Retinal dopamine mediates multiple dimensions of light-adapted vision

Abstract

Dopamine is a key neuromodulator in the retina and brain that supports motor, cognitive, and visual function. Here, we developed a mouse model on a C57 background in which expression of the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase, is specifically disrupted in the retina. This model enabled assessment of the overall role of retinal dopamine in vision using electrophysiological (electroretinogram), psychophysical (optokinetic tracking), and pharmacological techniques. Significant disruptions were observed in high-resolution, light-adapted vision caused by specific deficits in light responses, contrast sensitivity, acuity, and circadian rhythms in this retinal dopamine-depleted mouse model. These global effects of retinal dopamine on vision are driven by the differential actions of dopamine D1 and D4 receptors on specific retinal functions and appear to be due to the ongoing bioavailability of dopamine rather than developmental effects. Together, our data indicate that dopamine is necessary for the circadian nature of light-adapted vision as well as optimal contrast detection and acuity.

Figure 1.

Generation of retina-specific Th knock-out mice. A, Generation of conditional Th mice. Blue boxes, exons (Ex); gray arrow, negative selection marker Pgk-DTA; red arrow, neomycin resistance gene under the control of the Sv40 promoter (SvNeo); black ovals, loxP sites; green diamonds, Frt sites; black arrows depict Cre recombinase under the control of the Chx10 promoter (white box). Primers a plus b amplify 150 bp for the WT Th allele and 200 bp for the Th^lox allele, while primers a plus c yield a single ∼1000 bp band for homozygous mice, a single ∼900 bp band for WT mice, and two bands for hemizygous mice. B, Retinal dopamine levels in Ctrl (Th^lox/lox), hemizygous-Ctrl (Chx10-Cre:Th^lox/+), and homozygous-rTHKO mice (Chx10-Cre:Th^lox/lox) as measured by HPLC. HPLC was performed as described by (Ruan et al., 2008). Data are represented as means ± SEM (n = 4). C, TH-immunoreactive (TH⁺) cell number in the Ctrl (Th^lox/lox), Ctrl (Chx10-Cre:Th^lox/+), and rTHKO (Chx10-Cre:Th^lox/lox) mouse retinas. Immunocytochemistry was performed as described by (Ruan et al., 2008). Data are represented as means ± SEM (***p < 0.01, n = 4 mice). D, Brain dopamine (white bars), DOPAC (gray bars), and norepinephrine (black bars) levels measured in Ctrl (Th^lox/lox), Ctrl (Chx10-Cre:Th^lox/+), and rTHKO (Chx10-Cre:Th^lox/lox) by HPLC; no significant change.

Figure 2.

Dark-adapted ERG does not show a circadian response. A–B, Dark-adapted ERG, recorded during DD2, a-wave (triangles) and b-wave (circles) amplitudes at CT 6 (filled circles or triangles) or CT 18 (open circles or triangles) plotted as a function of log intensity in Ctrl (A) or rTHKO (B) mice. A three-way ANOVA revealed no significant effect of CT or genotype on either b-wave or a-wave amplitudes (b-wave: F_(1,29) = 0.19 and 0.28, respectively, p > 0.05; a-wave: F_(1,24) = 1.67 and 0.29, respectively, p > 0.05; n = 6 mice for each). Neither the b-wave implicit time nor the a-wave implicit time showed a significant difference between CT 6 and CT18 in both genotypes (data not shown).

Figure 3.

Light-adapted ERG amplitudes are circadian and controlled by retinal dopamine. A, B, Light-adapted ERG b-wave amplitudes at CT 6 (filled circles and triangles) or CT 18 (open circles and triangles) plotted as a function of light adaptation time in Ctrl or rTHKO mice during DD1 (A) or DD2 (B). A, A two-way interaction was shown for Genotype × Light Adaptation Time (F_(7,98) = 3.2, p < 0.01), such that Ctrl mice had significantly higher amplitudes compared with rTHKO mice at each level of light adaptation (p < 0.007, Bonferroni-corrected α). The Genotype × CT interaction was not significant (F_(1,57) = 0.9, p > 0.05). A main effect of CT showed a significant day/night difference during DD1 for both genotypes (F_(1,57) = 20.0, p < 0.01). B, In DD2, there was a significant two-way interaction for Genotype × CT (F_(1,36) = 7.4, p < 0.01). Significant day/night differences were observed for Ctrl mice (p < 0.025, Bonferroni-corrected α); however, day/night differences were not observed in rTHKO mice (p > 0.025, Bonferroni-corrected α).

Figure 4.

rTHKO mice displayed significant declines in visual parameters. A, Contrast sensitivity was significantly reduced in rTHKO mice at three of the six tested spatial frequencies (**p < 0.01 at 0.064, 0.092, and 0.103 cycles/degree, two-way ANOVA, n = 4–6 mice). B, Acuity threshold was significantly lower in rTHKO mice (*p < 0.05, two-way ANOVA, n = 4–8 mice) compared with control mice. All data are represented as means ± SEM.

