Optical measurement of synaptic glutamate spillover
and reuptake by linker optimized glutamate-sensitive
Samuel Andrew Hires*†, Yongling Zhu‡, and Roger Y. Tsien§¶
*Graduate Program in Neurosciences and§Department of Pharmacology and Howard Hughes Medical Institute, University of California at San Diego,
9500 Gilman Drive, La Jolla, CA 92093; and‡Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road,
La Jolla, CA 92037
Contributed by Roger Y. Tsien, December 19, 2007 (sent for review November 16, 2007)
Genetically encoded sensors of glutamate concentration are based
on FRET between cyan and yellow fluorescent proteins bracketing
a bacterial glutamate-binding protein. Such sensors have yet to
find quantitative applications in neurons, because of poor re-
sponse amplitude in physiological buffers or when expressed on
the neuronal cell surface. We have improved our glutamate-
sensing fluorescent reporter (GluSnFR) by systematic optimization
of linker sequences and glutamate affinities. Using SuperGluSnFR,
which exhibits a 6.2-fold increase in response magnitude over the
original GluSnFR, we demonstrate quantitative optical measure-
ments of the time course of synaptic glutamate release, spillover,
and reuptake in cultured hippocampal neurons with centisecond
temporal and spine-sized spatial resolution. During burst firing,
functionally significant spillover persists for hundreds of millisec-
onds. These glutamate levels appear sufficient to prime NMDA
receptors, potentially affecting dendritic spike initiation and compu-
tation. Stimulation frequency-dependent modulation of spillover
suggests a mechanism for nonsynaptic neuronal communication.
fluorescence resonance energy transfer ? hippocampal neurons ?
tern of synaptic release and propagation would provide insight
into diverse brain processes, including synaptic crosstalk, cere-
bral ischemia, and mechanisms of learning and memory. In
hippocampal slices, synaptic glutamate spillover to the dendrite
and neighboring synapses induces homeostatic regulation of
glutamate release through extrasynaptic mGluR activation (1),
limits synaptic independence (2), lengthens EPSC durations (3),
and permits heterosynaptic LTP/LTD (4). Spillover is a primary
means of chemical neurotransmission between mitral cells in the
rat olfactory bulb (5) and between climbing fibers and molecular
layer interneurons in cerebral cortex (6). Estimates of glutamate
concentration and dynamics in synaptic, extrasynaptic, and ex-
tracellular compartments have been made by NMDAR antag-
onist displacement (7), glutamate uptake inhibitor application
(2), whole ‘‘sniffer’’ cells (8), outside-out ‘‘sniffer’’ patch elec-
trodes (9, 10), patch recording of astrocyte synaptically evoked
transporter currents (STCs) (11), enzymatically coupled elec-
trochemical probes (12), enzymatically coupled metabolite im-
aging (13), and other methods. Although each method provided
a new perspective on glutamate action, all were hampered by a
lack of resolution in the spatial or temporal domains because of
single-site measurement, reliance on partially coupled or con-
founded currents, desensitizing receptors, or indirect and slow
Recently, the glutamate reporters glutamate-sensing fluores-
cent reporter (GluSnFR)?(14) and fluorescent indicator protein
for glutamate (FLIPE) (15) were constructed by linear genetic
fusions of the glutamate periplasmic binding protein GltI (also
known as ybeJ) with enhanced cyan fluorescent protein (ECFP)
lutamate is the primary excitatory neurotransmitter in the
brain, and precise measurement of its spatiotemporal pat-
and a yellow fluorescent protein, Citrine (16) or Venus (17).
These reporters provide a sensitive optical readout of glutamate
concentration in vitro by FRET-dependent changes in the CFP/
YFP emission ratio. When expressed on the surface of hip-
pocampal neurons, synaptic glutamate release was detectable?
(14, 15). However, quantitative measurements of rapid gluta-
mate transients have been hampered by the low signal-to-noise
ratio of these sensors when used in physiological buffers and by
suboptimal glutamate affinity.
