Complexins (Cplxs) regulate the speed and Ca(2+)-sensitivity of synaptic vesicle fusion. It has been shown that all four known Cplxs are present at mouse
retinal synapses--at conventional amacrine cell synapses (
Cplx 1 to
Cplx 3) and at photoreceptor and bipolar cell ribbon synapses (
Cplx 3 and
Cplx 4) [K. Reim et al. (2005) J. Cell Biol., 169, 669-680]. Electroretinographic recordings in
Cplx 3/
Cplx 4 double-knockout (DKO) mice showed perturbed transmission in the outer plexiform layer, and possible changes in the inner plexiform layer [K. Reim et al. (2009) J. Cell Sci., 122, 1352-1361]. In the present study, we examined the effects of the absence of
Cplx 3 and
Cplx 4 on
ganglion cell responses. We report that the lack of
Cplx 3 and
Cplx 4 differentially impacts the ON and OFF pathways. Under photopic conditions, the responses in the cone OFF pathway are largely unaffected, whereas the responses in the cone ON pathway are diminished in
Cplx 3/
Cplx 4 DKO mice. Under scotopic conditions, both ON and OFF response rates are reduced and high-sensitivity OFF responses are missing in
Cplx 3/
Cplx 4 DKO mice. The electrophysiological findings are corroborated by new immunocytochemical findings. We now show that rod spherules contain only
Cplx 4. However, both
Cplx 3 and
Cplx 4 co-localize in cone pedicles. In the inner plexiform layer,
Cplx 3 is present in rod bipolar cell terminals and in amacrine cell processes. Most importantly,
Cplx 3 is localized in the lobular appendages of AII amacrine cells, the sites of signal transmission from the primary rod pathway into the OFF pathway in the inner plexiform layer.