Content uploaded by Jianyong Tang
Author content
All content in this area was uploaded by Jianyong Tang on Apr 30, 2014
Content may be subject to copyright.
Two-photon single-cell optogenetic control of
neuronal activity by sculpted light
Bertalan K. Andrasfalvy, Boris V. Zemelman, Jianyong Tang, and Alipasha Vaziri
1
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147
Communicated by Charles V. Shank, Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, May 14, 2010 (received for review
April 6, 2010)
Recent advances in optogenetic techniques have generated new
tools for controlling neuronal activity, with a wide range of neu-
roscience applications. The most commonly used approach has
been the optical activation of the light-gated ion channel channelr-
hodopsin-2 (ChR2). However, targeted single-cell-level optogenetic
activation with temporal precessions comparable to the spike
timing remained challenging. Here we report fast (≤1 ms), selec-
tive, and targeted control of neuronal activity with single-cell
resolution in hippocampal slices. Using temporally focused laser
pulses (TEFO) for which the axial beam profile can be controlled
independently of its lateral distribution, large numbers of channels
on individual neurons can be excited simultaneously, leading to
strong (up to 15 mV) and fast (≤1 ms) depolarizations. Furthermore,
we demonstrated selective activation of cellular compartments,
such as dendrites and large presynaptic terminals, at depths up to
150 μm. The demonstrated spatiotemporal resolution and the se-
lectivity provided by TEFO allow manipulation of neuronal activity,
with a large number of applications in studies of neuronal micro-
circuit function in vitro and in vivo.
high-resolution neuronal stimulation
|
channelrhodopsin
|
temporal focusing
|
circuit mapping
|
electrophysiology
Artificial stimulation and inhibition of neuronal activity has
applications ranging from fundamental neurobiology ques-
tions (1–4) to potential clinical treatment of neuropsychiatric
disorders (5, 6). Historically, this task has been achieved mainly
by electrical stimulation. Yet the recent development of genetic
techniques for sensitizing neurons to optical stimulation (7–12)
and silencing (13–16) have, for the first time, provided cell type–
specific control of neuronal activity, which has been successfully
used to address significant biological questions (1–3, 17–19). In
the most widely used approach, genetically expressed light-gated
ion channels, such as channelrhodopsin-2 (ChR2) (20, 21), or ion
pumps, such as halorhodopsin or archaerhodopsin-3 (22, 23), and
optical methods for triggering their function are combined to
control neuronal activity. However, although optical activation of
ChR2 has been suitable to induce population activity in a large
number of neurons, directed, efficient, and fast stimulation of
single cells has not been feasible. The main reason for that is the
low channel conductance of ChR2 (21, 24). To achieve sufficient
depolarizations, a large number of channels have to be activated
nearly simultaneously, which typically extend over an area of tens
of square micrometers. Although conventional one-photon exci-
tation satisfies this requirement, light scattering and the extended
axial beam parameter of the excitation area lead typically to in-
evitable activation of neurons in an untargeted fashion. Some
success in addressing these issues has recently been reported (25,
26) using a two-photon scanning (27) approach. Although these
studies (25, 26) could show high spatial resolution, the necessary
time to activate a large surface area sufficient to fire action
potentials (APs) was ≈30 ms. Thus, neither the one-photon nor
the two-photon scanning excitation provides the necessary com-
bination of high spatial selectivity and the ability to stimulate
a membrane area that is large enough to produce simultaneous
and significant rapid depolarizations on a single neuron.
Results
ChR2 Activation by Two-Photon Temporal Focusing. We took a dif-
ferent approach for addressing the shortcomings of both the one-
photon and the two-photon method in a fundamentally unique
way and have demonstrated reliable single neuron–specific acti-
vation with temporal resolution (≤1 ms) in hippocampal slices.
This was done by effectively decoupling the lateral (i.e., the waist
size) and the axial (i.e., the Rayleigh length) beam parameters for
two-photon absorption, which are usually coupled for a Gaussian
beam, and thereby sculpting the spatial light distribution at the
focal plane. For a pulsed laser source, the decoupling can effec-
tively be achieved by using the spectrum of the pulse to control its
two-photon absorption probability in the axial direction. This has
been demonstrated in the technique of temporal focusing (28,
29), which has also found applications in widefield two-photon
imaging (30) and in 3D multilayer super-resolution microscopy
(31). Two-photon temporal focusing (TEFO) can be experi-
mentally realized by first spatially broadening a femtosecond
optical pulse using a diffraction grating. The spot on the grating is
then imaged onto the sample plane using a telescope that consists
of the microscope objective and an additional lens. This config-
uration leads to an axial geometry in which the pulse is broadened
everywhere in the sample except at the image plane, where the
spatially separated paths of the different frequency components
in the pulse meet again and the pulse reaches its minimum width.
