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L-2-Chloropropionic acid (L-CPA), when administered orally to rats, produces selective necrosis to the granule cell layer of the rat cerebellum which is delayed in onset, not appearing until 36-48 h after exposure. The present study was conducted to characterise the toxic effect of L-CPA in primary cell cultures of rat cerebellar granule cells in vitro. Exposure to L-CPA produced a time and concentration dependent loss in cerebellar granule cell viability. Mean 50% effective concentration (EC50) values for L-CPA toxicity were 18.3 +/- 0.3, 7.4 +/- 0.1, and 3.5 +/- 0.1 mM for 24, 48 and 72 h exposure respectively. Exposure for 24 h followed by a return to L-CPA free medium for 24 h was more toxic than exposure for 24 h alone. Cells maintained in culture for a longer duration were more susceptible to L-CPA-induced toxicity. The toxic effects of L-CPA could be partially or fully prevented by concomitant exposure of the cells to putative neuroprotective compounds. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (3 microM), afforded partial protection against L-CPA induced toxicity, whilst other glutamate receptor antagonists including, D(-)-2-amino-5-phosphopentanoic acid (D-AP5; 300 microM), D(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (D-CPP; 300 microM), 5,7-dichlorokynurenic acid (10 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 microM) were ineffective. The antioxidant, vitamin E (10 microM), afforded significant but incomplete protection from L-CPA toxicity. However when both MK-801 (3 microM) and vitamin E (10 microM) were present during L-CPA exposure, a greater degree of protection was observed than with either compound alone, although the combination failed to provide complete protection. Cyclosporin A, an inhibitor of the mitochondrial transition pore, also provided partial protection. By contrast, the free radical trapping agent, N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) provided concentration (1-10 mM) dependent protection against the L-CPA-induced toxicity, which was complete at 10 mM. Our findings suggest that free radical production may be involved in the mechanism of L-CPA-induced toxicity.

Whole-cell voltage clamp recordings have been used to identify and characterise inward currents mediated by native kainate receptors in rat cultured cerebellar granule cells. While the selective AMPA receptor antagonist GYKI 53655 (50 microM) completely abolished inward currents evoked by AMPA (10-100 microM) in the presence of cyclothiazide (100 microM), kainate evoked currents in cells pretreated with concanavalin A (Con A) always showed a component (35-140 pA, n = 13) resistant to blockade. The majority (73+/-7%, n = 5) of GYKI 53655-resistant kainate-evoked inward currents remained in the presence of 100 microM AMPA. However, these currents were reversibly blocked by the competitive AMPA/kainate receptor antagonist NBQX (100 microM). (2S, 4R)-4-methylglutamate (SYM 2081, 10 microM) evoked inward currents in Con A treated cells (15-60 pA, n = 7), which were resistant to complete blockade by GYKI 53655 (50 microM) but antagonised by NBQX (100 microM). Kainate-evoked responses in the presence of GYKI 53655 (50 microM) had linear or slightly outwardly rectifying current-voltage (I-V) relationships in all cells examined (n = 5) and were resistant to blockade by Joro spider toxin (JsTx, 1 microM; n = 5). These results provide evidence that rat cultured cerebellar granule cells express functional kainate receptors made up of subunits which are edited at the Q/R site, and that SYM 2081 is an agonist at these native kainate receptors with a greater selectivity than kainate itself.

ATP-sensitive channels were observed in isolated inside-out membrane patches from rat cultured central neurones. Two types of ATP-sensitive K+ channels were present in cortical neurones, one which had its open-state probability increased, the other its open-state probability decreased by application of ATP to the cytoplasmic membrane surface. Another, ATP-sensitive channel differing in ion conductance from all previously reported ATP-sensitive channels was also seen in patches from cortical neurones. This channel was nonselective with respect to Na+, K+ and Cl- ions and ATP produced a "flickery" type of block. The non-hydrolysable analogue, AMPPNP, did not mimic ATP and prevented ATP action. Preliminary experiments indicate that similar, but not, identical ATP-sensitive channels exist in cerebellar neurones.