in the brain may contribute to increase O2.- levels in the RTN being the outcome a chronic surge in central chemoreflex drive and the development/maintenance of altered breathing patterns. Overall, dysregulation of SOD2 and the resulting increase in O2.- levels in brainstem respiratory areas can disrupt normal respiratory control mechanisms and contribute to breathing dysfunction seen in certain disease conditions characterized by high oxidative stress.

Breathing stability during sleep and during hypercapnia is dependent on the retrotrapezoid nucleus in adult mice

Background: Eupneic breathing during sleep is determined by the chemical drive arising from central and peripheral chemoreceptors. The retrotrapezoid nucleus (RTN) contains chemoreceptor neurons that are proposed to be critical for ventilatory responses to CO 2 . RTN chemoreceptor neurons can be identified by expression of transcripts for Neuromedin B ( Nmb + ). We hypothesized that the ablation of RTN Nmb + neurons cause alveolar hypoventilation at rest and breathing instability during sleep. Normally, in intact subjects, increasing respiratory drive with hypercapnia produces a robust and more stable breathing pattern compared to resting condition. We furhter hypothesized that in the absence of RTN, hypercapnia will not increase breathing stability and the ventilatory response to CO 2 will be impaired. Methods: Bilateral microinjections of a designer cell-ablation virus, AAV5-Flex-Casp3-TEVP, were placed in the RTN in heterozygous Cre-positive (RTN lesion) and Cre-negative (controls) Nmb-Cre mice and EEG/neck EMG electrodes were implanted. One month later, mice were tested in an unrestrained plethysmography chamber for breathing and sleep/wake recordings in normoxic conditions. Mice were exposed to hyperoxic hypercapnia (F I CO 2 up to 0.09) to assess the central respiratory CO 2 chemoreflex after RTN lesion. Arterial blood gases were also collected in unrestrained unanesthetized conditions. Values are presented as mean ±SD. Results: Ablation of the RTN was complete (<1% of total RTN Nmb + neurons remaining in RTN lesion group) and selective based on counts of neighboring catecholaminergic and serotonergic neurons. Arterial PCO 2 was increased in RTN-lesion mice (n=6) compared to controls (n=7) (49 ± 5 vs. 39 ± 3 mmHg, p<0.001) and arterial PO 2 was reduced (75 ± 11 vs. 89 ± 7 mmHg, p=0.019). Minute-ventilation was reduced in RTN-lesion mice (n=13) compared to control (n=13) (1.7 ± 0.4 vs. 2.4 ± 0.3 mL/min/g, p<0.0001) due to a reduction in tidal volume that was most evident during NREM sleep. Breathing variability was increased in RTN-lesion mice (n=9) compared to controls (n=10) during NREM (22 ± 5 vs. 10 ± 1% of inter-breath variability, p<0.001) and REM sleep (37 ± 8 vs. 25 ±7 % of inter-breath variability, p<0.01) but not during wakefulness (28 ± 9 vs. 21 ±10 % of inter-breath variability, p=0.32). The ventilatory response to CO 2 was attenuated in RTN lesion mice compared to controls (3.2 ±1 vs. 10.4 ±2 mL/min/g at FICO2 0.09, p<0.0001).Finally, during hypercapnia, breathing was more variable in RTN-lesion mice (23 ±10 vs. 3.1 ±0.4 % of inter-breath variability, p<0.0001) compared to control. Summary and conclusion: In sum, RTN neurons contribute to resting alveolar ventilation and eupneic breathing, especially during sleep. The hypercapnic ventilatory response is severely blunted and hypercapnia does not increase breathing stability in the absence of RTN. We conclude that breathing stability during sleep and during hypercapnia is dependent on the RTN. This work is funded by National Institutes of Health, R01HL148004 to Stephen BG Abbott and R01HL108609 to Douglas A Bayliss. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Somatostatin-expressing parafacial neurons are CO2/H+ sensitive and regulate baseline breathing.

