396 | VOL.9 NO.4 | APRIL 2012 | nAture methods
local anesthetics effectively suppress pain sensation, but
most of these compounds act nonselectively, inhibiting
activity of all neurons. moreover, their actions abate slowly,
preventing precise spatial and temporal control of nociception.
We developed a photoisomerizable molecule, quaternary
ammonium–azobenzene–quaternary ammonium (QAQ), that
enables rapid and selective optical control of nociception.
QAQ is membrane-impermeant and has no effect on most cells,
but it infiltrates pain-sensing neurons through endogenous
ion channels that are activated by noxious stimuli, primarily
trPV1. After QAQ accumulates intracellularly, it blocks voltage-
gated ion channels in the trans form but not the cis form.
QAQ enables reversible optical silencing of mouse nociceptive
neuron firing without exogenous gene expression and can
serve as a light-sensitive analgesic in rats in vivo. Because
intracellular QAQ accumulation is a consequence of nociceptive
ion-channel activity, QAQ-mediated photosensitization is a
platform for understanding signaling mechanisms in acute
and chronic pain.
Optogenetic tools enable photoregulation of action potential firing
in neurons both in vitro and in vivo1 through the introduction
of exogenous genes. In contrast, small-molecule photoswitches
enable optical control of neuronal excitability without genetic
manipulation2,3. Photoswitch molecules confer light-sensitivity
on the intrinsic excitability of neurons within minutes4–6.
However, unlike optogenetic tools that can be promoter-targeted
for expression in particular types of neurons, photoswitches act
nonselectively on all neurons that are exposed to the molecule.
Depending on the scientific or biomedical application, it could
be a benefit or even a requirement to target photosensitivity to a
particular type of neuron.
Here we used a non-genetic strategy to target a photoswitch
molecule to pain-sensing (nociceptive) neurons. Nociceptive
neurons have been particularly inaccessible to selective electro-
physiological manipulation because both their peripheral sen-
sory endings and central synaptic terminals are quite small, either
embedded in the skin or interspersed with other neurons in the
spinal cord. Nociceptors are unique in possessing a high den-
sity of ion channels that respond directly or indirectly to noxious
stimuli7. For example, the capsaicin receptor TRPV1, which is
sensitive to noxious heat, protons and mediators of inflamma-
tion, is expressed in nociceptive neurons, but it is very sparsely
expressed elsewhere in the nervous system8. TRPV1 enters into
a pore-dilated state upon prolonged agonist activation, allowing
permeation of relatively large cations9. This property has been
exploited to deliver into nociceptors a membrane-impermeant
derivative of the local anesthetic lidocaine, QX-314 (ref. 10). The
selective entry and silencing of nociceptors by QX-314 gives this
molecule potential as a pain-selective local anesthetic10. However,
once QX-314 enters cells, it cannot escape, and silencing persists
for many hours11. The irreversibility of QX-314 precludes tem-
porally precise regulation of nociceptor activity.
Here we describe QAQ, a photoisomerizable molecule that con-
fers reversible light-sensitivity selectively onto neurons involved
in pain signaling, enabling rapid optical control of nociception
without genetic manipulation.
QAQ photosensitized voltage-gated ion channels
We developed QAQ, a photoswitch that reversibly suppresses
neuronal excitability by optically regulating voltage-gated Na+,
Ca2+ and K+ channels. QAQ has a central photoisomerizable
azobenzene coupled on both sides to quaternary ammonium
groups (Fig. 1a). Upon illumination with 380-nm light, the elon-
gated trans-QAQ converts to the bent cis form (Supplementary
Fig. 1a). Cis-QAQ spontaneously reverts to the trans form slowly
in the dark (Supplementary Fig. 1b), but this transition occurs
quickly (within milliseconds) in 500-nm light.
QAQ resembles lidocaine and its derivative QX-314 (Fig. 1b,c),
local anesthetics that block voltage-gated Na+, K+ and Ca2+
channels from the cytoplasmic side12,13. Lidocaine is a tertiary
amine that crosses the membrane in an uncharged state and
blocks ion channels after becoming protonated in the cytoplasm.
1Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, USA. 2Department of Chemistry, University of Munich, Munich,
Germany. 3Centre National de la Recherche Scientifique, Interdisciplinary Institute for Neuroscience, Unité Mixte de Recherche 5297 ‘Central Mechanisms of Pain
Sensitization’, Bordeaux, France. 4Université de Bordeaux, Bordeaux, France. 5Department of Chemical Engineering and Department of Bioengineering and The Helen
Wills Neuroscience Institute, University of California, Berkeley, California, USA. 6These authors contributed equally to this work. Correspondence should be addressed
to R.H.K. (firstname.lastname@example.org) or D.T. (email@example.com).
Received 15 SeptembeR 2011; accepted 26 JanuaRy 2012; publiShed online 19 febRuaRy 2012; doi:10.1038/nmeth.1897
rapid optical control of nociception with an
Alexandre Mourot1,6, Timm Fehrentz1,2,6, Yves Le Feuvre3,4, Caleb M Smith1, Christian Herold1, Deniz Dalkara5,
Frédéric Nagy3,4, Dirk Trauner2 & Richard H Kramer1
© 2012 Nature America, Inc. All rights reserved.
nAture methods | VOL.9 NO.4 | APRIL 2012 | 397
QX-314 contains a permanently charged quaternary ammonium,
preventing it from crossing the membrane. However, QX-314 is
a potent blocker of activity when introduced through a patch
pipette into the cytoplasm14.
To test whether QAQ can act like a photoregulated ion-channel
blocker, we made whole-cell recordings from NG108-15 cells, a
mouse neuroblastoma and rat glioma hybrid cell line that expresses
neuronal voltage-gated Na+ (Nav) channels15. When we delivered
QAQ into the cytoplasm through the patch pipette, it blocked most
of the Na+ current in the trans configuration, but blockade was
removed in 380-nm light (Fig. 1d). In contrast, bath application
of QAQ did not block (Supplementary Fig. 2) or photosensitize
the Na+ current (Fig. 1e), indicating that QAQ is membrane-
impermeant like QX-314 (ref. 10). Light-sensitive block of the Na+
current occurred at all membrane potentials tested (Fig. 1f). We
quantified block of trans-QAQ versus cis-QAQ by examining Na+
current during a train of depolarizing stimuli. In the trans form,
the amount of QAQ blockade is use-dependent, becoming more
complete with increasing duration or frequency of depolarization
(56% ± 10% block after 30 s, n = 7 cells, Fig. 1g). In contrast, cis-
QAQ decreased the current by 9.6% ± 0.1% (n = 7 cells), indistin-
guishable from control experiments with no QAQ (8.3% ± 0.1%,
n = 5 cells, P = 0.52 Student’s t-test). Photocontrol of Na+ current
could be elicited repeatedly and rapidly without decrement over
many minutes (Fig. 1h and Supplementary Fig. 3).
