Fast GCaMPS for improved tracking of neuronal activity

Article (PDF Available)inNature Communications 4:2170 · July 2013with125 Reads
DOI: 10.1038/ncomms3170 · Source: PubMed
Abstract
The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca(2+)-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca(2+)-chelating residues. Second, we find that off-responses to Ca(2+) are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca(2+) and by slow binding to the C-lobe at lower Ca(2+). We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of KD, allowing coexpression of multiple variants to span an expanded range of Ca(2+) concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.
ARTICLE
Received 1 Feb 2013 | Accepted 19 Jun 2013 | Published 18 Jul 2013
Fast GCaMPs for improved tracking of neuronal
activity
Xiaonan R. Sun
1,2,
*, Aleksandra Badura
1,2,
*, Diego A. Pacheco
1,2
, Laura A. Lynch
1,2
, Eve R. Schneider
1,2
,
Matthew P. Taylor
1,2
, Ian B. Hogue
1,2
, Lynn W. Enquist
1,2
, Mala Murthy
1,2
& Samuel S.-H. Wang
1,2
The use of genetically encodable calcium indicator proteins to monitor neuronal activity is
hampered by slow response times and a narrow Ca
2 þ
-sensitive range. Here we identify three
performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator
protein. First, we find that affinity is regulated by the calmodulin domain’s Ca
2 þ
-chelating
residues. Second, we find that off-responses to Ca
2 þ
are rate-limited by dissociation of the
RS20 domain from calmodulin’s hydrophobic pocket. Third, we find that on-responses are
limited by fast binding to the N-lobe at high Ca
2 þ
and by slow binding to the C-lobe at lower
Ca
2 þ
. We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and
show that they have a 200-fold range of K
D
, allowing coexpression of multiple variants to
span an expanded range of Ca
2 þ
concentrations. Finally, we show that Fast-GCaMPs track
natural song in Drosophila auditory neurons and generate rapid responses in mammalian
neurons, supporting the utility of our approach.
DOI: 10.1038/ncomms3170
1
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
2
Neuroscience Institute, Princeton University, Princeton,
New Jersey 08544, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.S.-H.W.
(email: sswang@princeton.edu).
NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | www.nature.com/naturecommunications 1
& 2013 Macmillan Publishers Limited. All rights reserved.
I
maging of intracellular Ca
2 þ
has assumed a central role in
cellular physiology
1
. Until recently, Ca
2 þ
has been imaged
with small-molecule fluorescent indicator dyes (for example,
fura-2 and Oregon Green BAPTA-1), which must be loaded into
single cells by pipette or by bulk-loading with low contrast of cell
populations. More recently, a promising approach has arisen in
the form of genetically encodable calcium indicator proteins
(GECIs)
2,3
, which are engineered proteins consisting of (i) a
Ca
2 þ
-sensing domain derived from calmodulin or troponin, (ii)
a peptide domain that binds the Ca
2 þ
-sensing domain and (iii)
one or more XFP domains whose fluorescence properties are
modulated by the Ca
2 þ
-sensing interaction. GECIs allow cell-
type-specific and long-term expression, and have been used to
image neuronal circuitry in flies, worms, fish and mammals.
Although in recent years the brightness and stability of GECIs
have improved, several design challenges remain. First, leading
GECIs have slow response kinetics (typically t
on
¼ 20 ms—1.4 s
and t
off
¼ 0.4–5 s)
4–7
compared with BAPTA-based indicators
(t
on
o1 ms and for OGB-1, t
off
¼ 7 ms). Physiological Ca
2 þ
signals can rise within 1 ms and fall in 10–100 ms in small
subcellular structures
8
, indicating that slow intramolecular GECI
dynamics can limit the ability to resolve spike times and firing
rate variations. Second, GECI binding cooperativity is high
(n
H
¼ 3–4), so that fluorescence signals change over a narrow
range of Ca
2 þ
concentration. For these two reasons, a GECI that
can detect single APs is susceptible to saturation during
continuous firing; more generally, any given GECI is expected
to exhibit most of its brightness change within a proscribed range
of firing rates
4,6,7
. Individual GECIs also do not span the
0.1–10 mMCa
2 þ
range over which synaptic plasticity and
neurotransmitter release are regulated
9–11
.
Here we report the results of a targeted, conservative approach
for modifying
Green fluorescent protein/Calmodulin protein
sensor (GCaMP), a GECI with low degradation, high per-
molecule brightness and large fluorescence changes
5,7,12
. Using
GCaMP3 as a starting scaffold, we developed a library of GCaMP
variants with a range of affinities and response rates. We found
that our variants termed Fast-GCaMPs show faster responses to
calcium events in both Drosophila and mammalian neurons.
Results
Design principles. Our principal goal was to generate acceler-
ated-response GCaMP variants with a variety of affinities. How-
ever, we also wished to avoid unintended reductions in maximum
brightness (F
max
) and dynamic range (R
f
). We therefore selected
target residues for alteration that have not been previously
identified to be involved in GFP chromophore stabilization, and
either participate in direct Ca
2 þ
chelation or are at the interface
between the calmodulin (CaM) hydrophobic pocket and
its binding partner, smooth muscle myosin light-chain kinase
peptide RS20 (often incorrectly called M13).
CaM contains four EF-hand ‘loop’ domains
13
, each containing
up to six residues that form a coordination cage of three acid
pairs (X, Y and Z; Fig. 1b). These residues are known to strongly
influence binding affinity
14–18
. To regulate affinity we designed
modifications that increased the number of acidic residues,
altered the acid pairings or substituted loop residues
with homologous sequences from troponin C
19
(Fig. 1c,
Supplementary Table S1). For specific attempts to lower the
affinity, we removed one or more acidic chelating residues
via Asp-Asn and Glu-Ala substitutions (Supplementary
Table S1)
15,16,18,19
.
To speed the kinetics of responses to changes in calcium, we
targeted internal binding steps. Interaction between GCaMP’s
RS20 peptide domain and its CaM domain is required for
cpEGFP CaMRS20
GCaMP
X
Y
Y
X
Z
Z
RS20
R RKWNK TG HAVR AIGR LSS
XYZY X Z
D K DGD G T I T TKEFKEAFSLF LGTVMRS I
LQDMINEV FLTMMARKED A DGDG T I D FPII
III
IV
I
Loop
I REAFRVF LRHVMT N LD K DGNG Y I SAAE
VDEM I REA FVQMMTAKD I DGDGQV N YEE
Helix
CaM
Helix
R
f
Normalized fluorescence
Relative F
max
Wavelength (nm)
0
0.5 1 1.5
0
0
440
460
480
500
520
540
560
1
0.8
0.6
0.4
0.2
5
10
15
20
25
EF
RS
Zero Ca
2+
High Ca
2+
ex
= 497 nm
em
= 512 nm
GCaMP3
41
322
333369
373
412
414
374
406442
358395431
42
47
51
55
Figure 1 | Targeted alteration of GCaMP3. (a) GCaMP consists of a cpEGFP fluorophore (green) flanked by an N-terminal RS20 peptide (yellow)
and a C-terminal CaM (grey). (b) In the folded state (PDB: 3EVR), RS20 (yellow) and CaM (grey) form an extensive intramolecular interface. Calcium ions
are coordinated through six amino acids (red) of the EF-hand loop (blue sphere: water; green sphere: Ca
2 þ
ion). (c) Targeted residues in the primary
sequence of the RS20 (yellow) and EF-hand (grey) domains with residues at the RS20-CaM interface coloured in blue and residues in the EF-hand loop
coloured in red. (d) Mean excitation and emission spectra of Fast GCaMPs. (e) For all 51 variants, dynamic range (R
f
¼ F
max
/F
min
) and maximum brightness
(F
max
) relative to GCaMP3 (grey line).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
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& 2013 Macmillan Publishers Limited. All rights reserved.
