Dysfunctional Light-Evoked Regulation of cAMP in Photoreceptors and Abnormal Retinal Adaptation in Mice Lacking Dopamine D4 Receptors

Dopamine is a retinal neuromodulator that has been implicated in many aspects of retinal physiology. Photoreceptor cells express dopamine D4 receptors that regulate cAMP metabolism. To assess the effects of dopamine on photoreceptor physiology, we examined the morphology, electrophysiology, and regulation of cAMP metabolism in mice with targeted disruption of the dopamine D4 receptor gene. Photoreceptor morphology and outer segment disc shedding after light onset were normal in D4 knock-out (D4KO) mice. Quinpirole, a dopamine D2/ D3/D4 receptor agonist, decreased cAMP synthesis in retinas of wild-type (WT) mice but not in retinas of D4KO mice. In WT retinas, the photoreceptors of which were functionally isolated by incubation in the presence of exogenous glutamate, light also suppressed cAMP synthesis. Despite the similar inhibition of cAMP synthesis, the effect of light is directly on the photoreceptors and independent of dopamine modulation, because it was unaffected by application of the D4 receptor antagonist L-745,870. Nevertheless, compared with WT retinas, basal cAMP formation was reduced in the photoreceptors of D4KO retinas, and light had no additional inhibitory effect. The results suggest that dopamine, via D4 receptors, normally modulates the cascade that couples light responses to adenylyl cyclase activity in photoreceptor cells, and the absence of this modulation results in dysfunction of the cascade. Dark-adapted electroretinogram (ERG) responses were normal in D4KO mice. However, ERG b-wave responses were greatly suppressed during both light adaptation and early stages of dark adaptation. Thus, the absence of D4 receptors affects adaptation, altering transmission of light responses from photoreceptors to inner retinal neurons. These findings indicate that dopamine D4 receptors normally play a major role in regulating photoreceptor cAMP metabolism and adaptive retinal responses to changing environmental illumination.Fil: Nir, Izhak. The University of Texas Health Science Center; Estados UnidosFil: Harrison, Joseph M.. The University of Texas Health Science Center; Estados UnidosFil: Haque, Rashidul. Emory University School of Medicine; Estados UnidosFil: Low, Malcolm J.. Oregon Health and Science University; Estados UnidosFil: Grandy, David K.. Oregon Health and Science University; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Iuvone, P. Michael. Emory University School of Medicine; Estados Unido

Ion transport modeling for retinal rod photoreceptor cells

"July 2010.""A Thesis presented to the Faculty of the Graduate School at the University of Missouri In Partial Fulfillment of the Requirements for the Degree Master of Science."Thesis supervisor: Dr. Jinglu Tan.In this study, a mathematical model is developed to describe the ion transport activities associated with the response of rod photoreceptor to light stimulus. In the model, the cell body is modeled as two capacitors connected via the connecting cilium. Roles of different ion channels during a photoreceptor light response are analyzed, and the relations between changes in ion concentration and response currents are assessed. Methods are developed for computing the membrane potential from ion concentrations and relating the material and electrical resistances. The steady state under different conditions can be uniquely defined with only three measured values. The model can effectively describe the rod photoreceptor response to different light stimuli. Model simulation of the a-wave for progressive narrowing of the connecting cilium corresponds well with published literature on hereditary retinal degeneration of Abyssinian cats. Reductions in amplitude and changes in the a-wave waveform are observed in different stages of the disease. Changes in the receptor response amplitude may not be measurable till the conductance of the connecting cilium is reduced to a comparable magnitude of the ion channels. The model can provide quantitative information of ionic activities, changes in ion concentrations and membrane voltage in the outer segment and the inner compartment. The ionic environment is found to be different between the outer segment and the inner compartment. During receptor response, changes in the outer segment appear to be stronger and quicker than those in the inner compartment. Reductions in the connecting cilium transport can reset the dark resting state.Includes bibliographical references (pages 87-91)

Effects of extracellular Ca++, K+, and Na+ on cone and retinal pigment epithelium retinomotor movements in isolated teleost retinas.

We have examined the effects of changes in extracellular ionic composition on cone and retinal pigment epithelium (RPE) retinomotor movements in cultured isolated teleost retinas. In vivo, the myoid portion of teleost cones contracts in the light and elongates in the dark; RPE pigment disperses in the light and aggregates in the dark. In vitro, cones of dark-adapted (DA) retinas cultured in constant darkness contracted spontaneously to their light-adapted (LA) positions if the culture medium contained greater than or equal to 10(-3)M Cao++. DA cones retained their long DA positions in a medium containing less than or equal to 10(-6)M Cao++. Low [Ca++]o (10(-5)-10(-7)M) also permitted darkness to induce cone elongation and RPE pigment aggregation. Light produced cone contraction even in the absence of Cao++, but the extent of contraction was reduced if [Ca++]o was less than 10(-3) M. Thus, full contraction appeared to require the presence of external Ca++. High [K+]o (greater than or equal to 27 mM) inhibited both light-induced and light-independent Ca++-induced cone contraction. However, low [Na+]o (3.5 mM) in the presence of less than or equal to 10(-6)M Cao++ did not mimic light onset by inducing cone contraction in the dark. High [K+]o also promoted dark-adaptive cone and RPE movements in LA retinas cultured in the light. All results obtained in high [K+]o were similar to those observed when DA or LA retinas were exposed to treatments that elevate cytoplasmic cyclic 3,5-adenosine monophosphate (cAMP) content

Absence of synaptic regulation by phosducin in retinal slices.

