Science - USA (2020-06-05)

Nelidovaet al.,Science 368 , 1108–1113 (2020) 5 June 2020 5of6

Fig. 4. NIR light responses in
the ex vivo human retina.
(A) Schematic of a human retinal
explant 8 weeks postmortem.
Long-term culture leads to
loss of outer segments and light
responses. (B) Top: Top view
of a human retina transduced
with both rTRPV1 and nanorods,
immunostained for TRPV1 (blue)
and overlaid with Hoechst
nuclear stain (gray). Bottom:
Cross section of the retina shown
in top image. Scale bars, 10mm.
(C) Number of rTRPV1-positive
(blue) photoreceptors per square
millimeter in human retinas
transduced with both rTRPV1
and nanorods or in control,
untransduced human retinas.
Each data point is collected
from a different region of
a retina (five regions per retina).
(D) Distribution of rTRPV1-positive
cells across retinal layers.
(E) Scanning electron microscopy
image of gold nanorods bound
to a rTRPV1-transduced human
retina. Scale bar, 1mm. Inset:
Transmission electron microscopy
image showing an anti-6x-His
antibody touching a gold nanorod
with its Fc domain. Scale bar,
20 nm. (F) Example calcium
responses recorded from human
retinal neurons in ONL, INL,
and GCL in response to full-field
NIR light. Vertical lines at bottom
indicate stimulus (915 nm,
100 ms) timing. Two-photon
images of OGB-1 filled cell bodies
(white circles), left of the response
curves. Scale bar, 10mm.
(G) Cumulative frequency of
responding neurons in different
layers of human retinas trans-
duced with both rTRPV1 and
nanorods (labs= 915 nm) or in
control, untransduced human
retinas. (H) Response amplitudes
(DF/F) by retinal layer. Increase
(↑) and decrease (↓) of calcium
signal are shown separately.
Number of neurons given as
ratio of responding neurons to
measured neurons. Log 10 light
intensity = 18.9.

B rTRPV1 C

ONL = 94.5 %

INL = 4.5 %

GCL = 1 %

rTRPV1 transduction specificity

D

ONL

INL

Human retina

0

1000

2000

3000

4000

5000

Nanorod binding

Human retina

Control

n = 3 explants

E

rTRPV1-nanorodsn = 3 explants

n = 3 explants

Antibody

Number of transduced photoreceptors per mm

2

rTRPV1

Cumulative frequenc

y o

f res

ponding

human retinal neurons (%)

GCL, rTRPV1-nanorods, n = 105 neurons

ONL, rTRPV1-nanorods, n = 101 neurons

INL, rTRPV1-nanorods, n = 118 neurons

G

A

Control, n = 325 neurons

F

ONL

INL

GCL

Postmortem human retina

1 s

20%

ΔF/F

GCL

Log 10 I 915 (photons cm-2 s-1)

15.4 16.0 18.1 18.4 18.9

1 2 3 1 2 3 1 2

rTRPV1-nanorods

3

ONL. OGB-1

INL. OGB-1

GCL. OGB-1

0

20

40

60

Log 10 I 915 (photons cm-2 s-1)

15 16 17 18 19

4

4

4

ONL

GCL

, rTRPV1-nanorods

ONL, rTRPV1-nanorods n = 15/101 neuronsINL, n = 53/105 neurons

rTRPV1-nanorods

n = 61/118 neurons

H

-20

-10

0

10

20

30

40

Human retinal neuron response

amplitude (%

ΔF/F)

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