GCaMP Ca
2 þ
sensing
20,21
. In studies of CaM dynamics,
disruptions to the peptide–CaM interaction lead to faster Ca
2 þ
dissociation
22
. Therefore, we generated point mutations that were
likely to disrupt the CaM–RS20 interface
23–25
(Fig. 1b,c).
Candidate GCaMP variants span a range of affinities. The 51
resulting variants were designated Fast-GCaMP-EF01 through
-EF31 (loop variants) and Fast-GCaMP-RS01 through -RS20
(CaM–RS20 interface variants). Variants had the same peak
excitation and excitation wavelengths as GCaMP3 (Fig. 1d;
l
ex
¼ 497 nm and l
em
¼ 512 nm in n ¼ 6 variants tested at high
Ca
2 þ
), as expected for changes to domains away from the GFP
core. To characterize variants in their originally synthesized form
we performed Ca
2 þ
titrations on purified protein to measure R
f
,
Ca
2 þ
dissociation constant (K
D
) and cooperativity (Hill coeffi-
cient or n
H
). Of these, 41 had a high/low calcium fluorescence
ratio (R
f
) of at least 6. Of those 41 variants, 18 had a maximum,
high-calcium brightness (F
max
) of at least 80% of that of GCaMP3
(Fig. 1e, Supplementary Table S3), for a yield rate of 18/51 ¼ 35%.
F
max
and R
f
were strongly correlated (Pearson’s correlation
r ¼þ0.85), indicating that these parameters were jointly altered
by perturbation of the high-fluorescence state. Changes in F
max
were accompanied by changes in both extinction coefficient and
estimated quantum yield (Table 1 and Supplementary Table 3).
One variant (Fast-GCaMP-EF20, K
D
¼ 6.1 mM) had a 1.37-fold
higher F
max
and a 1.09-fold higher extinction coefficient than
GCaMP3. In variants with altered F
max
, estimated quantum yield
tended to change to a greater extent than the extinction
coefficient (Table 1 and Supplementary Table S3). Sometimes
F
min
was also affected: one variant (Fast-GCaMP-EF15, K
D
¼ 1.4
mM) displayed a nearly 1.8-fold increase in R
f
through a
reduction in baseline brightness (Fig. 2a, Supplementary Table
S3).
Although full acidification of the XZ pairs in synthetic loop III
peptides has been reported to increase affinity
26
, in GCaMP3 this
change did not reduce K
D
(Fast-GCaMP-EF04, Supplementary
Table S3). Similarly, acidification of YZ pairs
18
did not increase
affinity when applied to loop II (Fast-GCaMP-EF02), loop IV
(Fast-GCaMP-EF03) or loops I and II (Fast-GCaMP-EF01,
Supplementary Table S3). Next we altered non-chelating
residues by recombining fragments of troponin C (TnC) with
the GCaMP3 CaM domain. In previous CaM–TnC chimeras,
replacements within the C-lobe (loops III and IV) increased
affinity
19,27
and accelerated off-binding
19
. To avoid interfering
with RS20 interactions, we avoided modifying the CaM helix
domains and only substituted up to six TnC residues in loop III
(Fast-GCaMP-EF05, residues 397-399; Fast-GCaMP-EF06,
residues 397-399 and 403-405, Fig. 1c, Supplementary Table
S1). Fast-GCaMP-EF06 was unchanged in affinity, but Fast-
GCaMP-EF05 showed a 1.6-fold improvement (K
D
¼ 155
±
7 nM,
95% CI, Supplementary Table S3) and reduced cooperativity
(n
H
¼ 2.0
±
0.3, 95% CI). Among all loop mutants, K
D
values
spanned a range from 0.16 to 6 mM (Fig. 2a,b), permitting the
monitoring of a wide range of [Ca
2 þ
]
free
.
Mutation of X- and Z-pairs has previously been shown to
influence magnesium affinity
15
. In Fast-GCaMP-EF05, -EF20 and
-RS06 variants, an IC
50,Mg
of 0.3–40 mM Mg
2 þ
was needed to
reduce fluorescence by 50%. The ratio IC
50,Mg
/K
D,Ca
was 2,000–
6,000, comparable to values of 2,000–4,000 for GCaMP3 and
GCaMP5G. Thus, in our variants the calmodulin domain’s ionic
selectivity remained intact.
The N-lobe is needed for a functional probe. To further explore
the participation of acid pairs in binding and fluorescence change,
we modified one loop at a time by progressively more disruptive
changes: (1) introducing three acid pairs (substitution with Asp);
(2) creating half-pairs by neutralizing acidic residues; (3)
neutralizing þ position residues and (4) neutralizing all acidic
residues (Fig. 2c, Supplementary Tables S1 and S3). We found
that all changes increased K
D
, while R
f
decreased only with dis-
ruptions to the N-lobe (Fig. 2c, Supplementary Table S3). When
all acidic residues were neutralized, the average R
f
of the N-lobe
mutants was 1.9
±
0.2 (mean
±
s.d.), compared with 12.6
±
5.2 for
C-lobe mutants (Supplementary Table S3). In addition, at the
N-lobe variant with highest K
D
(6.1 mM, EF20), R
f
was reduced.
In summary, strong calcium binding to loops I and II in com-
bination was necessary to give a functional GECI.
Table 1 | Biophysical properties of selected novel GCaMP3 variants.
Variant Mutations K
D
(lM) Relative F
max
R
f
Decay t
1/2
(ms) e
490nm
(M
1
cm
1
)
*
QY
w
OGB-1 0.24 14.0 5
GCaMP3 ref. 5 0.25 1.00 12.0 150 34,700
±
2,000 0.65
GCaMP5G T302L/R303P/D380Y 0.41 1.26
±
0.06 34.4 154 44,900
±
1,700 0.63
±
0.04
Loop variants
EF05 D397N/G398A/N399D 0.16 0.63
±
0.03 6.8 183 35,600
±
3,500 0.40
±
0.02
EF15 D431N/D433N/D435N 1.37 21.8 80
EF16 D431N/D433N/D435N/E442A 1.99 16.2 52
EF18 D435N 3.39 12.3 22
EF20 D362N/D366N 6.12 1.37
±
0.09 13.8 35 37,700
±
2,000 0.82
±
0.05
CaM–RS20 interface variants
RS06 M374Q 0.31 0.50
±
0.03 6.5 34 31,000
±
2,200 0.36
±
0.02
RS05 M373Q 0.47 8.5 27
RS08 T412Q/L414T 0.63 10.0 23
RS09 L414T 0.69 0.71
±
0.04 9.5 25 33,000
±
1,400 0.49
±
0.03
RS20 domain variants
RS12 DR40/DR41/DK42 0.90 11.1 22
RS14 DG47 1.19 10.7 10
RS15 DG47/DH48 1.78 10.6 7
K
D
(mM), e
490nm
, and relative F
max
were measured at 25 °C (pH 7.20) and decay t
1/2
(ms) was measured at 37 °C.
*Determined in 200 mM free Ca
2 þ
.
wValues normalized to GCaMP3 (ref. 7).
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& 2013 Macmillan Publishers Limited. All rights reserved.
Disruption of CaM–RS20 interactions accelerates kinetics.To
characterize response kinetics, we used stopped-flow fluorometry
with B1 ms steps in Ca
2 þ
concentration (Fig. 3a). For a step
down in free [Ca
2 þ
] from 10 mM to zero (o10 nM) (Fig. 3b),
most variants responded with a double exponential time course
(Supplementary Tables S4 and S5). We measured the decay half-
life (t
1/2
), as 150 ms (37 °C) and 344 ms (25 °C) for GCaMP3
and 5 ms (37 °C) for Oregon Green BAPTA-1 (OGB-1). Decay
responses of GCaMP5 variants
7
are comparable with those of
GCaMP3 (Supplementary Fig. S2A, D).
