, 2005). The detailed dissociation method is described in Supplemental Experimental Procedures. Dissociated E14.5 retinal neurons (1 × 105 cells) were plated on top of a confluent monolayer of control HEK293 cells, or a stable HEK293 cell line expressing either Sema5A or Sema5B, in 24-well plates and then cultured for 48 hr in culture medium (containing B-27 supplement, 2 mM L-Glutamine, 10 ng/ml ciliary neurotrophic factor [CNTF] [R&D Systems], 50 ng/ml brain-derived

neurotrophic factor [BDNF], 5 mM forskolin, 5 mg/ml insulin, 50 U/ml penicillin, and 50 μg/ml streptomycin), fixed in 4% PFA for 15 min, incubated with anti-βIII-tubulin (Promega; at 1:1000), followed by incubation with goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen; at 1:500), and then imaged for neurite outgrowth analysis. Neurite lengths of retinal neurons were quantified buy NVP-AUY922 using ImageJ plugin. ERG measurements were performed as previously described (Samuels et al., 2010). The amplitude of the a-wave was measured at 8 ms after flash presentation from the prestimulus

baseline. The amplitude of the b-wave was measured to the b-wave peak from the a-wave trough or, if no a-wave was present, from the baseline. The amplitude of individual oscillatory potentials was measured from the negative trough to the subsequent peak. The implicit times of the b-wave and individual oscillatory potentials were measured at the positive peak. RGC responses to a variety of light stimuli were recorded using a Multielectrode Array System (Multi Channel Systems; ALA Scientific Instruments, GW3965 research buy Farmingdale,

NY, USA). Dissections and recording conditions were previously described (Meister et al., 1994 and Ye et al., 2009). Visual stimuli were generated and presented using old MATLAB software (Natick, MA, USA; http://www.mathworks.com/), and the Psychophysics Toolbox extensions (Brainard, 1997 and Pelli, 1997). See Supplemental Experimental Procedures for a detailed description of the recordings and data analysis. OKR measurements were performed as previously described (Cahill and Nathans, 2008). The statistical significance of the differences between mean values among two or more groups was determined using Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s HSD test, respectively. The criterion for statistical significance was set at p < 0.05. Error bars are SEM. We thank K.-W. Yau and T. Xue for the Opn4Tau-LacZ/Tau-LacZ mice, M. Tessier-Lavigne for the PlexA3−/− mice, B. Howell for the Dab-1 antibody, and F. Haeseleer for the CaBP5 antibody. We also thank D. Kantor, S. Kozlov, C. Hawkins, and K. Takamiya for their assistance with the generation of the Sema5A−/− and Sema5B−/− mice. We thank M. Riccomagno, K. Mandai, S.-H. Wang, Y. Duan, and T. Tran for helpful suggestions and discussions throughout this project, D. Johnson for assistance with mouse experiments, and members of the Kolodkin laboratory for assistance.