Chemical Targeting of Voltage Sensitive Dyes to Specific Cells and Molecules in the Brain

Article abstract of DOI:10.1021/jacs.0c00861

Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. The impact of these highly lipophilic sensors has, however, been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a nongenetic molecular platform for cell- and molecule-specific targeting of synthetic VSDs in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of VSDs by dynamic encapsulation and high-affinity ligands to target the construct to specific neuronal cells utilizing only native components of the neurotransmission machinery at physiological expression levels. Dichloropane, a monoamine transporter ligand, enables targeting of dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. PFQX in conjunction with ligand-directed acyl imidazole chemistry enables covalent labeling of AMPA-type glutamate receptors in the same brain regions. Probe variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor-type dye respond to membrane potential changes in a similar manner to the parent dyes, as shown by whole-cell patch recording. We demonstrate the feasibility of optical voltage recording with our probes in brain tissue with one-photon and two-photon fluorescence microscopy and define the signal limits of optical voltage imaging with synthetic sensors under a low photon budget determined by the native expression levels of the target proteins. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.

Full text of DOI:10.1021/jacs.0c00861

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Article
Chemical Targeting of Voltage Sensitive Dyes to Specific Cells and
Molecules in the Brain
Tomas Fiala, Jihang Wang, Matthew Dunn, Peter Sebej, Se Joon Choi, Ekeoma C. Nwadibia,
Eva Fialova, Diana M. Martinez, Claire E. Cheetham, Keri J. Fogle, Michael J. Palladino,
Zachary Freyberg, David Sulzer,* and Dalibor Sames*
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ABSTRACT: Voltage sensitive fluorescent dyes (VSDs) are
important tools for probing signal transduction in neurons and
other excitable cells. The impact of these highly lipophilic sensors
has, however, been limited due to the lack of cell-specific targeting
methods in brain tissue or living animals. We address this key
challenge by introducing a nongenetic molecular platform for cell-
and molecule-specific targeting of synthetic VSDs in the brain. We
employ a dextran polymer particle to overcome the inherent
lipophilicity of VSDs by dynamic encapsulation and high-affinity
ligands to target the construct to specific neuronal cells utilizing
only native components of the neurotransmission machinery at
physiological expression levels. Dichloropane, a monoamine
transporter ligand, enables targeting of dense dopaminergic axons
in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. PFQX in conjunction with ligand-
directed acyl imidazole chemistry enables covalent labeling of AMPA-type glutamate receptors in the same brain regions. Probe
variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor-type dye respond to membrane potential
changes in a similar manner to the parent dyes, as shown by whole-cell patch recording. We demonstrate the feasibility of optical
voltage recording with our probes in brain tissue with one-photon and two-photon fluorescence microscopy and define the signal
limits of optical voltage imaging with synthetic sensors under a low photon budget determined by the native expression levels of the
target proteins. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific
imaging and pharmacology.
choice, design, and direct modification of the fluorophore,
enabling the tuning of photophysical and functional properties
(e.g., absorption and emission profile, photostability, bright-
ness, response magnitude (ΔF/F), and kinetics).8,16,17
Synthetic chemistry also allows for flexible design of sensors
for other imaging modalities (e.g., short wave infrared
(SWIR)18,19 fluorescence or photoacoustic (PA)20−22 imag-
ing).
Synthetic voltage sensors are, however, exceptionally
challenging to deliver to selected cell types due to their
inherent lipophilicity, which causes high background staining
and limited diffusibility in tissue. Successful attempts to target
synthetic VSDs to specific cell types have been scarce. For
INTRODUCTION
■
Monitoring signal transmission between and within the cells of
the nervous system has been and remains one of the key
challenges in neuroscience. While whole cell electrophysio-
logical recording techniques have provided unprecedented
insight into neuronal function and remain the standard
approach to probe membrane potential dynamics in excitable
cells,1,2 optical techniques have emerged as a powerful
complement to electrode-based methods. The development
of calcium sensitive dyes (CaSDs)3,4 and voltage sensitive dyes
(VSDs)5−8 has transformed neuroscience and cell biology at
large; however, the key shortcoming of synthetic sensors has
been the lack of targeting to specific cell types in brain tissue
and in vivo.9−11 Consequently, the advancement of protein
based calcium and voltage indicators have been intensely
pursued, as these genetically encoded tools can be targeted to
many neuronal cell types via cell-specific, promoter-based
genetic methods.12−15
Received: January 29, 2020
Synthetic dyes have several key advantages over fluorescent
proteins, mainly the availability of the entire chemical space for
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Figure 1. Components of the VoLDe targeting system. (a) Voltage sensitive dyes di-2-ANEPEQ (1)36,37 and dsRVF5 (2).38,39 (b) Dichloropane
(3), a potent DAT and NET ligand40,41 and PFQX (4), a selective AMPAR ligand.42 (c) A 3D model of the VoLDeMo targeting system. A di-2-
ANEP VSD (pink) and dichloropane ligand (green) are attached through short linkers to a dextran polymer carrier (blue). The dichloropane ligand
of the construct is shown bound to the dopamine transporter (gray; structure of Drosophila DAT44 was used for the graphic), and the lipophilic
VSD is inserted into the cell membrane. (d) VoLDeCAM targeting. The PFQX ligand (green) is attached to a dextran polymer carrier (blue) via an
acyl imidazole linker.43 Upon binding to AMPAR (gray; taken from ref. 45), a nearby nucleophilic amino acid residue (red) cleaves the acyl
imidazole linker, thus covalently binding the probe, deploying the VSD (pink), and expelling the ligand.
example, a chemo-genetic approach taking advantage of cell-
surface expression of alkaline phosphatase to dephosphorylate
and thus induce membrane localization of an electrochromic
aminonaphthyl-ethene-pyridinium (ANEP) VSD precursor
was reported.23−25 A similar strategy was adopted recently
using a porcine liver esterase to activate a fluorogenically silent
precursor of VoltageFluor, a photoinduced electron transfer
(PeT) based VSD.26 Ligand-tethering approaches were also
utilized in recent studies using the acyl carrier protein (ACP)-
tag27 or Spy-tag28 to target a Nile Red or a VoltageFluor VSD,
respectively, to neurons genetically engineered to express the
self-modifying enzyme. Targeting of rhodamine-based voltage
reporters using genetically encoded HaloTag in mouse brain
tissue has recently been introduced.29 However, no targeting
strategies have yet been introduced that can be performed in
the absence of genetic manipulation of the target cells in intact
tissue preparations ex or in vivo.
We have pursued chemical targeting strategies to deliver
synthetic fluorophores to specific neuronal cells and synaptic
release sites in intact brain tissue, utilizing native components
of the neurotransmission machinery. We developed fluorescent
false neurotransmitters (FFNs), probes that act as substrates of
relevant monoamine transporters, label dopaminergic or
noradrenergic neurons, and their synaptic vesicles, and enable
optical recording of transmitter release at individual synaptic
sites ex vivo and in vivo in rodents.30−35 For example, the probe
FFN102 labels dopaminergic release sites in the mouse
striatum with high selectivity, due to the selective transport
of FFN102 by the dopamine transporter (DAT) into
dopaminergic neurons.32 Encouraged by this approach, we
hypothesized that endogenously expressed membrane proteins
could enable the targeting of specific cell types in the brain
with a variety of sensors and actuators, including lipophilic
VSDs, by the means of high affinity ligands.
Targeting endogenously expressed components of the
neurotransmitter machinery to deliver lipophilic VSDs poses
a 2-fold challenge in comparison to the overexpressed chemo-
genetic handles (such as self-modifying enzymes). At the
targeting level, the native membrane protein accommodates a
relatively low number of sensor molecules, leading to
significant background even from low levels of nonspecific
staining. At the voltage recording level, the low number of
delivered VSD molecules limits their photon output, resulting
in a relatively weak functional signal. We sought to define the
limits of voltage recording with synthetic VSDs targeted to
specific neuronal populations in the brains of wild-type
animals. The embodiment of this goal required the use of
small carbohydrate polymers to accommodate the high
lipophilicity of VSDs and serve as a molecular carrier for
both the VSD and the targeting ligand.
