Marie E. Fina

Probing Neurochemical Structure and Function of Retinal ON Bipolar Cells with a Transgenic Mouse

Retinal ON bipolar cells make up about 70% of all bipolar cells. Glutamate hyperpolarizes these cells by binding to the metabotropic glutamate receptor mGluR6, activating the G‐protein Go1, 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 enhanced green fluorescent protein (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/mm2. We further sorted the fluorescent cells and created a pure ON bipolar cDNA library that was negative for photoreceptor unique genes. With this library, we determined expression of 27 genes of interest. We obtained positive transcripts for Go 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, and Gβ5; cGMP modulators: guanylyl cyclase (GC) 1α1, GC1β1, phosphodiesterase (PDE) 1C, and PDE9A; and channels: inwardly rectifying potassium channel Kir2.4, transient receptor potential TRPC2, and 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. J. Comp. Neurol. 510:484–496, 2008.

Regulation of KCC2 and NKCC during development: membrane insertion and differences between cell types.

The developmental switch of GABAs 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 KCC2s 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 GABAs action; additional events, possibly KCC2s 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. J. Comp. Neurol. 499:132–143, 2006.

Autoantibodies in Melanoma-Associated Retinopathy Target TRPM1 Cation Channels of Retinal ON Bipolar Cells

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 colocalized with GFP in Grm6-GFP transgenic mice, where GFP is expressed in all and only ON bipolar cells, and also colocalized 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.

Gβ3 Is Required for Normal Light ON Responses and Synaptic Maintenance

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 photoreceptors 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.

Cones Respond to Light in the Absence of Transducin β Subunit

Mammalian cones respond to light by closing a cGMP-gated channel via a cascade that includes a heterotrimeric G-protein, cone transducin, comprising Gαt2, Gβ3 and Gγt2 subunits. The function of Gβγ in this cascade has not been examined. Here, we investigate the role of Gβ3 by assessing cone structure and function in Gβ3-null mouse (Gnb3−/−). We found that Gβ3 is required for the normal expression of its partners, because in the Gnb3−/− cone outer segments, the levels of Gαt2 and Gγt2 are reduced by fourfold to sixfold, whereas other components of the cascade remain unaltered. Surprisingly, Gnb3−/− cones produce stable responses with normal kinetics and saturating response amplitudes similar to that of the wild-type, suggesting that cone phototransduction can function efficiently without a Gβ subunit. However, light sensitivity was reduced by approximately fourfold in the knock-out cones. Because the reduction in sensitivity was similar in magnitude to the reduction in Gαt2 level in the cone outer segment, we conclude that activation of Gαt2 in Gnb3−/− cones proceeds at a rate approximately proportional to its outer segment concentration, and that activation of phosphodiesterase and downstream cascade components is normal. These results suggest that the main role of Gβ3 in cones is to establish optimal levels of transducin heteromer in the outer segment, thereby indirectly contributing to robust response properties.

Differential function of Gγ13 in rod bipolar and ON cone bipolar cells

The G‐protein‐mediated metabotropic glutamate receptor 6 (mGluR6) signalling cascade is critical for light responses in all retinal ON bipolar cells. The present study evaluates the role of G‐protein subunit Gγ13 in ON bipolar cells by deleting the mouse Gng13 gene. Gγ13 is a partner of Gβ3 in all types of retinal ON bipolar cells, and contributes to generating the light response. Gγ13 also contributes to maintenance because, in its absence, the light response decreases with age. Three independent functionality assessments (i.e. light response as measured by electroretinogram b‐waves, localization of GTPase activating proteins and deterioration of the light response with age) show that the contribution of Gγ13 is much greater in rod bipolar cells than in ON cone bipolar cells. Our data also suggest that the contribution of Gγ13 to coupling mGluR6 to its effector is smaller than its contribution to maintaining GTPase activating protein localization.

mGluR6 Transcripts in Non-neuronal Tissues

To study mGluR6 expression, the authors investigated two transgenic mouse lines that express enhanced green fluorescent protein (GFP) under control of mGluR6 promoter. In retina, GFP was expressed exclusively in all ON bipolar cell types, either uniformly across all cells of this class (line 5) or in a mosaic (patchy) fashion (line 1). In brain, GFP was found in certain cortical areas, superior colliculus, axons of the corpus callosum, accessory olfactory bulb, and cells of the subcommissural organ. Outside the nervous system, GFP was seen in the corneal endothelium, testis, the kidney’s medulla, collecting ducts and parietal layer that surround the glomeruli, and B lymphocytes. Furthermore, RT-PCR showed that most tissues that expressed GFP in the transgenic mouse also transcribed two splice variants of mGluR6 in the wild-type mouse. The alternate variant was lacking exon 8, predicting a protein product of 545 amino acids that lacks the 7-transmembrane domains of the receptor. In cornea, immunostaining for mGluR6 gave strong staining in the endothelium, and this was stronger in wild-type than in mGluR6-null mice. Furthermore, calcium imaging with Fura-2 showed that application of L-AP4, an agonist for group III metabotropic glutamate receptors including mGluR6, elevated calcium in endothelial cells.

