ELECTROGENIC UPTAKE CONTRIBUTES A MAJOR COMPONENT OF THE DEPOLARIZING ACTION OF L-GLUTAMATE IN RAT HIPPOCAMPAL SLICES

1 A grease-gap technique has been used to measure d.c. potentials, in response to the application of excitatory amino acids and electrical stimulation of the Schaffer collateral-commissural pathway, in the CA1 region of rat hippocampal slices. The actions of L-glutamate (L-Glu) have been quantified and compared to those of structurally related compounds. 2 Perfusion of L-Glu (90 s applications) depolarized the tissue with a threshold of approximately 50-mu and a maximum response in excess of 10mM. L-Aspartate (L-Asp) produced a similar dose-response relationship. By comparison N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) were more potent excitants, producing dose-dependent depolarizations over the range 2-50-mu-M. 3 Application of the agonists depressed the amplitude of electrically-evoked synaptic responses; an effect that presumably reflects depolarization of neuronal tissue. However, for a given agonist-induced d.c. potential, L-Glu or L-Asp caused smaller depressions of synaptic responses than did either NMDA or AMPA. 4 The combined application of 50-mu-M D-2-amino-5-phosphonopentanoate (AP5) and 10-mu-M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) substantially depressed synaptic responses and antagonized responses to NMDA and AMPA producing mean (+/- s.e.) dose-ratios of 12.2 +/- 1.2 and 7.0 +/- 0.8, respectively. However, these compounds produced minimal antagonism of responses to L-Glu and L-Asp (dose-ratios of 1.5 +/- 0.1 and 1.5 +/- 0.2, respectively). 5 Responses to the stereoisomers of homocysteate (HCA) were compared over the range 50-mu-M to 10mM. D-HCA was approximately 3.6 times more potent than L-HCA and was antagonized to a greater extent by the combined application of 50-mu-M AP5 and 10-mu-M CNQX; the dose ratios were 8.7 +/- 0.8 and 5.1 +/- 0.9 for the D- and L- isomers, respectively. 6 The application of high doses of an excitant (e.g., 50-mM L-Glu or 5 mM D-HCA) caused an irreversible loss of sensitivity to NMDA and AMPA and abolished synaptic transmission. Responses to the other excitants were depressed by this excitotoxic lesion in the following order: D-HCA > L-HCA > L-Glu = L-Asp. In slices treated in this manner, L-Glu, L-Asp and L-HCA produced very similar dose-response curves. 7 Some slices were unresponsive to NMDA, AMPA and electrical stimulation from the onset of the experiment but had sensitivity to L-Glu, L-Asp and L-HCA similar to that of slices that had received an excitotoxic lesion. 8 Slices that were experimentally lesioned, such that they became unresponsive to synaptic stimulation, AMPA and NMDA, were depolarized by a variety of compounds when tested at 5 mM. The D- and L- enantiomers of Asp and Glu produced similar responses and were slightly more active than quisqualate and threo-hydroxy-L-aspartate (THLA). Glycine, L-serine, D-serine, L-alanine and 4-aminobutanoate (GABA) elicited responses 15-30% of the size, while L-lysine, alpha-amino-isobutanoate and L-proline produced depolarizations of less than 10% of the size of those induced by 5 mM L-Glu. NMDA and dihydrokainate were either inactive or induced small negative shifts in the d.c. potential. 9 We conclude that when hippocampal slices are perfused with L-Glu (or L-Asp) the main source of the depolarization is due to an interaction with an electrogenic carrier system.