ARTICLE
Received 1 Feb 2013 | Accepted 19 Jun 2013 | Published 18 Jul 2013
Xiaonan R. Sun1,2,*, Aleksandra Badura1,2,*, Diego A. Pacheco1,2, Laura A. Lynch1,2, Eve R. Schneider1,2, Matthew P. Taylor1,2, Ian B. Hogue1,2, Lynn W. Enquist1,2, Mala Murthy1,2 & Samuel S.-H. Wang1,2
The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca2 -sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we nd that afnity is regulated by the calmodulin domains Ca2 -chelating residues. Second, we nd that off-responses to Ca2 are rate-limited by dissociation of the
RS20 domain from calmodulins hydrophobic pocket. Third, we nd that on-responses are limited by fast binding to the N-lobe at high Ca2 and by slow binding to the C-lobe at lower
Ca2. 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 Ca2 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
Fast GCaMPs for improved tracking of neuronal activity
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: mailto:[email protected]
Web End [email protected] ).
NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 1
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
Imaging of intracellular Ca2 has assumed a central role in cellular physiology1. Until recently, Ca2 has been imaged with small-molecule uorescent 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 Ca2 -sensing domain derived from calmodulin or troponin, (ii)
a peptide domain that binds the Ca2-sensing domain and (iii) one or more XFP domains whose uorescence properties are modulated by the Ca2 -sensing interaction. GECIs allow cell-type-specic and long-term expression, and have been used to image neuronal circuitry in ies, worms, sh 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 ton 20 ms1.4 s
and toff 0.45 s)47 compared with BAPTA-based indicators
(tono1 ms and for OGB-1, toff 7 ms). Physiological Ca2
signals can rise within 1 ms and fall in 10100 ms in small subcellular structures8, indicating that slow intramolecular GECI dynamics can limit the ability to resolve spike times and ring rate variations. Second, GECI binding cooperativity is high (nH 34), so that uorescence signals change over a narrow
range of Ca2 concentration. For these two reasons, a GECI that can detect single APs is susceptible to saturation during continuous ring; more generally, any given GECI is expected to exhibit most of its brightness change within a proscribed range of ring rates4,6,7. Individual GECIs also do not span the0.110 mM Ca2 range over which synaptic plasticity and neurotransmitter release are regulated911.
Here we report the results of a targeted, conservative approach for modifying Green uorescent protein/Calmodulin protein
sensor (GCaMP), a GECI with low degradation, high per-molecule brightness and large uorescence changes5,7,12. Using GCaMP3 as a starting scaffold, we developed a library of GCaMP variants with a range of afnities and response rates. We found that our variants termed Fast-GCaMPs show faster responses to calcium events in both Drosophila and mammalian neurons.
ResultsDesign principles. Our principal goal was to generate accelerated-response GCaMP variants with a variety of afnities. However, we also wished to avoid unintended reductions in maximum brightness (Fmax) and dynamic range (Rf). We therefore selected target residues for alteration that have not been previously identied to be involved in GFP chromophore stabilization, and either participate in direct Ca2 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 domains13, 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 inuence binding afnity1418. To regulate afnity we designed modications that increased the number of acidic residues, altered the acid pairings or substituted loop residues with homologous sequences from troponin C19 (Fig. 1c, Supplementary Table S1). For specic attempts to lower the afnity, 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 GCaMPs RS20 peptide domain and its CaM domain is required for
GCaMP
RS20 R R KWN K T GH A V R A I GR L S S
X Y Z Y X Z
DKDGDGT I T T K E
F K E A F S L F
RS20
cpEGFP
CaM
41
42
47
51
55
Loop
CaM Helix
R f
Helix
322
I
333 369
L GT VMRS I
373
374
II
III
IV
358 395 431
L QDM I NE V
DADGDGT I D F P
E
F L TMMARK
412
414
I RE A F RV F
DKDGNGY I SAAE
406 442
L RHVMT N L
VDEM I RE A
D I DGDGQVNY E E
F VQMMT A K
[afii9838]ex= 497 nm [afii9838]em = 512 nm
1
0.8
0.6
0.4
0.2
Z
25 EF RS
Y
X
High Ca2+
20
GCaMP3
Z
15
Y
X
Normalized fluorescence
10
Zero Ca2+
5
0
0
440
460
480
500
520
540
560
0 0.5 1 1.5
Relative Fmax
Wavelength (nm)
Figure 1 | Targeted alteration of GCaMP3. (a) GCaMP consists of a cpEGFP uorophore (green) anked 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: Ca2 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 (Rf Fmax/Fmin) and maximum brightness
(Fmax) relative to GCaMP3 (grey line).