(Fig. S1 Aand B). The two-photon excitation probability in
TEFO is inversely proportional to the pulse width squared, which
leads to a depth of field ≈2 orders of magnitude shorter (31) than
the widefield one-photon epifluorescence technique. This allows
a sectioning capability close to a confocal or two-photon micro-
scope (27), while providing simultaneous excitation of an area
that is ≈3 orders of magnitude larger than the diffraction-limited
spot (Fig. S1C). Therefore, TEFO can be used for simultaneous
excitation of a thin disk-like volume in a biological sample. In
the present work we took advantage of this fact and demonstrated
that TEFO can be used for targeted single-cell excitation of
ChR2 with high spatiotemporal resolution in mouse and rat
hippocampal slices.
After confirming the above parameters of the excitation vol-
ume for our temporally focused beam (Figs. S1Band S2), we
investigated the activation of ChR2 expressed in cultured human
embryonic kidney (HEK293) (Fig. 1A) cells and measured
ChR2-mediated currents with whole-cell voltage clamp record-
ings. Fast (≤2 ms rise time) and large (≤1 nA) inward currents
Author contributions: B.K.A. designed and performed all electrophysiological experiments;
B.V.Z. prepared plasmids, designedAAV-ChR2-sfGFPand Cre recombinase-dependentrAAV-
FLEX-rev ChR2-sfGFP viruses, and helped with the manuscript; J.T. developed software for
data collection; A.V. designed and led research, built experimental setup, and performed
optical characterization experiments; B.K.A. and A.V. analyzed data; and B.K.A. and A.V.
wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. E-mail: vaziria@janelia.hhmi.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1006620107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1006620107 PNAS
|
June 29, 2010
|
vol. 107
|
no. 26
|
11981–11986
NEUROSCIENCEAPPLIED PHYSICAL
SCIENCES
mediated by ChR2 were detected using pulses between 1 and 100
ms (Fig. 1 Band C). We aimed to systematically identify the
optimal parameters for two-photon temporal focusing excitation
and investigated the activation of ChR2 as a function of laser
power, wavelength, and beam size (Fig. 1 D–I). Keeping a con-
stant beam diameter of 3.8 μm and pulse duration of 100 ms for
a range of wavelengths between 800 nm and 960 nm, we first
measured the peak amplitude and rise time of the inward current
as a function of laser power (Fig. 1 D–F). At each wavelength for
lower powers we observed a nonlinear dependence of ChR2 ac-
tivation on power, which is the signature of the two-photon effect
(27). This quadratic dependence on power is not evident at first
sight over the studied range of powers, because it is overshad-
owed by other parameters of the channel kinetics, such as satu-
ration and desensitization. However, under our experimental
conditions it could clearly be seen for the power range between
0 and 50 mW (Fig. 1D,Inset). At high powers the relationship
showed near-asymptotic behavior, likely indicating the saturation
of channel activation (26). These experiments revealed the
maximum two-photon activation efficiency to be at 880 nm, which
is slightly blue shifted compared with the double of the one-
photon absorption peak (Fig. 1F). Once the optimum wavelength
was identified, we examined the dependence of the response on
the beam size. Using 100-ms-long pulses at 880 nm we varied the
power at three different spot sizes (6 μm, 10 μm, and 14 μm). The
peak inward current increased with power for all beam sizes (Fig.
1G); however, by further reducing the spot size to 4 μm we could
show that the highest efficiency of activation (as shown by the
larger peak inward current and faster rise time; Fig. 1H) could be
achieved with the ≈6-μm spot (Fig. 1I). This fact reflects the in-
trinsic tradeoff between the quadratic dependence of the proba-
bility for opening a channel on the intensity, which increases with
decreasing spot size, and the total number of channels on the
illuminated surface area, which decreases with the spot size. The
induced peak current (or the total charge influx) is proportional
to the intensity squared and the area of illumination. Therefore,
for a constant photon number (or a constant power) the peak
current should be inversely proportional to the square of the pot
size (Fig. 1G,Inset). All cells were tested for activation of ChR2
with blue light (488 nm), which revealed comparable biophysical
parameters to TEFO excitation (Fig. S3 A–E).
