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Targeted Destruction of Photosensitive Retinal Ganglion
Cells with a Saporin Conjugate Alters the Effects of Light
on Mouse Circadian Rhythms
Didem Go
¨z
1
, Keith Studholme
2
, Douglas A. Lappi
4
, Mark D. Rollag
1
, Ignacio Provencio
1
, Lawrence P.
Morin
2,3
*
1Department of Biology, University of Virginia, Charlottesville, Virginia, United States of America, 2Department of Psychiatry, Stony Brook University, Stony Brook, New
York, United States of America, 3Program in Neuroscience, Medical Center, Stony Brook University, Stony Brook, New York, United States of America, 4Advanced
Targeting Systems, San Diego, California, United States of America
Abstract
Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated
by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive,
expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit
for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting
of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this
immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult
mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is
significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously
observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to
show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/
dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit
for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a
technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any
species subject to the existence of appropriate anti-melanopsin antibodies.
Citation: Go
¨z D, Studholme K, Lappi DA, Rollag MD, Provencio I, et al. (2008) Targeted Destruction of Photosensitive Retinal Ganglion Cells with a Saporin
Conjugate Alters the Effects of Light on Mouse Circadian Rhythms. PLoS ONE 3(9): e3153. doi:10.1371/journal.pone.0003153
Editor: Ernest Greene, University of Southern California, United States of America
Received July 28, 2008; Accepted August 11, 2008; Published September 5, 2008
Copyright: ß2008 Goz 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.
Funding: NIH grant R01 NS22168 to LPM NIH grant R01 NS052112 to IP The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the mansuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: lawrence.morin@stonybrook.edu
Introduction
The hypothalamic suprachiasmatic nuclei (SCN) comprise the
primary circadian pacemaker in mammals and are responsible for
the generation of nearly all daily rhythms in physiology and
behavior. Timing of such rhythms is set by the daily photoperiod.
The retinohypothalamic tract (RHT) arises from a subset of retinal
ganglion cells and conveys photic information to the SCN through
the optic nerve. Most of these retinal ganglion cells express
melanopsin photopigment (Opn4) and are intrinsically photosen-
sitive [1,2]. These intrinsically photosensitive retinal ganglion cells
(ipRGCs) project to numerous brain regions, in addition to the
SCN [3].
Mice lacking either the classical photoreceptors (rods and cones)
[4,5] or melanopsin photopigment [6–8] exhibit relatively normal
entrainment of circadian rhythms to the light:dark photoperiod,
masking, and constriction of the pupil in response to ocular
illumination. However, the absence of both classical photorecep-
tors and melanopsin eliminates all three responses to light [9,10].
The purpose of these studies was to determine whether photic
information received by classical photoreceptors requires the
presence of ipRGCs in order to modify non-image-forming visual
responses. Toward this end, we developed a saporin-based
immunotoxin (UF008/SAP) that specifically ablates ipRGCs in
the fully-differentiated adult retina. Importantly, this approach can
be modified to target melanopsin-expressing cells in any animal
model, even those genetically intractable, provided that the
appropriate targeting antibodies are available.
Results
Specific Targeting of ipRGCs
In order to show specific ablation of melanopsin expressing cells,
RGC-5 cells stably expressing melanopsin were exposed to the
UF008/SAP conjugate. After 4 days of exposure, the cells were
killed in a dose-dependent manner. The highest concentration of
UF008/SAP (1000 pg UF008/SAP /ml) caused maximum cell
death whereas the same concentration of the non-immunized
IgG/SAP conjugate did not cause any cell death (Figure 1). Also,
RGC-5 cells that do not express melanopsin were not affected by
the UF008/SAP conjugate, even at the highest dose tested.
Injection of the UF008/SAP conjugate into the vitreous of adult
C57BL/6J mouse eyes killed ipRGCs in a dose-dependent manner
(Figure 2A and B), the curve becoming asymptotic at approximately
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400 ng/eye. All control retinas from various dose groups had similar
melanopsin cell densities and were combined. Relative to the
average control retina, about 57% of melanopsin cells were killed by
400 ng UF008/SAP per eye which was not significantly different
than the cell death achieved with the highest dose group (800 ng/
eye). Representative images from each dose group (Figure 2A) show
the decreasing number of immunopositive cells in retinas exposed to
increasing UF008/SAP amounts. A time course study showed that
maximal melanopsin cell death occurs about 2 weeks after 400 ng
UF008-SAP injection (Figure 2C). Each group was significantly
different from controls. Retinas of the 4 week group that received
400 ng/eye UF008/SAP were analyzed for regional differences in
the efficacy of the conjugate. Analysis of peripheral to central
regional variation in UF008/SAP-dependent melanopsin cell death
in the mouse retina revealed that the density of remaining cells was
uniform indicating no regional differences (Figure 3).
There are various ways of assessing retinal health, but relative
thickness of the outer nuclear layer (ONL) has been used
previously as a direct measurement of the degree of photoreceptor
cell death [11,12]. Figure 4A and B shows that neither relative
ONL nor relative inner nuclear layer (INL) thickness of retinas
from mice receiving 800 ng/eye UF008/SAP differed from
corresponding measurements from PBS-injected retinas. Because
there was no significant effect of the highest dose, no effects were
expected with the lower doses.
Exposure of the retina to UF008/SAP does not affect its tissue
morphology, as non-melanopsin cells remain intact. Cellular
analysis in Figure 4C confirms that UF008/SAP (400 ng/eye)
causes the loss of intrinsically photoreceptive ganglion cells
(ipRGCs), but does not appear to alter other structural
characteristics of the mouse retina. A normal distribution of glial
fibrillary acid protein (GFAP; labels Muller cells), calbindin-
(CALB; labels horizontal cells) and choline acetyltransferase
(ChAT; labels cholinergic amacrine cells) immunoreactivity was
observed in both control and UF008/SAP injected eyes.
