Retinal ON bipolar cells comprise about 70% of all bipolar cells. Glutamate hyperpolarizes these cells by binding to the metabotropic glutamate receptor mGluR6, activating the G-protein G o1 , and closing an unidentified cation channel. To facilitate investigation of ON bipolar cells, we here report on the production of a transgenic mouse (Grm6-GFP) in which EGFP, under control of mGluR6 promoter, was expressed in all and only ON bipolar cells. We used the mouse to determine density of ON bipolar cells, which in central retina was 29,600 cells/mm 2 . We further sorted the fluorescent cells and created a pure ON bipolar cDNA library that was negative for photoreceptor unique genes. Using this library, we determined expression of 27 genes of interest. We obtained positive transcripts for: G o interactors: regulators of G-protein signaling (RGS), Ret-RGS1 (a variant of RGS20), RGS16, RGS7, purkinje cell protein 2 (PCP2, also called L7 or GPSM4), synembryn (RIC-8), LGN (GPSM2), RAP1GAP, Gβ5; cGMP modulators: guanylyl cyclase (GC) 1α1, GC1β1, phosphodiesterase (PDE) 1C, PDE9A; and channels: inwardly rectifying potassium channel Kir2.4, transient receptor potential TRPC2, sperm-specific cation channels CatSper 2-4. The following transcripts were not found in our library: AGS3 (GPSM1), RGS10, RGS19 (GAIP), calbindin, GC1α2, GC1β2, PDE5, PDE2A, amiloride-sensitive sodium channel ACCN4, and CatSper1. We then localized Kir2.4 to several cell types, and showed that in ON bipolar cells, the channel concentrates in their dendritic tips. The channels and modulators found in ON bipolar cells likely shape their light response. Additional uses of the Grm6-GFP mouse are also discussed.

In darkness, glutamate released from photoreceptors activates the metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells. This activates the G protein G o , which then closes transient receptor potential melastatin 1 (TRPM1) channels, leading to cells' hyperpolarization. It has been generally assumed that deleting mGluR6 would render the cascade inactive and the ON bipolar cells constitutively depolarized. Here we show that the rod bipolar cells in mGluR6-null mice were hyperpolarized. The slope conductance of the current-voltage curves and the current noise were smaller than in wild type. Furthermore, while in wild-type rod bipolar cells, TRPM1 could be activated by local application of capsaicin; in null cells, it did not. These results suggest that the TRPM1 channel in mGluR6-null rod bipolar cells is inactive. To explore the reason for this lack of activity, we tested if mGluR6 deletion affected expression of cascade components. Immunostaining for G protein subunit candidates G␣ o , G␤ 3 , and G␥ 13 showed no significant changes in their expression or distribution. Immunostaining for TRPM1 in the dendritic tips was greatly reduced, but the channel was still present in the soma and primary dendrites of mGluR6-null bipolar cells, where a certain fraction of TRPM1 appears to localize to the plasma membrane. Consequently, the lack of TRPM1 activity in the null retina is unlikely to be due to failure of the channels to localize to the plasma membrane. We speculate that, to be constitutively active, TRPM1 channels in ON bipolar cells have to be in a complex, or perhaps require an unidentified factor. rod bipolar; metabotropic glutamate receptor 6-null; G protein cascade; transient receptor potential melastatin 1; regulator of G protein signaling IN RETINA, AN INCREMENT OF light intensity hyperpolarizes the photoreceptor and sets off two opposing signals in the OFF and ON bipolar cells. In darkness, glutamate released by photoreceptors depolarizes OFF bipolar cells and hyperpolarizes ON bipolar cells. The key steps in this ON bipolar cell "sign inverting" cascade are as follows: glutamate activates the metabotropic glutamate receptor 6 (mGluR6) (Akazawa et al.

Heterotrimeric G-proteins, comprising Gα and Gβγ subunits, couple metabotropic receptors to various downstream effectors and contribute to assembling and trafficking receptor based signaling complexes. A G-protein β-subunit, Gβ3 plays a critical role in several physiological processes as a polymorphism in its gene is associated with a risk factor for several disorders. Retinal ON bipolar cells express Gβ3, and they provide an excellent system to study its role. In the ON bipolar cells, mGluR6 inverts the photoreceptor’s signal via a cascade in which glutamate released from photoreceptors closes the TRPM1 channel. This cascade is essential for vision since deficiencies in its proteins lead to complete congenital stationary night blindness. Here we report that Gβ3 participates in the G-protein heterotrimer that couples mGluR6 to TRPM1. Gβ3 deletion in mouse greatly reduces the light response under both scotopic and photopic conditions, but it does not eliminate it. In addition, Gβ3 deletion causes mislocalization and downregulation of most cascade elements and modulators. Furthermore, Gβ3 may play a role in synaptic maintenance since in its absence, the number of invaginating rod bipolar dendrites is greatly reduced, a deficit that was not observed at 3 weeks, the end of the developmental period.

Melanoma-associated retinopathy (MAR) is characterized by night blindness, photopsias, and a selective reduction of the electroretinogram b-wave. In certain cases, the serum contains autoantibodies that react with ON bipolar cells, but the target of these autoantibodies has not been identified. Here we show that the primary target of autoantibodies produced in MAR patients with reduced b-wave is the TRPM1 cation channel, the newly identified transduction channel in ON bipolar cells. Sera from two well characterized MAR patients, but not from a control subject, stained human embryonic kidney cells transfected with the TRPM1 gene, and Western blots probed with these MAR sera showed the expected band size (~180 kDa). Staining of mouse and primate retina with MAR sera revealed immunoreactivity in all types of ON bipolar cells. Similar to staining for TRPM1, staining with the MAR sera was strong in dendritic tips and somas and was weak or absent in axon terminals. This staining co-localized with GFP in Grm6-GFP transgenic mice, where GFP is expressed in all and only ON bipolar cells, and also co-localized with Gαo, a marker for all types of ON bipolar cells. The staining in ON bipolar cells was confirmed to be specific to TRPM1 because MAR serum did not stain these cells in a Trpm1−/− mouse. Evidence suggests that the recognized epitope is likely intracellular, and the sera can be internalized by retinal cells. We conclude that the vision of at least some patients with MAR is compromised due to autoantibody-mediated inactivation of the TRPM1 channel.

The developmental switch of GABA's action from excitation to inhibition is likely due to a change in intracellular chloride concentration from high to low. Here we determined if the GABA switch correlates with the developmental expression patterns of KCC2, the chloride extruder K+-Cl- cotransporter, and NKCC, the chloride accumulator Na+-K+-Cl- cotransporter. Immunoblots of ferret retina showed that KCC2 upregulated in an exponential manner similar to synaptophysin (a synaptic marker). In contrast, NKCC, which was initially expressed at a constant level, upregulated quickly between P14 and P28, and finally downregulated to an adult level that was greater than the initial phase. At the cellular level, immunocytochemistry showed that in the inner plexiform layer KCC2's density increased gradually and its localization within ganglion cells shifted from being primarily in the cytosol (between P1-13) to being in the plasma membrane (after P21). In the outer plexiform layer, KCC2 was detected as soon as this layer started to form and increased gradually. Interestingly, however, KCC2 was initially restricted to photoreceptor terminals, while in the adult it was restricted to bipolar dendrites. Thus, the overall KCC2 expression level in ferret retina increases with age, but the time course differs between cell types. In ganglion cells the upregulation of KCC2 by itself cannot explain the relatively fast switch in GABA's action; additional events, possibly KCC2's integration into the plasma membrane and downregulation of NKCC, might also contribute. In photoreceptors the transient expression of KCC2 suggests a role for this transporter in development.