, 2003, Johnson et al., 2003, Sherry et al., 2003 and Stella et al., 2008) and is required for synaptic glutamate release (Hnasko et al., 2010 and Stuber et al., 2010), we mated ET33-Cre mice with mice that carry floxed alleles of VGLUT2 (Hnasko et al., 2010) in order to generate mice lacking VGLUT2 specifically in ipsilateral-projecting RGCs. In mice, VGLUT2 protein is expressed at low levels at P0 and increases dramatically

over the first postnatal week (Sherry et al., 2003 and Stella et al., 2008). We found that Cre expression in ET33-Cre mice starts embryonically at least as early as embryonic day 18 (Figure S1C) and when we cultured RGCs from postnatal day 3 (P3) ET33-Cre mice expressing either wild-type or floxed VGLUT2 and immunostained them on P5, we found that VGLUT2 immunofluorescence intensity was nearly absent from the ET33-Cre::VGLUT2flox/flox RGCs (Figures S2A–S2G). To determine if retinogeniculate transmission PF 01367338 was reduced in ET33-Cre::VGLUT2flox/flox mice, we measured electrophysiological responses of dLGN neurons in response to optic tract stimulation. We prepared brain slices containing the optic tract and dLGN, which allowed

us to stimulate RGC axons and record postsynaptic responses in whole-cell voltage-clamped dLGN neurons (Chen and Regehr, 2000: Koch and Ullian, 2010). The optic tract contains axons from both eyes, so by removing one eye from young mice and allowing the severed RGC axons to LY2157299 price degenerate we were able to prepare slices that contained either contralateral or ipsilateral axons, but not both (Figures 2A and 2B). We also injected CTb into the intact eye to visualize its projections in the slice, thus allowing proper targeting of the recording and stimulating electrodes (Figure 2B). Recordings were performed on P5 and P10. Stimulation of contralateral RGC axons in P5 slices produced postsynaptic NMDAR-mediated responses in every dLGN neuron tested, regardless of genotype. Indeed, the size of the contralateral NMDAR-mediated these responses was

indistinguishable between Cre-expressing and Cre-negative slices (Figures 2C and 2E; VGLUT2flox/flox = 1006 ± 138.69 pA, n = 11 and ET33-Cre::VGLUT2flox/flox = 1102 ± 176.1 pA, n = 11; p > 0.05 by Student’s t test). By contrast, when ipsilateral RGC axons were stimulated, dLGN neurons in ET33-Cre::VGLUT2flox/flox slices often failed to respond (11 responses out of 24 cells) and response sizes were reduced by ∼55% (Figures 2D and 2F; VGLUT2flox/flox mice = 343.75 ± 59.21 pA, n = 19 and ET33-Cre::VGLUT2flox/flox mice = 157.49 ± 40.51 pA, n = 22; p = 0.014 by Mann-Whitney U test). AMPAR-mediated responses showed similar results (Figures S2H–S2M). Next we assessed retinogeniculate transmission in slices from P10 mice, an age when ongoing spontaneous activity continues to refine and maintain eye-specific retinogeniculate projections (Chapman, 2000 and Demas et al., 2006).