Synthesis and biological evaluation of bis-CNB-GABA, a photoactivatable neurotransmitter with low receptor interference and chemical two-photon uncaging properties

Article abstract of DOI:10.1021/ja411082f

Photoactivatable "caged" neurotransmitters allow optical control of neural tissue with high spatial and temporal precision. However, the development of caged versions of the chief vertebrate inhibitory neurotransmitter, γ-amino butyric acid (GABA), has be

Full text of DOI:10.1021/ja411082f

Article
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Open Access on 01/14/2015
Synthesis and Biological Evaluation of Bis-CNB-GABA, a
Photoactivatable Neurotransmitter with Low Receptor Interference
and Chemical Two-Photon Uncaging Properties
Diana D. Shi,†,‡,§ Federico F. Trigo,∥ Martin F. Semmelhack,‡ and Samuel S.-H. Wang*,§
‡
§
†Department of Psychology, Department of Chemistry, Department of Molecular Biology and Neuroscience Institute, Princeton
University, Princeton, New Jersey 08544, United States
∥Laboratoire de Physiologie Cer
́ ́ ́
ebrale, Centre National de la Recherche Scientifique et Universite Paris Descartes, 75006 Paris,
France
S
* Supporting Information
ABSTRACT: Photoactivatable “caged” neurotransmitters allow
optical control of neural tissue with high spatial and temporal
precision. However, the development of caged versions of the
chief vertebrate inhibitory neurotransmitter, γ-amino butyric acid
(GABA), has been limited by the propensity of caged GABAs to
interact with GABA receptors. We describe herein the synthesis
and application of a practically useful doubly caged GABA analog, termed bis-α-carboxy-2-nitrobenzyl-GABA (bis-CNB-GABA).
Uncaging of bis-CNB-GABA evokes inward GABAergic currents in cerebellar molecular layer interneurons with rise times of 2
ms, comparable to flash duration. Response amplitudes depend on the square of flash intensity, as expected for a chemical two-
photon uncaging effect. Importantly, prior to uncaging, bis-CNB-GABA is inactive at the GABAA receptor, evoking no changes in
holding current in voltage-clamped neurons and showing an IC50 of at least 2.5 mM as measured using spontaneous GABAergic
synaptic currents. Bis-CNB-GABA is stable in solution, with an estimated half-life of 98 days in the light. We expect that bis-
CNB-GABA will prove to be an effective tool for high-resolution chemical control of brain circuits.
upstream from intracellular voltage and second messenger
signaling, caged neurotransmitters allow for a remarkable
degree of specificity in chemical modulation of neural activity.
The use of caged neurotransmitters offers important advantages
over other established methods. Notably, it is possible to use
patterned photostimulation techniques to achieve stimulation
at many arbitrary locations in parallel;1,2 microelectrode-based
methods are not amenable to this type of task. Moreover,
neurotransmitter uncaging offers a useful alternative to
optogenetic approaches3 because uncaging does not require
gene delivery, is neurotransmitter-specific, and uses different
wavelengths of light than those employed in optogenetics.
GABA (γ-amino butyric acid) is the chief vertebrate
inhibitory neurotransmitter and is therefore an important
target for caging. The ideal caged GABA neurotransmitter
should exhibit a number of properties, including: (1) inertness
at the receptor; (2) high combined extinction coefficient and
quantum yield; (3) ability to undergo rapid cleavage to unveil
the active neurotransmitter with few side products; and (4)
excellent chemical stability in aqueous solution. To date, a
number of caged GABA-based compounds have been
developed that satisfy many of these criteria: α-carboxy-2-
nitrobenzyl (CNB)-, 4-carboxymethoxy-5,7-dinitroindolinyl
(CDNI)-, 1,3-bis(dihydroxyphosphoryloxy)propan-2-yloxy]-7-
INTRODUCTION
■
Over the past two decades, caged neurotransmitters have
emerged as a useful tool for the high-resolution, electrode-free
chemical stimulation of single neurons or neural circuits. These
probe compounds are prepared via covalent appendage of a
light-sensitive protecting groupthe cageto a signaling
molecule. With the cage in place, the signaling molecule is
unable to activate its receptor. Upon delivery of a pulse of light,
the cage is rapidly cleaved to reveal the active signaling
molecule (Figure 1, top). When introduced into sliced or intact
living brain tissue, caged neurotransmitters may activate
neurotransmitter pathways at defined locations with micro-
meter and millisecond precision. Because they act one level
Figure 1. Top, conventional uncaging scheme for GABA. Bottom,
chemical two-photon uncaging of double-caged GABA.
