IPSCs and EPSCs (11 of 32 cells). Therefore, just
43%of SCN neurons receiving direct input
from ipRGCs receive inhibitory ipRGC input
(Fig. 3D). The synaptic latency of EPSCs and
IPSCs elicited in SCN neurons was not sig-
nificantly different, which suggests that both
types of postsynaptic currents arise from mono-
synaptic input from ipRGCs (Fig. 3E). When
possible, we mapped the location of recorded
SCN neurons (fig. S8) and immunolabeled
recorded cells for vasoactive intestinal pep-
tide (VIP) (Fig. 3, B and C). A higher proportion
of VIP+ neurons in the SCN received mono-
synaptic ipRGC input (59%) compared with
VIP−neurons (33%) (Fig. 3F). A larger percent-
age of VIP+ neurons received purely excitatory
ipRGC input (41%) compared with VIP−neu-
rons (15%), and a similar proportion of VIP+
and VIP−neurons received inhibitory ipRGC
input (Fig. 3F).
Bath application of 2,3-Dioxo-6-nitro-1,2,3,4-
tetrahydrobenzo[f]quinoxaline-7-sulfonamide
(NBQX) and D-(-)-2-Amino-5-phosphonopentanoic
acid (D-APV) abolished the evoked EPSCs (Fig. 3,
G and H) in SCN neurons receiving excitatory
and inhibitory ipRGC input, but it did not affect
IPSC amplitudes. This further confirms that elic-
ited IPSCs were likely not a result of a disynaptic
inhibition arising from evoked glutamatergic
ipRGC inputs onto a GABAergic interneuron.
Subsequent bath application of gabazine (SR-
95531) abolished the remaining light-evoked
IPSCs in SCN neurons (Fig. 3, G and H).
To assess how GABA release by ipRGCs in-
fluences non–image-forming visual behavior,
we crossedOpn4Cremice ( 20 ) withGad2fx/fx
mice ( 21 ) to knock outGad2specifically in
ipRGCs (Opn4Cre/+; Gad2fx/fx, referred to as
Gad2cKO). These animals showed normal
ipRGC projections and visual acuity, which
indicates that this manipulation does not
affect the development of the visual system
(figs. S9 and S10). We measured the PLR of
Gad2cKO animals compared with littermate
controls (Opn4+/+; Gad2fx/fx)acrossarangeof
light intensities and measured the irradiance-
response relationship (Fig. 4, A to C). PLR
amplitude and kinetics were both unaffected
at bright light intensities inGad2cKO animals
(Fig. 4C and fig. S11), and baseline pupil size in
darkness was also unchanged (Fig. 4, A and B).
However, at low light levels,Gad2cKO mice
showed significantly stronger pupil constric-
tion (Fig. 4C) than that of their littermate
controls, although the kinetics were not sig-
nificantly different (fig. S11).
We next tested whether these changes in
PLR were caused by a lack ofGad2in ipRGCs
or, potentially, by developmental effects of
Gad2excision early in development. We knocked
out theGad2gene specifically in RGCs of
adultGad2fx/fxmice through intravitreal de-
livery of an AAV that drives Cre expression
under an RGC-specific promoter (AAV2/SNCG-
Cre-HA) ( 22 ) (fig. S12 and methods). Less than
5% of amacrine cells were labeled in well-
infected areas (fig. S12B). We then measured
the PLR in response to dim (10.4 log photons
cm−^2 s−^1 ) and bright (13.4 log photons cm−^2 s−^1 )
light stimuli. Mice in whichGad2was knocked
out in RGCs exhibited significantly more sensi-
tive PLR in response to dim light but not bright
light (fig. S12, C and D) compared with con-
trol animals. These results suggest that these
changes in PLR are not due to developmental
defects fromGad2gene excision.
To determine whether GABA release by
ipRGCs also influences circadian photoentrain-
ment, we tracked voluntary wheel-running
activity of littermate controls andGad2cKO
mice across multiple light levels. Mice were
first exposed to a 12-hour–12-hour light-dark
(LD) cycle with bright, 100-lux light during
the light phase. We then performed a 6-hour
phase advance of the LD cycle at 4-week in-
tervals and simultaneously lowered the light
intensity at each shift, first to 1.5 and then to
0.2 lux (Fig. 4D and fig. S13). The rate of re-
entrainment in response to both 6-hour phase
advances was the same inGad2cKO com-
pared to control animals (fig. S14). However,
Gad2cKO animals had significantly higher
circadian amplitudes in LD cycles at low (1.5
and 0.2 lux) but not high (100 lux) light levels
(Fig. 4E). This indicates that the circadian
photoentrainment ofGad2cKO animals re-
mains more robust at lower light levels and is
relatively insensitive to decreases in environ-
mental light levels. There were no significant
differences betweenGad2cKO and control
animals in total daily activity, activity onset
time, or activity onset variability (fig. S15).
Instead,Gad2cKO animals exhibited signif-
icantly less activity in the light phase at 1.5
and 0.2 lux, which likely accounts for their
increased circadian amplitude at low light lev-
els relative to controls (Fig. 4F).
Our results reveal a GABAergic circuit orig-
inating in the retina that decreases the sen-
sitivity of the non–image-forming visual system
at low light levels. Recent reports have shown
that the ipRGCs providing input to non–image-
forming brain regions are highly sensitive to
dim light ( 23 – 25 ), and yet the non–image-
forming behaviors driven by these inputs are
relatively insensitive to light compared with
image-forming vision ( 26 , 27 ). The mecha-
nisms underlying this discrepancy between
sensitive cellular inputs to non–image-forming
brain regions and relatively insensitive behav-
ioral outputs had remained a mystery. Our
results suggest that a subpopulation of ipRGCs
may serve to actively dampen the sensitivity
of the non–image-forming visual behaviors
by releasing the inhibitory neurotransmitter
GABA, providing a circuit mechanism for this
discrepancy. For the PLR, these inputs serve
to maximize light entry into the eye at lowlight levels. For circadian behaviors, these in-
puts likely prevent unnecessary adjustments
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ACKNOWLEDGMENTS
We thank T. Bozza, R. Allada, and M. Gallio for helpful comments
on the manuscript; S. Hattar for the gift ofOpn4Cremice; and
Q. Wu for the gift ofGad2fxmice.Funding:This work was funded by
a Klingenstein-Simons Fellowship in the Neurosciences to T.M.S., a
Sloan Research Fellowship to T.M.S., NIH grant 1DP2EY022584 to
T.M.S, NIH T32 EY025202 to support T.S., and NIH F31 EY030360-01
to T.S.Author contributions:Conceptualization, T.S., J.Y.L., and
T.M.S.; Investigation, T.S., J.Y.L., N.W.H., Y.O., and T.M.S.; Formal
analysis, T.S.; Software, J.C.C.; Writing–original draft, T.S. and T.M.S.;
Writing–reviewing and editing, T.S., J.Y.L., and T.M.S.; Visualization,
T.S., J.Y.L., N.W.H., J.C.C., and T.M.S.; Resources, J.C.C., S.B., and H.N.;
and Funding acquisition, T.S. and T.M.S.Competing interests:The
authors declare no competing interests.Data and materials
availability:All data are available in the manuscript or the
supplementary materials.SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/368/6490/527/suppl/DC1
Materials and Methods
Figs. S1 to S15
References ( 28 – 30 )
MDAR Reproducibility Checklist7 June 2019; resubmitted 31 January 2020
Accepted 13 March 2020
10.1126/science.aay3152SCIENCEsciencemag.org 1 MAY 2020•VOL 368 ISSUE 6490 531
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