Temporal Resolution of TEFO. These results motivated us to in-
vestigate the potential of this method as a tool with high spa-
tiotemporal resolution for neuronal circuit mapping and other
neuroscience studies. We turned to hippocampal slices and used
different types of ChR2-expressing neurons: CA1 pyramidal
cells (CA1PCs) in Thy1-ChR2-YFP transgenic mice (32) and
parvalbumin-positive interneurons (PV-INs) in PV-Cre mice
(33) that were infected with a Cre-dependent ChR2-GFP con-
0
200
Peak current (pA)
400
600
800
E
HI
F
ABC
100 pA
20 ms
D
control
820 nm
488 nm
6810
Spot size (µm)
41412
0
4
Rise time (ms)
8
12
16
20
0
200
Peak current (pA)
400
600
100 200
Power (mW)
0400300 0
10
Rise time (ms)
20
30
100 200
Power (mW)
400300
40
50
G
120
160
Peak current (pA)
200
240
280
840 880
Wavelength (nm)
800 960920
0
200
Peak current (pA)
400
600
100 200
Power (mW)
0 400300
0
8
Rise time (ms)
12
16
100 200
Power (mW)
400300
20
24
1
10
100
ms
100 pA
20 ms
6µm
10 µm
14 µm
4
0
24
0
12
400200
800 nm
880 nm
800
840
880
920
960
nm
100
200
300
6 8 10 12 14
Spot size (µm)
Peak current (pA)
05025
30
60
90
Fig. 1. Characterization of the biophysical properties of TEFO activation of ChR2 in HEK293 cells. (A) Superimposed fluorescent and differential interference
contrast (DIC) image of a HEK293 cell during recording. (B) Representative traces of induced inward current by one-photon excitation of ChR2 (blue), TEFO
excitation of ChR2-expressing (red), and of control uninfected cell (black), using 100-ms-long pulses. (C) Representative traces of TEFO-activated ChR2-
mediated inward current with different pulse durations. (Dand E) Peak current amplitude (D) and rise time (E) at different wavelengths measured as
a function of average power at the sample for a ≈3.8-μm spot and 100-ms duration (n= 22 cells). Inset in Dshows the nonlinear dependence of peak inward
current on the power at lower power levels, which is the result of two-photon excitation of ChR2. (F) Wavelength-dependency of TEFO excitation of ChR2 (n=
16 cells). Inset in Femphasizes the differences in activation efficiency of 800-nm and 880-nm light. (Gand H) Peak current (G) and rise time (H) as a function of
spot size at 880-nm wavelength and 100-ms duration (n= 13 cells). Inset in Gshows the induced peak current with the TEFO for a constant photon number
(constant power at 200 mW) for different spot sizes. Maximum amplitude and fastest rise time could be achieved with a spot size of ≈5–6μm(I). Dots and
error bars represent means ±SEM.
11982
|
www.pnas.org/cgi/doi/10.1073/pnas.1006620107 Andrasfalvy et al.
taining adeno-associated virus (ChR2-GFP-AAV) (34) (Materi-
als and Methods and SI Materials and Methods). First we aimed to
determine the efficacy and basic biophysical characteristics of
our technique to explore the optimal parameters for different
types of applications. Somatic current-clamp recordings were
performed in ChR2-expressing neurons while the soma was il-
luminated by a temporally focused spot of ≈5-μm diameter with
1-, 2-, 5-, 10-, and 100-ms pulse duration at 880 nm with ≈460-
mW power at the sample (Fig. 2A). Similar to HEK293 cells, in
both types of neurons fast (≈1–5 ms) and large (≈5–15 mV)
ChR2-mediated depolarizations could be evoked using TEFO
excitation (Fig. 2A). At a holding potential of −80 mV (to pre-
vent generation of APs), the ChR2-induced membrane de-
polarization continuously increased with pulse duration and
reached a saturation value of ≈15–20 mV for pulses longer than
≈10 ms (Fig. 2B). In these experiments we used a static beam
path and moved the specimen to position the temporally focused
spot for targeting a certain area. However, to produce fast spa-
tiotemporal activation patterns, it would be advantageous to be
able to move the excitation spot on a submillisecond time scale
to any desired number of target points. To achieve this we in-
tegrated the temporal focusing beam into a microscope equipped
with a pair of laser scanning mirrors (Materials and Methods and
SI Materials and Methods). We tested this technical advance by
recording from CA1PCs and subiculum pyramidal cells (SubPCs)
in Thy1-ChR2-YFP mice and scanning variable spots in the slice
on and around the somata and dendrites of the patched cells. As
expected, ChR2-mediated responses were detected only when
the excitation spot was placed precisely on the soma or the
dendrite, but no response was evoked when the beam was placed
away from the recorded cell (Fig. 2C). Using very short pulsed
illuminations (0.1 ms) with short interpulse intervals (0.1 ms) at
multiple points positioned on the soma, we could effectively
increase the activated surface area and decrease the necessary
time to achieve the desired depolarization level to evoke APs
within a submillisecond time scale (Fig. 2D). (Further advantages
of the multiple-spot activation along single dendrites are dis-
cussed in Fig. S4).
Spatial Resolution of TEFO. Precise spatial resolution of excitation
was indicated by the elimination of ChR2-mediated de-
polarization when the beam was moved a few microns away from
the dendrite (Fig. 3 Aand B). Given that dendrites provide
a spatially confined and a much smaller excitable region (≈1–2
μm diameter dendritic compartments) than the soma, they were
ideal for precise measurement of the spatial resolution of our
TEFO technique. We used thin apical dendrites (50–400 μm
from the soma, in 50–160 μm depth) and quantitatively com-
pared the spatial resolution of our techniques with that acquired
with 488-nm light. We displaced the temporally focused spot
relative to the dendrite in 3D while recording voltage signals at
the soma (Fig. 3 A–D). When the TEFO excitation spot was
moved laterally (x-direction) or axially (z-direction), the peak
depolarization dropped off sharply to 50% of the peak response
after ≈10 μm and was almost eliminated at ≈20 μm distance [Fig.
3B,C, and D(red trace)]. However, in comparison, responses
induced by 488-nm light only dropped off by ≈50% at ≈50 μm
away from the dendrite (x-direction) [Fig. 3B(blue trace)] and
remained almost unaltered at 100 μm above or below the den-
drite (z-direction) (Fig. 3 Cand D). This result demonstrates a
5-fold improvement in the lateral excitation precession and at
least a 10-fold improvement in the axial excitation precession.