Immunocytochemical evidence that monkey rod bipolar cells use GABA.

Certain bipolar cells in most species immunostain for GABA or its synthesizing enzyme glutamic acid decarboxylase. However, it is unknown whether they actually release GABA and, if so, from which cellular compartment and by what release mechanism. We investigated these questions in monkey retina where rod bipolar cells immunostain for GABA. We found that rod bipolar cells immunostain for one isoform of GAD (GAD65) in their somas, dendrites and axon terminals. Near the fovea, the somatic stain of rod bipolar cells is weaker than that of horizontal cells but, at the periphery, it is stronger. Staining for the vesicular GABA transporter in monkey rod bipolar cells is negative. However, staining for the GABA transporter GAT3 is positive in the soma and primary dendrites (but not in the axon terminals). Staining for GAT3 is also positive in horizontal cells. Double staining of rod bipolar cells and the alpha subunit of the GABAA receptor reveals scarce GABAA puncta that appose rod bipolar dendrites. We conclude that monkey rod bipolar cells use GABA and discuss the possibility that they tonically release GABA from their dendrites using a reverse action of GAT3.

Ret-PCP2 colocalizes with protein kinase C in a subset of primate ON cone bipolar cells

Purkinje cell protein 2 (PCP2), a member of the family of guanine dissociation inhibitors and a strong interactor with the G‐protein subunit Gαo, localizes to retinal ON bipolar cells. The retina‐specific splice variant of PCP2, Ret‐PCP2, accelerates the light response of rod bipolar cells by modulating the mGluR6 transduction cascade. All ON cone bipolar cells express mGluR6 and Gαo, but only a subset expresses Ret‐PCP2. Here we test the hypothesis that Ret‐PCP2 contributes to shaping the various temporal bandwidths of ON cone bipolar cells in monkey retina. We found that the retinal splice variants in monkey and mouse are similar and longer than the cerebellar variants. Ret‐PCP2 is strongly expressed by diffuse cone bipolar type 4 cells (DB4; marked with anti‐PKCα) and weakly expressed by midget bipolar dendrites (labeled by antibodies against Gαo, Gγ13, or mGluR6). Ret‐PCP2 is absent from diffuse cone bipolar type 6 (DB6; marked with anti‐CD15) and blue cone bipolar cells (marked with anti‐CCK precursor). Thus, cone bipolar cells that terminate in stratum 3 of the inner plexiform layer (DB4) express more Ret‐PCP2 than those that terminate in strata 3 + 4 (midget bipolar cells), and these in turn express more than those that terminate in stratum 5 (DB6 and blue cone bipolar cells). This expression pattern approximates the arborization of ganglion cells (GC) with different temporal bandwidths: parasol GCs stratifying near stratum 3 are faster than midget GCs stratifying in strata 3 + 4, and these are probably faster than the sluggish GCs that arborize in stratum 5. J. Comp. Neurol. 518:1098–1112, 2010.

Kir2.4 Surface Expression and Basal Current Are Affected by Heterotrimeric G-Proteins

Background: Basal activity of the inward rectifying potassium channel Kir2.4 is important for a variety of neuronal functions. Results: Pertussis toxin-sensitive Gα subunits reduce basal current and surface expression of Kir2.4, whereas Gβγ increases them. Conclusion: Heterotrimeric G-proteins regulate the surface expression of potassium channels. Significance: This study extends the role of G-protein subunits in modulating neuronal physiology by regulating the expression of channel proteins. Kir2.4, a strongly rectifying potassium channel that is localized to neurons and is especially abundant in retina, was fished with yeast two-hybrid screen using a constitutively active Gαo1. Here, we wished to determine whether and how Gαo affects this channel. Using transfected HEK 293 cells and retinal tissue, we showed that Kir2.4 interacts with Gαo, and this interaction is stronger with the GDP-bound form of Gαo. Using two-electrode voltage clamp, we recorded from oocytes that were injected with Kir2.4 mRNA and a combination of G-protein subunit mRNAs. We found that the wild type and the inactive mutant of Gαo reduce the Kir2.4 basal current, whereas the active mutant has little effect. Other pertussis-sensitive Gα subunits also reduce this current, whereas Gαs increases it. Gβγ increases the current, whereas m-phosducin, which binds Gβγ without affecting the state of Gα, reduces it. We then tested the effect of G-protein subunits on the surface expression of the channel fused to cerulean by imaging the plasma membranes of the oocytes. We found that the surface expression is affected, with effects paralleling those seen with the basal current. This suggests that the observed effects on the current are mainly indirect and are due to surface expression. Similar results were obtained in transfected HEK cells. Moreover, we show that in retinal ON bipolar cells lacking Gβ3, localization of Kir2.4 in the dendritic tips is reduced. We conclude that Gβγ targets Kir2.4 to the plasma membrane, and Gαo slows this down by binding Gβγ.