Here, we report a chemical platform which enables the
targeting of lipophilic VSDs to monoaminergic projections and
glutamatergic synapses in brain tissue. Our Voltage sensor−
Ligand−Dextran probes, or VoLDe probes, integrate three
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Figure 2. Design and synthesis of DAT/NET-targeted fluorescent probes. (a) Dichloropane ligand bearing a Boc-4-aminobutyl linker (5) and its
transformation into probe 6. (b) Schematic illustration of ligand−dye fusion concepts. Left: a short linker separates the dye (pink) and the ligand
(black anchor) targeting a membrane protein of interest (red). Right: The dye and the ligand are separated by a polymer carrier (blue) that enables
insertion of the dye into the cell membrane (gray). (c) Synthesis of azidopropyl-carbamate-dextran(6000) (8-w). Number w reflects the degree of
dextran functionalization with azidopropyl ligands (average number of azide functionalities per polymer molecule; see Supporting Information for
details). (d) One-pot assembly of VoLDeMo via click chemistry by the reaction of N3-dextran 8-w (w = 3.25 or 2.15) with a 1:2 mixture of VSD 9
or 10 and ligand 11. Note: Dextran branching is omitted for clarity. Linker substitution on dextran is shown only in position 2; however, it is likely
that positions 3, 4, and terminal 6 are also functionalized to a lower extent (see ref. 50 and Supporting Information).
modalities into one multifunctional construct to achieve this
goal. We selected two VSDs for this exploratory study: di-2-
ANEPEQ (1), a well-established electrochromic hemicyanine
VSD36,37 and dsRVF5 (disulfo Rhodol VoltageFluor-5, 2), a
disulfonated38 version of a state-of-the-art VSD (Figure 1a).39
As a second componentthe targeting ligandwe used the
tropane analog dichloropane (3) as a potent ligand of
dopamine and norepinephrine transporters (DAT and NET
respectively)40,41 and 6-pyrrolyl-7-trifluoromethyl-quinoxaline-
2,3-dione (PFQX, 4) as a ligand for the α-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid (AMPA)-type glutamate
receptor (AMPAR; Figure 1b).42 Both the VSD and the
ligand are appended to a dextran polymer carrier that
compensates for the dye’s lipophilicity and decreases non-
specific labeling in cell cultures and in the mammalian brain.
Our probes enable not only cell-type specific but also
molecule-specific targeting of VSDs by selectively binding the
target membrane proteins. We demonstrate noncovalent
targeting of DAT and NET using dichloropane (3) as a high
affinity monoamine transporter ligand (“VoLDeMo” probe,
Figure 1c) and irreversible covalent labeling of the AMPAR by
the means of the “chemical AMPAR modification” (CAM)
strategy developed by Hamachi and co-workers43 (“VoLDe-
CAM” probe, Figure 1d).
DAT’s 3D structure,44,46 we hypothesized that introducing an
N-Boc-4-aminobutyl linker instead of the methyl group on the
bridgehead nitrogen of dichloropane (3) would largely
conserve its affinity to monoamine transporters. Indeed,
enantiomerically pure dichloropane analog 5 (Figure 2a)
blocked APP+ (an established DAT substrate47) uptake by
HEK-293 cells stably transfected with human DAT (hDAT-
HEK cells) with an IC50 of (11
1) nM (Figure S1). We
further synthesized rhodamine-labeled dichloropane (6, Figure
2a),48 which strongly labeled hDAT-HEK cells in a coronal
fashion, compared to null transfected cells, and the labeling
was efficiently blocked by the DAT inhibitor, nomifensine49
(Figure S2). In addition, probe 6 blocked APP+ uptake by
hDAT-HEK cells with an IC50 of (25 1) nM (Figure S3),
comparable in potency to dichloropane analog 5. This system
was employed as a positive control in subsequent VSD
targeting studies.
Design, Synthesis, and Characterization of a Non-
genetic Platform for Targeting VSDs to Monoaminergic
Neurons. Targetable probe 6 is schematically illustrated in
Figure 2b-left. The same concept cannot be applied to VSD
targeting, since a short linker would not enable insertion of the
fluorophore into the cell membrane. A long linker and/or
polymer carrier is needed to separate the ligand and the VSD
(Figure 2b-right) and render the construct functional. The first
logical choice for a long ligand−VSD separator was a
poly(ethylene glycol) (PEG) chain. PEG is a widely used
polymer in chemical biology due to its solubility, synthetic
RESULTS
■
Establishment of Dichloropane as a Targeting
Ligand. On the basis of previous reports and examination of
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1
Figure 3. Proposed conformational behavior and targeting model of VoLDeMo. (a) H NMR (500 MHz, D2O) of di-2-ANEP-VoLDeMo (12),
zoomed in aromatic region (6.2−9.2 ppm). Broadened peaks reflect restrained conformational freedom of the lipophilic VSD which is shielded by
the dextran carrier. Green asterisks−signals of the dichloropane ligand which are significantly sharper, reflecting more conformational freedom of
the ligand. (b) 1H NMR (500 MHz, DMSO-d6) of di-2-ANEP-VoLDeMo (12), zoomed in aromatic region (6.2−9.2 ppm). Sharp peaks indicate
high conformational flexibility of the construct in the more solubilizing environment of DMSO. (c) Targeting model of VoLDeMo. Top: in bulk
aqueous environment, the VSD (pink) is encapsulated by the dextran carrier (blue spheres) from the solvent. Bottom: after binding of the
dichloropane ligand (green) to DAT (gray), the equilibrium is shifted and the VSD partitions into the cell membrane.
accessibility, and presumed low protein binding.51 It has been
successfully applied previously for delivery of dye mole-
cules.27,28,52 We therefore constructed a dichloropane−
PEG(3400)−di-2-ANEP construct (S1, Figure S4) and tested
its targeting specificity in hDAT-HEK cells. However, DAT-
independent cell labeling as well as internalization of construct
S1 was observed (Figure S4). We thus concluded that PEG
was unable to compensate for the lipophilic character of di-2-
ANEP and render it targetable to specific membrane proteins.
We next turned our attention to dextran, a bacterial
polysaccharide composed of glucopyranose units connected
through α-1,6-glycosidic linkages with occasional 1,3- and less
frequently 1,2- and 1,4-branching.53 Dextran was an attractive
choice due to its clinical use, biocompatibility, slow
degradation in mammals, and high diffusion rates in tissue
including brain.54−56 On the basis of our exploratory studies,
we chose dextran(6000) (avg. Mw = 6000 Da) as a
compromise between a large-enough hydrophilic surface to
compensate for the lipophilicity of the VSD and a relatively
small size for sufficient diffusion in tissue (data not shown).
We developed a functionalization protocol enabling attach-
ment of 3-azidopropyl linker groups to dextran using activated
carbamate 7 to provide azidopropyl-carbamate-dextran(6000)
(8-w, Figure 2c). This chemical labeling allowed for
straightforward control of dextran coverage (average number
of azide functionalities per polymer molecule − w; see
Supporting Information for detailed protocol and discussion
of this parameter) with N3-terminated linkers. Next, we used
strain-promoted azide−alkyne cycloaddition click chemistry to
attach functionalized di-2-ANEP (9) or dsRVF5 VSD (10) and
dichloropane ligand (11). The position of the linker
terminated with the clickable bicyclo[6.1.0]non-4-yn-9-ylmeth-
yl (BCN) group was chosen based on the expected orientation
of the VSD in the cell membrane. The ethylene−quaternary
ammonium headgroup of di-2-ANEPEQ (1) was substituted
with a flexible butylene linker terminated with carbamate-
bound BCN in the functionalized dye 9 (linker attached via the
pyridinium nitrogen). Similarly, dsRVF5 10 was functionalized
with BCN at the pyrrolidine group, which faces the
extracellular space upon membrane localization of the VSD.