Received: October 30, 2013
Published: January 14, 2014
© 2014 American Chemical Society
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nitroindoline (DPNI)-, 4-methoxy-5,7-dinitroindolinyl
(MDNI)-, and ruthenium-bipyridine-triphenylphosphine-
(RuBi-GABA) have high combined extinction coefficient and
quantum yield4,5 and cleave rapidly to generate neuro-
transmitters with few side products. All of these caged GABA
compounds are chemically stable in aqueous solution on time
scales of weeks or longer.6−8 However, in their caged form, they
are not inactive. CNB-, CDNI-, DPNI-, and MDNI-caged
GABA compounds are antagonists of GABAA receptors,6−8 as
are RuBi-GABA,9 the related compound RuBi-glutamate,10 and
4-methoxy-7-nitroindolinyl (MNI)-glutamate.8 The practical
limit at which these compounds can be used without interfering
with neural circuit function is so low (<200 μM) that they
cannot be used to attain the near-millimolar concentrations that
occur locally during synaptic transmission.11,12 Presumably,
these caged GABA compounds present residual receptor-
binding epitopes such as amine7,8,13 or carboxylate.10,14 In
specific cases such as RuBi, a phosphine moiety is also
suspected to cause antagonistic effects.9
We sought to address the problem of receptor antagonism by
adopting a “double-caging” strategy (Figure 1, bottom).
Incorporation of two cages at different positions on a
neurotransmitter has been shown to offer several advan-
tages.15,16 First, release of the caged substrate is proportional to
the square of the flash energy, creating a nonlinear effect
resembling two-photon excitation17 and therefore improving
spatial resolution. Moreover, a transmitter molecule modified at
two locations is less likely to interact with its receptor than a
single-caged analog, due to its reduced structural resemblance
to the original transmitter.16 We envisioned modifying GABA
at both the acid and amine positions with CNB, a readily
synthesized cage with good water solubility.13 Caging at the N
position would be achieved via direct modification of GABA,14
rather than through the use of a carbamate linker, which leads
to slow uncaging kinetics.18 We describe herein the synthesis
and evaluation of a doubly caged GABA analog, bis-CNB-
GABA, 4. This compound is anticipated to emerge as a
powerful tool for high-resolution control of brain circuits.
this synthetic route is comparable in ease to that of a single-
caged GABA. Thus, bis-CNB-GABA is readily accessible from
commercially available materials.
Bis-CNB-GABA exhibits maximal absorbance at 262 nm (ε =
7550 M−1 cm−1). Using mono-O-CNB-GABA as a reference
standard to quantify conversion, the quantum yield (using 254
nm light) was 0.15 at the O-position and 0.032 at the N-
position; these values are the same as the quantum yields
determined at 308 nm excitation for the corresponding mono-
CNB-GABA compounds.13,14 Finally, bis-CNB-GABA is
soluble in pH 7.0 phosphate buffer solution up to 17 mM, at
levels comparable to bis-CNB-glutamate.15
Effects of Uncaging to Evoke Currents in Molecular
Layer Interneurons. The biological properties of our
synthetic bis-CNB-GABA were evaluated using whole-cell
patch recording from cerebellar molecular layer interneurons.
Molecular layer interneurons receive excitatory glutamatergic
synapses as well as inhibitory GABAergic synapses that show a
pronounced level of spontaneous activity (gray traces in Figure
2a). The effects of bis-CNB-GABA on these synaptic currents
were tested. In the presence of bis-CNB-GABA, photolysis,
using either a UV high-intensity LED focused to the back focal
plane of the objective or a minimized 405 nm laser spot,
produced currents (Figure 2b−d) that were reversibly
eliminated in the presence of 3 μM of the GABAA receptor
antagonist, gabazine (Figure 2e).
