Edmund M. Talley

Distribution of α2C‐adrenergic receptor‐like immunoreactivity in the rat central nervous system

The distribution of α2C‐adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70‐amino‐acid polypeptide portion of the third intracellular loop of the α2C‐AR fused to glutathione‐S‐transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]‐photoaffinity‐labeled α2‐AR and by immunohistochemical labeling of COS cells expressing the individual rat α2‐AR subtypes. In both cases the antibody recognized only the α2C‐AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, α2C‐AR‐like immunoreactivity (α2C‐AR‐LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of α2C‐LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

TASK-1 is a highly modulated pH-sensitive 'leak' K(+) channel expressed in brainstem respiratory neurons.

Central respiratory chemoreceptors adjust respiratory drive in a homeostatic response to alterations in brain pH and/or P(CO(2)). Multiple brainstem sites are proposed as neural substrates for central chemoreception, but molecular substrates that underlie chemosensitivity in respiratory neurons have not been identified. In rat brainstem neurons expressing transcripts for TASK-1, a two-pore domain K(+) channel, we characterized K(+) currents with kinetic and voltage-dependent properties identical to cloned rat TASK-1 currents. Native currents were sensitive to acid and alkaline shifts in the same physiological pH range as TASK-1 (pK approximately 7.4), and native and cloned pH-sensitive currents were modulated similarly by neurotransmitters and inhalational anesthetics. This pH-sensitive TASK-1 channel is an attractive candidate to mediate chemoreception because it is functionally expressed in respiratory-related neurons, including airway motoneurons and putative chemoreceptor neurons of locus coeruleus (LC). Inhibition of TASK-1 channels by extracellular acidosis can depolarize and increase excitability in those cells, thereby contributing to chemoreceptor function in LC neurons and directly enhancing respiratory motoneuronal output.

Motoneurons express heteromeric TWIK-related acid-sensitive K+ (TASK) channels containing TASK-1 (KCNK3) and TASK-3 (KCNK9) subunits

Background potassium currents carried by the KCNK family of two-pore-domain K+ channels are important determinants of resting membrane potential and cellular excitability. TWIK-related acid-sensitive K+ 1 (TASK-1, KCNK3) and TASK-3 (KCNK9) are pH-sensitive subunits of the KCNK family that are closely related and coexpressed in many brain regions. There is accumulating evidence that these two subunits can form heterodimeric channels, but this evidence remains controversial. In addition, a substantial contribution of heterodimeric TASK channels to native currents has not been unequivocally established. In a heterologous expression system, we verified formation of heterodimeric TASK channels and characterized their properties; TASK-1 and TASK-3 were coimmunoprecipitated from membranes of mammalian cells transfected with the channel subunits, and a dominant negative TASK-1(Y191F) construct strongly diminished TASK-3 currents. Tandem-linked heterodimeric TASK channel constructs displayed a pH sensitivity (pK ∼7.3) in the physiological range closer to that of TASK-1 (pK ∼7.5) than TASK-3 (pK ∼6.8). On the other hand, heteromeric TASK channels were like TASK-3 insofar as they were activated by high concentrations of isoflurane (0.8 mm), whereas TASK-1 channels were inhibited. The pH and isoflurane sensitivities of native TASK-like currents in hypoglossal motoneurons, which strongly express TASK-1 and TASK-3 mRNA, were best represented by TASK heterodimeric channels. Moreover, after blocking homomeric TASK-3 channels with ruthenium red, we found a major component of motoneuronal isoflurane-sensitive TASK-like current that could be attributed to heteromeric TASK channels. Together, these data indicate that TASK-1 and TASK-3 subunits coassociate in functional channels, and heteromeric TASK channels provide a substantial component of background K+ current in motoneurons with distinct modulatory properties.

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.

TASK channel deletion in mice causes primary hyperaldosteronism

When inappropriate for salt status, the mineralocorticoid aldosterone induces cardiac and renal injury. Autonomous overproduction of aldosterone from the adrenal zona glomerulosa (ZG) is also the most frequent cause of secondary hypertension. Yet, the etiology of nontumorigenic primary hyperaldosteronism caused by bilateral idiopathic hyperaldosteronism remains unknown. Here, we show that genetic deletion of TWIK-related acid-sensitive K (TASK)-1 and TASK-3 channels removes an important background K current that results in a marked depolarization of ZG cell membrane potential. Although TASK channel deletion mice (TASK−/−) adjust urinary Na excretion and aldosterone production to match Na intake, they produce more aldosterone than control mice across the range of Na intake. Overproduction of aldosterone is not the result of enhanced activity of the renin–angiotensin system because circulating renin concentrations remain either unchanged or lower than those of control mice at each level of Na intake. In addition, TASK−/− mice fail to suppress aldosterone production in response to dietary Na loading. Autonomous aldosterone production is also demonstrated by the failure of an angiotensin type 1 receptor blocker, candesartan, to normalize aldosterone production to control levels in TASK−/− mice. Thus, TASK−/− channel knockout mice exhibit the hallmarks of primary hyperaldosteronism. Our studies establish an animal model of nontumorigenic primary hyperaldosteronism and identify TASK channels as a possible therapeutic target for primary hyperaldosteronism.

