Enhanced exocytosis in mossy fibers after CQ injection.

Hippocampal slices (400- µm thickness) were prepared 2 h after i.p. injection of CQ (30 mg/kg). To measure the decrease in FM4-64 fluorescence intensity in mossy fiber terminals (boutons), mossy fiber terminals were doubly stained with ZnAF-2 and FM4-64. ZnAF-2 staining was done under an optimal condition to stain mossy fiber terminals clearly, unlike the ZnAF-2 staining condition of Fig. 4. The terminals strongly stained with ZnAF-2 were determined as region of interest. To observe presynaptic activity, tetanic stimulation at 10 Hz for 30 s, which was shown by a shaded bar, was delivered to mossy fibers and then single strong stimulation at 100 Hz for 18 s was delivered to the same position. The activity-dependent component of FM4-64 signal was measured for each punctum (1 s) by subtracting its residual fluorescence intensity measured by the strong electrical stimulation. The data (mean ± SEM) represent the ratio (%) of each FM4-64 intensity to the basal FM4-64 intensity before tetanic stimulation at 10 Hz for 30 s, which was averaged and expressed as 100% (vehicle, n = 10; CQ, n = 12). SL, stratum lucidum; PCL, CA3 pyramidal cell layer. *, p<0.05, vs. vehicle.