0246 81012
0
5
10
15
20
Pulse duration (ms)
Peak depolarization (mV)
2 mV
20 ms
1
2
5
10
100
ms 488
nm
880 nm
10 mV
AB
4
2
1
3
0.1 s
2 mV
D
1 3 2 4
C
50 ms
20 mV
50 ms
2 mV
0.1 ms
3x
5x
4x
20 µm
//
//
20 µm
Fig. 2. Temporal resolution of TEFO activation of ChR2 in hippocampal
neurons of acute brain slices. (A) Representative traces and summary graph
(B)(n= 5) of depolarization evoked by 1- to 100-ms-long TEFO pulses at the
somata of hippocampal neurons. (A, Inset) Representative response to 488-
nm light. (C) Single focal section of a ChR2-expressing CA1PC loaded with
Alexa594 in hippocampal slice from Thy1-ChR2-YFP mouse, with numbered
TEFO illumination spots indicated by red dots. Bottom traces show mem-
brane potential responses to 2-ms-long stimulations at the spots indicated in
C. Note that somatic stimulation fires the cell reliably, whereas dendritic
stimulation results in smaller depolarization, and stimulation at a spot out-
side of the cell area has no effect. (D) CA1PC soma with five two-photon
temporal focusing spots indicated by dots. (Lower) Response to 1-ms-long
TEFO excitation at a single spot (black) as well as to 0.1-ms-long two-photon
temporal focusing illuminations at three to five spots with 0.1-ms intervals.
Individual excitations are also shown (Lower, interval = 300 ms). Note that
scale bar for the uppermost trace differs from that related to the four lower
traces. Depolarization was more effective with multiple short illuminations
than that obtained with a single 1-ms pulse, despite the shorter total exci-
tation time.
02040
Lateral distance (µm)
050100
Axial distance (µm)
-50-100
0
0.2
0.4
0.6
0.8
1.0
0
0.2
Normalized depol.
0.4
0.6
0.8
1.0
1.2
1 mV
50 ms
20 µm
B
C
A
Normalized depol.
0
50
100
20
-20
-100 µm
-50
D
2 mV
20 ms
488 nm
880 nm
488 nm
880 nm
100 %
50 %
488 nm
880 nm
Fig. 3. Spatial resolution of TEFO activation of ChR2. (A) Stack image of
a CA1PC loaded with Alexa594, showing location of laterally moved TEFO
activation spots. (B) Lateral localization of response induced by 488-nm light
(blue, n= 4) and TEFO (red, n= 3). (C) Axial localization of response induced
by 488-nm light (blue) and TEFO with 50% (≈130 mW, red) and 100% (≈260
mW, black) power (dashed lines indicate Gaussian fit). (Inset) Individual
responses at the focal plane of the dendrite (color-coded respectively). (D)
Representative responses to 488-nm (blue) and TEFO-evoked (red) responses
at different distances above and below the dendrite.
Andrasfalvy et al. PNAS
|
June 29, 2010
|
vol. 107
|
no. 26
|
11983
NEUROSCIENCEAPPLIED PHYSICAL
SCIENCES
The axial resolution could be further improved by decreasing the
applied power by 50% (Fig. 3C). This is a strong indication, as
also discussed in a recent publication (26), that saturation of
ChR2 in the center of the target plane leads to an additional
contribution of the out-of-focus regions to the evoked response.
Depth Penetration. We next quantified how the efficacy of TEFO
excitation depended on the depth in the tissue. The maximum
depolarization evoked by single 1-ms pulses, placed on somata
located at variable depths in the slice, showed a linear attenuation
with depth (Fig. 4A). However, as demonstrated above, the total
induced depolarization could be increased by using the multispot
temporal focusing excitation approach. As a result, when a mul-
tispot temporal focusing excitation pattern with pulsed illumina-
tion durations of 0.1–0.2 ms and interpulse intervals of 0.1 ms was
used, the efficiency of the excitation was increased. As shown in
Fig. 4A, this led for the studied cells to a more than 2-fold in-
crease of depth of excitation for the same total illumination du-
ration. This dependence of the spatiotemporal patterning of
excitation was used to induce depolarizations that were strong
enough to evoke APs even at the apical tuft at >150 μm depth,
where depolarization induced by a single short-pulse illumination
was barely detectable (Fig. 4B).
Induction of Suprathreshhold Activity. In current clamp, at the
resting membrane potential (−58 to −67 mV), we set out to
determine the parameters necessary to induce the minimal de-
polarization that was sufficient to reach AP threshold in a given
cell of all three neuron types included in our study (Fig. 5A). We
used either one spot with variable pulse durations (PV-INs and
two of six CA1PCs; total t = 1–5 ms), or five spots with sub-
millisecond pulses (four of six CA1PC, and all SubPC, total t =
0.1–0.5 ms ×5). In all cells tested (n= 13), total pulse duration
of ≤5 ms evoked suprathreshold depolarization and AP firing
(PV-INs: 3.0 ±1.2 ms, n= 3; CA1PCs: 2.5 ±0.8 ms, n=6;
SubPCs: 1.3 ±0.1 ms, n= 4; Fig. 5A). We next tested the ability
of our method to induce high-frequency firing in PCs and PV-
INs. We found that all cell types usually fired APs at the first few
stimulations in the train. Subsequently, PCs responded to the
train of stimuli only intermittently (Fig. 5 B–D). Increasing pulse
duration partially overcame AP failures and improved firing
fidelity (Fig. 5C). This reduced firing probability is the conse-
quence of the spatial confinement of the TEFO excitation
leading to an inactivation of ChR2 at higher frequencies, an
effect previously also observed for blue light excitation (10, 32,
35, 36). Only recently a new genetic manipulation of ChR2 (12)
could overcome the ChR2 inactivation problem at higher fre-
quencies of stimulation.
