1. Alkaline extracellular pH transients evoked by afferent stimulation, and local pressure ejection of glutamate and gamma-aminobutyric acid (GABA), were studied in the CA1 region of rat hippocampal slices. Amino acid-evoked responses were obtained by use of a dual micromanipulator, with the tip of a double-barreled pH-sensitive microelectrode positioned 50 microns from a pressure ejection pipette. 2. At 31 degrees C, in Ringer solutions buffered with 26 mM HCO3- and 5% CO2, mean extracellular pH in submerged 300-microns slices was 7.15 +/- 0.12 (n = 27 slices), at a tissue depth of approximately 150 microns. In Ringer buffered with 35 mM HCO3- and 5% CO2, extracellular pH was 7.29 +/- 0.10 (n = 19 slices). 3. Repetitive stimulation of the Schaffer collaterals caused an extracellular alkaline shift in stratum oriens, pyramidale, and radiatum, averaging 0.05 +/- 0.03 pH units among all regions (n = 138), with a maximum response of 0.16 pH units. Alkaline transients of similar appearance were obtained by local ejection of glutamate (0.01-0.12 pH units, n = 110) and GABA (0.01-0.18 pH units, n = 137). Control ejection of these amino acids into dilute agar caused only small acid shifts. 4. Superfusion of 100 microM picrotoxin abolished the GABA-evoked alkaline shift but failed to inhibit the Schaffer collateral- and glutamate-evoked alkalinizations. 5. Superfusion of 10(-5)-10(-3) M acetazolamide acidified the baseline by 0.05-0.10 pH units and amplified the Schaffer collateral- and glutamate-evoked alkaline shifts.(ABSTRACT TRUNCATED AT 250 WORDS)
December 19, 1991) ABSTRACT Excitatory synaptic transmission has been associated with a rapid alkalinition of the brain extracellular space. These pH shifts are markedly increased by acetazolamide, an inhibitor of carbonic anhydrase. Although this effect can be readily explained by inhibition of extracellular carbonic anhydrase, this enzyme has been considered strictly intracellular in the central nervous system. To determine whether these alkaline shifts are regulated by extracellular carbonic anhydrase, we studied the effects of a membrane impermeant, dextran-bound inhibitor of this enzyme. Extraceflular alkaline transients, measured with pH-sensitive microelectrodes, were generated in the CAl region of rat hippocampal slices by repetitive electrical stimulation of Schaeffer collateral fibers or by local ejection of glutamate. More direct lkalinizations were elicited by focal ejection of NaOH in the vicinity of a pH microelectrode. These pH transients were reversibly enhanced by addition of the dextran-bound inhibitor. We conclude that there is significant carbonic anhydrase activity in the extracellular space of the brain. We postulate that this enzyme functions in the regulation and modulation of extracellular pH transients associated with neuronal activity.The enzyme carbonic anhydrase (CA) catalyzes the reversible hydration of CO2 and plays an important role in the function of erythrocytes and secretory epithelia (1). In the central nervous system, CA is abundant (2) and has been localized predominantly within glia (3-5) and choroid plexus (6). However, apart from its role in the secretion of cerebral spinal fluid (7,8), the functions of CA in the brain have remained obscure.While most forms of CA are intracellular, an extracellular isoform exists in several tissues, including kidney, lung, and muscle (9). Although there is no morphological evidence for extracellular CA in the brain, inhibitors of CA have been shown to have a large effect on the behavior of extracellular pH. Sulfonamide derivatives, such as acetazolamide, greatly increased the alkaline shifts associated with synaptic transmission (10-13). These effects could not be explained by enhancement of electrophysiological responses, since corresponding field potentials were unaffected. However, it remained unclear whether the action of acetazolamide was principally intracellular or extracellular, as this agent can readily penetrate cell membranes (1).It was subsequently noted that the effects of acetazolamide could be simply explained by a decrease in the extracellular buffering capacity (14). Experimental manipulation of the extracellular buffering power supported this view: synaptically evoked alkaline shifts were enhanced when bicarbonatebuffered saline was replaced by poorly buffered solutions containing Hepes (15,16). Although a role of metabolically generated CO2 could not be ruled out, the amplification and persistence of these alkalinizations in Hepes-buffered media suggested that shifts in CO2 and bicarbonate were not immediately respo...
1. The pharmacology of synaptically evoked extracellular alkaline shifts was studied in the CA1 area of rat hippocampal slices. 2. Stimulus-evoked alkalinizations were unaffected by 2-amino-5-phosphonovalerate (APV) (20 microM). 3. 6-Cyano-7-nitro-nitroquinoxaline-2,3-dione (CNQX) (10 microM) inhibited the alkalinizations. In the continued presence of CNQX, an APV-sensitive, picrotoxin-insensitive, alkaline shift was elicited in low Mg2+ media. 4. Antidromic stimulation produced small alkaline shifts in comparison with orthodromic activation. 5. Our results demonstrate that in the hippocampal CA1 region, synaptically evoked alkalinizations can arise through both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. These responses cannot be explained by cell firing per se.
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