Local anesthetics are used to silence the activity of sensory
neurons, which have a variety of voltage-gated Na+ channels,
including tetrodotoxin-sensitive and -resistant types7. Whole-
cell recordings from rat trigeminal ganglion neurons showed that
both channel types could be photoregulated by intracellular QAQ
(Supplementary Fig. 4).
QAQ also photoregulated voltage-gated Ca2+ channels. We
recorded from HEK-293 cells stably expressing voltage-gated
Ca2+ channel Cav2.2 and from GH3 cells, a rat pituitary tumor
cell line expressing L-type calcium channels16. In both cell types,
internal trans-QAQ blocked the Ca2+ current, but blockade was
removed in 380-nm light (Fig. 1i and Supplementary Fig. 5a).
Photoregulation of both Ca2+ channels was rapid, occurred
at all voltages tested and exhibited little decrement over time
(Supplementary Fig. 5b–f).
Voltage-gated K+ channels were also sensitive to QAQ. We
recorded from HEK-293 cells expressing the inactivation-removed
Shaker K+ channel17 and again observed robust photoregulation,
with current blocked by trans-QAQ and unblocked by converting
the molecule to the cis form (Fig. 1j). QAQ block at 500 nm was
voltage-dependent, increasing with depolarization, as observed
with other quaternary ammonium molecules5 (Supplementary
Fig. 6a). QAQ photosensitized other voltage-gated K+ channels
exogenously expressed in HEK-293 cells as well as native K+
current in hippocampal neurons (Supplementary Fig. 6b–j).
Photoregulation of K+ channels occurred rapidly and without
decrement over time (Supplementary Fig. 6k,l).
Hence whereas QAQ was normally membrane-impermeant,
it photosensitized current flowing through voltage-gated Na+,
Ca2+ and K+ channels when introduced into the cell (Fig. 1k).
Intracellular QAQ photosensitized many but not all K+ channels;
inward-rectifier (Kir) and hyperpolarization-activated cyclic
nucleotide–gated (HCN) channels were unaffected by QAQ
(Supplementary Fig. 7). Intracellular QAQ did not photoregulate
current through N-methyl-d-aspartic acid (NMDA) and non-
NMDA receptors (Supplementary Fig. 8).
QAQ enabled photocontrol of neuronal excitability
Because it imparted light-sensitive block on voltage-gated Na+,
K+ and Ca2+ channels, QAQ should have a strong influence on
the electrical excitability of neurons. To examine the net effect of
Figure 1 | Intracellular QAQ photosensitized
voltage-gated ion channels. (a–c) Chemical
structures of cis and trans QAQ (a), lidocaine (b)
and QX-314 (c). kBT, thermal energy of
relaxation, with kB denoting the Boltzman
constant and T, the temperature. (d) Na+
current in cells with intracellular QAQ (100 µM).
Depolarization was from −70 mV to −10 mV.
Photoswitching, as defined by (current
(I)380 nm − I500 nm)/I380 nm, was 60.5 ± 5.8%
(n = 4 cells) (e) Na+ current in cells with
extracellular QAQ (1 mM). Photoswitching was
1.4 ± 1.3% (n = 7 cells). (f) Current versus
membrane voltage (Vm) of peak Na+ current.
(g) Na+ current in cells with intracellular
QAQ (100 µM) and repetitive depolarizing
pulses (1 Hz). Control with no QAQ is shown.
(h) Reversibility of Na+ current photoswitching.
(i) Cav2.2 current using intracellular QAQ
(100 µM). Depolarizing pulse was from
−60 mV to +10 mV. Photoswitching was 60.5 ±
10.5% (n = 3 cells). (j) Shaker K+ channel
current using intracellular QAQ (100 µM).
Depolarizing pulse was from −70 mV to +40
mV. Photoswitching was 60.3 ± 8.6% (n = 4 cells). (k) Percentage photoswitching of currents through voltage-gated Na+ (Nav), voltage-gated Ca2+ (Cav)
and voltage-gated K+ (Kv) channels. ‘Neuronal’, Na+ channels from NG108-15 cells; ‘sensory’, Na+ channels from rat trigeminal ganglion neurons; TTXR,
tetrodotoxin-resistant; L-type, voltage-gated channels from GH3 cells; Cav2.2, Kv2.1, Kv3.1 and Kv4.2 were expressed in HEK-293 cells; ‘hippocampal’,
K+ channels from primary hippocampal cultures (n = 3–13 cells, error bars, s.e.m.). NG108-15 cells were examined in d–h, and HEK-293 cells in i,j.
© 2012 Nature America, Inc. All rights reserved.
398 | VOL.9 NO.4 | APRIL 2012 | nAture methods
internal QAQ on action-potential firing we carried out current
clamp recordings from dissociated rat hippocampal neurons in
culture. Current pulses of increasing amplitude elicited a pro-
gressive increase in the number of action potentials when QAQ
was in the cis configuration (Fig. 2a). However, when QAQ was
converted to the trans form, neurons fired a single spike at the
onset of stimulation, but did not fire additional spikes even with
the largest current pulse tested, consistent with use-dependent
blockade of Na+ channels (Fig. 2a,b). The amplitude of the first
spike decreased and its half-width increased when we switched
from 380 nm to 500 nm light, in agreement with both Na+ and
K+ channels being blocked (Fig. 2c). Higher internal concen-
tration of QAQ (≥200 µM) eliminated all spikes at 500 nm
(data not shown).