For Fast-GCaMP-EF variants and GCaMP3 itself, we observed
an approximately reciprocal relationship between K
D
and t
1/2,decay
(25 °C, Fig. 3c, curve), reminiscent of the close relationship
between affinity and off-binding observed for BAPTA-based
indicators
28
. These responses were comparable to those of
GCaMP3 and GCaMP5 variants (Supplementary Fig. S2A).
However, Fast-GCaMP-RS variants did not follow this
relationship (Fig. 3c), instead showing decay responses up to
6.5-fold faster (Fig. 3b, c) than EF variants of a comparable K
D
(for decay t
1/2
values measured at 37 °C see Supplementary Fig. S1
and Supplementary Table S5). Mutations at the CaM surface of
the interface (M373Q, RS05 and M374Q, RS06, Fig. 3c,
Supplementary Table S2) accelerated off-responses by 5.5- and
4.4-fold, respectively, without significant loss of affinity or R
f
.In
the RS20 domain, the L414T mutation (Fast-GCaMP-RS08 and
Fast-GCaMP-RS09, Fig. 3c, Supplementary Table S2) accelerated
the decay response while increasing K
D
by 2.5-fold.
Rise times were also accelerated in RS mutations. The rise t
1/2
decreased with increasing D[Ca
2 þ
] (Fig. 3d). In comparison to
GCaMP3, RS06 showed a 60% increase in rise rate at D[Ca
2 þ
]
¼ 100 nM and a 100% increase at D[Ca
2 þ
] ¼ 570 nM, indicating
faster on-responses at physiological concentrations. No improve-
ments in rise responses were detected in GCaMP5 variants
7
(Supplementary Fig. S2B,C). For calcium concentrations above
(and sometimes below) 2 mM, on-responses were faster than the
dead time (B1 ms) of the instrument (Fig. 3e). In summary,
hydrophobic residues at the CaM–RS20 interface are rate-limiting
in both on- and off-responses. For monitoring in vivo Ca
2 þ
transients we selected RS05, RS06, RS08 and RS09 (Table 1).
Several features of the on-responses indicated the presence of a
combination of fast and slow processes (Fig. 3e): first, rise
responses at all values of D[Ca
2 þ
] had at least two exponential
components; second, rise kinetics were not saturated at
concentrations for which equilibrium fluorescence was near-
maximal and third, the first data point after the mixing dead time
(B1 ms) was increasingly elevated from baseline with increasing
values of D[Ca
2 þ
]. For example, for GCaMP3, within the dead
time the fluorescence change was 10% complete at D[Ca
2 þ
] ¼
K
D
¼ 250 nM, 50% at D[Ca
2 þ
] ¼ 1 mM and 65% at D[Ca
2 þ
]
¼ 10 mM (Fig. 3f). Similar observations were made for all tested
variants, with half-maximal amplitudes of the pre-dead-time
phase appearing at concentrations of 41 mM. These observations
are consistent with the existence of a rapid low-affinity binding
step that can drive transition to a high-fluorescence state.
Imaging sensory-evoked Ca
2 þ
activity in Drosophila . As our
first test of in vivo performance, we expressed variants in
Drosophila melanogaster (Fig. 4a) and optically monitored
responses to sound stimuli along the antennal nerve, in a subset
of mechanosensory neurons (Johnston’s organ neurons, JONs;
Fig. 4a,b). JON population activity as assessed by field potential
recording is highly reproducible between stimulus trials
29
.We
analyzed small regions of interest (ROIs) comprising B5 axons
per ROI. We used two types of song stimuli: a 10-s natural
courtship song, containing both sine and pulse song (Fig, 4c), and
synthetic song pulse trains (Fig. 4h).
We expected that on average, higher-affinity variants would
generate larger signals to the same courtship song. For Fast-
GCaMP-EF05 and another high-affinity variant, Fast-GCaMP-
RS06 (Fig. 4d), more ROIs showed measurable responses than
GCaMP3 and the overall distribution of responses was shifted to
larger values of peak DF/F
0
(Fig. 4e; Fast-GCaMP-RS06,
P ¼ 0.0025; Fast-GCaMP-EF05, Po10
6
, Kolmogorov–
Smirnov test). Low-affinity variants (Fast-GCaMP-RS09, -EF15
and -EF18) generated little to no fluorescence increase, whereas
the highest-affinity variant (Fast-GCaMP-EF05) outperformed
GCaMP3 by 3.7-fold (Fig. 4f) and GCaMP5G by 2.4-fold. In
summary, Fast-GCaMP variants retained their calcium-sensitive
reporting properties in the form of increased fluorescence change.
To test the song response speeds for two variants (Fast-
GCaMP-EF05 and Fast-GCaMP-RS06), we estimated the rising
and falling t
1/2
in ROIs with a response signal-to-noise ratio
(SNR) of at least 2. Compared to GCaMP3 and GCaMP5G, Fast-
GCaMP-RS06 performed 3–4 times faster, whereas Fast-GCaMP-
EF05 performed only 1.4–1.6-fold faster (Table 2 and Fig. 4g).
Rise and decay times for the new variants tended to be less
variable (Fast-GCaMP-RS06: CV
rise
¼ 0.36, CV
decay
¼ 0.40; Fast-
GCaMP-EF05: CV
rise
¼ 0.23, CV
decay
¼ 0.35) than for GCaMP3
(CV
rise
¼ 1.02, CV
decay
¼ 0.45) and GCaMP5G (CV
rise
¼ 0.31,
CV
decay
¼ 0.68).
R
f
R
f
012345678
0
5
10
15
20
25
K
D
(μM)
GCaMP3
EF15
EF16
EF18
EF20
Log
10
([Ca
2+
]/M)
OGB-1
EF05
EF16GCaMP3
RS06
RS09
EF20
EF15
–8
–7.5
–7
–6.5
–6
–5.5
–5
–4.5
0
5
10
15
20
EF05
GCaMP3
OGB-1
RS06
EF15
EF16
EF20
RS09
ΔF/F
0
0
5
10
15
20
25
Three
acid
pairs
No
paired
acids
No +
position
acids
No
acids
Loops III/IV
GCaMP3
Loops I/II
Loops III/IV
Loops I/II
Figure 2 | Equilibrium fluorescence properties of purified GCaMP3 variants. (a)Ca
2 þ
titration of GCaMP3, OGB-1 and six novel GECIs. Solid curves
represent fits to the Hill equation. Horizontal bars represent the Ca
2 þ
-sensitive range (5–95% of total fluorescence change). (b) Dynamic range as a
function of K
D
. Each data point represents one variant. Open circles: N-lobe variants. Closed circles: C-lobe variants. (c) The dependence of the dynamic
range on the combination of loop acidic residues and the EF-hand site. Three acid pairs: all residues at chelating positions are acidic residues. No paired
acids: one/two acidic residues are removed to eliminate acid pairs. No þ position acids: acidic residues at þ X/ þ Y/ þ Z positions are neutralized to N/N/
N (N: Asparagine). No acids: all Asp and Glu are replaced with Asn and Ala, respectively.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
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We also tested responses to trains of 10 song pulses with
240 ms interpulse intervals (IPI) and 20 song pulses with 120, 60
and 30 ms IPI (Fig. 4h). In ROIs that responded (SNR42) to low
frequency stimulation (10 pulses at 240 ms IPI), the peak DF/F
0
was 3.8
±
1.3% for GCaMP3 (mean
±
s.d., n ¼ 2 responding out of
45 ROIs), whereas the peak of Fast-GCaMP-EF05 and Fast-
GCaMP-RS06 responses were 15.9
±
6.0% (n ¼ 28 responding out
of 35 ROIs) and 14.0
±
3.7% (n ¼ 12 responding out of 34 ROIs)
respectively, comparable to GCaMP5G (15.4
±
5.8%, n ¼ 27
responding out of 92 ROIs; Fig. 4i). Such a performance
improvement over GCaMP3 for stimuli spaced at 240 ms is
consistent with the high affinity and the faster response of the two
Fast-GCaMP variants. Responses to short, high-frequency pulses
(20 pulses at 30 ms IPI) were also larger for the new variants and
GCaMP5G (Fast-GCaMP-EF05, 35
±
16%; Fast-GCaMP-RS06,
39
±
22%; and GCaMP5G, 37
±
24% versus GCaMP3, 11
±
5%;
mean
±
s.d.; Fig. 4i), with Fast-GCaMP-RS06 showing an
average off-response time of t
1/2
¼ 0.25
±
0.01 s (Supplementary
Figure S3). Lastly, we measured responses to single synthetic
pulses and found that none of the GECIs tested generated
measurable signals. Thus, high affinity (Fast-GCaMP-EF05) and
fast response (Fast-GCaMP-RS06) were associated with improved
responses to both natural song and synthetic song pulse trains.