The sensors and ligand equipped with the strained alkyne
moiety were reacted with the azide functionalized dextran 8-w
to assemble the final di-2-ANEP-VoLDeMo (12) and dsRVF5-
VoLDeMo (13) constructs, respectively (Figure 2d). The
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Figure 4. Labeling specificity and membrane potential sensitivity of VoLDeMo probes in hDAT-HEK cells. (a, d) Brightfield (BF, top row) and
corresponding fluorescence (Fluo, bottom row) images of HEK cells labeled with di-2-ANEP- (panel a) and dsRVF5-VoLDeMo (panel d) probes
(100 nM dextran/74 nM or 63 nM VSD, respectively). Labeling was compared in hDAT-HEK cells (left column), hDAT-HEK cells preincubated
with the DAT inhibitor nomifensine (2 μM; middle column), and null-transfected HEK cells (right column). All fluorescence images are equally
contrasted for each VoLDeMo probe. Note: bright spots in some fluorescence images likely correspond to nonspecifically labeled dead cells or
cellular fragments. (b, e) Representative concatenated traces of the fractional change in fluorescence of di-2-ANEP- (panel b) and dsRVF5-
VoLDeMo (panel e) probes (100 nM dextran/74 nM or 63 nM VSD, respectively) versus time of a voltage-clamped HEK cell, held at −60 mV and
then subjected to 100 ms voltage holdings ranging from −100 mV (blue trace) to +80 mV (red trace) in 30 mV intervals. (c, f) Plots of percent
fluorescence change of di-2-ANEP- (panel c) and dsRVF5-VoLDeMo (panel f) probes (100 nM dextran/74 nM or 63 nM VSD, respectively)
versus membrane voltage. ΔF/F per 100 mV = (−4.7 1.1)%, SNR = 4.5 0.5, n = 5; mean SEM for di-2-ANEP-VoLDeMo (12). ΔF/F per
100 mV = (13.1 3.7)%, SNR = 14.2 2.9, n = 5; mean SEM for dsRVF5-VoLDeMo (13).
coverage of the dextran polymer with ligands (y) and VSDs (z)
was chosen as follows: to minimize the number of polymer
molecules which would carry two or more lipophilic VSDs
(increasing the probability of nonspecific binding or
fluorescence self-quenching due to short-range interactions),
we aimed for y < 1; to ensure that a vast majority of polymer
molecules that carry a VSD also carry at least one dichloropane
ligand, we aimed for z > 1 and y:z = 1:2. di-2-ANEP-
VoLDeMo (12) was obtained with a coverage of 0.74 ANEP/
the structure is likely folded in aqueous environments, wherein
the lipophilic dye is concealed by the dextran polymer to
minimize the unfavorable VSD−water interactions. Restricted
flexibility of the VSD in such a folded state results in
broadened peaks. Signals belonging to the dichlorophenyl
group of the dichloropane ligand show significantly less
broadening (green asterisks in Figure 3a and Figure S8a)
reflecting less shielding by dextran. Only in the presence of a
less polar (and aprotic dipolar) environment (DMSO-d6) the
VoLDeMo probe favors an unfolded conformation. We
therefore propose the following targeting model for the
VoLDeMo sensors: in the bulk extracellular environment, the
construct exists predominantly in a folded state with the VSD
minimally exposed to the aqueous environment, which results
in low nonspecific binding. Association of the dichloropane
ligand with DAT or NET increases the effective concentration
of the VoLDeMo construct at the cell membrane; the
conformational equilibrium is shifted, and the VSD partitions
into the membrane (Figure 3c).
VoLDeMo Selectively Labels DAT-Expressing Cells
and Enables Optical Recording of Membrane Potential
Changes. hDAT-HEK cells were incubated with di-2-ANEP-
VoLDeMo (12; 100 nM dextran/74 nM VSD) or dsRVF5-
VoLDeMo (13; 100 nM dextran/63 nM VSD) for 5 or 15 min,
respectively, at room temperature and washed, resulting in
membrane-localized fluorescence staining. Upon pre- and
cotreatment with the DAT inhibitor, nomifensine (2 μM),
1
polymer and 1.38 ligand/polymer as determined by H NMR
(Figure S5). dsRVF5-VoLDeMo (13) was synthesized twice: a
coverage of 0.63 dsRVF5/polymer and 1.39 ligand/polymer
was obtained for sample 1 and 0.74 dsRVF5/polymer and 1.45
ligand/polymer was obtained for sample 2 (Figure S6 and S7).
Different concentration values of the VoLDeMo probes can
be reported with respect to the dextran polymer, VSD or
ligand, based on the coverages above. For example, a 100 nM
di-2-ANEP-VoLDeMo (12) with respect to dextran is ∼74 nM
with respect to the VSD (see Supporting Information for
details). Concentration values with respect to both dextran and
VSD are specified in subsequent experiments.
1
A significant difference was observed between the H NMR
spectra of VoLDeMo probes in D2O and DMSO-d6. While
broad signals were observed in the aromatic region in D2O
(Figure 3a, Figure S8a), much sharper peaks were observed in
DMSO-d6 (Figure 3b, Figure S8b). This observation provides
a clue about the conformational behavior of these constructs:
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Figure 5. Targeting of VoLDeMo probes to catecholamine axons in mouse brain slices. (a) Nissl stained sagittal slice of a mouse brain (image
credit: Allen Institute61,62) with a schematic representation of a dopaminergic neuron originating in the SNc and highly arborizing in the dSTR.
The blue rectangle indicates the approximate region where the images in panels b and c were acquired via two-photon excitation microscopy. (b, c)
Representative images of striatal slices labeled with di-2-ANEP and dsRVF5 probes, respectively. Left: striatal slice incubated with the targeted di-2-
ANEP-VoLDeMo (12; 100 nM dextran/74 nM VSD, 30 min, 15 μm depth from the tissue surface) or dsRVF5-VoLDeMo (13; 100 nM dextran/
63 nM VSD, 30 min, 15 μm depth), respectively; middle: an analogous experiment with pre- and coincubation with the DAT/NET blocker
nomifensine (2 μM); right: striatal slice incubated with untargeted di-2-ANEPEQ (1, 100 nM, 30 min, 15 μm depth) or dsRVF5 (2, 100 nM, 30
min, 15 μm depth). (d) Nissl stained sagittal slice of a mouse brain (image credit: Allen Institute61,62) with a schematic representation of a
noradrenergic neuron originating in the LC with sparse axonal projections in the CTX. The blue rectangle indicates the approximate region in the
somatosensory cortex where the images in panels e and f were acquired via two-photon excitation microscopy. (e, f) Representative images of
cortical slices labeled with di-2-ANEP and dsRVF5 probes, respectively. Left: cortical slice incubated with the targeted di-2-ANEP-VoLDeMo (12;
100 nM dextran/74 nM VSD, 30 min, 15 μm depth) or dsRVF5-VoLDeMo (13; 100 nM dextran/63 nM VSD, 30 min, 15 μm depth), respectively.
Middle: an analogous experiment with pre- and coincubation with nomifensine (2 μM). Right: cortical slice incubated with untargeted di-2-
ANEPEQ (1, 100 nM, 30 min, 15 μm depth) or dsRVF5 (2, 100 nM, 30 min, 15 μm depth). Images within the same panel were contrasted
equally. ANEP probes 1 and 12 were excited at 920 nm and emission collected at 570−610 nm. dsRVF5 probes 2 and 13 were excited at 820 nm
and emission collected at 570−610 nm.
the membrane staining of hDAT-transfected cells was
significantly diminished (20.2 5.7 fold decrease, n = 30
cells from 3 experiments, P = 0.0013 for di-2-ANEP-
VoLDeMo (12) and 3.4 0.5 fold decrease, n = 30 cells
to hDAT-HEK cells and null-HEK cells as a stock solution in
H2O vs DMSO (Figure S10). Coronal staining of null-HEK
cells (off-target staining) was lower by a factor of 3.7 0.4
compared to labeling of hDAT-HEK cells (n = 15 cells from 3
experiments, P = 0.0001) when an aqueous solution of 13
(stock solution: 50 μM VSD, final concentration: 50 nM VSD)
was applied. In contrast, addition of DMSO solution of 13 at
the same concentration resulted in a 2.2 0.8 fold decrease in
coronal staining of null-HEK cells compared to hDAT-HEK
cells (n = 15 cells from 3 experiments, P = 0.094). These
findings support our targeting model of VoLDeMo probes
proposed above (Figure 3c): the labeling selectivity is greater
when an aqueous solution of dsRVF5-VoLDeMo (13) is
added to the cells where the VSD is shielded by the dextran,
while direct application of a DMSO solution of the probe
where the VSD is not sufficiently encapsulated (assuming slow
folding kinetics) results in inferior labeling selectivity.