Intramolecular FRET reporter responses have been dramat-
ically improved by restricting the rotational freedom of the
GluSnFR response magnitude for neuronal measurements, we
performed a comprehensive, mammalian cell based screen of
linker truncations of linearly fused constructs, and circularly
permuted fluorescent protein substitution. To optimize sensor
affinity, we rationally mutated GltI residues known to coordi-
nate ligand binding. We have identified a greatly improved
variant of GluSnFR, SuperGluSnFR, which exhibits 44% change
in emission ratio upon glutamate binding with a dissociation
constant (Kd) of 2.5 ?M when expressed on the extracellular
surface of neurons.
We have used SuperGluSnFR to directly measure the time
course of glutamate propagation after synaptic release. We
demonstrate that submicromolar glutamate persists along the
dendritic surface for hundreds of milliseconds after coordinated
synaptic release. Spillover concentration is strongly modulated
by stimulation number and frequency. Active uptake and buff-
ering by neuronal and glial glutamate transporters appears
insufficient to prevent extrasynaptic NMDA receptor activation
after bursts of synaptic release. Furthermore, uptake transporter
capacity may regulate the dependence of extrasynaptic gluta-
mate signaling on action potential frequency and provide an
avenue for nonsynaptic neuron and astrocyte communication.
The initial construct design of GluSnFR bracketed the mature
length GltI domain with truncated ECFP (AA1–228) and full-
research; S.A.H. analyzed data; and S.A.H. and R.Y.T. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database [accession no. EU422995 (SuperGluSnFR)].
†Present address: 19700 Helix Drive, Janelia Farm Research Campus, Ashburn, VA 20147.
¶To whom correspondence should be addressed. E-mail: email@example.com.
?Hires SA, Zhu Y, Stevens CF, Tsien RY, Thirty-Fourth Annual Meeting of the Society for
Neuroscience, October 23–27, 2004, San Diego, CA.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
March 18, 2008 ?
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length Citrine (Fig. 1a). Later experiments anchored GluSnFRs
to the extracellular surface of mammalian cells by fusion to a
truncated PDGF receptor (Fig. 1b). Minimal linkers were used
to maximize interdomain rigidity. This first GluSnFR was des-
ignated GluSnFR0N0C, where the subscript indicates the amino
acids truncated from the N and C termini of mature GltI before
fusion. When dilute purified GluSnFR0N0Cin 50 mM Tris buffer
was excited at 420 nm, its emission spectrum showed two peaks
corresponding to CFP and YFP emissions, whose ratio was
modestly sensitive to glutamate (Fig. 1c). Trypsin digestion
extinguished the 526-nm YFP peak and raised the 476-nm CFP
peak by 15.2%, indicating a FRET efficiency of 13.2% [sup-
porting information (SI) Fig. 6a]. Application of increasing
glutamate increased the emission ratio from ?0.66 to ?0.77,
with a maximum ratio change (?Rmax) of 18% and apparent Kd
of ?150 nM (Fig. 1d). Replacing Tris buffer with HBSS reduced
?Rmaxto 7% (Fig. 1 c and d). Addition of NaCl to Tris buffer
caused a concentration-dependent reduction in ?Rmaxto 0% in
50 mM Tris plus 1 M NaCl (Fig. 1e). Substitution of 100 mM
Na-gluconate or KCl for 100 mM NaCl reduced ?Rmaxequally,
indicating a general effect of buffer ionic strength rather than
substrate interactions with a specific ion (data not shown).
Because neurons require buffers with ionic strength of ?0.15,
observed ?Rmax in mammalian cells with surface-displayed
reporters had been limited to ?10% (15).
Glutamate-dependent ratio changes were partially reversible
by glutamate deamination by glutamate-pyruvate transaminase
(SI Fig. 6b). Titrations with aspartate and glutamine gave Kd
values of ?700 nM and ?30 ?M, respectively, consistent with
fluorescently labeled GltI (21). Application of 1 mM serine,
arginine, and sucrose had no effect (data not shown).
