Catherine W. Morgans

TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells

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.

The nob2 mouse, a null mutation in Cacna1f : Anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses

Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the alpha1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.

Photoreceptor calcium channels: insight from night blindness.

The genetic locus for incomplete congenital stationary night blindness (CSNB2) has been identified as the CACNA1f gene, encoding the alpha 1F calcium channel subunit, a member of the L-type family of calcium channels. The electroretinogram associated with CSNB2 implicates alpha 1F in synaptic transmission between retinal photoreceptors and bipolar cells. Using a recently developed monoclonal antibody to alpha 1F, we localize the channel to ribbon active zones in rod photoreceptor terminals of the mouse retina, supporting a role for alpha 1F in mediating glutamate release from rods. Detergent extraction experiments indicate that alpha 1F is part of a detergent-resistant active zone complex, which also includes the synaptic ribbons. Comparison of native mouse rod calcium currents with recombinant alpha 1F currents reveals that the current-voltage relationship for the native current is shifted approximately 30 mV to more hyperpolarized potentials than for the recombinant alpha 1F current, suggesting modulation of the native channel by intracellular factors. Lastly, we present evidence for L-type alpha 1D calcium channel subunits in cone terminals of the mouse retina. The presence of alpha 1D channels in cones may explain the residual visual abilities of individuals with CSNB2.

TRPM1: The endpoint of the mGluR6 signal transduction cascade in retinal ON-bipolar cells

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.

Rod bipolar cells and horizontal cells form displaced synaptic contacts with rods in the outer nuclear layer of the nob2 retina

The nob2 mouse carries a null mutation in the Cacna1f gene, which encodes the pore‐forming subunit of the L‐type calcium channel, Cav1.4. The loss of the electroretinogram b‐wave in these mice suggests a severe reduction in transmission between photoreceptors and second‐order neurons in the retina and supports a central role for the Cav1.4 calcium channel at photoreceptor ribbon synapses, to which it has been localized. Here we show that the loss of Cav1.4 leads to the aberrant outgrowth of rod bipolar cell dendrites and horizontal cell processes into the outer nuclear layer (ONL) of the nob2 retina and to the formation of ectopic synaptic contacts with rod photoreceptors in the ONL. Ectopic contacts are predominantly between rods and rod bipolar cells, with horizontal cell processes also present at some sites. Ectopic contacts contain apposed pre‐ and postsynaptic specializations, albeit with malformed synaptic ribbons. Cone photoreceptor terminals do not participate in ectopic contacts in the ONL. During retinal development, ectopic contacts appear in the days after eye opening, appearing progressively farther into the ONL at later postnatal stages. Ectopic contacts develop at the tips of rod bipolar cell dendrites and are less frequently associated with the tips of horizontal cell processes, consistent with the adult phenotype. The relative occurrence of pre‐ and postsynaptic markers in the ONL during development suggests a mechanism for the formation of ectopic synaptic contacts that is driven by the retraction of rod photoreceptor terminals and neurite outgrowth by rod bipolar cell dendrites. J. Comp. Neurol. 500:286–298, 2007.

Localization of nyctalopin in the mammalian retina

Complete X‐linked congenital stationary night blindness (CSNB1) is a hereditary visual disease characterized by abnormalities in both the dark‐ and light‐adapted electroretinogram, consistent with a defect in synaptic transmission between photoreceptors and ON‐bipolar cells. The gene responsible for CSNB1, NYX, encodes a novel, leucine‐rich repeat protein, nyctalopin. Consistent with its predicted glycosylphosphatidylinositol linkage, we show that recombinant nyctalopin is targeted to the extracellular cell surface in transfected HEK293 cells. Within the retina, strong nyctalopin immunoreactivity is present in the outer plexiform layer, the site of the photoreceptor to bipolar cell synapses. Double labelling of nyctalopin and known synaptic proteins in the outer plexiform layer indicate that nyctalopin is associated with the ribbon synapses of both rod and cone terminals. In the inner plexiform layer, nyctalopin immunoreactivity is associated with rod bipolar cell terminals. Our findings support a role for nyctalopin in synaptic transmission and/or synapse formation at ribbon synapses in the retina.

