action potentials of DA neurons, as shown in
current-clamp traces. Trains of NIR illumination
at 10 to 50 Hz elicited multiple spikes (Fig. 2E).
The spike probability showed no significant dependence on the frequency of the NIR light (Fig. 2F).
We next tested the in vivo utility of UCNP-
mediated NIR upconversion optogenetics. We
sensitized VTA DA neurons of TH-Cre mice to
transcranial NIR stimulation through viral de-
livery of ChR2 followed by bilateral UCNP injec-
tion (Fig. 3A). Anesthetized mice were exposed
to transcranial pulsed NIR irradiation (15-ms
pulses at 20 Hz, 3 s every 3 min for 30 min, 3.0-W
peak power, 15-m W average power) delivered
from an optical fiber (200 mm in diameter)
placed 2 mm above the skull (1.4-W/mm2 NIR
on the skull surface). NIR-activated DA neurons
were mapped by imaging the expression of c-Fos
(Fig. 3, B and C, and fig. S13). Neuronal exci-
tation was only triggered by NIR light in ChR2-
transfected mice in the presence of UCNPs, as
indicated by the significantly higher propor-
tion of c-Fos–positive cells in areas where UCNP
injection and ChR2 expression overlapped. We
injected UCNPs to just one side of the VTA and
observed NIR-induced c-Fos expression only
in the injected hemisphere (fig. S14). We also
observed up-regulation of c-Fos expression in
the ventral striatum (fig. S15), which receives
inputs from VTA DA neurons (31), indicating
NIR-evoked excitation of postsynaptic structures
of the targeted neurons. Control mice with UCNP
injection, ChR2 expression, or NIR stimulation
alone showed no significant c-Fos expression
in either VTA or ventral striatum.
We evaluated the real-time efficacy of NIR-evoked excitation of VTA DA neurons with fast-scan cyclic voltammetry (FSCV) (Fig. 3D). Striatal
DA transients reflect the phasic spike activity
of VTA DA neurons (31) and have therapeutic
implications for the treatment of major depression. In nomifensine-pretreated mice with both
UCNP injection and ChR2 expression in VTA, we
detected DA release that was temporally locked
to transcranial NIR stimulation (15-ms pulses at
20 Hz, 700-m W peak power) (Fig. 3F). After a 2-s
NIR stimulation, striatal DA release lasted for
more than 15 s and peaked at ~5 s after light
onset (Fig. 3, F and G). We detected no significant DA release in control mice with omission
of NIR stimulation, UCNP injection, or ChR2
expression (Fig. 3, G to I). We compared the
efficacy of NIR with blue light in evoking DA
release by VTA DA neurons (Fig. 3E). The tip
of an optic fiber transmitting NIR or blue light
was positioned at various distances from the
VTA target for optogenetic activation of DA
neurons. When illuminating from a distance of
0.5 mm, NIR and blue light triggered similar
amounts of DA release in ventral striatum (Fig.
3E and fig. S16). However, transcranial application of blue light did not result in striatal DA
release (Fig. 3, E and I). Furthermore, NIR stimulation showed significantly slower attenuation
in DA release with the increase of the distance
from fiber tip to VTA (Fig. 3E).
We next expanded the application of in vivo
upconversion optogenetics to multiple modes
of neural control, including inhibition, as well
as to different brain regions. First, we developed
green-emitting UCNPs to match the maximum
absorption of rhodopsins that hyperpolarize neurons, such as NpHR and Arch, to achieve noninvasive neuronal inhibition. The emission of
UCNPs was tuned to ~540 nm by codoping Er3+
and Yb3+ into the Na YF4 host lattice (Fig. 4A and
fig. S1). We then assayed the ability of Na YF4:Yb/
Er@SiO2 UCNPs to inhibit hippocampal activity during chemically induced hyperexcitability.
Arch was virally expressed in excitatory neurons
in the CA1 and dentate gyrus (DG) regions of
682 9 FEBRUARY 2018 • VOL 359 ISSUE 6376 sciencemag.org SCIENCE
Fig. 3. Transcranial NIR stimulation of VTA DA neurons in vivo. (A) In vivo experimental scheme for
transcranial NIR stimulation of the VTA in anesthetized mice. (B) Confocal images of the VTA after
transcranial NIR stimulation under different conditions. Extensive NIR-driven c-Fos (red) expression was
observed only in the presence of both UCNPs (blue) and ChR2 expression (labeled with EYFP, green).
Scale bars: 100 mm. (C) Percentage of c-Fos–positive neurons within cell population indicated by DAPI
(4′,6-diamidino-2-phenylindole), corresponding to the four conditions presented in (B) (n = 3 mice each,
F3,8 = 10.40, P < 0.01). (D) Scheme of in vivo FSCV to measure DA transients in ventral striatum during
NIR stimulation of the VTA. (E) Relative DA signals in ventral striatum under NIR and blue-light stimulation of
the VTA as a function of the distance from the light source to the VTA target. (F) A trace of background-subtracted current measured by FSCV in the ventral striatum of a nomifensine-pretreated mouse in
response to transcranial NIR stimulation of the VTA (15-ms pulses at 20 Hz, 700-m W peak power). Vertical
dashed lines marked by a horizontal orange line in between indicate the start and end of 2-s transcranial
NIR stimulation. (G and H) Transient DA concentrations in ventral striatum in response to transcranial
VTA stimulation under different conditions. Each color corresponds to a condition shown in (I). Significant
DA release temporally locked to NIR stimulation was detected only in the presence of both UCNPs and
ChR2 expression. (I) Cumulative DA release within 15 s after the start of transcranial stimulation under the
five conditions presented in (G) and (H) (F4,10 = 32.93, P < 0.0001). Data are presented as mean ± SEM.