Large Boutons Activation by TEFO. Our early data showing the lack
of effect of glutamate receptor blockers on ChR2-evoked de-
polarization in Thy1-ChR2-YFP mice (Materials and Methods
and Fig. 2) suggested that excitatory axons of ChR2-expressing
principal cells, innervating the recorded cell, are not activated by
two-photon temporal focusing within our standard parameter
range. Further investigations of excitatory axonal activation in
the CA1 region confirmed this fact (Fig. S5). Although we could
not evoke synaptic responses from Schaffer collaterals, we hy-
pothesized that certain types of terminals/axons with more fa-
vorable properties could be excited by TEFO. Synaptic boutons
of PV-INs are possible candidates, because they are relatively
large and they form multiple contacts on target cells (37), thus
these could provide more surface area to the temporally focused
two-photon illumination. To investigate that, we recorded from
(ChR2-negative) CA1PCs that were innervated by ChR2-
expressing PV-INs (mostly basket, axo-axonic, and bistratified
cells) in AAV-ChR2-GFP virus-infected PV-Cre mice (Materials
and Methods and SI Materials and Methods) in the presence of
ionotropic glutamate receptor antagonists. The temporally fo-
cused beam spot was first placed around the soma and proximal
dendrites of the recorded CA1PC, overlapping with the location
of ChR2-expressing boutons provided by PV-INs. Our experi-
mental conditions were optimized to detect GABAergic post-
A0.1 ms
1 ms
0.1 ms x 5
0
5
10
15
Depolarization (mV)
0.2 ms
0.2 ms x 5
Depth of soma (µm)
-80-100 -60 -40
50 ms
20 mV
10 x 3
50 ms
0.5 mV
10 µm
B
Fig. 4. Dependence of induced depolarization on tissue depth. (A) De-
pendence of induced depolarization at the somata of CA1PCs from Thy1-
ChR2-YFP mice on tissue depth and spatiotemporal patterning of TEFO ex-
citation for three different pulsed illumination durations and interpulse
times. Red, single 1-ms pulses (n=6,fitted linearly); open black dot and
squares (total n= 3), single submillisecond pulses; filled circle and black dots
(total n= 3), 5×0.1-ms spatiotemporally patterned excitation. Note that
repeating the 0.1-ms pulses five times on the same cells (filled black dot and
squares, total excitation time ≤1 ms) yields a higher total depolarization
compared with the single-spot 1-ms excitation. This can be used to induce
bigger depolarizations at larger tissue depths. (B) Ten pulses (0.1 ms long)
repeated three times with 0.1-ms intervals at the indicated locations in the
apical tuft (distance from soma ≈400 μm, depth in slice ≈150 μm) evoked
sufficient depolarization to evoke somatic AP (upper trace), whereas
responses to single activations were almost undetectable (bottom traces).
10 mV
50 ms
AB
C
10 mV
20 ms
20 Hz
50 Hz
100 Hz
D
20 m
V
20 ms
20 mV
20 ms
20 Hz
50 Hz
100 Hz
20 Hz
20 Hz
50 Hz
0
5
10
15
Depolarization (mV)
0
2
4
6
8
Pulse duration (ms)
CA1 Subiculum
2 ms
5 ms
2 ms
0.2 ms x 5
PV-IN PCPC
5 ms CA1-PC Sub-PC
PV-IN
Fig. 5. Fast and reliable AP responses by TEFO ChR2 excitation. (A) ChR2-
mediated depolarization efficiently evoked APs in CA1 PV-INs (n=3)and
pyramidal cells (PC) in CA1 (n= 6) and subiculum (n= 4). ChR2-induced de-
polarization was measured by hyperpolarizing the soma as necessary to
prevent AP firing. Graphs indicate the difference between resting mem-
brane potential and AP threshold (green circles), amplitude of ChR2-induced
suprathreshold depolarization (red squares), and the corresponding pulse
duration (black squares) for each cell type. (B) Action potentials evoked in
ChR2-expressing PV-IN by 1-ms-long pulses at 20–100 Hz. Insets: Sub-
threshold responses at hyperpolarized holding potential. Similar results
were obtained in n= 3 cells. (Cand D) Representative responses of a CA1PC
(C,n= 7) and a subiculum PC (D,n= 6) to 20-, 50-, or 100-Hz stimulation with
different pulse durations using single or multiple spots with different illu-
mination time.
