Dysfunctional light-evoked regulation of cAMP in photoreceptors and abnormal retinal adaptation in mice lacking dopamine D4 receptors

Abstract

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.

Fig. 1.

Ultrastructure of photoreceptors in D4KO mice. Rod photoreceptors with normal morphology of outer segments (ROS) are seen. The distal ends of the photoreceptor outer segments are in close proximity to the RPE cells (PE). Magnification, 3610×.

Fig. 2.

Disc shedding and phagocytosis in D4KO mice. Shedding of outer segment discs and phagocytosis by the RPE are evident by the presence of shed rod outer segment disk packet (arrow) engulfed by RPE microvilli (M). Magnification, 17,290×.

Fig. 3.

Regulation of cAMP levels in WT retina in a medium containing exogenous glutamate. Isolated retinas were incubated in the presence of 10 mm glutamate, as described in Materials and Methods, to functionally isolate the photoreceptor response to light. Retinas were incubated in the dark, in the presence of quinpirole alone (Q; 1 μm), or with quinpirole and the specific D4 receptor antagonist l-745,870 (Q+L; 10 μm). Control retinas were incubated in dark in the presence of neither drug. Quinpirole significantly decreased cAMP levels (p < 0.001); this effect of quinpirole was antagonized byl-745,870 (p < 0.001). The effect of the antagonist on cAMP levels in light was analyzed in retinas that were incubated in the light (2 min dark plus 7 min light) in the presence of l-745,870 (L) at 10 μm and 50 μm. Control retinas (C) were incubated in light without the drug. Light significantly decreased cAMP levels (p< 0.001), but the effect of light was unaffected byl-745,870 (p < 0.616 for 10 μm; p < 0.503 for 50 μm). N = 5–6/group.

Fig. 4.

Regulation of cAMP by light in WT and D4KO retinas in the presence of exogenous glutamate. Isolated retinas were incubated in a medium containing glutamate and IBMX. Retinas were incubated in the dark for 9 min, or for 2 min dark plus 7 min light. Two-factor ANOVA revealed a significant interaction of genotype and treatment (F = 15.438; p < 0.001). Light significantly reduced cAMP levels in WT retinas (p < 0.001) but not in D4KO retinas (p = 0.412). N = 5–6/group.

Fig. 5.

Regulation of cAMP in the dark in WT and D4KO retinas by quinpirole. Isolated retinas were incubated in medium containing glutamate with quinpirole (1 μm) for 9 min. Control retinas were incubated without quinpirole. Two-factor ANOVA revealed a significant interaction of genotype and treatment (p = 0.03). Multiple comparison by the Student–Newman–Keuls test indicated that quinpirole significantly reduced cAMP levels in WT retinas (p = 0.001) but not in D4KO retinas (p = 0.606).N = 5–6/group.

Fig. 6.

ERGs recorded from 12 hr dark-adapted mice. The a-wave amplitudes (A), b-wave amplitudes (B), and b-wave implicit times (C) of the ERGs of D4KO and WT mice are plotted as a function of flash illuminance presented during the dark after 12 hr of dark adaptation. The lower limb of the function near a-wave threshold is not illustrated because the next lower flash illuminance available was too dim to produce an a-wave reliably. Error bars ± SEM. N = 8 (D4KO) and 9 (WT). ERGs produced by stimulation of each mouse with the standard flash (9 scotopic cd · sec⁻¹ · m⁻¹) in the dark after 12 hr of dark adaptation were averaged across the eight D4KO and nine WT controls separately and are presented graphically (D) and with an expanded time scale focusing on the a-wave (E).

Fig. 7.

ERGs recorded in the first 20 min of light adaptation. The b-wave amplitudes (A) and implicit times (B) of the ERGs of D4KO and WT mice are plotted as a function of time during light adaptation to a rod desensitizing ganzfeld background (58 cd/m). There is a gradual increase of b-wave amplitude during the 20 min light adaptation period and an initial decrease in b-wave implicit time in the first 6 min of light adaptation for both the D4KO and the WT mice. B-wave amplitudes are markedly depressed in D4KO mice compared with WT controls at all times during light adaptation. Error bars: 95% confidence interval. N = 6 per group. ERGs produced by stimulation of each mouse with the standard flash after 10 min adaptation to a rod desensitizing ganzfeld background were averaged across the six D4KO and six WT controls separately and are presented graphically (C).

Fig. 8.

ERGs recorded in the dark after turning off the lighted background. The a-wave amplitudes (A), b-wave amplitudes (B), and b-wave implicit times (C) of the ERGs of D4KO and WT mice are plotted as a function of time during dark adaptation after previous light adaptation for 20 min to a rod desensitizing, ganzfeld background. There is a gradual increase of a-wave amplitude during the 12 min dark adaptation period for both the D4KO and the WT mice. The a-wave and b-wave amplitudes of D4KO mice were reduced from 2 to 10 min of dark adaptation compared with WT controls. The b-wave implicit times of D4KO mice were longer than those of WT mice. Error bars ± SEM.N = 6 per group. ERGs produced by stimulation of each mouse with the standard flash at 10 min in the dark after 20 min light adaptation were averaged across D4KO and WT controls separately and are presented graphically (D) and with an expanded time scale focusing on the a-wave (E).