from 3 experiments, P < 0.0001 for dsRVF5-VoLDeMo (13),
two-tailed unpaired t-test). Additionally, coronal staining was
not observed with null-transfected HEK cells under identical
conditions (8.4 1.5 fold decrease in fluorescence intensity
with respect to hDAT-HEK cells, n = 30 cells from 3
experiments, P = 0.0018 for di-2-ANEP-VoLDeMo (12) and
6.0 0.6 fold decrease, n = 30 cells from 3 experiments, P <
0.0001 for dsRVF5-VoLDeMo (13), two-tailed unpaired t-
test), confirming that binding to DAT is required for
localization of the probe to the surface of the membrane
(Figure 4a,d, Figure S9). Higher signal to background ratio
observed for di-2-ANEP-VoLDeMo (12) compared to
dsRVF5-VoLDeMo (13) is likely a result of the lower
lipophilicity of the di-2-ANEP VSD with respect to dsRVF5,
which results in a lower extent of nonspecific background
staining. Interestingly, we observed significant difference in
labeling selectivity when dsRVF5-VoLDeMo (13) was added
Next, we quantified the binding of VoLDeMo probes to
hDAT. Both di-2-ANEP-VoLDeMo (12) and dsRVF5-
VoLDeMo (13) inhibited APP+ uptake into hDAT-HEK
cells with the following IC50 values: (74
3) nM for 12,
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corresponding to (55 3) nM with respect to the VSD and
(103 5) nM with respect to the ligand; (45 2) nM for 13,
corresponding to (34 1) nM VSD and (63 2) nM ligand
(Figure S11), showing only a minor loss in DAT inhibitory
potency (as a measure of binding affinity) compared to those
of dichloropane derivative 5 and rhodamine probe 6.
prevent membrane crossing, while maintaining voltage
sensitivity and showing higher photostability.
VoLDeMo Probes Deliver VSDs to Dopamine and
Norepinephrine Axons in Mouse Brain Tissue. The next
key test for the targeted constructs was the examination of
labeling specificity in acute mouse brain tissue. We specifically
focused on two regions with vastly different densities of
catecholamine axons, the dorsal striatum and somatosensory
cortex (Figure 5). The dorsal striatum (dSTR) receives dense
dopaminergic axonal projections from neurons originating in
the substantia nigra compacta (SNc) in the midbrain that
highly arborize at their target (Figure 5a).60 Striatal brain slices
(living slice preparations; see Supporting Information)
incubated with di-2-ANEP-VoLDeMo (12, 100 nM dextran/
74 nM VSD, 30 min) exhibited a string-like, punctate pattern
of fluorescent labeling (Figure 5b, Figure S14a), typical for
dopaminergic axons in this region.32 Quantitative colocaliza-
tion with an established dopamine probe FFN102 gave a high
We further verified that di-2-ANEP and dsRVF5 dyes
remain voltage sensitive when attached to dextran as a part of
VoLDeMo probes. hDAT-HEK cells labeled with di-2-ANEP-
VoLDeMo (12; 100 nM dextran/74 nM VSD) or dsRVF5-
VoLDeMo (13; 100 nM dextran/63 nM VSD) were whole-cell
voltage clamped. The cells were first held at −60 mV to mimic
the resting membrane potential of neurons, then subjected to
DC holding potential steps ranging from −100 mV to +80 mV
in 30 mV intervals, and imaged at 200 Hz (Figure 4b,e). In the
case of di-2-ANEP-VoLDeMo (12), we observed a decrease in
fluorescence intensity following depolarization of the mem-
brane potential, which is common for electrochromic VSDs
when excited at the red edge of their absorption band.57 When
the fractional fluorescence change was plotted against the
membrane potential, a linear plot (R2 = 0.998) with a ΔF/F
per 100 mV = (−4.7 1.1)% was obtained (n = 5, Figure 4c).
This result was comparable to what was previously
demonstrated for ANEP class dyes.57 For dsRVF5-VoLDeMo
(13), we observed increased fluorescence intensity with
increasing membrane potential, as expected for the PeT-
based class of VSDs.39 The plot of fractional fluorescence
change vs membrane potential was found to be linear (R2 =
level of colocalization (78.0%
3.5%, n = 3, Figure S15),
which was significantly higher than the respective scrambled
control (17.3% 2.3%, P < 0.0001, two-tailed unpaired t-test).
Furthermore, a high level of colocalization was observed with
green fluorescent protein (GFP) expressed under the control
of the tyrosine hydroxylase (TH, a marker of catecholamine
neurons) promoter in the striatum of TH-GFP mice (Figure
S16a). As expected, preincubation and coincubation with
nomifensine (2 μM) diminished the targeting of dopamine
axons and resulted in predominantly diffuse background
labeling, indicating that the axonal labeling is dependent on
DAT (Figure 5b). To further demonstrate the high degree of
VoLDeMo selectivity, we incubated striatal brain slices with
untethered di-2-ANEPEQ (1, 100 nM, 30 min). As expected, a
dramatically contrasting pattern corresponding to nonspecific
fluorescent labeling of cell membranes was observed (Figure
5b).
We next tested the labeling of striatal slices with dsRVF5-
VoLDeMo (13) bearing the more lipophilic and larger rhodol-
based VSD. Remarkably, dSTR brain slices incubated with
dsRVF5-VoLDeMo (13; 100 nM dextran/63 nM VSD, 30
min) exhibited a similar string-like pattern of fluorescent
labeling as its di-2-ANEP counterpart (Figure 5c, Figure
S14b). Preincubation and coincubation with nomifensine (2
μM) produced a diffuse background staining (Figure 5c). The
high degree of dsRVF5-VoLDeMo (13) selectivity in striatal
slice was further demonstrated by comparison to untethered
dsRVF5 (2, 100 nM, 30 min, Figure 5c), which labeled many
cell membrane compartments.
To our knowledge, these are the first examples of selective
delivery of diffusible VSDs to specific neuronal cell types in
mammalian brain tissue via a purely chemical approach
without any genetic manipulation as well as the first direct
targeting to specific axonal projections and release sites in
brain tissue with such probes.
The somatosensory cortex (CTX), unlike the dSTR,
contains a far lower density of catecholaminergic axons,
which we recently reported are mostly noradrenergic
extensions expressing NET34 originating in the locus coeruleus
(LC) (Figure 5d).63 These sparse projections represent a more
challenging target for VoLDeMo probes, due to the far lower
ratio of target noradrenergic cells to other nontarget cells and
tissue components. Despite this, cortical brain slices incubated
with di-2-ANEP-VoLDeMo (12; 100 nM dextran/74 nM
VSD, 30 min) exhibited a distinctive fluorescent labeling
0.999) with a ΔF/F per 100 mV = (13.1
Figure 4f).
3.7)% (n = 5,
Under whole-cell voltage clamp mode, hDAT-HEK cells
were also subjected to simulated trains of rapid depolarizations
to explore the temporal resolution of VoLDeMo probes in
resolving AC voltage changes. Cells labeled with di-2-ANEP-
VoLDeMo (12; 100 nM dextran/74 nM VSD) or dsRVF5-
VoLDeMo (13; 100 nM dextran/63 nM VSD) were held at
−60 mV and subjected to bursts of ten individual voltage
spikes of 2 ms length and 100 mV magnitude to resemble the
membrane action potential of a typical neuron. The ten voltage
spikes within one burst were applied at 100 Hz in frequency.
At 1 kHz imaging rate, both VoLDeMo probes 12 and 13 were
able to detect and distinctively resolve individual voltage spikes
within one burst (Figure S12).
We compared the photostability of VoLDeMo probes 12
and 13 with that of the corresponding untethered VSDs 1 and
2, respectively. While no significant difference in photostability
was observed between dsRVF5-VoLDeMo (13) and dsRVF5
(2) (540 nm illumination; Figure S13b), di-2-ANEP-
VoLDeMo (12) was significantly more photostable than di-
2-ANEPEQ (1) (450 nm illumination; Figure S13a). It has
been previously shown that polymeric or macrocyclic materials
including polysaccharides can increase the photostability of
fluorescent dyes by decreasing the availability of the excited
chromophore to the attack of nucleophilic species from the
surrounding environment.58,59 We hypothesize that the
dextran polymer carrier may be playing this protective role
in the case of di-2-ANEP-VoLDeMo (12). In addition,
although the core chromophore in probes 12 and 1 is
identical, we note that the photostability of di-2-ANEPEQ (1)
could be affected by the charged quaternary ammonium group
in direct vicinity of the dye. The dextran-tethered di-2-ANEP-
VoLDeMo (12) does not require this second charged group to
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Figure 6. Two-photon fluorescence recording of evoked activity in striatal dopaminergic axons. (a) Representative two-photon fluorescence image
of a striatal slice labeled with dsRVF5-VoLDeMo (13; 676 nM dextran/500 nM VSD, 30 min, 35 μm depth from the tissue surface). (b)
Representative image of a single two-photon spiral scan frame from the recording of evoked activity in panels c and d, demonstrating the field of
view and spatial resolution. (c) Fluorescence trace recorded at 30 Hz from striatal slices labeled with dsRVF5-VoLDeMo (13; 676 nM dextran/500
nM VSD, 30 min) with electric stimulation (25 pulses at 50 Hz, period highlighted in yellow). The trace is normalized to an analogous experiment
conducted in the presence of TTX (1 μM) and is an average from multiple trials (n = 100 from 10 slices). A two-point moving average was applied
to smooth the trace. (d) Mean fluorescence ( SEM) of the voltage recording from panel c during the stimulation and prestimulation periods.
