On the peripheral and central chemoreception and control of breathing: an emerging role of ATP

Peripheral and central respiratory chemoreceptors are ultimately responsible for maintenance of constant levels of arterial   P _O2  ,   P _CO2  and [H⁺], protecting the brain from hypoxia and ensuring that the breathing is always appropriate for metabolism. The aim of this discussion is to shed some light on the potential mechanisms of chemosensory transduction – the process which links chemosensory mechanisms to the central nervous mechanisms controlling breathing. Recent experimental data suggest that the purine nucleotide ATP acts as a common mediator of peripheral and central chemosensory transduction (within the carotid body and the medulla oblongata, respectively). In response to a decrease in   P _O2  (hypoxia) oxygen-sensitive glomus cells of the carotid body release ATP to activate chemoafferent fibres of the carotid sinus nerve which transmit this information to the brainstem respiratory centres. In response to an increase in   P _CO2/[H⁺]  (hypercapnia) chemosensitive structures located on the ventral surface of the medulla oblongata rapidly release ATP, which acts locally within the medullary respiratory network. The functional role of ATP released at both sites is similar – to evoke adaptive enhancement in breathing. Understanding the mechanisms of ATP release in response to chemosensory stimulation may prove to be essential for further detailed analysis of cellular and molecular mechanisms underlying respiratory chemosensitivity.     

Stimulus-specific signaling pathways in rabbit carotid body chemoreceptors

The carotid body is an arterial chemosensory organ which responds to multiple natural and pharmacological stimuli, including hypoxia and nicotine. Numerous studies have investigated the initial molecular events which activate chemosensory type I cells in the carotid body, but less attention has been focused on later steps in the transduction cascade, which mediate neurotransmitter release from type I cells and excitation of chemoreceptor afferent fibers in the carotid sinus nerve. In the present study, we examined the effects of a highly specific inhibitor of calcium/calmodulin-dependent kinase II, KN-62, and a calmodulin inhibitor, trifluoperazine, on carotid sinus nerve activity and catecholamine release evoked from rabbit carotid bodies superfused in vitro. KN-62 did not alter sinus nerve activity and catecholamine release evoked by hypoxia, but this agent significantly reduced nerve activity and neurotransmitter release evoked by 100 microM nicotine. Trifluoperazine (10 microM), likewise inhibited activity evoked by nicotine, as well as hypoxia. Basal levels of nerve activity and catecholamine release (established in superfusate equilibrated with 100% O2) were unaffected by all drug treatments. Separate biochemical experiments showed that Ca2+/calmodulin-dependent incorporation of 32P into carotid body particulate proteins is significantly reduced following incubation of intact carotid bodies in nicotine, but not following exposure to hypoxia. Our observations suggest that excitation of the carotid body by diverse stimuli may involve the activation of distinct, stimulus-specific transduction pathways. Furthermore, these data correlate with our previous findings which showed that hypoxia, on the one hand, and nicotine on the other, evoke the preferential release of either dopamine or norepinephrine, respectively, from carotid bodies incubated in vitro.     

Purines, the carotid body and respiration

The carotid body is essential to detecting levels of oxygen in the blood and initiating the compensatory response. Increasing evidence suggests that the purines ATP and adenosine make a key contribution to this signaling by the carotid body. The glomus cells release ATP in response to hypoxia. This released ATP can stimulate P2X receptors on the carotid body to elevate intracellular Ca(2+) and to produce an excitatory response. This released ATP can be dephosphorylated to adenosine by a series of extracellular enzymes, which in turn can stimulate A(1), A(2A) and A(2B) adenosine receptors. Levels of extracellular adenosine can also be altered by membrane transporters. Endogenous adenosine stimulates these receptors to increase the ventilation rate and may modulate the catecholamine release from the carotid sinus nerve. Prolonged hypoxic challenge can alter the expression of purinergic receptors, suggesting a role in the adaptation. This review discusses evidence for a key role of ATP and adenosine in the hypoxic response of the carotid body, and emphasizes areas of new contributions likely to be important in the future.     

