Sensitization of focal AMPA-evoked calcium responses in membrane microdomains. AMPA-evoked calcium transients were measured along dendritic branches using the ratiometric calcium probe Fura-2 at the rate of 10 image pairs per second. Focal calcium bursts evoked by (
a) AMPA (20 μ M) were ( b) suppressed when EtOH remained present in the bathing media, and ( c) enhanced during EtOH WD. ( d) Quantitation of AMPA-evoked calcium transients showing the median amplitudes of calcium responses for the indicated conditions. ( e–g) Representative images for the indicated conditions showing (from top to bottom) pseudocolor images of baseline and AMPA-evoked calcium transients, immunofluorescent staining of the same dendrite for GM1, GluR1 and the merged images. Lower tracings show baseline and AMPA-evoked calcium responses for the indicated regions. ( h–k) Representative traces of AMPA-evoked calcium transients evoked after a 2 min pre-exposure to vehicle or EtOH were inhibited by the selective calcium-permeable AMPAR antagonist, Naspm trihydrochloride (Naspm, 50 μ M). ( l) Quantification of AMPA-evoked calcium transients showing the median amplitudes of calcium currents evoked under the indicated conditions. ( m) Representative immunoblots of rat hippocampal neurons treated with forskolin (Forsk, 20 μ M) and IBMX (50 μ M). ( n) Quantification of surface GluR1 and total GluR1 after treatment of forskolin and IBMX. ( o) Representative traces of AMPA-evoked calcium responses in neurons pretreated with forskolin and IBMX and then continuously exposed to EtOH. ( p) Quantification of AMPA-evoked calcium responses for the indicated conditions. Data are median±s.d. ***P<0.001; <0.01; ##P <0.001. AMPA, ###P n-2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid; AMPAR, AMPA receptor; EtOH, ethanol; IBMX, 3-isobutyl-1-methylxanthine; WD, withdrawal.