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. 2017 Oct 12;6:e31307. doi: 10.7554/eLife.31307

Figure 2. ON and OFF responses in VG3-AC neurites differ in preferred stimulus size, but are equally transient.

(A) Ca2+ responses of ROIs at different imaging depths to contrast steps in spots of different size. Spot diameters are noted above the bars indicating stimulus timing. The black traces (shaded areas) show the mean (±SEM) responses of VG3-AC somata (n = 8). The color-coded traces (shaded areas) indicate the mean (±SEM) responses of ROIs at different IPL depths (21%: n = 306, purple; 29%: n = 456, blue; 37%: n = 336, sky; 43%: n = 367, green; 50%: n = 700, lime; 60%: n = 588, olive). (B) The distributions of ON (white) and OFF (dark gray) receptive field center sizes of VG3-AC neurite ROIs. ON receptive field centers were larger than OFF receptive field centers (p<10−99, Wilcoxon rank sum test) (C) Receptive field center sizes (mean ±SEM) of ON (open circles) and OFF (filled circles) responses as a function of IPL depth. Because small response amplitudes led to rejection of >50% of ON responses of ROIs from 21–37% IPL depth (s. Material and methods), we restrict comparisons to 43–60% IPL depth. At all these depths, ON receptive field center sizes were larger than OFF receptive field center sizes (43%: p<10−8, 50%: p<10−9, 60%: p<10−9, Wilcoxon rank sum test with multiple comparison correction using the Benjamini–Hochberg procedure). Even without image segmentation, using the average activity of each image plane a single data point, ON receptive field centers were larger than OFF receptive field centers (p<10−3, Wilcoxon rank sum test, total: n = 61, 21%: n = 6; 29%: n = 13; 37%: n = 7; 43%: n = 6; 50%: n = 12; 60%: n = 17). (D) The distributions of transience indices of ON (white) and OFF (dark gray) responses of VG3-AC neurite ROIs did not differ significantly (p=0.925, Wilcoxon rank sum test). (E) Summary data (mean ± SEM) of transience indices of ON (open circle) and OFF (filled circle) responses as a function of IPL depth. Due to the high rejection rate of ON responses from 21 to 37% IPL depth, comparisons were restricted to 43–60% IPL depth. Transience indices of ON response is marginally lower than those of OFF responses at 43% (p<0.05), but were not significantly different at 50% (p=0.82) and 60% (p=0.05) IPL depth (Wilcoxon rank sum test with multiple comparison correction using the Benjamini–Hochberg procedure). We are not sure what accounts for the greater response transience observed in VG3-AC neurites vs. somata (p<10−4, Wilcoxon rank sum test). One possibility is that inhibitory synaptic inputs favor neurites and abbreviate responses.

Figure 2.

Figure 2—figure supplement 1. Depth-dependent shift in contrast preferences of VG3-AC neurites is robust across stimulus sizes.

Figure 2—figure supplement 1.

(A, B) Summary data (mean ± SEM) of polarity indices as a function of IPL depth, calculated from responses to a single spot size (A), diameter: 100 μm, s. Figure 1D) or from responses to all stimulus sizes (B), diameters: 20 μm – 800 μm). In (B), polarity indices differed between different IPL depths (p<10−65, Kruskal-Wallis one-way ANOVA). ROIs at 21% and 29% IPL depth were more biased to OFF responses than at other depths (p<10−7 for 37–60%). ROIs at 60% IPL depth were more biased to ON responses than ROIs from 21 to 51% (p<10−7 for 21–44%; p<0.01 for 51%).

Figure 2—figure supplement 2. VG3-AC neurites respond selectively to small stimuli.

Figure 2—figure supplement 2.

(A, B) The distribution of size selectivity indices calculated as the ratio of the difference between responses (A: OFF, B: ON) to small (diameter: 100 μm) and large (diameter: 400 μm) and the sum of these responses in VG3-AC neurite ROIs. The median of size selectivity indices were 0.97 and 0.95 for OFF and ON responses, respectively. For presentation purposes, negative size selectivity indices are not shown (1.8% and 5.4% of ROIs for OFF and ON responses, respectively).

Figure 2—figure supplement 3. Scan rates did not limit measurement of VG3-AC neurite response transience.

Figure 2—figure supplement 3.

(A) Ca2+ transients of ROIs recorded at different scan rates from two different IPL depths. The bar at the top indicates the stimulus timing. Black traces (shaded areas) show the mean (±SEM) responses of VG3-AC neurites scanned at 9.5 Hz; and teal traces (shaded areas) show the mean (±SEM) responses of VG3-AC neurites scanned at 37.9 Hz. (40% at 9.5 Hz: n = 126; 40% at37.9 Hz: n = 67; 54% at 9.5 Hz: n = 62; 54% at 37.9 Hz: n = 28.) (B) Summary data (mean ± SEM) of transience indices as a function of IPL depth for responses scanned at 9.5 Hz (black) and 37.9 Hz (teal). The response transience of ROIs was not significantly different between frequencies (p=0.22, main effect of frequency, three-way ANOVA). The interactions between frequency and contrast (p=0.83), frequency and depth (p=0.11), and among frequency, contrast and depth (p=0.31) were not significantly different.