Promyelinating drugs promote functional recovery in an autism spectrum disorder mouse model of Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is an autism spectrum disorder caused by autosomal dominant mutations in the human transcription factor 4 gene (TCF4). One pathobiological process caused by murine Tcf4 mutation is a cell autonomous reduction in oligodendrocytes and myelination. In this study, we show that the promyelinating compounds, clemastine, sobetirome and Sob-AM2 are effective at restoring myelination defects in a Pitt-Hopkins syndrome mouse model. In vitro, clemastine treatment reduced excess oligodendrocyte precursor cells and normalized oligodendrocyte density. In vivo, 2-week intraperitoneal administration of clemastine also normalized oligodendrocyte precursor cell and oligodendrocyte density in the cortex of Tcf4 mutant mice and appeared to increase the number of axons undergoing myelination, as EM imaging of the corpus callosum showed a significant increase in the proportion of uncompacted myelin and an overall reduction in the g-ratio. Importantly, this treatment paradigm resulted in functional rescue by improving electrophysiology and behaviour. To confirm behavioural rescue was achieved via enhancing myelination, we show that treatment with the thyroid hormone receptor agonist sobetirome or its brain penetrating prodrug Sob-AM2, was also effective at normalizing oligodendrocyte precursor cell and oligodendrocyte densities and behaviour in the Pitt-Hopkins syndrome mouse model. Together, these results provide preclinical evidence that promyelinating therapies may be beneficial in Pitt-Hopkins syndrome and potentially other neurodevelopmental disorders characterized by dysmyelination.

Stroke-Induced Respiratory Dysfunction Is Associated With Cognitive Decline.

BACKGROUND: Respiratory dysfunction is a common complication of stroke, with an incidence of over 60%. Despite the high prevalence of stroke-induced respiratory dysfunction, how disordered breathing influences recovery and cognitive outcomes after ischemic stroke is unknown. We hypothesized that stroke induces chronic respiratory dysfunction, breathing instability, and apnea in mice, which would contribute to higher mortality and greater poststroke cognitive deficits. METHODS: Mice were subjected to a 60-minute transient middle cerebral artery occlusion or permanent distal middle cerebral artery occlusion. Whole body plethysmography was performed on C57BL/6 young (2-3 months) and aged (20 months) male and female mice. Animals were exposed to a variety of gas conditions to assess the contribution of peripheral and central chemoreceptors. A battery of cognitive tests was performed to examine behavioral function. RESULTS: Middle cerebral artery occlusion led to disordered breathing characterized by hypoventilation and apneas. Cognitive decline correlated with the severity of disordered breathing. Distal permanent middle cerebral artery occlusion, which produces a smaller cortical infarct, also produced breathing disorders and cognitive impairment but only in aged mice. CONCLUSIONS: Our data suggest that poststroke apnea is associated with cognitive decline and highlights the influence of aging on breathing disorders after stroke. Therefore, the treatment of respiratory instability may be a viable approach to improving cognitive outcomes after stroke.

Update on vascular control of central chemoreceptors.

At least four mechanisms have been proposed to elucidate how neurons in the retrotrapezoid (RTN) region sense changes in CO2 /H+ to regulate breathing (i.e., function as respiratory chemosensors). These mechanisms include: (1) intrinsic neuronal sensitivity to H+ mediated by TASK-2 and GPR4; (2) paracrine activation of RTN neurons by CO2 -responsive astrocytes (via a purinergic mechanism); (3) enhanced excitatory synaptic input or disinhibition; and (4) CO2 -induced vascular contraction. Although blood flow can influence tissue CO2 /H+ levels, there is limited understanding of how control of vascular tone in central CO2 chemosensitive regions might contribute to respiratory output. In this review, we focus on recent evidence that CO2 /H+ -induced purinergic-dependent vasoconstriction in the ventral parafacial region near RTN neurons supports respiratory chemoreception. This mechanism appears to be unique to the ventral parafacial region and opposite to other brain regions, including medullary chemosensor regions, where CO2 /H+ elicits vasodilatation. We speculate that this mechanism helps to maintain CO2 /H+ levels in the vicinity of RTN neurons, thereby maintaining the drive to breathe. Important next steps include determining whether disruption of CO2 /H+ vascular reactivity contributes to or can be targeted to improve breathing problems in disease states, such as Parkinson's disease.