Stimulus-specific signaling pathways in rabbit carotid body chemoreceptors

The carotid body is an arterial chemosensory organ which responds to multiple natural and pharmacological stimuli, including hypoxia and nicotine. Numerous studies have investigated the initial molecular events which activate chemosensory type I cells in the carotid body, but less attention has been focused on later steps in the transduction cascade, which mediate neurotransmitter release from type I cells and excitation of chemoreceptor afferent fibers in the carotid sinus nerve. In the present study, we examined the effects of a highly specific inhibitor of calcium/calmodulin-dependent kinase II, KN-62, and a calmodulin inhibitor, trifluoperazine, on carotid sinus nerve activity and catecholamine release evoked from rabbit carotid bodies superfused in vitro. KN-62 did not alter sinus nerve activity and catecholamine release evoked by hypoxia, but this agent significantly reduced nerve activity and neurotransmitter release evoked by 100 μM nicotine. Trifluoperazine (10 μM), likewise inhibited activity evoked by nicotine, as well as hypoxia. Basal levels of nerve activity and catecholamine release (established in superfusate equilibrated with 100% O₂) were unaffected by all drug treatments. Separate biochemical experiments showed that Ca²⁺/calmodulin-dependent incorporation of ³²P into carotid body particulate proteins is significantly reduced following incubation of intact carotid bodies in nicotine, but not following exposure to hypoxia.Our observations suggest that excitation of the carotid body by diverse stimuli may involve the activation of distinct, stimulus-specific transduction pathways. Furthermore, these data correlate with our previous findings which showed that hypoxia, on the one hand, and nicotine on the other, evoke the preferential release of either dopamine or norepinephrine, respectively, from carotid bodies incubated in vitro.     

The Carotid Sinus Nerve—Structure,Function, and Clinical Implications

Interest has been renewed in the anatomy and physiology of the carotid sinus nerve (CSN) and its targets (carotid sinus and carotid body, CB), due to recent proposals of surgical procedures for a series of common pathologies, such as carotid sinus syndrome, hypertension, heart failure, and insulin resistance. The CSN originates from the glossopharyngeal nerve soon after its appearance from the jugular foramen. It shows frequent communications with the sympathetic trunk (usually at the level of the superior cervical ganglion) and the vagal nerve (main trunk, pharyngeal branches, or superior laryngeal nerve). It courses on the anterior aspect of the internal carotid artery to reach the carotid sinus, CB, and/or intercarotid plexus. In the carotid sinus, type I (dynamic) carotid baroreceptors have larger myelinated A-fibers; type II (tonic) baroreceptors show smaller A- and unmyelinated C-fibers. In the CB, afferent fibers are mainly stimulated by acetylcholine and ATP, released by type I cells. The neurons are located in the petrosal ganglion, and centripetal fibers project on to the solitary tract nucleus: chemosensory inputs to the commissural subnucleus, and baroreceptor inputs to the commissural, medial, dorsomedial, and dorsolateral subnuclei. The baroreceptor component of the CSN elicits sympatho-inhibition and the chemoreceptor component stimulates sympatho-activation. Thus, in refractory hypertension and heart failure (characterized by increased sympathetic activity), baroreceptor electrical stimulation, and CB removal have been proposed. Instead, denervation of the carotid sinus has been proposed for the “carotid sinus syndrome.” Anat Rec, 302:575–587, 2019. © 2018 Wiley Periodicals, Inc.     

Pronounced depression by propofol on carotid body response to CO2 and K+-induced carotid body activation

Propofol is a commonly used anesthetic agent, and it attenuates hypoxic ventilatory response in humans. Propofol reduce in vivo and in vitro carotid body responses to hypoxia as well as to nicotine in experimental animals. In the present study we examined the effects of propofol on carotid body responses to hypercapnia and K(+)-induced carotid body activation and compared these effects with hypoxia in an in vitro rabbit carotid body preparation. Hypoxia, hypercapnia and potassium increased the carotid sinus nerve activity and propofol attenuated the chemoreceptor responses to all three stimuli. However, the magnitude of propofol-induced attenuation was greater for hypercapnic and K(+)-induced carotid body activation compared to the hypoxic response. These observations suggest that propofol-induced attenuation of the hypoxic response is partly secondary to depression of chemoreceptor response to hypercapnia inhibiting the synergistic interactions between O(2) and CO(2) and may involve CO(2)/H(+) sensitive K(+) channels.     

O2 sensing at the mammalian carotid body: why multiple O2 sensors and multiple transmitters?

