Daniel K. Mulkey

Expression of Phox2b by Brainstem Neurons Involved in Chemosensory Integration in the Adult Rat

Central congenital hypoventilation syndrome is caused by mutations of the gene that encodes the transcription factor Phox2b. The syndrome is characterized by a severe form of sleep apnea attributed to greatly compromised central and peripheral chemoreflexes. In this study, we analyze whether Phox2b expression in the brainstem respiratory network is preferentially associated with neurons involved in chemosensory integration in rats. At the very rostral end of the ventral respiratory column (VRC), Phox2b was present in many VGlut2 (vesicular glutamate transporter 2) mRNA-containing neurons. These neurons were functionally identified as the respiratory chemoreceptors of the retrotrapezoid nucleus (RTN). More caudally in the VRC, many fewer neurons expressed Phox2b. These cells were not part of the central respiratory pattern generator (CPG), because they were typically cholinergic visceral motor neurons or catecholaminergic neurons (presumed C1 neurons). Phox2b was not detected in serotonergic neurons, in the A5, A6, and A7 noradrenergic cell groups nor within the main cardiorespiratory centers of the dorsolateral pons. Phox2b was expressed by many solitary tract nucleus (NTS) neurons including those that relay peripheral chemoreceptor information to the RTN. These and previous observations by others suggest that Phox2b is expressed by an uninterrupted chain of neurons involved in the integration of peripheral and central chemoreception (carotid bodies, chemoreceptor afferents, chemoresponsive NTS neurons projecting to VRC, RTN chemoreceptors). The presence of Phox2b in this circuit and its apparent absence from the respiratory CPG could explain why Phox2b mutations disrupt breathing automaticity during sleep without causing major impairment of respiration during waking.

TASK channels determine pH sensitivity in select respiratory neurons but do not contribute to central respiratory chemosensitivity.

Central respiratory chemoreception is the mechanism by which the CNS maintains physiologically appropriate pH and PCO2 via control of breathing. A prominent hypothesis holds that neural substrates for this process are distributed widely in the respiratory network, especially because many neurons that make up this network are chemosensitive in vitro. We and others have proposed that TASK channels (TASK-1, K2P3.1 and/or TASK-3, K2P9.1) may serve as molecular sensors for central chemoreception because they are highly expressed in multiple neuronal populations in the respiratory pathway and contribute to their pH sensitivity in vitro. To test this hypothesis, we examined the chemosensitivity of two prime candidate chemoreceptor neurons in vitro and tested ventilatory responses to CO2 using TASK channel knock-out mice. The pH sensitivity of serotonergic raphe neurons was abolished in TASK channel knock-outs. In contrast, pH sensitivity of neurons in the mouse retrotrapezoid nucleus (RTN) was fully maintained in a TASK null background, and pharmacological evidence indicated that a K+ channel with properties distinct from TASK channels contributes to the pH sensitivity of rat RTN neurons. Furthermore, the ventilatory response to CO2 was completely retained in single or double TASK knock-out mice. These data rule out a strict requirement for TASK channels or raphe neurons in central respiratory chemosensation. Furthermore, they indicate that a non-TASK K+ current contributes to chemosensitivity of RTN neurons, which are profoundly pH-sensitive and capable of driving respiratory output in response to local pH changes in vivo.

Regulation of Ventral Surface Chemoreceptors by the Central Respiratory Pattern Generator