As anticipated,15 the current amplitude evoked by photolysis
of bis-CNB-GABA was related to laser flash energy by a square
relationship (Figure 2d; log−log slope = 2.2 0.1, n = 3 cell
bodies). By contrast, the relationship for mono-O-CNB-GABA
was close to linear (log−log slope = 1.3 0.1, n = 3). For these
measurements, evoked currents were normalized to the
maximum current observed in the same neuron. Moreover,
the integrated evoked current over time was proportional to the
square of the laser energy. These power laws are consistent
with a process in which each molecule of bis-CNB-GABA must
cumulatively undergo two uncaging reactions in order to
release an active GABAA agonist. This result is predicted by
localized, nonlinear release of GABA at the laser’s focus spot
and is in line with the localized release and spatial resolution
seen in previous applications of chemical two-photon
uncaging.15,16
RESULTS
■
Bis-CNB-GABA: Synthesis and Physical Properties. The
synthesis of bis-CNB-GABA is outlined in Scheme 1.
Laser-Evoked GABA Responses. To test whether bis-
CNB-GABA-evoked responses resemble physiological events in
their kinetics, we measured the kinetic properties of flash-
evoked responses. For responses comparable in size with
spontaneous inhibitory postsynaptic currents (IPSCs), the 10−
90% rise time was 2.2 0.6 ms (n = 10); somewhat longer than
the flash duration of 1.0 ms. This rise time is consistent with the
dark-reaction time (1.5 ms) for the slower cage, N-mono-CNB-
GABA.14 The falling t1/2 was 24.2 8.2 ms (n = 10). These
rates approach those of spontaneous events and are among the
fastest described for other GABA cages. In some recordings
(for example, Figure 2a responses at sites 1 and 3), the kinetics
of laser-evoked events were nearly indistinguishable from those
of spontaneous IPSCs, perhaps because of short electrotonic
distances between the uncaging site and the recording
electrode. In summary, photouncaging of bis-CNB-GABA was
sufficiently rapid to mimic synaptic events.
Scheme 1. Synthesis of Bis-CNB-GABA
Nitrophenylacetic acid (1) was converted to its t-butyl ester,
then brominated with NBS and AIBN to generate benzyl
bromide 2. The latter was used to alkylate GABA
simultaneously at both the amine and the acid positions (3);
a final deprotection generated bis-CNB-GABA (4) as a tan
Effects of Bis-CNB-GABA on Endogenous Synaptic
Communication. In order to evaluate the viability of bis-
CNB-GABA as a probe compound, we made three measures of
the potential undesirable effects of both mono- and bis-caged
1
powder. This material was shown by H NMR analysis to be
>99% pure and to contain <0.2% residual GABA. A portion of
the product was further purified by preparative HPLC. Notably,
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spontaneous currents. No difference in induced holding current
was seen between raw product and HPLC-purified product.
We next measured the effects of bis-CNB-GABA on
spontaneous IPSCs (Figure 3b). At 1 mM, mono-O-CNB-
GABA (IC50 = 28 μM; ref 6) triggered a dramatic decrease in
the rate of spontaneous IPSCs (not shown), presumably due to
inhibition of GABAA receptors in the recorded neuron and/or
of activity in presynaptically connected MLIs. In contrast,
addition of bis-CNB-GABA served to reduce GABA current
amplitudes by approximately one-third (Figure 3b, top; 0.4−2.0
mM; ratio of amplitudes during drug/control: 0.68 0.17, n =
14). The concentration dependence of the reduction yielded an
estimated IC50 of 2.5 mM (Figure 3c). This IC50 is, in fact, a
lower bound; the material tested was crude product, which may
contain minute amounts of mono-CNB-GABA. Bis-CNB-
GABA therefore exhibits at least 100-fold lower affinity
compared to its mono-caged analog. Moreover, the 10−90%
rise time of IPSCs was unaffected by bis-CNB-GABA
application (control, 0.44 0.13 ms, vs bis-CNB-GABA, 0.44
0.11 ms), in contrast with DPNI-GABA, which prolongs rise
times.7 Taken together, these findings are consistent with the
hypothesis that bis-CNB-GABA shows virtually no antagonist
activity at submillimolar concentrations.