Characterization of Fast-GCaMPs in mammalian neurons.As
our second in vivo functional test, we used two preparations to
assess performance in mammalian neurons (Fig. 5a and Table 2).
Rat superior cervical ganglion neurons (SCGN) were cultured for
9–14 days before infection with the common neuroanatomical
tracing strain pseudorabies virus (PRV) Bartha-expressing
GCaMP3 or Fast-GCaMP-EF05, -RS05 or -RS09. Responses to
extracellular stimulation were imaged from axonal protrusions
using two-photon microscopy in line scan mode (2 ms per line).
Normalized
response
0
0.5
1
0
0
10
[Ca
2+
]
(μM)
50 100
Time (ms)
150
GCaMP3
EF25
EF19
RS06
RS08
OGB-1
0
0.1
0.2
0.3
0.4
0.5
0.6
Decay t
1/2
02468
K
D
(μM)
EF
RS
GCaMP3
OGB-1
Excitation
Loading
ports
Syringe A
Syringe B
Observation
cell
Rise t
1/2
(s)
[Ca
2+
] step (μM)
GCaMP3
EF05
RS06
0
0.2 1
0.4
0.8
1.2
1.6
0
2
4
6
8
Time (s) Time (ms)
0 0.5 1
0
2
ΔF/F
0
Δ[Ca
2+
]
(
μ
M)
0
10
20
0
0.2
0.4
0.6
0.8
1
Fraction of signal
Δ
[Ca
2+
] (μM)
0.1 1 10
GCaMP3
EF06
RS09
EF09
Dead time
Dead time (~1 ms)
Post-mixing
Post-mixing
Figure 3 | Stopped-flow measurement of calcium off- and on-responses from Fast-GCaMPs. (a) Stopped-flow fluorimeter. (b) The fluorescence
decay response of selected variants at 37 °C to a step in [Ca
2 þ
]
free
from 10 mMtoo10 nM. Traces are scaled to the baseline by bi-exponential fit and to
the maximum fluorescence intensity at [Ca
2 þ
]
free
¼ 10 mM. (c) Relationship between off-response t
1/2
and K
D
(at 25 °C) for variants with mutations
at the loop domain (EF variants) or at the RS20–CaM interface (RS variants). The solid line represents a reciprocal curve through the GCaMP3 data point.
(d) Calcium-dependence of the rise responses (t
1/2
) for GCaMP3, EF05, and RS06 at 25 °C. Values smaller than the dead time are plotted as 1 ms.
(e) Rise response traces of GCaMP3 to different sizes of [Ca
2 þ
] steps from 0. Data are shown at 1-s (left) and 20-ms (right) timescales. The first data
point of each DF/F
0
trace represents the magnitude of fluorescence change during the dead time. The magnitude of DF/F
0
during the post-mixing phase is
the difference between A
0
(estimated through bi-exponential fitting) and the first recorded point. (f) The magnitude of the signal change during the
pre-mixing/dead time (black) and post-mixing (grey) phases as a fraction of the full signal amplitude.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170 ARTICLE
NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | www.nature.com/naturecommunications 5
& 2013 Macmillan Publishers Limited. All rights reserved.
Mouse layer 2/3 (L2/3) pyramidal neurons were imaged in brain
slices from 14–21-day-old mice following embryonic in utero
electroporation of GCaMP3, GCaMP5G or Fast-GCaMP-EF05,
-RS06 or -EF13. Action potentials were evoked by current injection
in whole-cell patch recordings (typical responses, Fig. 5b,c).
In mammalian neurons, peak DF/F
0
scaled inversely with
in vitro K
D
(Fig. 5d), with the largest responses for the highest-
affinity variant (Fast-GCaMP-EF05, n ¼ 10 SCGN and n ¼ 7 L2/3
pyramidal neurons) and no detectable response for the EF13
variant (n ¼ 6 neurons), similar to performance in Drosophila
(Fig. 5d, curve). The peak DF/F
0
for GCaMP5G (n ¼ 5 neurons)
was variable but on average greater than both GCaMP3 and
Fast-GCaMP variants. In L2/3 neurons expressing GCaMP3,
GCaMP5G, Fast-GCaMP-EF05 and Fast-GCaMP-RS06, we
observed a strong correlation between number of evoked action
potentials and peak fluorescence (Fig. 5c).
To quantify response times in both SCGN (Fig. 5e) and L2/3
neurons (Fig. 5f), we estimated time to first response (t
response
)
and decay t
1/2
(Table 2). We calculated t
response
as the time to
reach an SNR of 42. In SCG neurons, Fast-GCaMP-EF05, -RS05
and -RS09 all had response and decay times shorter than
GCaMP3 and GCaMP5G. In L2/3 neurons, Fast-GCaMP-RS06
and Fast-GCaMP-EF05 also had shorter response/decay times
than GCaMP3 and GCaMP5G, with the exception of the EF05
response time. Taken together, our results show that the increased
sensitivity and speed of Fast-GCaMP-EF and Fast-GCaMP-RS
Pulse trains
Song response
Speaker
5 μm
20 pulses at 30 ms
20% ΔF/F
0
10% ΔF/F
0
1 s
1 s
10 pulses at 240 ms
GCaMP3
EF05
RS06
GCaMP5G
30 ms IPI
240 ms IPI
Peak signal (ΔF/F
0
)
0
0.5
1
0
0.2
0.4
GCaMP3
EF05
RS06
GCaMP5G
Responding ROIs
0.2
0
0.4
0.6
0.8
1
Response time (s)
Rise
Deca
y
0
1
0.5 0.5
2
5
1
2
5
0
GCaMP3
EF05
RS06
GCaMP5G
GCaMP3
EF05
RS06
GCaMP5G
GCaMP3
EF05
RS06
GCaMP5G
RS06
EF05
GCaMP3
Cumulative fraction
Peak ΔF/F
0
0123
0
0.5
1
0 0.5 1 1.5
0
1
2
Peak ΔF/F
0
Cuvet K
D
(μM)
GCaMP5G
GCaMP3
EF05
EF15
RS08
RS06
{
*
GCaMP5G
1 s
RS06 EF05
GCaMP3
GCaMP5G
Courtship song
Normalized ΔF
0
1
2-P
Figure 4 | Responses of Fast-GCaMPs in Drosophila. (a) Two-photon imaging of responses to sound in Drosophila antennal nerve. (b) Expression of the
EF05 variant in antennal nerve axons 2 days after eclose (scale bar, 5 mM). (c) Normalized example responses to D. melanogaster courtship song. Full scale
corresponds to a DF/F
0
range of GCaMP3 70%, GCaMP5G 60%, EF05 100%, and RS06 50%. ( d) Fraction of responding ROIs (‘*’ represents Po0.005 by
Fisher’s exact test, GCaMP3, n ¼ 95 ROIs, four animals; GCaMP5G, n ¼ 92 ROIs, seven animals; EF05, n ¼ 83 ROIs, three animals; RS06, n ¼ 56 ROIs, 3
animals; error bars, s.e.m.). (e) Cumulative distribution of peak fluorescence amplitudes (peak D F/F
0
). (f) Peak DF/F
0
. The dependence of peak DF/F
0
on K
D
(black line) is calculated using n
H
¼ 3 (error bar: s.e.m.; RS08: filled black circle; EF15: open black circle). (g) Rise (left) and decay (right) times (t
1/2
)of
song-responsive ROIs for GCaMP3 (rise, n ¼ 53 ROIs; decay n ¼ 39 ROIs), GCaMP5G (rise, n ¼ 72 ROIs; decay, n ¼ 69 ROIs), EF05 (rise, n ¼ 63 ROIs;
decay, n ¼ 54 ROIs) and RS06 (rise, n ¼ 46 ROIs; decay, n ¼ 34 ROIs). (h) Example fluorescence responses to trains of sound pulses (black). (i) Responses
(DF/F
0
) to sound pulses at 33 Hz (left, 30 ms IPI) and 4.2 Hz (right, 240 ms IPI). Line segments represent means. GCaMP3 (grey), GCaMP5G (black), EF05
(cyan) and RS06 (red).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
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& 2013 Macmillan Publishers Limited. All rights reserved.