Carotid bodies are the sensory organs for detecting systemic hypoxia and the ensuing reflexes prevent the development of tissue/cellular hypoxia. Although every mammalian cell responds to hypoxia, O2 sensing by the carotid body is unique in that it responds instantaneously (within seconds) to even a modest drop in arterial PO2. Sensing hypoxia in the carotid body requires an initial transduction step involving O2 sensor(s) and transmitter(s) for subsequent activation of the afferent nerve ending. This brief review focuses on: (a) whether the transduction involves 'single' or 'multiple' O2 sensors; (b) the identity of the excitatory transmitter(s) responsible for afferent nerve activation by hypoxia; and (c) whether inhibitory transmitters have any functional role. The currently proposed O2 sensors include various haem-containing proteins, and a variety of O2-sensitive K+ channels. It is proposed that the transduction involves an ensemble of, and interactions between, haem-containing proteins and O2-sensitive K+-channel proteins functioning as a 'chemosome'; the former for conferring sensitivity to wide range of PO2 values and the latter for the rapidity of the response. Hypoxia releases both excitatory and inhibitory transmitters from the carotid body. ATP is emerging as an important excitatory transmitter for afferent nerve activation by hypoxia. Whereas the inhibitory messengers act in concert with excitatory transmitters like a 'push-pull' mechanism to prevent over excitation, conferring the 'slowly adapting' nature of the afferent nerve activation during prolonged hypoxia. Further studies are needed to test the interactions between putative O2 sensors and excitatory and inhibitory transmitters in the carotid body.     

Histaminergic and dopaminergic traits in the human carotid body

Carotid body (CB) chemoreceptors are the main sensors detecting systemic hypoxia. Studies in animals revealed that dopamine and histamine may serve as transmitters between the chemoreceptor cells and the afferent nerve. To gain insight whether histamine and dopamine could play a role in the human CB and thus be important for the understanding of breathing disorders, we have investigated the chemosensory traits in human CBs from nine subjects of different ages obtained at autopsy. Immunohistochemistry revealed expression of histidine decarboxylase, vesicular monoamine transporter 2, histamine receptors 1 and 3 in virtually all chemosensory cells within the glomeruli of different ages. By contrast, catecholaminergic traits (tyrosine hydroxylase and vesicular monoamine transporter 1) were only detected in a subset of CB chemosensory cells at each age group while dopamine D2 receptors were expressed in the great majority of them. Our data suggest that histamine along with catecholamines may serve as transmitters between chemoreceptor cells and the afferent nerve in humans as well.     

Inhibition in carotid body chemoreceptors mediated by D-2 dopaminoceptors: Antagonism by benzamides

Inhibition of chemosensory nerve impulses in the cat is evoked by dopamine (DA) applied to carotid body chemoreceptors. Pharmacological characterization of the dopaminoceptors involved in this action was determined through their blockade with benzamides, selective antagonists of D-2 receptors. Both metoclopramide and sulpiride were effective blockers of DA-induced chemosensory inhibition. Furthermore, both drugs induced an immediate increase in the frequency of carotid nerve chemosensory impulses, suggesting the presence of previous tonic inhibition of chemoreceptor discharges by endogenous DA released from glomus cells.     

Evidence for functional,inhibitory, histamine H3 receptors in rat carotid body Type I cells

The Type I cells are the sensory elements of the carotid bodies and play a critical role in defining the ventilatory response to hypoxia and hypercapnia. Type I cells release multiple neurotransmitters during a chemosensory stimulus resulting in increased firing of the carotid sinus nerve and modification of the breathing pattern. While much is known about the actions of individual neurotransmitters in this system, very little is known about how multiple neurotransmitters may integrate to shape the output of the carotid body. Recent data has indicated that the neurotransmitter histamine does not excite isolated Type I cells despite being released during hypoxia and its receptors being present on the Type I cells. Here the hypothesis that histamine might modulate an excitatory neurotransmitter such as acetylcholine was tested. Using calcium imaging techniques it was found that histamine attenuated calcium signaling events initiated by the muscarinic acetylcholine receptor agonist acetyl-β-methylcholine via an H3 receptor mediated mechanism. In summary, these results suggest that when acetylcholine and histamine are co-released from Type I cells in response to chemostimuli, histamine may attenuate or modulate the excitatory presynaptic actions of acetylcholine.     

