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The effect of anoxia and ischemia on the release of amino acid transmitters from cerebellar slices induced by veratridine or high [K+] was studied. Synaptic specificity was tested by examining the tetradotoxin (TTX)-sensitive and the Ca2+-dependent components of stimulated release. Evoked release of endogenous amino acids was investigated in addition to more detailed studies on the stimulated efflux of preloaded [14C]GABA and D-[3H]aspartate (a metabolically more stable anologue of acidic amino acids). [14C]GABA release evoked by either method of stimulation was unaffected by periods of up to 35 min of anoxia and declined moderately by 45 min. In contrast, induced release of D-[3H]Asp increased markedly during anoxia to a peak at about 25 min, followed by a decline when anoxia was prolonged to 45 min. Evidence was obtained that the increased evoked efflux of D'[3H]Asp from anoxic slices was not due to impaired reuptake of the released amino acid and that it was completely reversible by reoxygenation of the slices. Results of experiments examining the evoked release of endogenous amino acids in anoxia were consistent with those obtained with the exogenous amino acids. Only 4 of the 10 endogenous amino acids studied exhibited TTX-sensitive veratridine-induced release under aerobic conditions (glutamate, aspartate, GABA, and glycine). Anoxia for 25 min did not affect the stimulated efflux of these amino acids with the exception of glutamate, which showed a significant increase. Compared with anoxia, effects of ischemia on synaptic function appeared to be more severe. Veratridine-evoked release of [14C]GABA was already depressed by 10 min and that of D-[3H[Asp showed a modest elevation only a 5 min. Stimulated release of D-Asp and labelled GABA declined progressively after 5 min. These findings were compared with changes in tissue ATP concentrations and histology. The latter studies indicated that in anoxia the earliest alterations are detectable in glia and that nerve terminals were the structures by far the most resistant to anoxic damage. The results thus indicated that evoked release of amino acid transmitters in the cerebellum is compromised only by prolonged anoxia in vitro. In addition, it would appear that the stimulated release of glutamate is selectively accentuated during anoxia. This effect may have a bearing on some hypoxic behavioral changes and, perhaps, also on the well-known selective vulnerability of certain neurons during hypoxia.

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Preparations of structurally preserved cerebellar perikarya (cells) were found to express high‐affinity transport systems for glutamate but not for certain putative transmitter substances (including monoamines, glycine and taurine) and non‐transmitter amino acids. The characteristics of the high‐affinity glutamate transport system were similar to those of other preparations of brain tissue: [3H]glutamate uptake by the cells was Na+‐dependent and was inhibited competetively by other acidic amino acids. The rank order of apparent affinities of the carrier for acidic amino acids was L‐aspartate > L‐glutamate > D‐aspartate ≫ D‐glutamate (the affinity for D‐glutamate being over two orders of magnitude lower than for the other three amino acids). Comparison of high‐affinity [3H]glutamate uptake in preparations enriched in different cell types showed that although the affinities are similar (2‐4 fiM), the rate is outstandingly high in astrocytes (Vmax 18 nmol/min per mg protein). Significantly, uptake into the putatively glutamatergic granule cells was very low. These observations were supported by autoradiographic findings which showed that the predominant sites of [3H]glutamate uptake in cerebellar cultures enriched in interneurones are the astrocytes. Furthermore, the Vmax in cultures enriched in astrocytes was as high as that in separated astrocytes. Thus, it seems that the principal cell type involved in acidic amino acid uptake in the cerebellum is the astrocyte, and this must be taken into consideration when high‐affinity uptake is used as a marker for glutamatergic transmitter systems. Furthermore, the selective cellular distribution of glutamate transport sites, together with the uneven distribution of enzymes related to glutamate metabolism observed previously, indicates that a metabolic interaction takes place between the different cell types, supporting the current hypothesis on metabolic compartmentation in the brain.

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