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Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). The exact role of LRIT3 in ON-bipolar cell signaling cascade remains to be elucidated. Recently, we have characterized a novel mouse model lacking Lrit3 [no b-wave 6, (Lrit3(nob6/nob6) )], which displays similar abnormalities to patients with cCSNB with LRIT3 mutations. Here we compare the localization of components of the ON-bipolar cell signaling cascade in wild-type and Lrit3(nob6/nob6) retinal sections by immunofluorescence confocal microscopy. An anti-LRIT3 antibody was generated. Immunofluorescent staining of LRIT3 in wild-type mice revealed a specific punctate labeling in the outer plexiform layer (OPL), which was absent in Lrit3(nob6/nob6) mice. LRIT3 did not co-localize with ribeye or calbindin but co-localized with mGluR6. TRPM1 staining was severely decreased at the dendritic tips of all depolarizing bipolar cells in Lrit3(nob6/nob6) mice. mGluR6, GPR179, RGS7, RGS11 and Gβ5 immunofluorescence was absent at the dendritic tips of cone ON-bipolar cells in Lrit3(nob6/nob6) mice, while it was present at the dendritic tips of rod bipolar cells. Furthermore, peanut agglutinin (PNA) labeling was severely reduced in the OPL in Lrit3(nob6/nob6) mice. This study confirmed the localization of LRIT3 at the dendritic tips of depolarizing bipolar cells in mouse retina and demonstrated the dependence of TRPM1 localization on the presence of LRIT3. As tested components of the ON-bipolar cell signaling cascade and PNA revealed disrupted localization, an additional function of LRIT3 in cone synapse formation is suggested. These results point to a possibly different regulation of the mGluR6 signaling cascade between rod and cone ON-bipolar cells., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Purpose: Protein kinase C α (PKCα) is abundantly expressed in rod bipolar cells (RBCs) in the retina, yet the physiological function of PKCα in these cells is not well understood. To elucidate the role of PKCα in visual processing in the eye, we examined the effect of genetic deletion of PKCα on the ERG and on RBC light responses in the mouse., Methods: Immunofluorescent labeling was performed on wild-type (WT), TRPM1 knockout, and PKCα knockout (PKC-KO) retina. Scotopic and photopic ERGs were recorded from WT and PKC-KO mice. Light responses of RBCs were measured using whole-cell recordings in retinal slices from WT and PKC-KO mice., Results: Protein kinase C alpha expression in RBCs is correlated with the activity state of the cell. Rod bipolar cells dendrites are a major site of PKCα phosphorylation. Electroretinogram recordings indicated that loss of PKCα affects the scotopic b-wave, including a larger peak amplitude, longer implicit time, and broader width of the b-wave. There were no differences in the ERG a- or c-wave between PKCα KO and WT mice, indicating no measurable effect of PKCα in photoreceptors or the RPE. The photopic ERG was unaffected consistent with the lack of detectable PKCα in cone bipolar cells. Whole-cell recordings from RBCs in PKC-KO retinal slices revealed that, compared with WT, RBC light responses in the PKC-KO retina are delayed and of longer duration., Conclusions: Protein kinase C alpha plays an important modulatory role in RBCs, regulating both the peak amplitude and temporal properties of the RBC light response in the rod visual pathway.

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma and the presence of autoantibodies that label neurons in the inner retina. The visual symptoms and electroretinogram (ERG) phenotype characteristic of MAR resemble the congenital visual disease caused by mutations in TRPM1, a cation channel expressed by both melanocytes and retinal bipolar cells. Four serum samples from MAR patients were identified as TRPM1 immunoreactive by 1. Labeling of ON-bipolar cells in TRPM1+/+ but not TRPM1-/- mouse retina, 2. Labeling of TRPM1-transfected CHO cells; and 3. Attenuation of the ERG b-wave following intravitreal injection of TRPM1-positive MAR IgG into wild-type mouse eyes, and the appearance of the IgG in the retinal bipolar cells at the conclusion of the experiment. Furthermore, the epitope targeted by the MAR autoantibodies was localized within the amino-terminal cytoplasmic domain of TRPM1. Incubation of live retinal neurons with TRPM1-positive MAR serum resulted in the selective accumulation of IgG in ON-bipolar cells from TRPM1+/+ mice, but not TRPM1-/- mice, suggesting that the visual deficits in MAR are caused by the uptake of TRPM1 autoantibodies into ON-bipolar cells, where they bind to an intracellular epitope of the channel and reduce the ON-bipolar cell response to light.

