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RESEARCH ARTICLE
Retinofugal Projections from Melanopsin-
Expressing Retinal Ganglion Cells Revealed by
Intraocular Injections of Cre-Dependent Virus
Anton Delwig
1
, DeLaine D. Larsen
1
, Douglas Yasumura
1
, Cindy F. Yang
2
, Nirao M. Shah
2
,
David R. Copenhagen
1,3
*
1Department of Ophthalmology, UCSF, San Francisco, California, United States of America, 2Department
of Anatomy, UCSF, San Francisco, California, United States of America, 3Department of Physiology, UCSF,
San Francisco, California, United States of America
*cope@phy.ucsf.edu
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 spe-
cifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno-
associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phos-
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-
jections 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 contralat-
eral innervation. As validation of our approach, we found projection patterns largely corre-
sponded 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 neu-
ral pathway for aversive responses to light in neonates. In addition, projections to the acces-
sory 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 func-
tions. 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.
PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 1/14
OPEN ACCESS
Citation: Delwig A, Larsen DD, Yasumura D, Yang
CF, Shah NM, Copenhagen DR (2016) Retinofugal
Projections from Melanopsin-Expressing Retinal
Ganglion Cells Revealed by Intraocular Injections of
Cre-Dependent Virus. PLoS ONE 11(2): e0149501.
doi:10.1371/journal.pone.0149501
Editor: Tudor C Badea, NIH/NEI, UNITED STATES
Received: July 27, 2015
Accepted: February 2, 2016
Published: February 19, 2016
Copyright: © 2016 Delwig et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was funded by R01NS049488
(NMS) National Institute of Neurological Disease and
Stroke, NIH R01NS083872 (NMS) National Institute
of Neurological Disease and Stroke, NIH
R01EY02317 (DRC) National Eye Institute, NIH
R01EY01869 (DRC) National Eye Institute, NIH
T32EY007120 (AD) National Eye Institute, NIH
P30EY002162 (Matthew LaVail, PI), Core Grant,
National Eye Institute, NIH, Benign Essential
Blepharospasm Research Foundation (DRC), www.
blepharospasm.org/, That Man May See, UCSF
Introduction
Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye have been recently recog-
nized as important mediators of non-image forming visual responses, such as circadian photo-
entrainment and pupillary light responses, in many mammalian species [1]. Published maps of
central projections from mRGCs [2–5] have provided an important groundwork for formulat-
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
genetic Cre-lox system (Opn4
cre
::AP
loxP
and Opn4
cre
::GFP
loxP
;[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
reporter genes in cells across many brain areas of Opn4
cre
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)
reporter into Opn4
cre
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 (4x10
12
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.
Retinofugal Projections from mRGCs
PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 2/14
(DRC), thatmanmaysee.org/, and Research to
Prevent Blindness (DRC and Dept. of Ophthalmology,
UCSF), www.rpbusa.org/. The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
The following animals were used in this study: 1) C57BL/6J wild-type mice (Jackson Labora-
tory); 2) mice homozygous for Opn4
cre
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 Opn4
cre
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 CO
2
and transcardially perfused with 10
ml HEPES-buffered saline (HBS; 8.2 g/l NaCl, 6 g/l HEPES, 0.1 g/l Na
2
HPO
4
, pH to 7.4 with
NaOH) followed by 20 ml cold 4% paraformaldehyde (PFA) in HBS. All subsequent solutions
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 Opn4
cre
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
Retinofugal Projections from mRGCs
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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 MgCl
2
). 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 tetra-
zolium, 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 fol-
lowed 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 Technolo-
gies, 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.