11984
|
www.pnas.org/cgi/doi/10.1073/pnas.1006620107 Andrasfalvy et al.
synaptic hyperpolarization (holding potential, −60 to −65 mV;
reversal potential of chloride, −80 mV). Strikingly, using pulse
durations of 1–2 ms, we could evoke inhibitory synaptic responses
of 0.5- to 4.0-mV amplitude that were completely eliminated by
aspecific GABAA receptor antagonist (SR-95531, 10 μM, n=3)
(Fig. 6A). Increasing the pulse duration did not lead to any further
increase of the inhibitory postsynaptic potential (IPSP) amplitude,
suggesting that local synapses making contact with the post-
synaptic soma were activated in an all-or-none fashion (Fig. 6B).
One-photon excitation at 488 nm resulted in ≈6–9-mV hyperpo-
larization (Fig. 6 Aand B), presumably because the longer axial
size of the beam and scattering lead to activation of significantly
more synapses. To examine whether activation of boutons elicited
APs in the axon, we tried to evoke postsynaptic responses at
different spatial locations of the activation beam. We marked 5
spots around the recorded CA1PC soma (Fig. 6 C–E,Soma),10
spots in stratum pyramidale (Fig. 6 C–E, Pyr), 10 spots in proximal
stratum radiatum (Fig. 6 C–E, Rad), and finally 5 spots on a
ChR2-expressing interneuron soma (Fig. 6 C–E, IN). In all of the
locations, synchronous activation of the spots evoked IPSPs in the
postsynaptic cell, and the responses were eliminated by applica-
tion of tetrodotoxin (TTX, 0.5 μM; Fig. 6D). Notably, the latency
of the responses varied depending on the distance and activation
time of the excitation spot (Fig. 6E), further indicating that axonal
APs were elicited and propagated to evoke the synaptic responses.
These results altogether demonstrate that the TEFO technique is
efficient to directly activate the large (≈2–3μm diameter) synaptic
boutons (or population of boutons) arising from PV-INs (37).
Discussion
Although the use of genetically expressed ChR2 in different types
of neurons has become widely popular in recent years, one-
photon excitation does not allow for spatial precision of neuronal
activation using this method. Recognizing this caveat, recent
studies reported the possibility of two-photon excitation of ChR2
using a scanning approach in cultured neurons (26). Although the
spatial resolution indeed dramatically improved with two-photon
excitation, the long scanning time necessary for sufficient channel
activation and consequent depolarization significantly com-
promises the temporal precision of neuronal activation (≈30 ms
for evoking an AP). The TEFO technique, which does not require
scanning, provides reliable activation of cells within ≈1–3msand
thereby provides ≈15 times faster activation than the scanning
approach. This improvement practically implies that only the two-
photon temporal focusing approach allows quasi-synchronous
activation of neurons and their different cellular compartments in
future studies. The combination of TEFO with a conventional
dual galvanometer–based scanning system, as described in our
study, makes repositioning of the excitation spot fast (<0.2 ms to
any point in a 100-μmfield) and convenient. The precise spatial
and temporal control of firing activity of individual or a desired
number of individually selected cells, especially when combined
with selective expression of ChR2 in particular cell populations,
opens up the possibility (among others) for detailed, high-
throughput studies of connectivity and dynamics of small- and
large-scale neuronal networks and assessment of the functional
consequences of their activation both in vitro and in vivo.
Although the current TEFO technique can reliably stimulate
a few APs, further advances of the technique, such as 3D light
sculpting and development of ChR2 mutants with higher con-
ductance or less desensitization (35), are expected to enable pre-
cise induction of longer AP trains in a wide range of frequencies.
Beside the reliable generation of APs at the soma, an important
aspect of our study is the demonstration of the possibility for
spatially and temporally precise activation of certain cellular
compartments, such as dendrites and large synaptic boutons. This
feature can be exploited in many applications, for example for
studying dendritic properties and integration without or combined
with uncaging methods (38), or for detailed investigation of the
spatiotemporal characteristics and role of inhibition by genetically
labeled interneuron types. On the other hand, the lack of activa-
tion of excitatory synaptic terminals, such as Schaffer collaterals,
shows that different types of synaptic boutons, presumably having
diverse anatomical and biophysical properties, can exhibit differ-
ent levels of excitability by the TEFO technique.
The lack of axonal activation, at least under these experimental
conditions and within the explored wide parameter range, can be
crucial in studies in which target neurons lie within a densely
intermingled network of thin ChR2-expressing axons with small
boutons, and therefore makes the TEFO technique a powerful
tool for studies such as circuit mapping. On the other hand, the
ability to specifically activate axons with TEFO could be useful,
for example, for mapping long-range connections in the brain.