Anatomical evaluation of the RHT terminal plexus in the SCN
was performed in mice that had received unilateral UF008/SAP
injections. The lesioned eye was given an intra-vitreous injection of
Figure 1. A saporin/anti-melanopsin(UF008) conjugate destroys cultured RGC-5 cells in a dose-dependent manner. Cultures of RGC-5
cells, either stably expressing or not expressing mouse melanopsin, were exposed to a saporin conjugate (UF008/SAP) for 4 days. The concentrations
of conjugates are shown above each column and the experiments were done in triplicate. Each panel represents a randomly selected field from a
single well.
doi:10.1371/journal.pone.0003153.g001
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cholera toxin subunit B (CT-B) conjugated to Alexa488 and the
intact eye was injected with CT-B/Alexa594 conjugate. Ipsi- and
contralateral retinas contribute equally to mouse SCN innervation
[13], allowing the contributions of lesioned and intact retinas to be
compared. Ablation of ipRGCs yielded an unexpected topography
of remaining SCN retinal innervation. Fluorophore-conjugated CT-
B labeled a dense corona of terminals along the lateral and ventral
SCN border, with very sparse innervation centrally and medially.
The normal retinal input occupied the central/medial area, as well
as overlapping the more lateral terminal corona (Figure 5).
Behavioral Experiments
Having revealed there are no significant morphological
differences between retinas dissected from UF008/SAP injected
and control eyes, we subjected mice to a visual cliff task, in order to
test general visual function. Figure 6 shows that bilaterally sighted
and injected mice differ slightly in their responses to a visual cliff
test, but both groups perform significantly above 50% chance
levels. Mice were also given a light-dark preference test that
revealed no difference between UF008/SAP-injected and control
mice with respect to the time spent in the dark or the number of
times the mice entered the lighted chamber (65.263.6 vs
71.665.5% time in the dark and (279653 vs 185640 entries
into the light for UF008/SAP and controls, respectively; the
corresponding values for 9 blind controls were 49.764.3 and
316645, both values significantly different from the other groups,
p,.02). When the comparison with controls was limited to the 6
mice that were free-running under LD, there was also no
difference between groups.
Mice in the rhythm regulation studies were unilaterally injected
with 400 ng UF008/SAP or IgG/SAP and contralaterally
enucleated (to reduce overall variability in ipRGC ablation) under
Figure 2.
In vivo
injection of UF008/SAP rapidly destroys ipRGCs in a dose-dependent manner. UF008/SAP-dependent killing of ipRGCs
in adult C57BL/6J mouse retina. A) Selected retinal flat-mount images (106magnification) of PBS and UF008/SAP injected eyes from each dose
group. B) Left eyes of mice (n =5 per dose group) were injected with increasing doses of the UF008/SAP conjugate (25, 50, 100, 200, 400 and 800 ng/
eye). Right eyes were injected with PBS to serve as controls. (1-way ANOVA with Tukey’s Multiple Comparison Test, ** p,0.01, *** p,0.001). C) Mice
(n = 4 per time group) were bilaterally injected with 400 ng of UF008/SAP conjugate per eye or 400 ng/eye of IgG/SAP (saporin conjugated to a
‘‘nonsense’’ rabbit IgG). At one week post-injection, the remaining number of melanopsin cells in the UF008/SAP injected retinas was significantly less
than the controls, but the maximum melanopsin cell death was achieved at 2 weeks post-injection (1-way ANOVA with Tukey’s Multiple Comparison
Test, * p,0.05, *** p,0.001). The results for each experimental group in B and C were normalized relative to the data from the contralateral control
eyes.
doi:10.1371/journal.pone.0003153.g002
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LD12:12 conditions before being subjected to various lighting
regimes. Figure 7A shows actograms representative of behavior
across four different lighting conditions. None of the mice lost
entrainment in LD12:12 as an immediate consequence of UF008/
SAP treatment. When switched to a graded photoperiod in which
the irradiance gradually declined to zero (LD15G:9; Figure 7B), all
animals entrained, but with markedly different phase angles
between groups (Figure 7C). Large differences in phase angle of
entrainment were related to the correspondingly large differences
in irradiance at the time of activity onset (Figure 7B). In constant
darkness (DD), the circadian periods of the two groups did not
differ. In constant light (LL), the circadian period of UF008/SAP
mice did not change, whereas for control mice the period
lengthened, as expected (Figure 7D). When the mice were
returned from LL to the original LD12:12 (without regard to the
phase of the individual mice), 9 of 10 UF008/SAP mice required
16 or more days to re-entrain (median = 24 days), whereas every
control animal required 14 or fewer days (median = 3 days).
Subsequent histology showed that, on the average, UF008/SAP
treatment yielded approximately 80% loss of melanopsin cells.
Among the UF008/SAP-treated mice, the 3 individuals with the
highest densities of remaining ipRGCs required 0, 16 and 16 days
to re-entrain (corresponding to 81, 27 and 21 cells/mm
2
,
respectively). Four of the UF008/SAP-injected mice never re-
entrained. The density of melanopsin expressing RGCs averaged
15.2 cells/mm
2
in these four animals.
Figure 7E shows that constant light exposure induces a
suppression of total wheel-running activity (masking) in control
(IgG/SAP) mice, but not in UF008/SAP injected mice. Entrained,
UF008/SAP-injected mice had relatively normal masking in
response to a 1 hr light pulse, but recovered more slowly than
expected at the end of the pulse (Figure 8). In contrast, UF008/
SAP mice that fail to entrain also do not show masking to a 1 hr
light pulse administered about 30 minutes after activity onset.
Discussion
Saporin is a type I ribosome inactivating protein that was
initially identified in the common soapwort plant (Saponaria
officinalis) [14]. It depurinates a nucleotide in 28S ribosomal
RNA resulting in a conformational change that prevents
elongation factors from associating with the ribosome, thereby
irreversibly arresting protein synthesis at the translocation step.
