nature neurOSCIenCe  VOLUME 14 | NUMBER 3 | MARCH 2011 387
t e C h n I C a l r e p O r t S
Optogenetics is a technique for controlling subpopulations
of neurons in the intact brain using light. This technique has
the potential to enhance basic systems neuroscience research
and to inform the mechanisms and treatment of brain injury
and disease. Before launching large-scale primate studies, the
method needs to be further characterized and adapted for use
in the primate brain. We assessed the safety and efficiency of
two viral vector systems (lentivirus and adeno-associated virus),
two human promoters (human synapsin (hSyn) and human
thymocyte-1 (hThy-1)) and three excitatory and inhibitory
mammalian codon-optimized opsins (channelrhodopsin-2,
enhanced Natronomonas pharaonis halorhodopsin and the step-
function opsin), which we characterized electrophysiologically,
histologically and behaviorally in rhesus monkeys (Macaca
mulatta). We also introduced a new device for measuring in vivo
fluorescence over time, allowing minimally invasive assessment
of construct expression in the intact brain. We present a set of
optogenetic tools designed for optogenetic experiments in the
non-human primate brain.
Systems neuroscience relies mainly on recordings of neural activity
and its correlation with behavior. It also uses pharmacological mani­
pulations to perturb the neural system and thereby to establish causal
relationships between neural activity and behavior. These manipula­
tions can be targeted to specific cell types and are very powerful1,
but they act on a time scale of minutes, whereas neurons act on a
time scale of milliseconds. Electrical stimulation can be used for
temporally more precise, but not cell type–specific, manipulations.
Electrical stimulation also does not allow highly controlled inhibi­
tion and causes electrical interference that hampers the simultaneous
electrical recording of neural signals from the same site. Optogenetics
addresses these challenges by introducing into neurons light­sensitive
proteins that regulate the ion conductance of the membrane. These
proteins, encoded by microbial opsin genes, are derived from sources
such as archaebacteria and algae. They allow optical excitation2,3 or
inhibition4,5 of specific neuron types based on their expression or
projection patterns. Moreover, optogenetics allows simultaneous
artifact­free electrical recording of action potentials6–8.
Optogenetics has been applied in a multitude of behavioral and
electrophysiological studies in rodents6,9–13, and an initial study
in rhesus monkeys has been successful14. However, three main
challenges and constraints remain before optogenetic techniques are
ready for broad application in non­human primate science, including
neural prosthetics research15.
First, it is necessary to characterize the extent, efficiency, toler­
ance and pattern of opsin expression in non­human primate cortex
to facilitate scientific interpretation of results, and to minimize poten­
tial risks. Viral vectors, promoters and opsins are the three relevant
agents that need to be tested. In addition, the amount of laser power
applied to the brain is of central interest as too much power can
lead to thermal damage16–18. Second, the reliability of optogenetic
stimulation and its effect on neural activity and behavior need to be
tested to aid in the design of future experiments, and to maximize
the chances of experimental success. Third, standard histological
approaches, which are useful for analyzing expression patterns, can
be performed only after completion of experiments. Experiments
with behaviorally trained monkeys typically span months or years
and result in extremely valuable experimental subjects. This makes
standard histological evaluation less attractive. A method is needed
that allows repeated in vivo fluorescence measurements in the non­
human primate brain to determine expression levels and to find the
opsin­expressing sites, which can be distant from the injection site
because of axonal or trans­synaptic trafficking19.
Here we address these three challenges with a panel of optogenetic
tools applied in rhesus macaques and tested with single­unit and
local field potential electrophysiology. We also compare the effects
of optical, electrical and combined opto­electronic stimulation in
motor cortex on passive behavior, and show in vivo and ex vivo fluo­
rescence measurements.
