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Fig. 1 Substance P diurnally regulates transmission of the visual signal (A) Multiphoton section through the eye of a zebrafish larva (7 days post fertilization [dpf]) expressing the glutamate reporter iGluSnFR in a subset of bipolar cells. (B) Timing of measurements and NK1R manipulations. The light-dark cycle was 14 h light/10 h dark. (C) Examples of iGluSnFR signals from an individual synapse in the morning, before (green) and after (blue) injection of the NK1R antagonist L-733,060 (∼20 nM). In each case, the top trace shows the iGluSnFR signal and the lower trace the estimated quanta. Modulation of light intensity at bottom. (D) Contrast-response (CR) functions in ON synapses in the morning, before (green, C1/2 = 29%, n = 9) and after injection of L-733,060 (blue, C1/2 = 46%, n = 8). C1/2 values interpolated from fits to Hill function. Each point shows the mean ± SEM. (E) As (D), but OFF synapses. C1/2 = 57% in control (n = 20) and 54% after injection of L-733,060 (n = 15). (F) Examples of iGluSnFR signals from an individual synapse in the afternoon, before (pink) and after (blue) injection of L-733,060. (G) Contrast-response function in ON synapses in the afternoon, before (green, C1/2 = 39%, n = 5) and after (blue, C1/2 = 39%, n = 5) injection of L-733,060. (H) As (G), but OFF synapses (n = 5, C1/2 = 51% before, C1/2 = 39% after). Note the lack of effect of antagonizing endogenous substance P in the afternoon (p > 0.6; KS test). (I) Examples of iGluSnFR signals recorded in the afternoon from an individual OFF synapse before (pink) and after (black) injection of substance P (∼20 nM). (J) Contrast-response function measured in ON bipolar cell synapses in the afternoon before (green, n = 20 synapses, C1/2 = 44%) and after intravitreal injection of substance P in the PM (black, n = 9, C1/2 = 60%). (K) Contrast-response function in OFF synapses in the afternoon before (pink, C1/2 = 53%, n = 26) and after intravitreal injection of substance P (black, C1/2 = 61%, n = 16). See also Figure S1.