Science - USA (2020-05-01)

of RGCs that innervate non–image-forming

structures—liketheSCN,IGL,vLGN,andOPN

shell—and are required for circadian photo-

entrainment and the PLR ( 11 , 16 , 17 ). To test

whetherGad2+ RGCs are ipRGCs, we intra-

vitreally injectedGad2-IRES-Cremice with

an AAV expressing a Cre-dependent mCherry

reporter (AAV2/hSyn-DIO-mCherry) and im-

munolabeled these retinas for melanopsin

(Fig. 1E). Of the melanopsin immunoreactive

cells, 12% were mCherry+ (167 of 1437 cells

from seven retinas), and the proportion of

mCherry+ ipRGCs was highest (31%) in the

dorsal-temporal quadrant of the retinas (Fig. 1,

F and G). The individual densities ofGad2+

cells or of melanopsin immunopositive cells

528 1 MAY 2020•VOL 368 ISSUE 6490 sciencemag.org SCIENCE

(^1) Department of Neurobiology, Northwestern University,
Evanston, IL, USA.^2 Northwestern University Interdepartmental
Neuroscience Program, Northwestern University, Chicago, IL,
USA.^3 Microsoft, Redmond, WA, USA.^4 University Grenoble
Alpes, Inserm, U1216, Grenoble Institut Neurosciences, 38000
Grenoble, France.^5 Department of Ophthalmology, Feinberg
School of Medicine, Northwestern University, Chicago, IL, USA.
*Present address: Department of Neurobiology, Duke University
Medical Center, Durham, NC, USA.
†Corresponding author. Email: [email protected]
Fig. 2. ipRGCs expressGad2.
(A) In situ hybridization for
Opn4 (green) andGad2
(magenta). ONL, outer nuclear
later; INL, inner nuclear layer;
GCL, ganglion cell layer; DAPI,
4 ′,6-diamidino-2-phenylindole.
(B)Gad2+(top) andGad2−
(bottom) ipRGCs from (A). See
figs. S5 and S7 for how the
proportion ofGad2+ipRGCs
was estimated. (C) Strategies
for labeling ipRGC axons in
the SCN. Black text indicates
that reporter mice, in which
the synaptophysin-tdT fusion
protein was expressed in
the presence of Cre recombi-
nase (Syp-tdT or Ai34),
were intravitreally injected
with the pgk-Cre virus. Gray
text indicates thatOpn4Cre/+
animals were intravitreally
injected with a virus driving
Cre-dependent expression of
Chrimson-tdT. (D) ipRGC
terminals of Syp-tdt mice
intravitreally injected
with pgk-Cre virus (top) and
immunolabeled for GAD65
(magenta) and synapsin
to label axon terminals (cyan).
Bottom panels show magnified
images of ipRGC axon
terminals that were GAD65
immunoreactive (boxes 1
and 2) and GAD65 negative
(box 3). (E) Percentage
of ipRGC terminals that were
GAD65 immunoreactive
(IR).n= 5 Syp-tdT mice
injected with pgk-Cre virus,
n=4Opn4Cre/+mice injected
with FLEX-Chrimson-tdT
virus, andn=3Opn4Cre/+;
Gad2fx/fxmice injected with
FLEX-Chrimson-tdT virus.
Error bars indicate SD.
10μm
Syp-tdT (ipRGCs)
Gad65
Synapsin
Box 1
Box 3
Box 2
Box 1
Box 2
Box 3
Syp-tdT Gad65 Synapsin
% Gad65-IR axon terminals
Syp-tdT mice
w/ Cre virus
Rotation control

C

E

D

Opn4

Cre/+

Opn4

Cre/+

;

Gad2

fx/fx

Suprachiasmatic

nucleus (SCN)

Syp-tdT (Ai34) or

Opn4Cremice

AAV2/pgk-Cre or

Syn-FLEX-Chrimson-tdT

0

5

10

15

20

A B

In situ hybridization on retinal sections

A

Syp-tdT

100μm

Immunohistochemistry on ipRGC axon terminals

DAPI Opn4 Gad2

Example of Gad2+ ipRGC (~26%)

Example of Gad2- ipRGC

DAPI Opn4 Gad2 Merged

DAPI Opn4 Gad2 Merged

30μm 10μm

GCL

INL

ONL

0

5

10

15

20

Gad2+

Gad2-

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