Results
Labeling melanopsin retinal ganglion cells
We previously reported light-driven, melanopsin-dependent activation of neurons in the cen-
tral amygdala and posterior thalamus in neonatal mice using immunolocalization of immediate
early gene expression [12]. To further understand the contribution of melanopsin-expressing
retinal ganglion cells (mRGCs) in the eye to non-image forming responses, we decided to look
more carefully at projections of mRGCs to these and other brain areas. When we crossed
Opn4
cre
mice [5] with mice carrying floxed tdTomato reporter [8], we found tdTomato in neu-
rons of the inner retina, presumed to be mRGCs. We also found tdTomato in many areas of
the brain that are not known to be direct targets of retinal ganglion cells, such as the somato-
sensory cortex and cerebellum (S1 Fig). Similar extra retinal expression of Opn4
cre
driven
reporter was also observed by Ecker et al.[5].
To limit the expression of Opn4
cre
driven reporter to retinofugal projections from mRGCs
and to maximize the labeling of mRGCs with weak melanopsin expression, we created an
adeno-associated virus (AAV) vector with floxed human placental alkaline phosphatase gene
(AAV-flex-plap, Fig 1A; see methods). The intravitreal injection of AAV-flex-plap into Opn4
cre
mice leads to robust labeling of many mRGCs around the site of injection (Fig 1B). The signal
was always specific to the injected eye and was never observed in the contralateral, non-injected
eye (Fig 1C). This result confirms that the virus did not spread outside the injected eye and that
the labeling is specific to the virally encoded PLAP and not a result of endogenous alkaline
phosphatase activity. We found that control injections of AAV-flex-plap into the vitreous of
wild-type mice did not result in any labeling (n = 2, data not shown) thereby confirming that
the PLAP signal is specific to Opn4
cre
expressing retinal cells.
Retinofugal Projections from mRGCs
PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 4/14
Brain areas innervated by retinal mRGCs
Two to eight weeks after the intravitreal injection of AAV-flex-plap, the brains were sectioned
coronally and processed to visualize PLAP. A representative coronal series is presented in Figs
2and 3. The following sections describe our findings (summarized in Tables 1and 2). For each
case analyzed the entire series of sections was scored for the presence or absence of PLAP-posi-
tive axons in each brain area. Adjacent sections processed for cytochrome oxidase staining and
ToPro3 counterstains were used to identify architectonic boundaries in the sections. The
Mouse Brain in Stereotaxic Coordinates [13] and Allen Mouse Brain Atlas [14] were used to
identify regions and nuclei in the stained sections. For AOS anatomy we also referred to Giolli
et al. [15].
Basal forebrain and hypothalamus. The most rostral targets of mRGCs that we observed
in the mouse brain were the horizontal limb of the diagonal band (HDB, 1/5), the lateral preop-
tic area (LPO, 3/5) and the medial preoptic area (MPO, 3/5; Fig 2A and 2B). The sparse labeling
was always on the contralateral side. Further caudally, single mRGCs axons were found in the
ventrolateral preoptic area (VLPO, 2/5) and the anterior amygdala (AA, 5/5), again always on
the contralateral side (Fig 2B and 2C). Dense bilateral staining was observed in the suprachias-
matic nucleus (SCN, 5/5; Fig 2D). Caudal to the SCN, midline areas tended to be bilaterally
innervated whereas lateral targets were innervated contralaterally. Sparse bilateral labeling was
seen in retrochiasmatic nucleus (RCh, 5/5; Fig 2E). Sparse contralateral projections were
observed in the medial preoptic area (MPO, 3/5), lateral hypothalamus (LHA, 5/5), supraoptic
nucleus (SO, 5/5) and medial amygdala (MeA, 5/5; Fig 2D–2F). A novel finding of this study
was the observation of sparse innervation of the central amygdala (CeA, 5/5; Fig 2E and 2F).