ΔF/F = (0.12 0.03)% (n = 100 from 10 slices, P < 0.0001, two-tailed unpaired t-test).
pattern (Figure 5e, Figure S14c) typical of noradrenergic axons
in this brain area.34 There appeared to be no prominent
nonspecific uptake of the probes by resident cell bodies, either
in the cortex or striatum. Similar to the striatum, a high level of
colocalization was observed between di-2-ANEP-VoLDeMo
(12) labeling and TH-GFP in the cortex (Figure S16b).
Similar control experiments were employed as in the striatal
slices: preincubation and coincubation with nomifensine (2
μM) nearly abolished the string-like labeling (Figure 5e); and
incubation of a cortical brain slice with untethered di-2-
ANEPEQ (1, 100 nM, 30 min) produced a pattern
corresponding to nonspecific fluorescent labeling of the brain
tissue (Figure 5e). Consistent with appropriate targeting,
dsRVF5-VoLDeMo (13; 100 nM dextran/63 nM VSD, 30
min) produced a sparse string-like fluorescent labeling pattern
in the cortical slice (Figure 5f, Figure S14d), albeit with slightly
higher background than di-2-ANEP-VoLDeMo (12; Figure
5e). This construct also showed occasional bright punctate or
tubular objects larger than the axonal strings or varicosities,
which we ascribe to nonspecific labeling and/or clustering of
the probe. Despite the combination of a low density of
noradrenergic projections in somatosensory cortex together
with the higher lipophilicity of the dsRVF5 VSD, a dramatic
contrast was observed compared to images obtained with
nomifensine (2 μM) inhibition and unmodified dsRVF5 (2,
100 nM, 30 min, Figure 5f).
Both di-2-ANEP- and dsRVF5-VoLDeMo constructs
showed good diffusion in living tissue, capable of carrying
the embodied lipophilic VSDs to depths typically used in our
laboratories for imaging acute slices under standard exper-
imental conditions. Diffusion to depths below 30 μm in the
striatum could be achieved using higher probe concentrations
while maintaining high labeling specificity (Figure S17). In
addition, the less lipophilic di-2-ANEP-VoLDeMo (12)
generally penetrated deeper than dsRVF5-VoLDeMo (13)
under the same conditions.
VoLDeMo probes also exhibited clear, ligand-dependent
labeling in whole adult brains from Drosophila melanogaster.
Incubation of ex vivo preparations of whole living fly brains
with VoLDeMo probes resulted in a punctate labeling pattern,
partially colocalizing with a dopaminergic marker, which was
abolished by inhibition with dichloropane derivative 5 or when
the nontargeted di-2-ANEPEQ (1) was used (Figure S18 and
Drosophila Brain section in the Supporting Information). This
demonstrates the versatility of the VoLDeMo probes across
different species and experimental preparations.
dsRVF5-VoLDeMo Enables Recording of Evoked
Activity in Striatal Dopaminergic Axons. Compared to
protein-based genetically encoded voltage indicators (GEVIs)
or hybrid chemo-genetic voltage sensors relying on overex-
pressed membrane proteins, VoLDeMo probes are targeted
only to a relatively small population of natively expressed
monoamine transporters. Therefore, the ultimate challenge for
VoLDeMo probes was to obtain functional recordings in ex
vivo rodent brain slices with the limited photon output from
these targeted sensors. We labeled mouse striatal slices with
dsRVF5-VoLDeMo (13; 676 nM dextran/500 nM VSD, 30
min; Figure 6a). Next, we performed electric stimulation (25
pulses at 50 Hz during a 0.5 s period) while recording two-
photon fluorescence images with spiral scanning (Figure 6b) at
30 Hz. This slow scanning approach does not provide
recording of single action potentials but does enable recording
of overall activity of the selected region (5.5 × 103 μm2 in
Figure 6b). Indeed, we observed increase in fluorescence
during the stimulation period upon averaging multiple trials
(ΔF/F = (0.12
0.03)%, mean
SEM, n = 100 from 10
slices, P < 0.0001, two-tailed unpaired t-test) when referenced
to an analogous experiment where tetrodotoxin (TTX, 1 μM, n
= 50 from 5 slices) was used to block the propagation of action
potentials (Figure 6c,d; Figure S19). Since a stimulation-
dependent artifact (baseline-drift) was observed for all
measurements (Figure S19c), traces shown in Figure 6c are
represented as the difference between the measurement in the
absence and in the presence of TTX: a normalization strategy
which accounts for the stimulation-dependent drift while
leaving the voltage-dependent signal unaffected. To our
knowledge, this is the first example of optical voltage recording
from a specifically targeted neuronal population in brain tissue
without any genetic manipulation as well as the first such
recording specific to axons.
Dextran is Compatible with Ligand-Directed Acyl
Imidazole Chemistry. A potential drawback of VoLDeMo
design is the inherent perturbation of the system by the
blockage of an important transporter protein upon ligand
binding. Recent development in ligand-directed covalent
labeling strategies provided tools to circumvent this issue.64
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Figure 7. Dextran is compatible with ligand-directed acyl imidazole chemistry. (a) Synthesis of BCN-carbonate-dextran(6000) (15-w). Number w
reflects the degree of dextran functionalization with BCN ligands (average number of BCN functionalities per polymer molecule, see Supporting
Information for details). (b) Reaction of 15-2.95 with model acyl imidazole 16. (c) 1H NMR (500 MHz, DMSO-d6, c = 11 mM) monitoring of the
reaction in panel (b) (diagnostic region 6.6 to 9.0 ppm). Three sets of signals (a, b and c as indicated in the structure) are well separated between
the starting material (16, designated as *) and the product (17, designated as #). Dotted lines are a visual aid for peaks indicating disappearance of
the starting material (16) signals over time. The bottom spectrum represents 1H NMR (500 MHz, DMSO-d6, c = 11 mM) of ω-N-acetylhistamine
(AcHA), the expected product of acyl imidazole decomposition.
Among these, ligand-directed acyl imidazole (LDAI) chemistry
developed by Hamachi and co-workers65 stood out as a highly
efficient labeling strategy which has been employed in covalent
labeling of membrane proteins in brain tissue.43,66 We
therefore sought to investigate whether the cleavable acyl
imidazole linker is compatible with a dextran polymer, a
potentially nucleophilic polyol.
the expected product of acyl imidazole decomposition, was
observed (Figure 7c). Acyl imidazoles covalently attached to
dextran are therefore stable in DMSO-d6 solution at room
temperature at least on the time scale of months. We then
removed the DMSO-d6 solvent under high vacuum, redis-
1
solved product 17 in D2O, and monitored its stability by H
NMR (Figure S20, Table S1). While formation of AcHA was
observed in the aqueous environment, a low extent of
decomposition was observed in aqueous environment on the
time scale of hours (16% decomposition at 3 h, 40% at 24 h).
We therefore concluded that acyl imidazoles covalently
attached to dextran are sufficiently stable and thus compatible
with a majority of labeling and imaging experiments.
VoLDeCAM Covalently Targets Voltage Sensitive
Dyes to AMPA Receptors in Brain Tissue. We employed
our VoLDe platform to covalently target the AMPA receptor
(AMPAR), a major ionotropic glutamate receptor in the
central nervous system, using the “Chemical AMPAR
Modification” (CAM) strategy developed by Hamachi and
co-workers (Figure 1d).43 Although AMPAR is broadly
expressed in many neuronal types, it represents an attractive
target due to its synaptic localization.67 The distribution and
localization of AMPAR is distinct from that of monoamine
transporters targeted by VoLDeMo probes, representing yet
another challenge for the targeting selectivity of the VoLDe
platform. Striatal slices colabeled with di-2-ANEP-VoLDeMo
(12) and CAM2(Fl) (a fluorescent probe composed of an
AMPAR selective PFQX ligand, an acyl imidazole linker and
The N3 functional group, compared to its BCN click
chemistry partner, is significantly more robust for incorpo-
ration into a broad range of molecular architectures, including
acyl imidazoles. We therefore decided to reverse the click
partners for these studies, using BCN-functionalized dex-
tran(6000) and N3-functionalized ligand and probe. We used a
similar strategy as introduced in Figure 2c to incorporate BCN
groups to the dextran polymer by a carbonate linkage (Figure
7a). While we were still able to achieve good control over
dextran coverage with BCN groups (w in compound 15-w),
only 72−84% of the introduced groups were available for
further functionalization by click chemistry (see Supporting
Information for detailed discussion of this phenomenon).
