RESEARCH ARTICLE Retinofugal Projections from Melanopsin- Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-Dependent Virus Anton Delwig1, DeLaine D. Larsen1, Douglas Yasumura1, Cindy F. Yang2, Nirao M. Shah2, David R. Copenhagen1,3* 1 Department of Ophthalmology, UCSF, San Francisco, California, United States of America, 2 Department of Anatomy, UCSF, San Francisco, California, United States of America, 3 Department of Physiology, UCSF, San Francisco, California, United States of America * [email protected] Abstract To understand visual functions mediated by intrinsically photosensitive melanopsin- OPEN ACCESS expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections Citation: Delwig A, Larsen DD, Yasumura D, Yang from these cells into the brain. Initial studies reported that melanopsin is expressed only in CF, Shah NM, Copenhagen DR (2016) Retinofugal retinal ganglion cells within the eye. However, recent studies in Opn4-Cre mice revealed Projections from Melanopsin-Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-mediated marker expression in multiple brain areas. These discoveries complicate the Cre-Dependent Virus. PLoS ONE 11(2): e0149501. use of melanopsin-driven genetic labeling techniques to identify retinofugal projections spe- doi:10.1371/journal.pone.0149501 cifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno- Editor: Tudor C Badea, NIH/NEI, UNITED STATES associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phos- Received: July 27, 2015 phatase) 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 pro- Accepted: February 2, 2016 jections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in Published: February 19, 2016 the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures Copyright: © 2016 Delwig et al. This is an open tended to be bilaterally innervated, whereas the lateral structures received mostly contralat- access article distributed under the terms of the eral innervation. As validation of our approach, we found projection patterns largely corre- Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any sponded with previously published results; however, we have also identified a few novel medium, provided the original author and source are targets. Our discovery of projections to the central amygdala suggests a possible direct neu- credited. ral pathway for aversive responses to light in neonates. In addition, projections to the acces- Data Availability Statement: All relevant data are sory optic system suggest that mRGCs play a direct role in visual tracking, responses that within the paper and its Supporting Information files. were previously attributed to other classes of retinal ganglion cells. Moreover, projections to Funding: This work was funded by R01NS049488 the zona incerta raise the possibility that mRGCs could regulate visceral and sensory func- (NMS) National Institute of Neurological Disease and tions. However, additional studies are needed to investigate the actual photosensitivity of Stroke, NIH R01NS083872 (NMS) National Institute of Neurological Disease and Stroke, NIH mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" R01EY02317 (DRC) National Eye Institute, NIH with very sensitive reporters that uncover low levels of expression. Light-evoked signaling R01EY01869 (DRC) National Eye Institute, NIH from these cells must be shown to be of sufficient sensitivity to elicit physiologically relevant T32EY007120 (AD) National Eye Institute, NIH responses. P30EY002162 (Matthew LaVail, PI), Core Grant, National Eye Institute, NIH, Benign Essential Blepharospasm Research Foundation (DRC), www. blepharospasm.org/, That Man May See, UCSF PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 1/14 Retinofugal Projections from mRGCs (DRC), thatmanmaysee.org/, and Research to Introduction Prevent Blindness (DRC and Dept. of Ophthalmology, UCSF), www.rpbusa.org/. The funders had no role in Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye have been recently recog- study design, data collection and analysis, decision to nized as important mediators of non-image forming visual responses, such as circadian photo- publish, or preparation of the manuscript. entrainment and pupillary light responses, in many mammalian species [1]. Published maps of – Competing Interests: The authors have declared central projections from mRGCs [2 5] have provided an important groundwork for formulat- that no competing interests exist. ing hypotheses related to the physiological and behavioral responses modulated by mRGCs. These previous studies relied on indirect labeling of mRGCs with an antibody against pituitary adenylate cyclase-activating peptide [2], or on low titer of intravitreal AAV-GFP [3], or on the direct labeling of all melanopsin-expressing cells using a reporter gene (Opn4-lacZ;[4]) or the cre loxP cre loxP genetic Cre-lox system (Opn4 ::AP and Opn4 ::GFP ;[5, 6]). The Cre-lox based genetic approach has been the most sensitive technique to reveal the diversity of mRGCs subtypes and their central targets. However, this approach also revealed extra retinal expression of floxed cre reporter genes in cells across many brain areas of Opn4 mice including cerebral cortex, thala- mus and brainstem [5] that are not thought to express melanopsin. Melanopsin has also been recently found in the iris of mice using immunohistochemistry [7]. These findings complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs In this present study, we employed an alternative genetic method to specifically label mRGCs in the eye and to further increase the labeling of mRGCs with weak melanopsin expression. Delivery of a Cre-dependent reporter using a recombinant adeno-associated virus (AAV) has become a popular tool to label genetically defined cells. This method offers two advantages over the systemic Cre-lox genetic reporter approach. First, it does not reveal the his- toric pattern of expression thereby eliminating the report of transient expression in cells during early development. Second, it delivers multiple copies of the Cre-dependent reporter per infected cell thereby increasing the chance of recombination in cells with weak Cre expression. Therefore, we decided to further investigate the central targets of mRGCs in the brain by intra- vitreal injection of AAV carrying floxed human placental alkaline phosphatase (PLAP) cre reporter into Opn4 mice [5]. Here we present the results of these tracing studies. 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 regu- latory element (WPRE) and bovine growth hormone polyadenylation (pA) sequence to gener- ate 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. 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 Commit- tee (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. PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 2/14 Retinofugal Projections from mRGCs 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. doi:10.1371/journal.pone.0149501.g001 The following animals were used in this study: 1) C57BL/6J wild-type mice (Jackson Labora- cre tory); 2) mice homozygous for Opn4 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 cre injections were made (11