For almost 30 years the ion channel that initiates the ON visual pathway in vertebrate vision has remained elusive. Recent findings now indicate that the pathway, which begins with unbinding of glutamate from the metabotropic glutamate receptor 6 (mGluR6), ends with the opening of the transient receptor potential (TRP)M1 cation channel. As a component of the mGluR6 signal transduction pathway, mutations in TRPM1 would be expected to cause congenital stationary night blindness (CSNB), and several such mutations have already been identified in CSNB families. Furthermore, expression of TRPM1 in both the retina and skin raises the possibility that a genetic link exists between certain types of visual and skin disorders.

The rate-limiting step in the recovery of the photoreceptor light response is the hydrolysis of GTP by transducin, a reaction that is accelerated by the RGS9-Gbeta5 complex, and its membrane anchor, R9AP. Similar complexes, including RGS7, RGS11, and Gbeta5, are found in retinal ON-bipolar cell dendrites. Here, we present evidence that R9AP is also expressed in the dendritic tips of ON-bipolar cells. Immunofluorescent staining for R9AP revealed a punctate pattern of labeling in the outer plexiform layer, where it colocalized with mGluR6. In photoreceptors, R9AP is required for proteolytic stability of the entire regulator of G protein signaling complex, and we found that genetic deletion of R9AP also results in a marked reduction in the levels of RGS11 and Gbeta5 in the bipolar cell dendrites; the level of RGS7 was unaffected, suggesting the presence of another interaction partner to stabilize RGS7. To determine the effect of R9AP deletion on the response kinetics of ON-bipolar cells, we compared the electroretinogram (ERG) between wild-type and R9AP-deficient mice. The ERG b-wave, reflecting ON-bipolar cell activity, was delayed and larger in the R9AP-deficient mice. Our data indicate that R9AP is required for stable expression of RGS11-Gbeta5 in ON-bipolar cell dendrites. Furthermore, they suggest that the RGS11-Gbeta5-R9AP complex accelerates the initial ON-bipolar cell response to light.

Purpose: The retinal ON-bipolar cell (ON-BPC) light response is initiated upon deactivation of the metabotropic glutamate receptor mGluR6 and the G protein Go. G protein-based signaling cascades are typically accelerated by interaction of the G protein alpha subunit with a member of the regulator of G protein signaling (RGS) protein family. The goal of this study was to determine whether RGS7 and/or -11 serve this function in retinal ON-BPCs., Methods: Retinas from mice lacking RGS11 (RGS11(-/-)), or with a deletion mutation in RGS7 (RGS7(Delta/Delta)), or both, were compared to wild-type (WT) by immunofluorescence confocal microscopy. The retinal light response was measured with the electroretinogram (ERG). The kinetics of simulated light responses from individual rod bipolar cells were recorded by whole-cell patch-clamp electrophysiology., Results: Levels of the R7 RGS interaction partners, Gbeta5 and R9AP, were reduced in the outer plexiform layer of the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. ERG recordings demonstrated a delay in the rising phase of the ERG b-wave, larger photopic b-wave amplitudes, and increased scotopic threshold response sensitivity in the RGS11(-/-) and RGS7(Delta/Delta)/RGS11(-/-) mice. The ERG measured from the RGS7(Delta/Delta) retina was normal. Patch-clamp recordings of chemically simulated light responses of rod BPCs revealed a 25-ms delay in the onset of the ON-BPC response in the RGS7(Delta/Delta)/RGS11(-/-) mouse compared with the WT., Conclusions: RGS11 plays a role in the deactivation of Galphao, which precedes activation of the depolarizing current in ON-BPCs. RGS7 must also serve a role as changes in RGS7(Delta/Delta)/RGS11(-/-) mice were greater than those in RGS11(-/-) mice.