The high glutamate affinity (150 nM Kd) of GluSnFR0N0Cmight
rates of ?108M?1s?1(22), implying a koffof ?15 s?1for this
GluSnFR that would filter the response decay to brief impulses of
glutamate. We reduced the glutamate affinity by site-directed
mutagenesis of key glutamate binding residues (21), which pro-
duced a range of glutamate Kd(T93A, E26A, and E26D were 300
nM; S73T was 2.5 ?M; R25K was 20 ?M; and E26R was 700 ?M).
Mutations are numbered from start of the mature GltI product.
S73T was preferred for neuronal measurements.
To maximize response magnitude, we screened a linker-
truncation library of GluSnFRs on the surface of mammalian
cells in HBSS. GluSnFRs with N- and C-terminal truncations of
GltI(S73T) (Fig. 2a) fused to a truncated PDGF receptor (Fig.
1 a and b) expressed spatially uniformly on the extracellular
surface of mammalian cells (Fig. 2b). Constructs were scored by
efficiency of membrane targeting and ratio change between zero
and saturating [glutamate] (Fig. 2c). The best mutant,
GluSnFR8N5C(S73T), had a dramatically lower glutamate-free
CFP/YFP ratio and a 44% average ?Rmax(n ? 18), a 6.2-fold
improvement over the 7.1% average of GluSnFR0N0C(Fig. 2 d
and e). We refer to this construct as SuperGluSnFR. In vitro tests
of soluble SuperGluSnFR demonstrated ?Rmaxof 46% and 34%
in 50 mM Tris and Ringer’s solution, respectively (Fig. 2f). The
be due to the increased conformational freedom of free solution.
Glutamate titration curves of SuperGluSnFR expressed on
HEK293 or HeLa cells demonstrated a 2.5 ?M apparent Kdand
a 1.0 Hill coefficient (Fig. 2g).
To test the selectivity of SuperGluSnFR for glutamate, a panel
of glutamate receptor agonists and antagonists consisting of 2,3-
2-amino-5-phosphonovaleric acid (APV), ?-amino-3-hydroxy-5-
methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartic
acid (NMDA), kainic acid (KA), (?/?)-trans-1-amino-1,3-
cyclopentane dicarboxylic acid (ACPD), and (RS)-?-methyl-4-
carboxyphenylglycine (MCPG) was sequentially applied to
SuperGluSnFR transfected HEK293 cells. No tested compound
?M NMDA, 25 ?M ACPD, and 250 ?M MCPG each caused a
Addition of 100 ?M DL-threo-?-benzyloxyaspartate (TBOA) or 1
mM ?-aminobutyric acid had no effect (data not shown).
We used SuperGluSnFR to detect glutamate on the surface of
cultured dissociated hippocampal neurons. After transfection, pro-
tein was localized on the extracellular surface of the neuron with
even distribution across dendritic spines and shaft and significant
intracellular fluorescence confined to the soma or occasional tiny
inclusions in the dendritic shaft (Fig. 3a). The glutamate affinity of
SuperGluSnFR in neurons was determined by bath changes of
Ringer’s solution (2 mM Ca2?, 1.3 mM Mg2?, 25 ?M NBQX, and
50 ?M APV) with increasing glutamate from 0 to 100 ?M and was
identical to the affinity in HEK293 cells (Fig. 2g). Background
TBOA in the titration buffer was required to prevent local deple-
tion of glutamate by astrocyte-mediated uptake (SI Fig. 7 a–d).
We tested the ability of SuperGluSnFR to resolve electrically
evoked glutamate transients with high spatial resolution. A brief
train of 10 field stimulations was delivered at 30 Hz while a small
segment of SuperGluSnFR expressing dendrite was imaged at
high (?150) magnification. Each pulse in the train was designed
to evoke a single action potential (AP) across all neurons (23).
During burst stimulation, a rapid transient increase in CFP/YFP
ratio was observed across all areas of the dendrite (Fig. 3a).