As a third and final test of the synaptic effects of bis-CNB-
GABA, we measured its impact on spontaneous glutamatergic
postsynaptic currents (EPSCs) recorded from MLIs. With this
configuration, no change in the amplitude of the EPSCs was
observed (Figure 3b, bottom) even with concentrations of bis-
CNB-GABA up to 2 mM (Figure 3e, p = 0.4, Spearman rank
order correlation test). In summary, at concentrations of 1 mM,
our doubly caged bis-CNB-GABA was found to have minimal
or no effects on holding current, IPSCs, or EPSCs.
Spontaneous Rate of Hydrolysis of Bis-CNB-GABA vs
Mono-O-CNB-GABA. We next sought to study the hydrolytic
stability of bis-CNB-GABA under normal handling conditions.
Accordingly, we prepared samples of bis-CNB-GABA and
mono-O-CNB-GABA in aqueous buffer (23 °C, 12 mM, pH =
7.4), stored the samples under fluorescent room lights, and
determined accumulation of deprotected GABA at 1 day
intervals.
The mechanism of photodecaging of the CNB group is an
active area of study; several pathways are available to caged
amines and caged carboxylate derivatives (for discussion see ref
21). The dominant final photoproduct of bis-CNB-GABA was
GABA, as identified using 1H NMR (Figure 4a). The combined
amount of monodecaging (N-CNB-GABA) and double-
decaging (GABA) photoproduct was quantified using the
integrated multiplet at 2.15 ppm, with dimethoxyethane
(singlet peaks at 3.20 and 3.45 ppm) as a standard. Over the
first 8 days, we found that bis-CNB-GABA produced
photoproduct spontaneously at an approximately linear rate
(Figure 4b), 1.5 0.1%/day (n = 4 runs), for an extrapolated
half-life of t1/2 = 98 days (95% CI, 92 to 105 days). Mono-O-
CNB-GABA produced GABA at a similar rate of 1.0 0.2%/
day (n = 2 runs) or t1/2 = 138 days (95% CI, 116−169 days),
which is consistent with a prior report of <1% conversion in the
dark at 24 h,13 but not consistent with another report.8
Figure 2. Physiological responses to photolysis of bis-CNB-GABA. (a)
A cerebellar molecular layer interneuron visualized using Alexa 488 in
the patch recording electrode solution. Bis-CNB-GABA (0.6 mM) was
photolyzed with a 405 nm laser spot in 3 different locations (indicated
by 1−3). Laser-evoked GABAergic currents are shown on the right
panel. Gray traces show individual sweeps. Black traces are averages.
The gray sweep at bottom indicates the laser flash (1 ms duration,
intensity 5 mW) as recorded using a photodiode. (b) Bis-CNB-GABA
(1.4 mM) was photolyzed with a 365 nm LED at progressively higher
flash energies (0.25−1.1 mW, 5−50 ms, 1.25−55 μJ). (c) Same
experiment as (b) with mono-CNB-GABA (50 μM). (d) Normalized
current as a function of relative LED flash energies plotted on a log−
log scale. (e) Laser-evoked whole-cell current recorded in the absence
and presence of 3 μM gabazine, a GABAA receptor antagonist. Note
that a larger flash energy was used in the presence of gabazine.
GABA analogs. First, we monitored changes in whole-cell
holding current under voltage clamp, while caged GABA
compounds were applied by bath application or by local
perfusion (Figure 3).19 Under these conditions, mono-O-CNB-
GABA (0.1 mM) led to increases of 66 47 pA (mean SD, n
= 6) in inward holding current at −60 mV (Ihold; Figure 3a,d).
