, 2011). To specifically manipulate L4 function, we replaced the Gal4 drivers with either half of the split-Gal4 system ( Luan et al., 2006) and obtained a splitL4-Gal4 line (L40980-VP16AD, L40987-Gal4DBD) that was expressed only in L4 and in no other neurons ( Figures 2J–2L).
To generate tools that would allow independent manipulations of L4 and other cell types using different binary expression systems, Crizotinib order we also replaced the Gal4 in the L4 drivers with two other transcription factors, LexA and QF ( Lai and Lee, 2006 and Potter et al., 2010). The L40987-LexA, L40987-QF, and L40980-QF lines recapitulated the expression pattern of their Gal4 progenitors ( Figures 2M–2O). L40987-QF was additionally expressed in trachea, which, however, did not interfere with our experiments. We first sought to determine the visual response properties of L4 (Figures 3A and 3B). We measured in vivo calcium signals from L4 terminals in medulla layers M2 and M4 (Figures 3B and 3C) using two-photon imaging of the genetically encoded calcium indicator TN-XXL ( Figures 3D–3G) ( Mank et al., 2008 and Reiff et al., 2010). When presented
with alternating increases and decreases in light intensity, the average ratiometric calcium signal of all cells decreased when the light was on and increased when the light was off, in both layers M2 OSI-906 mw and M4 ( Figure 3D and data not shown). This is consistent with L4 hyperpolarizing to brightening and depolarizing to darkening ( Douglass and Strausfeld, 1995). Very similar calcium signals were seen when either Gal4 or QF transcription factors were used to drive TN-XXL expression ( Figure 3D; see Experimental Procedures). Next we tested whether
L4 displays direction-selective responses to motion. In response to a narrow bright bar, moving on a dark background at 10°/s, L4 terminals responded with an initial decrease in calcium signal associated with the light increment when the bar reached their receptive field, followed by an increase in calcium signal Tolmetin as the bar left the receptive field (Figure 3E). Using bars that moved either horizontally or vertically, we found no signs of direction selectivity (Figure 3E). Similar results were obtained for bars moving at 20°/s and 50°/s (data not shown). To characterize the response properties of L4 under continuous, dynamic stimulation, we used a rapidly flickering, uniform-field stimulus with Gaussian distributed intensity changes. Using linear-filter estimation procedures, we extracted the temporal linear filter that best captured the calcium response as a function of time (Chichilnisky, 2001 and Sakai et al., 1988). This linear filter had a large negative lobe consistent with a sign inversion of the input contrast (Figure S3), results that are similar to those previously described for L2 (Clark et al., 2011).