Addition of 100 ?M TBOA increased the peak response and
dramatically prolonged the recovery toward baseline glutamate
levels (Fig. 3b), indicating a glutamate-specific response. Low-
ering extracellular calcium to 0.1 mM and raising magnesium to
5 mM reversibly abolished responses to field stimulation, indi-
construction of GluSnFR for protein purification (Upper) or mammalian cell
surface display (Lower). His-6 is a hexahistidine protein purification tag. Ig-?
GluSnFR in ligand-free state. (c) In vitro emission spectra changes of soluble
GluSnFR0N0Cto 1 mM glutamate in 50 mM Tris buffer (black) or HBSS (red). (d)
Titration curves of soluble GluSnFR0N0Cin 50 mM Tris (black triangle) or HBSS
(red square). (e) Maximum ratio change of GluSnFR0N0C(circle) decreases with
increasing ionic strength of buffer.
In vitro characterization of prototype GluSnFR constructs. (a) Genetic
www.pnas.org?cgi?doi?10.1073?pnas.0712008105 Hires et al.
cating the glutamate source was synaptic release (data not
The average area of an active zone in hippocampal culture has
been estimated at 0.027 ?m2(24) contributing to ?2% of the
total dendritic surface area. Furthermore, the high speed of
intersynaptic glutamate diffusion (D ? 0.76 ?m2/ms) has been
predicted to produce a relatively smooth glutamate distribution
for timescales of ?5 ms (25). Even if a significant proportion of
the functional synapses are made directly on the dendritic shaft,
the GluSnFR signal, particularly when integrated ?33-ms inter-
vals, should primarily report glutamate levels arising from
synaptic spillover and pooling.
Because spike number may affect the spatial distribution of
spillover, we reduced the stimulus to a single AP. The signal to
noise ratio was insufficient to resolve individual responses
without significant spatial averaging. Therefore, we averaged 30
single AP stimulations. This response also had very broad spread
across dendrites, indicating that spillover affects spines and
extrasynaptic areas to a similar extent (Fig. 3c). Although the
trial-averaged spread was homogeneous, because of the stochas-
tic nature of synaptic release (?0.3 vesicles/synapse at 2 mM
Ca2?, 1.3 mM Mg2?) (26), there may have been a greater
heterogeneity on individual trials that we were unable to resolve
because of signal/noise or camera speed.
We assessed the temporal resolution of SuperGluSnFR by
high-speed imaging (770 fps) of glutamate transients evoked by
a single AP. Twenty-seven CFP/YFP traces of single AP stim-
ulation on a single neuron’s dendrite were averaged and cor-
rected by a fit bleach curve of a stimulation free trace (Fig. 4a).
Individual responses were clearly resolvable (Fig. 4a gray). The
trial-averaged ratio was converted to estimated glutamate con-
centration (Fig. 4b), using titration curves (Fig. 2g). The 20–80%
rise time was 6.6 ms, peak glutamate concentration was 720 nM
and the time to half decay was 40 ms from peak (Fig. 4b). Low
camera resolution and binning prevented a spatial analysis of the
For SuperGluSnFR to accurately report glutamate spillover
decay time course, the rate of ligand unbinding, koff, must exceed
the rate of decay of ligand at the neuronal surface. We were
uncertain how increasing the sensor Kdto 2.5 ?M would affect
the kinetics, so we estimated them, using a numerical model of
glutamate release and uptake. Total release amount and
GluSnFR binding constants were allowed to vary while holding
other parameters fixed. Glutamate release was assumed to be
instantaneous and homogeneous with minimal buffering from
GluSnFR. Because FRET ratio is a sublinear function of glu-
tamate concentration (Fig. 2g), spatial averaging may underes-
timate the concentration of the initial portion (?5 ms) of the
heterogeneously distributed synaptic release transient. Asyn-
chronous release, or buffering by GluSnFR itself would also
lower the apparent kinetic rates. Thus, the best-fit parameters of
kon? 3.0 ? 107M?1?s?1and koff? 75 s?1(SI Fig. 8a) serve only
as a lower bound. Even at this lower bound, SuperGluSnFR
kinetics are sufficiently rapid to capture the essential waveform
of spillover glutamate decay beyond the first 10 ms after synaptic
To determine how active glutamate reuptake regulates
spillover, we imaged glutamate during a set of six field
stimulation conditions. Trains of 1 AP, 10 AP–15 Hz and 10
AP–30 Hz were delivered in Ringer’s with or without 100 ?M
TBOA (Fig. 5a). TBOA increased the peak glutamate con-
centration during stimulation and greatly slowed the decay
back to baseline. In the single-AP case, TBOA increased the
average peak [glutamate] at 67 ms from 270 nM to 440 nM and
increased the time to half decay from 90 ms to 140 ms (Fig. 5b).
