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

Abstract

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 adeno-associated 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.

Melanopsin expression in the cornea

“…The rationale for this approach is that the PLAP would only be observed in those cells that were both acutely transduced by the intraocularly injected AAV-flex-plap and were simultaneously expressing Opn4 cre . We readily observed PLAP staining in the retinas (Delwig et al, 2016) and in the corneas and anterior regions of the injected eyes. We saw prominent PLAP staining in both the nerves in the cornea (Fig.…”

“…In an attempt to validate the tdTomato corneal results and to determine whether melanopsin was being actively expressed in the corneas of adult mice, we injected an adeno-associated virus (AAV) with the floxed human placental alkaline phosphatase (PLAP) gene (AAV-flex-plap) into the vitreal cavity of Opn4 cre mice (Delwig et al, 2016). The rationale for this approach is that the PLAP would only be observed in those cells that were both acutely transduced by the intraocularly injected AAV-flex-plap and were simultaneously expressing Opn4 cre .…”

“…Melanopsin-expressing cells in the eyes of adult mice were labeled by intravitreal injection of the Cre-dependent human placental alkaline phosphatase (PLAP) reporter (AAV-flex-plap; Plasmid #80422, Addgene, Cambridge, MA; Delwig et al, 2016). Mice were anesthetized with isoflurane and topical administration of proparacaine (0.5%; Bausch & Lomb).…”

“…For the primary staining, we used two antimelanopsin antibodies: (1) UF008, Advanced Targeting Systems (http://antibodyregistry.org/search?q=uf008) at 1:5000 for 3 days at +4°C; and (2) an affinity purified rabbit antimelanopsin polyclonal antibody raised against the first 15 amino acids of the N-terminus of melanopsin (Tu et al, 2005). Secondary, tertiary, and DAB stainings were done as previously described (Delwig et al, 2016). …”

How Does Light Regulate Mood and Behavioral State?

“…M1 cells project to the suprachiasmatic nucleus (SCN), the shell of the olivary pretectal nucleus (OPN), the intergeniculate leaflet (IGL), and the lateral geniculate nucleus (vLGN) as well as to limbic and other nonvisual thalamic nuclei such as the medial amygdala and peri-habenular region (pHb) [17]. Non-M1 ipRGCs project to the SCN, but also to the core of the OPN, to mood-and pain-modulating regions such as the periaqueductal gray (PAG) and amygdala, and to traditionally visual brain areas such as the dorsal LGN (dLGN) and the superior colliculus (SC) [12,[18][19][20][21]. Brn3b-positive M1 cells project to the OPN and are essential for driving the pupillary light reflex [22].…”