Type I ribosome inactivating proteins lack the lectin-like B chain
characteristic of type II ribosome inactivating proteins such as
ricin. This peptide facilitates cell entry. Therefore, saporin and
other type I ribosome inactivating proteins must be conjugated to
moieties that specify their targeting and internalization. Conjuga-
tion to ligands of cell surface receptors or to antibodies that
recognize cell surface epitopes have been the most common
strategies. For example, a substance P/saporin conjugate has been
used to specifically ablate inhibitory hippocampal neurons in the
rat [15]. 192-IgG/saporin, a conjugate with an antibody specific
for the low-affinity neurotrophin receptor, p75
NTR
, has been used
extensively to lesion cholinergic neurons in the basal forebrain
[16,17].
The feasibility of using saporin conjugates to ablate specific
retinal cell types was demonstrated in rat and ferret [18,19]. A
conjugate consisting of an antibody raised against the vesicular
acetylcholine transporter covalently linked to saporin was injected
intravitreally. The number of cholinergic amacrine cells in the
retina was dramatically reduced.
In this study, we pursued a similar approach to specifically
target and kill melanopsin cells. Our saporin conjugate, UF008/
SAP, eliminated most of the melanopsin-expressing cells in a dose-
and time-dependent manner. Furthermore, the cell killing was
uniform across the retina. The percentage of killed cells varied
from about 60% to 80% across experiments, and may have
depended upon the exact method of injection. The number of
remaining cells showing melanopsin immunoreactivity may be an
overestimation of functional melanopsin cells as they differed in
appearance from those in PBS injected eyes; labeling intensity was
reduced in many of the residual cells and the dendrites appeared
shorter and broken, suggesting a functionally compromised state.
Other than the loss of melanopsin cells, intravitreal injection of
UF008/SAP did not cause any obvious alteration in retinal
morphology. The thicknesses of the outer and inner nuclear layers
were not diminished compared to controls, suggesting the absence
of non-specific cell death. Furthermore, the dendrites arising from
the cholinergic amacrine cells retained an appropriate position
within the inner plexiform layer suggesting that no drastic
reorganization within this synaptic layer had occurred in the face
of melanopsin cell loss. Finally, an analysis of visual competency
through the use of the visual cliff and light-dark preference tasks
indicates that conjugate-injected animals do not suffer any gross
defects in visual perception.
Figure 3. Destruction of ipRGCs by UF008/SAP depends on dose, but not on location within the retina. Ablation of ipRGCs by UF008/
SAP is dose-dependent, but not related to retinal eccentricity. Each triplet of bars represents data from ‘‘peripheral’’ (P), ’’middle’’ (M), and ‘‘central’’
(C) fields, respectively. (2-way ANOVA with Bonferroni post-tests).
doi:10.1371/journal.pone.0003153.g003
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Nevertheless, robust effects of UF008/SAP-induced melanopsin
cell loss were seen upon examination of certain non-image forming
visual responses. These included a greatly impaired ability to entrain
properly to the prevailing photoperiod and loss of masking responses
to light. In particular, lesioned mice transferred to a ‘‘dusk
entrainment’’ paradigm, in which the transition from light to dark
occurs gradually, adopted a vastly different phase angle of
entrainment. The data support the view that animals with a
compromised circadian photosensory system are less sensitive to light
as indicated by the fact that they entrain to dusk transitional light
only after much longer exposure to much brighter light than
necessary for entrainment of control animals injected with a non-
specific IgG/SAP conjugate. The number of remaining anti-
melanopsin immunoreactive cells was clearly related to the phase
angle of entrainment, although the exact relationship is not yet clear.
The absence of a difference in free running periods of the
control and UF008/SAP treatment groups in DD suggests that
immunotoxin damage does not impact the circadian clock per se,
but rather exerts its specific actions by modifying photic input
pathway to the circadian system. In this respect, the failure of
UF008/SAP-treated mice to lengthen their circadian periods in
LL is the second important indicator that the circadian visual
system is impaired in its response to light. This is in sharp contrast
to results obtained from melanopsin null mice which continue to
exhibit LL-dependent period lengthening(albeit diminished by
about 60%) [20]. The UF008/SAP-injected animals more closely
resembled Opn4
2/2
;rd/rd mice, which are melanopsin-null, lack
rod and cone photoreceptors and show no period lengthening
under LL. The results indicate that rods and cones require the
presence of some minimal number of ipRGCs in order to
Figure 4. UF008/SAP kills ipRGCs without apparent damage to general retinal morphology. UF008/SAP does not induce changes in gross
retinal morphology. Eyes injected with 800 ng/eye of UF008/SAP conjugate did not differ ( 2-way ANOVA with Bonferroni post-test) from the PBS-
injected control eyes in their relative (A) ONL or (B) INL thickness. (open bars, control eyes; black bars, UF008/SAP-injected). C) Cryostat sections
through control retina (left column) and through UF008/SAP treated retina (right column). Red label (all images) - Melanopsin immunoreactive (IR)
cells (*) and processes (arrowheads); Green label (Upper Row) - GFAP-IR glia at the base of the ganglion cell layer; Green label (Middle Row) - CALB-IR
cells (arrows) in the outer and inner nuclear layers; Green label (Bottom row) - ChAT-IR amacrine cells (arrows) in the inner nuclear and ganglion cell
layers between which are the two ChAT-IR terminal zones of the inner plexiform layer. Blue label - The nuclear stain, DAPI, most clearly reveals cells in
the outer nuclear layer of the upper and lower images. Note the absence of melanopsin-IR staining in treated retinas (right column). Abbreviations of
retinal layers: g – ganglion cell; inl – inner nuclear; ipl – inner plexiform (on and off sublayers); onl – outer nuclear; opl – outer plexiform; p –
photoreceptor. Bar = 20 mm.
doi:10.1371/journal.pone.0003153.g004
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influence the ability to entrain with a normal phase angle and the
period lengthening response to LL.
