Retinal ganglion cells were analyzed in term of contrast sensitivity, only. For this purpose we designed three different protocols of stimulation. Luminance (irradiance) sensitivity was assessed by stimulating the dark-adapted fish with a series of flashes (4 × 3 s flashes at 6 s intervals) at nine different light intensities, ranging between 11 pW/mm2 and 110 nW/mm2 with 0.5 log unit steps. The sequence of light intensities was randomized to reduce habituation artifacts in the recordings. Maximum light intensity, 110 nW/mm2, is equivalent to 3.3 × 1011 photons/mm2 × s−1. Contrast sensitivity AZD9291 manufacturer was assessed by stimulating the dark-adapted fish with a series

of 10 s light oscillations at 5 Hz around a constant light level (55 nW/mm2) at 10 different levels of contrast, ranging from 10% to 100% of the constant light level. Finally, frequency sensitivity was assessed by stimulating the dark-adapted fish with a series of 10 s light oscillations around a constant light level (55 nW/mm2) at 90% contrast at 14 different frequencies, ranging from 0.2 to 25 Hz. Image sequences were acquired at 10 Hz (256 × 100 pixels per frame, 1 ms per line) for the irradiance and contrast experiments and at 40 Hz

(256 × 25 pixels per frame, 1 ms per line) for frequency experiments. The stimulation of the olfactory bulb was obtained by bath application of the amino acid methionine (Sigma) 1 mM, as in Maaswinkel and Li (2003). To manipulate Crenolanib dopamine signaling in the retina we injected neuroactive drugs into the the eye. Final concentrations of the drugs were calculated by diluting the injected concentration into the free volume of the eye. The volume of a typical 9 dpf old zebrafish eye was assessed by three-dimensional reconstruction of the eye chamber and the lens through two-photon microscopy scanning. The final volume was estimated as the

difference between the total eye volume and the volume of the lens core. We calculated a total free volume of ∼500 μm3. Given a typical injected volume of 10 μl, the final dilution factor can be approximated to 1:50. Dopamine receptors were activated by injection of the long-lasting dopamine receptor ligand [3H] 2-amino-6,7-dihydroxy 1,2,3,4-tetrahydronapthalene (ADTN) (Sigma) 10 μM, as in Li and Dowling (2000b). Dopamine action on postsynaptic targets was prevented by injection of the strong dopamine D1 receptor antagonist SCH 23390 (Sigma) 2 nM, as in Huang et al. (2005) or the selective dopamine D2 receptor antagonist sulpiride (Sigma), as in Lin and Yazulla (1994) and Mora-Ferrer and Gangluff (2000). Finally, the level of dopamine in the circuit was frozen by injection of the dopamine release and reuptake inhibitor vanoxerine (Santa Cruz Biotechnology) 2 μM, as in Schlicker et al. (1996).