Barosensory neurons in the ventrolateral medulla in rabbits and their responses to various afferent inputs from peripheral and central sources

In 55 anesthetized and paralyzed adult rabbits, 161 spontaneously active neurons which responded to electrical stimulation of A-fibers of the aortic nerve were found within the ventrolateral medulla (VLM). They were termed barosensory VLM neurons, since the aortic nerve A-fibers were considered to consist exclusively of afferents from arterial baroreceptors. Forty percent of barosensory VLM neurons tested (49/123) were activated antidromically by stimulation of the dorsolateral funiculus indicating that they send descending bulbospinal projections. Spontaneous discharges of barosensory VLM neurons were invariably inhibited by stimulation of aortic nerve A-fibers. Ninety-three percent of 80 neurons tested also responded to stimulation of aortic nerve C-fibers, a mixture of barosensory and nonbarosensory afferents. Natural stimulation of carotid sinus baroreceptors by an intravenous injection of phenylephrine in 19 vagotomized rabbits with aortic nerves disrupted inhibited spontaneous activity of all the 50 barosensory VLM neurons tested. By contrast, pharmacological stimulation of right or left carotid body chemoreceptors by close arterial injection of NaCN into the carotid sinus augmented activity of 93% of barosensory VLM neurons tested (41/44). The neuronal response was always greater to stimulation of chemoreceptors in the contralateral carotid sinus. Seven out of 8 barosensory VLM neurons tested (88%) were orthodromically excited by stimulation of the posterior hypothalamic area. In 74% of the 97 neurons examined in 29 vagotomized animals, a distinct respiratory-related rhythm, locked to that of phrenic nerve activity, was discerned. Thus, spontaneous activity of barosensory VLM neurons is inhibited by afferent inputs from aortic and carotid sinus baroreceptors, but is excited by incoming signals from carotid body chemoreceptors and the posterior hypothalamic area. It is also subject to the influence of the central mechanism generating the respiratory rhythm.     

Is ATP a suitable co-transmitter in carotid body arterial chemoreceptors?

A review is presented on carotid body ATP content, effects and release, receptors involved and results of their block by purinergic antagonists, and the possibility of cholinergic-purinergic co-transmission in the carotid body. Glomus cells release ACh and ATP upon physiological stimulation. Both agents and their agonists have chemo-excitatory actions and their combined effects disappear upon blocking n-ACh and P2X receptors. Both ACh and ATP also are capable of exciting the somata of chemosensory neurons of petrosal ganglia. Although a combined cholinergic-purinergic block suppresses the chemosensory activity in neurons co-cultured with glomus cells and some carotid body preparations in vitro, basal chemosensory activity and chemosensory responses to hypoxic stimuli persist in cat carotid body preparations in situ and in vitro. Therefore, ATP is an effective excitatory agent for carotid body chemosensory activity, although less potent than ACh; their joint participation may contribute to -- but does not entirely explain -- the transfer of chemoreceptor excitation from glomus cells to sensory endings in carotid body.     

Inferior cardiac nerve activity in the cat during occlusion of the mesenteric artery.

A number of studies in this and other laboratories using hemodynamic and pharmacologic evidence have suggested that occlusion of the mesenteric artery evokes a pressor reflex initiated by mesenteric baroreceptors. To provide additional evidence in support of this hypothesis, neurophysiological recordings were made of inferior cardiac nerve activity during mesenteric artery occlusion (MAO). The results indicate that MAO enhances inferior cardiac nerve activity in the cat, providing that the carotid sinus nerves have been cut. Cutting of the mesenteric nerves further facilitates cardiac nerve activity and abolishes the response to mesenteric artery occlusion. The evidence suggests that MAO evokes a reflex sympathetic discharge which is subject to override by the carotid sinus depressor reflex. The afferent limb of the reflex is characterized by a tonic depressor outflow from the mesenteric pressure receptors.     

Neurotransmitters in carotid body development

This review examines the possible role of neurotransmitters present in the carotid body on the functional expression of chemosensory activity during postnatal development. In particular, dopamine, acetylcholine, adenosine and neuropeptides are reviewed. Evidence to date shows involvement of these transmitters in signal transmission from the chemoreceptor cells to chemosensory afferent fibers of the sinus nerve, with clear age- or maturation-dependence of some aspects. However, it remains unresolved whether these neurotransmitters, some of which are expressed in the carotid body before birth, are directly involved in the maturation of the functional properties of the carotid chemoreceptors in sensing oxygen or other stimuli during postnatal development.     