Peak glutamate levels, particularly for the single AP cases, are
likely underestimates because of the camera’s broad temporal
integration window. In the 10-AP cases, glutamate levels
reached an apparent steady state after 4AP of stimulation with
between ECFP and Citrine. Truncation sites are indicated with thin vertical lines. The truncations for the original GluSnFR0N0Cand best responding construct
GluSnFR8N5Care noted in bold lines. (b) FRET channel showing clean extracellular membrane expression on transfected HEK293 cells. (c) Map of the truncation
(d) (Left) HEK293 cells transfected with the nonoptimized GluSnFR0N0C. (Right) The best responder GluSnFR8N5C. Color bar values encompass the same range of
relative CFP/YFP change for both constructs. (e) Ratio change between glutamate free and 100 ?M glutamate solutions of HBSS for GluSnFR0N0C(n ? 9 fields)
titration curves of GluSnFR8N5CHEK/HeLa cells in Ringer’s (squares and black dashed line; n ? 20) and neurons in Ringer’s plus 100 ?M TBOA (diamonds and red
line; n ? 5). (Scale bars: 10 ?m.)
Optimization of GluSnFR response on mammalian cell surface. (a) Amino acid sequence of GltI N-terminal (Upper) and C-terminal (Lower) truncations
Hires et al.
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active uptake, while they continued to rise throughout the
entire TBOA stimulation. TBOA raised peak [glutamate] from
540 to 1,200 nM and 830 to 1,320 nM, whereas half decay
increased from 160 to 650 ms and 140 to 390 ms for 15 and 30
Hz respectively. Doubling stimulation frequency from 15 Hz to
30 Hz gave a 60% enhancement of peak [glutamate] with active
uptake, whereas only a 10% enhancement with uptake
blocked. Thus, active reuptake may serve multiple purposes, to
recycle glutamate and to permit AP frequency dependent
signaling by modulation of spillover concentration.
To explore the effect of our measured glutamate transients
on NMDA receptor activation, we made a simple numerical
model of glutamate dynamics for 2 s after onset of field
stimulation (Fig. 5c, SI Fig. 8 b–e, and SI Scheme 1). After a
single action potential, only 1.4% of NR2A, 2.4% of NR2B,
and 3.7% of NR2D receptors were doubly bound by glutamate
(Fig. 5d). However, the prolonged spillover from a burst of
10AP at either 15 or 30 Hz caused a sustained activation of 20
or 22% NR2A, 36 or 34% NR2B, and 50 or 45% of NR2D
Sensor Optimization. FRET sensor optimization is an area of active
research, with numerous recent reports on improved fluorescent
techniques. Optimization of GluSnFR demonstrates the delicate
sensitivity of FRET reporters to their constituents. The fitness
landscape of GluSnFR linkers was surprisingly peaked. A single
construct, GluSnFR8N5C, of the 176 tested was far superior to all
response as linkers were truncated, the process was nonmonotonic,
limiting the predictability of this design strategy. We attempted
systemic substitution of fluorescent proteins (FPs) and circularly
permuted FPs to improve response, including the fluorescent
protein variants ECFP-A206K, Cerulean (27), CyPet, YPet (28),
Venus (17) and cpVenus (M145, 157, 173, 195). Unexpectedly,
substitutions of improved components to linker-optimized GluSn-
FRs reduced either the quality of the reporter’s surface expression
or response magnitude. This is likely due to linker sequences being
already highly tuned for the specific chromophore orientations and
subtle electrostatic interactions of the GltI domain and the FP pair.