Further technical developments, such as combination of our
current method with holographic techniques (39), are being
considered to allow targeted and specific stimulation of axonal
compartments with sculpted light. In summary, our method of
two-photon excitation of ChR2 by temporal focusing, especially
A
C
E*
1 mV
20 ms
1 mV
10 ms
PC soma
Pyr
IN
B
IN
Soma
Pyr
Rad
20 µm
1 mV
40 ms
SR-95531
880 nm
488 nm
-4
Hyperpolarization (mV)
-2
0
048
Pulse duration (ms)
1026 12
-6
1 mV
300 ms
IN
2 mV
300 ms
Soma
Rad
Pyr
2 mV
300 ms
+TTX
+TTX
*
*
*
*
*
*
+TTX
*********
**********
*****
*
*
x5
x5
x10
x10
x10
x5
x5
*****
D
Fig. 6. Large inhibitory synaptic terminals are activated by TEFO ChR2 ex-
citation. (A) Representative traces of optically evoked hyperpolarizing
postsynaptic potentials in a CA1PC innervated by ChR2-expressing PV-INs
(red, TEFO excitation; blue, 488-nm excitation) by targeting the soma. SR-
95531 (10 μM) blocked responses with both types of stimulations (black
traces, representative for n= 3 cells). (B) Peak hyperpolarization evoked by
TEFO (red) did not depend on pulse duration (n= 6). Excitation at 488 nm
(blue) evoked larger responses than TEFO excitation (n= 5). Dots and error
bars represent means ±SEM. (C) Stack image showing a recorded CA1PC and
indicating locations of series of TEFO illumination spots at the soma (5 spots),
stratum pyramidale (Pyr; 10 spots), stratum radiatum (Rad; 10 spots), and at
the soma of a PV-IN (IN; 5 spots). (D) Representative traces showing
responses to illumination at the locations indicated in C, using 100-ms
stimulus intervals (green), 0.1-ms stimulus intervals (red), and 0.1-ms stimulus
intervals in the presence of 0.5 μm TTX (black). (E) Overlaid traces evoked by
stimulation at the soma, in stratum pyramidale (Pyr), and at the soma of the
PV-IN (IN). Note the differences in response latency and the stepwise rise of
the response evoked in stratum pyramidale.
Andrasfalvy et al. PNAS
|
June 29, 2010
|
vol. 107
|
no. 26
|
11985
NEUROSCIENCEAPPLIED PHYSICAL
SCIENCES
when combined with genetic and electrophysiological techniques,
has broad potential to contribute to our understanding of the
cellular and network basis of neuronal functions.
Materials and Methods
Optical Setup. Please refer to SI Materials and Methods for a detailed de-
scription. Briefly, a tunable 140-fs pulsed laser was used as the source for
temporal focusing. After passing through a variable zoom telescope the
beam was diffracted by a diffraction grating. The spot on the grating was
imaged via a telescope consisting of an achromatic lens and the microscope
objective onto the specimen plane. For the multispot excitation experiments
the temporally focused beam was integrated into the scan head of a com-
mercial two-photon scanning microscope.
Virus Preparation and Injection. Cre recombinase–dependent virus was as-
sembled, harvested, and purified as previously described (40) and injected
into the hippocampal CA1 region of parvalbumin-Cre (33) mice and hippo-
campal CA3 region of Sprague-Dawley rats.
Hippocampal Slice Preparation. Transverse slices were prepared as previously
described (41), according to methods approved by the Janelia Farm Institu-
tional Animal Care and Use Committee.
ACKNOWLEDGMENTS. We thank J. Magee for supporting this project;
A. Losonczy, J. Makara, and K. Svoboda for numerous insightful discussions,
experimental suggestions, and comments on the manuscript; A. Losonczy for
testing cell type-specific viruses, for sample preparation, and for help with
the analysis; and D. O’Connor and G. Paez for sample preparation.
1. Sohal VS, Zhang F, Yizhar O, Deisseroth K (2009) Parvalbumin neurons and gamma
rhythms enhance cortical circuit performance. Nature 459:698–702.
2. Cardin JA, et al. (2009) Driving fast-spiking cells induces gamma rhythm and controls
sensory responses. Nature 459:663–667.
3. Tsai HC, et al. (2009) Phasic firing in dopaminergic neurons is sufficient for behavioral
conditioning. Science 324:1080–1084.
4. Luo L, Callaway EM, Svoboda K (2008) Genetic dissection of neural circuits. Neuron 57:
634–660.
5. Gradinaru V, Mogri M, Thompson KR, Henderson JM, Deisseroth K (2009) Optical
deconstruction of parkinsonian neural circuitry. Science 324:354–359.
6. Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K, de Lecea L (2007) Neural
substrates of awakening probed with optogenetic control of hypocretin neurons.
Nature 450:420–424.
7. Zemelman BV, Lee GA, Ng M, Miesenböck G (2002) Selective photostimulation of
genetically chARGed neurons. Neuron 33:15–22.
8. Zemelman BV, Nesnas N, Lee GA, Miesenbock G (2003) Photochemical gating of
heterologous ion channels: Remote control over genetically designated populations
of neurons. Proc Natl Acad Sci USA 100:1352–1357.
9. Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH (2004) Light-activated ion
channels for remote control of neuronal firing. Nat Neurosci 7:1381–1386.
10. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale,
genetically targeted optical control of neural activity. Nat Neurosci 8:1263–1268.
11. Li X, et al. (2005) Fast noninvasive activation and inhibition of neural and network
activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc Natl Acad
Sci USA 102:17816–17821.