Next, we reacted 15-2.95 with model N3-functionalized acyl
imidazole 16 in DMSO-d6 (Figure 7b) while following the
reaction progress by 1H NMR (Figure 7c). The reaction
reached 68% conversion within 30 min when the first NMR
spectrum was taken and was fully completed within 6 h. The
reaction mixture containing product 17 sealed in an NMR
cuvette at room temperature was further monitored for 31 days
during which no formation of ω-N-acetylhistamine (AcHA),
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Figure 8. Synthesis of dsRVF5-VoLDeCAM and its targeting in brain tissue. (a) One-pot assembly of dsRVF5-VoLDeCAM (21) and the
corresponding ligand-free control probe (22) via click chemistry by reacting BCN-dextran 15-2.95 with a 1:3 mixture of VSD 18 and the PFQX-
AcIm ligand 19 or the dummy ligand 20, respectively. Note: Dextran branching is omitted for clarity. Linker substitution on dextran is shown only
in position 2. (b, d) Nissl stained sagittal slices of a mouse brain (image credit: Allen Institute61,62). Highlighted in red are brain regions−
somatosensory cortex (CTX) and dorsal striatum (dSTR) − from which images in panels c and e, respectively, were taken. (c, e) Representative
two-photon excitation microscopy images of cortical (c) and striatal (e) slices labeled with dsRVF5-VoLDeCAM. Left: slice incubated with the
targeted dsRVF5-VoLDeCAM (21; 71 nM dextran/50 nM VSD; 2 h; 30 μm depth). Middle: an analogous experiment with pre- and coincubation
with the AMPAR blocker NBQX (10 μM). Right: the corresponding slice incubated with the untargeted control probe 22 (71 nM dextran/50 nM
VSD; 2 h; 30 μm depth). Images within the same panel were contrasted equally. The probes were excited at 820 nm and emission collected at
570−610 nm. Scale bars are 20 μm.
fluorescein as a polar fluorophore)43 demonstrated the contrast
between the string-like pattern of DAT labeling and the
punctate AMPAR labeling (Figure S21).
We combined BCN-dextran 15-2.95 with N3-terminated
dsRVF5 VSD (18) and the PFQX ligand with a suitable acyl
imidazole linker43 (PFQX-AcIm, 19), providing the targetable
dsRVF5-VoLDeCAM probe (21, Figure 8a). The reaction was
carried out in DMSO-d6 with 1H NMR monitoring, using 0.95
equiv of total N3 (18 + 19) with respect to BCN (Figure S22).
Without excess reagents, the reaction mixture provided pure
product upon completion and could be directly used for
imaging. Similarly as for the VoLDeMo probes, we used 0.7
equiv of dsRVF5 with respect to dextran(6000) to avoid the
formation of polymer molecules carrying two or more VSDs. A
1:3 dsRVF5/PFQX-AcIm ratio was used to ensure sufficient
ligand coverage. Analogously, we also synthesized control
probe 22, containing a dummy ligand in place of PFQX-AcIm
(Figure 8a, Figure S23).
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Figure 9. Two-photon fluorescence recording of evoked activity in AMPAR-expressing cortical neurons. (a) Representative two-photon
fluorescence image of a cortical slice labeled with dsRVF5-VoLDeCAM (21; 143 nM dextran/100 nM VSD, 2 h, 35 μm depth from the tissue
surface). (b) Representative image of a single two-photon spiral scan frame from the recording of evoked activity in panels c and d, demonstrating
the field of view and spatial resolution. (c) Fluorescence trace recorded at 30 Hz from cortical slices labeled with dsRVF5-VoLDeCAM (21; 143
nM dextran/100 nM VSD, 2 h) with electric stimulation (25 pulses at 50 Hz, period highlighted in yellow). The trace is normalized to an
analogous experiment conducted in the presence of TTX (1 μM) and is an average from multiple trials (n = 50 from 5 slices). A two-point moving
average was applied to smoothen the trace. (d) Mean fluorescence ( SEM) of voltage recording from panel c during the stimulation and
prestimulation periods. dsRVF5-VoLDeCAM (21) exhibits a ΔF/F of (0.28 0.02)% (n = 50 from 5 slices, P < 0.0001, two-tailed unpaired t-test).
Figure 10. One-photon fluorescence voltage recording from patch-clamped cortical neurons. Mouse cortical slices were labeled with dsRVF5-
VoLDeCAM (21; 143 nM dextran/100 nM VSD, 2 h), and layer V cortical neurons were subjected to whole-cell patch clamp. (a) Schematic of the
stimulation profile used in this experiment. (b) One-photon fluorescence recording (50 Hz) of labeled cortical neurons subjected to the stimulation
profile in panel a (blue) or constantly held at a resting potential of −60 mV (gray). Depolarization period highlighted in yellow. Averaged traces
(n = 90 depolarization steps from 18 neurons) smoothened by a 5-point moving average. (c) dsRVF5-VoLDeCAM (21) exhibited a significant
fluorescence increase (ΔF/F per 100 mV = (0.36 0.06)%, n = 90 depolarization holdings from 18 neurons; mean SEM) during the 100 mV
depolarization step (yellow in panel b) compared to resting potential (P < 0.0001, two-tailed unpaired t-test). (d) An insignificant difference in
fluorescence was recorded during the corresponding timeframes when the neuron was constantly held at −60 mV (n = 90 periods from 18 neurons,
P = 0.11, two-tailed unpaired t-test). Error bars represent SEM. (e) For each neuron subjected to five 100 mV depolarization waves, average
fluorescence from the depolarization periods (red, also red in panel a) was compared to the average fluorescence from the resting periods (green,
also green in panel a). A blue line connects corresponding data points. For 17 out of 18 neurons, increase in fluorescence was observed. (f)
Analogous representation for neurons constantly held at −60 mV. Increase in fluorescence was observed in 7 cases, while decrease was observed in
11 cases.
As the CAM labeling strategy has been established in brain
tissue,43 we investigated the use of our VoLDeCAM probes in
mouse cortical and striatal slices. In contrast to the monoamine
transporters targeted by the VoLDeMo probes, AMPAR is
highly abundant in the nervous system. Consequently, a dense
punctate labeling pattern was observed upon labeling cortical
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or striatal neurons with dsRVF5-VoLDeCAM (21; 71 nM
dextran/50 nM VSD, 2 h; Figure 8b−e), comparable to that of
the control probe CAM2(Fl) (Figure S21).43 Potentially due
to the increased density of AMPAR, longer incubation times
(2 h) were required for sufficient penetration of dsRVF5-
VoLDeCAM (21) through brain tissue than for VoLDeMo
probes (30 min). The labeling was strongly diminished upon
coincubation with the AMPAR blocker NBQX or using the
ligand-free control probe 22, demonstrating ligand-dependent
delivery of dsRVF5 (Figure 8c,e).
experiment in the presence of 1 μM TTX (vide supra and
Figure S27).
dsRVF5-VoLDeCAM Enables One-Photon Fluores-
cence Recording of Voltage Holdings in Patch Clamped
Cortical Neurons. We then sought to employ dsRVF5-
VoLDeCAM (21) to obtain recordings of membrane potential
changes from single neurons under voltage clamp conditions.