This inward current presumably arises either via indirectly
evoked increases in excitation20 or from direct activation of
GABAA receptors (by residual free GABA in the caged
compound solution or by mono-O-CNB-GABA, which may
itself have partial agonist activity). Similar, though less
pronounced, results have been observed with DPNI-GABA
(see Figure 3a and ref 7). In contrast, application of bis-CNB-
GABA (raw product, 0.4−2.0 mM) evoked no detectable
change in the holding current (Figure 3d; ratio of holding
DISCUSSION
■
current drug/control = 1.0
0.3, n = 14, p = 0.7, Mann−
As described above, we have identified a novel, double-caged
bis-CNB-GABA that is highly resistant to pre-uncaging
interactions with GABAA receptors. Importantly, of a wide
range of structurally diverse caged GABA analogs, bis-CNB-
Whitney test) or in its standard deviation, which is a measure of
steady-state channel noise (Figure 3d, from 3.0 0.5 to 3.2
0.8 pA, n = 14, p = 0.3) measured during periods of no
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Figure 3. Quantification of unwanted effects of caged GABA. (a) Voltage clamp recordings from cerebellar interneurons exposed to caged GABA.
Upper trace, 1 mM bis-CNB-GABA; middle trace, 0.1 mM mono-O-CNB-GABA; bottom trace, 1 mM DPNI-GABA. Right, expanded traces
illustrating the detailed effects on steady-state holding current and fluctuations in holding current. (b) Effects of caged GABA on spontaneous IPSCs
and excitatory postsynaptic currents (EPSCs). IPSCs and EPSCs were identified and separated based on kinetic criteria. Left, box plots of
spontaneous postsynaptic current amplitudes in control conditions and in the presence of 1 mM bis-CNB-GABA. Boxes show interquartile range
and whiskers show full range of values. Right, individual traces (gray) and average (block) of detected spontaneous IPSCs and EPSCs. (c)
Dependence of spontaneous IPSC amplitude on bis-CNB-GABA concentration. The curve indicates a fit with KD = 2.5 0.2 mM, nH = 0.93 0.09.
(d) Comparison of effects of mono-O-CNB-GABA (0.1 mM) and bis-CNB-GABA (1.0 mM, except for 0.4 mM for IPSCs) on spontaneous IPSC
amplitude, standard deviation of holding current (noise), and holding current (Ihold). (e) Spontaneous EPSC size was unaffected by bis-CNB-GABA
at all concentrations tested.
Figure 4. Accumulation of GABA in ambient room light. (a) 1H NMR data used to quantify GABA accumulation. Top, bis-CNB-GABA before light
exposure. Arrows denote methylene peaks of bis-CNB-GABA. Peaks at ∼3.45 and ∼3.2 ppm in each spectrum correspond to dimethoxyethane, used
as an internal standard. Bottom, the teal overlay indicates the postphotolysis spectrum of bis-CNB-GABA after 17 days of exposure to ambient
fluorescent light. Arrows denote the two visible peaks corresponding to a combination of GABA and mono-N-CNB-GABA photoproducts. Note the
lack of extraneous peaks in the 0−4 ppm range after light exposure. (b) In aqueous solutions in the light, accumulation of photoproducts from bis-
CNB-GABA (black) and GABA from mono-CNB-GABA (gray). Error bars indicate SD.
GABA exhibits the highest half-maximal concentration (IC50)
of GABAA antagonistic activity (Table 1).
Table 1. Comparative properties of caged GABA and
glutamate compounds in blocking synaptic GABAA currents
It has been suggested that some cage groups may themselves
antagonize GABAA receptors;9 if this were the case, then a
double-caged-GABA analog might be expected to show an
increased ability to block GABAA receptors. Our findings
demonstrate the opposite and support the view that when CNB
is used as the cage group, an exposed carboxyl or amine is a key
factor in residual receptor interaction.
Although it is useful to compare the relative inertness of bis-
CNB-GABA as a receptor antagonist with caged compounds
already in use, such as DPNI-GABA or CDNI-GABA, a more
appropriate comparison from a structure−function standpoint
is with the mono-O-CNB-GABA analog. Such a comparison
clearly reveals the advantages of adding a second cage of similar
caged compound
IC50
stability
a
mono-CNB-GABA
28 μM
0.1−0.3 mM
0.5 mM
t1/2 = 138 days
stable in dark
stable in dark
stable in dark
b
RuBi-glutamate, GABA
c
DPNI-GABA
d
CDNI-GABA
0.6 mM
e
bis-CNB-GABA
≥2.5 mM
t1/2 = 98 days
a
b
O-CNB-GABA in hippocampal dentate neurons (ref 6). 0.3 mM for
RuBi-glutamate (ref 10). Assumes possible RuBi-GABA effect, which
has only been tested at 20 μM (ref 9). Cerebellar interneurons (ref
7). Interpolated from Figure S2 in ref 8. This work. t1/2 was
c
d
e
measured under room light.