12. Gunaydin LA, et al. (2010) Ultrafast optogenetic control. Nat Neurosci 13:387–392.
13. Lechner HAE, Lein ES, Callaway EM (2002) A genetic method for selective and quickly
reversible silencing of Mammalian neurons. J Neurosci 22:5287–5290.
14. Slimko EM, McKinney S, Anderson DJ, Davidson N, Lester HA (2002) Selective
electrical silencing of mammalian neurons in vitro by the use of invertebrate ligand-
gated chloride channels. J Neurosci 22:7373–7379.
15. Karpova AY, Tervo DGR, Gray NW, Svoboda K (2005) Rapid and reversible chemical
inactivation of synaptic transmission in genetically targeted neurons. Neuron 48:
727–735.
16. HanX, Bo ydenES (2007) Multi ple-coloro pticalact ivation,s ilencing, and desynchronization
of neuralactivity, with single-spike temporal resolution. PLoS ONE 2:e299.
17. Huber D, et al. (2008) Sparse optical microstimulation in barrel cortex drives learned
behaviour in freely moving mice. Nature 451:61–64.
18. Petreanu L, Huber D, Sobczyk A, Svoboda K (2007) Channelrhodopsin-2-assisted
circuit mapping of long-range callosal projections. Nat Neurosci 10:663–668.
19. Varga V, et al. (2009) Fast synaptic subcortical control of hippocampal circuits. Science
326:449–453.
20. Nagel G, et al. (2003) Channelrhodopsin-2, a directly light-gated cation-selective
membrane channel. Proc Natl Acad Sci USA 100:13940–13945.
21. Nagel G, et al. (2005) Channelrhodopsins: Directly light-gated cation channels.
Biochem Soc Trans 33:863–866.
22. Lanyi JK (1986) Halorhodopsin: A light-driven chloride ion pump. Annu Rev Biophys
Biophys Chem 15:11–28.
23. Chow BY, et al. (2010) High-performance genetically targetable optical neural
silencing by light-driven proton pumps. Nature 463:98–102.
24. Feldbauer K, et al. (2009) Channelrhodopsin-2 is a leaky proton pump. Proc Natl Acad
Sci USA 106:12317–12322.
25. Schoenenberger P, Grunditz A, Rose T, Oertner TG (2008) Optimizing the spatial
resolution of Channelrhodopsin-2 activation. Brain Cell Biol 36:119–127.
26. Rickgauer JP, Tank DW (2009) Two-photon excitation of channelrhodopsin-2 at
saturation. Proc Natl Acad Sci USA 106:15025–15030.
27. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence
microscopy. Science 248:73–76.
28. Zhu GH, van Howe J, Durst M, Zipfel W, Xu C (2005) Simultaneous spatial and
temporal focusing of femtosecond pulses. Opt Express 13:2153–2159.
29. Oron D, Tal E, Silberberg Y (2005) Scanningless depth-resolved microscopy. Opt
Express 13:1468–1476.
30. Tal E, Oron D, Silberberg Y (2005) Improved depth resolution in video-rate line-
scanning multiphoton microscopy using temporal focusing. Opt Lett 30:1686–1688.
31. Vaziri A, Tang J, Shroff H, Shank CV (2008) Multilayer three-dimensional super
resolutionimaging of thick biological samples.Proc Natl Acad Sci USA 105:20221–20226.
32. Arenkiel BR, et al. (2007) In vivo light-induced activation of neural circuitry in
transgenic mice expressing channelrhodopsin-2. Neuron 54:205–218.
33. Hippenmeyer S, et al. (2005) A developmental switch in the response of DRG neurons
to ETS transcription factor signaling. PLoS Biol 3:e159.
34. Atasoy D, Aponte Y,Su HH, Sternson SM (2008) AFLEX switch targets Channelrhodopsin-
2 to multiple cell types for imaging and long-range circuit mapping. J Neurosci 28:
7025–7030.
35. Lin JY, Lin MZ, Steinbach P, Tsien RY (2009) Characterization of engineered
channelrhodopsinvariantswithimprovedpropertiesand kinetics.Biop hys J 96:1803–1814.
36. Wang H, et al. (2007) High-speed mapping of synaptic connectivity using
photostimulation in Channelrhodopsin-2 transgenic mice. Proc Natl Acad Sci USA 104:
8143–8148.
37. Cobb SR, et al. (1997) Synaptic effects of identified interneurons innervating both
interneurons and pyramidal cells in the rat hippocampus. Neuroscience 79:629–648.
38. Lutz C, et al. (2008) Holographic photolysis of caged neurotransmitters. Nat Methods
5:821–827.
39. Papagiakoumou E, de Sars V, Oron D, Emiliani V (2008) Patterned two-photon
illumination by spatiotemporal shaping of ultrashort pulses. Opt Express 16:
22039–22047.
40. Grieger JC, Choi VW, Samulski RJ (2006) Production and characterization of adeno-
associated viral vectors. Nat Protoc 1:1412–1428.
41. Losonczy A, Makara JK, Magee JC (2008) Compartmentalized dendritic plasticity and
input feature storage in neurons. Nature 452:436–441.
11986
|
www.pnas.org/cgi/doi/10.1073/pnas.1006620107 Andrasfalvy et al.