RESULTS
We injected two monkeys at seven different sites with four different
constructs (Fig. 1). These constructs included the membrane channel
channelrhodopsin­2 (ChR2)2, which activates neurons when driven
with blue light; the chloride pump enhanced Natronomonas pharaonis
halorhodopsin (eNpHR2.0)5, which inhibits spiking when driven with
yellow or green light; and a step­function opsin (SFO), which is a
mutated version of channelrhodopsin (hChR2(C128S))20 that puts
neurons in a state of increased excitability for many seconds after a
brief blue light pulse. This last effect can be reversed by a brief pulse
1Department of Bioengineering, Stanford University, Stanford, California, USA. 2Neurosciences Program, Stanford University, Stanford, California, USA.
3Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA. 4Howard Hughes Medical Institute, Stanford University, Stanford,
California, USA. 5Department of Electrical Engineering, Stanford University, Stanford, California, USA. 6Present address: Department of Electrical Engineering and
Computer Science, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA. Correspondence should be addressed to K.D. (deissero@stanford.edu) or
K.V.S. (shenoy@stanford.edu).
Received 4 October 2010; accepted 29 December 2010; published online 30 January 2011; doi:10.1038/nn.2749
An optogenetic toolbox designed for primates
Ilka Diester1, Matthew T Kaufman2, Murtaza Mogri1, Ramin Pashaie1,6, Werapong Goo1, Ofer Yizhar1,
Charu Ramakrishnan1, Karl Deisseroth1–4 & Krishna V Shenoy1,2,5
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388  VOLUME 14 | NUMBER 3 | MARCH 2011 nature neurOSCIenCe
t e C h n I C a l r e p O r t S
of yellow light. Our promoter choices included two human promot­
ers. hSyn has been implicated in the regulation of neurotransmitter
release at synapses, particularly at glutamatergic and GABAergic syn­
apses21. hThy-1 is a gene for a cell­surface protein, and was originally
discovered as a thymocyte antigen. It is also present on the axonal
processes of neurons22. As primate­appropriate viral vectors, we chose
adeno­associated virus (AAV) serotype 2 pseudotyped with serotype 5
(here referred to as AAV5). We injected 1 µl of virus each millimeter
from the cortical surface to a depth of 6–10 mm (normal to the brain
surface), to test infections across all cortical layers and taking into
account potential cortical folding. Monkey D was injected with AAV5­
hSyn­ChR2­EYFP along a line through motor and somatosensory
cortex, as well as with AAV5­hThy­1­ChR2­EYFP and AAV5­hThy­1­
eNpHR2.0­EYFP in motor cortex. Monkey B was injected with AAV5­
hThy­1­ChR2­EYFP in somatosensory cortex. All AAV5 vectors had
a titer of 1012 particles per ml. We also injected one site in monkey B
with a lentivirus carrying hSyn-SFO-EYFP in parietal cortex with
a titer of 109–1010 particles per ml. Between weeks 5 and 12 after
viral vector injection, we optically stimulated the injected sites while
simultaneously recording neural activity. We also monitored potential
effects of the optical stimulation on passive motor behavior, and com­
pared and combined optical stimulation with electrical stimulation to
explore effects on behavior. Five months after injection (monkey D)
and four months after injection (monkey B), we assessed expression
levels and patterns first by in vivo fluorescence measurement and
subsequently with standard histological methods.
Optogenetic inhibition
During the period between 5 and 12 weeks after injection of the
eNpHR2.0 vector, we illuminated tissue with green (561 nm) or yellow
(594 nm) light. Neurons responded with a rapid reduction in firing rate
to pulse trains or continuous green light with latencies of 1–3 ms (Fig. 2a
and Supplementary Fig. 1a,b). Power densities ranged from 3 mW
mm−2 to 255 mW mm−2, measured at the tip of the 200­µm diameter
fiber, which produced estimated power densities of 0.34 mW mm−2
to 27 mW mm−2 at the site of electrical recordings (the electrode tip
typically led the fiber by 300 µm; see Online Methods for calculation
details and Supplementary Fig. 1c). For a quantitative analysis of
how individual neurons responded to light, we performed a χ2­test
(criterion P < 0.01, χ2 = 3.8415, 1 degree of freedom) comparing
baseline activity with activity during illumination. At the eNpHR2.0­
expressing site we found that 38% (55/144) of all recorded single
units and 22% (7/32) of all multi­units significantly changed their
firing rate in response to green light (see Supplementary Table 1).
