FREQUENCY-DEPENDENT ACTIONS OF BENZODIAZEPINES ON GABA(A) RECEPTORS IN CULTURED MURINE CEREBELLAR GRANULE CELLS

1. Miniature IPSCs recorded from cultured murine cerebellar granule ce lls increased in halfwidth and amplitude following application of the benzodiazepine (BDZ) Flunitrazepam (Flu, 1 mu M). The increase in the half-width was much greater than that in the amplitude. 2. Five-millis econd applications of 1 mM GABA to nucleated outside-out patches elici ted rapidly rising biexponentially decaying responses that resembled I PSCs. Flu had no effect on the amplitude of such responses, but consis tently slowed their deactivation by similar to 50 %. This effect was r eversed by Flu washout or application of the BDZ antagonist Ro15-1788. The partial inverse agonist Ro15-4513 speeded deactivation and depres sed peak current amplitude bg 23 +/- 12%. 3. The EC50 for GABA was bet ween 45 and 50 mu M. At submaximally effective agonist concentrations, Flu increased response amplitude and slowed response deactivation. Bo th effects were present in all cells taken from young cultures (4-7 da ys in vitro) but the latter was absent in 55% of the neurones obtained from older cultures (14-27 days in vitro). 4. With 120 ms application s of 20 mu M GABA, responses activated monoexponentially (time constan t, 39.8 +/- 2.8 ms) and deactivated biexponentially (time constants, 4 0.4 +/- 2.1 and 251 +/- 15 ms). Application of Flu slowed both activat ion and deactivation. The latter effect arose from an increased contri bution of the slower component of decay. 5. Desensitization of respons es to 1 mM GABA was biexponential, with time constants of 47 +/- 11, a nd 479 +/- 49 ms. Flu speeded desensitization by decreasing both fast and slow time constants. GABA(A) receptor desensitization consistently slowed subsequent deactivation. No significant relationship between t he level of desensitization and the amount of slowing of deactivation produced by Flu was found. 6. Responses to paired 5 ms applications of 1 mM GABA indicated that the slowing of deactivation and the speeding of desensitization produced by Flu combine to generate a marked frequ ency dependence in the actions of this BDZ. Thus when compared with co ntrol responses, GABA-induced charge transfer was only enhanced by Flu during the first of two successive agonist applications.