We inserted ECFP into the putative transmembrane loops of
GltI, similar to FLI81PE reported by Deuschle et al. (18). When
to glutamate, but this and all tested ECFP insertion mutants fail to
express properly on the surface of mammalian cells. Incorporation
of superfolding GFP mutations (29) into the inserted ECFP
improved folding and trafficking of many insertion mutants. How-
(Magnification: ?150.) (Scale bar: 10 ?m.) Spatially resolved percent ratio
change before (Left), during (Middle), and after (Right) a 10-AP, 30-Hz field
intensity-modulated. Temporal averages of 10 frames are indicated by bars
above the traces in 4b, spatially filtered with a 5-pixel wide (466 nm eq.)
two-dimensional wiener filter to reduce noise. (b) Averaged ratio change
TBOA (red). (c) Spatially resolved ratio change to average of 30 single AP
stimuli. (Far Left) FRET as in a (Upper Left to Lower Right) sequential frames
of response (33 ms per frame). Stimulation of one AP occurs with slight jitter
Spatial resolution of glutamate measurement after synaptic release.
CFP/YFP emission ratio of a SuperGluSnFR expressing dendritic arbor during
single action potential field stimulus. Individual traces are in gray; average
traces are in black (n ? 27). (b) Corresponding glutamate concentration
measurements after calibration.
Temporal resolution of SuperGluSnFR response. (a) Normalized
field stimulation in Ringer’s solution (solid line) or with 100 ?M TBOA added
(open shapes, dotted line) to block active glutamate uptake. (b) The decay
phase of a scaled to the maximum height to highlight the relative glutamate
to observed levels. Average SuperGluSnFR responses to field stimulation in
Ringer’s solution or in 100 ?M TBOA (black). Model of instantaneous gluta-
integration (red). (d) Model of glutamate double-bound NR2A (black), NR2B
(blue), and NR2D (red) NMDA receptors in response to 1 AP, 10 AP–15 Hz, and
10 AP–30 Hz stimulation in Ringer’s.
Time course of glutamate release and uptake. (a) Average responses
www.pnas.org?cgi?doi?10.1073?pnas.0712008105 Hires et al.
ever, our best-case ?Rmaxwhen expressed on cell surface was only
4% (data not shown). Further improvements of sensor response
may be possible by FP substitution or insertion but will require a
rescreening of many linker combinations for that pair.
Screening of single circularly permuted fluorescent protein
insertions into GltI may produce a single wavelength glutamate
sensor analogous to camgaroos (19). A single-FP GluSnFR
could be of practical use in more challenging preparations, such
as 2-photon in vivo imaging, when using multiple optical sensors
simultaneously or when quantitative calibration is not a priority.
SuperGluSnFR represents a major improvement over other
optical indicators of glutamate. No previous membrane-tethered
ratio change for glutamate or any other substrate. Another
recently reported glutamate sensor, EOS (30), requires purifi-
cation of recombinant protein followed by thiol-mediated dye
labeling and cell surface immobilization through biotinylation.
The apparent glutamate off-rate of EOS is on the order of
hundreds of milliseconds. In contrast, SuperGluSnFR is genet-
ically expressed, allowing cell-specific or subcellular targeting,
has been quantitatively calibrated, and has adequate response
size, sensitivity and kinetic rates to resolve single action poten-
tials. Future versions of SuperGluSnFR may be genetically
targeted to the active zone by fusion to specific synaptic proteins
or targeting motifs, raising the possibility of direct comparison
of synaptic vs. extrasynaptic glutamate dynamics. For synaptic
targeting, a GluSnFR variant with lower glutamate affinity, such
as R25K or E26, would be desired to prevent sensor saturation
in the synaptic cleft.