Glutamatergic neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating breathing in response to tissue CO2/H+. The RTN and greater parafacial region may also function as a chemosensing network composed of CO2/H+-sensitive excitatory and inhibitory synaptic interactions. In the context of disease, we showed that loss of inhibitory neural activity in a mouse model of Dravet syndrome disinhibited RTN chemoreceptors and destabilized breathing (Kuo et al., 2019). Despite this, contributions of parafacial inhibitory neurons to control of breathing are unknown, and synaptic properties of RTN neurons have not been characterized. Here, we show the parafacial region contains a limited diversity of inhibitory neurons including somatostatin (Sst)-, parvalbumin (Pvalb)-, and cholecystokinin (Cck)-expressing neurons. Of these, Sst-expressing interneurons appear uniquely inhibited by CO2/H+. We also show RTN chemoreceptors receive inhibitory input that is withdrawn in a CO2/H+-dependent manner, and chemogenetic suppression of Sst+ parafacial neurons, but not Pvalb+ or Cck+ neurons, increases baseline breathing. These results suggest Sst-expressing parafacial neurons contribute to RTN chemoreception and respiratory activity.

Histamine Activates Chemosensitive Neurons in the Retrotrapezoid Nucleus

Histaminergic neurons of the posterior hypothalamic tuberomammillary nucleus (TMN) are pH-sensitive and contribute to CO2/H+-dependent behaviors including arousal and respiratory activity. Considering histaminergic neurons project to several brainstem respiratory centers including the retrotrapezoid nucleus (RTN), and since chemosensitive RTN neurons are an important source of CO2/H+-dependent respiratory drive, we wondered whether and how histamine contributes to RTN chemoreception at the cellular level and in anesthetized rats. In brain slices obtained from rat pups (7-12 days old), chemosensitive RTN neurons were identified in cell-attached voltage-clamp mode by their characteristic firing response to CO2 (≥ 0.8 Hz increase in activity in response to 10% CO2). We found that most chemosensitive RTN neurons showed a dose dependent increase in activity in response to bath application of histamine dihydrochloride (HD: 1-25 μM). The increased activity of RTN neurons by HD (25 mM) was blunted by bath application of the H1 receptor antagonist DPH (diphenhydramine hydrochloride; 100 µM) (1.1 ± 0.1 Hz to -0.06 ± 0.2 Hz; n = 7) or the H2 receptor antagonist ranitidine (ranitidine hydrochloride; 10 µM) (1.0 ± 0.2 Hz to 0.3 ± 0.3 Hz; n = 5). At the whole animal level, we measured mean arterial pressure (MAP) and diaphragm muscle activity (DIAEMG) in urethane-anaesthetized, vagotomized and artificial ventilated male Wistar rats. Unilateral injection of HD (25 mM - 50 nl) into the RTN increased DIAEMG amplitude (17.41 ± 3.8 vs. saline: 0.55 ± 1.9%) and DIAEMG frequency (4.72 ± 1.7 vs -1.8 ± 1.5) without changes MAP. Bilateral injections of DPH (0.5 mM) into the RTN decreased DIAEMG amplitude (-62.6 ± 14.5. vs. saline: -1.1 ± 1.3%), DIAEMG frequency (-66.4 ± 13.4 vs. saline: -1.5 ± 1.9%) and MAP (-43.67 ± 8,4 vs. saline: -3.2 ± 1.7 mmHg). Despite the strong inhibitory effect of DPH on baseline breathing, the hypercapnic ventilatory response was preserved in urethane-anesthetized rats under these experimental conditions. These results suggest histamine signaling via H1 and H2 receptors modulates activity of RTN neurons and respiratory activity.

Astrocytes within the retrotrapezoid nucleus maintain irregular breathing patterns in heart failure: role of P2X7 receptors