5-HT7 receptors expressed in the mouse parafacial region are not required for respiratory chemosensitivity.

A brainstem homeostatic system senses CO2 /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 CO2 /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, CO2 -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 CO2 -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 CO2 -evoked firing activity in RTN neurons or on CO2 -stimulated breathing in mice. KEY POINTS: Neurons of the retrotrapezoid nucleus (RTN) are intrinsic CO2 /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 CO2  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 CO2 /H+ -stimulation of RTN neuronal activity or CO2 -stimulated breathing. These data do not support a role for 5-HT7 receptors in respiratory chemosensitivity mediated by RTN neurons.

Kir 5.1-dependent CO2 /H+ -sensitive currents contribute to astrocyte heterogeneity across brain regions.

Astrocyte heterogeneity is an emerging concept in which astrocytes within or between brain regions show variable morphological and/or gene expression profiles that presumably reflect different functional roles. Recent evidence indicates that retrotrapezoid nucleus (RTN) astrocytes sense changes in tissue CO2/ H+ to regulate respiratory activity; however, mechanism(s) by which they do so remain unclear. Alterations in inward K+ currents represent a potential mechanism by which CO2 /H+ signals may be conveyed to neurons. Here, we use slice electrophysiology in rats of either sex to show that RTN astrocytes intrinsically respond to CO2 /H+ by inhibition of an inward rectifying potassium (Kir ) conductance and depolarization of the membrane, while cortical astrocytes do not exhibit such CO2 /H+ -sensitive properties. Application of Ba2+ mimics the effect of CO2 /H+ on RTN astrocytes as measured by reductions in astrocyte Kir -like currents and increased RTN neuronal firing. These CO2 /H+ -sensitive currents increase developmentally, in parallel to an increased expression in Kir 4.1 and Kir 5.1 in the brainstem. Finally, the involvement of Kir 5.1 in the CO2 /H+ -sensitive current was verified using a Kir5.1 KO rat. These data suggest that Kir inhibition by CO2 /H+ may govern the degree to which astrocytes mediate downstream chemoreceptive signaling events through cell-autonomous mechanisms. These results identify Kir channels as potentially important regional CO2 /H+ sensors early in development, thus expanding our understanding of how astrocyte heterogeneity may uniquely support specific neural circuits and behaviors.

The retrotrapezoid nucleus and the neuromodulation of breathing.

Breathing is regulated by a host of arousal and sleep-wake state-dependent neuromodulators to maintain respiratory homeostasis. Modulators such as acetylcholine, norepinephrine, histamine, serotonin (5-HT), adenosine triphosphate (ATP), substance P, somatostatin, bombesin, orexin, and leptin can serve complementary or off-setting functions depending on the target cell type and signaling mechanisms engaged. Abnormalities in any of these modulatory mechanisms can destabilize breathing, suggesting that modulatory mechanisms are not overly redundant but rather work in concert to maintain stable respiratory output. The present review focuses on the modulation of a specific cluster of neurons located in the ventral medullary surface, named retrotrapezoid nucleus, that are activated by changes in tissue CO2/H+ and regulate several aspects of breathing, including inspiration and active expiration.

Isoflurane inhibits a Kir4.1/5.1-like conductance in neonatal rat brainstem astrocytes and recombinant Kir4.1/5.1 channels in a heterologous expression system.

