DOI: 10.7272/q6rf5rzx
title: Retinofugal projections from melanopsin-expressing retinal ganglion cells revealed by intraocular injections of Cre-dependent virus

description: 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.
description: Methods
 
 Generating AAV-flex-plap This virus was generated using standard
 sub-cloning with a modified pAAV-MCS backbone. The cDNA encoding human
 placental alkaline phosphatase (PLAP, NM_001632) was flanked by loxP
 (Fig.1A, open triangles) and lox2722 (Fig. 1A, closed triangles) sites to
 yield the flex-plap transgene. This transgene was inserted in reverse
 orientation into a modified pAAV-MCS plasmid 3’ to a CMV promoter and 5’
 to a woodchuck hepatitis virus post-transcriptional regulatory element
 (WPRE) and bovine growth hormone polyadenylation (pA) sequence to generate
 pAAV-flex-plap. High titer virus of serotype 2/1 (4x1012 IU/mL) was
 generated from this plasmid at the University of North Carolina, Chapel
 Hill Vector Core facility. Fig 1. Selective labeling of mRGCs. (A) Map of
 the AAV-flex-plap vector that expresses alkaline phosphatase in a
 Cre-dependent manner (see methods for detailed description). (B, C)
 Representative flat-mount retinas from an Opn4cre mouse that received
 intravitreal injection into the right eye. (B) Retina from the injected
 right eye. (C) Retina from the non-injected left eye. Scale bar: 200 um.
 
 Animals Mice were housed in an AALAC-accredited pathogen-free animal
 facility with ad libitum access to food and water and with a 12-hour
 light-dark cycle with lights on at 7AM and off at 7PM. The University of
 California, San Francisco Institutional Animal Care and Use Committee
 (IACUC) specifically approved this study. The protocols, animal care
 procedures and the experimental methods meet all of the guidelines on the
 care and use of laboratory animals by the U.S. Public Health Service. 
 
 The following animals were used in this study: 1) C57BL/6J wild-type mice
 (Jackson Laboratory); 2) mice homozygous for Opn4cre allele (gift from
 Samar Hattar [5]), which express Cre under the melanopsin (Opn4) promoter;
 and 3) mice homozygous for Ai14 allele (Jackson Lab [8]), which is a
 Cre-dependent tdTomato reporter. Mice were genotyped by PCR with
 allele-specific primers [5]. 
 
 Intravitreal injections The age of the mice ranged from P38 to P96 at the
 time of injection. Mice were anesthetized with Isoflurane and topical
 administration of proparacaine (0.5%; Bausch & Lomb). The pupils were
 dilated by topical administration of phenylephrine (2.5%; Bausch &
 Lomb) and atropine sulfate (1%; Bausch & Lomb) eye drops. A 32-gauge
 Hamilton syringe was used to inject 2 microliters of AAV-flex-plap into
 the superior part of the vitreous of right eyes. A total of 13 injections
 were made (11 into Opn4cre mice and 2 into C57BL/6J wild-type mice). No
 PLAP signal was detected in the retinas of wild-type mice. Visually
 detectible PLAP labeling was examined and quantified in the brains of 5
 animals. 
 
 Tissue processing for PLAP histochemistry PLAP histochemistry was
 performed as previously described [9, 10]. Two to eight weeks after the
 intravitreal injection the mice were euthanized by CO2 and transcardially
 perfused with 10 ml HEPES-buffered saline (HBS; 8.2 g/l NaCl, 6 g/l HEPES,
 0.1 g/l Na2HPO4, pH to 7.4 with NaOH) followed by 20 ml cold 4%
 paraformaldehyde (PFA) in HBS. All subsequent solutions were prepared with
 HBS unless otherwise noted. Brains and eyes were dissected and post-fixed
 in 4% PFA for 3-4 hours at 4°C. The brains were embedded in 3% agar and
 cut on a vibratome (Model 3000, Vibratome Company) into 100 μm sections.
 The retina was dissected from the fixed eyes and flat-mounted. Following
 rinsing in HBS, the endogenous alkaline phosphatase was heat-inactivated
 by incubation at 72°C for 1 hour. Tissue was then washed twice in buffer 1
 (100 mM Tris pH 7.5, 150 mM NaCl) and then twice again in buffer 2 (100 mM
 Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2). PLAP reporter was visualized by
 incubating tissue in buffer two with BCIP (5-bromo-4-chloro-3-indolyl
 phosphate, 0.2 mg/ml) and NBT (nitro blue tetrazolium, 1 mg/ml) at room
 temperature for 1 to 12 hours. PLAP reaction was monitored and stopped
 before background staining became excessive by rinsing 3 times in 1mM EDTA
 followed by post-fixation in 4% PFA for 1 hour. To remove background
 staining, the tissue was cleared by immersing in ethanol series
 (30%-70%-95%-100%-95%-70%-30%) for 1-3 minutes in each series and the
 subsequent wash in the HBS. Brain sections were counterstained (see
 below). Brain sections and whole mount retinas were mounted on microscope
 slides, briefly rinsed in distilled water and cover slipped using
 Aqua/Poly mount (Polysciences, Cat. # 18606).
 
 Counterstaining To visualize brain nuclei, all brain sections were
 counterstained with ToPro3 (Life Technologies, Cat. # T3605), a
 fluorescent nuclear stain, at 1:5,000 dilutions. To better visualize
 thalamic nuclei, alternate sections were processed for Cytochrome Oxidase
 staining [11] by immersing slices in Cytochrome Oxidase staining solution
 (30 mg Cytochrome C, 20 mg Catalase, 50 mg DAB in 100 ml PBS) for 2-4
 hours at room temperature.