Thalamus and habenula. Single mRGC axons were observed innervating contralateral
bed nucleus of the stria terminalis (BST, 4/5) and bilateral central medial nucleus (CM, 2/5; Fig
2C and 2D). More caudally, mRGCs axons were found in the contralateral lateral dorsal
nucleus (LD, 5/5) and central lateral nucleus (CL, 5/5; Fig 3A). We did not observe projections
into the lateral habenula (LHb). The terminals of mRGCs were localized in the nearby CL (Fig
3A), with a single case that also extended into the medial dorsal nucleus (MD, 1/5). Lateral pos-
terior nucleus (LP, 5/5) received contralateral projections from mRGCs of medium density
(Fig 3B–3E). We observed dense bilateral innervation of the dorsal lateral geniculate nucleus
(dLG, 5/5) and dense mostly contralateral innervation of the ventral lateral geniculate nuclei
(vLG, 5/5) and intergeniculate leaflet (IGL, 5/5; Fig 3B–3E). Sparse contralateral innervation
was observed in the zona incerta (ZI, 5/5; Fig 3B–3E). We did not observe projections
from mRGCs to the ventral posterior nucleus (VP) or posterior thalamic nuclear group
(Po; Fig 3B–3E).
Midbrain. Sparse contralateral innervation was observed in the periaqueductal gray
(PAG, 3/5; Fig 3D). Olivary pretectal nucleus (OPT, 5/5) received dense innervation by
mRGCs with most projections going to the contralateral side (Fig 3C–3E). Other visual pretec-
tal nuclei received bilateral innervation of medium density. They included the anterior, medial
and posterior pretectal nuclei (APT, MPT, and PPT, 5/5) and the nucleus of the optic tract
(NOT, 5/5; Fig 3D and 3E).
Accessory optic system and the superior colliculus. Another novel finding was the obser-
vation of dense contralateral innervation by mRGCs of the accessory optic nuclei including
dorsal, lateral, and medial terminal nuclei (DT, LT, and MT 5/5; Fig 4). The superior colliculus
(SC, 5/5) received dense, mostly contralateral, innervation (Fig 4).
Retinofugal Projections from mRGCs
PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 5/14
Fig 2. Central targets of mRGCs in the brain. Representative coronal sections (rostral to caudal) from the brains of Opn4
cre
mice with intravitreal injection
of AAV-flex-plap into the right eye. Left panels in each row are low-power images of brain sections processedto visualize alkaline phosphate. The next panel
in each row is an image of an adjacent brain section stained for Cytochrome Oxidase. Some insets are composite montages of several images (D, E, F). See
Retinofugal Projections from mRGCs
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Table 1 for nomenclature. Scale bar: 200 μm (inset, 100 μm). Ipsilateral and contralateral sides of the brains are labeled. Note that HDB and MPA areas
appear on the “contralateral”, right side of the section. Since these sections are rostral to the chiasm, one might think fibers in HDB and MPA should be on the
ipsilateral side. However, we have traced these fibers back to the chiasm and find that they have indeed crossed over from the ipsilateral side and projected
in the rostral direction.
doi:10.1371/journal.pone.0149501.g002
Fig 3. Central targets of mRGCs in the brain. Representative coronal sections (rostral to caudal) from the brains of Opn4
cre
mice with intravitreal injection
of AAV-flex-plap into the right eye. Left panels in each row are low-power images of brain sections processedto visualize alkaline phosphate. The next panel
in each row is an image of an adjacent brain section stained for Cytochrome Oxidase. See Table 1 for nomenclature. Scale bar: 200 μm (inset, 100 μm).
doi:10.1371/journal.pone.0149501.g003
Retinofugal Projections from mRGCs
PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 7/14
Discussion
In the present study, we used viral delivery of a Cre-dependent reporter to label melanopsin-
expressing retinal ganglion cells (mRGCs) in the mouse eye and to determine what central
Table 1. Abbreviations used in the text and in the figures.