Irregular breathing (IB) patterns are a hallmark of heart failure (HF). The mechanisms underlying these alterations are not known. Interestingly, it has been described that purinergic signaling (PurS) in the retrotrapezoid nucleus (RTN), a main central chemoreceptor area, modulate ventilatory chemoreflex control in healthy conditions. However, nothing is known about the role of the RTN on IB in HF nor the precise molecular mechanisms underpinning the development/maintenance of IB in HF. Accordingly, we aimed to determine in HF rats: i) the contribution of the RTN on IB; ii) the alterations in PurS in the RTN; iii) the activation level of RTN chemoreceptor neurons/astrocytes; iv) the effects of chronic RTN chemogenetic manipulation using DREADDs on the maintenance of IB; and v) if astrocyte‐targeted expression of the P2X7 receptor (P2X7r) normalizes breathing. Sprague‐Dawley rats underwent volume overload to induce HF. IB was assessed by whole‐body plethysmography. RTN micropunches were used to determine neuron/astrocyte activation, P2X7r gene/protein expression and ATP levels. Adeno‐associated vector (AAV) expressing an excitatory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) or an AAV carrying the P2X7r under the control of GFAP promoter were stereotaxically injected into the RTN of HF rats. Compared to control rats, HF rats (Sham vs. HF, p<.05) displayed increases in the apnea/hypoapnea index (AHI) (3.5±1 vs. 8.7±2 events/hr), breath‐to‐breath interval variability (SD1: 38.5±8 vs. 83.6±8 ms; SD2: 48.5±5 vs. 107.4±10 ms) and in the coefficient of variation (CV) of VTamplitudes (8.7±2 vs. 14.3±3 %). In addition, both ATP levels (78±2 vs. 21±10 pmoles/100μg of protein) and P2X7r gene/protein expression were reduced in the RTN of HF rats. Importantly, we found that P2X7r expression was restricted only to astrocytes within the RTN. No chronic activation of RTN neurons were found in HF as evidenced by no changes in fosB expression compared to control rats. Contrarily, GFAP expression was significantly lower in the RTN from HF rats compared to controls. Chronic chemogenetic activation of RTN astrocytes in HF increases ATP levels (28±3 vs. 71±4 pmoles/100μg of protein; HF vs. HFDREADD; p<.05, respectively) and improve breathing regularity (Irregularity score: 25±3 vs. 12±1%; HF vs. HFDREADD; p<.05, respectively). Finally, increasing the expression of P2X7r in RTN astrocytes markedly reduced AHI (8.7±2 vs. 3,7±1 events/hr; HF vs. HFP2X7r; p<.05, respectively) and decreases breath‐to‐breath (SD1 83.6±8 vs. 53.6±4 ms; SD2 107.4±10.1 vs 68.5±5.0 ms; HF vs. HFP2X7r; p<.05, respectively) and VTvariability (CV: 25±1 vs. 19±2; HF vs. HFP2X7r; p<.05, respectively). Our results show that the RTN is necessary for the maintenance of IB in HF and that RTN astrocytes play a pivotal role on breathing rhythm regulation by a mechanism encompassing ATP release and the P2X7r.Support or Funding InformationSupported by FONDECYT 1180172 and the basal Center of Excellence in Aging and Regeneration (AFB 170005).

Episodic stimulation of central chemoreceptor neurons elicits disordered breathing and autonomic dysfunction in volume overload heart failure.

Enhanced central chemoreflex (CC) gain is observed in volume overload heart failure (HF) and is correlated with autonomic dysfunction and breathing disorders. The aim of this study was to determine the role of the CC in the development of respiratory and autonomic dysfunction in HF. Volume overload was surgically created to induce HF in male Sprague-Dawley rats. Radiotelemetry transmitters were implanted for continuous monitoring of blood pressure and heart rate. After recovering from surgery, conscious unrestrained rats were exposed to episodic hypercapnic stimulation [EHS; 10 cycles/5 min, inspiratory fraction of carbon dioxide () 7%] in a whole body plethysmograph for recording of cardiorespiratory function. To determine the contribution of CC to cardiorespiratory variables, selective ablation of chemoreceptor neurons within the retrotrapezoid nucleus (RTN) was performed via injection of saporin toxin conjugated to substance P (SSP-SAP). Vehicle-treated rats (HF+Veh and Sham+Veh) were used as controls for SSP-SAP experiments. Sixty minutes post-EHS, minute ventilation was depressed in sham animals relative to HF animals (ΔV̇e: −5.55 ± 2.10 vs. 1.24 ± 1.35 mL/min 100 g, P < 0.05; Sham+Veh vs. HF+Veh). Furthermore, EHS resulted in autonomic imbalance, cardiorespiratory entrainment, and ventilatory disturbances in HF+Veh but not Sham+Veh rats, and these effects were significantly attenuated by SSP-SAP treatment. Also, the apnea-hypopnea index (AHI) was significantly lower in HF+SSP-SAP rats compared with HF+Veh rats (AHI: 5.5 ± 0.8 vs. 14.4 ± 1.3 events/h, HF+SSP-SAP vs. HF+Veh, respectively, P < 0.05). Finally, EHS-induced respiratory-cardiovascular coupling in HF rats depends on RTN chemoreceptor neurons because it was reduced by SSP-SAP treatment. Overall, EHS triggers ventilatory plasticity and elicits cardiorespiratory abnormalities in HF that are largely dependent on RTN chemoreceptor neurons.