The rat retrotrapezoid nucleus (RTN) contains neurons described as central chemoreceptors in the adult and respiratory rhythm-generating pacemakers in neonates [parafacial respiratory group (pfRG)]. Here we test the hypothesis that both RTN and pfRG neurons are intrinsically chemosensitive and tonically firing neurons whose respiratory rhythmicity is caused by a synaptic feedback from the central respiratory pattern generator (CPG). In halothane-anesthetized adults, RTN neurons were silent below 4.5% end-expiratory (e-exp) CO2. Their activity increased linearly (3.2 Hz/1% CO2) up to 6.5% (CPG threshold) and then more slowly to peak ∼10 Hz at 10% CO2. Respiratory modulation of RTN neurons was absent below CPG threshold, gradually stronger beyond, and, like pfRG neurons, typically (42%) characterized by twin periods of reduced activity near phrenic inspiration. After CPG inactivation with kynurenate (KYN), RTN neurons discharged linearly as a function of e-exp CO2 (slope, +1.7 Hz/1% CO2) and arterial pH (threshold, 7.48; slope, 39 Hz/pH unit). In coronal brain slices (postnatal days 7–12), RTN chemosensitive neurons were silent at pH 7.55. Their activity increased linearly with acidification up to pH 7.2 (17 Hz/pH unit at 35°C) and was always tonic. In conclusion, consistent with their postulated central chemoreceptor role, RTN/pfRG neurons encode pH linearly and discharge tonically when disconnected from the rest of the respiratory centers in vivo (KYN treatment) and in vitro. In vivo, RTN neurons receive respiratory synchronous inhibitory inputs that may serve as feedback and impart these neurons with their characteristic respiratory modulation.

Retrotrapezoid nucleus: a litmus test for the identification of central chemoreceptors.

Central chemoreception is the mechanism by which arterial blood P(CO2) is detected by the CNS to regulate breathing. Two main theories have been proposed to account for the phenomenon. The distributed chemosensitivity theory argues that pH sensitivity is a widespread attribute of brainstem neurones and that central chemoreception results from the cumulative effects of pH on countless neurones. The specialized chemoreceptor theory envisions the existence of small and specialized populations of CNS cells (chemoreceptors) that are unique in their ability to detect very small pH fluctuations and, via specific connections, regulate a respiratory network that is itself unresponsive to pH. The recently identified CO2-sensitive neurones of the retrotrapezoid nucleus (RTN) seem to possess most of the attributes that one would expect of such chemoreceptors. In this review we also suggest that many fewer medullary neurones are intrinsically responsive to CO2 in vivo than might have been anticipated from prior experimentation in vitro. The properties of RTN neurones provide renewed support for the specialized chemoreceptor theory of central chemoreception, proposed in the early 1960s. However, many uncertainties remain, especially as regards the molecular mechanisms of chemoreception, the type of cell that actually detects pH in vivo (neurone, glia or others) and the number and location of bona fide central chemoreceptors.Smith JC, Morrison DE, Ellenberger HH, Otto MR & Feldman JL (1989). Brainstem projections to the major respiratory neuron populations in the medulla of the cat. J Comp Neurol 281, 69–96. Solomon IC, Edelman NH & O’Neal MH III (2000). CO2/H+ chemoreception in the cat pre-Botzinger complex in vivo. J Appl Physiol 88, 1996–2007. Spyer KM, Dale N & Gourine AV (2004). ATP is a key mediator of central and peripheral chemosensory transduction. Exp Physiol 89, 53–59. Teppema LJ, Veening JG, Kranenburg A, Dahan A, Berkenbosch A & Olievier C (1997). Expression of c-fos in the rat brainstem after exposure to hypoxia and to normoxic and hyperoxic hypercapnia. J Comp Neurol 388, 169–190. Veasey SC, Fornal CA, Metzler CW & Jacobs BL (1995). Response of serotonergic caudal raphe neurons in relation to specific motor activities in freely moving cats. J Neurosci 15, 5346–5359. Voipio J & Ballanyi K (1997). Interstitial PCO2 and pH, and their role as chemostimulants in the isolated respiratory network of neonatal rats. J Physiol 499, 527–542. Wang JQ, Kon J, Mogi C, Tobo M, Damirin A, Sato K et al. (2004). TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor. J Biol Chem 279, 45626–45633. Washburn CP, Bayliss DA & Guyenet PG (2003). Cardiorespiratory neurons of the rat ventrolateral medulla contain TASK-1 and TASK-3 channel mRNA. Respir Physiol Neurobiol 138, 19–35. Washburn CP, Sirois JE, Talley EM, Guyenet PG & Bayliss DA (2002). Serotonergic raphe neurons express TASK channel transcripts and a TASK-like pHand halothane-sensitive K+ conductance. J Neurosci 22, 1256–1265. Wellner-Kienitz MC, Shams H & Scheid P (1998). Contribution of Ca2+-activated K+ channels to central chemosensitivity in cultivated neurons of fetal rat medulla. J Neurophysiol 79, 2885–2894. Weston MC, Stornetta RL & Guyenet PG (2004). Glutamatergic neuronal projections from the marginal layer of the rostral ventral medulla to the respiratory centers in rats. J Comp Neurol 473, 73–85. Xu H, Cui N, Yang Z, Qu Z & Jiang C (2000). Modulation of kir4.1 and kir5.1 by hypercapnia and intracellular acidosis. J Physiol 524, 725–735.