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structure to the first. In this context, we have shown that adding
a second cage to mono-CNB-GABA dramatically reduces
receptor antagonism, by a factor of 100. We predict that other
forms of N,O-bis-caged GABA compounds would exhibit
comparable reduction of antagonism compared to their O-
caged analogs. Because DPNI-GABA and CDNI-GABA
incorporate carboxyl-modifying groups, preparation of bis-
caged analogs of these compounds would require modification
of the N-position with CNB or another cage. Such a “hybrid”
caged GABA should similarly exhibit minimal receptor activity.
As described above, modification of GABA at the amino
position by direct attachment of CNB affords uncaging
activity at GABAA receptors, which limits the concentrations
that can be employed. We describe herein the synthesis and
evaluation of bis-CNB-GABA, the first caged GABA that takes
advantage of chemical two-photon uncaging, achieving non-
linear localized release of GABA and a significant decrease in
GABAA receptor antagonism prior to photolysis. Bis-CNB-
GABA is a powerful advanced optical probe that may be used
to study GABAergic inhibitory effects with a degree of
resolution that permits the probing of single-synapse
communication and neuronal integration.
ASSOCIATED CONTENT
■
responses consistent with a dark reaction time of 1.5 ms.14
A
S
* Supporting Information
previous approach had made use of a carbamate linker, which
generates neurotransmitter in ∼7 ms via a carbamate
intermediate,18 out of concern that direct attachment would
yield unwanted non-GABA side products. Our results
demonstrate that, in fact, direct attachment can lead to efficient
GABA production, as measured by NMR, and rapid photolysis
as measured by the time course of photolyzed currents.
Experimental procedures and spectral data are provided. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
Importantly, the high speed of uncaging obtained with bis-
CNB-GABA allows for a more highly focused chemical two-
photon effect and, accordingly, micrometer-to-submicrometer
localization in biological experiments. For a dark reaction
longer than ∼0.2 ms, spatial resolution of uncaging for a
diffraction-focused beam is limited by the distance that a caged
compound diffuses before it produces agonist. For bis-CNB-
GABA, dark reaction times of 28 μs13 and 1.5 ms and a
diffusion constant of D = 0.3 μm2/ms would predict a root-
mean-square spread of <x>1/2 = √(6·D·t)=0.2 and 1.6 μm,
respectively. As a beam passes through brain tissue and
becomes less focused due to scattering, diffraction- and
diffusion-based limits might not be reached.
sswang@princeton.edu
Present Address
†107 Avenue Louis Pasteur, Box 102, Boston, MA 02115.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH R01 NS045193. F.F.T. is
supported by a Chaire d’Excellence from the University Paris
Descartes and the CNRS. We thank Laura Miller, Jeffrey
Garber, and David Ogden for help and comments.
Caged compounds in solution are usually handled in room
light, during which both spontaneous degradation and
photolysis can occur. Under these conditions the rate of O-
position degradation was similar for mono-O-CNB-GABA and
bis-CNB-GABA. These findings are consistent with good
stability at the carboxylate position, as previously reported,13
and with higher stability at the amino position. However, these
results are not consistent with a claim of t1/2 = 17 h for mono-
CNB-GABA in a study that did not report methods.8 During a
week in the light, we estimate the production of N-CNB-GABA
to be 10%; the accumulation of GABA during that period
should therefore be <1%. Due to the possibility of accumulation
of mono-CNB-GABA isomers in solution, bis-CNB-GABA
should be kept dry before use, for instance through aliquoting
of solutions in distilled water followed by lyophilization. It is of
note that purification steps involving aqueous solution, such as
preparative HPLC, might require a trade-off in the form of
accumulated mono-CNB-GABA or GABA. We found the use
of crude product, without HPLC purification, to be effective in
biological experiments; accordingly, crude-product level purity
may be acceptable for many biological experiments.
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