Autonomic innervation of the carotid body: role in efferent inhibition

The carotid body (CB) is a chemosensory organ that monitors blood chemicals and initiates compensatory reflex adjustments to maintain homeostasis. The 'afferent' sensory discharge induced by changes in blood chemicals, e.g. low PO(2) (hypoxia), is relayed by carotid sinus nerve (CSN) fibers and has been well studied. Much less is known, however, about a parallel autonomic (parasympathetic) 'efferent' pathway that is the source of CB inhibition. This pathway is the focus of this review which begins with a historical account of the early findings and links them to more recent data on the source of this innervation, and the role of endogenous neurotransmitters in efferent inhibition. We review evidence that these autonomic neurons are embedded in 'paraganglia' within the glossopharyngeal (GPN) and CSN nerves, and for the role of nitric oxide (NO) in mediating efferent inhibition. Finally, we discuss recent data linking the action of hypoxia and a key CB neurotransmitter, i.e. ATP, to potential mechanisms for activating this efferent pathway.     

Carotid body chemosensory activity and ventilatory chemoreflexes in cats persist after combined cholinergic-purinergic block

Acetylcholine (ACh) and ATP have been proposed as excitatory co-transmitters operating at synapses between glomus cells and sensory nerve endings of the carotid body (CB). To test such hypothesis, we performed experiments on cats under pentobarbitone anesthesia and breathing spontaneously. Cholinergic and purinergic agonists and antagonists were given into one common carotid artery. Chemoreflex ventilatory changes initiated from the ipsilateral CB or chemosensory activity from the ipsilateral carotid nerve were recorded. Agonists ACh, nicotine, epibatidine, ATP, betagamma-methylene-ATP and gammaS-ATP induced transient chemoreflex enhancements of ventilation or increased chemosensory activity. When given in combination, mecamylamine and suramin suppressed both nicotine- and ATP-induced ventilatory chemoreflexes or chemosensory responses. However, neither chemoreflex hyperventilation induced by brief hypoxic exposures or steady-state hypoxic levels, nor chemosensory excitation elicited by these maneuvers were eliminated. Asphyxia-induced chemosensory excitation was not reduced by combined blockade of ACh and ATP receptors. Furthermore, ventilatory or chemosensory depression evoked by 100% O2 tests was unmodified, thus evidencing that basal chemosensory drive in normoxia was not suppressed by combined cholinergic-purinergic blockade. Therefore, although ACh and ATP may participate in chemoexcitation of the CB, their involvement fails to explain the origin of chemosensory discharges from synaptic transmission between glomus cells and chemosensory nerve endings of the CB.     

Evidence for the involvement of purinergic signalling in the control of respiration.

The ventrolateral medulla has a critical role in the generation and patterning of respiration via an extensive network of respiratory neurones. We have investigated the effects of activating purinergic P2 receptors within the ventrolateral medulla of the anaesthetised rat on the overall pattern of respiratory activity. In addition, using immunohistochemical techniques, we have identified the subtypes of P2X receptors in the ventrolateral medulla. Unilateral microinjection of ATP into the ventrolateral medulla reduced in a dose-dependent manner, or abolished, resting phrenic nerve discharge recorded as an indication of central inspiratory drive. ATP also elicited increases in blood pressure and variable changes in heart rate. These effects were mimicked by microinjection of the P2X receptor agonist α,β-methylene ATP into the ventrolateral medulla. Whilst microinjection of suramin, a P2 receptor antagonist, had no effect on resting cardiorespiratory variables it blocked the respiratory and cardiovascular effects of ATP microinjected into the ventrolateral medulla. Immunohistochemical staining using IgG antibodies showed that P2X₁, P2X₂, P2X₅ and P2X₆, but not P2X₃, P2X₄ or receptor subunits were localised in the rostral ventrolateral medulla.Our results indicate that several P2X receptor subtypes are localised within areas of the ventrolateral medulla that are important for cardiorespiratory control (including the pre-Bötzinger and Bötzinger complexes), and that activation of these receptors can have profound effects on both the cardiovascular and the respiratory networks. Our pharmacological data suggest that different P2X subunits in this region may co-assemble to form hetero-oligomeric assemblies as well as homomultimers within this region.     

