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Keywords:

  • patch clamp;
  • aspartate;
  • glutamate uptake;
  • ionotropic glutamate receptors;
  • Purkinje cells;
  • cerebellar interneurons;
  • slice cultures

Abstract

The effects of glutamate, aspartate and N-methyl-d-aspartate (NMDA) on Purkinje cells and interneurons were investigated in cerebellar slice cultures using the whole-cell configuration of the patch-clamp technique. l-Glutamate and l-aspartate induced inward currents in Purkinje cells voltage-clamped at -60 mV. In standard external solution, the amplitude of the responses induced by these two amino-acids was a linear function of the membrane potential. l-Aspartate-induced currents were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective antagonist of non-NMDA receptors. NMDA, a selective agonist of NMDA receptors, had no effect of its own on the excitability of Purkinje cells, but was effective in blocking the responses induced by aspartate in Purkinje cells in a voltage-independent manner. In contrast, d-(–)-2-amino-5-phosphonovaleric acid (d-APV), a selective antagonist of NMDA receptors, had no effect on aspartate-induced responses. d-Aspartate also induced responses in Purkinje cells, and the amplitude of these responses was a linear function of the membrane potential. Currents induced by l- and d-aspartate were inhibited by dihydrokainate, a glutamate uptake blocker. In sodium-free external solution, glutamate still induced outward currents in Purkinje cells, whereas l- and d-aspartate no longer evoked any current. When sodium was replaced by lithium in the external medium, no change in the holding current could be detected in Purkinje cells maintained at -60 mV; moreover, in this bathing medium l-aspartate no longer evoked any current whereas glutamate-induced responses were still present. In contrast, interneurons were sensitive to both NMDA and aspartate applications, and these responses were antagonized by d-APV. In addition, aspartate still induced an outward current in sodium-free external solution. This study presents rather direct evidence in favour of l-aspartate as being a very selective NMDA receptor agonist in the cerebellum. l-Aspartate-induced currents in Purkinje cells are not due to activation of mixed NMDA/non-NMDA receptors, but are probably due to the release of l-glutamate induced by aspartate through glutamate uptake.