SUMMARY Astrocytes perform crucial supportive functions, including neurotransmitter clearance, ion buffering and metabolite delivery. They can also influence blood flow and neuronal activity by releasing gliotransmitters in response to intracellular Ca2+ transients. However, little is known about how astrocytes are engaged during different behaviors in vivo. Here we demonstrate that norepinephrine primes astrocytes to detect changes in cortical network activity. We show in mice that locomotion triggers simultaneous activation of astrocyte networks in multiple brain regions. This global stimulation of astrocytes was inhibited by alpha-adrenoceptor antagonists and abolished by depletion of norepinephrine from the brain. Although astrocytes in visual cortex of awake mice were rarely engaged when neurons were activated by light stimulation alone, pairing norepinephrine release with light stimulation markedly enhanced astrocyte Ca2+ signaling. Our findings indicate that norepinephrine shifts the gain of astrocyte networks according to behavioral state, enabling astrocytes to respond to local changes in neuronal activity.

belong to the Deg/ENaC super family of ion channels (1). Members of this super gene family form Na ϩ -selective ion channels (P Na /P K , 8 -100) that can be blocked by amiloride (IC 50 , 0.2-20 M). All family members show some common hallmarks including two hydrophobic domains, short intracellular N and C termini, and a large extracellular loop containing conserved cysteines. Channels of this gene family probably form tetramers (2, 3).To date, six different members of the ASIC subfamily have been cloned (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4), which are encoded by four genes. ASIC1a and ASIC1b are alternative splice products of the ASIC1 gene (4, 5). Splicing exchanges approximately the first third of the protein, including the first transmembrane domain and the proximal part of the large ectodomain. In contrast, the C-terminal twothirds are identical. We will use the term ASIC1 when we do not refer to a specific splice variant. All ASICs with the exception of ASIC4 (6) are expressed in sensory neurons of the dorsal root ganglion. Proposed functions in sensory neurons include peripheral pain perception (1,7,8) and mechanotransduction (9, 10). Although some evidence suggests that some of the native H ϩ -gated currents in sensory neurons are mediated by heteromeric ASICs (11, 12), part of these currents are probably mediated by homomeric ASIC1 and ASIC3 (8, 11).ASIC1a is expressed in sensory neurons and throughout the brain (13), whereas ASIC1b is specifically expressed in sensory neurons (5). Both subunits are Na ϩ -selective (P Na /P K Ϸ 10 -15), and only ASIC1a has a low Ca 2ϩ permeability (P Na /P Ca Ϸ 15) (4). ASIC1a and ASIC1b form rapidly activating and completely desensitizing ion channels ( act , ϳ10 msec; desens , ϳ1 s) (4). The expression of ASIC1 in small diameter, capsaicinsensitive sensory neurons (5, 11, 14, 15) has led to the proposal that ASIC1a mediates excitation during tissue acidosis, which accompanies inflammation and ischemia. However, complete desensitization of ASIC1 makes it difficult to imagine how this channel can sense H ϩ signals during inflammation and ischemia when the pH persistently falls.Here we show that both ASIC1a and ASIC1b undergo steady-state inactivation. The steady-state inactivation curve for ASIC1b is shifted by 0.25 pH units to more acidic values as compared with ASIC1a, showing that ASIC1b can operate at a more acidic resting pH. pH activation is shifted by 0.7 pH units. Differences in the sensitivity of activation and inactivation by protons are intimately linked, as both are controlled by only two amino acids in the ectodomain. These two amino acids are exchanged by alternative splicing. Moreover, we show that Ca 2ϩ , Mg 2ϩ , and spermine, when applied during the steadystate, shift the steady-state inactivation curves of both ASIC1a and ASIC1b. This leads to a potentiation of the current. Modulation by di-and polyvalent cations may be a means to adapt ASIC1 activity to changes in the extracellular concentration of these ions. Our results show that ASIC1b...

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 ∼ 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.

Acid-sensing ion channels (ASICs) 3 are cation channels that are gated by extracellular H ϩ (1, 2). A rise in the H ϩ concentration opens ASICs, and the continued presence of H ϩ desensitizes them. Desensitization has time constants from less than 100 ms in fish ASICs (3, 4) to several seconds in mammalian ASIC2a (5).ASIC subunits have a simple topology: short cytoplasmic tails, two transmembrane domains (TM1 and TM2), and a large (Ͼ350 amino acids) extracellular region (6). The recently determined crystal structure of a chicken ASIC1 deletion mutant (7) reveals a trimeric arrangement, which is characterized by a high degree of asymmetry in the hexahelical transmembrane region.This structure was obtained at acidic pH and most likely represents a desensitized-like conformation and therefore does not allow direct identification of the H ϩ sensor. The extracellular region is composed of five subdomains, which are connected to the membrane-spanning region via an apparently flexible wrist. As predicted (8), this region is stabilized by cysteine bridges formed by 14 conserved cysteines. The structure also confirms electrophysiological experiments suggesting that the second transmembrane domain (9 -11) and a pre-TM1 domain (12, 13) contribute to the ion pore, although residues from TM1 also line the pore.In mammals, four asic genes code for at least six ASIC subunits (ASIC1a, 1b, 2a, 2b, 3, and 4) (2, 14), which assemble into homo-or heterooligomeric channels. Among them, homomeric ASIC1a and ASIC3 are the most H ϩ

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