Fig. 4.
Diazepam triggers release of Ca2+ from the intracellular stores which is required for internalisation of GABAARs and prevented by Ro 15-1788, bicuculline, thapsigargin and U-73122. Time lapse imaging of intracellular Ca2+ in Fluo-4-labelled cortical neurones treated with diazepam (D; 1 μM) alone (a, inset: representative images before and after diazepam addition; scale bars = 20 μm), or in the presence of Ro15-1788 (Ro, 25 μM; b) or bicuculine (Bic, 50 μM; c), shown as a fluorescence ratio Ft/F0, and d quantified at the peak of response to diazepam in dendrites and somas (d; mean ± s.e.m.; ANOVA/Bonferonni post-hoc test; *p < 0.05; n = 5 neurones in each group from 2 independent experiments). e Diazepam (D; 1 μM)-dependent increase in intracellular Ca2+ (insert: representative images before and after diazepam addition; scale bars = 20 μm) is inhibited by thapsigargin (T; 2 μM; f) and U-73122 (U; 10 μM; g). h Ft/F0 was quantified at the peak of response to diazepam in dendrites and somas of labelled cortical neurons (mean ± s.e.m.; ANOVA/Bonferonni post-hoc test; *p < 0.05; n = 4 neurones in each group from 2 independent experiments). i–k Diazepam (D; 1 μM)-dependent internalisation of GABAARs in neurones (left), and Diazepam (D; 1 μM)/Isoguvacine (I; 5 μM)-dependent internalisation of GABAARs in α1/β2/γ2myc-HEK293 cells (right) are prevented by thapsigargin (T; 2 μM; i) and U-73122 (U; 10 μM; j), but insensitive to EGTA (E; 1 mM; k). Changes in surface GABAARs were measured by cell surface ELISA using β2/3-specific antibody in neurones or myc-antibody in α1/β2/γ2myc-HEK293 cells and presented in graphs as mean ± s.e.m. Statistical analysis was done using ANOVA with Bonferonni post-hoc test; *p < 0.05 (n = 7 thapsigargin, n = 5 U-73122, n = 9 EGTA independent experiments)