Light, when administered during an animal’s night, acutely
suppresses activity, a phenomenon known as ‘‘masking’’ [21]. In
addition to its effect on circadian period, LL exposure typically
reduces the total amount of nocturnal locomotion. Such activity
reduction was observed in control mice, but not in UF008/SAP
injected mice. When the UF008/SAP mice were re-exposed to a
standard LD12:12 photoperiod, 5 of 9 failed to re-entrain, a
further indicator of diminished circadian system photosensitivity.
The locomotor activity of mice that did not entrain animals was
also completely unresponsive to a 1 hr light pulse during the early
subjective night that produced the expected massive masking in
controls. Light pulse-induced masking by the UF008/SAP-treated
mice that were entrained was reasonably normal, unlike the LL-
induced masking which occurred in all treated mice. This suggests
that a greater number of ipRGCs must be lost in order to block re-
entrainment and pulse-induced masking than is necessary to block
either LL-induced period lengthening or masking.
Anatomical Considerations
Three separate investigations using different methods have now
demonstrated that intact melanopsin cells are necessary in order
for the mouse non-image forming visual system to function
properly ([22,23] and this report). Cell ablation greatly reduces the
size of the RHT terminal field. In all likelihood, it is this specific
change that accounts for the absence of light-induced phase shifts
and masking. However, an alternative explanation is possible.
Melanopsin cells have dendro-dendritic synaptic contacts with
amacrine cells [24,25] and may transmit photic information to
Figure 5. UF008/SAP greatly reduces retinal projections to the suprachiasmatic nucleus and intergeniculate leaflet. CT-B tracing of
RHT projections remaining after intravitreal UF008/SAP injection shows a terminal field densest along the lateral, ventrolateral and ventral SCN
border, but greatly reduced in, or absent from, the dorso-central SCN. B) In the IGL, the contralateral projection from the UF008/SAP-injected retinais
largely absent. C) There are no readily apparent differences with respect to the remaining retinal innervation of the OPT. The red label in all parts of
the figure identifies terminals from the contralateral, undamaged retina. Abbreviations: APT – anterior pretectal n.; CPT – commissural pretectal n.;
DLGc – dorsal lateral geniculate n;, contralateral; IGLc – intergeniculate leaflet, contralateral; IGLi – intergeniculate leaflet, ipsilateral; MPT – medial
pretectal n.; NOT – nucleus of the optic tract; PPT – posterior pretectal n.; RHT – retinohypothalamic tract; VLGc – ventral lateral geniculate n.,
contralateral. (Ipsi- and contra- are referenced with respect to the injected with UF008/SAP).
doi:10.1371/journal.pone.0003153.g005
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them [26]. Therefore, loss of melanopsin cells might be
interrupting necessary transmission of information to the SCN
that would pass through amacrine cells in contact with non-
melanopsin-containing retinal ganglion cells that also project to
the SCN.
The present data (Figure 7C) suggest the possibility that there
may be a sharply delineated minimum melanopsin cell density that
is required for normal non-image forming visual responses.
Potential melanopsin cell photoreceptive network properties may
be lost when cell density falls below approximately 25 cells/mm
2
.
A similar, sharp threshold effect on rat working memory has been
observed following saporin-induced destruction of basal forebrain
cholinergic cells [27]. In that instance, memory was impaired only
if an approximate 75% cell destruction threshold was exceeded.
The mouse RHT provides bilaterally symmetrical innervation to
the SCN [28]. This characteristic has enabled us to compare, in
individual mice, the terminal distributions from a normal retina with
those from an ipRGC-ablated retina. The results indicate a
specialized topography of the RHT terminal field that remains after
melanopsin cell cell death. Whether the particular distribution of
terminals observed is of functional importance remains to be seen.
However, it is not associated with any previously described sector of
the mouse SCN based on distributions of peptidergic cell types, clock
gene expression or afferent terminal fields.
The thalamic intergeniculate leaflet (IGL) is a component of the
circadian system particularly noteworthy because of the robust
projection it provides to the SCN. The IGL also receives direct
photic input from a predominantly contralateral retinal projection
[29]. In mice sustaining UF008/SAP-induced ablation of
melanopsin cells, retinal projections to the contralateral IGL are
virtually eliminated. This suggests that all or nearly all retinal
projections to the contralateral IGL arise from melanopsin cells. A
functional implication concerns the role of the IGL as a mediator
of circadian system response to tonic light exposure. In the
hamster, loss of the IGL reduces the period lengthening effect of
LL by approximately the same amount as loss of melanopsin
photopigment does in the mouse [20,30]. Classical photoreceptors
account for the remaining effect of LL on period length and may
exert their action directly on the SCN via the RHT.
Our results are consistent with those from two earlier studies
(Table 1) employing different methods to reach similar conclusions
about the essential role played by melanopsin-expressing retinal
ganglion cells in the regulation of certain non-image forming visual
responses [22,23]. All three clearly indicate that the absence of
melanopsin cells causes a major deficiency of entrainment to LD
cycles and a lack of period lengthening in LL. Collectively, the three
sets of data demonstrate that melanopsin cells are not necessary for
at least rudimentary image forming vision as indicated by response
to a wide variety of visual tasks. Unexpectedly, our light-dark
preference test, which is unlikely to involve either image formation
or optomotor reflexes, was performed equally well by both sighted
and melanopsin cell deficient mice, implying that this is behavior
relies on different neural mechanisms than used for simple
irradiance detection (cf., [31].
With respect to circadian rhythm regulation, our results are
most similar with those of Hatori and colleagues [22] who also
used a procedure that acutely killed melanopsin cells in adult mice.
In contrast, Guler and colleagues [23] observed effects (longer
circadian period in DD, gradual loss of melanopsin cell
projections) that are likely the consequence of altered circadian
system development. The inducible lesion procedure applicable to
adult mice developed by Hatori et al. appears to be the most
consistently successful of the three methods used to kill melanopsin
cells. In particular, all mice with diphtheria toxin activated
melanopsin cell death rapidly lost entrainment and exhibited free-
running rhythms. Nevertheless, as is true for the other procedures,
this method also fails to kill all melanopsin cells.
The advantages of the UF008/SAP procedure are two-fold.
