We gave single uncaging pulses to spines of different sizes and measured the postsynaptic response of the spines, namely the uEPSC amplitude, by whole-cell patch electrophysiology (voltage-clamp) and compared that with the volume of the spine head. We found, in agreement with

published data (Asrican et al., 2007, Matsuzaki et al., 2004 and Smith et al., 2003), that the uEPSC amplitude measured at a spine was directly proportional to its volume (Figures S3A–S3E). Because this relationship held across cells within a slice (Figures S3B–S3E) (Smith et al., 2003), the initial postsynaptic strength of a spine 3-MA concentration is calculable from a spine volume-to-uEPSC amplitude calibration function obtained from a different cell within the slice.

Thus, we recorded the uEPSC amplitude of different spines along a dendritic branch from a cell (cell 1) that neighbored the cell of interest buy MK-8776 (cell 2) to determine a spine volume-to-uEPSC amplitude calibration function. We then induced STC at cell 2 by stimulating two spines, L1 (GLU+FSK stimulation) and L2 (GLU+FSK stimulation in the presence of anisomycin), on an analogous branch in cell 2 and followed this with whole-cell recordings and single-spine stimulations of L1 and L2 and neighboring spines from cell 2 at the end of the experiment to determine the stimulated and neighboring spines’ strengths. We estimated the potentiation of L1 and L2 by normalizing the final uEPSC amplitude to the initial uEPSC amplitude estimated from the initial spine volume of L1 and L2 using the previously calculated calibration function from cell 1. We found that both L1 and L2 underwent potentiation (Table 1; Figures S3F and S3G), whereas PD184352 (CI-1040) there was no change in neighboring spines, both in terms of volume and estimated uEPSC amplitude. Combining these data with the data from Figure 1B;

Figures S1B–S1D and data from the literature (Tanaka et al., 2008), we conclude that similar to the case for E-LTP (Asrican et al., 2007, Harvey and Svoboda, 2007 and Matsuzaki et al., 2004), the spine volume increases that we observed at the spines to which the GLU+FSK stimulation was given and also at the spines in which STC occurred are indicative of a change in potentiation. Thus, we used spine volume change as a measure of both L-LTP and E-LTP for the remainder of the experiments. Analogously to data obtained from a population of synapses using field recordings and stimulation (Frey and Morris, 1997), we found that GLU+FSK stimulation at one spine (L1) followed by GLU stimulation (which normally induces only E-LTP) at a second spine (E2; Figure 2E) resulted in L-LTP expression not only at L1 but also at E2, again demonstrating STC in our single-spine stimulation system (Figure 2F; Figure S2A).