QAQ affected spiking but had little or no effect on the rest-
ing properties of these neurons. Neither the input resistance nor
the membrane potential changed over time as QAQ diffused into
the neuron (Fig. 2d,e). Moreover, light had no effect on these
parameters, in agreement with QAQ not affecting Kir and HCN
channels, two channels that have a role in setting the resting
membrane potential of neurons. Firing threshold in these neurons
was the same with (195 pA ± 31 pA; ± s.e.m.) and without (161 pA ±
12 pA, P = 0.44 Student t-test) QAQ in the pipette and was not
affected by changing the wavelength of light (Fig. 2f).
QAQ entered cells through nociceptive ion channels
QAQ is normally membrane impermeant, so it does not photo-
sensitize most cells. However, we asked whether QAQ could be
delivered into cells without requiring dialysis through a patch
electrode. This strategy involves using nociceptive pore channels
as a conduit for QAQ entry. We transfected HEK-293 cells with
the gene encoding the Shaker K+ channel, which we used as
an indicator of intracellular QAQ accumulation. We first used
TRPV1, a channel whose pore dilates after exposure to its agonist
capsaicin9. Control cells treated with capsaicin showed no QAQ-
mediated photosensitization (Fig. 3a). However, cells expressing
TRPV1 showed photosensitization of Shaker current, but only
when we applied QAQ on the cells in conjunction with capsaicin.
We then tested two other TRP channels, TRPA1 and TRPM8,
but found no significant loading through either TRPA1 (7.3% ±
4.0%; s.e.m., n = 11 cells, P = 0.39, Student t-test) or TRPM8
(−0.3% ± 1.4%; s.e.m., n = 3 cells, P = 0.81, Student t-test) channels
after these channels were activated with allyl isothiocyanate
(30 µM) or menthol (30 µM), respectively (data not shown).
Some ionotropic receptors for ATP (P2X receptors) also exhibit
pore dilation upon prolonged activation18,19. Therefore we tested
whether P2X7 channels could be used as a conduit for QAQ
entry. Control HEK-293 cells treated with ATP showed no QAQ-
mediated photosensitization (Fig. 3b). However, cells expressing
P2X7 receptors showed photosensitization of Shaker current, but
only when we applied QAQ on the cells in conjunction with ATP.
The amount of K+ channel photosensitization was nearly the same
5 min and 30 min after ATP application, suggesting that QAQ
equilibrated quickly in the cell.
To test whether QAQ can enter into neurons through dilat-
ing pore channels, we recorded from cultured rat hippocampal
neurons. QAQ alone had no effect on endogenous voltage-gated
K+ current (Fig. 3c). However, we could bestow light-sensitivity
to neurons that exogenously expressed P2X7 receptors, by
treating them with QAQ and ATP. A cell death assay showed
that there was no toxicity resulting from this treatment
(Supplementary Fig. 9).
Figure 2 | Intracellular QAQ as a
photoswitchable inhibitor of neuronal
activity. (a) Action potential (AP)
firing in dissociated rat hippocampal
neurons with 100 µM QAQ in the patch
pipette under illumination with 380-nm
or 500-nm light. Firing was elicited with
current injections (Iinj.) of increasing
amplitude (35-pA steps). (b) Number
of action potentials elicited by incremental
current injections at indicated wavelengths.
(c) Action potential shape in 380-nm
and 500-nm light. (d) Input resistance
directly after (0 min; P = 0.86) and
~15 min after establishing whole-cell
mode, in 380-nm and 500-nm light (P = 0.93).
(e) Resting membrane potential at 0 min
(P = 0.47) and ~15 min after establishing whole-cell mode in 380-nm and 500-nm light (P = 0.47). (f) Firing threshold ~15 min after establishing
whole-cell mode in 380-nm and 500-nm light (P = 0.5). All error bars, s.e.m.; Student t-test; n = 4–6 cells.
Number of APs
Input resistance (MΩ)
Membrane potential (mV)
15 min, 380 nm
15 min, 500 nm
Firing threshold (pA)
Figure 3 | TRPV1 channels and P2X7 receptors as a conduit for QAQ entry
into cells. (a) Percentage photoswitching of Shaker K+ channels in cells
expressing (+) or not expressing (−) TRPV1 channels and treated with or
without capsaicin (1 µM) in conjunction with QAQ (30 min, 1 mM);
n = 10 cells for each condition. (b) Same experiment as in a using P2X7
receptors (P2X7R) instead of TRPV1 channels. ATP (1 mM) was applied
for 5 min or 30 min to activate the P2X7 receptors during QAQ loading;
n = 3–5 cells for each condition. (c) Percentage photoswitching of
native K+ channels in hippocampal neurons. Control neurons or neurons
expressing P2X7R were treated for 30 min with QAQ (100 µM) plus ATP
(2.5 mM). Error bars, ± s.e.m. (n = 12 cells).
© 2012 Nature America, Inc. All rights reserved.
nAture methods | VOL.9 NO.4 | APRIL 2012 | 399
TRPV1 channels are crucial for nociception in peripheral sen-
sory neurons7,8. The presence of endogenous TRPV1 channels
suggests that QAQ might enter nociceptive neurons without
requiring exogenous gene expression. We examined the effect of
QAQ on three different parts of nociceptive neurons; their cell
bodies, located in the dorsal root ganglion (DRG), their synaptic
terminals, located in the spinal cord, and their sensory nerve end-
ings, located in the periphery.
Photosensitization of neurons in intact drGs
We developed a system to record and analyze many mouse DRG
neurons at once while simultaneously photoregulating their elec-
trical activity (Fig. 4a and Online Methods). We used a three-
dimensional multielectrode array containing 60 pin-shaped
electrodes. We controlled the isomeric state of QAQ with a light
source positioned under the multielectrode array; the array
was transparent to 380-nm and 500-nm light. We positioned a
suction electrode on the peripheral nerve of the DRG and used
an external stimulation unit to elicit action potentials, which
could be recorded as extracellular signals by electrodes of the
multielectrode array (Fig. 4b). We focused on action potentials
resulting from slowly conducting C fibers (<2.5 m s−1), which
specifically belong to nociceptive neurons. With the stimulat-
ing electrode 10–15 mm from the recording electrodes, spikes
attributable to these neurons appeared >4 ms after the onset of
After treatment with QAQ, individual electrodes recorded
spikes that could be silenced by switching from 380-nm to 500-nm
light, consistent with QAQ photosensitization (Fig. 4c). This
suggests that there must be some basal activity of QAQ-
permeant channels in DRG neurons. We plotted the activity of
24 neurons on a raster plot (Fig. 4d). Trains of stimuli at 10 Hz
elicited trains of action potentials, which could be photo-
regulated by switching from 380-nm to 500-nm light. At both
wavelengths, the number of spikes diminished within a train of
stimuli, but the extent of spike-train accommodation was much
greater in 500-nm light.