Anatomical Area Abbreviation
anterior amygdala AA
anterior hypothalamic area AH
anterior pretectal nuclei APT
anteroventral nucleus AV
bed nucleus of the stria terminalis BST
central amygdala CeA
central lateral nucleus CL
central medial nucleus CM
dorsal terminal nucleus of the accessory optic tract DT
dorsal lateral geniculate nucleus dLG
internal capsule ic
intergeniculate leaflet IGL
lateral dorsal nucleus LD
lateral hypothalamus LHA
lateral habenular nucleus LHb
Lateral posterior nucleus LP
lateral preoptic area LPO
lateral terminal nucleus of the accessory optic tract LT
medial dorsal nucleus MD
medial amygdala MeA
medial geniculate nucleus MG
medial habenular nucleus MHb
medial preoptic area MPO
medial pretectal nuclei MPT
medial terminal nucleus of the accessory optic tract MT
horizontal limb of the diagonal band HDB
nucleus of the optic tract NOT
Olivary pretectal nucleus OPT
periaqueductal gray PAG
posterior commissure Pc
Posterior thalamic nucleus Po
posterior pretectal nuclei PPT
paraventricular hypothalamus PA
retrochiasmatic nucleus RCh
Superior colliculus SC
suprachiasmatic nucleus SCN
supraoptic nucleus SO
stria terminalis st
ventral lateral geniculate nuclei vLG
ventrolateral preoptic area VLPO
ventral posterior thalamic nucleus VP
zona incerta ZI
doi:10.1371/journal.pone.0149501.t001
Retinofugal Projections from mRGCs
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targets in the brain they innervate. 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 innerva-
tion. These results are in accord with the previously published results [2–5]. The following sec-
tions highlight the novel targets and discrepancies with the previous studies. A caveat of the
intravitreal viral delivery of the reporter is that this technique does not label all mRGCs in the
eye. It labels only a subset of mRGCs near the site of injection (Fig 1B). Therefore, this tech-
nique only samples the projections by mRGCs from one region of the retina; there might be
additional brain regions innervated by mRGCs that were not labeled by our injections. Another
caveat is that PLAP labeling in various brain areas does not necessarily represent synapses in
these regions; PLAP labeled fibers could represent fibers of passage.
Table 2. Compilation of frequency of innervation by PLAP-positive fibers.
# Of cases % Of cases
Basal Forebrain and Hypothalamus
NDB 1 20%
MPO 3 60%
LPO 3 60%
VLPO 2 40%
SO 5 100%
BST 4 80%
LHA 5 100%
RCh 5 100%
SCN 5 100%
AA 5 100%
MeA 5 100%
CeA 5 100%
Thalamus and Habenula
CM 2 40%
LD 5 100%
CL 5 100%
MD 1 20%
LP 5 100%
dLG 5 100%
vLG 5 100%
IGL 5 100%
ZI 5 100%
Midbrain
PAG 3 60%
OP 5 100%
APN 5 100%
MPT 5 100%
PPT 5 100%
NOT 5 100%
SC 5 100%
DT 5 100%
LT 5 100%
MT 5 100%
doi:10.1371/journal.pone.0149501.t002
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A critical question is whether we were viewing axon terminals targeted to different brain
regions or might the PLAP-positive fibers be axons of passage? Based on our impression
that the axons ended in a particular region we assumed this was an area targeted by mRGCs.
However to substantiate this assertion we would have to do high-resolution microscopy and
antibody labeling. Axon terminals should be enriched for synaptic proteins such as
VGLUT2 or synapsin, or show structural specializations usually associated with synaptic
boutons.
Central amygdala
A novel finding of this study is sparse but consistent innervation of the central amygdala (Fig
2E and 2F). These findings suggest that the light-induced neural activation in CeA of the neo-
natal mice [12] may be due to direct activation of neurons in CeA by mRGCs. The extent of
direct vs. indirect effect of mRGCs on neural activation in CeA remains unknown. We specu-
late that if this retina-amygdala pathway is conserved in humans, it may be the neural mecha-
nism of aversive responses to light in neonatal and preterm infants [16,17].