Low-voltage-activated calcium channel subunit expression in a genetic model of absence epilepsy in the rat

The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbred strain of rats that display many of the characteristics of human absence epilepsy. In these rats, reciprocal thalamocortical projections play a critical role in the generation of spike-and-wave discharges that characterize absence seizures. When compared to those of the non-epileptic control strain, juvenile animals of the GAERS strain reportedly possess higher-amplitude T-type calcium currents in neurons of the thalamic reticular nucleus (nRt). We hypothesized that differences in calcium currents seen between GAERS and controls result from differences in expression of genes for low-voltage-activated calcium channels. Quantitative in situ hybridization was used to compare expression of alpha1G, alpha1H, alpha1I, and alpha1E calcium channel subunit mRNAs from adult and juvenile animals of the two strains. We found higher levels of alpha1H mRNA expression in nRt neurons of juvenile animals (34.9+/-2. 3 vs. 28.4+/-1.8 grains/10(3) pixels, p<0.05), perhaps accounting in part for earlier reports of elevated T-type current amplitude in those cells. In adult GAERS animals, we found elevated levels of alpha1G mRNA in neurons of the ventral posterior thalamic relay nuclei (64.8+/-3.5 vs. 53.5+/-1.7 grains/10(3) pixels, p<0.05), as well as higher levels of alpha1H mRNA in nRt neurons (32.6+/-0.8 vs. 28.2+/-1.6 grains/10(3) pixels, p<0.05). These results suggest that the epileptic phenotype apparent in adult GAERS may result in part from these significant, albeit small ( approximately 15-25%), elevations in T-type calcium channel mRNA levels.

Two-Pore-Domain (Kcnk) Potassium Channels: Dynamic Roles in Neuronal Function

Leak K+ currents contribute to the resting membrane potential and are important for modulation of neuronal excitability. Within the past few years, an entire family of genes has been described whose members form leak K+ channels, insofar as they generate potassium-selective currents with little voltage- and time-dependence. They are often referred to as “two-pore-domain” channels because of their predicted topology, which includes two pore-forming regions in each subunit. These channels are modulated by a host of different endogenous and clinical compounds such as neurotransmitters and anesthetics, and by physicochemical factors such as temperature, pH, oxygen tension, and osmolarity. They also are subject to long-term regulation by changes in gene expression. In this review, the authors describe multiple roles that modulation of leak K+ channels play in CNS function and discuss evidence that members of the two-pore-domain family are molecular substrates for these processes.

Neuromodulation of hypoglossal motoneurons: cellular and developmental mechanisms.

Hypoglossal motoneurons (HMs) in the caudal brainstem have a respiratory-related activity pattern and contribute to control of upper airway resistance. In this review, we focus primarily on signalling mechanisms utilized by neurotransmitters to enhance HM excitability. In particular, we consider: (1) the membrane depolarization induced by a number of different putative transmitters [thyrotropin-releasing hormone (TRH), serotonin (5-HT), norepinephrine (NE)]; and (2) the inhibition of a calcium-dependent spike after hyperpolarization (AHP) by 5-HT and its effect on firing behavior. Potential functional consequences on HM behavior of these different neurotransmitter effects is discussed. In addition, we describe postnatal changes in transmitter effects and suggest potential cellular mechanisms to explain those developmental changes. Most of the data discussed are derived from in vitro electrophysiological recordings performed in preparations from neonatal and adult rats.

HCN Subunit-Specific and cAMP-Modulated Effects of Anesthetics on Neuronal Pacemaker Currents

General anesthetics have been a mainstay of surgical practice for more than 150 years, but the mechanisms by which they mediate their important clinical actions remain unclear. Ion channels represent important anesthetic targets, and, although GABAA receptors have emerged as major contributors to sedative, immobilizing, and hypnotic effects of intravenous anesthetics, a role for those receptors is less certain in the case of inhalational anesthetics. The neuronal hyperpolarization-activated pacemaker current (Ih) is essential for oscillatory and integrative properties in numerous cell types. Here, we show that clinically relevant concentrations of inhalational anesthetics modulate neuronal Ih and the corresponding HCN channels in a subunit-specific and cAMP-dependent manner. Anesthetic inhibition of Ih involves a hyperpolarizing shift in voltage dependence of activation and a decrease in maximal current amplitude; these effects can be ascribed to HCN1 and HCN2 subunits, respectively, and both actions are recapitulated in heteromeric HCN1-HCN2 channels. Mutagenesis and simulations suggest that apparently distinct actions of anesthetics on V1/2 and amplitude represent different manifestations of a single underlying mechanism (i.e., stabilization of channel closed state), with the predominant action determined by basal inhibition imposed by individual subunit C-terminal domains and relieved by cAMP. These data reveal a molecular basis for multiple actions of anesthetics on neuronal HCN channels, highlight the importance of proximal C terminus in modulation of HCN channel gating by diverse agents, and advance neuronal pacemaker channels as potentially relevant targets for clinical actions of inhaled anesthetics.

Motoneuronal TASK Channels Contribute to Immobilizing Effects of Inhalational General Anesthetics

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+ 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+ 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.