Dopamine receptor loss of function is not protective of rd1 rod photoreceptors in vivo

Abstract

Purpose: The retinal degeneration (rd1) mouse undergoes a rapid loss of rod photoreceptors due to a defect in the cGMP-phosphodiesterase gene. We have previously demonstrated that dopamine (DA) antagonists or DA depletion blocks photoreceptor degeneration and that DA is necessary for photoreceptor degeneration in the rd1 mouse retinal organ culture model. Antagonists for either D1- or D2-family DA receptors are protective in rd1 organ cultures.

Methods: To determine whether photoreceptor survival can be increased in vivo in the rd1 mouse, we used both a pharmacological and a genetic approach. The pharmacological approach involved three techniques to administer 6-hydroxydopamine (6-OHDA) in an attempt to deplete DA in postnatal mouse retina in vivo. As a genetic alternative, DA receptor signaling was inactivated by crossbreeding rd1 mice to D1, D2, D4, and D5 knockout mice to create four lines of double mutants.

Results: Pharmacological DA depletion was incomplete due to the limiting size of the postnatal mouse eye and the lethality of systemic inhibition of DA signaling. In all four lines of double mutants, no increase in rod photoreceptor survival was observed. To determine whether protection of rd1 photoreceptors by inhibition of dopaminergic signaling is a result of conditions specific to the organ culture environment, we grew in vitro retinas from the four lines of double mutant mice for four weeks. Again, no increase in photoreceptor survival was seen. Finally, three triple mutants were generated that lacked two DA receptors (D1/D2; D1/D4; and D2/D4) on a rd1 background. In all three cases, rod photoreceptors were not protected from degeneration.

Conclusions: The dramatic protection of rd1 rod photoreceptors by inhibition of DA signaling in organ culture has not been reproduced in vivo by either a pharmacological approach, due to technical limitations, or by genetic manipulations. The possible role of compensatory effects during retinal development in DA receptor deficient mice is considered.

Figure 1

Dopamine receptor deletion does not alter photoreceptor cell survival in rd1 retinas in vivo. Retinas from wild-type (A), rd1/rd1 (B), D1^−/−, rd1/rd1 (C), D2^−/−, rd1/rd1 (D), D4^−/−, rd1/rd1 (E), and D5^−/−, rd1/rd1 (F) mice were harvested at postnatal day 21. The ONL of rd1/rd1 retinas was reduced to a monolayer of photoreceptors, regardless of DR genotype. Abbreviations: outer segments (OS); inner segments (IS); outer nuclear layer (ONL); outer plexiform layer (OPL); inner nuclear layer (INL); inner plexiform layer (IPL); ganglion cell layer (GCL). The scale bar represents 10 μm.

Figure 2

Dopamine receptor deletion does not alter photoreceptor cell survival in rd1 retinal organ cultures. Retinas from D1^−/−, rd1/+ (A), D1^+/−, rd1/rd1 (B), D1^−/−, rd1/rd1 (C), D2^−/−, rd1/rd1 (D), D4^−/−, rd1/rd1 (E), and D5^−/−, rd1/rd1 (F) mice were harvested at postnatal day 2 and grown in organ culture for 27 days in vitro. Wild-type retinas maintained approximately five to six rows of cells in the ONL (A), while the ONL of untreated rd1/rd1 cultures, regardless of DR genotype, was reduced to approximately two to three rows (B-F). Quantitative analysis of photoreceptor survival as measured by ONL thickness is shown in (G). Black bars represent the genotypes shown in A-F and additional controls. Grey bars represent organ cultures from D2^−/−, D4^−/−, rd1/rd1 triple mutant mice treated with drugs as labeled. The number of cultures is indicated on the column for each condition; error bars indicate standard deviation. No significant difference is seen in the ONL thickness among rd1 organ cultures, regardless of DR genetic background or treatment. Similarly, no difference is seen among rd1 heterozygous control organ cultures. Abbreviations: outer segments (OS); inner segments (IS); outer nuclear layer (ONL); outer plexiform layer (OPL); inner nuclear layer (INL); inner plexiform layer (IPL); ganglion cell layer (GCL). The scale bar represents 10 μm.

Figure 3

Deletion of multiple dopamine receptors does not alter photoreceptor cell survival in rd1 retinas in vivo. Retinas from D1^−/−, D2^−/−, rd1/rd1 (A), D1^−/−, D4^−/−, rd1/rd1 (B), D2^−/−, D4^−/−, rd1/rd1 (C) mice were harvested at postnatal day 21. The ONL of rd1/rd1 retinas was reduced to a monolayer of photoreceptors, regardless of DR genotype. Abbreviations: retinal pigment epithelium (RPE); outer nuclear layer (ONL); outer plexiform layer (OPL); inner nuclear layer (INL); inner plexiform layer (IPL); ganglion cell layer (GCL). The scale bar represents 10 μm.