Zona incerta
We found consistent projections of mRGCs to the zona incerta (Fig 3B–3E). It is one of the
limbic nodes and is implicated in the regulation of water and food intake, reproductive behav-
iors and cardiovascular activity [18]. Additionally, the zona incerta is involved in processing
pain by controlling the transmission of signals from the spinothalamic tract to the posterior
thalamus [19]. In primates, zona incerta is also implicated in controlling saccades via GABAer-
gic projections to the superior colliculus [20]. Altogether, our findings suggest that mRGCs
may play role in regulating these non-image forming responses.
Fig 4. Projections of mRGCs to accessory optic system. Four representative coronal sections show the
projections of mRGCs to the accessory optic system and the superior colliculus. Note these sections
contained dark crystal-like puncta we believe are salt crystals that were not adequately washed out during
slide preparation. Based on color and positional differences, we felt comfortable differentiating between these
artifacts and the purple/blue PLAP reaction products. Scale bar: 200 μm.
doi:10.1371/journal.pone.0149501.g004
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Posterior thalamus
An electrophysiological study by Noseda et al.[21] of light-responsive neurons in LD, LP, VP
and Po in rats suggested a direct input from mRGCs to these thalamic regions. We were able to
detect projections from mRGCs to LD and LP but not to VP or Po (Fig 3A–3E) in mice. The
observed lack of innervation in VP and Po does not necessarily prevent neurons in these brain
areas from receiving light signals originating from mRGCs. The dendritic fields of VP and Po
neurons are large enough (350 μm; [22]) to receive direct visual input from nearby visual tha-
lamic areas that are heavily innervated by mRGCs. Additionally, the absence of direct innerva-
tion of Po and VP that we report here could reflect differences between mice and rats.
Accessory optic system (AOS)
An unexpected finding of our study is the heavy innervation of the accessory optic system by
mRGCs. We saw dense projections to all AOS nuclei including OT, DT and MT (Figs 3E and
4) suggesting that mRGCs contribute to tracking responses. Consistent with this suggestion, an
M4 subtype of mRGCs was recently found to contribute to the contrast sensitivity of the opto-
kinetic response [23]. However, in contrast to our findings, Hoxd10 retinal ganglion cells,
which include many types of AOS projecting directionally selective cells in the retina [24], do
not express melanopsin and are not intrinsically photosensitive as determined by immunola-
beling with anti-melanopsin antibody and whole-cell electrophysiology (personal communica-
tion with Maureen Stabio and David Berson). Additionally, the previous study by Ecker et al.
[5] using the same Opn4
cre
mouse strain and a genetic reporter did not detect any signal in the
AOS. One possible resolution of the apparent disparity between our results and that of Ecker
et al. is that AAV-mediated delivery of Cre-dependent reporter may result in multiple copies of
viral genomes per infected cell, thereby increasing the chance of detecting cells with low level
of melanopsin expression. It is possible that this increased sensitivity revealed novel AOS pro-
jecting subtypes of mRGCs that were not previously described. There are a number of addi-
tional experiments that need be carried out to further investigate the contribution of mRGCs
to visual tracking: (a) analysis of labeled mRGCs to see if they include directionally selective
retinal ganglion cells; and (b) retrograde labeling of retinal ganglion cells projecting to MT and
OT nuclei followed by analysis of melanopsin expression and of their intrinsic
photosensitivity.
Retinal photoreceptors
We found examples of PLAP positive cones in pAAV-flex-plap transfected retinas (S2 Fig).
These results corroborate previous studies reporting melanopsin-driven reporter expression in
photoreceptors (Ecker et al. 2010). A critical question from a developmental standpoint is
when is Opn4 active? In this present study we find PLAP expression in retinas injected after
postnatal day 30. Whether melanopsin photopigment contributes to detection of light in the
Opn4-expressing photoreceptors will require further study.
