Supplementary Figure 3: TPE stimulation evokes GCaMP3 transients consistent with action potentials (APs) and opsin-mediated depolarization in awake mice.
From: Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields
a. GCaMP3 ΔF/F values vs. stimulation pulse number. Somatic ΔF/F values for 6 neurons stimulated at 5, 10, and 20 Hz (16 ms per pulse), measured 500 ms after pulse-train onset (values for each cell are normalized to the peak response; average of 3-7 trials per data point). The monotonic relationship between ΔF/F and stimulation pulse number is consistent with a regime in which ΔF/F values also scale approximately linearly with AP number (assuming 1 AP per pulse; Tian et al., Nat. Methods 6, 875-881 [2009]). Inset: sample traces from one neuron stimulated with 10 pulses at 5, 10, and 20 Hz (each trace is a 5-trial average). Colored underlines indicate the corresponding stim. train period. Dashed line indicates the time at which values ΔF/F values were measured (500 ms after stim. onset). b. Histogram of measured GCaMP3 fluorescence transient half-decay times following offset of a photostimulation epoch (τ1/2 calculated from single-exponential decay fits). Following stim. offset, transients evoked in cells returned to resting levels with off-kinetics (τ1/2 = 375+/-196 ms; mean +/- s.d.) in the range observed in vivo during trains of electrically stimulated APs (τ1/2 = 384+/-76 ms for 10 APs; Tian et al., Nat. Methods 6, 875-881 [2009]). c. Peak GCaMP3 transient amplitude during raster-scanning photostimulation of a cell using the spatial focusing path (SF in Fig. 1, main text) shown for different TPE raster-scan periods, which varied by changing the number of lines in a raster-scan, and which were repeated over an interval of 512 ms. The dashed line indicates the approximate C1V1(t/t) inactivation time-constant (τoff = 40-50 ms; Mattis et al., Nat. Methods 9, 159-172 [2012]; Prakash et al., Nat. Methods 9, 1171-1179 [2012]). Faster-scanning photostimulation trials (Ts< τoff) produced larger-amplitude responses than slower-scanning trials (Ts> τoff; n=31 target cells; values for each cell normalized by maximum amplitude in that cell). This relationship is a signature of membrane depolarization mediated by scanning recruitment of opsin probes (Rickgauer and Tank, PNAS 106, 15025-15030 [2009]; Prakash et al., Nat. Methods 9, 1171-1179 [2012]; Packer et al., Nat. Methods 9, 1202-1205 [2012]). Inset: Exemplary ΔF/F traces from one cell illustrating this relationship (colors indicate scan periods of same-color dots in panel; bars indicate s.d.).