Principal component analysis is a common method in multi-
electrode array recordings for sorting spikes belonging to differ-
ent neurons, depending on their stereotypical spike waveforms.
However, QAQ modulated voltage-gated channels underlying
action potentials, changing the spike waveform as a consequence
of photoswitching (Fig. 4e). Therefore, instead of assigning spikes
to different units, which could be particularly error-prone in this
circumstance, we devised an analytical method that involves inte-
grating the signal over a ‘poststimulus’ time window (Fig. 4e and
After treating the DRG with a high concentration of QAQ
(1 mM for 30 min), we observed a dramatic decrease in the
integrated signal upon switching from 380-nm to 500-nm light
(Fig. 4f). At a lower concentration of QAQ (0.3 mM for 5 min),
the integrated signal decreased to a lesser extent upon switch-
ing to 500-nm light. The weak photoswitching imparted on the
DRG neurons by mild QAQ treatment suggests that little QAQ
accumulated in the cells.
If the TRPV1 channel is the main conduit for QAQ
entry, blocking or eliminating TRPV1 channels should
reduce QAQ loading and consequently the amount of
photosensitization. Consistent with this, we found that
tetrahydro-pyrazine1(2H)-carboxamide (BCTC), a TRPV1
antagonist that inhibits acid- and capsaicin-induced activa-
tion, considerably reduced DRG photosensitization (Fig. 4g).
In Trpv1−/− mice, photosensitization was also strongly reduced
but not completely abolished. Ruthenium red, a nonselective
TRP channel pore blocker, entirely prevented photosensitiza-
tion. Taken together, these data suggest that TRPV1 channels
are the main entry route for QAQ into DRGs.
Figure 4 | Photosensitization of intact
DRGs recorded with a three-dimensional
multielectrode array (MEA). (a) Experimental
setup. (b) Mouse DRG placed onto the MEA
(scale bar, 200 µm). Extracellular recordings
are shown superimposed on each electrode.
(c) Signals recorded from one electrode
(under 380-nm and 500-nm light) after
30-min treatment with 1 mM QAQ.
(d) For simultaneous recording of 24 units,
shown are a raster plot of spiking under
10-Hz stimulation (top) and average firing
rate (bottom; 100 ms time bins). (e) Signals
from a single electrode during a train of
50 stimuli (stim; 10 Hz). The first response
is shown in black; the last in violet or green.
The signal was integrated over a poststimulus
time window represented by the colored box.
(f) Average integrated signal over five
light cycles, from a DRG treated with 1 mM
QAQ for 30 min or 0.3 mM QAQ for 5 min.
(g) Quantification of photosensitization
with the following treatment conditions,
normalized to photosensitization with QAQ alone (0.3 mM for 5 min, dotted line): BCTC (1 µM), 22 ± 18%, P = 0.03; Trpv1−/− mice, 34 ± 8% photosensitization,
P = 0.005; ruthenium red (RR, 10 µM), 7 ± 18% photosensitization, P = 0.006; capsaicin (1 µM), 172 ± 30% photosensitization, P = 0.05; Bradykinin
(BK; 1 µM), 192 ± 30% photosensitization, P = 0.02; and electrical stimulation (stim.: 5-s trains of 1-ms stimuli at 10 Hz, repeated every 30 s for 5 min),
138 ± 21% photosensitization, P = 0.02. Error bars ± s.e.m.; Mann-Whitney U test (n = 3–5 DRGs).
Normlaized photoswitching gf
Firing rate (Hz)
QAQ 1 mM, 30 min
QAQ 0.3 mM, 5 min
Integrated signal (µV.s)
© 2012 Nature America, Inc. All rights reserved.
400 | VOL.9 NO.4 | APRIL 2012 | nAture methods
This system can be used as a platform for assessing the activity
of TRP channels in the intact DRG in response to various
stimuli. We treated the ganglia with capsaicin during QAQ
loading, followed by thorough washing with normal saline.
Capsaicin is a selective agonist of TRPV1, and as expected,
it increased QAQ loading and therefore photosensitization
(Fig. 4g). Bradykinin is a neuropeptide that promotes
pain hypersensitivity and inflammation20. We found that
Bradykinin also promoted QAQ-mediated DRG photosensitiza-
tion, consistent with a signaling cascade that leads to activation
of TRP channels21.
Direct electrical stimulation of sensory neuron axons in
the peripheral nerve also promoted DRG photosensitization
(Fig. 4g), indicating enhanced QAQ entry during the stimulation
period. Action potential firing may directly promote QAQ entry
into TRPV1-containing neurons, but TRPV1 channels are only
weakly voltage-sensitive22. In addition, action potential firing
may promote DRG somata to release neuro-inflammatory trans-
mitters, and these may indirectly lead to activation of nociceptive
channels, a positive feedback mechanism that could contribute to
prolonged hypersensitivity and chronic pain.
Photosensitization of neurons in spinal cord slices
TRPV1 is abundantly expressed throughout the entire length of
nociceptive neurons, including the central terminals in the spinal
cord, but it is thought to be largely absent from non-nociceptive
sensory neurons7. The central terminals of nociceptive neu-
rons are located in laminae I–II of the dorsal horn of the spi-
nal cord, whereas the terminals of non-nociceptive neurons are
located in laminae III–IV (ref. 7). If QAQ loading is selective for
nociceptors, it should photosensitize only the subset of sensory
neurons that terminate in laminae I–II.
