INTERACTIONS OF 2,3-BENZODIAZEPINES AND CYCLOTHIAZIDE AT AMPA RECEPTORS - PATCH-CLAMP RECORDINGS IN CULTURED NEURONS AND AREA CA1 IN HIPPOCAMPAL SLICES

1 The 2,3-benzodiazepines GYKI 52466, GYKI 53405 and GYKI 53655 antago nized AMPA-induced currents in cultured superior colliculus neurones i n a non use-dependent manner (steady state IC(50)s: GYKI 52466 9.8+/-0 .6 mu M; GYKI 53405 3.1+/-0.6 mu M; GYKI 53655 0.8+/-0.1 mu M). 2 High er concentrations of all three antagonists slowed the onset kinetics a nd quickened the offset kinetics of AMPA-induced currents indicative o f an allosteric interaction with the AMPA recognition site. 3 Cyclothi azide (3-300 mu M) dramatically slowed desensitization of AMPA-induced currents and potentiated steady state currents (EC(50) 10.0+/-2.5 mu M) to a much greater degree than peak currents. Both tau(on) and tau(o ff) were also increased by cyclothiazide in a concentration-dependent manner (EC(50): tau(on) 42.1+/-4.5 mu M; tau(off) 31.6+/-6.6 mu M). 4 Cyclothiazide (10-100 mu M) shifted the concentration-response curves of the 2,3-benzodiazepines to the right. For example, with 10 mu M cyc lothiazide the IC(50)s Of GYKI 52466 and GYKI 53405 on steady-state AM PA-induced currents were 57.9+/-9.5 and 41.6+/-1.5 mu M, respectively. 5 GYKI 53405 and GYKI 52466 concentration-dependently reversed the ef fects of cyclothiazide (100 mu M) on offset kinetics (GYK1 53405 IC50 16.6+/-4.2 mu M). However, the 2,3-benzodiazepines were unable to rein troduce desensitization in the presence of cyclothiazide and even conc entration-dependently slowed the onset kinetics of AMPA responses furt her (GYKI 53405 EC(50) 8.0+/-2.8 mu M). 6 GYKI 52466 decreased the pea k amplitude of hippocampal area CAI AMPA receptor-mediated excitatory postsynaptic currents (e.p.s.cs) (IC50 10.8+/-0.8 mu M) with no appare nt effect on response kinetics. Cyclothiazide prolonged the decay time constant of AMPA receptor-mediated e.p.s.cs (EC(50) 35.7+/-6.5 mu M) with less pronounced effects in slowing e.p.s.c. onset kinetics and in creasing e.p.s.c. amplitude. 7 Cyclothiazide (330 mu M) shifted the co ncentration-response curve for the effects of GYKI 52466 on AMPA recep tor-mediated e.p.s.c. peak amplitude to the right (GYKI 52466 IC50 26. 9+/-9.4 mu M). Likewise, GYKI 52466 (30-100 mu M) shifted the concentr ation-response curve for the effects of cyclothiazide on AMPA receptor -mediated e.p.s.c. decay time constants to the right. 8 In conclusion, cyclothiazide and the 2,3-benzodiazepines seem to bind to different s ites on AMPA receptors but exert strong allosteric interactions with o ne another and with other domains such as the agonist recognition site . The interactions of GYKI 52466 and cyclothiazide on AMPA receptor-me diated e.p.s.cs in area CA1 of hippocampal slices provide evidence tha t the decay time constant of these synaptic events are not governed by desensitization.