Adenosine Signaling through A1 Receptors Inhibits Chemosensitive Neurons in the Retrotrapezoid Nucleus.

A subset of neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating depth and frequency of breathing in response to changes in tissue CO2/H+. The activity of chemosensitive RTN neurons is also subject to modulation by CO2/H+-dependent purinergic signaling. However, mechanisms contributing to purinergic regulation of RTN chemoreceptors are not entirely clear. Recent evidence suggests adenosine inhibits RTN chemoreception in vivo by activation of A1 receptors. The goal of this study was to characterize effects of adenosine on chemosensitive RTN neurons and identify intrinsic and synaptic mechanisms underlying this response. Cell-attached recordings from RTN chemoreceptors in slices from rat or wild-type mouse pups (mixed sex) show that exposure to adenosine (1 µM) inhibits chemoreceptor activity by an A1 receptor-dependent mechanism. However, exposure to a selective A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, DPCPX; 30 nM) alone did not potentiate CO2/H+-stimulated activity, suggesting activation of A1 receptors does not limit chemoreceptor activity under these reduced conditions. Whole-cell voltage-clamp from chemosensitive RTN neurons shows that exposure to adenosine activated an inward rectifying K+ conductance, and at the network level, adenosine preferentially decreased frequency of EPSCs but not IPSCs. These results show that adenosine activation of A1 receptors inhibits chemosensitive RTN neurons by direct activation of a G-protein-regulated inward-rectifier K+ (GIRK)-like conductance, and presynaptically, by suppression of excitatory synaptic input to chemoreceptors.

Bicarbonate directly modulates activity of chemosensitive neurons in the retrotrapezoid nucleus

Changes in CO2 result in corresponding changes in both H+ and HCO3- and despite evidence that HCO3- can function as an independent signalling molecule, there is little evidence suggesting HCO3- contributes to respiratory chemoreception. We show that HCO3- directly activates chemosensitive retrotrapezoid nucleus (RTN) neurons. Identifying all relevant signalling molecules is essential for understanding how chemoreceptors function, and because HCO3- and H+ are buffered by separate cellular mechanisms, having the ability to sense both modalities adds additional information regarding changes in CO2 that are not necessarily reflected by pH alone. HCO3- may be particularly important for regulating activity of RTN chemoreceptors during sustained intracellular acidifications when TASK-2 channels, which appear to be the sole intracellular pH sensor, are minimally active. Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO2 /H+ . The retrotrapezoid nucleus (RTN) is an important site of respiratory chemoreception. Mechanisms underlying RTN chemoreception involve H+ -mediated activation of chemosensitive neurons and CO2 /H+ -evoked ATP-purinergic signalling by local astrocytes, which activates chemosensitive neurons directly and indirectly by maintaining vascular tone when CO2 /H+ levels are high. Although changes in CO2 result in corresponding changes in both H+ and HCO3- and despite evidence that HCO3- can function as an independent signalling molecule, there is little evidence suggesting HCO3- contributes to respiratory chemoreception. Therefore, the goal of this study was to determine whether HCO3- regulates activity of chemosensitive RTN neurons independent of pH. Cell-attached recordings were used to monitor activity of chemosensitive RTN neurons in brainstem slices (300μm thick) isolated from rat pups (postnatal days 7-11) during exposure to low or high concentrations of HCO3- . In a subset of experiments, we also included 2',7'-bis(2carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF) in the internal solution to measure pHi under each experimental condition. We found that HCO3- activates chemosensitive RTN neurons by mechanisms independent of intracellular or extracellular pH, glutamate, GABA, glycine or purinergic signalling, soluble adenylyl cyclase activity, nitric oxide or KCNQ channels. These results establish HCO3- as a novel independent modulator of chemoreceptor activity, and because the levels of HCO3- along with H+ are buffered by independent cellular mechanisms, these results suggest HCO3- chemoreception adds additional information regarding changes in CO2 that are not necessarily reflected by pH.