We labeled mouse cortical slices with dsRVF5-VoLDeCAM
(21; 143 nM dextran/100 nM VSD, 2 h) and performed
whole-cell patch-clamp recordings of layer V pyramidal
neurons. We held the neuron at −60 mV in the voltage
clamp mode and subjected it to five 500 ms, 100 mV
depolarization steps to +40 mV (Figure 10a) while recording
one-photon fluorescence from an ROI surrounding the
neuronal body. We also performed control experiments
where neurons were constantly held at −60 mV for 7 s (the
entire duration of optical recording). Upon averaging record-
ings from n = 18 neurons, a clear increase in fluorescence
intensity was observed during the depolarization steps (Figure
10b,c), while no statistically significant change from baseline
was observed in the control experiment at constant potential
(Figure 10b,d). A depolarization-dependent increase in
fluorescence was observed in 17 of 18 neurons (Figure 10e),
while a 7/11 increase/decrease ratio was observed in the
control experiment at constant potential (Figure 10f). These
results demonstrate that VoLDeCAM probes can be used to
perform voltage recordings from individual neurons under
voltage clamp conditions. We note that AMPAR targeting
produces a dense label of neuropil surrounding the patch-
clamped neuron (see labeling pattern in Figure 8c) which
contributes strongly to the background and likely reduces the
magnitude of the signal.
Next, we investigated whether labeling with dsRVF5-
VoLDeCAM (21) affects the native function of AMPAR by
whole-cell patch-clamp recordings from layer V pyramidal
neurons in the prefrontal cortex. While selective labeling was
observed in this region when incubating cortical slices with
dsRVF5-VoLDeCAM (21) for 2 h at 10, 50, and 100 nM
(concentrations with respect to VSD, Figure S24), we
observed concentration-dependent attenuation of electrically
evoked excitatory postsynaptic currents (eEPSCs; Figure S25)
by 32%, 58%, and 86%, respectively, which persisted even after
washing (continuous perfusion with artificial cerebrospinal
fluid, ACSF) of the slice for 1 h. We also recorded spontaneous
excitatory postsynaptic currents (sEPSCs) of cortical slices
labeled with dsRVF5-VoLDeCAM (21). sEPSC frequency was
attenuated in a concentration-dependent manner (Figure
S26a,b,e), while sEPSC amplitude was affected only negligibly
(Figure S26c−e). This indicates that although partial blockage
of AMPAR occurs, the remaining receptors are fully functional.
Several factors could lead to such attenuation. Since 2.1 equiv
of PFQX ligand per dextran polymer have been used to
prepare dsRVF5-VoLDeMo (21), multivalent species are
formed which could block AMPAR with a second ligand
molecule even after covalent attachment. We also observed
that high concentrations (1 μM) of PFQX derivative S2
(Figure S26a) attenuated the sEPSC frequency under the same
loading conditions (2 h incubation at ambient temperature
followed by ACSF perfusion; Figure S26a). This suggests that
at high ligand concentrations, the internalization of AMPAR, a
well-established phenomenon,68 likely occurs. The ligand-free
control probe 22, loaded at high concentration which leads to
nonspecific membrane labeling (1 μM, 2 h), also partially
attenuated sEPSC frequency (Figure S26a). This suggests that
loading the cell membrane with dextran-bound dsRVF5 can
partially affect the excitability of cortical neurons in a ligand-
independent manner. Although dsRVF5-VoLDeCAM (21)
partially perturbs the native function of AMPAR in cortical
tissue, the receptors nevertheless retain a significant fraction of
their activity at probe concentrations that provide clear and
selective labeling.
DISCUSSION
■
Here, we introduce a targeting platform to enable the delivery
of lipophilic voltage sensitive dyes to specific neuronal cell
types in brain tissue without genetic manipulation. Our system
consists of three components combined into a macromolecular
sensor: (1) A fluorescent indicator of membrane potential
(lipophilic voltage sensitive dye). The classical electrochromic
di-2-ANEP36,37 and state-of-the-art dsRVF538,39 probes were
chosen as model VSDs. (2) A small molecule ligand that binds
strongly to the target membrane protein from the extracellular
side. The monoamine transporter ligand dichloropane40,41 and
the AMPAR ligand PFQX42 (the latter in conjunction with a
linker for ligand-directed acyl imidazole covalent labeling43)
were used. (3) A polar polymer carrier represented by the
polysaccharide dextran that compensates for the lipophilicity of
the VSD by dynamic encapsulation, thus limiting nonspecific
binding of the probe to cell membranes while enabling
diffusion through tissue.
dsRVF5-VoLDeCAM Enables Two-Photon Fluores-
cence Recording of Evoked Activity in Cortical
Neurons. Given the ability to record optical changes driven
by evoked activity in striatal dopaminergic axons with dsRVF5-
VoLDeMo (13), we tested whether dsRVF5-VoLDeCAM
(21) enables similar analyses from AMPAR-expressing cortical
neurons. We labeled mouse cortical slices with dsRVF5-
VoLDeCAM (21; 143 nM dextran/100 nM VSD, 2 h; Figure
9a). Electric stimulation (25 pulses at 50 Hz during a 0.5 s
period) while recording two-photon fluorescence images with
spiral scanning (Figure 9b) at 30 Hz resulted in an increase in
fluorescence with a ΔF/F of (0.28 0.02)% (n = 50 from 5
slices, P < 0.0001, two-tailed unpaired t-test; Figure 9c,d). The
trace shown in Figure 9c is normalized to an analogous
In contrast to previous approaches which were successful
only in targeting VSDs in cell23−25 or neuronal cultures26−28
while demanding high expression levels of genetically encoded
protein targeting modalities, we designed VoLDe probes to
target specific cell types in native brain tissue of wild-type
rodent brain slices and whole Drosophila brains using
endogenous membrane proteins at physiological expression levels.
The delivery of a relatively small number of dye molecules
dictated by native expression levels of the protein target poses
a great challenge in terms of both targeting selectivity and
voltage sensitivity.
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Remarkably, the level of selectivity observed with VoLDeMo
for dopaminergic axons in the striatum is comparable to that of
the genetically encoded GFP labeling under the control of the
TH promoter, and to the small molecule, highly hydrophilic
FFN102 probe.32 A similarly high selectivity was observed with
VoLDeCAM targeting AMPAR-expressing terminals in the
cortex and striatum. Although the binding selectivity of VoLDe
probes is governed by a high affinity ligand, the dextran
polymer carrier plays a crucial role in the targeting process.
The high polarity of the dextran polymer compensates for the
lipophilicity of the VSD, while its flexibility (and to some
extent amphiphilic character) likely enables dynamic encapsu-
lation of the dye, leading to relatively low background staining
and high diffusion through the brain tissue. Such dynamic
behavior will be studied further to optimize the targeting
capacity and tissue diffusion of small polymers. Especially
dsRVF5-VoLDeMo (13) will benefit from further optimization
of the polymer carrier to improve the labeling specificity in
highly challenging environments with sparse distribution of
target cells and projections (such as noradrenergic axons in the
cortex; see Figure 5f).
Although state-of-the-art synthetic voltage sensors are
superior to GEVIs in many photophysical parameters such as
voltage sensitivity,69,70 photostability,71 and photon output,72
VoLDe probes cannot compete with overexpressed GEVIs or
hybrid chemo-genetic tools73 in recording single action
potentials. Here, we provide an initial examination of the
limits of optical voltage imaging with synthetic sensors
delivered to protein targets at native expression levels. Both
VoLDeMo and VoLDeCAM probes were successfully used to
record electrically evoked activity in striatal dopaminergic
axons and cortical AMPA-expressing neurons, respectively,
after signal averaging. In addition, VoLDeCAM was used to
record depolarization steps in individual cortical neuronal
bodies after averaging multiple trials. These results are, to our
knowledge, the first examples of optical voltage recordings
from specific cell types in brain tissue targeted via a pure
chemical approach, without any genetic manipulation. This
proof of concept study delineates the boundaries of targeted
voltage imaging without the use of genetic manipulation and
provides a baseline for future improvement of sensors, imaging
software, and hardware to enable high fidelity optical voltage
recordings in wild-type species. Specifically, the VoLDe
platform suffers primarily from the low photon budget defined
by the small number of protein targets to which the probe is
delivered. Consequently, fast voltage imaging at kHz rates in
brain tissue is currently impossible and compromises in spatial
resolution were necessary to provide sufficient signal (see
Figure 6b, where a single pixel comprises 30 μm2, losing the
resolution of individual ∼1 μm2 dopamine release sites).