Functional Significance. Measurements of glial synaptic trans-
porter currents (STCs) indicate that the bulk of synaptically
released glutamate is rapidly buffered and internalized by glu-
tamate transporters. Estimates of spillover glutamate time
course, using STCs, primarily mediated by EAAT2 in the
hippocampus, have provided an evolving range of estimates of
clearance time in the low tens of milliseconds (31). Recently,
deconvolution analysis, using partially blocked STC time
courses, has suggested that the true clearance rate of glutamate
is faster than the STC decay rate, with an exponential ?decayof
5.8 ms in P12–14 and 0.75 ms in adult hippocampal slices (32).
to prolong NMDAR-mediated EPSCs by tens to hundreds of
milliseconds in a stimulus intensity-dependent manner (3). Pos-
sible mechanisms include increased activation of extrasynaptic
NR2B and NR2D subunit-containing receptors (33) or cooper-
ative action of glutamate at neighboring synapses (2). Further-
more, extended activation of NMDARs can trigger dendritic
calcium spikes in the basal dendrites of cortical and CA1
pyramidal neurons (34, 35). Priming of the NMDARs by residual
glutamate has been suggested as the activation mechanism, but
direct measurements of these glutamate transients have not been
SuperGluSnFR imaging may reconcile the discrepancy be-
tween STC estimates of millisecond glutamate clearance rates
and prolonged NMDAR activation. After bursts of electrical
stimulation, our imaging shows long, slowly decaying glutamate
transients persist across both dendritic spines and shaft. These
are of submicromolar concentration, but are sufficient to dra-
matically enhance NMDAR activation for hundreds of millisec-
onds in a stimulus strength-dependent manner (Fig. 5d). These
glutamate transients may be missed in STC recordings, because
STCs are confounded by a small slowly decaying potassium
conductance, which is difficult to perfectly compensate (31).
Given the estimated EAAT2 glutamate Kd of 18 ?M (36),
transporter currents at submicromolar extracellular glutamate
concentrations are probably small enough to be obscured by
errors associated with the potassium conductance. Although
SuperGluSnFR measurements are insensitive to the first few
milliseconds of spillover, because of sensor kinetics and heter-
ogeneous initial glutamate distribution, their calibration, sensi-
tivity, and spatial resolution provide a valuable complement to
the fine temporal resolution of STCs.
The ease of imaging SuperGluSnFR in a reduced culture
preparation forced some tradeoffs in potential physiological
relevance. Spillover decay time course may vary from culture to
brain slice because of differences in the density of synapses,
transporters and buffering agents, temperature, neuropil geom-
etry, and access to bath. We drove synaptic release with spatially
broad field stimulations to heterogeneous circuits, which caused
simultaneous release from many synaptic neighbors likely lead-
ing to pooling of extrasynaptic glutamate. In vivo, neuronal
activity is generally sparser, and glutamate uptake may be more
efficient, so spillover and NMDAR priming may be more
temporally and spatially constrained. Nonetheless, our imaging
results in culture are congruent with numerous observations of
the functional consequences of spillover in slice (2, 3, 34, 35).
Direct observation of glutamate propagation with GluSnFRs in
slice models (37) or via 2-photon imaging and/or microendos-
copy (38) in vivo would more directly address the extent and
significance of glutamate spillover.
Steady-state spillover concentration during burst stimula-
tion is strongly modulated by stimulation frequency (Fig. 5a).
This suggests that nonsynaptically connected neurons or as-
trocytes may be able to estimate the firing rate of neighboring
neurons by the degree of activation of high-affinity extrasyn-
aptic glutamate receptors. This could allow induction of a
measured amount of heterosynaptic LTD (4), homeostatic
regulation (1), or vasoregulation (39). These effects would be
sensitive to modulation of glutamate uptake by changes in
astrocyte membrane potential, internal glutamate concentra-
tion, or other means.