Inhibition of the hypercapnic ventilatory response by adenosine in the retrotrapezoid nucleus in awake rats

The brain regulates breathing in response to changes in tissue CO2/H+ via a process called central chemoreception. Neurons and astrocytes in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors. The role of astrocytes in this process appears to involve CO2/H+-dependent release of ATP to enhance activity of chemosensitive RTN neurons. Considering that in most brain regions extracellular ATP is rapidly broken down to adenosine by ectonucleotidase activity and since adenosine is a potent neuromodulator, we wondered whether adenosine signaling contributes to RTN chemoreceptor function. To explore this possibility, we pharmacologically manipulated activity of adenosine receptors in the RTN under control conditions and during inhalation of 7–10% CO2 (hypercapnia). In urethane-anesthetized or unrestrained conscious rats, bilateral injections of adenosine into the RTN blunted the hypercapnia ventilatory response. The inhibitory effect of adenosine on breathing was blunted by prior RTN injection of a broad spectrum adenosine receptor blocker (8-PT) or a selective A1-receptor blocker (DPCPX). Although RTN injections of 8PT, DPCPX or the ectonucleotidase inhibitor ARL67156 did not affected baseline breathing in either anesthetized or awake rats. We did find that RTN application of DPCPX or ARL67156 potentiated the respiratory frequency response to CO2, suggesting a portion of ATP released in the RTN during high CO2/H+ is converted to adenosine and serves to limit chemoreceptor function. These results identify adenosine as a novel purinergic regulator of RTN chemoreceptor function during hypercapnia.

Fluorocitrate-mediated depolarization of astrocytes in the retrotrapezoid nucleus stimulates breathing.

Evidence indicates that CO2/H+-evoked ATP released from retrotrapezoid nucleus (RTN) astrocytes modulates the activity of CO2-sensitive neurons. RTN astrocytes also sense H+ by inhibition of Kir4.1 channels; however, the relevance of this pH-sensitive current remains unclear since ATP release appears to involve CO2-dependent gating of connexin 26 hemichannels. Considering that depolarization mediated by H+ inhibition of Kir4.1 channels is expected to increase sodium bicarbonate cotransporter (NBC) conductance and favor Ca2+ influx via the sodium calcium exchanger (NCX), we hypothesize that depolarization in the presence of CO2 is sufficient to facilitate ATP release and enhance respiratory output. Here, we confirmed that acute exposure to fluorocitrate (FCt) reversibly depolarizes RTN astrocytes and increased activity of RTN neurons by a purinergic-dependent mechanism. We then made unilateral injections of FCt into the RTN or two other putative chemoreceptor regions (NTS and medullary raphe) to depolarize astrocytes under control conditions and during P2-recepetor blockade while measuring cardiorespiratory activities in urethane-anesthetized, vagotomized, artificially ventilated male Wistar rats. Unilateral injection of FCt into the RTN increased phrenic (PNA) amplitude and frequency without changes in arterial pressure. Unilateral injection of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS, a P2-receptor antagonist) into the RTN dampened both PNA amplitude and frequency responses to FCt. Injection of MRS2179 (P2Y1-receptor antagonist) into the RTN did not affect the FCt-induced respiratory responses. Fluorocitrate had no effect on breathing when injected into the NTS or raphe. These results suggest that depolarization can facilitate purinergic enhancement of respiratory drive from the RTN.NEW & NOTEWORTHY Astrocytes in the retrotrapezoid nucleus (RTN) are known to function as respiratory chemoreceptors; however, it is not clear whether changes in voltage contribute to astrocyte chemoreception. We showed that depolarization of RTN astrocytes at constant CO2 levels is sufficient to modulate RTN chemoreception by a purinergic-dependent mechanism. These results support the possibility that astrocyte depolarization can facilitate purinergic enhancement of respiratory drive from the RTN.

In vitro characterization of noradrenergic modulation of chemosensitive neurons in the retrotrapezoid nucleus.