variants are associated with improved reporting performance
when expressed in situ.
Discussion
By making targeted changes in Ca
2 þ
-sensing components of
GCaMP3, we have generated a series of variants termed Fast-
GCaMPs. Fast GCaMPs respond to Ca
2 þ
with up to 20-fold
improved kinetics and have affinities spanning the range of
intracellular neuronal Ca
2 þ
signals while retaining their per-
molecule brightness.
Recent research leading to improved GCaMP variants involved
screening thousands of mutants generated by exhaustive
mutagenesis to yield improvements in proteolytic stability and
per-molecule fluorescence
5,7
. Another effort has led to similar
brightness improvements and modest kinetic improvements
7
.
Although a combinatorial approach can be effective at
maximizing one parameter at a time, parameters such as K
D
,
decay response and rise response present a challenge because they
are often linked to one another. Our results demonstrate that
functional parameters of a GECI can be engineered without
losing existing beneficial features, and can lead to kinetic
Table 2 | In vivo response times of Fast-GCaMPs.
Variant Protein Drosophila Mammalian
t
1/2
(off) SCGN L2/3
25 °C37°C t
rise
t
decay
t
response
t
decay
t
response
t
decay
GCaMP3 344 150 946
±
133 770
±
55 136
±
36 795
±
162 146
±
10 316
±
13
GCaMP5G 351 154 631
±
23 1,070
±
87 202
±
42 291
±
17
Fast-GCaMP-EF05 357 183 594
±
18
w
559
±
27
w
91
±
7* 365
±
30* 143
±
15 254
±
13
z
Fast-GCaMP-RS05 42 27 75
±
9* 248
±
78*
Fast-GCaMP-RS06 63 34 231
±
26
w
269
±
20
w
——81
±
6
w
173
±
40
z
Fast-GCaMP-RS09 51 25 82
±
11 210
±
19*
Measurements on purified protein were performed using stopped-flow fluorometry. Drosophila measurements show the t
1/2
at the start and after the termination of courtship song at 25 °C. Mammalian
measurements show the time to first response (t
response
) and decay time (t
1/2
) to trains of 5–10 impulses or action potentials at 35 °C. All values are in ms and are expressed as mean
±
s.e.m.
*Po0.05, wPo0.01, zPo0.002, smaller values compared with GCaMP3 by paired t-test; for SCGN: GCaMP3 n ¼ 7, EF05 n ¼ 10, RS05 n ¼ 7, RS09 n ¼ 5; for L2/3: GCaMP3 n ¼ 3, GCaMP5G n ¼ 5, EF05
n ¼ 7 and RS06 n ¼ 5.
GCaMP3 Alexa 594
SCG neuron
20 μm
L2/3 neuron
20 μm
20 μm
Response time (s)
0.5
0.8
0
1
0.3
0.2
0.1
SCG neurons
0
0.4
0.2
0.6
RS06
GCaMP3
EF05
RS05
1
2
GCaMP3
EF05
RS05
RS09
RS09
Decay time (s)
120 mV
0.25 s
Cuvet K
D
(μM)
0
1
2
Peak ΔF/F
0
0 0.5 1 1.5
L2/3
SCG
EF05
GCaMP5G
GCaMP3
RS05
RS06
RS09
EF13
Drosophila
3
L2/3 neurons
GCaMP3
EF05
RS06
Decay time (s)
0.4
0
0.2
0.5
0.3
0.1
GCaMP5G
0
0.05
0.1
0.3
0.2
0.15
GCaMP3
EF05
RS06
Response time (s)
GCaMP5G
0.55
EF05
RS06
GCaMP3
Number of spikes
Peak ΔF/F
0
(%)
0
25
10
0
100
200
300
30
15
5
20
GCaMP5G
EF05
GCaMP3
GCaMP5G
Normalized ΔF
0
1
Figure 5 | Responses of fast GCaMPs in superior cervical ganglion and neocortical pyramidal neurons. (a) Top left: Epifluorescence image of layer 2/3
pyramidal neuron expressing GCaMP3 in acute brain slice form postnatal day 16 mouse. Top right: Same cell filled with Alexa Fluor 594 hydrazide by
whole-cell patch electrode (depicted by drawing). Bottom: two-photon image of cultured mouse SCGN expressing GCaMP3 8 h post-infection. The white
line represents the axonal scan location. The outline depicts the location of the stimulation electrode. (b) Normalized example traces of fluorescence
responses to action potentials elicited by a depolarizing current step in layer 2/3 pyramidal neurons at 35 °C when monitored with GCaMP3 (grey),
GCaMP5G (black), EF05 (cyan), and RS06 (red). Full scale corresponds to a DF/F
0
range of GCaMP3 120%, GCaMP5G 500%, EF05 120% and RS06 50%.
(c) In layer 2/3 pyramidal neurons, the peak DF/F
0
was correlated with the number (4 or greater) of evoked spikes. Peak DF/F
0
is the mean of three-spike
bins and error bars represent s.e.m.. Open circles: 100 ms depolarization step; closed circles: 1 s depolarization step. (d) Relationship between peak DF/F
0
and the affinity of the variant for both SCGN (open circles) and layer 2/3 pyramidal neurons (closed circles; for SCGN: GCaMP3, n ¼ 7; EF05, n ¼ 10; RS05,
n ¼ 7, RS09, n ¼ 5; for L2/3: GCaMP3, n ¼ 3; GCaMP5G, n ¼ 5; EF05, n ¼ 7; RS06, n ¼ 5). Error bars, s.e.m. (e) Kinetics of Ca
2 þ
-mediated fluorescence
responses. SCGN processes for GCaMP3 (grey), EF05 (cyan), RS05 (orange) and RS09 (pink). (f) Layer 2/3 pyramidal neurons for GCaMP3 (grey),
GCaMP5G (black), EF05 (cyan), RS06 (red). Line segments indicate means.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170 ARTICLE
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& 2013 Macmillan Publishers Limited. All rights reserved.
improvements beyond previous fast-responding GECIs including
TN-XL and GCaMP1.6 (decay t ¼ 240–260 ms)
30,31
.
For kinetic optimization, our quantitative evaluation of Fast-
GCaMP variants required an evaluation method in which cellular
calcium dynamics are not a rate-limiting factor. The time course
of cellular fluorescence signals is limited by both calcium
dynamics and probe response kinetics. As an illustration of why
these factors matter, a recent GCaMP optimization effort
12
showed an improvement in physiological off-responses from
t
1/2
¼ 0.6 s using GCaMP3 to t
1/2
¼ 0.4 s using their variants of
GCaMP6 and GCaMP8. However, those measurements were
done in slice cultures in which calcium removal mechanisms were
slower than in acute slices, as evidenced by the slow GCaMP3
responses. Our fastest physiological off-response times were
several times faster, t
1/2
¼ 0.1–0.2 s for RS06 and RS09, and we
found that stopped-flow measurements on purified protein were
faster still, with t
1/2
¼ 7–30 ms. In addition, the variant of
GCaMP6 produced by Ohkura et al.