Phosducin is an abundant photoreceptor protein that binds G-protein βγ subunits and plays a role in modulating synaptic transmission at photoreceptor synapses under both dark-adapted and light-adapted conditions in vivo. To examine the role of phosducin at the rod-to-rod bipolar cell (RBC) synapse, we used whole-cell voltage clamp recordings to measure the light-evoked currents from both wild-type (WT) and phosducin knockout (Pd(-/-)) RBCs, in dark- and light-adapted retinal slices. Pd(-/-) RBCs showed smaller dim flash responses and steeper intensity-response relationships than WT RBCs, consistent with the smaller rod responses being selectively filtered out by the non-linear threshold at the rod-to-rod bipolar synapse. In addition, Pd(-/-) RBCs showed a marked delay in the onset of the light-evoked currents, similar to that of a WT response to an effectively dimmer flash. Comparison of the changes in flash sensitivity in the presence of steady adapting light revealed that Pd(-/-) RBCs desensitized less than WT RBCs to the same intensity. These results are quantitatively consistent with the smaller single photon responses of Pd(-/-) rods, owing to the known reduction in rod G-protein expression levels in this line. The absence of an additional synaptic phenotype in these experiments suggests that the function of phosducin at the photoreceptor synapse is abolished by the conditions of retinal slice recordings

Outer Retinal Structure in Best Vitelliform Macular Dystrophy

Importance Demonstrating the utility of adaptive optics scanning light ophthalmoscopy (AOSLO) to assess outer retinal structure in Best vitelliform macular dystrophy (BVMD). Objective To characterize outer retinal structure in BVMD using spectral-domain optical coherence tomography (SD-OCT) and AOSLO. Design, Setting, and Participants Prospective, observational case series. Four symptomatic members of a family with BVMD with known BEST1 mutation were recruited at the Advanced Ocular Imaging Program research lab at the Medical College of Wisconsin Eye Institute, Milwaukee. Intervention Thickness of 2 outer retinal layers corresponding to photoreceptor inner and outer segments was measured using SD-OCT. Photoreceptor mosaic AOSLO images within and around visible lesions were obtained, and cone density was assessed in 2 subjects. Main Outcome and Measure Photoreceptor structure. Results Each subject was at a different stage of BVMD, with photoreceptor disruption evident by AOSLO at all stages. When comparing SD-OCT and AOSLO images from the same location, AOSLO images allowed for direct assessment of photoreceptor structure. A variable degree of retained photoreceptors was seen within all lesions. The photoreceptor mosaic immediately adjacent to visible lesions appeared contiguous and was of normal density. Fine hyperreflective structures were visualized by AOSLO, and their anatomical orientation and size were consistent with Henle fibers. Conclusions and Relevance The AOSLO findings indicate that substantial photoreceptor structure persists within active lesions, accounting for good visual acuity in these patients. Despite previous reports of diffuse photoreceptor outer segment abnormalities in BVMD, our data reveal normal photoreceptor structure in areas adjacent to clinical lesions. This study demonstrates the utility of AOSLO for understanding the spectrum of cellular changes that occur in inherited degenerations such as BVMD. Photoreceptors are often significantly affected at various stages of inherited degenerations, and these changes may not be readily apparent with current clinical imaging instrumentation

Localization of zinc transporter-3 (ZnT-3) in mouse retina

AbstractStudies of the central nervous system have localized the zinc-transporter-3 (ZnT-3) protein to synaptic vesicles containing glutamate and zinc. We have examined the distribution of the ZnT-3 protein in the light-adapted mouse retina using immunohistochemical techniques. Light microscopic analysis of 15–30-μm retinal sections revealed a rich band of ZnT-3 protein in the region of the outer limiting membrane and photoreceptor inner segments. ZnT-3 reactivity was also present in the outer plexiform, inner nuclear, inner plexiform, and ganglion cell layers. The outer nuclear layer and photoreceptor outer segments did not exhibit ZnT-3 immunoreactivity. In the light-adapted murine retina, ZnT-3 appears localized in regions which have been found reactive for ionic zinc

Functional Characterization and Molecular Cloning of the K+-dependent Na+/Ca2+ Exchanger in Intact Retinal Cone Photoreceptors

Light-dependent changes in cytoplasmic free Ca2+ are much faster in the outer segment of cone than rod photoreceptors in the vertebrate retina. In the limit, this rate is determined by the activity of an electrogenic Na+/Ca2+ exchanger located in the outer segment plasma membrane. We investigate the functional properties of the exchanger activity in intact, single cone photoreceptors isolated from striped bass retina. Exchanger function is characterized through analysis both of the electrogenic exchanger current and cytoplasmic free Ca2+ measured with optical probes. The exchanger in cones is K+ dependent and operates both in forward and reverse modes. In the reverse mode, the K+ dependence of the exchanger is described by binding to a single site with K1/2 about 3.6 mM. From the retina of the fish we cloned exchanger molecules bassNCKX1 and bassNCKX2. BassNCKX1 is a single class of molecules, homologous to exchangers previously cloned from mammalian rods. BassNCKX2 exists in four splice variants that differ from each other by small sequence differences in the single, large cytoplasmic loop characteristic of these molecules. We used RT-PCR (reverse transcriptase polymerase chain reaction) of individual cells to identify the exchanger molecule specifically expressed in bass single and twin cone photoreceptors. Each and every one of the four bassNCKX2 splice variants is expressed in both single and twin cones indistinguishably. BassNCKX1 is not expressed in cones and, by exclusion, it is likely to be an exchanger expressed in rods