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO2/H(+) changes and serve as an integration center for other autonomic centers, including brain stem noradrenergic neurons. Norepinephrine (NE) contributes to respiratory control and chemoreception, and, since disruption of NE signaling may contribute to several breathing disorders, we sought to characterize effects of NE on RTN chemoreception. All neurons included in this study responded similarly to CO2/H(+) but showed differential sensitivity to NE; we found that NE activated (79%), inhibited (7%), or had no effect on activity (14%) of RTN chemoreceptors. The excitatory effect of NE on RTN chemoreceptors was dose dependent, retained in the presence of neurotransmitter receptor blockers, and could be mimicked and blocked by pharmacological manipulation of α1-adrenergic receptors (ARs). In addition, NE-activation was blunted by XE991 (KCNQ channel blocker), and partially occluded the firing response to serotonin, suggesting involvement of KCNQ channels. However, in whole cell voltage clamp, activation of α1-ARs decreased outward current and conductance by what appears to be a mixed effect on multiple channels. The inhibitory effect of NE on RTN chemoreceptors was blunted by an α2-AR antagonist. A third group of RTN chemoreceptors was insensitive to NE. We also found that chemosensitive RTN astrocytes do not respond to NE with a change in voltage or by releasing ATP to enhance activity of chemosensitive neurons. These results indicate NE modulates subsets of RTN chemoreceptors by mechanisms involving α1- and α2-ARs.

α1- and α2-adrenergic receptors in the retrotrapezoid nucleus differentially regulate breathing in anesthetized adult rats.

Norepinephrine (NE) is a potent modulator of breathing that can increase/decrease respiratory activity by α1-/α2-adrenergic receptor (AR) activation, respectively. The retrotrapezoid nucleus (RTN) is known to contribute to central chemoreception, inspiration, and active expiration. Here we investigate the sources of catecholaminergic inputs to the RTN and identify respiratory effects produced by activation of ARs in this region. By injecting the retrograde tracer Fluoro-Gold into the RTN, we identified back-labeled catecholaminergic neurons in the A7 region. In urethane-anesthetized, vagotomized, and artificially ventilated male Wistar rats unilateral injection of NE or moxonidine (α2-AR agonist) blunted diaphragm muscle activity (DiaEMG) frequency and amplitude, without changing abdominal muscle activity. Those inhibitory effects were reduced by preapplication of yohimbine (α2-AR antagonist) into the RTN. Conversely, unilateral RTN injection of phenylephrine (α1-AR agonist) increased DiaEMG amplitude and frequency and facilitated active expiration. This response was blocked by prior RTN injection of prazosin (α1-AR antagonist). Interestingly, RTN injection of propranolol (β-AR antagonist) had no effect on respiratory inhibition elicited by applications of NE into the RTN; however, the combined blockade of α2- and β-ARs (coapplication of propranolol and yohimbine) revealed an α1-AR-dependent excitatory response to NE that resulted in increase in DiaEMG frequency and facilitation of active expiration. However, blockade of α1-, α2-, or β-ARs in the RTN had minimal effect on baseline respiratory activity, on central or peripheral chemoreflexes. These results suggest that NE signaling can modulate RTN chemoreceptor function; however, endogenous NE signaling does not contribute to baseline breathing or the ventilatory response to central or peripheral chemoreceptor activity in urethane-anesthetized rats.