Linker-optimized GluSnFRs may be adapted for a number of
other neuronal applications beyond measuring synaptic spill-
over. The selectivity of GluSnFRs for true glutamate over
standard glutamatergic agonists and antagonists should facili-
tate dissection of feedback loops where GluRs modulate release
or uptake. SuperGluSnFR is currently being used in studies of
glutamate release from astrocytes. GluSnFRs could be used as
a calibration tool to assess the propagation of glutamate in
uncaging or iontophoresis experiments, as a specific marker of
presynaptic modulation in studies of LTP or LTD, or in mapping
the functional connectivity of the brain. More clinically relevant
uses may include GluSnFR imaging in the screening of drug
candidates for glutamate release, transporter or receptor mod-
ulation, or in models of glutamate excitotoxicity in cerebral
Materials and Methods
Sensor Construction and in Vitro Characterization. Genomic DNA from Esch-
erichia coli was isolated by chemical lysis and cartridge purification. GltI
was amplified from the genomic DNA by PCR and subcloned into the
bacterial expression vector pRSETB (Invitrogen). To make GluSnFR0N0C-
pRSETB, GltI was ligated into the AKAR2-pRSETBPKA reporter (40) at the
SphI and SacI sites between ECFP and Citrine. This construct was expressed
in bacteria, extracted by chemical lysis (B-PER II; Pierce), purified by 6-His
Ni-NTA gel column filtration (Qiagen), and dialyzed. Spectroscopy was
performed as described in ref. 41. Ligand-induced ratio changes were
assayed by progressive glutamate addition to an albumin-coated quartz
cuvette containing 10 nM GluSnFR in 50 mM Tris or HBSS (pH 7.40). For
mammalian cell surface expression, an ECFP-GltI fragment and Citrine
fragment were amplified from GluSnFR0N0C-pRSETBby PCR with new BglII
and PstI restriction sites flanking the N and C termini of the combined
fragment. These were triple ligated into a pDisplay vector (Invitrogen)
between the BglII and PstI sites to make GluSnFR0N0C-pDisplay.
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Library Construction and Screening. Affinity mutations were made in GltI-
pRSETB, using QuikChange (Qiagen); transferred to GluSnFR0N0C-pRSETBby
digestion and ligation at the SphI and SacI sites; and assayed in vitro as above.
GltI(S73T) was incorporated into GluSnFR0N0C-pDisplay by digestion and liga-
tion at SphI and SacI sites. Glutamate affinity was assayed by bath changes of
HBSS with increasing [glutamate] on transfected HEK293 and HeLa cells.
Preliminary large truncations of the N and C termini of GltI indicated that
deletions beyond the first putative ?-helix element of the N and C termini
(data not shown) caused misfolding. Therefore, the library was limited to 176
combinations of deletions of 0–15 aa of the N terminus and 0–10 of the C
terminus of GltI. Truncation combinations were amplified with Phusion poly-
merase (NEB) and purified with 96-well PCR cleanup cartridges (Qiagen).
Truncations were digested with SphI and SacI, ligated into the GSFR0N0C-
media. Two colonies of each transformation were cultured and miniprepped.
Proper insert length was checked for all by analytical restriction digests.
HEK293 or HeLa cells were seeded on 96-well culture plates, grown for one
day, and transfected with one of the 176 ? 2 minipreps. Two days after
transfection, glutamate responses we measured by thorough bath exchanges
with 0 and 100 ?M [glutamate] HBSS. Repeated optical measurements were
made on selected fields in each well with a 20? air objective and a motorized
stage. A random library sample and all large responders were sequenced.
0N0C, 8N0C and 8N5C truncations underwent confirmatory imaging in trans-
fected HEK293 cells on 12-mm coverslips in a Warner imaging chamber with
a 40? oil-immersion objective.
Additional Methods. See SI Text.
ACKNOWLEDGMENTS. We thank P. Steinbach for imaging assistance and M.
Lin, M. Ha ¨usser, C. Stevens, J. Isaacson, and J. Diamond for helpful discussions.
the Howard Hughes Medical Institute.
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