Figure 3.

Direct imaging of a fluorescent steroid analogue suggests sequestration by γ-cyclodextrin explains reduced GABA responses. (a) Comparison of the effect of γ-cyclodextrin on equimolar concentrations of C11-NBD 3α5αP and 3α5αP. (a1) An oocyte responded to GABA (2 μM) and co-application of GABA and 2 μM C11-NBD 3α5αP. γ-Cyclodextrin (0.5 mM) partially reversed the effect of the tagged steroid. (a2) By contrast, 3α5αP (2 μM) more dramatically potentiated the GABA response, but γ-cyclodextrin more fully reversed the potentiation. (b) Summary of the protocol in (a) performed on four oocytes. Potentiation refers to the fold increase in GABA response, with 1.0 representing a doubling. The inhibition of 3α5αP potentiation was significantly larger than inhibition of C11-NBD 3α5αP potentiation (P< 0.05). (c) Imaging of C11-NBD 3α5αP applied to neurons in culture in the absence (two left panels, two right panels) and presence (two middle panels) of 0.5 mM γ-cyclodextrin. The reduction in fluorescence of the cells during co-application of steroid and cyclodextrin was consistent with sequestration of steroid by cyclodextrin. Images were taken after a 5 min pre-equilibration with steroid (left two panels), 15 s after addition of γ-cyclodextrin to the steroid-containing solution (γ-CD; middle panels) and 15 s after re-applying steroid alone. Interval between successive panels in the same experimental condition was 5 s.