Noradrenergic modulation of chemosensitive neurons in the retrotrapezoid nucleus

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO2/H+ changes, and serve as an integration center for other autonomic centers including brainstem noradrenergic neurons. Considering that application of norepinephrine (NE) into the RTN strongly modulates breathing (see abstract by Oliveira et al.) and disruption of NE signaling has been implicated in breathing disorders associated with Rett syndrome, sleep‐disordered breathing and multiple system atrophy, we sought to characterize the effects of NE on chemosensitive RTN neurons. Cell‐attached recordings were used to identify RTN chemoreceptors in brainstem slices (300 μm thick) isolated from rat pups (P7–11 days postnatal) by their characteristic response to CO2; i.e., low basal activity in 5% CO2 (0.2 ± 0.1 Hz) and high activity in 10% (1.8 ± 0.1 Hz) or 15% CO2 (2.1 ± 0.1 Hz ). We found that bath application of NE (1 μM) increased activity by 1.1 ± 0.1 Hz in 48 of 66 cells (73%), decreased activity by 1.0 ± 0.1 Hz in 6 of 66 cells (9%), or had no effect on activity in 12 of 66 (18%) of cells. Interestingly, basal activity but not CO2/H+‐sensitivity differed between each group based on their NE response; e.g., basal activity and firing response to 10% CO2 was 0.4 ± 0.1 and 1.2 ± 0.1 Hz (NE‐activated), 1.1 ± 0.3. and 1.1 ± 0.1 Hz (NE‐inhibited) and 0.03 ± 0.01 and 1.1 ± 0.01 Hz (NE‐insensitive). The excitatory effect of NE on RTN chemoreceptors was dose dependent (EC50 of 235 nM), retained during synaptic blockade (high Mg+2/low Ca2+) and could be mimicked by an α1‐receptor agonist (phenylephrine; 10 μM) and blocked with a α1‐receptor antagonist (prazosin; 1 μM). The inhibitory effect of NE on RTN chemoreceptors was retained during synaptic blockade and blunted by a α2‐receptor antagonist (idazoxan; 1 μM). A subset of NE‐activated cells were inhibited by NE in the presence of prazosin, whereas some NE‐inhibited cells showed an excitatory response to NE in the presence of idazoxan. A third group of RTN chemoreceptors did not respond to NE under control conditions or during hypercapnia, and these cells also did not respond to phenylephrine. In addition, whole‐cell voltage clamp (Vhold= −80 mV; TTX) recordings from CO2/H+‐sensitive RTN astrocytes show the absence of an NE‐sensitive current. These results indicate that i) RTN chemoreceptors are intrinsically sensitive to NE; ii) NE can increase or decrease chemoreceptor activity by α1‐ and α2‐receptor dependent mechanisms, respectively; and iii) NE modulates discrete subsets of RTN chemoreceptors based on basal activity and the differential contributions of α1‐α2‐receptors.Support or Funding InformationNIH Grant :HL104101

Influence of CO2 and HCO3− on Basal Activity and Acid Sensitivity of Chemosensitive Neurons in the Retrotrapezoid Nucleus

Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO2/H+. A region of the brainstem called the retrotrapezoid nucleus (RTN) is an important site of chemoreception. The mechanisms of RTN chemoreception involve direct H+‐mediated activation of chemosensitive neurons, and indirect modulation of chemosensitive neurons by CO2/H+‐dependent ATP‐purinergic signaling. Recent evidence identifies astrocytes as the source of CO2/H+‐evoked ATP release, possibly by a mechanism involving CO2‐mediated opening of connexin 26 hemichannels. However, the extent to which ATP contributes to RTN chemoreception is questionable; some studies report that ATP release from astrocytes is required for chemosensitive RTN neurons to sense changes in CO2/H+, whereas other studies showed that blocking ATP receptors in the RTN decreased (~ 30%) but did not eliminate CO2/H+‐sensitivity of RTN chemoreceptors. Based on evidence that CO2 is required for ATP release by RTN astrocytes, we chose to investigate the contribution of purinergic to RTN chemoreception by comparing the firing responses of chemosensitive RTN neurons to graded acidifications in the presence and absence of CO2 and HCO3−. Cell‐attached recordings were used to identify RTN chemoreceptors in brainstem slices (300 μm thick) isolated from rat pups (P7–11 days postnatal) incubated in a bicarbonate (26 mM) buffered solution by their characteristic response to CO2; i.e., low basal activity in 5% CO2 (pH=7.3; 0.7 ± 0.1 Hz) and high activity in 10% CO2 (pH=7.0; 1.7 ± 0.3 Hz) or 15% CO2 (pH=6.9; 2.2 ± 0.4 Hz). After returning to control conditions (5% CO2; pH=7.3) switching to a bicarbonate‐free HEPES buffered solution (pH=7.3) decreased baseline activity by 0.5 ± 0.4 Hz (T3=5.175, p=0.014; paired t‐test). In HEPES buffer, acidifications to pH=7.0 and pH=6.9 increased activity by 0.65 ± 0.2 Hz and 0.8 ± 0.2 Hz, respectively. In some cases, during prolonged exposure to HEPES baseline activity increased to near control levels, suggesting that removal of CO2/HCO3− from the perfusate only transiently suppressed chemoreceptor activity. These results suggest that RTN chemoreceptors respond more vigorously to hypercapnic acidosis compared to the same acidification in HEPES buffer. This finding is consistent with the possibility that CO2‐evoked ATP release from astrocytes contributes in part to RTN chemoreception. These results also suggest that HCO3− controls baseline activity of RTN chemoreceptors, possibly by influencing intracellular pH or regulating HCO3−‐sensitive K+ channels (i.e., KCNQ).Support or Funding InformationNIH Grant HL104101

The retrotrapezoid nucleus as a central brainstem area for central and peripheral chemoreceptor interactions.

