A simple method for electrical field stimulation of cultured granule cells

A method suitable for electrical field stimulation of superfused primary cultures of cerebellar granule cells is described. A microchamber of about 0.5 ml was obtained by closing the culture dishes with a perspex plug equipped with stimulating electrodes and inlet-outlet tubing. Two-minute trains of electrical pulses (alternate polarity, 2-ms duration; 100 mA intensity; 10 V drop between electrodes; frequency 5, 10 and 20 Hz) applied to cultures kept at 27 degrees C, elicited a D-[3H]aspartate outflow which was frequency related, [Ca2+]0 dependent, tetrodotoxin sensitive. Moreover 2 trains of 10 Hz pulses (S1 and S2) at 30-min intervals caused an S2/S1 ratio equal or near to one, thus demonstrating that a steady-state condition had been achieved. The NMDA antagonist DL-2-amino-5-phosphonopentanoic acid (AP5) but not the non-NMDA antagonist, 6-cyano-7-nitroquinozaline-2,3-dione (CNQX), added before S2, significantly increased the electrically evoked tritium efflux, suggesting that the endogenous transmitter released during electrical stimulation activated an NMDA-mediated negative feed-back. This technique of electrical field stimulation seems particularly feasible to study the extent and time course of drug effects on spontaneous and evoked D-[3H]aspartate outflow.