Effects of combined cholinergic-purinergic block upon cat carotid body chemoreceptors in vitro

Since acetylcholine (ACh) and ATP have been proposed as excitatory co-transmitters at synapses between glomus cells and sensory nerve endings of the carotid body (CB), we tested such hypothesis by studying the effects of combined cholinergic-purinergic block on the chemosensory activity recorded from cat's carotid bodies perfused and/or superfused in vitro. The preparations were bathed with Tyrode's solution, either normoxic (PO2=98.5+/-13.5 Torr) or hypoxic (PO2=31.8+/-5.2 Torr), and the frequency of chemosensory impulses (fchi) was recorded from the carotid (sinus) nerve. Dose-response curves for fchi increases evoked by intra-stream boluses of acetylcholine, nicotine and ATP were studied. A combination of mecamylamine 2 microM and suramin 50 microM, applied through the perfusate or superfusate, suppressed nicotine- and ATP-induced increases in fchi, but the basal chemosensory activity in normoxia and the chemosensory excitation elicited by hypoxic superfusion were preserved, although variably reduced in most preparations. Thus, in spite of the excitatory effects provoked by applying ACh and ATP to the perfused/superfused CB in vitro, a co-release of these substances cannot account entirely for the chemosensory excitation induced by hypoxic stimulation of the CB.     

Restoration of reflex ventilatory response to hypoxia after removal of carotid bodies in the cat

In mammals there are two sets of peripheral arterial chemoreceptors, the carotid bodies innervated by the sinus branch of the glossopharyngeal nerve and the aortic bodies innervated by the vagus nerves. The afferent impulse discharge from both receptors increases during hypoxia and there is a reflexly mediated increase in ventilation (hypoxic hyperventilation). In the present study we tested this response by exposing anesthetized cats to decreased inspired O₂ concentration before and up to 315 days after bilateral resection of the carotid bodies. Acutely after removing the carotid bodies, hypoxic hyperventilation was abolished. This observation supports the view that the reflex pathway from the aortic body receptors normally contributes minimally to hypoxic hyperventilation. Subsequently, there was a restoration of hypoxic hyperventilation. Restoration was significant 30–43 days after removing the carotid bodies, it reached 70% of the preoperative value at 93–111 days and was essentially complete in terminal experiments 260–315 days after carotid body resection. In terminal experiments, hypoxic hyperventilation was not affected by recutting the regenerated carotid sinus nerves but was abolished completely by bilateral transection of the cervical vagosympathetic trunks. The restored ventilatory response was due predominantly to an increase in rate of breathing while an increase in tidal volume was predominant before carotid body resection. Resting ventilation breathing room air was not consistently decreased after carotid body resection while expired CO₂ was elevated from day 20 to day 111 and at the preoperative level in terminal experiments.It is concluded that restoration of hypoxic hyperventilation in the cat after carotid body resection is mediated by the reflex pathway from aortic body chemoreceptors. The possible contribution of chemo-receptive regenerated carotid sinus nerve axons was excluded. It is suggested that restoration may be a consequence of the central reorganization of chemoreceptor afferent pathways consequent to interruption of the carotid body reflex pathway and that as a result the ‘gain’ of the aortic body ventilatory chemoreflex is enhanced.     

CO2, brainstem chemoreceptors and breathing.

The regulation of breathing relies upon chemical feedback concerning the levels of CO2 and O2. The carotid bodies, which detect O2, provide tonic excitation to brainstem respiratory neurons under normal conditions and dramatic excitation if O2 levels fall. Feedback for CO2 involves the carotid body and receptors in the brainstem, central chemoreceptors. Small increases in CO2 produce large increases in breathing. Decreases in CO2 below normal can, in sleep and anesthesia, decrease breathing, even to apnea. Central chemoreceptors, once thought localized to the surface of the ventral medulla, are likely distributed more widely with sites presently identified in the: (1) ventrolateral medulla; (2) nucleus of the solitary tract; (3) ventral respiratory group; (4) locus ceruleus; (5) caudal medullary raphé; and (6) fastigial nucleus of the cerebellum. Why so many chemoreceptor sites? Hypotheses, some with supporting data, include the following. Geographical specificity; all regions of the brainstem with respiratory neurons contain chemoreceptors. Stimulus intensity; some sites operate in the physiological range of CO2 values, others only with more extreme changes. Stimulus specificity; CO2 or pH may be sensed by multiple mechanisms. Temporal specificity; some sites respond more quickly to changes on blood or brain CO2 or pH. Syncytium; chemosensitive neurons may be connected via low resistance, gap junctions. Arousal state: sites may vary in effectiveness and importance dependent on state of arousal. Overall, as judged by experiments of nature, and in the laboratory, central chemoreceptors are critical for adequate breathing in sleep, but other aspects of the control system can maintain breathing in wakefulness.