12
and GCaMP8 did not
have shorter rising t
1/2
than GCaMP3, whereas our Fast GCaMPs
showed faster-rising responses than GCaMP3, both in Drosophila
and in neocortical L2/3 neurons. Although these measurements
all point toward our variants having the fastest responses, direct
comparison of GCaMP kinetic performance will ultimately
require either stopped-flow measurements or an expression
system in which calcium signals are extremely rapid (for
example, single spikes in unbuffered dendritic spines
8
).
Our finding of a submillisecond response for calcium steps
4K
D
is consistent with previous observations on GCaMP1.6
(ref. 32) and calmodulin itself
33
. GCaMP may therefore have a
low-affinity binding state capable of rapid transition to a high-
fluorescence state. A likely rapid-binding candidate is the low-
affinity pair of sites at the N-domain
33
, an idea that is consistent
with our observation that chelation by N-domain loops I and II is
necessary to generate a functional probe.
A second target for perturbation was the interaction between
CaM and its target. Upon Ca
2 þ
binding, CaM must interact with
RS20 to allow a conformational change to a high-fluorescence
state. Ca
2 þ
dissociation from the high-fluorescence state is
energetically unfavored because RS20 binding increases Ca
2 þ
affinity
22
. Consistent with this concept is the recent observation
that alterations in a linker domain led to both strongly increased
affinity and considerable slowing of off-responses, indicating that
bound Ca
2 þ
is effectively trapped
6
. The relatively bright GECI
YC-Nano15 has high affinity, making it useful for detecting single
action potentials
34
; however, high affinity is accompanied by
extremely slow off-kinetics, precluding the tracking of successive
spikes occurring at high frequency. The same difficulty is
apparent for the faster GCaMP5 family of GECIs
7
. Our
findings demonstrate the converse point: perturbations to
CaM—RS20 interactions decreased affinity and led to
considerable speeding of off-responses.
We constructed a molecular dynamics model based on our
observations. Several conditions had to be satisfied: (1) based on
our results, the elimination of Ca
2 þ
binding in any EF-hand
loop resulted in reduced Ca
2 þ
affinity, indicating cooperative
interactions among the four EF-hand sites (Supplementary Tables
S2 and S3). (2) Deletion of residues from the RS20 peptide can
severely disrupt probe activity (Supplementary Tables S2 and S3),
indicating a necessary role for RS20 in reaching both high
fluorescence and high Ca
2 þ
affinity. (3) Elimination of either
loop I or loop II leads to a significant reduction in R
f
(Fig. 2c),
indicating a necessity for Ca
2 þ
binding to both sites of the
N-lobe to achieve protection of the chromophore and conforma-
tional changes that lead to high fluorescence. (4) The elimination
of Ca
2 þ
binding to loop III or loop IV led to reduced Ca
2 þ
affinity and left the R
f
intact (Fig. 2b), indicating that the C-lobe is
required for high-affinity Ca
2 þ
binding but not for chromophore
protection. (5) Based on our discovery of the fast, submillisecond
rise response and experimental evidence described by Faas
et al.
33
, binding of Ca
2 þ
to the N-lobe occurs on a
submillisecond timescale, with lower affinity than the slower-
binding C-lobe (Figs 4a and 5b).
We propose a kinetic model in which GCaMP has two
pathways to a high-fluorescence state (Fig. 6). Loops I and II (N-
lobe) begin in a low-affinity (1 mM) state, while loops III and IV
(C-lobe) begin in a high-affinity (250 nM) state. In ‘C-like’
activation for small Ca
2 þ
transients, Ca
2 þ
would bind to the
C-lobe with slow kinetics. The bound C-lobe then acts via
interactions with the RS20 domain to increase the calcium affinity
of the N-lobe
22
. After the N-lobe binds to Ca
2 þ
, the entire CaM–
RS20 complex shifts in conformation
20
, leading to reduced
chromophore-solvent access, leading to a high-fluorescence state.
The submillisecond responses we observe suggest a second
possible kinetic pathway, in which high calcium levels can drive
rapid binding to the low-affinity state of the N-lobe, which then
would be sufficient to drive the CaM–RS20 conformational shift
and chromophore protection. In this ‘N-like’ mode, C-lobe
binding to Ca
2 þ
is not required, as evidenced by the fact that
elimination of loop III or IV Ca
2 þ
binding sites leaves a
functional (albeit low-affinity) probe. Finally, after removal of
calcium, off-responses are limited in part by dissociation of the
CaM–RS20 interface domain followed by Ca
2 þ
unbinding.
RS20
Large calcium step
N-like activation C-like activation
Small calcium step
Slow binding of
Ca
2+
to C-lobe
Fast binding
to N-lobe
Slow binding
to C-lobe
Fast binding of
Ca
2+
to N-lobe
NC
CN
cpEGFP
cpEGFP CN
cpEGFP
cpEGFP N CCNcpEGFP
Figure 6 | A functional model for GCaMP molecular dynamics.
A functional model for intramolecular interactions between cyclically
permuted GFP (cpGFP; grey and green), calmodulin N (loops I and II;
yellow) and C (loops III and IV;dark yellow)-lobes and RS20 (red) domains
interacting with calcium ions (blue). High affinity for Ca
2 þ
is indicated by
deeper sockets in the N- and C-lobes.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
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& 2013 Macmillan Publishers Limited. All rights reserved.
A fruitful approach for future improvement would be to take
advantage of high-throughput design methods, with which our
approach is complementary. The mutations reported here can be
integrated with recently reported high-R
f
variants
7
. The residues
altered for high response and high per-molecule fluorescence
reside in different probe domains
7
, opening the possibility of
combinatorial approaches for continued improvement of
performance. Another possibility that does not require further
design improvements is to co-express multiple variants of
differing affinities. Coexpression can expand detection range
35
.
Such a combination of GECIs would give performance that had
lower apparent cooperativity than any single GECI.
Methods
Fast-GCaMP variant synthesis. Point mutations to GCaMP3 were generated
using the QuikChange II Site-Directed Mutagenesis Kit and Primer Design
Program (Agilent Technologies). Coding regions were PCR amplified to attach
restriction enzyme site linkers and cloned into the NotI and XbaI sites of the
pET28b (Novagen) expression vector without removing the N terminus hex-
ahistidine tag. BL21(DE3) E. coli (New England Biolabs, Ipswich, MA, USA) was
transformed and starter cultures were grown in 10 ml LB medium supplemented
with 50 mg l
1
kanamycin shaken at 225 r.p.m. at 37 °C. After 4 hours, each starter
culture was added to 1 l of LB medium and maintained to OD ¼ 1.0 at 600 nm, the
temperature reduced to 25 °C, and protein expression induced with 1 mM IPTG for
12–16 h. Cells were collected by centrifugation at 4500 r.p.m. at 4 °C and resus-
pended in anti-serine-protease wash buffer (25 mM Tris-Cl, 500 mM NaCl, 20 mM
imidazole, 1 mM phenylmethylsulfonyl fluoride, pH 8.0) and lysed by two passes
through the Emulsiflex C3 homogenizer (Avestin, Ottawa, ON, Canada). Cell
debris were removed by 40 000 g centrifugation for 1 h at 4 °C. Clear lysates were
purified by loading onto nickel-nitrilotriacetic acid Superflow resin columns
(Qiagen) followed by 40 ml wash buffer. Protein was eluted with 500 mM imidazole
and concentrated with 10 kDa Amicon Ultra centrifugal filters (Millipore), passed
through a PD-10 desalting column (GE Healthcare), and resuspended in storage
buffer (30 mM MOPS, 100 mM KCl, pH 7.20). Protein aliquots were frozen in
ethanol chilled with dry ice and stored at 80 °C.