We treated spinal cord slices with QAQ and recorded syn-
aptic responses in dorsal horn neurons triggered by electrical
stimulation of the dorsal root (Fig. 5a). In lamina II, the average
excitatory postsynaptic current (EPSC) amplitude was reduced
by switching from 380-nm to 500-nm light, whereas light had
no effect for EPSCs recorded in laminae III–IV (Fig. 5b,c).
These results are consistent with preferential photosensitization
of nociceptive neurons by QAQ (Fig. 5d).
To distinguish between a pre- versus postsynaptic effect of
QAQ, we recorded spontaneous EPSCs in lamina II neurons
and analyzed the cumulative distribution of amplitudes and inter-
event intervals. The amplitude of these EPSCs was unaffected by
the wavelength of light but the frequency of these EPSCs was
decreased by 500-nm light (Fig. 5e–g) in six of eight cells. A change
in EPSC amplitude indicates a postsynaptic alteration in neuro-
transmitter receptor function, whereas a change in frequency usu-
ally indicates a change in presynaptic neurotransmitter release.
QAQ-mediated photosensitization also impacted polysynaptic
pathways on the spinal cord. Trains of stimuli generated a strong
inward current that persisted for several seconds after the mono-
synaptic EPSCs should have decayed (Fig. 5h). Switching from
380-nm to 500-nm light caused a dramatic reduction in the
amplitude of this current. Switching back to 380-nm light largely
restored the initial amplitude of the response (Fig. 5i).
In some, but not all lamina II neurons, QAQ photosensitized
not only the presynaptic inputs but also intrinsic voltage-gated
channels K+ currents (Supplementary Fig. 10a). In contrast,
there was little photosensitization of K+ channels in lamina III–IV
neurons. Photosensitization of lamina II neurons was elimi-
nated by BCTC (Supplementary Fig. 10b). These results suggest
that TRPV1 channels enabling QAQ entry are present and
active in lamina II neurons to a much greater extent than in
lamina III–IV neurons.
In vivo photoregulation of peripheral nerve endings
If QAQ effectively photosensitizes nociceptive neurons, exposure
to light should alter pain sensation in vivo. We explored this possi-
bility by testing the pain-avoidance (nocifensive) blinking response
that is elicited by mechanical stimulation of the cornea in rats using
the von Frey hair test23 (Online Methods). The cornea is densely
innervated with nociceptors24 that mediate the blink response23.
Free nerve endings are only a few micrometers below the surface,
and the cornea is transparent, facilitating optical control. To enable
QAQ entry into nociceptor nerve endings, we topically applied
Figure 5 | Photosensitization of spinal cord
slices. (a) Schematic of a spinal cord slice with
a whole-cell patch recording from neurons
either in lamina II or laminae III–IV and using
electrical stimulation of the dorsal root (stim.).
(b,c) Postsynaptic responses recorded in a
lamina II (b) and a laminae III–IV neuron (c),
in response to single stimuli to the dorsal root.
(d) Percentage photoswitching of the integrated
current exhibited by inputs to laminae II
(35 ± 9%, n = 8 cells) and III–IV neurons
(−1.5 ± 9.4%, n = 10 cells, *P = 0.01).
(e,f) Cumulative probability distributions of
EPSC amplitude (e) and frequency (f) recorded
in a lamina II neuron in 380-nm and 500-nm
light. (g) Average light-elicited EPSC frequency
in 380-nm, 500-nm and again in 380-nm light
(37.5 ± 11.3% photoswitching, n = 8 cells).
(h) Polysynaptic responses recorded from a lamina II neuron in response to a 20 Hz train of stimuli to the dorsal root. (i) Quantification of average
responses to train stimulation (50 Hz for 500 µs) in 11 neurons in 380-nm, 500-nm and again in 380-nm light. Synaptic responses after the stimulus
train were integrated and normalized to the initial amplitude in 380-nm light (n = 11 cells). In all experiments described in this figure, slices were
treated with 1 mM QAQ for 20 min before recording. Error bars, ± s.e.m.; Student t-test.
frequency (% of control)
0 10 20 30 40 50
050 100 150 200
© 2012 Nature America, Inc. All rights reserved.
nAture methods | VOL.9 NO.4 | APRIL 2012 | 401
QAQ with capsaicin in one eye and capsaicin alone in the contra-
lateral eye. We immobilized the rats by mild sedation with xylazine
and ketamine at low doses that do not interfere with nocifensive
blinking25. The von Frey hair test in ambient light showed that
the normalized blink threshold was about fivefold higher in the
eye treated with QAQ plus capsaicin compared to the eye treated
with capsaicin alone (Fig. 6a). Moreover, 380-nm light decreased
the normalized blink threshold in the eye treated with QAQ
plus capsaicin (Fig. 6b). The decrease in blink sensitivity caused
by QAQ was completely removed by exposure to 380-nm light
(Fig. 6c). Taken together, these results show that QAQ can serve
as a local anesthetic that can be turned off with light.
Microbial light-sensitive ion transporters, including halorho-
dopsin26 and archaerhodopsin-3 (ref. 27), have been used as opto-
genetic inhibitors of neuronal activity. Genes encoding these proteins
can be promoter-targeted to subpopulations of neurons1. However,
for several reasons, nongenetic optical control of nociception with
QAQ may be preferable to optogenetic methods.
Unlike optogenetic tools that overpower the natural activity of
cells, QAQ acts on endogenous ion channels that underlie initia-
tion and propagation of action potentials. Hence QAQ suppresses
electrical excitability at its source. Because the ion-transport rate
of transporters is much slower than ion flux through channels,
optical silencing with halorhodopsin and archaerhodopsin-3
requires very high expression1. Exogenous expression can be
achieved by injecting viral vectors into the appropriate part of
the nervous system1, but expression requires days to weeks and
is restricted to neurons that are exposed to an adequate titer of
virus. Optogenetic expression can result in permanent genetic
alteration of neurons, which may not be necessary or desirable
for the acute regulation of pain signaling, either for scientific
or biomedical applications. In contrast, QAQ-mediated photo-
sensitization occurs within minutes and persists only until the
molecule dissipates, either by being metabolized inside the cell
or diffusing away from targeted neurons. Because QAQ is a small
molecule, it diffuses readily through tissue and presumably gains
access to all neurons that have ion channels that permit its entry
into the cytoplasm.