Figure 5.

Retinal bioluminescence rhythms persist in retina-specific dopamine-deficient mice. Retinal explants sampled from Ctrl:Per2^Luc (black lines) or rTHKO:Per2^Luc (red lines). Data shown were baseline corrected by calculating a 24 h moving average of the raw data, and then the deviation from the moving average was plotted as a function of days in culture.

Figure 6.

l-DOPA treatment rescues visual phenotype in dopamine-deficient mice. A, B, Light-adapted ERG analysis of Ctrl (filled circles), rTHKO (open circles), l-DOPA-treated rTHKO (filled triangles), and l-DOPA withdrawn (WD) rTHKO (open triangles) mice showed that l-DOPA treatment partially restored the b-wave amplitude at CT6 and CT18. A significant two-way interaction was found for Genotype (with treatment) × CT (F_(3,90) = 25.4, p < 0.01). Significant day/night differences were revealed for Ctrl, rTHKO, and rTHKO-l-DOPA groups using post hoc comparisons (CT6 vs CT18 for each tested group; p < 0.001, p < 0.01, and p < 0.01, respectively), with no difference detected in the rTHKO-WD group. It is noted that the rTHKO group in this experiment, under this analysis, displayed a significant day/night difference; however, its response amplitude is significantly decreased compared with the day/night difference of the Ctrl group (Bonferroni-corrected alpha, p < 0.001). C, Visual contrast sensitivity was preserved with l-DOPA treatment in rTHKO mice when compared with the same mice that had l-DOPA treatment withdrawn from their regimen (*p < 0.05; two-way ANOVA, post hoc student Newman–Kuels; n = 4–8 mice). D, l-DOPA treatment maintained visual acuity threshold levels in rTHKO mice (*p < 0.05; t test; n = 6–8 mice). All data are represented as means ± SEM.

Figure 7.

Dopamine receptor signaling supports the amplitude and circadian rhythm of the light-adapted b-wave ERG response. A, During DD1 a three-way ANOVA revealed significant main effects of Light Adaptation (F_(7,94) = 35.7, p < 0.01), Genotype (F_(1,68) = 117.2, p < 0.01), and CT (F_(1,27) = 83.8, p < 0.01). B, During DD2 similar significant main effects were observed, with a significant two-way interaction found for Genotype × CT (F_(1,18) = 24.3, p < 0.01). Post hoc analysis (Tukey's HSD) revealed significant differences for the WT (circles) CT6 group versus all other groups, including WT CT18 (p < 0.01). Also, the WT CT18 group showed greater amplitude than the D4RKO group (triangles) at either CT (p < 0.01 for both). For D4RKO mice, there was no significant difference in b-wave amplitudes between CT6 and CT18 (p > 0.05). C, D1RKO mice retained significant circadian regulation of the light-adapted ERG as measured by two-way ANOVA (p < 0.001, n = 3–6); however, there was an apparent drop in amplitude compared with wild-type mice in other experiments. D, A significant two-way interaction was found for Treatment Group × CT (F_(1,14) = 13.9, p < 0.05). Post hoc analysis showed a significant day/night difference for the rTHKO group treated with PD168077 (filled and open triangles; p < 0.01); however, the same genotype of mouse treated with saline displayed no difference (filled and open circles; p = 0.89). Data are represented as means ± SEM.

Figure 8.

Dopamine receptors modulate contrast sensitivity and visual acuity threshold in mice. A, D4RKO mice displayed significant decreases of contrast sensitivity detection at all spatial frequencies tested (*p < 0.001 at 0.031, 0.064, 0.092, 0.103, 0.192, and 0.272 cycles/degree, Student–Newman–Keuls post hoc test, n = 4–6 mice). B, Contrast sensitivity measurements in D1RKO mice were similar to WT controls, except at 0.103 and 0.192 cycles/degree (*p < 0.05; Student–Newman–Keuls, n = 4–6 mice) where significant increases were observed. C, Injecting rTHKO mice with SKF38393 did not increase contrast sensitivity levels; however, injection of PD168077 significantly increased contrast sensitivity levels in rTHKO mice (filled triangles; *p < 0.001 at 0.064, 0.092, and 0.103 cycles/degree; Student–Newman–Keuls method, n = 4–6 mice). D, Visual acuity levels were lower in D1RKO mice and mice deficient in retinal dopamine, as measured by increasing spatial frequency of a sine-wave gradient, as compared with WT controls and D4RKO mice (*p = 0.002; n = 4 mice). Intraperitoneal injection of SKF38393 significantly increased visual acuity in rTHKO mice (**p < 0.001; Student–Newman–Keuls method, n = 4–6 mice), while PD168077 failed to have any effect on acuity in mice lacking retinal dopamine. Data are represented as means ± SEM.