Serotonergic Neurons Activate Chemosensitive Retrotrapezoid Nucleus Neurons by a pH-Independent Mechanism

Serotonin activates respiration and enhances the stimulatory effect of CO2 on breathing. The present study tests whether the mechanism involves the retrotrapezoid nucleus (RTN), a group of medullary glutamatergic neurons activated by extracellular brain pH and presumed to regulate breathing. We show that the RTN is innervated by both medullary and pontine raphe and receives inputs from thyrotropin-releasing hormone (TRH) and substance P-expressing neurons. Coexistence of serotonin and substance P in terminals within RTN confirmed that lower medullary serotonergic neurons innervate RTN. In vivo, unilateral injection of serotonin into RTN stimulated inspiratory motor activity, and pH-sensitive RTN neurons were activated by iontophoretic application of serotonin or substance P. In brain slices, pH-sensitive RTN neurons were activated by serotonin, substance P, and TRH. The effect of serotonin in slices was ketanserin sensitive and persisted in the presence of glutamate, GABA, glycine, and purinergic ionotropic receptor antagonists. Serotonin and pH had approximately additive effects on the discharge rate of RTN neurons, both in slices and in vivo. In slices, serotonin produced an inward current with little effect on conductance and had no effect on the pH-induced current. We conclude that (1) RTN receives input from multiple raphe nuclei, (2) serotonin, substance P, and TRH activate RTN chemoreceptors, and (3) excitatory effects of serotonin and pH are mediated by distinct ionic conductances. Thus, RTN neurons presumably contribute to the respiratory stimulation caused by serotonergic neurons, but serotonin seems without effect on the cellular mechanism by which RTN neurons detect pH.

Retrotrapezoid nucleus and parafacial respiratory group

The rat retrotrapezoid nucleus (RTN) contains about 2000 Phox2b-expressing glutamatergic neurons (ccRTN neurons; 800 in mice) with a well-understood developmental lineage. ccRTN neuron development fails in mice carrying a Phox2b mutation commonly present in the congenital central hypoventilation syndrome. In adulthood, ccRTN neurons regulate the breathing rate and intensity, and may regulate active expiration along with other neighboring respiratory neurons. Prenatally, ccRTN neurons form an autonomous oscillator (embryonic parafacial group, e-pF) that activates and possibly paces inspiration. The pacemaker properties of the ccRTN neurons probably vanish after birth to be replaced by synaptic drives. The neonatal parafacial respiratory group (pfRG) may represent a transitional phase during which ccRTN neurons lose their group pacemaker properties. ccRTN neurons are activated by acidification via an intrinsic mechanism or via ATP released by glia. In summary, throughout life, ccRTN neurons seem to be a critical hub for the regulation of CO(2) via breathing.