First, we have shown that complete ablation of retinal ganglion
cells is not required to observe massive deficits in non-image
forming visual behavior of mice, such as circadian rhythm
entrainment and masking. Second, this technique has broad
potential applicability across species, limited only by the
availability of appropriate antibodies that can be conjugated to
saporin, creating an melanopsin cell-specific immunotoxin.
Finally, this is the first report of a functional immunotoxin made
with an antibody to a G protein-coupled receptor. Previous toxins
to G protein-coupled receptors have relied on the ligand for
targeting and internalization of a toxin [32–34]. Antibody
targeting of cell-surface proteins has become an effective tool in
cancer therapy [35–37], and these data indicate that targeting G
protein-coupled receptors with antibodies may likewise be useful.
Methods
Generation of UF008/SAP immunotoxin
UF008, an anti-melanopsin polyclonal antibody raised in rabbit
against the 15 N-terminal extracellular amino acids of mouse
melanopsin, was covalently conjugated to saporin (Advanced
Targeting Systems, San Diego, California). Synthesis was as
described previously [38].
Validation of UF008/SAP immunotoxin specificity
The RGC-5-mWT-2 cell line was established by stably
transfecting RGC-5 cells using calcium phosphate precipitation
[39] with a ScaI linearized vector containing the mOpn4 open
reading frame (gi:14349304) in parental vector pcDNA3.1/Zeo(+)
(Invitrogen, Carlsbad, CA). Melanopsin expression was confirmed
by immunocytochemistry. The RGC-5 cell line was a generous gift
Figure 6. UF008/SAP treatment has little effect on visual cliff
performance. Both UF008/SAP and IgG/SAP injected groups perform
quite accurately in the visual cliff test, but there is a small deficit in the
UF008/SAP treated group (p,0.05, Mann-Whitney test).
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Figure 7. Depletion of ipRGCs greatly alters entrainment, period lengthening in LL and masking by LL. Running records of a control
and 3 UF008/SAP-treated mice showing phase angle of entrainment adopted in response to the gradual offset photoperiod, period during DD,
during LL and re-entrainment (animals #139, 135) or failure to re-entrain (animals #122, 133) to LD12:12 (grayed area indicates darkness). The phase
angle of entrainment (W) during the gradual offset photoperiod is indicated for each individual (ZT12 = light completely off). B) Daily irradiance
pattern recorded with a Gigahertz-Optik P-9710-2 universal optometer measured at cage level during the gradual offset light-dark paradigm. C)
Relationship between remaining ipRGC density and the stable phase angle of entrainment of adopted by UF008/SAP injected mice and controls
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of Dr. Neeraj Agarwal [40]. It was derived by transformation of
neonatal retinal cells with psi2 E1a, a retroviral vector containing
the 12S E1A adenoviral oncogene. RGC-5 cells express mRNA
markers characteristic of retinal ganglion cells. Cells were
maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented
with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA).
Triplicate cultures were exposed to increasing concentrations of
the UF008/SAP immunotoxin ranging from 0 to 1000 pg/ml.
Controls were exposed to 1000 pg/ml of the nonsense immuno-
globulin conjugate IgG/SAP which consists of a general non-
specific anti-rabbit IgG covalently conjugated to saporin. In
addition, RGC-5 cells not expressing mOpn4 were exposed to 0 or
1000 ng/ml of UF008/SAP or 1000 ng/ml of IgG/SAP. After 4
days in standard culture conditions, representative images of the
triplicate cultures were visualized and captured under Hoffman
modulation contrast on an Olympus CK2 inverted microscope
fitted with a camera mount.
Animals
C57BL/6J male mice (Jackson Laboratories, Bar Harbor, ME)
were used in this study. All the experimental procedures were
carried out in accordance with Association for Assessment of
Laboratory Animal Care policies and approved by the University
of Virginia Animal Care and Use Committee or by the Stony
Brook University Institutional Animal Care and Use Committee.
Eye Injections
Injections (2 ml/eye) were performed in animals anesthetized
with isoflurane and eyes topically anesthetized with one drop of
0.5% proparacaine (Akorn Inc, Buffalo Grove, IL). Eyelids were
gently retracted with fingers, allowing the eyeball to protrude. A
Hamilton syringe with a 30 gauge needle was used to make the
injection at the level of the ora serrata into the posterior chamber
of the eye and needle was left in place for about 2 minutes after the
injections. The lids were then slowly released allowing the eyeball
to retract back into the orbit. Animals were systemically
administered a mouse ketoprofen analgesic mixture after the
injections.
Dose/response to UF008/SAP
Three month old C57BL/6J male mice were divided into six
dose groups (n= 5) The right eye of each animal was injected with
2ml phosphate buffered saline (PBS) vehicle and the left eye was
injected with 25, 50, 100, 200, 400 or 800 ng of UF008/SAP
suspended in 2 ml PBS vehicle. All animals were sacrificed by CO
2
asphyxiation 4 weeks post-injection. Eyes from 1 animal per group
Figure 8. Masking response to a light pulse is absent in mice that lose entrainment to LD12:12. Masking is nearly normal by UF008/SAP
injected mice in response to a 1 hr light pulse. However, the animals recovered more slowly than controls. There is no effect of treatment or of time,
but there is a significant interaction (1 way ANOVA with repeated measures, p,.004). In mice that lost entrainment subsequent to UF008/SAP
treatment, a 1 hr light pulse failed to induce masking.
doi:10.1371/journal.pone.0003153.g008
during the gradual offset photoperiod. Note the cluster of controls and the outlier animal (green-filled circle), which was injected with the saporin
conjugate, but has not lost its ipRGCs. A second outlier animal (red-filled circle) had a normal phase angle of entrainment despite greatly reduced
ipRGC density. D) There was no difference in period in circadian period during DD between UF008/SAP and IgG/SAP injected mice, but in LL, IgG/SAP
injected mice significantly lengthened their periods (p,0.001; paired t test), becoming significantly different from UF008/SAP injected animals
(p,.001, unpaired t test) which did not show any period lengthening in response to LL. E) LL induced masking was absent in UF008/SAP injected
mice. Revolutions per day for the last 5 days in DD were compared to those during the initial 5 days of LL. For UF008/SAP mice, revolutions during DD
and LL did not differ; for controls, revolutions/24 hr dropped by about 40% (p,.001; paired t tests).
doi:10.1371/journal.pone.0003153.g007
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PLoS ONE | www.plosone.org 9 September 2008 | Volume 3 | Issue 9 | e3153
were sectioned whereas eyes from the remaining 4 animals were
used for retinal flat-mounts.
