Within the NFL, we identified dyad synapses, which are characterized by glutamatergic bipolar cell endings onto AC and RGC dendrites. These contacts were characterized by a presynaptic ribbon surrounded by synaptic vesicles in the bipolar ending, an enlarged synaptic cleft, and prominent postsynaptic densities in both members of the dyad ( Figure 4F). Thus in fat3KOs, ACs form stable synapses in ectopic locations that are maintained into adulthood. Altogether, the ultrastructural evidence, presence of synaptic proteins, and recruitment of bipolar cell endings indicate that ectopic AC dendrites produce bona fide plexiform layers in fat3KOs. Therefore, we refer

to the new layer in the INL as the outer misplaced plexiform layer (OMPL), and the layer inside of the GCL as the inner misplaced plexiform layer (IMPL). The addition of two new plexiform layers is accompanied by a striking re-organization of the cellular layers AZD2281 ic50 in fat3KO retinas. First, the OMPL creates a break at the level of the Müller glia cell bodies that separates

the majority of ACs from the remainder of the INL ( Figures 5A and 5B). Second, and more unexpectedly, the GCL is thicker than in control retinas, with a ∼45% increase in total cell number ( Figure 5K). The additional cells are not RGCs, as demonstrated by expression of the RGC marker Brn3 ( Figures 5C, 5D, and 5K). Instead, there is a significant increase in the number of displaced ACs in the GCL of fat3KOs compared with littermate controls ( Figures 5E and 5F). Because there is no change R428 in vivo in total AC number between genotypes ( Figure 5K), we conclude that the increase in GCL content reflects changes in AC distribution rather than proliferation. Consistent with this finding, we also observed a ∼50% reduction in the frequency of calretinin-positive PtdIns(3,4)P2 ACs in the mutant INL ( Figures 3A and 3B). These changes in retinal lamination could reflect an additional function for Fat3 in migration or could be secondary to the presence of the IMPL and OMPL. To distinguish between these possibilities, we asked whether specific classes of ACs are affected using two

general markers: the transcription factor Bhlhb5, which is present in populations of GABAergic ACs and off-cone bipolars (Feng et al., 2006), and EBF, which is expressed by glycinergic ACs with the exception of the AIIs (Voinescu et al., 2009). The AII cells were marked by Dab1 (Rice and Curran, 2000) and the cholinergic starburst ACs by ChAT. We found that GABAergic AC distribution is specifically disrupted by loss of fat3, with a significant proportion of Bhlhb5-positive cells mislocalized in the GCL or trapped within the IPL ( Figure 5G-H,K). In contrast, glycinergic ACs and the starburst cells, which are equally divided between the INL and GCL in WT retina, are properly distributed in fat3KOs ( Figures 5G–5K).