From (Equation 13) and (Equation 16) we VX-809 manufacturer have equation(Equation 19) dNoutdt=dFdt⋅119⋅Fvqwhich can be differentiated to obtain the relation stated in the main text: equation(Equation 20) Vexo(t)=a[dFdt+(kendo⋅(F(t)−b))]where a=1/19⋅Fvqa=1/19⋅Fvq and b=Ntotal⋅Fvq⋅(1+20⋅αmin).b=Ntotal⋅Fvq⋅(1+20⋅αmin). In the absence of exocytosis, there are unquenched pHluorin molecules on the surface membrane, equivalent to a fraction αmin of all vesicles.

This surface fraction can be measured by quenching with acid (Granseth et al., 2006). For ON terminals, the minimal surface fluorescence is reached in the dark, and for OFF terminals, in bright light. These measurements were carried out in intact zebrafish by changing the pH of the bathing medium from 7.4 to 3.2. Averaged measurements are shown

in Figure 3C. The surface fraction (αmin) was then calculated as (ΔF/19.7)/FpH3.2. The relative fluorescence of an ON terminal in darkness decreased to 0.84 during acid quenching of surface pHluorin, from which αmin = 0.97%. The relative fluorescence of an OFF terminal in bright light decreased to 0.91, from which αmin = 0.51%. Because the measuring error in these experiments was high, we used an average value of αmin = 0.8% for both ON and OFF terminals. We also estimated αmin in dissociated bipolar BGB324 ic50 cells with giant synaptic terminals using epifluorescence microscopy, as shown in Figure S3. The value we obtained αmin = 1.7% was somewhat higher than the value we obtained in vivo. The average density of vesicles in a bipolar cell terminal was calculated as ∼1050 per μm3 using electron micrographs of retinal slices 80 nm thick (Schmitt and Dowling, 1999; Figure 3A). Ntotal was then calculated for each terminal by multiplying the density by the volume of the terminal (TVol). TVol was not measured by full 3D reconstruction of each terminal, but by assuming that the optical section we were imaging contained the center of the terminal, which was shaped spherically. To minimize errors in this estimate,

the thickness of the optical section was increased to ∼2.5 μm by Parvulin reducing the numerical aperture of the IR beam used for multiphoton imaging. The average diameter of a bipolar cell terminal in these images was about 3 μm, which is very similar to estimates made from electron micrographs. We thank Aude Derevier for help with immunofluorescence and Mario Dorostkar for support with the SARFIA software. Ellen Schmitt and John Dowling provided us with EM pictures of zebrafish bipolar cells. Support for this work was provided by the Medical Research Council, the Wellcome Trust, and an E.U. Marie Curie fellowship to B.O. “
“Excitatory transmission at the central synapses is primarily mediated by the amino acid glutamate (Edmonds et al., 1995).