The retrotrapezoid nucleus as a central brainstem area for central and peripheral chemoreceptor interactions.

Cholinergic control of ventral surface chemoreceptors involves Gq/inositol 1,4,5-trisphosphate-mediated inhibition of KCNQ channels.

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. 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.

The retrotrapezoid nucleus stimulates breathing by releasing glutamate in adult conscious mice.

The retrotrapezoid nucleus (RTN) is a bilateral cluster of neurons located at the ventral surface of the brainstem below the facial nucleus. The RTN is activated by hypercapnia and stabilises arterial Pco2 by adjusting lung ventilation in a feedback manner. RTN neurons contain vesicular glutamate transporter-2 (Vglut2) transcripts (Slc17a6), and their synaptic boutons are Vglut2-immunoreactive. Here, we used optogenetics to test whether the RTN increases ventilation in conscious adult mice by releasing glutamate. Neurons located below the facial motor nucleus were transduced unilaterally to express channelrhodopsin-2 (ChR2)-enhanced yellow fluorescent protein, with lentiviral vectors that employ the Phox2b-activated artificial promoter PRSx8. The targeted population consisted of two types of Phox2b-expressing neuron: non-catecholaminergic neurons (putative RTN chemoreceptors) and catecholaminergic (C1) neurons. Opto-activation of a mix of ChR2-expressing RTN and C1 neurons produced a powerful stimulus frequency-dependent (5-15 Hz) stimulation of breathing in control conscious mice. Respiratory stimulation was comparable in mice in which dopamine-β-hydroxylase (DβH)-positive neurons no longer expressed Vglut2 (DβH(C) (re/0);;Vglut2(fl/fl)). In a third group of mice, i.e. DβH(+/+);;Vglut2(fl/fl) mice, we injected a mixture of PRSx8-Cre lentiviral vector and Cre-dependent ChR2 adeno-associated virus 2 unilaterally into the RTN; this procedure deleted Vglut2 from ChR2-expressing neurons regardless of whether or not they were catecholaminergic. The ventilatory response elicited by photostimulation of ChR2-positive neurons was almost completely absent in these mice. Resting ventilatory parameters were identical in the three groups of mice, and their brains contained similar numbers of ChR2-positive catecholaminergic and non-catecholaminergic neurons. From these results, we conclude that RTN neurons increase breathing in conscious adult mice by releasing glutamate.

Molecular underpinnings of ventral surface chemoreceptor function: focus on KCNQ channels.

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.

HCN channels contribute to serotonergic modulation of ventral surface chemosensitive neurons and respiratory activity.

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) provide a CO2/H(+)-dependent drive to breathe and function as an integration center for the respiratory network, including serotonergic raphe neurons. We recently showed that serotonergic modulation of RTN chemoreceptors involved inhibition of KCNQ channels and activation of an unknown inward current. Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are the molecular correlate of the hyperpolarization-activated inward current (Ih) and have a high propensity for modulation by serotonin. To investigate whether HCN channels contribute to basal activity and serotonergic modulation of RTN chemoreceptors, we characterize resting activity and the effects of serotonin on RTN chemoreceptors in vitro and on respiratory activity of anesthetized rats in the presence or absence of blockers of KCNQ (XE991) and/or HCN (ZD7288, Cs(+)) channels. We found in vivo that bilateral RTN injections of ZD7288 increased respiratory activity and in vitro HCN channel blockade increased activity of RTN chemoreceptors under control conditions, but this was blunted by KCNQ channel inhibition. Furthermore, in vivo unilateral RTN injection of XE991 plus ZD7288 eliminated the serotonin response, and in vitro serotonin sensitivity was eliminated by application of XE991 and ZD7288 or SQ22536 (adenylate cyclase blocker). Serotonin-mediated activation of RTN chemoreceptors was blocked by a 5-HT7-receptor blocker and mimicked by a 5-HT7-receptor agonist. In addition, serotonin caused a depolarizing shift in the voltage-dependent activation of Ih. These results suggest that HCN channels contribute to resting chemoreceptor activity and that serotonin activates RTN chemoreceptors and breathing in part by a 5-HT7 receptor-dependent mechanism and downstream activation of Ih.