Fig. 3.

Activation of group I mGlu receptors potentiates KARs in rat cortical cultures. (A) Ca²⁺-dependent fluorescent signal of cortical cultures loaded with Fluo-4 was monitored. With use of the integrated fluidics of the FLEXStation, a bolus of HBSS was delivered at 60 seconds, resulting in a miniscule reduction in the fluorescent signal. At 180 seconds, addition of ionomycin (100 µM) or thapsigargin (5 µM) resulted in a long-lasting increase in the [Ca²⁺]_i. Data are the mean of eight replicates from a single plate. (B) in cultures pretreated with NMDA, AMPA, and GABA_A receptor blockers (10 µM MK801, 100 µM d-AP5, 100 µM GYKI 52466, and 100 µM bicuculline), a similar rapid increase in fluorescence was detected after application of DHPG (50 µM) and AMPA (50 µM). The DHPG-induced potentiation of the AMPA response was blocked by preincubation with 4 µM MPEP. Data are the mean of eight replicates from a single plate. Bars indicate the duration of drug exposures. (C) MPEP (4 µM, n = 6) and Ro 320432 (4 µM, n = 6) prevented DHPG-induced KAR potentiation. Data are the mean and standard error of paired t tests (*P < 0.05). (D) in cultures pretreated with NMDA, AMPA, kainite, and GABA_A receptor blockers (10 µM MK801, 100 µM d-AP5, 500 µM CNQX, 500 µM NBQX, 100 µM GYKI 52466, and 100 µM bicuculline), a rapid increase in fluorescence was detected after application of KCl (12 mM) and DHPG (50 µM). Activation of group I mGlu receptors by DHPG slightly reduced the KCl-induced calcium transient. Data are the mean of eight replicates from a single plate. Bars indicate the duration of drug exposures. The panel below shows that the calcium transients elicited by KCl were not potentiated by DHPG or PMA (n = 12 experiments from two independent culture preparations). (E) in cultures pretreated with NMDA, AMPA, and GABA_A receptor blockers (10 µM MK801, 100 µM d-AP5, 100 µM GYKI 52466, and 100 µM bicuculline), a similar rapid increase in fluorescence was detected after application of AMPA (50 µM). AMPA-induced increase in intracellular calcium was increased by exposure to the KAR potentiator ConA in a dose-dependent manner. Data are the mean S.E. of four experiments from a single culture. (F) In cultures pretreated with NMDA, AMPA, and GABA_A receptor blockers, the AMPA-induced increase in fluorescent counts was significantly blocked by a high (100 µM) but not a low (1.5 µM) concentration of CNQX. (G) in cultures pretreated with NMDA, AMPA, and GABA_A receptor blockers, a similar rapid increase in fluorescence was detected after application of the KAR agonist SYM2081. (H) the SYM2081-induced increase in intracellular calcium was dose-dependent. Data are the mean of the peak calcium signal and standard error of three experiments from two independent cultures.