QAQ has potential value as both a scientific and a clinical tool
for controlling nociception. Because it selectively accumulates in
nociceptors, QAQ could selectively inhibit pain signaling while
sparing other sensory modalities and therefore could function
as a targeted analgesic. This is similar to the recently proposed
therapeutic use of QX-314 (ref. 10). However, QAQ has the added
feature of being rapidly controllable with light. In vivo photo-
control would require delivery of sufficient QAQ and projec-
tion of sufficient light onto target neuronal tissues. Because it is
doubly charged, QAQ is unlikely to cross the blood-brain bar-
rier. But our results show that QAQ penetrates into spinal cord
slices and intact DRGs, so if injected, QAQ should have access
to other neural structures. Implanted fiber-optic systems such as
those developed for deep brain photocontrol of neurons express-
ing optogenetic tools28 could be adapted for controlling QAQ
administered to internal neural structures (for example, spinal
roots or DRGs). QAQ might also be controlled by an external
light source after topical administration (for example, for treat-
ing corneal pain).
QAQ-mediated photosensitization could facilitate mapping of
nociceptive circuit connections mediated by fast conventional
synapses or by slow neuromodulatory neurotransmitters that may
contribute to central sensitization and pain hypersensitivity20.
Our synaptic studies in spinal cord were limited to full-field
regulation of presynaptic activity, but higher-resolution photo-
control should be possible by projecting through a microscope
small spots or patterns of light, for example, to target presynaptic
axons or terminals.
Finally QAQ provides insight into the activity status of ion
channels implicated in pain and inflammation. Previously, the
activity of nociceptive ion channels has been studied almost
exclusively in isolated neurons that had been enzymatically and
mechanically dissociated from DRGs21. This disruptive procedure
could alter the activity and expression of these channels29. QAQ
enabled investigation of nociceptive ion-channel activity in
undisrupted neural structures. Moreover, QAQ photosensitized
regions of a nociceptor that are largely inaccessible to electrodes.
Hyperalgesia in both inflammatory and neuropathic pain is
associated with upregulation of TRP channel gene expression in
peripheral nociceptors7, but the activation status of these channels
in chronic pain is unknown. Because QAQ-mediated photosen-
sitivity is a consequence of the cumulative activity of nociceptive
channels, it serves as an ultrasensitive reporter that provides new
insights about when and where these channels are active, in both
physiological and pathological conditions.
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturemethods/.
Note: Supplementary information is available on the Nature Methods website.
We thank M.R. Banghart and M. Kienzler for helping synthesize QAQ, S. Scott
for his help with computer programming, D. Bautista for helpful comments,
A. Nicke (Max Planck Institute of Brain Research) for the P2X7 receptor clone,
A. Blatz (Photoswitch Biosciences, Inc.) for HEK-293 cells stably expressing
Cav2.2 and F. Ory for artistic input. This work was supported by US National
Institutes of Health grants MH088484 to R.H.K. and PN2 EY018241 to R.H.K.
and D.T. (the University of California Berkeley Nanomedicine Development
Center) and by the Center for Integrated Protein Science, Munich to D.T.
Threshold for eye blink (g)
Before Dark 380 nm
Change in threshold
Change in threshold
Figure 6 | QAQ optically regulated nociception in vivo, in live rats.
(a) Change in blink threshold in the eye treated with capsaicin (10 µM) plus
QAQ (20 mM), normalized to the contralateral eye treated with capsaicin
alone. Experiments were performed in ambient light. Average increase was
5.1-fold (black dot; n = 22 rats, P = 0.007). (b) Change in blink threshold in
the eye treated with capsaicin (10 µM) plus QAQ (20 mM) after illumination
with 380-nm light. Average decrease was to 0.5 the value in the dark
(black dot; n = 9 rats, P = 0.03). (c) Group data for eye-blink threshold
experiments. Numbers in parentheses indicate the total number of rats
together with number of rats that did not respond to the maximum force
applied (1 g). Error bars, ± s.e.m.; Student t-test; *P < 0.05; **P < 0.01.
© 2012 Nature America, Inc. All rights reserved.
402 | VOL.9 NO.4 | APRIL 2012 | nAture methods
A.M. and R.H.K. wrote the paper. A.M., T.F., Y.L.F., C.M.S., F.N., D.T. and.
R.H.K. designed experiments. AM., T.F., Y.L.F., C.M.S. and C.H. performed
electrophysiological experiments and analyzed data. A.M. and D.D. performed
in vivo experiments.
comPetinG FinAnciAl interests
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemethods/.
reprints and permissions information is available online at http://www.nature.
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General. All animals were housed in the centralized animal
facilities as assigned by the University of California Berkeley
and were provided food and water ad libitum. Animal care
and experimental protocols were approved by the University
of California Berkeley Animal Care and Use Committee.
All chemicals were purchased from Sigma-Aldrich, except
tetrahydro-pyrazine1(2H)-carboxamide) and AITC (allyl isothio-
cyanate) that were purchased from Tocris.
QAQ synthesis and spectroscopic characterization. QAQ was
synthetized as previously described5. UV-vis spectra of QAQ were
measured using a smartSpec Plus spectrophotometer (Bio-Rad)
in combination with illumination using the Polychrome V (Till
Photonics), through an optic fiber positioned perpendicular to
the detection beam of the spectrophotometer.
Cell culture. HEK-293 cells were cultured under standard con-
ditions (Dulbecco’s modified Eagle medium (DMEM) contain-
ing 10% FBS). We grew GH3 cells in F-12K medium containing
15% horse serum and 2.5% FBS. NG108-15 cell medium con-
tained 95% DMEM mixed with HAT (0.1 mM hypoxanthine,
400 nM aminopterin and 0.016 mM thymidine) and 5% FBS.