2 NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170 ARTICLE
GCaMP Ca2 sensing20,21. In studies of CaM dynamics, disruptions to the peptideCaM interaction lead to faster Ca2 dissociation22. Therefore, we generated point mutations that were likely to disrupt the CaMRS20 interface2325 (Fig. 1b,c).
Candidate GCaMP variants span a range of afnities. The 51 resulting variants were designated Fast-GCaMP-EF01 through -EF31 (loop variants) and Fast-GCaMP-RS01 through -RS20 (CaMRS20 interface variants). Variants had the same peak excitation and excitation wavelengths as GCaMP3 (Fig. 1d; lex
497 nm and lem 512 nm in n 6 variants tested at high
Ca2 ), as expected for changes to domains away from the GFP core. To characterize variants in their originally synthesized form we performed Ca2 titrations on puried protein to measure Rf,
Ca2 dissociation constant (KD) and cooperativity (Hill coefcient or nH). Of these, 41 had a high/low calcium uorescence ratio (Rf) of at least 6. Of those 41 variants, 18 had a maximum, high-calcium brightness (Fmax) of at least 80% of that of GCaMP3
(Fig. 1e, Supplementary Table S3), for a yield rate of 18/51 35%.
Fmax and Rf were strongly correlated (Pearsons correlation r 0.85), indicating that these parameters were jointly altered
by perturbation of the high-uorescence state. Changes in Fmax
were accompanied by changes in both extinction coefcient and estimated quantum yield (Table 1 and Supplementary Table 3). One variant (Fast-GCaMP-EF20, KD 6.1 mM) had a 1.37-fold
higher Fmax and a 1.09-fold higher extinction coefcient than GCaMP3. In variants with altered Fmax, estimated quantum yield tended to change to a greater extent than the extinction coefcient (Table 1 and Supplementary Table S3). Sometimes Fmin was also affected: one variant (Fast-GCaMP-EF15, KD 1.4
mM) displayed a nearly 1.8-fold increase in Rf through a reduction in baseline brightness (Fig. 2a, Supplementary Table S3).
Although full acidication of the XZ pairs in synthetic loop III peptides has been reported to increase afnity26, in GCaMP3 this change did not reduce KD (Fast-GCaMP-EF04, Supplementary
Table S3). Similarly, acidication of YZ pairs18 did not increase afnity 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 CaMTnC chimeras, replacements within the C-lobe (loops III and IV) increased afnity19,27 and accelerated off-binding19. 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 afnity, but Fast-GCaMP-EF05 showed a 1.6-fold improvement (KD 1557 nM,
95% CI, Supplementary Table S3) and reduced cooperativity (nH 2.00.3, 95% CI). Among all loop mutants, KD values
spanned a range from 0.16 to 6 mM (Fig. 2a,b), permitting the monitoring of a wide range of [Ca2 ]free.
Mutation of X- and Z-pairs has previously been shown to inuence magnesium afnity15. In Fast-GCaMP-EF05, -EF20 and -RS06 variants, an IC50,Mg of 0.340 mM Mg2 was needed to reduce uorescence by 50%. The ratio IC50,Mg/KD,Ca was 2,000
6,000, comparable to values of 2,0004,000 for GCaMP3 and GCaMP5G. Thus, in our variants the calmodulin domains ionic selectivity remained intact.
