Central chemoreception is the mechanism by which CO(2)/pH sensors regulate breathing in response to tissue pH changes. There is compelling evidence that pH-sensitive neurons in the retrotrapezoid nucleus (RTN) are important chemoreceptors. Evidence also indicates that CO(2)/H(+)-evoked adenosine 5'-triphosphate (ATP) release in the RTN, from pH-sensitive astrocytes, contributes to chemoreception. However, mechanism(s) by which RTN astrocytes sense pH is unknown and their contribution to chemoreception remains controversial. Here, we use the brain slice preparation and a combination of patch-clamp electrophysiology and immunohistochemistry to confirm that RTN astrocytes are pH sensitive and to determine mechanisms by which they sense pH. We show that pH-sensitive RTN glia are immunoreactive for aldehyde dehydrogenase 1L1, a marker of astrocytes. In HEPES buffer the pH-sensitive current expressed by RTN astrocytes reversed near E(K(+)) (the equilibrium potential for K(+)) and was inhibited by Ba(2+) and desipramine (blocker of Kir4.1-containing channels), characteristics most consistent with heteromeric Kir4.1-Kir5.1 channels. In bicarbonate buffer, the sodium/bicarbonate cotransporter also contributed to the CO(2)/H(+)-sensitive current in RTN astrocytes. To test the hypothesis that RTN astrocytes contribute to chemoreception by a purinergic mechanism, we used fluorocitrate to selectively depolarize astrocytes while measuring neuronal activity. We found that fluorocitrate increased baseline activity and pH sensitivity of RTN neurons by a P2-receptor-dependent mechanism, suggesting that astrocytes may release ATP to activate RTN chemoreceptors. We also found in bicarbonate but not HEPES buffer that P2-receptor antagonists decreased CO(2) sensitivity of RTN neurons. We conclude that RTN astrocytes sense CO(2)/H(+) in part by inhibition of a Kir4.1-Kir5.1-like current and may provide an excitatory purinergic drive to pH-sensitive neurons.

To understand the role of mitochondrial uncoupling protein (UCP) in regulating insulin signaling and glucose homeostasis, we created transgenicDrosophila lines with targeted UCP expression in insulin producing cells (IPCs). Increased UCP activity in IPCs results in decreased steady state Ca2+ levels in IPCs as well as decreased PI3K activity and increased FoxO nuclear localization in periphery. This reduced systemic insulin signaling is accompanied by a mild hyperglycemia and extended life span. To test the hypothesis that ATP-sensitive potassium (KATP) channels may link changes in metabolic activity (e.g., glucose mediated ATP production or UCP-mediated ATP reduction) with insulin secretion, we characterized the effects of glucose and a specific KATP channel blocker, glibenclamide on membrane potential in adult IPCs. Exposure to glucose depolarizes membrane potential of IPCs and this effect is mimicked with glibenclamide, suggesting that KATP channels contribute to the mechanism whereby IPCs sense changes in circulating sugar. Further, as demonstrated in mammalian β-pancreatic cells, high glucose initiates a robust Ca2+ influx in adult IPCs. The presence of functional KATP channels in adult IPCs is further substantiated by in situ hybridization detecting the transcript for the sulfonylurea receptor (Sur) subunit of the KATP channel in those cells. Quantitative expression analysis demon-strates a reduction in transcripts for both Sur and the inward rectifying potassium channel (Kir) subunits when IPCs are partially ablated. In summary, we have demonstrated a role for UCP in adult Drosophila IPCs in influencing systemic insulin signaling and longevity by a mechanism that may involve KATP channels.

We sought to understand mechanisms underlying glucose sensing in Drosophila melanogaster. We found that adult insulin producing cells (IPCs) respond to glucose and glibenclamide with a burst-like pattern of activity. Under control 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 Ca 2+ imaging confirms that IPCs respond to glucose and glibenclamide with increased intracellular Ca 2+ . These results provide the first detailed characterization of electrical properties of adult Drosophila IPCs and suggest that these cells sense glucose by a mechanism similar to mammalian pancreatic β cells.

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 O 2 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 (P O 2 = 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 O 2 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.

Using RNA interference, we have selectively perturbed neurotransmitter-related features of the larval swimming behavior of Oikopleura dioica, a tunicate with a central nervous system comprising about 130 neurons. We injected dsRNA into fertilized eggs to knockdown the expression of the genes, respectively, encoding ChAT (choline acetyltransferase) and GAD (glutamic acid decarboxylase), enzymes critical for the biosynthesis of acetylcholine and GABA. These two neurotransmitters have conserved roles during evolution, particularly within chordate motor systems, where they mediate respectively neuromuscular and central inhibitory signals. In Oikopleura, interference with ChAT expression prevented the normal bidirectional, propagating tail movement characteristic of swimming, permitting only repeated unilateral tail bends. Proper swimming was never observed, and the resting period between episodes of activity was lengthened. This phenotype is most likely caused by the reduction of transcription observed for both the targeted ChAT gene and the VAChT gene (Vesicular Acetylcholine Transporter), both genes being transcribed from the same operon. Interference with GAD expression led to an uncoordinated version of swimming with a spiral movement trajectory, but with episodes similar in duration and cycle frequency to those of normal swimming. Our results suggest locomotor functions for ChAT and GABA that are more subtle than previously proposed for tunicates and opens the way for a genetic dissection of Oikopleura neuronal circuits, which are likely to be among the most simplified in the chordate phylum.