Cells were plated on poly(l-lysine) (0.1 mg/ml) treated cover-
lips in a density of 12,000 cells per cm2 for electrophysiological
measurements. Dissociated hippocampal neuronal preparations
were performed from neonatal Sprague-Dawley rats according
to standard procedures4. Hippocampi were dissected, dissoci-
ated and cells were plated on poly(l-lysine)-coated coverslips
at a density of 100,000/cm2. We grew hippocampal neurons
in minimum essential medium containing 5% FBS, 20 mM
glucose, B27 (Invitrogen), glutamine and Mito+ Serum Extender
(BD Biosciences). Trigeminal ganglion (TG) neurons from neo-
natal rats were prepared as previously described30. TGs were
dissected and neurons were dissociated (with collagenase and
trypsin) and plated on poly(l-lysine)-coated coverslips. We
grew TG neurons in minimum essential medium containing
5% horse serum, MEM vitamins (Invitrogen), glutamine and
penicillin-streptomycin. HEK-293 cells were transfected using
calcium phosphate precipitation and measured after 24–48 h
(ref. 4). GH3 and NG108-15 cells were recorded 24 h after plating.
Hippocampal neurons were transfected 7 d after plating and
measured 10–14 d after plating. TG cells were measured 12–48 h
Dorsal root ganglia (DRG) preparation. Mice, C57/BL6 wild
type or Trpv1−/− aged 1–6 months of either sex, were deeply
anesthetized with isoflurane and killed by cervical dislocation.
The spinal column and surrounding muscle, from the sacral to
cervical regions, was removed from the mouse and dissected in
cold (4 °C) artificial cerebral spinal fluid (ACSF) (124 mM NaCl,
4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 26 mM NaHCO3, 20 mM
glucose, 2 mM sodium pyruvate and 0.4 mM ascorbic acid,
pH 7.3) equilibrated with 95% O2 and 5% CO2. A laminectomy
was performed from the thorax to the sacrum, and the spinal
cord was gently removed, exposing the DRGs. The DRGs and
attached nerves (10–20 mm long) were removed from the lumbar
region and incubated for at least 30 min at room temperature
(18–22 °C) in an oxygenation chamber on a nitrocellulose
membrane (Sartorius Stedim Biotech) moistened with ACSF.
Spinal cord slice preparation. C57/BL6 mice were deeply anes-
thetized with isoflurane and quickly beheaded. The spinal column
and surrounding muscles were removed and dissected in ice-
cold oxygenated low calcium, low magnesium ACSF (101 mM
NaCl, 3.8 mM KCl, 18.7 mM MgCl2, 1.3 mM MgSO4, 1.2 mM
KH2PO4, 10 mM HEPES, 1 mM CaCl2 and 1 mM glucose). After
laminectomy, the spinal roots were cut, the spinal cord was gently
removed, and its lumbar part was placed into a small agarose
block. We prepared 300-µm-thick slices using a Leica VTS 1000
vibratome. The slices were then transferred in warm (31 °C)
ASCF equilibrated with 95% O2 and 5% CO2 for at least 1 h before
starting patch-clamp recordings.
Whole-cell electrophysiology. Patch clamp recordings of
mammalian cells were performed at room temperature. Bath
solution for K+ current contained 138 mM NaCl, 1.5 mM KCl,
1.2 mM MgCl2, 2.5 mM CaCl2, 1 µM tetrodotoxin (for hippo-
campal neurons only), 5 mM HEPES and 10 mM glucose. Bath
solution for Na+ current contained NaCl 145 mM, CdCl2 0.5 mM,
CaCl2 2 mM, HEPES 5 mM and glucose 5 mM. Bath solution
for Ca2+ current contained NaCl 138 mM, KCl 5.4 mM, MgCl2
0.8 mM, BaCl2 20 mM, tetrodotoxin 1 µM (for GH3 cells only),
HEPES 10 mM and glucose 5 mM. Bath solution for current
clamp experiments contained NaCl 138 mM, KCl 1.5 mM, MgCl2
1.2 mM, CaCl2 2.5 mM, HEPES 5 mM and glucose 10 mM. Pipette
solution for K+ current contained NaCl 10 mM, K+ gluconate
135 mM, HEPES 10 mM, MgCl2 2 mM, MgATP 2 mM, EGTA
1 mM. Pipette solution for Na+ current contained NaCl 30 mM,
CsCl 100 mM, HEPES 10 mM, MgCl2 2 mM, CaCl2 1 mM, MgATP
2 mM, NaGTP 0.05 mM, EGTA 10 mM and glucose 5 mM. Pipette
solution for Ca2+ current contained CsCl 120 mM, HEPES 20 mM,
CaCl2 1 mM, MgATP 2 mM, EGTA 11 mM and glucose 5 mM.
Pipette solution for current clamp experiments contained NaCl
38 mM, potassium gluconate 97 mM, HEPES 20 mM, MgATP
4 mM, NaGTP 0.35 mM and EGTA 0.35 mM. All solutions were
adjusted to pH 7.4. Electrophysiological measurements were
performed with an Axopatch 200A (Molecular Devices) or a
Patch-Clamp PC505B (Warner) amplifier. Patch pipettes resist-
ances were 2–4 MΩ. Sodium channel currents in NG108-15 cells
and calcium channel currents in GH3 cells were corrected by P/N
leak subtraction. pClampex 8.2 software (Molecular Devices) in
combination with a Digidata 1200 interface (Molecular Devices)
were used to create and apply pulse protocols. Voltage clamp
recordings were low-pass–filtered at 2 kHz and current clamp
measurements were low-pass filtered at 5 kHz. Illumination of
cells was based on a xenon lamp either in combination with nar-
row band-pass filters or with a monochromator Polychrome V,
as described previously5. For direct internal application through
the patch pipette, QAQ was dissolved to a final concentration
of 100 µM. Measurements were started after 5–10 min of equi-
libration time for HEK-293, NG108-15, GH3 cells and TG
neurons, and after 15–20 min for hippocampal neurons. For
bath incubation, cells were incubated with QAQ (classically
1 mM) in the presence or absence of agonist (1–2.5 mM ATP
or 1 µM capsaicin) at 37 °C in the dark. Loading solution is
similar to K+ current recording solution but with no calcium.
© 2012 Nature America, Inc. All rights reserved.
doi:10.1038/nmeth.1897 Download full-text
Treated coverslips were rinsed with regular calcium-containing
recording solution before measurement.
