Figure 6.

Ca²⁺ uncaging reveals a decreased intrinsic release kinetics of the remaining FRP vesicles (ves). A, Presynaptic Ca²⁺ currents (I_Ca; top), and EPSCs (bottom) in response to a 20 Hz train followed by Ca²⁺ uncaging. B, Presynaptic [Ca²⁺]_i as imaged by fura-2FF (top) and EPSCs (bottom) in response to a flash applied to the naive synapse (black traces) and to the predepressed synapse (red traces). The predepressed EPSC was scaled to the peak of the naive EPSC (dashed red trace; note the slower rise phase of the predepressed EPSC). C, Transmitter release rates (inset) and cumulative (cum.) release traces of the EPSCs shown in B. Double exponential fits of the cumulative release traces are shown as gray dashed lines. Note the slower time constant of the fast release component (τ_fast) despite the similar post-flash [Ca²⁺]_i step obtained in these examples (see B). The cumulative release trace following the 20 Hz train was scaled (see red dashed trace) to match the amplitude of the fast release component (A_fast) under control conditions. D, Plot of the fast release time constants obtained from double exponential fits to cumulative release traces evoked by Ca²⁺ uncaging (see C). Time constants of control flash responses and flashes following the conditioning 20 Hz train are shown as filled and open symbols, respectively. The relationship between fast time constants and [Ca²⁺]_i in response to control flashes was fitted with a one-pool model of Ca²⁺ binding and vesicle fusion (black line; see Materials and Methods). The x-axis plots [Ca²⁺]_i (in μm) as in E. E, Plot of the fold change between the fast release time constants and the fit to the control data of the fast release time constants. Note that while the control data (filled symbols) fluctuate around a value of 1 as expected (filled symbols), the τ_fast values following the 20 Hz trains (open symbols) show a robust ∼1.5–2 fold slowing. The average values of all relative τ_fast values are shown as red average symbols for the control data and following the 20 Hz trains (red closed and open symbols, respectively). F, Average fast-time constants (n = 7 cell pairs) separated for two ranges of postflash [Ca²⁺]_i values (left, 5–8 μm; right, 8 – 17.5 μm), both under control conditions (gray bars), and following 20 Hz trains (open bars). Note the significant slowing of τ_fast for all paired comparisons (p = 0.002 and 0.01 for 5–8 and 8–17.5 μm [Ca²⁺]_i, respectively).