Significantly brighter VSDs as well as improved instrumenta-
tion enabling more efficient photon collection and noise
attenuation would promote the practical applicability of
VoLDe probes. Nevertheless, nongenetic targeting of probes
is desirable especially in models of brain disorders which often
require multiple genetic modifications,74 making further
manipulation in order to install GEVIs challenging. Overex-
pressed GEVIs can further face a variety of issues including
toxicity or increased membrane capacitance.16
for studying many physiological processes such as behaviorally
relevant changes in circuit activity. For example, midbrain
dopamine cell bodies fire tonically at ∼4 Hz with super-
imposed bursts at ∼20 Hz that are triggered by environmental
stimuli.75 The ability to measure changes due to burst firing
promises a means to image the physiologically appropriate
“language” of these neurons, and to do so from axons, the sites
responsible for neurotransmitter release, is a goal that to date
has eluded the field.
The modular design of dextran-based VoLDe probes offers
many possibilities in terms of the potential applications and
impact. Regarding the payload, we first focused on VSDs, since
these sensors report on membrane potential changesa
fundamental process in the nervous systemand are excep-
tionally challenging to deliver to specific cells and neurites due
to their high lipophilicity. In this sense, VSDs represent a high
bar for testing the targeting efficacy of the designed molecular
delivery constructs. Our findings indicate that the dextran
platform promises a means to accommodate a wide variety of
lipophilic cargo molecules. Imaging modalities such as
photoacoustic (PA) imaging20−22 and short-wave infrared
(SWIR) fluorescence imaging18,19 also utilize lipophilic
synthetic imaging agents, while few genetically encoded PA
probes76,77 and no genetically encoded SWIR probes have
been developed to date. The concept of the VoLDe targeting
platform may therefore offer a promising solution to cell
specific targeting of PA and SWIR probes in the brain. Our
platform may also be applied to include actuators, signaling
modulators, and drugs, thus unlocking chemical and chemo-
genetic approaches to cell specific imaging and pharmacol-
ogy.78
In terms of the cell-targeting component of VoLDe probes,
dichloropane and PFQX can be substituted for a variety of
ligands, unlocking the possibilities of targeting phenotype/
pathology-associated cell surface molecules. Furthermore, the
VoLDe system will be readily adaptable to chemo-genetic
targeting using covalent tagging strategies (e.g., SNAP-tag,79
Halo-tag,80 or TMP-tag81) by substituting the ligand for a
suitable electrophile. In the current version of the VoLDeMo
system, targeting DAT or NET by the dichloropane ligand
perturbs the experimental system by blocking these native
neurotransmitter transporters. This is a general problem with
receptor ligands, as the agonist/antagonist activity of these
compounds perturbs the system under study. We tackled this
issue in the VoLDeCAM system by employing a recently
developed ligand-directed acyl imidazole covalent labeling
approach,43 which we show is compatible with the potentially
nucleophilic dextran polymer carrier. Although full “trace-
lessness” was not achieved in VoLDeCAM, partial retention of
native AMPAR function was achieved under conditions which
provided clear and specific labeling with our sensor. We
hypothesize that improvement of the traceless labeling strategy
will be possible by developing more specific functionalization
of the dextran polymer, which will not lead to stochastic
multivalent species but rather well-defined assemblies contain-
ing one ligand molecule per polymer carrier.
For in vivo applications, a major issue is the delivery of
exogenous diffusible chemical probes to the brain. In small
laboratory animals, local injection into brain parenchyma is
feasible.34 In the long term, less invasive methods are required
for research in animals and to pave the way toward clinical
applications: one of the major rationales for developing
chemically targeted imaging and pharmacological agents.
In the present recordings with the VoLDe probes in brain
tissue, we record voltage changes that occur more slowly than
single action potentials (∼1 ms), specifically in the range of 10
to 1000 ms. This temporal regime could nevertheless be useful
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Article
These may include intrathecal administration that circumvents
the blood-brain barrier (BBB) or intravenous administration
coupled with BBB penetration approaches, based on either
temporary physical disruption of BBB (e.g., focused ultrasound
methods) or exploitation of native transporter systems.
In summary, VoLDe probes represent the first nongenetic
platform for targeting lipophilic voltage sensitive dyes to
specific neuronal cells and their axons in brain tissue. We show
that voltage recording from specific cell populations in wild-
type mammalian brain tissue is feasible and define the frontier
of optical voltage recording with probes targeted to
endogenous membrane proteins at their native expression
levels. Although many issues remain to be addressed in terms
of functional parameters and practical applicability of targeted
VSDs, small carbohydrate polymers show great promise as the
molecular platform for cell-selective imaging and pharmacol-
ogy. We have demonstrated the feasibility of a chemical
targeting approach that will be applicable to other lipophilic
sensors, actuators, or drugs.
Diana M. Martinez − Department of Psychiatry, Columbia
University, New York, New York 10027, United States
Claire E. Cheetham − Department of Neurobiology, University
of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
Keri J. Fogle − Department of Pharmacology & Chemical
Biology and Pittsburgh Institute of Neurodegenerative Diseases
(PIND), University of Pittsburgh, Pittsburgh, Pennsylvania
15260, United States
Michael J. Palladino − Department of Pharmacology &
Chemical Biology and Pittsburgh Institute of Neurodegenerative
Diseases (PIND), University of Pittsburgh, Pittsburgh,
Pennsylvania 15260, United States
Zachary Freyberg − Department of Psychiatry and Department
of Cell Biology, University of Pittsburgh, Pittsburgh,
Pennsylvania 15213, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c00861
Author Contributions
¶T.F. and J.W. contributed equally to this work.
METHODS
■
Notes
All experimental procedures are described in detail in the Supporting
Information.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
ASSOCIATED CONTENT
* Supporting Information
. The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c00861.
■
■
sı
This paper is dedicated to the memory of James H.
Handelman (The G. Harold & Leila Y. Mathers Charitable
Foundation). We thank Prof. Rafael Yuste (Department of
Biological Sciences, Columbia University) and Dr. Yuki Bando
(Department of Biological Sciences, Columbia University;
current address: Department of Organ and Tissue Anatomy,
Hamamatsu University School of Medicine, Japan) for their
generous help with electrophysiology experiments in cell
cultures. We also thank Christopher Hwu (Department of
Chemistry, Columbia University) for assistance with DAT
inhibition experiments, Matthew Ding (Department of
Chemistry, Columbia University) for assistance with com-
pound synthesis, and Dr. Brandon Fowler (Department of
Chemistry, Columbia University) for HRMS analysis. T.F. is
the recipient of the Alfred Bader Fellowship in Organic
Chemistry. This work was supported by the G. Harold & Leila
Y. Mathers Charitable Foundation (to D. Sames), The
National Institute of Mental Health (NIMH) of the National
Institutes of Health (NIH), grant R01MH122470 (to D.
Sames and D. Sulzer), the Collaborative and Multidisciplinary
Pilot Research (CaMPR) award (to D.M.M.), the National
Institute on Drug Abuse (NIDA) of NIH, grant R01DA07418
(to D. Sulzer) and the JPB Foundation (to D. Sulzer). The
work in Drosophila brain was supported by John F. and Nancy
A. Emmerling Fund of The Pittsburgh Foundation (to Z.F.),
start-up funds from the University of Pittsburgh Department of
Psychiatry (to Z.F.), start-up funds from the University of
Pittsburgh Department of Neurobiology (to C.E.C.), and
funding from the NIH: R21AG059386 (to M.J.P.),
R01GM108073 (to M.J.P.), and R21NS095614 (to M.J.P.).
Experimental details including synthetic schemes and
protocols, excitation and emission spectra, infrared
spectra, NMR spectra, protocols for biological character-
izations, and data analysis (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Dalibor Sames − Department of Chemistry and
NeuroTechnology Center, Columbia University, New York, New
York 10027, United States; orcid.org/0000-0001-6911-
2260; Email: ds584@columbia.edu
David Sulzer − Department of Psychiatry, Department of
Psychiatry, and Department of Pharmacology, Columbia
University, New York, New York 10027, United States;
Department of Molecular Therapeutics, New York Psychiatric
Institute, New York, New York 10032, United States;
Email: ds43@cumc.columbia.edu
Authors
Tomas Fiala − Department of Chemistry, Columbia University,
New York, New York 10027, United States
Jihang Wang − Department of Chemistry, Columbia University,
New York, New York 10027, United States
Matthew Dunn − Department of Chemistry, Columbia
University, New York, New York 10027, United States
̌
Peter Sebej − Department of Chemistry, Columbia University,
New York, New York 10027, United States; orcid.org/
0000-0003-0317-0630
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Ekeoma C. Nwadibia − Department of Chemistry, Columbia
University, New York, New York 10027, United States
Eva Fialova − Department of Chemistry, Columbia University,
New York, New York 10027, United States
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