Science - USA (2019-01-04)

(although not POR) (Fig. 1E). If visual responses
in POR depended on a geniculate input relayed
via other visual areas, rather than on a collicular
input, optogenetic activation of GABAergic SC
neurons could reduce putative geniculate-

mediated visual responses in POR. We therefore

blocked neuronal activity in SC with tetrodotoxin

(TTX) injected in the stratum opticum of SC (Fig. 3,

G and H, and fig. S11). Again, we ensured that the

receptive field of the TTX injection site matched

the receptive field of the recording site in POR.

TTX application abolished visually evoked re-

sponses in POR to both moving dots (Fig. 3H)

(94.46 ± 3.18% average decrease ± SEM in

visually evoked firing rate;P< 0.0001,n=44,

Beltramoet al.,Science 363 ,64–69 (2019) 4 January 2019 4of6

Fig. 3. POR is driven by
the colliculo-cortical
pathway.(A)Anterograde
trans-synaptic tracing.
AAV1.hSyn.Cre was
injected in SC and AAV8.
CAG.Flex.GFP was injected
in caudal pulvinar. AAV1.
CAG.TdTomato injected in
V1 was used to identify
higher visual areas.
Delineated cortical areas
are as shown in fig. S8.
(B) (Left) Double labeling
of axons targeting POR.
Red, V1 afferents (for POR
localization); green,
caudal pulvinar afferents.
(Inset) GFP expression
in the caudal pulvinar.
(Middle) Magnification of
the region marked by
the rectangle in the left
panel (GFP channel) and
summary distribution
of GFP fluorescence
(caudal pulvinar afferents)
along POR cortical depth
(five animals). (Right)
Fluorescence density
distribution of trans-
synaptically labeled caudal
pulvinar axons across
cortical areas. Green bars,
normalized averages
and SEM (five animals)
across 10 distinct cortical
areas. For each animal, the
fluorescence density of
each area was normalized
to that for POR. Each
symbol corresponds to a
different animal. Black
triangle, experiment on
the left. (C) SC silencing
and POR recording.
The visual stimulus was
a dark dot moving at a
speed of 30° per second along the two cardinal and two oblique axes for
0.67 or 3 s (i.e., the time required to cover, at 30° per second, trajectories of
20° or 90° of visual space, respectively). (D)(Left)RasterplotandPSTH
of a POR unit under control conditions (black) and during SC silencing (blue).
(Middle) Aligned average PSTH for 96 isolated units (five animals). The
average PSTH was generated by aligning the response of each isolated
POR unit to the center of the“SC response interval”for the same stimulus
(time 0 s) (see materials and methods for a description of the moving dot
analysis; see also fig. S9). (Right) Scatter plot of firing rates of POR units,
averaged over a 450-ms window (i.e., the response window; see materials and
methods description of the moving dot analysis) within the SC response
interval. Green data point, example on the left. (EandF) Results of experiments

similar to those shown in (C) and (D) but with a circular drifting grating patch

as the stimulus (diameter: 20°; 44 isolated units; two animals). The average

PSTH was aligned to the visual stimulus onset. Scatter plot, neuronal responses

measured during stimulation period (0.9 s), as in Fig. 1D. (G) SC silencing

and POR recording. The visual stimulus was a dark dot moving at 30° per

second along a straight trajectory of 20° of visual space (stimulus duration,

0.67s). The superficial layers of SC were silenced via local application of TTX.

(H) (Left) Raster plot and PSTH of an isolated POR unit under control

conditions (black) and ~20 min after the beginning of TTX infusion in SC

(red). The gray portion of the raster plot indicates the initial ~20 min of TTX

infusion. (Middle) Average PSTH, aligned to the visual stimulus onset, for

44 isolated units (three animals). (Right) Scatter plot as in (D).

0

4

8

Time from stimulus onset (s)

Firing rate (Hz)

0

2

8

4

6

0

2

4

6

8

Firing rate (Hz)

Firing rate (Hz)^0

0.2

0.4

0.6

0.1

0.2

0.3

0.4

0.5

-2 -1 0 +1 +2

0

Normalized firing rate

Normalized firing rate

Time from the center of

“SC response interval” (s)

POR single unit

POR single unit

POR single unit

Time from

TTX inj.(min)

0

+10

+20

+30

+40

-10

-20

-2 0 246

0

2

4

6

-2

-2 0 246

-2

0

2

4

6

0

10 30

-2^0246

-2

0

2

4

6

10 30

25 75

SC

Laser

POR

Rec.

Moving dot

0.6

10

12

8

Drifting grating

POR

Rec.

0.8

POR firing rate-laser SC ON (Hz)

POR firing rate-laser SC OFF (Hz)

POR firing rate-laser SC ON (Hz)

Time from stimulus onset (s) POR firing rate-laser SC OFF (Hz)

POR control firing rate (Hz)

POR firing rate after TTX injection (Hz)

300 μm

POR Fluorescence DensityNormalized

0.2 0.6 1

L1

L2/3

L4

L5

L6

GFP

POR average

POR average

/ GFP / Td-TomatoDAPI

Caudal pulvinar

axons (GFP)

B

D

F

SC

TTX

Moving dot

POR

Rec.

H

C

E

G

A

Rostral

Pulv

SC

AAV1-hSyn-Cre

Anterograde

transynaptic

AAV8-CAG

Flex-GFP

AAV1-CAG TdTomato

P

LILM

AL

AM

A

RL

POR

PM

V1

Caudal

TEp

TEa Aud

1mm

V1

POR

Bregma: -4.45mm

150 μm

Pulv

(caudal)

Bregma: -3.07mm

Bregma: -4.45mm

Normalized Fluorescence Density

(^0) PORLITEa TEpA/RLLM AL AMP PM
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
Normalized firing rate
0.8
-1 -0.5 0 +0.5 +1
POR average
-2-2-1-1 00 +1+1+2+2+3+3
-1 -0.5 0 +0.5 +1
-1 -0.5 0 +0.5 +1 -1 -0.5 0 +0.5 +1
Time from stimulus onset (s)
Time from stimulus onset (s)
Time from stimulus onset (s)
SC
Laser
RESEARCH | REPORT
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