Moreover, our ability to observe PLAP-positive photoreceptors is informative with respect
to finding an AAV serotype (2/1) that is capable of infecting photoreceptors when injected into
the vitreous. This has importance for devising gene therapy interventions in outer retinal
degenerative diseases. Viral transfection of rods and cones following an injection different
AAV serotypes into the vitreous is uncommon. Of 7 different serotypes tested by Hellstrom
et al.[25] only AAV2/3 and AAV2/5 seemed efficacious for transducing rods and cones. Our
findings show that AAV2/1 can also transduce photoreceptors, suggesting AAV2/1 has proper-
ties similar to AAV2/3 or AAV2/5. Further study is required to quantitatively compare trans-
duction percentages of these different serotypes.
Retinofugal Projections from mRGCs
PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 11 / 14
Two issues using AAV based markers deserve consideration. 1. An important concern that
arises from the use of highly sensitive reporters is the relationship between the labeling and the
physiologically relevant level of melanopsin expression. As labeling techniques become pro-
gressively more sensitive, with an ability to mark ever-lower levels of expression, a question
arises as at what point the labeling becomes excessive and is not physiologically relevant. It is
possible that our technique labeled RGCs with low levels of melanopsin expression that may
not be sufficient to confer intrinsic photosensitivity or a significant biological function. Further
studies are needed to show that the labeled mRGCs are indeed intrinsically photosensitive.
2. An important question that arises is whether the virus we used transfected selective clas-
ses of mRGC. The intersectional genetic strategy used by Ecker et al.[5] showed that Opn4-cre
was virtually ubiquitously expressed, however, we cannot assess whether our PLAP-flex-plap
virus infected all subtypes of mRGC. Differences in the density of staining in target regions
identified by intravitreal injections of a rAAV-GFP (Gooley, et al,[3]) and ours is consistent
with the notion that different viruses may not infect all classes of ipRGC. Hellstrom etal.[25]
illustrated evidence for selective transduction of subsets of single classes of retinal cell by differ-
ent AAV serotypes. In conclusion we cannot say whether selective classes of ipRGC were trans-
fected by our AAV-flex-plap injections.
In summary, our findings highlight sparse but diffuse innervation of multiple limbic areas
in the brain suggesting that light activation of mRGCs may contribute to regulation of multiple
homeostatic responses in the animal including sleep, feeding, reproductive behaviors, cardio-
vascular function, alertness, mood, pain and memory. Additionally, projections to the acces-
sory optic system suggest that mRGCs play role in saccades and visual tracking, responses that
were previously attributed to other classes of retinal ganglion cells.
Supporting Information
S1 Fig. Extra retinal expression of Opn4
cre
driven reporter. Representative examples of
Opn4
cre
-driven expression of Ai14, a floxed tdTomato fluorescent reporter, in the brain of P21
mouse (Opn4
cre
::Ai14). Widespread expression of tdTomato is observed in numerous areas
including (A) the somatosensory cortex, (B) thalamus, and (C) cerebellum. Scale bar: 100 um.
(TIFF)
S2 Fig. AAV-mediated labeling of Opn4
cre
cells in the retina. Three representative retinal
slices from Opn4
cre
mouse with intravitreal injection of AAV-flex-plap. PLAP staining was
maximized to reveal finer details of PLAP labeling in a sparse population of cones, albeit at the
expense of saturating PLAP signal in the retinal ganglion cells and in the inner plexiform layer.
Abbreviations: GC—ganglion cell layer; IPL—inner plexiform layer; INL—inner nuclear layer;
OPL—outer plexiform layer; ONL—outer nuclear layer; OS—outer segments.
(TIFF)
Acknowledgments
The authors thank Dr. David Berson and Dr. Maureen Stabio for helpful comments on the
manuscript and discussion on mRGC projections to AOS.
Author Contributions
Conceived and designed the experiments: AD DDL CFY NMS DRC. Performed the experi-
ments: AD DDL DY CFY. Analyzed the data: AD DDL CFY. Contributed reagents/materials/
analysis tools: CFY NMS DDL AD. Wrote the paper: AD DRC DDL CFY NMS.
Retinofugal Projections from mRGCs
PLOS ONE | DOI:10.1371/journal.pone.0149501 February 19, 2016 12 / 14
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