Retinofugal Projections from Melanopsin-Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-Dependent Virus

To understand visual functions mediated by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections from these cells into the brain. Initial studies reported that melanopsin is expressed only in retinal ganglion cells within the eye. However, recent studies in Opn4-Cre mice revealed Cre-mediated marker expression in multiple brain areas. These discoveries complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adenoassociated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phosphatase) that was injected into the vitreous of Opn4-Cre mouse eyes. The labeling observed in the brain of these mice was necessarily restricted specifically to retinofugal projections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures tended to be bilaterally innervated, whereas the lateral structures received mostly contralateral innervation. As validation of our approach, we found projection patterns largely corresponded with previously published results; however, we have also identified a few novel targets. Our discovery of projections to the central amygdala suggests a possible direct neural pathway for aversive responses to light in neonates. In addition, projections to the accessory optic system suggest that mRGCs play a direct role in visual tracking, responses that were previously attributed to other classes of retinal ganglion cells. Moreover, projections to the zona incerta raise the possibility that mRGCs could regulate visceral and sensory functions. However, additional studies are needed to investigate the actual photosensitivity of mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" with very sensitive reporters that uncover low levels of expression. Light-evoked signaling from these cells must be shown to be of sufficient sensitivity to elicit physiologically relevant responses.