Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H+ and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2, a pH-sensitive K+ channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel and essential central mediators of respiratory chemosensitivity.

SummaryThe retrotrapezoid nucleus (RTN) contains non-cholinergic non-catecholaminergic glutamatergic neurons that express the transcription factor Phox2b (chemically coded or "cc" RTN neurons). These cells regulate breathing and may be central chemoreceptors. Here we explore their ultrastructure and their acid-sensitivity using two novel BAC eGFP-Phox2b transgenic mice (B/G, GENSAT JX99) in which respectively 36% and 100% of the cc RTN neurons express the transgene in complete or partial anatomical isolation from other populations of eGFP neurons.All but one eGFP-labeled RTN neuron recorded in these mice were acid-activated in slices. These cells contained VGLUT2 mRNA and 50% contained preprogalanin mRNA (determined by singlecell PCR in the B/G mouse). Two neuronal subgroups were revealed which differed in discharge rate at pH 7.3 (Type I ~2; Type II ~4 Hz) and the degree of alkalization that silenced the cells (Type I: 7.4 -7.6; Type II: 7.8 -8.0). Medial to the RTN, C1 neurons recorded in a tyrosinehydroxylase-GFP mouse were pH-insensitive between pH 6.9 -7.5.Ultrastructural studies demonstrated that eGFP-labeled RTN neurons were surrounded by numerous capillaries and were often in direct contact with glial cells, pericytes and the basement membrane of capillaries. Terminals contacting large proximal eGFP-dendrites formed mainly symmetric, likely inhibitory, synapses. Terminals on more distal eGFP-dendrites formed preferentially asymmetric, presumably excitatory, synapses.In sum, C1 cells are pH-insensitive whereas cc RTN neurons are uniformly acid-sensitive. The RTN neurons receive inhibitory and excitatory synaptic inputs and may have unfettered biochemical interactions with glial cells and the local microvasculature.

Fast-spiking (FS) cells are a prominent subtype of neocortical γ-aminobutyric acidergic interneurons that mediate feed-forward inhibition and the temporal sculpting of information transfer in neural circuits, maintain excitation/inhibition balance, and contribute to network oscillations. FS cell dysfunction may be involved in the pathogenesis of disorders such as epilepsy, autism, and schizophrenia. Mature FS cells exhibit coordinated molecular and cellular specializations that facilitate rapid responsiveness, including brief spikes and sustained high-frequency discharge. We show that these features appear during the second and third postnatal weeks driven by upregulation of K(+) channel subunits of the Kv3 subfamily. The low membrane resistance and fast time constant characteristic of FS cells also appears during this time, driven by expression of a K(+) leak current mediated by K(ir)2 subfamily inward rectifier K(+) channels and TASK subfamily 2-pore K(+) channels. Blockade of this leak produces dramatic depolarization of FS cells suggesting the possibility for potent neuromodulation. Finally, the frequency of FS cell membrane potential oscillations increases during development and is markedly slower in TASK-1/3 knockout mice, suggesting that TASK channels regulate FS cell rhythmogenesis. Our findings imply that some of the effects of acidosis and/or anesthetics on brain function may be due to blockade of TASK channels in FS cells.

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 CO 2 /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 , and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO 2 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.

General anesthetics cause sedation, hypnosis, and immobilization via CNS mechanisms that remain incompletely understood; contributions of particular anesthetic targets in specific neural pathways remain largely unexplored. Among potential molecular targets for mediating anesthetic actions, members of the TASK subgroup [TASK-1 (K2P3.1) and TASK-3 (K2P9.1)] of background K ϩ channels are appealing candidates since they are expressed in CNS sites relevant to anesthetic actions and activated by clinically relevant concentrations of inhaled anesthetics. Here, we used global and conditional TASK channel single and double subunit knock-out mice to demonstrate definitively that TASK channels account for motoneuronal, anesthetic-activated K ϩ currents and to test their contributions to sedative, hypnotic, and immobilizing anesthetic actions. In motoneurons from all knock-out mice lines, TASK-like currents were reduced and cells were less sensitive to hyperpolarizing effects of halothane and isoflurane. In an immobilization assay, higher concentrations of both halothane and isoflurane were required to render TASK knock-out animals unresponsive to a tail pinch; in assays of sedation (loss of movement) and hypnosis (loss-of-righting reflex), TASK knock-out mice showed a modest decrease in sensitivity, and only for halothane. In conditional knock-out mice, with TASK channel deletion restricted to cholinergic neurons, immobilizing actions of the inhaled anesthetics and sedative effects of halothane were reduced to the same extent as in global knock-out lines. These data indicate that TASK channels in cholinergic neurons are molecular substrates for select actions of inhaled anesthetics; for immobilization, which is spinally mediated, these data implicate motoneurons as the likely neuronal substrates.