Typically, responsive neurons decreased their firing rates (Fig. 2b and
Supplementary Fig. 2a). Only fifteen cells responded with an increase
in firing rate, presumably due to disinhibitory network effects (that
is, optical inhibition of a neuron that inhibited the neuron under
observation). To investigate further whether the firing rate increase
was an indirect network effect or based on the stimulation of axons
originating from ChR2­expressing sites we stimulated the eNpHR2.0­
expressing site with blue light (Supplementary Figs. 2b and 3). We
found that 17 units (single and multi­units) responded to blue light
with an increase in firing rates (and 5 units with a decrease). There
was a trend toward longer latencies (6–7 ms as opposed to the 2–3 ms
latencies at ChR2­expressing sites; see below), which suggested that
an indirect network effect was responsible, but short latency responses
also occurred. Simultaneously with single­unit recordings, we mea­
sured local field potentials (LFPs). LFP deflections followed stimula­
tion frequencies (Supplementary Fig. 4), and the polarity of LFP
deflections caused by green or yellow light was positive with a nega­
tive rebound, as expected from the underlying ion flow. Blue light did
not cause LFP modulations.
To test whether eNpHR2.0 expression had an effect on neuronal
activity we compared baseline firing rates (that is, without optical stim­
ulation) of light responsive and light unresponsive single units. Light
responsive neurons did not differ significantly from light unrespon­
sive neurons in their spontaneous activity (Wilcoxon rank­sum test;
Viral vector
a
b
5 weeks
Monkey D
Dorsal
Anterior
Ventral
Posterior
Premotor
cortex
Posterior parietal
cortex
Somatosensory
cortex
Motor
cortex
Somatosensory
cortex
Monkey B
Excitatory opsin
Inhibitory opsin
Bistable, long-lasting
excitatory opsin
AAV5-hSyn-ChR2-EYFP AAV5-hThy-1-ChR2-EYFP AAV5-hThy-1-ChR2-EYFPLenti-hSyn-SFO-EYFP
AAV5-hThy-1-eNpHR2.0-EYFP
Optical stimulation and electrical recordings: 5–12 weeks after injection
In vivo uorescence measurements: 11–12 weeks after injection
Optical stimulation
Neural recording+
–
Electrode + optical ber
(optrode)
Opsin-XFP–expressing area
llluminated area
In vivo
uorescence measurements
Figure 1 Schematic overview of preparation.
(a) Left, injection device; right, stimulation,
recording and in vivo fluorescence detector
outline. A standard recording grid guided an
injection needle to the desired location. Small
quantities (1 µl) of viral vectors carrying the
opsin-fluorophore fusion gene were injected at
different depths and sites in cortex. Starting
5 weeks after injections we stimulated the
injected sites optically and simultaneously
recorded electrical neural activity using
a combination of an optical fiber and an
electrode (optrode) guided by the same grid as
used for the injections. During the last week
of the experiment we also measured in vivo
fluorescence. (b) Injection sites and viral vectors
in monkeys D and B. Monkey D was injected at
five different sites with three different constructs.
Along a line through motor (AP 16 mm, ML 6 mm
and AP 11 mm, ML 6 mm) and somatosensory
cortex (AP 7 mm, ML 6 mm) we injected AAV5-
hSyn-ChR2-EYFP. More laterally, we injected
at two different sites in motor cortex, AAV5-
hThy-1-eNpHR2.0-EYFP and AAV5-hThy-1-
ChR2-EYFP (AP 11 mm, ML 10 mm and AP
16 mm, ML 10 mm, respectively). Monkey B
was injected with AAV5-hThy-1-ChR2-EYFP in
somatosensory cortex (AP 6 mm, ML 14 mm)
and with Lenti-hSyn-SFO-EYFP in parietal
cortex (AP 2 mm, ML 14 mm).
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