Generation, identification and functional characterization of the nob4 mutation of Grm6 in the mouse

We performed genome-wide chemical mutagenesis of C57BL/6J mice using N-ethyl-N-nitrosourea (ENU). Electroretinographic screening of the third generation offspring revealed two G3 individuals from one G1 family with a normal a-wave but lacking the b-wave that we named nob4. The mutation was transmitted with a recessive mode of inheritance and mapped to chromosome 11 in a region containing the Grm6 gene, which encodes a metabotropic glutamate receptor protein, mGluR6. Sequencing confirmed a single nucleotide substitution from T to C in the Grm6 gene. The mutation is predicted to result in substitution of Pro for Ser at position 185 within the extracellular, ligand-binding domain and oocytes expressing the homologous mutation in mGluR6 did not display robust glutamate-induced currents. Retinal mRNA levels for Grm6 were not significantly reduced, but no immunoreactivity for mGluR6 protein was found. Histological and fundus evaluations of nob4 showed normal retinal morphology. In contrast, the mutation has severe consequences for visual function. In nob4 mice, fewer retinal ganglion cells (RGCs) responded to the onset (ON) of a bright full field stimulus. When ON responses could be evoked, their onset was significantly delayed. Visual acuity and contrast sensitivity, measured with optomotor responses, were reduced under both photopic and scotopic conditions. This mutant will be useful because its phenotype is similar to that of human patients with congenital stationary night blindness and will provide a tool for understanding retinal circuitry and the role of ganglion cell encoding of visual information.

Gβ5–RGs complexes co-localize with mGluR6 in retinal ON-bipolar cells

The time course of G‐protein‐coupled responses is largely determined by the kinetics of GTP hydrolysis by the G protein α 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 Gαo, 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 Gαo‐selective RGS proteins RGS7 and RGS11, with their obligate binding partner, Gβ5, 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 Gβ5 shifts away from the dendritic tips, implying a functional relationship with mGluR6. The precise co‐localization of Gβ5–RGS7 and Gβ5–RGS11 with mGluR6, and the dependence of localization on the presence of mGluR6, suggests that Gβ5–RGS7 and Gβ5–RGS11 function specifically in the mGluR6 signal transduction pathway, where they may stimulate the GTPase activity of Gαo, thus accelerating the ON‐bipolar cell light response, in a manner analogous to the acceleration of photoreceptor light responses by the Gβ5–RGS9‐1 complex.

Calcium-induced calcium release contributes to synaptic release from mouse rod photoreceptors.

We tested whether calcium-induced calcium release (CICR) contributes to synaptic release from rods in mammalian retina. Electron micrographs and immunofluorescent double labeling for the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) and synaptic ribbon protein, ribeye, showed a close association between ER and synaptic ribbons in mouse rod terminals. Stimulating CICR with 10 microM ryanodine evoked Ca(2+) increases in rod terminals from mouse retinal slices visualized using confocal microscopy with the Ca(2+)-sensitive dye, Fluo-4. Ryanodine also stimulated membrane depolarization of individual mouse rods. Inhibiting CICR with a high concentration of ryanodine (100 microM) reduced the electroretinogram (ERG) b-wave but not a-wave consistent with inhibition of synaptic transmission from rods. Ryanodine (100 microM) also inhibited light-evoked voltage responses of individual rod bipolar cells (RBCs) and presumptive horizontal cells recorded with perforated patch recording techniques. A presynaptic site of action for ryanodines effects is further indicated by the finding that ryanodine (100 microM) did not alter currents evoked in voltage-clamped RBCs by puffing the mGluR6 antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), onto bipolar cell dendrites in the presence of the mGluR6 agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4). Ryanodine (100 microM) also inhibited glutamatergic outward currents in RBCs evoked by electrical stimulation of rods using electrodes placed in the outer segment layer. Together, these results indicate that, like amphibian retina, CICR contributes to synaptic release from mammalian (mouse) rods. By boosting synaptic release in darkness, CICR may improve the detection of small luminance changes by post-synaptic neurons.

Gbeta5-RGS complexes co-localize with mGluR6 in retinal ON-bipolar cells.

The time course of G‐protein‐coupled responses is largely determined by the kinetics of GTP hydrolysis by the G protein α 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 Gαo, 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 Gαo‐selective RGS proteins RGS7 and RGS11, with their obligate binding partner, Gβ5, 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 Gβ5 shifts away from the dendritic tips, implying a functional relationship with mGluR6. The precise co‐localization of Gβ5–RGS7 and Gβ5–RGS11 with mGluR6, and the dependence of localization on the presence of mGluR6, suggests that Gβ5–RGS7 and Gβ5–RGS11 function specifically in the mGluR6 signal transduction pathway, where they may stimulate the GTPase activity of Gαo, thus accelerating the ON‐bipolar cell light response, in a manner analogous to the acceleration of photoreceptor light responses by the Gβ5–RGS9‐1 complex.