For spinal slice electrophysiology, slices were placed in a recod-
ing chamber bathed with warmed (31 °C) ACSF (NaCl 130.5 mM;
KCl 2.4 mM, CaCl2 2.4 mM, NaHCO3 19.5 mM, MgSO4 1.3 mM,
KH2PO4 1.2 mM, HEPES 1.25 mM and glucose 10 mM, pH 7.4)
equilibrated with 95% O2 and 5% CO2. Electrophysiological mea-
surements were performed under the control of an Olympus BX51
microscope using an Axoclamp 2B (Molecular devices). Patch
pipettes (7–11 MΩ) were filled with appropriate pipette solution
(potassium gluconate 120 mM, KCl 20 mM, CaCl2 0.1 mM, MgCl2
1.3 mM, EGTA 1 mM, HEPES 10 mM, GTP 0.1 mM, cAMP
0.2 mM, leupeptin 0.1 mM, Na2ATP 3 mM and d-manitol 77
mM, pH 7.3). Illumination of preparations was performed using
two different wavelength diodes (380 nm and 500 nm) control-
led by transistor-transistor logic (TTL) pulses. A glass suction
electrode connected to Master-8 (A.M.P.I.) stimulator was used
to stimulate dorsal roots. Non-nociceptive primary afferent fibers
were specifically recruited using low-threshold stimulations
(50 µs, less than 100 µA), whereas nociceptive fibers were recruited
using high-intensity stimulations (500 µs, more than 250 µA).
Multielectrode array recordings. A DRG was placed onto a
three-dimensional multielectrode array (MEA) chip (MEA60
200 3D GND, Ayanda Biosystems) and secured in place with a
‘harp’ made from dialysis membrane stretched over thick plati-
num wire and bonded with super glue; the wire was U-shaped
to allow the nerve to exit without being crushed. The MEA chip
was mounted on an MEA1060-Up amplifier (Multi Channel
Systems) and placed on the stage of an IX71 inverted microscope
(Olympus). The nerve was led into a manipulator-mounted glass
suction electrode of appropriate size driven by a DS2 stimulus
isolator (Digitimer) triggered by pClamp v10.0 software through
a Digidata 1440A data acquisition system (Molecular Devices).
Except during drug incubations, the MEA chamber was continu-
ously perfused with oxygenated ACSF at ~2 ml/min. Recordings
were performed at ~30 °C.
Before the drug incubation, the DRG was checked for response
to stimulation at 1 Hz. If the signal was acceptable, the MEA
chamber solution was replaced with oxygenated ACSF containing
QAQ with or without other drugs and incubated for 5 min. When
using blockers, the DRG was preincubated with the blocker
for 5 min before the application of QAQ with the blocker. The
DRG was then washed with ACSF for 10 min before performing
Recordings were done at a stimulation rate of 10 Hz while illu-
minating the DRG with 380-nm or 500-nm light. Each experiment
consisted of 5 cycles of 30 s under 380-nm light followed by 30 s
under 500-nm light. The DRG was stimulated with 1-ms pulses
at 10 Hz for the last 5 s under each wavelength of light, allowing
25 s to recover from adaptation in between stimulation episodes.
Illumination was provided by a U-LH100HGAPO mercury lamp
(Olympus) through a 4× objective, resulting in intensities of
17–28 mW/mm2. Filters for 380 nm and 500 nm were switched
by a Lambda 10-3 system (Sutter Instrument Company) under
the control of Metamorph v18.104.22.168 software (Molecular Devices).
Evoked responses were recorded at 20 kHz with MC_Rack
v4.0 software (Multi Channel Systems). Pictures were taken
using Metamorph with a CoolSNAP HQ2 camera (Photometric)
connected to the microscope.
Multielectrode array data analysis. Data were recorded in 40-ms-
long sweeps synced to stimulation pulses, so that the stimulation
produced an artifact at the beginning of the sweeps. Evoked spikes
were detected by a negative threshold manually set beyond the
noise level. For each detected spike, the first millisecond before
the peak and the two milliseconds after were extracted into
text files by MC_DataTool software (Multi Channel Systems),
for processing with a custom Matlab (MathWorks) program.
Our custom Matlab program calculated the area under each
spike to the threshold level. A region of interest (ROI) was also set
manually for each recording to exclude the stimulus artifact. The
total integrated area of all spikes was calculated for each sweep,
and averaged over the five cycles in each wavelength (Fig. 4e,f).
This averaged area per sweep was summated over the 5 s of stimu-
lation to quantify the total evoked response in each wavelength of
light. For each active channel, the normalized photosensitization
was calculated as (area380nm − area500nm) / (area380nm + area500nm).
Channels with excessively small and/or irregular signals were
conservatively culled. The per-channel photosensitization values,
generated from at least three separate DRGs per drug condition,
were pooled by condition and compared for significance using a
Mann-Whitney U test (5% significance level).
Cornea-evoked reflex blinks. Sprague-Dawley rats (3–6 weeks
old of either sex) were sedated using intraperitoneal injection
of xylazine (9 mg/kg) and ketamine (60 mg/kg). We placed rats
on a warming pad, and we intiated behavioral testing when rat
spontaneous movements ceased but while pinching the rat’s paw
with a pair of forceps elicited a brisk withdrawal reflex. We used a
series of von Frey hairs, nylon fibers of increasing diameter, which
we pressed against the cornea to impart increasing force with high
accuracy. We held von Frey hairs perpendicular to the cornea for
~2 s, or until a blink initiated, using progressive increase in force
from 8 mg to a maximal value of 1 g. Stimuli were presented three
times for each stiffness, at intervals of several seconds. Both eyes
were tested, and a positive response was noted if the rat blinked
two or three times for a given force. Capsaicin (10 µM) was then
topically applied on one cornea using a pipette (10 µl volume), and
the contralateral cornea was treated with a mixture of capsaicin
(10 µM) and QAQ (20 mM). Von Frey testing was done again
10–15 min after drug application. Immediately after von Frey test-
ing, light was applied using an LED (Prizmatix, λmax = 385 nm,
30 mW/cm2) for 1 min and von Frey testing was done again.
Statistical analysis. Unless otherwise noted, all data are presented
as ± s.e.m. and statistics were analyzed using a Student t-test.
30. McKemy, D.D., Neuhausser, W.M. & Julius, D. Identification of a cold
receptor reveals a general role for TRP channels in thermosensation.
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© 2012 Nature America, Inc. All rights reserved.