If present, ribbons in = 3; = 5; 0.0001, Wilcoxon rank sum test). Horizontal Cell-Ablated Mice Display a Retraction of Rod Terminals and Disrupted Ribbon Assembly The low OPL thickness in valueconditional knock-out mice, in which horizontal cells are partially mislocalized in the inner retina. without horizontal cells, the dendrites of rod bipolar cells never entered rod terminals. Furthermore, rods displayed significantly fewer and shorter presynaptic ribbons, suggesting that glutamate release is decreased, which coincided with significantly reduced expression of postsynaptic proteins (mGluR6, GPR179) in rod bipolar cells. Collectively, our findings uncover that horizontal cells are indeed necessary guideposts for rod bipolar cells. Whether horizontal cells release diffusible guidance cues or provide structural guidance by expressing specific cell adhesion molecules remains to be seen. conditional knock-out mice, in which horizontal cells are partially mispositioned to the inner retina. In addition, Wu et al. (2013) reported that photoreceptor terminals of knock-out mice, that lack 80% of horizontal cells, contained less invaginations and displayed a loss of the classic triadic organization of postsynaptic processes. These defects were already present at P16, shortly after triad formation is completed in wild-type mice, suggesting that horizontal cells might play an important role in the assembly of photoreceptor ribbon synapses. However, it still remains unclear to which extent synaptic contacts between photoreceptors and ON bipolar cells are formed in the absence of horizontal cells, as the removal of horizontal cells from the OPL has never been complete and synapse assembly has never been studied during development. In the present study, we investigated the role of horizontal cells in the assembly of the rod-to-rod bipolar cell synapse by specifically ablating horizontal cells IL18 antibody from the early postnatal mouse retina via diphtheria toxin receptor (DTR)-mediated cell knock-out. We monitored the formation of the rod synapse in the absence of horizontal cells using quantitative electron microscopy Carboxypeptidase G2 (CPG2) Inhibitor and immunohistochemistry. Our analysis revealed that invaginating (rod) ON bipolar cell dendrites were completely absent from horizontal cell-deficient rod terminals. Furthermore, synaptic ribbon assembly was disrupted and the expression of the postsynaptic proteins mGluR6 and GPR179 at the dendritic tips of rod bipolar cells was strongly reduced. These findings demonstrate that horizontal cells are critical for synapse formation between rods and rod bipolar cells. Materials and Methods Animals The generation of Cx57-DTRfrtCre mice has been described previously (Sonntag et al., 2012). Cx57-DTRfrtCre mice can be obtained from the European Mouse Mutant Archive. Animals were housed on a 12 h light/dark cycle with water and food ad libitum. For the experiments, mice of either sex were used. All procedures were performed in accordance with the law on animal protection (= 3C6 for each developmental stage) and = 3C6 for each developmental stage) mice were washed in 0.1 M PB (3 10 min) and cryoprotected with 30% sucrose in 0.1 M PB overnight at 4C. The following day, tissue was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek) and sectioned vertically at 20 m using a Leica CM1860 cryostat. Cryosections were blocked with 5% ChemiBLOCKER (Millipore), 0.3% Triton X-100 and 0.02% NaN3 Carboxypeptidase G2 (CPG2) Inhibitor in 0.1 M PB for 1 h at RT and incubated with primary antibodies in blocking solution overnight at 4C. A list of primary antibodies is given in Table 1. After washing in 0.1 M PB (3 10 min), sections were incubated with secondary antibodies in blocking solution for 2 h at RT, washed again in 0.1 M PB (3 10 min) and mounted in Vectashield (Vector Laboratories). Secondary antibodies used were from donkey or goat and conjugated to either Alexa 488 or Alexa 568 (1:600, Thermo Fisher Scientific). TABLE 1 List of primary antibodies used in this study. = Carboxypeptidase G2 (CPG2) Inhibitor 3 for P8, = 4 for P11, = 5 for P15, = 3 for P21) and = 3 for P8, = 4 for P11, = 3 for P15, = 2 for P21) retinae, we measured the distance from outer nuclear layer (ONL) to inner nuclear layer (INL) somata at 10 locations per animal using the line tool in Fiji. For comparison of rod synaptogenesis in = 4 for P11, = 3 for P15) and = 4 for P11, = 5 for P15), we analyzed between 746 and 1177 rod.