Measurements on purified protein
. To measure the properties of purified
Fast-GCaMP proteins, the Ca
2 þ
buffer used was 10 mM complexometry grade
EGTA (Sigma-Aldric h) for steady-state mea sure ments and 2 mM BAPTA
(Molecular Probes) for kinetic measurements. Vario us free Ca
2 þ
concentrations
were generated by mixing of high-Ca
2 þ
(Ca
2 þ
plus EGTA or BAPTA) and
zero-Ca
2 þ
(EGTA or BAPTA alone) solutions
36
obtained from a commercial
source (Invitrogen, Gran d Island, NY) or made according t o the method of
Neher
42
. Free Ca
2 þ
concentrations were verified through titration of Fura-2
and Fura-4F (Molecular Probes). Free Ca
2 þ
concentrations were calculated
using MaxChelator (C. Patton, maxchelator.stanford.edu) assuming an ionic
strength of 0.15 N for 10 mM K
2
H
2
EGTA, 100 mM KCl and 30 mM KM OPS
(pH 7.20).
Excitation and emission spectra were measured on a FluoroLog 3 spectro-
fluorometer (Horiba Jobin Yvon Inc., Edison, NJ, USA). For calcium-dependence,
steady-state measurements were made using an F-2500 fluorescence spectro-
photometer (488 nm excitation, 509 nm emission) running FL Solutions version
4.1 softwa re (Hitachi, Japan) at 23 °C using 0.5–2 mM of purified protein
suspended in either zero-C a
2 þ
bufferorhigh-Ca
2 þ
buffer, followed by
reciproc al dilution with the other buffer to re ach free Ca
2 þ
concentrations
between 0.01 and 10 mM. Calcium/magnesium s electivity was measured by
competition assay in which 0.01–100 mM MgCl
2
was added to a solution
containing 0.16–0.27 mMfreeCa
2 þ
.
Extinction coefficients (e
490nm
) were estimated using the absorption coefficient
A
490nm
at saturating [Ca
2 þ
]
free
¼ 39 mM. A
490nm
is largely suppressed under
conditions of low calcium and is eliminated by protein denaturation. Molar protein
concentration was determined by measuring absorbance at 280 nm (A
280nm
); in the
cases listed in Table 1, concentrations were further confirmed using A
447nm
after
alkali denaturation with 0.1 M NaOH for 3 minutes to eliminate fluorescence and
generate an absorption band with e
447nm
¼ 44 000 M
1
cm
1
(ref. 38). Quantum
yield (QY) was determined by scaling F
max
/e
490nm
proportionally using QY ¼ 0.65
for GCaMP3 (ref. 7) as a benchmark.
Stopped-flow measurements were performed at 25 °Cor37°C with the AutoSF-
120 and Stopflow version 1.0.1830 data acquisition software (KinTek, Austin, TX)
using 488 nm xenon arclamp monochromator excitation and 525/40 nm filter
(Chroma Technologies, Brattleboro, VT, USA) emission. The mixing dead time
was o1 ms. Each shot consisted of 20 ml of reactant from each chamber. At least
five shots were averaged and analyzed for 1–2 exponential components. Traces
were fitted to a double exponential f(t) ¼ A
0
þ A
1
exp( k
1
t) þ A
2
exp( k
2
t). To
estimate the decay half-life (t
1/2
), f(0) was used in compensating for the instrument
dead time and A
0
was used as the equilibrium fluorescence intensity.
Imaging in Drosophila
. Fast-GCaMP variants (Fast-GCaMP-EF05, -RS06, -RS08
and -EF15) were cloned into the pJFRC-MUH (Addgene) plasmid containing 20
UAS (GAL4 DNA binding domains). The transgene was inserted into the genome
via PhiC31 integration (Rainbow Transgenic Flies, Inc., Camarillo, CA) into the
attP2 landing of D. melanogaster. GCaMP5G was inserted into the attP40 landing
site, which, when compared with the attP2 site, does not show differences in
expression pattern/level
37
. Injected flies were crossed to a w
1118
strain and the
significant transformants were identified. The Fru
P1
Gal4 driver
38
was used to
express GCaMP3, GCaMP5G, and all variants. Within the antennal nerve,
therefore, both Fru þ olfactory and mechanosensory (JO) neurons expressed
GCaMP; however, only JO neurons should respond to auditory stimuli. 20 UAS-
GCaMP3 also in attP2 was used as an expression-level-matched control
37
.
Optical r ecordings were collected from 2-day-old virgin females. Flies were
mounted ventral side up, with the dorsal side of the head, including antennae,
protruding from the bottom surface of the platform
23
. The antennal nerve was
imaged via dissection through the pr oboscis. To reduce motion, some muscles
surrounding t he brain were removed. A ntennal nerve axons were imaged on a
Zeiss LSM 710 two-photon laser scanning microscope at 15–16 frames per second
using a 20 water immersion objective (NA ¼ 1.0) at 256 256 resolution
using puls ed 920 nm excitation (Coherent) and for detection, GaAsP
photomultiplier tubes (Hamamatsu, Hamamatsu City, Japan) with 500–550 nm
emission lters (Chroma Technologies, Brattleboro, VT). Laser intensity was
maintained at o14 mW. Two types of sound stimuli w ere delivered via a
calibrated sound delivery system
29
: 10 seconds of wild-type fly song and synthetic
song pulse trains of 10–20 pulses separated by IPIs of 240, 120, 60 or 30 ms.
Stimulus trials were separated b y at least 20 s. S ound intensity (measured as
particle velocity) reached 5.29 mm s
1
during natural song and 6.25 mm s
1
for
synthetic song pulse trains. Peak DF/F
0
was calculated during the 5 s following
stimulu s onset. Decay phase t
1/2
was e stimated through mono-exponential fitting
for the first 5 s after t ermination of the song. ROIs were selected from a single
imaging plane.
Imaging of superior cervical ganglion neurons
. PRV expressing GCaMP
variants were constructed by homologous re combination as previously
described
39
. Proper fluorescent protein expression was assessed by
epifluorescence microscopy.
SCGNs were cultured from the lower mandible of embryonic rats
40
for 9–14
days. At 37 °C, SCGNs were either AM-loaded with 1–5 mM of fluorescent Ca
2 þ
indicator Oregon Green 488 BAPTA-1/AM (Life Technologies, Grand Island, NY)
for 30 min or incubated for 60 min with 5 ml of viral stock (10
8
PFU ml
1
) added
to 3 ml of neurobasal media supplemented with B27, glutamine and nerve growth
factor, followed by incubation in PRV-free media for 6–9 h.
SCGNs were imaged at B35 °C using a custom-built two-photon laser scanning
microscope using pulsed 830 nm (OGB-1) or 920 nm (GCaMP) excitation from
a Ti:sapphire laser (Mira 900, Coherent). Excitation power was kept at o15 mW
at the backplane of the objective ( 40, NA 0.8 IR-Achroplan; Carl Zeiss,
Thornwood, NY). Line scans (500 Hz) were made from neurites between 1 and 2
cell-diameters away from the soma. Data acquisition was controlled by ScanImage
r3.6.1 (ref. 41). SCGNs were stimulated extracellularly by glass pipets filled with
aCSF with tip diameters of B5 mM placed B0.5 cell-diameter away from the soma
using 50-Hz pulse trains (1 ms, 5 V).