Eyes were excised and hemisected. Posterior eyecups containing
the lens were immersed into freshly prepared 4% paraformalde-
hyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS
and kept at 4uC for 24 hours. For flat-mounts, lenses were
removed and retinas dissected from eyecups after which they were
spread on a filter paper. All immunohistochemical procedures on
flatmounts were carried out in 1.5 ml microfuge tubes. For retinal
sections, eyecups were cryo-protected in 30% sucrose in 0.1 M PB
overnight at 4uC, embedded and frozen in a prepared (3% gelatin,
30% egg albumin in distilled H
2
O) or commercially available
(Tissue-Tek OCT 4583 Compound, Sakura Finetek, Torrance,
CA) mounting medium, and sectioned in a cryostat at 16 mm
thickness. Sections were thaw-mounted onto gelatin/chromium-
coated glass microscope slides, air dried, and stored frozen (220uC
or 280uC) until further processing.
Flat-mounted retinas and sections were washed 3 times (10 min
each) in tris-buffered saline (TBS) (Quality Biological, Gaithes-
burg, MD) and blocked for 30 minutes in 6% normal goat serum
(Vector Laboratories, Burlingame, CA) in TBS. Blocking was
followed by 3 washes with TBS followed by incubation at 4uC for
24 hrs in a 1:2500 dilution of the primary anti-mouse melanopsin
antiserum (UF006) in a TBS buffer containing 1% BSA, 0.25%
carrageenan lambda, and 0.3% Triton X-100. Tissues were then
washed 3 times with TBS and incubated for 1 hr at room
temperature in Cy3-conjugated anti-rabbit IgG secondary anti-
body (Jackson Immunoresearch, West Grove, PA) diluted 1:500 in
incubating buffer, followed by three TBS washes. Flat mounts
were removed from the filter paper and transferred to glass slides.
Sections and flat-mounts were mounted in DAPI-containing
Vectashield (Vector Laboratories, Burlingame, CA), coverslipped
and sealed.
Grayscale images were captured on a Zeiss epifluorescence
microscope equipped with a SPOT charge-coupled device
camera. Photoshop 6.0 (Adobe Systems, San Jose, CA) was used
to enhance image files for brightness and contrast. In each
quadrant of the flat-mount, three images corresponding to an area
of 0.61 mm
2
were captured sequentially, from the periphery to the
center (optic nerve), for a total of 12 images/retina. These three
images were termed ‘‘peripheral’’, ‘‘middle’’ and ‘‘central’’. The
cell count in each of the 12 frames was converted to cell number/
mm
2
. Selected images were pseudocolored with Photoshop to
reflect the long wavelength emission fluorescence of the Cy3
fluorophore.
Time course to UF008/SAP
Three month old C57BL/6J male mice were divided into five
groups (n= 4). Four groups received bilateral ocular injections of
UF008/SAP (400 ng/eye) and were killed 1, 2, 3 or 7.5 weeks
post-injection. The fifth group was bilaterally injected with the
nonsense immunoglobulin conjugate IgG/SAP as a control and
killed at 7.5 weeks. All animals were sacrificed by CO
2
asphyxiation 4 weeks post-injection. Eyes from 1 animal per
Table 1. Effect of physical destruction of melanopsin-containing retinal ganglion cells: comparison of results from three studies
using different methods.
Present Data Reference [23] Reference [22]
METHOD
Anti-Opn4 antibody conjugated to saporin
injected in adults
Opn4
aDTA
mouse Opn4
Cre/+
;R26
iDTR/+
mouse adult
inducible lesion with diphtheria toxin
MEASUREMENT RESULT
Entrainment Yes, with advanced onset; about half fail to
re-entrain
Most do not entrain, the rest have advanced
onset
Absent
Circadian period (DD) Normal Longer Normal
Circadian period (LL) Same as DD Shorter than DD Same as DD
Phase shift to light – Absent –
Masking Absent
1
Reduced Absent
Gross retinal structure Normal Normal Normal
OPN4 cells remaining 18–40% 3–17% ,10%
Residual SCN innervation Absent centrally; reduced elsewhere Substantially reduced over 1 yr; more so in
homozygotes
Nearly absent
Residual IGL innervation Nearly absent Disappears over 1 yr –
Residual OPT innervation Robust (CTb) ipRGC input reduced and disappears over 1 yr –
Pupillary light reflex – Absent at low irradiance
2
Absent at low irradiance
Visual acuity – Slight decrease
3
Visual cliff Normal – Normal
Visual learning task – Normal –
Light/dark preference Normal – –
ERGs (scotopic & photopic) – Normal Normal
Optomotor nystagmus – Normal –
1
– but masking was nearly normal in UF008/SAP treated mice that re-entrained to LD12:12.
2
– at high irradiance, most mice had normal constriction; 3 were reduced by about 50% and these 3 mice failed to re-entrain, unlike the other 6.
3
– possibly attributable to pupil size.
doi:10.1371/journal.pone.0003153.t001
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PLoS ONE | www.plosone.org 10 September 2008 | Volume 3 | Issue 9 | e3153
group were sectioned whereas eyes from the remaining animals
were used for retinal flat-mounts. Tissues were processed as
described previously for the dose/response studies.