All inhalation anesthetics used clinically including isoflurane can suppress breathing; since this unwanted side effect can persist during the postoperative period and complicate patient recovery, there is a need to better understand how isoflurane affects cellular and molecular elements of respiratory control. Considering that astrocytes in a brainstem region known as the retrotrapezoid nucleus (RTN) contribute to the regulation of breathing in response to changes in CO2/H+ (i.e., function as respiratory chemoreceptors), and astrocytes in other brain regions are highly sensitive to isoflurane, we wanted to determine whether and how RTN astrocytes respond to isoflurane. We found that RTN astrocytes in slices from neonatal rat pups (7-12 days postnatal) respond to clinically relevant levels of isoflurane by inhibition of a CO2/H+-sensitive Kir4.1/5.1-like conductance [50% effective concentration (EC50) = 0.8 mM or ~1.7%]. We went on to confirm that similar levels of isoflurane (EC50 = 0.53 mM or 1.1%) inhibit recombinant Kir4.1/5.1 channels but not homomeric Kir4.1 channels expressed in HEK293 cells. We also found that exposure to CO2/H+ occluded subsequent effects of isoflurane on both native and recombinant Kir4.1/5.1 currents. These results identify Kir4.1/5.1 channels in astrocytes as novel targets of isoflurane. These results suggest astrocyte Kir4.1/5.1 channels contribute to certain aspects of general anesthesia including altered respiratory control.NEW & NOTEWORTHY An unwanted side effect of isoflurane anesthesia is suppression of breathing. Despite this clinical significance, effects of isoflurane on cellular and molecular elements of respiratory control are not well understood. Here, we show that isoflurane inhibits heteromeric Kir4.1/5.1 channels in a mammalian expression system and a Kir4.1/5.1-like conductance in astrocytes in a brainstem respiratory center. These results identify astrocyte Kir4.1/5.1 channels as novel targets of isoflurane and potential substrates for altered respiratory control during isoflurane anesthesia.

Volatile Anesthetics Activate a Leak Sodium Conductance in Retrotrapezoid Nucleus Neurons to Maintain Breathing during Anesthesia in Mice.

BACKGROUND: Volatile anesthetics moderately depress respiratory function at clinically relevant concentrations. Phox2b-expressing chemosensitive neurons in the retrotrapezoid nucleus, a respiratory control center, are activated by isoflurane, but the underlying mechanisms remain unclear. The hypothesis of this study was that the sodium leak channel contributes to the volatile anesthetics-induced modulation of retrotrapezoid nucleus neurons and to respiratory output. METHODS: The contribution of sodium leak channels to isoflurane-, sevoflurane-, and propofol-evoked activity of Phox2b-expressing retrotrapezoid nucleus neurons and respiratory output were evaluated in wild-type and genetically modified mice lacking sodium leak channels (both sexes). Patch-clamp recordings were performed in acute brain slices. Whole-body plethysmography was used to measure the respiratory activity. RESULTS: Isoflurane at 0.42 to 0.50 mM (~1.5 minimum alveolar concentration) increased the sodium leak channel-mediated holding currents and conductance from -75.0 ± 12.9 to -130.1 ± 34.9 pA (mean ± SD, P = 0.002, n = 6) and 1.8 ± 0.5 to 3.6 ± 1.0 nS (P = 0.001, n = 6), respectively. At these concentrations, isoflurane increased activity of Phox2b-expressing retrotrapezoid nucleus neurons from 1.1 ± 0.2 to 2.8 ± 0.2 Hz (P < 0.001, n = 5), which was eliminated by bath application of gadolinium or genetic silencing of sodium leak channel. Genetic silencing of sodium leak channel in the retrotrapezoid nucleus resulted in a diminished ventilatory response to carbon dioxide in mice under control conditions and during isoflurane anesthesia. Sevoflurane produced an effect comparable to that of isoflurane, whereas propofol did not activate sodium leak channel-mediated holding conductance. CONCLUSIONS: Isoflurane and sevoflurane increase neuronal excitability of chemosensitive retrotrapezoid nucleus neurons partly by enhancing sodium leak channel conductance. Sodium leak channel expression in the retrotrapezoid nucleus is required for the ventilatory response to carbon dioxide during anesthesia by isoflurane and sevoflurane, thus identifying sodium leak channel as a requisite determinant of respiratory output during anesthesia of volatile anesthetics.

Cholinergic neurons in the pedunculopontine tegmental nucleus modulate breathing in rats by direct projections to the retrotrapezoid nucleus.

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 CO2 /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 CO2 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 (VT ) but otherwise increased respiratory rate (fR ) and net respiratory output as indicated by an increase in ventilation (VE ). 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.

Epilepsy-Associated KCNQ2 Channels Regulate Multiple Intrinsic Properties of Layer 2/3 Pyramidal Neurons.

