An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.

We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2^+/−; Crx^−/− retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx^−/− retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2^+/−; Crx^−/− retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.

Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms underlying this targeting and intracellular trafficking of transient receptor potential (TRP) channels remain poorly understood, and identifying proteins involved in these processes will provide insight into underlying mechanisms. Vision is dependent on the normal function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1) channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization to the synapse.

Crx, an Otx-like homeobox gene, is expressed primarily in the photoreceptors of the retina and in the pinealocytes of the pineal gland. The CRX homeodomain protein is a transactivator of many photoreceptor/pineal-specific genes in vivo, such as rhodopsin and the cone opsins. Mutations inCrx are associated with the retinal diseases, cone–rod dystrophy-2, retinitis pigmentosa, and Leber9s congenital amaurosis, which lead to loss of vision. We have generated transgenic mice, using 5′- and/or 3′-flanking sequences from the mouse Crxhomeobox gene fused to the β-galactosidase (lacZ) reporter gene, and we have investigated the promoter function of the cell-specific and developmentally regulated expression ofCrx. All of the independent transgenic lines commonly showed lacZ expression in the photoreceptor cells of the retina and in the pinealocytes of the pineal gland. We characterized the transgenic lines in detail for cell-specific lacZexpression patterns by 5-bromo-4-chloro-3-indolyl β-d-galactoside staining and lacZimmunostaining. The lacZ expression was observed in developing and developed photoreceptor cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with thelacZ and opsin antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of Crx during retinal development. This study demonstrates that the mouse Crx5′-upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of Crx in photoreceptor cells.