Previous studies have implicated extracellular carbonic anhydrases (CAs) in buffering the alkaline pH shifts that accompany neuronal activity in the rat and mouse hippocampus. CAs IV and XIV both have been proposed to mediate this extracellular buffering. To examine the relative importance of these two isozymes in this and other physiological functions attributed to extracellular CAs, we produced CA IV and CA XIV knockout (KO) mice by targeted mutagenesis and the doubly deficient CA IV͞XIV KO mice by intercrossing the individual null mice. Although CA IV and CA XIV null mice both are viable, the CA IV nulls are produced in smaller numbers than predicted, indicating either fetal or postnatal losses, which preferentially affect females. CA IV͞XIV double KO mice are also produced in fewer numbers than predicted and are smaller than WT mice, and many females die prematurely before and after weaning. Electrophysiological studies on hippocampal slices on these KO mice showed that either CA can mediate buffering after synaptic transmission in hippocampal slices in the absence of the other, but that eliminating both is nearly as effective as the CA inhibitor, benzolamide, in blocking the buffering seen in the WT mice. Thus, both CA IV and CA XIV contribute to extracellular buffering in the central nervous system, although CA IV appears to be more important in the hippocampus. These individual and double KO mice should be valuable tools in clarifying the relative contributions of each CA to other physiological functions where extracellular CAs have been implicated. interstitial pH ͉ mouse hippocampus ͉ targeted mutagenesis

Ion-selective microelectrodes (ISMs) have been used extensively in neurophysiological studies. ISMs selective for H(+) and Ca(2+) are notable for their sensitivity and selectivity, but suffer from a slow response time, and susceptibility to noise because of the high electrical resistance of the respective ion exchange cocktails. These drawbacks can be overcome by using a "coaxial" or "concentric" inner micropipette to shunt the bulk of the ion exchanger resistance. This approach was used decades ago to record extracellular [Ca(2+)] transients in cat cortex, but has not been subsequently used. Here, we describe a method for the rapid fabrication of concentric pH- and Ca(2+)-selective microelectrodes useful for extracellular studies in brain slices or other work in vitro. Construction was simplified compared with previous implementations, by using commercially available, thin-walled borosilicate glass, drawing an outer barrel with a rapid taper (similar to a patch pipette), and by use of a quick and reliable silanization procedure. Using a piezoelectric stepper to effect a rapid solution change, the response time constants of the concentric pH and Ca(2+)-electrodes were 14.9 +/- 1.3 and 5.3 +/- 0.90 ms, respectively. Use of these concentric ISMs is demonstrated in rat hippocampal slices. Activity-dependent, extracellular pH, and [Ca(2+)] transients are shown to arise two- to threefold faster, and attain amplitudes two- to fourfold greater, when recorded by concentric versus conventional ISMs. The advantage of concentric ISMs for studies of ion transport and ion diffusion is discussed.

Cultured astrocytes do not succumb to hypoxia/zero glucose for up to 24 h, yet astrocyte death following injury can occur within 1 h. It was previously demonstrated that astrocyte loss can occur quickly when the gaseous and interstitial ionic changes of transient brain ischemia are simulated: After a 20-40-min exposure to hypoxic, acidic, ion-shifted Ringer (HAIR), most cells died within 30 min after return to normal saline (i.e., "reperfusion"). Astrocyte death required external Ca2+ and was blocked by KB-R7943, an inhibitor of reversed Na+-Ca2+ exchange, suggesting that injury was triggered by a rise in [Ca2+]i. In the present study, we confirmed the elevation of [Ca2+]i during reperfusion and studied the role of Na+-Ca2+ and Na+-H+ exchange in this process. Upon reperfusion, elevation of [Ca2+]i was detectable by Fura-2 and was blocked by KB-R7943. The low-affinity Ca2+ indicator Fura-FF indicated a mean [Ca2+]i rise to 4.8+/-0.4 microM. Loading astrocytes with Fura-2 provided significant protection from injury, presumably due to the high affinity of the dye for Ca2+. Injury was prevented by the Na+-H+ exchange inhibitors ethyl isopropyl amiloride or HOE-694, and the rise of [Ca2+]i at the onset of reperfusion was blocked by HOE-694. Acidic reperfusion media was also protective. These data are consistent with Na+ loading via Na+-H+ exchange, fostering reversal of Na+-Ca2+ exchange and cytotoxic elevation of [Ca2+]i. The results indicate that mechanisms involved in pH regulation may play a role in the fate of astrocytes following acute CNS injuries.

Carbonic anhydrase (CA) activity in the brain extracellular space is attributable mainly to isoforms CA4 and CA14. In brain, these enzymes have been studied mostly in the context of buffering activity-dependent extracellular pH transients. Yet evidence from others has suggested that CA4 acts in a complex with anion exchangers (AEs) to facilitate Cl Ϫ -HCO 3 Ϫ exchange in cotransfected cells. To investigate whether CA4 or CA14 plays such a role in hippocampal neurons, we studied NH 4 ϩ -induced alkalinization of the cytosol, which is mitigated by Cl Ϫ entry and HCO 3 Ϫ exit. The NH 4 ϩ -induced alkalinization was enhanced when the extracellular CAs were inhibited by the poorly permeant CA blocker, benzolamide, or by inhibitory antibodies specific for either CA4 or CA14. The NH 4 ϩ -induced alkalinization was also increased with inhibition of anion exchange by 4,4*-diisothiocyanostilbene-2,2*-disulfonic acid, or by eliminating Cl Ϫ from the medium. No effect of benzolamide was seen under these conditions, in which no Cl Ϫ -HCO 3 Ϫ exchange was possible. Quantitative PCR on RNA from the neuronal cultures indicated that AE3 was the predominant AE isoform. Single-cell PCR also showed that Slc4a3 (AE3) transcripts were abundant in isolated neurons. In hippocampal neurons dissociated from AE3-null mice, the NH 4 ϩ -induced alkalinization was much larger than that seen in neurons from wild-type mice, suggesting little or no Cl Ϫ -HCO 3 Ϫ exchange in the absence of AE3. Benzolamide had no effect on the NH 4 ϩ -induced alkalinization in the AE3 knock-out neurons. Our results indicate that CA4 and CA14 both play important roles in the regulation of intracellular pH in hippocampal neurons, by facilitating AE3-mediated Cl Ϫ -HCO 3 Ϫ exchange.