KCNQ2 potassium channels are critical for normal brain function, as both loss-of-function and gain-of-function KCNQ2 variants can lead to various forms of neonatal epilepsy. Despite recent progress, the full spectrum of consequences as a result of KCNQ2 dysfunction in neocortical pyramidal neurons is still unknown. Here, we report that conditional ablation of Kcnq2 from mouse neocortex leads to hyperexcitability of layer 2/3 (L2/3) pyramidal neurons, exhibiting an increased input resistance and action potential frequency, as well as a reduced medium afterhyperpolarization (mAHP), a conductance partly mediated by KCNQ2 channels. Importantly, we show that introducing the KCNQ2 loss-of-function variant KCNQ2I205V into L2/3 pyramidal neurons using in utero electroporation also results in a hyperexcitable phenotype similar to the conditional knock-out. KCNQ2I205V has a right-shifted conductance-to-voltage relationship, suggesting loss of KCNQ2 channel activity at subthreshold membrane potentials is sufficient to drive large changes in L2/3 pyramidal neuronal excitability even in the presence of an intact mAHP. We also found that the changes in excitability following Kcnq2 ablation are accompanied by alterations at action potential properties, including action potential amplitude in Kcnq2-null neurons. Importantly, partial inhibition of Nav1.6 channels was sufficient to counteract the hyperexcitability of Kcnq2-null neurons. Therefore, our work shows that loss of KCNQ2 channels alters the intrinsic neuronal excitability and action potential properties of L2/3 pyramidal neurons, and identifies Nav1.6 as a new potential molecular target to reduce excitability in patients with KCNQ2 encephalopathy. SIGNIFICANCE STATEMENT: KCNQ2 channels are critical for the development of normal brain function, as KCNQ2 variants could lead to epileptic encephalopathy. However, the role of KCNQ2 channels in regulating the properties of neocortical neurons is largely unexplored. Here, we find that Kcnq2 ablation or loss-of-function at subthreshold membrane potentials leads to increased neuronal excitability of neocortical layer 2/3 (L2/3) pyramidal neurons. We also demonstrate that Kcnq2 ablation unexpectedly leads to a larger action potential amplitude. Importantly, we propose the Nav1.6 channel as a new molecular target for patients with KCNQ2 encephalopathy, as partial inhibition of these channels counteracts the increased L2/3 pyramidal neuron hyperexcitability of Kcnq2-null neurons.

Bicarbonate directly modulates activity of chemosensitive neurons in the retrotrapezoid nucleus.

KEY POINTS: 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. ABSTRACT: 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 (300 µm 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.

MeCP2 deficiency results in robust Rett-like behavioural and motor deficits in male and female rats.

Since the identification of MECP2 as the causative gene in the majority of Rett Syndrome (RTT) cases, transgenic mouse models have played a critical role in our understanding of this disease. The use of additional mammalian RTT models offers the promise of further elucidating critical early mechanisms of disease as well as providing new avenues for translational studies. We have identified significant abnormalities in growth as well as motor and behavioural function in a novel zinc-finger nuclease model of RTT utilizing both male and female rats throughout development. Male rats lacking MeCP2 (Mecp2ZFN/y) were noticeably symptomatic as early as postnatal day 21, with most dying by postnatal day 55, while females lacking one copy of Mecp2 (Mecp2ZFN/+) displayed a more protracted disease course. Brain weights of Mecp2ZFN/y and Mecp2ZFN/+ rats were significantly reduced by postnatal day 14 and 21, respectively. Early motor and breathing abnormalities were apparent in Mecp2ZFN/y rats, whereas Mecp2ZFN/+ rats displayed functional irregularities later in development. The large size of this species will provide profound advantages in the identification of early disease mechanisms and the development of appropriately timed therapeutics. The current study establishes a foundational basis for the continued utilization of this rat model in future RTT research.

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.

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

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.

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 (ß-AR antagonist) had no effect on respiratory inhibition elicited by applications of NE into the RTN; however, the combined blockade of α2- and ß-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 ß-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.

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.

Independent purinergic mechanisms of central and peripheral chemoreception in the rostral ventrolateral medulla.

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.

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.

Purinergic signalling contributes to chemoreception in the retrotrapezoid nucleus but not the nucleus of the solitary tract or medullary raphe.

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.