Effects of exposure to the immunotoxin on the
morphology of the retina
Assessment of outer and inner nuclear layer
thickness. Retinal sections from the 800 ng/eye dose group
of the dose/response study (and the corresponding PBS control)
were assessed for the thickness of the outer and inner nuclear layer
as indicators of retinal health. Five midsagittal sections containing
the optic nerve (or sections within 32 mm of the optic nerve) were
analyzed in three non-overlapping microscope fields, according to
their eccentricity (‘‘central’’, ‘‘middle’’ and ‘‘peripheral’’).
Axiovision Software of the Zeiss epifluorescence microscope was
used to measure outer nuclear, inner nuclear and total retinal
thickness in each of these areas. The thickness of the nuclear layers
were normalized to total retinal thickness at each eccentricity, for
both control and UF008/SAP injected eyes.
Assessment of retinal architecture. Mice were anesthetized
(100 mg/kg ketamine plus 10 mg/kg xylazine, intraperitoneally)
and were transcardially perfused with saline followed by 4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, with
sodium m-periodate (0.01 M) and lysine (0.075 M) added. Eyes
were removed, hemisected, and immersed in the same fixative for
1 hour and rinsed with PBS for 30 min. Posterior eyecups were
cryoprotected, sectioned, mounted, and stored as described above.
For double-labeling experiments, retinal sections were thawed
at room temperature for 20 min., washed three times in PBS,
pH 7.4, for 10 minutes, and then blocked in 2% normal donkey
serum in PBS for 20 minutes. Tissue sections were incubated for
24 to 48 hours at 4uC in a mixture of a rabbit polyclonal anti-
melanopsin antiserum (UF007; 1:2000 final dilution) and either
mouse anti-glial fibrillary acidic protein (GFAP), 1:500; goat anti-
choline acetyltransferase (ChAT), 1:750 (Chemicon International,
Temecula, CA) or mouse anti-calbindin (CALB), 1:1000 (Sigma-
Aldrich, St. Louis, MO). UF007 identifies melanopsin-containing
retinal ganglion cells in hamster [41]. About 53% of UF007
positive retinal ganglion cells are b-gal positive in the heterozygous
b-gal mouse (Baver, Sollars & Pickard, personal communication),
very similar to what is seen using the UF006 antiserum [42].
Primary antibodies were diluted in an incubation solution
containing 0.3% Triton X-100 and 5% normal donkey serum.
After washing in PBS for 45 minutes and blocking in 2% donkey
serum the slides were incubated for 45 min. at 37uC in a mixture
of donkey anti-rabbit Texas Red and donkey anti-mouse or anti-
goat FITC secondary antibodies (JacksonImmunoResearch Labs,
West Grove, PA) diluted 1:150 in incubation solution. After final
washes in PBS sections were coverslipped with ProlongGold
(Invitrogen, Carlsbad, CA) and stored at 4uC in the dark.
Assessment of retinal projections. Mice (N = 4) were
anesthetized with ketamine (100 mg/kg; Wyeth Pharmaceuticals,
Madison, NJ) and xylazine (10 mg/kg; Ben Venue Laboratories,
Bedford, OH) about 14 days after a unilateral injection of 400 ng
UF008/SAP. At this time, the saporin-injected eye received an
intravitreal injection of cholera toxin, B subunit, (CT-B)
conjugated to Alexa488 and the other eye was injected with
CT-B conjugated to Alexa594 (each in 2 mlof1mg/ml of CT-B in
0.9% saline with 2% DMSO; both tracers from Molecular Probes,
Eugene, OR). After 3 days transport time, the mice were deeply
anesthetized with ketamine/xylazine (as above) and perfused with
physiological saline and 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4. Brains were removed, post-fixed
overnight at 4uC, cryoprotected in 20% sucrose for 24–48 hr,
then sectioned in the coronal plane at 30 mm on a freezing stage
microtome. Four series of free floating adjacent sections were cut
from the optic chiasm caudally through the pretectal region and
collected in 0.01 M phosphate-buffered saline-azide (pH 7.4). One
series was immediately mounted on gelatin-coated glass slides, air
dried and coverslipped with Krystalon (Diagnostic Systems, Inc.,
Gibbstown, NJ).
Photomicrographic false color images were obtained using
epifluorescence and a black and white digital camera (Zeiss
AxioCam) attached to a Zeiss Axioplan 2 microscope controlled
by Zeiss software (Carl Zeiss, Oberkochen, Germany). Images
were shot at 10 or 206magnification, with composites made of
the pretectal region. Images were adjusted with Corel Photo-Paint
12 for brightness and contrast and assembled into figures with
CorelDraw 12 (Corel Corp., Ottawa, Ontario, Canada).
Behavioral Experiments
Rhythm study 1. For this study of entrainment and period or
masking response to constant lighting conditions, each mouse was
placed in a cage with a running wheel inside an enclosure in which
white LED lighting was computer-controlled. Each enclosure
contained five mouse cages with an array (OSRAM OS-CM01E-
W) of 9 LEDs above each cage. All arrays per enclosure were
controlled via D-A voltage computer output to an LED dimmer
(OSRAM OT DIM) with its outputs connected to the arrays.
Steady-state maximal irradiance was about 1 uW/cm
2
. Wheel
revolutions were monitored by computer, stored as revolutions/
min, then summed across each 5 min interval (288/day) and
plotted in raster format using custom software (WinCollectRT,
Stony Brook University).
When rhythms were stable, each mouse was deeply anesthetized
with ketamine and xylazine (as above) and unilaterally enucleated.
Under the anesthesia, the remaining eye was injected intravitreally
with 400 ng UF008/SAP conjugate (N = 10) or 400 ng rabbit
IgG/SAP conjugate (N = 10; 2 additional uninjected mice were
included as controls) per 2 ml saline. At the conclusion of the study,
the animals were anesthetized, perfused, and their retinas taken for
histology using wholemount and cryostat sections.
