Targeted Destruction of Photosensitive Retinal Ganglion Cells with a Saporin Conjugate Alters the Effects of Light on Mouse Circadian Rhythms

Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies

UF008/SAP greatly reduces retinal projections to the suprachiasmatic nucleus and intergeniculate leaflet.

<p>CT-B tracing of RHT projections remaining after intravitreal UF008/SAP injection shows a terminal field densest along the lateral, ventrolateral and ventral SCN border, but greatly reduced in, or absent from, the dorso-central SCN. B) In the IGL, the contralateral projection from the UF008/SAP-injected retina is largely absent. C) There are no readily apparent differences with respect to the remaining retinal innervation of the OPT. The red label in all parts of the figure identifies terminals from the contralateral, undamaged retina. Abbreviations: APT – anterior pretectal n.; CPT – commissural pretectal n.; DLGc – dorsal lateral geniculate n;, contralateral; IGLc – intergeniculate leaflet, contralateral; IGLi – intergeniculate leaflet, ipsilateral; MPT – medial pretectal n.; NOT – nucleus of the optic tract; PPT – posterior pretectal n.; RHT – retinohypothalamic tract; VLGc – ventral lateral geniculate n., contralateral. (Ipsi- and contra- are referenced with respect to the injected with UF008/SAP).</p

Destruction of ipRGCs by UF008/SAP depends on dose, but not on location within the retina.

<p>Ablation of ipRGCs by UF008/SAP is dose-dependent, but not related to retinal eccentricity. Each triplet of bars represents data from “peripheral” (P), ”middle” (M), and “central” (C) fields, respectively. (2-way ANOVA with Bonferroni post-tests).</p

Masking response to a light pulse is absent in mice that lose entrainment to LD12∶12.

<p>Masking is nearly normal by UF008/SAP injected mice in response to a 1 hr light pulse. However, the animals recovered more slowly than controls. There is no effect of treatment or of time, but there is a significant interaction (1 way ANOVA with repeated measures, p<.004). In mice that lost entrainment subsequent to UF008/SAP treatment, a 1 hr light pulse failed to induce masking.</p

UF008/SAP treatment has little effect on visual cliff performance.

<p>Both UF008/SAP and IgG/SAP injected groups perform quite accurately in the visual cliff test, but there is a small deficit in the UF008/SAP treated group (p<0.05, Mann-Whitney test).</p