The time resolution of the Cm measurement is the period of f1, which we varied from 5.12 to 0.32 ms, corresponding to 195 to 3125 Hz. Comparisons between single-sine and dual-sine-wave methods showed no differences for membrane responses to

100 ms depolarizations to −20 mV. Single-sine values of 90 ± 15 fF (n = 12) were obtained as compared to 110 ± 24 fF for dual-sine wave measurements from the same cell population. The same two-sine wave technique (implemented in jClamp) has been used previously, but only as a Kinase Inhibitor Library cell assay before and after measurement which gave similar results to the single-sine method (Edmonds et al., 2004 and Thoreson et al., 2004). Details of methods and control data are presented in Figures S1–S3. The time between stimuli was varied based on the previous stimulus but was never less than 2 min and typically varied between 5 and 10 min to ensure appropriate time for reaching equilibrium. Swept-field confocal high-speed (SFC) calcium Trichostatin A price imaging was performed as previously described (Beurg et al., 2009). The SFC (Prairie Technologies, Middleton WI) was coupled to a Redshirt camera. Fluo 4ff was

used as the indicator, chosen to both limit effects on release properties and serve to localize the calcium source. Images were captured at 125 fps with a 35 mm slit. A 100 × dipping lens with an added 1.25 magnification gave a final pixel size of ∼350 nm. Ribbons were identified by using the Ctbp2 peptide tagged with rhodamine (Zenisek et al., 2003). Data was analyzed by selecting 3 × 3 pixel regions uniformly encompassing Ctbp2-labeled regions. Image planes were selected to help isolate individual synapses to ensure individual already synapses were being investigated. Data were included based on several parameters. Leak currents needed to be less than 50 pA at −85 mV and series resistance (uncompensated) needed to

be stable and below 15 M Ω. As electrode capacitance compensation is critical for the accurate use of the two-sine wave methodology, bath height and electrode filling were kept low to limit stray capacitance. Unless otherwise stated, data are presented as mean ± standard deviation with number of samples (n) given. Where appropriate Student’s t tests, two-tailed, were performed to assess significance; p values and correlation coefficients (r2) are listed with data. Isolated papillae were incubated for 1 hr at 4°C in external solution containing 4% formaldehyde and 0.1% Triton X-100. After washing four times for 10 min at room temperature in external solution containing 0.1% Triton X-100, papillae were incubated for 30 min in the same solution supplemented with 5% bovine serum albumin. Specimens were then incubated with primary antisera diluted at 1:250 to 1:500 in the same solution overnight at 4°C. Antisera included those against Ctbp2 (rabbit polyclonal, #1869), Ribeye (rabbit polyclonal, #1846), and PSD-95 (mouse monoclonal, Abcam #2723, concentrated at 3.