Imaging of cortical L2/3 neurons
. L2/3 progenitor cells were transfected via
in utero electroporation in timed-pregnant E-15 Swiss Webster mice (strain
B6.129-Calb1tm1Mpin/J, The Jackson Laboratories, Bar Harbor, ME) with
plasmids expressing GECIs under the CAGS promoter. At P14–21, 250 mM-thick
cortical brain slices were prepared at in ice-cold artificial CSF (aCSF) containing
(in mM) 126 NaCl, 3 KCl, 1 NaH
2
PO
4
,20°D-glucose, 25 NaHCO
3
, 2 CaCl
2
and
1 MgCl
2
and saturated with 95% O
2
/5% CO
2
. Slices were preincubated at 34 °C
for 40–60 min and then kept at room temperature. For recording, slices were
transferred to an immersion-type recording chamber perfused at 2–4 ml min
1
with aCSF solution saturated with 95% O
2
/5% CO
2
at B35 °C. L2/3 pyramidal
neurons were shadowpatched with borosilicate patch recording electrodes
(6–9 MO) filled with a solution containing (in mM, pH to 7.30 with KOH) 133
methanesulfonic acid, 7.4 KCl, 0.3 MgCl
2
,3Na
2
ATP and 0.3 Na
3
GTP, 290 mOsm.
Electrophysiological signals were acquired with an Axopatch 200B amplifier and
Clampex 8.0 software (Axon Instruments, Foster City, CA, USA). After whole-cell
break-in, cells were held in current clamp mode (holding currents at 65 mV were
50 to 400 pA) and series resistances were 15–30 MO. Series resistance was
monitored periodically and compensated by balancing the bridge. Spiking was
induced through injection of current pulses at various amplitudes and durations
and individual trials were separated by at least 10 s. L2/3 neurons were imaged
using a custom-built two-photon laser scanning microscope using pulsed 830 nm
(OGB-1) or 920 nm (GCaMP) excitation from a Ti:sapphire laser (Mira 900,
Coherent). Excitation power was kept below 15 mW at the backplane of the
objective ( 40, NA 0.8 IR-Achroplan; Carl Zeiss, Thornwood, NY). Line scans
(500 Hz) were made from dendrites at least 1 cell-diameter away from the soma.
Data acquisition was controlled by ScanImage r3.6.1 (ref. 41).
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170 ARTICLE
NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | www.nature.com/naturecommunications 9
& 2013 Macmillan Publishers Limited. All rights reserved.
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Acknowledgements
This work was supported by NIH R01 NS045193, (S.S.-H.W.) RC1 NS068414 (L.W.E./
S.S.-H.W.), and P40 RR18604 and NS060699 (L.W.E.), a McKnight Technological
Innovations Award (S.S.-H.W.), a W.M. Keck Foundation Distinguished Young Inves-
tigator award (S.S.-H.W.), an Alfred P. Sloan Research Fellowship, Klingenstein,
McKnight, and NSF CAREER Young Investigator awards (M.M.), and an American
Cancer Society Postdoctoral Research Fellowship (M.P.T./I.B.H.). We thank Smita Patel
for advice and equipment access for stopped-flow fluorimetry, Fred Hughson for plas-
mids and protein purification materials, Loren Looger and Jasper Akerboom for advice
and GCaMP constructs, Timothy Tayler for assistance with establishing Drosophila lines,
Fred Hughson for advice and the gift of BL21(DE3) E. coli, and Steve Lin, Daniel Chang,
Tamar Friling, and Yulia Lampi for assistance in experiments.
Author contributions
X.R.S., L.W.E., M.M. and S.S.-H.W. designed the project and experiments; X.R.S. and
S.S.-H.W. designed the sensors; X.R.S. and L.A.L. performed protein purification and
equilibrium measurements; X.R.S. performed stopped-flow measurements; D.A.P. per-
formed imaging in Drosophila; A.B. and E.R.S. performed brain slice imaging; M.P.T. and
I.B.H. created recombinant PRV strains; X.R.S., M.P.T. and I.B.H. cultured SCGNs; A.B.
performed SCGN imaging; X.R.S. performed equilibrium and kinetics data analysis;
X.R.S., A.B. and D.A.P. performed Drosophila data analysis; A.B. analyzed slice and
SCGN data; X.R.S. and S.S.-H.W. generated the molecular dynamical model; X.R.S. and
S.S.-H.W. led the project. X.R.S., A.B. and S.S.-H.W. wrote the paper.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Sun, X. R. et al. Fast GCaMPs for improved tracking of neuronal
activity. Nat. Commun. 4:2170 doi: 10.1038/ncomms3170 (2013).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
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& 2013 Macmillan Publishers Limited. All rights reserved.
    • "Our data seem to contrast with another Ca +2 imaging work arguing that neighboring neurons' responses are highly similar (Issa et al. 2014). However, those measurements used GCamp3 as an indicator, which does not detect single spikes so critical in our dataset (Sun et al. 2013). We thus argue that the Issa et al. paper could be biased to neurons with higher spike rates missing on sparsely responsive neurons and resulting in an underestimation of the true depth of cortical heterogeneity. "
    [Show abstract] [Hide abstract] ABSTRACT: In the auditory system, early neural stations such as brain stem are characterized by strict tonotopy, which is used to deconstruct sounds to their basic frequencies. But higher along the auditory hierarchy, as early as primary auditory cortex (A1), tonotopy starts breaking down at local circuits. Here, we studied the response properties of both excitatory and inhibitory neurons in the auditory cortex of anesthetized mice. We used in vivo two photon-targeted cell-attached recordings from identified parvalbumin-positive neurons (PVNs) and their excitatory pyramidal neighbors (PyrNs). We show that PyrNs are locally heterogeneous as characterized by diverse best frequencies, pairwise signal correlations, and response timing. In marked contrast, neighboring PVNs exhibited homogenous response properties in pairwise signal correlations and temporal responses. The distinct physiological microarchitecture of different cell types is maintained qualitatively in response to natural sounds. Excitatory heterogeneity and inhibitory homogeneity within the same circuit suggest different roles for each population in coding natural stimuli.
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    • "researchers! to! simultaneously!visualize!neural!connectivity!and!activity![30,31]!(e.g.,!Figure!1D).! Originally,! the! fluorescent! "
    [Show abstract] [Hide abstract] ABSTRACT: In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.
    Full-text · Article · Nov 2015
    • "These observations support the notion that enteric glia, similar to those in the peripheral and central nervous systems, may not just sense, but also modulate neurotransmission (Robitaille, 1998). Using genetically-encoded calcium indicators such as GCaMP3 (Tian et al., 2009; Wilms and Häusser, 2009; Akerboom et al., 2012; Yamada and Mikoshiba, 2012; Zariwala et al., 2012; Sun et al., 2013) expressed in various enteric neurons and glia (Boesmans et al., 2015; Foong et al., 2015), we show here that the behavior of these cells and their role in colonic motor patterns can be readily analyzed in the undissected isolated colon using low-power imaging of GCaMP3. The specificity and quality of labeling of neurons and glia with GCaMP3 allows a much better understanding of how motor behaviors emerge from the activity of individual neurons in the gut both in situ and in vivo. "
    [Show abstract] [Hide abstract] ABSTRACT: Genetically encoded Ca2+ indicators (GECIs) have been used extensively in many body systems to detect Ca2+ transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca2+ indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We bred several GCaMP3 mice: (1) Wnt1-GCaMP3, all enteric neurons and glia; (2) GFAP-GCaMP3, enteric glia; (3) nNOS-GaMP3, enteric nitrergic neurons; and (4) ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC), using low power Ca2+ imaging. In this preliminary study, we observed neuronal and glial cell Ca2+ transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100–1000 active cells) through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca2+ transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca2+ transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca2+ transients in some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural activity but continued long after neural activity had waned. With these new tools an unprecedented level of detail can be recorded from the enteric nervous system (ENS) with minimal manipulation of tissue. These techniques can be extended in order to better understand the roles of particular enteric neurons and glia during normal and disordered motility.
    Full-text · Article · Nov 2015
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