For the photoreceptors to function

For the photoreceptors to function, recycling of visual pigments by isomerization and oxidation, from all-trans retinol to 11-cis retinol, and to 11-cis retinal, is essential (Parker and Crouch, 2010). The first essential step of isomerization to form 11-cis retinol is catalyzed by RPE65 in RPE for rod visual pigments (Redmond et al., 1998) and in Muller Cy5.5 hydrazide for cone visual pigments (Travis et al., 2005). The further oxidation to restore 11-cis retinal is then catalyzed by retinol dehydrogenases (RDHs), which are distributed in retina and RPE in a number of isoforms with overlapping activities (Maeda et al., 2007; Parker and Crouch, 2010). Because the grafted ONLs often form rosettes with their outer segments being separated from the RPE, isomerization of all-trans retinol may be inhibited. Since the treatment of 9-cis retinyl acetate (9-cis retinol acetate) restored the impaired visual function in Rpe65 mice (Maeda et al., 2009), we supplied 9-cis retinol acetate when we performed a behavior test with light-only signals to supply a substantial amount of the source of 11-cis retinal, although not the direct competent form. Partly because SAS was a time-consuming test, we did not perform the experiments with or without the drug treatment, or the tests after washout, so we were unable to determine the effects of 9-cis retinol acetate supplementation. Treatment of 9-cis retinol acetate had no effect on unsuccessful transplantation or non-treated rd1 mice, so the change in SAS results after transplantation is not due to the protective effect by the treatment. In electrophysiological evaluation using MEA, we carefully evaluated the graft-originated responses not simply by their presence or absence but by their functional properties. The presence of mERG responses were hardly detected in rd1 host retinas at 7 weeks or older, and, even in earlier degeneration, all the wave components were eliminated by mGluR6 blockade by L-AP4 (Fujii et al., 2016). After transplantation, marked light-responsive mERGs were recorded in the grafted area in all the samples tested, although the amplitudes were smaller than those of wild-type retina and the wave patterns were more variable, indicating that graft is responsive to light. The presence of the remaining negative-wave components after mGluR6 blockade implies that these responses may originate either from the graft photoreceptors or OFF components of host second neurons, which were both absent in rd1 host retinas before transplantation (Fujii et al., 2016). Furthermore, we were able to record significant light-responsive spikes from the host RGC layer, most of which were clustered as transient ON patterns. With our MEA system, approximately 20 cells are on one electrode in the RGC layer, half of which are reportedly amacrine cells (Jeon et al., 1998). Possible flaws in interpreting these RGC recordings include (1) residual responses from the host (cone) photoreceptors, (2) light-dependent RGC responses from intrinsically photosensitive retinal ganglion cells (iRGCs) (Berson et al., 2002; Hattar et al., 2002; Pickard and Sollars, 2012), and (3) spikes from graft cells, including graft RGCs, that may have been exposed on the host RGC surface layer. As we have described, the possibility of residual host transient ON function is unlikely. The possibility of iRGC activities can also be discounted, because the L-AP4 blockage of mGluR6 demonstrated that the RGC responses were postsynaptic. We also observed that in the area where light-responsive RGCs were recorded, the RGC and inner layers of host retina was present by histological examination, leaving little possibility that these RGC responses were mostly contributed by exposed graft cells. Interestingly, the channels that elicit RGC responses and those with evident mERG responses are not completely the same, such as seen in TP-6 in Figure S4A, which indicated that the former may represent the graft status such as the presence of graft photoreceptor outer segments in correct orientation, whereas the latter may represent the location of RGCs that eventually received input from graft light response. A few RGC responses detected outside the graft margin, as shown in Figure 4B, may also support that the responses derive from the host RGCs that may include the graft area in their receptive fields.