AMP-activated protein kinase inhibits TREK channels

AMP‐activated protein kinase (AMPK) is a serine/threonine kinase activated by conditions that increase the AMP : ATP ratio. In carotid body glomus cells, AMPK is thought to link changes in arterial O2 with activation of glomus cells by inhibition of unidentified background K+ channels. Modulation by AMPK of individual background K+ channels has not been described. Here, we characterize effects of activated AMPK on recombinant TASK‐1, TASK‐3, TREK‐1 and TREK‐2 background K+ channels expressed in HEK293 cells. We found that TREK‐1 and TREK‐2 channels but not TASK‐1 or TASK‐3 channels are inhibited by AMPK. AMPK‐mediated inhibition of TREK involves key serine residues in the C‐terminus that are also known to be important for PKA and PKC channel modulation; inhibition of TREK‐1 requires Ser‐300 and Ser‐333 and inhibition of TREK‐2 requires Ser‐326 and Ser‐359. Metabolic inhibition by sodium azide can also inhibit both TREK and TASK channels. The effects of azide on TREK occlude subsequent channel inhibition by AMPK and are attenuated by expression of a dominant negative catalytic subunit of AMPK (dnAMPK), suggesting that metabolic stress modulates TREK channels by an AMPK mechanism. By contrast, inhibition of TASK channels by azide was unaffected by expression of dnAMPK, suggesting an AMPK‐independent mechanism. In addition, prolonged exposure (6–7 min) to hypoxia (= 11 ± 1 mmHg) inhibits TREK channels and this response was blocked by expression of dnAMPK. Our results identify a novel modulation of TREK channels by AMPK and indicate that select residues in the C‐terminus of TREK are points of convergence for multiple signalling cascades including AMPK, PKA and PKC. To the extent that carotid body O2 sensitivity is dependent on AMPK, our finding that TREK‐1 and TREK‐2 channels are inhibited by AMPK suggests that TREK channels may represent the AMPK‐inhibited background K+ channels that mediate activation of glomus cells by hypoxia.

Anesthetic Activation of Central Respiratory Chemoreceptor Neurons Involves Inhibition of a THIK-1-Like Background K+ Current

At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO2/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b–eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of an Na+-dependent cationic current and inhibition of a background K+ current. Single-cell reverse transcription-PCR analysis of dissociated green fluorescent protein-labeled RTN neurons revealed expression of THIK-1 (TWIK-related halothane-inhibited K+ channel, K2P13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na+, and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO2 levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated primarily by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions.

Glucose increases activity and Ca2+ in insulin-producing cells of adult drosophila

We sought to understand the mechanisms underlying glucose sensing in Drosophila melanogaster. We found that insulin-producing cells (IPCs) of adult Drosophila respond to glucose and glibenclamide with a burst-like pattern of activity. Under controlled conditions IPCs have a resting membrane potential of −62±4 mV. In response to glucose or glibenclamide, IPCs generate action potentials at a threshold of −36±1.4 mV with an amplitude of 46±4 mV and width of 9.3±1.8 ms. Real-time Ca2+ imaging confirms that IPCs respond to glucose and glibenclamide with increased intracellular Ca2+. These results provide the first detailed characterization of electrical properties of IPCs of adult Drosophila and suggest that these cells sense glucose by a mechanism similar to mammalian pancreatic &bgr; cells.

Characterization of the chemosensitive response of individual solitary complex neurons from adult rats

We studied the CO(2)/H(+)-chemosensitive responses of individual solitary complex (SC) neurons from adult rats by simultaneously measuring the intracellular pH (pH(i)) and electrical responses to hypercapnic acidosis (HA). SC neurons were recorded using the blind whole cell patch-clamp technique and loading the soma with the pH-sensitive dye pyranine through the patch pipette. We found that SC neurons from adult rats have a lower steady-state pH(i) than SC neurons from neonatal rats. In the presence of chemical and electrical synaptic blockade, adult SC neurons have firing rate responses to HA (percentage of neurons activated or inhibited and the magnitude of response as determined by the chemosensitivity index) that are similar to SC neurons from neonatal rats. They also have a typical response to isohydric hypercapnia, including decreased DeltapH(i), followed by pH(i) recovery, and increased firing rate. Thus, the chemosensitive response of SC neurons from adults is similar to the chemosensitive response of SC neurons from neonatal rats. Because our findings for adults are similar to previously reported values for neurons from neonatal rats, we conclude that intrinsic chemosensitivity is established early in development for SC neurons and is maintained throughout adulthood.