The ON pathway of the visual system, which detects increases in light intensity, is established at the first retinal synapse between photoreceptors and ON-bipolar cells. Photoreceptors hyperpolarize in response to light and reduce the rate of glutamate release, which in turn causes the depolarization of ON-bipolar cells. This ON-bipolar cell response is mediated by the metabotropic glutamate receptor, mGluR6, which controls the activity of a depolarizing current. Despite intensive research over the past two decades, the molecular identity of the channel that generates this depolarizing current has remained elusive. Here, we present evidence indicating that TRPM1 is necessary for the depolarizing light response of ON-bipolar cells, and further that TRPM1 is a component of the channel that generates this light response. Gene expression profiling revealed that TRPM1 is highly enriched in ON-bipolar cells. In situ hybridization experiments confirmed that TRPM1 mRNA is found in cells of the retinal inner nuclear layer, and immunofluorescent confocal microscopy showed that TRPM1 is localized in the dendrites of ON-bipolar cells in both mouse and macaque retina. The electroretinogram (ERG) of TRPM1-deficient (TRPM1(-/-)) mice had a normal a-wave, but no b-wave, indicating a loss of bipolar cell response. Finally, whole-cell patch-clamp recording from ON-bipolar cells in mouse retinal slices demonstrated that genetic deletion of TRPM1 abolished chemically simulated light responses from rod bipolar cells and dramatically altered the responses of cone ON-bipolar cells. Identification of TRPM1 as a mGluR6-coupled cation channel reveals a key step in vision, expands the role of the TRP channel family in sensory perception, and presents insights into the evolution of vertebrate vision.

Photoreceptor degenerations can trigger morphological alterations in second-order neurons, however, the functional implications of such changes are not well known. We conducted a longitudinal study, using whole-cell patch-clamp, immunohistochemistry and electron microscopy to correlate physiological with anatomical changes in bipolar cells of the rd10 mouse - a model of autosomal recessive retinitis pigmentosa. Rod bipolar cells (RBCs) showed progressive changes in mGluR6-induced currents with advancing rod photoreceptor degeneration. Significant changes in response amplitude and kinetics were observed as early as postnatal day (P)20, and by P45 the response amplitudes were reduced by 91%, and then remained relatively stable until 6 months. These functional changes correlated with the loss of rod photoreceptors and mGluR6 receptor expression. Moreover, we showed that RBCs make transient ectopic connections with cones during progression of the disease. At P45, ON-cone bipolar cells (ON-CBCs) retain mGluR6 responses for longer periods than the RBCs, but by about 6 months these cells also strongly downregulate mGluR6 expression. We propose that the relative longevity of mGluR6 responses in CBCs is due to the slower loss of the cones. In contrast, ionotropic glutamate receptor expression and function in OFF-CBCs remains normal at 6 months despite the loss of synaptic input from cones. Thus, glutamate receptor expression is differentially regulated in bipolar cells, with the metabotropic receptors being absolutely dependent on synaptic input. These findings define the temporal window over which bipolar cells may be receptive to photoreceptor repair or replacement.

The time course of G-protein-coupled responses is largely determined by the kinetics of GTP hydrolysis by the G protein alpha subunit, which is accelerated by interaction with regulator of G-protein signaling (RGS) proteins. Light responses of ON-bipolar cells of the vertebrate retina require rapid inactivation of the G protein Galphao, which is activated in the dark by metabotropic glutamate receptor, mGluR6, in their dendritic tips. It is not yet known, however, which RGS protein(s) might be responsible for rapid inactivation kinetics. By immunofluorescence and co-immunoprecipitation, we have identified complexes of the Galphao-selective RGS proteins RGS7 and RGS11, with their obligate binding partner, Gbeta5, that are localized to the dendritic tips of murine rod and cone ON-bipolar cells, along with mGluR6. Experiments using pre- and post-synaptic markers, and a dissociated bipolar cell preparation, clearly identified the location of these complexes as the ON-bipolar cell dendritic tips and not the adjacent photoreceptor terminals or horizontal cell dendrites. In mice lacking mGluR6, the distribution of RGS11, RGS7 and Gbeta5 shifts away from the dendritic tips, implying a functional relationship with mGluR6. The precise co-localization of Gbeta5-RGS7 and Gbeta5-RGS11 with mGluR6, and the dependence of localization on the presence of mGluR6, suggests that Gbeta5-RGS7 and Gbeta5-RGS11 function specifically in the mGluR6 signal transduction pathway, where they may stimulate the GTPase activity of Galphao, thus accelerating the ON-bipolar cell light response, in a manner analogous to the acceleration of photoreceptor light responses by the Gbeta5-RGS9-1 complex.