This transport occurs via the NaHCO3 cotransporter (NBC, SLC4a4) (Bevensee et al., 2000; Boyarsky et al., 1993; Pappas and

Ransom, 1994; Schmitt et al., 2000), a protein that is highly expressed in astrocytes (Cahoy et al., 2008). In addition, astrocytes also express other HCO3−-relevant enzymes such as carbonic anhydrase (Cahoy et al., 2008). We reasoned that HCO3−-sensitive sAC, if present in astrocytes, could provide an important link for coupling neuronal activity to the metabolic protection provided by the breakdown of glycogen and subsequent release of lactate from astrocytes. Here we show that in the brain, HCO3−-sensitive sAC is highly expressed in astrocytes. HCO3− activation of this enzyme, by either high [K+]ext or aglycemia, increases intracellular cAMP, which leads AZD6738 to glycogen breakdown and the delivery of lactate to neurons for use as an energy substrate. We used several approaches to determine whether HCO3−-sensitive sAC is expressed in the brain and, if so, in which cell types it resides. Immunohistochemical staining showed that GFAP-labeled astrocyte somata and major processes, including endfeet, expressed sAC (using R21, anti-sAC monoclonal antibody) (Figure 1A,

top), whereas MAP-2-labeled neuronal somata and dendrites revealed no specific sAC staining (Figure 1B). As a control for the specificity of labeling, DAPT concentration immunohistochemical staining using R21 in the presence of a sAC blocking peptide that corresponds to the epitope identified by R21 (Hallows et al., 2009) showed no sAC labeling in rat brain slices (Figure 1A, bottom). Western

blotting (with R21 antibody) results confirmed that sAC protein was expressed in both rat brain slices and cultured astrocytes (Figure 1C) and, in the presence of sAC-blocking peptide, antigen-antibody interaction was disrupted (Figure 1C). RT-PCR results confirmed that sAC mRNA was expressed in both rat brain slices and cultured astrocytes (Figure 1D). Several splice variants of sAC have been reported in different Calpain tissues (Farrell et al., 2008). Using further RT-PCR experiments with cultured astrocytes, we demonstrated that astrocytes expressed all the different reported splice variants of sAC. These include sAC, which is encoded by exons 1–5 (see Figure S1A available online), sACsomatic, which has a unique start site upstream of exons 5–13 (Farrell et al., 2008) (Figure S1B), sACfl, which is encoded by all 32 of the known exons (Buck et al., 1999; Jaiswal and Conti, 2001) (indicated by the top band in Figure S1C), and sACt, which is encoded by exons 9–13 but skips exon 12, resulting in an early stop codon (indicated by the bottom band in Figure S1C). Finally, we used immunoelectron microscopy to examine the distribution of sAC in the hippocampus region of wild-type and Sacytm1Lex/Sacytm1Lex (genetic deletion of the exon 2 through exon 4 catalytic domain, sAC-C1 knockout [KO]) mice (Esposito et al., 2004; Hess et al.