Prior to the surgery, lighting was LD12:12, with lights on at
0300 and lights off at 1500. Animals were unilaterally enucleated
under LD12:12, allowed to recover for about 2 weeks, then the
remaining eye was injected with 400 ng UF008/SAP or IgG/SAP
(2 mL) and animals remained in LD12:12 for about 2 additional
weeks. At this time, because no animals had lost entrainment or
adopted a new phase angle of entrainment, all were exposed to a
photoperiod in which irradiance gradually declined to zero. This
afforded the animals an opportunity to establish a new phase angle
indicative of the amount of light exposure necessary to achieve
stable entrainment. When entrainment stabilized, each animal was
exposed to about 4 weeks DD, followed by 3 weeks in LL, followed
by re-exposure to LD12:12. During LD12:12, animals were given
1 hr light pulses of 1 uW/cm
2
white LED light beginning
approximately 2 hr after activity onset.
Rhythm study 2. Masking in response to a light pulse was
tested in mice that either remained entrained to the prevailing
photoperiod or lost entrainment as a result of UF008/SAP
treatment. In this study, mice were placed in cages with running
wheels on shelves in a general animal housing room that provided
lighting from overhead fluorescent fixtures producing an LD12:12
photoperiod and yielding about 20 mW/cm
2
irradiance at cage
level. Mice were allowed to entrain, then anesthetized as described
above, unilaterally enucleated and given an injection of 400 ng
UF008/SAP or IgG/SAP into the remaining eye. Locomotor
rhythms were followed for 2 weeks while the animals remained
Entrainment Requires ipRGCs
PLoS ONE | www.plosone.org 11 September 2008 | Volume 3 | Issue 9 | e3153
exposed to LD12:12 except for a single night-time light exposure
for a test of masking. All mice also were subsequently tested for
response to a visual cliff and for responses in a light/dark
preference test.
Masking. Rhythm study 1 demonstrated that UF008/SAP
treatment causes a marked drop in sensitivity to light, as measured
by the phase angle of entrainment. These mice failed to re-entrain
following exposure to constant lighting conditions. In Rhythm
study 2, all animals were housed under LD12:12, but some failed
to entrain after simultaneous unilateral enucleation and UF008/
SAP injection into the remaining eye. This study of masking was
conducted to determine whether the mice that had lost
entrainment to light had also masking response to a standard
light stimulus. Therefore, a 1 hr light pulse consisting of general
room light exposure (20 mW/cm
2
) was initiated at ZT13 for
entrained mice or at CT13 to mice that were not entrained. A
repeated measures ANOVA was used to compare masking by
UF008/SAP and IgG/SAP groups
Visual cliff test. Mice were bilaterally injected with UF008/
SAP or IgG/SAP (400 ng for each). About 2 wks later, animals
were handled daily for several days before beginning the visual cliff
test which was conducted in an arena under ordinary laboratory
fluorescent light (about 100 mW/cm
2
). The arena consisted of a
59655629 cm LWH open-top box with black plastic sides and a
6.4 mm thick clear plastic bottom placed on a desk. Beneath one
half of the floor was a black and white checkerboard pattern of
2 cm squares; the other half extended off a desk, about 77 cm
above the laboratory floor. Resting on the middle of the floor,
dividing the arena into non-cliff (checkerboard) side and cliff side
was black plastic beam (2.9 cm high63.5 cm wide).
Each mouse was lifted by its tail and lowered until all 4 limbs
were firmly on the middle part of the beam when it was released.
At this moment, the trial was initiated and continued until 5 min
had elapsed or the mouse stepped off the center beam with all 4
paws placed one side of the beam. The mouse was then placed
back in its cage. The arena walls, floor and beam were cleaned
using diluted alcohol on a fresh paper towel between tests. The
procedure was repeated ten times for each mouse.
Light/dark preference test. The same mice used for the
visual cliff test were used for the light/dark preference test begun
approximately 2 wks later. The test apparatus consisted of two
similar chambers, each about 29631630 cm LWH connected by
a 5.5 cm square opening. The walls were covered with a pale
flooring tile and the floor covered with absorbent paper. A single
food pellet was placed in each chamber and a spigot from an
externally mounted water bottle protruded into each chamber
through the wall opposite the between-chamber opening. One
chamber was designated the ‘‘dark side’’ and was covered by an
opaque sheet of black plastic. Most of the ‘‘light side’’ was similarly
covered by opaque black plastic. However, a central,
11.5616.5 cm area was removed as a viewport for a Microsoft
LifeCam VX-6000 video camera positioned about 11.5 cm above
and focused on the chamber floor. The camera was set into, and
was surrounded by, opaque black plastic which completed the
light side top. Parallel and adjacent to each long side of the
viewport, and attached to the underside of the roof, was a Phillips
F4T5 Softwhite fluorescent fixture that provided about 100 mW/
cm
2
white light continuously. In the dark side, irradiance was
0.17 mW/cm
2
directly opposite the between-chamber opening
and was darkest (0.04 mW/cm
2
) in the corners nearest the
opening.
The camera was set to black and white mode with contrast and
brightness adjusted to bleach nearly all visible patterns in the
image. Thus, when a pigmented mouse entered the chamber, the
number of pixels detected as black per video image increased
dramatically (factor of about 50). Custom software detected and
counted the number of transitions from dark side to light side and
determined the percentage of each minute the animal spent in the
light side.
The exposed surfaces of each chamber were sponged clean,
washed with water, then dried with paper towels before each test.
An animal was placed in the dark side chamber about 1–2 hr prior
to lights off in the room where it was normally housed and
removed 20–22 hr later. Data collected during the five hour
interval beginning at the time of expected activity onset were used
for analysis. The number of dark to light side transitions during the
4 hr interval ranged from 68 to 388 (median = 174), indicating
that all animals had the opportunity for a large amount of light
sampling.
Author Contributions
Conceived and designed the experiments: DG MDR IP LPM. Performed
the experiments: DG KMS IP LPM. Analyzed the data: DG KMS IP
LPM. Contributed reagents/materials/analysis tools: DAL IP. Wrote the
paper: DG KMS DAL MDR IP LPM.
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PLoS ONE | www.plosone.org 13 September 2008 | Volume 3 | Issue 9 | e3153