The N-lobe is needed for a functional probe. To further explore the participation of acid pairs in binding and uorescence change, we modied 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 position residues and (4) neutralizing all acidic
residues (Fig. 2c, Supplementary Tables S1 and S3). We found that all changes increased KD, while Rf decreased only with disruptions to the N-lobe (Fig. 2c, Supplementary Table S3). When all acidic residues were neutralized, the average Rf of the N-lobe mutants was 1.90.2 (means.d.), compared with 12.65.2 for C-lobe mutants (Supplementary Table S3). In addition, at the N-lobe variant with highest KD (6.1 mM, EF20), Rf was reduced.
In summary, strong calcium binding to loops I and II in combination was necessary to give a functional GECI.
Table 1 | Biophysical properties of selected novel GCaMP3 variants.
Variant Mutations KD (lM) Relative Fmax Rf Decay t1/2 (ms) e490nm (M 1 cm 1)* QYw OGB-1 0.24 14.0 5 GCaMP3 ref. 5 0.25 1.00 12.0 150 34,7002,000 0.65 GCaMP5G T302L/R303P/D380Y 0.41 1.260.06 34.4 154 44,9001,700 0.630.04
Loop variantsEF05 D397N/G398A/N399D 0.16 0.630.03 6.8 183 35,6003,500 0.400.02 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 1.370.09 13.8 35 37,7002,000 0.820.05
CaMRS20 interface variantsRS06 M374Q 0.31 0.500.03 6.5 34 31,0002,200 0.360.02 RS05 M373Q 0.47 8.5 27 RS08 T412Q/L414T 0.63 10.0 23 RS09 L414T 0.69 0.710.04 9.5 25 33,0001,400 0.490.03
RS20 domain variantsRS12 DR40/DR41/DK42 0.90 11.1 22
RS14 DG47 1.19 10.7 10 RS15 DG47/DH48 1.78 10.6 7
K (mM), e , and relative F were measured at 25 C (pH 7.20) and decay t (ms) was measured at 37 C. *Determined in 200 mM free Ca2 .
wValues normalized to GCaMP3 (ref. 7).
NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 3
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
20 EF05
GCaMP3
25 EF15
EF16
EF18
EF20
Loops I/II
25 Loops I/II
Loops III/IV
Loops III/IV
GCaMP3
RS06
EF15 EF16 EF20
RS09
20
15
20 GCaMP3
F/F 0
10
OGB-1
15
15
R f
R f
5
10
10
0
5
5
8
7.5
7
6.5
6
5.5
5
4.5
0
0 1 2 3 4 5 6 7 8
Log 10([Ca2+]/M)
OGB-1 EF05
EF16
GCaMP3 RS06
RS09
EF20
0 Three acid pairs
No paired acids
No + position acids
No acids
KD (M)
EF15
Figure 2 | Equilibrium uorescence properties of puried GCaMP3 variants. (a) Ca2 titration of GCaMP3, OGB-1 and six novel GECIs. Solid curves represent ts to the Hill equation. Horizontal bars represent the Ca2 -sensitive range (595% of total uorescence change). (b) Dynamic range as a function of KD. 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 position acids: acidic residues at X/ Y/ Z positions are neutralized to N/N/
N (N: Asparagine). No acids: all Asp and Glu are replaced with Asn and Ala, respectively.
Disruption of CaMRS20 interactions accelerates kinetics. To characterize response kinetics, we used stopped-ow uorometry with B1 ms steps in Ca2 concentration (Fig. 3a). For a step down in free [Ca2 ] 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 (t1/2), 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 variants7 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 KD and t1/2,decay
(25 C, Fig. 3c, curve), reminiscent of the close relationship between afnity and off-binding observed for BAPTA-based indicators28. 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 to6.5-fold faster (Fig. 3b, c) than EF variants of a comparable KD (for decay t1/2 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- and4.4-fold, respectively, without signicant loss of afnity or Rf. 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 KD by 2.5-fold.
Rise times were also accelerated in RS mutations. The rise t1/2
decreased with increasing D[Ca2 ] (Fig. 3d). In comparison to GCaMP3, RS06 showed a 60% increase in rise rate at D[Ca2 ]
100 nM and a 100% increase at D[Ca2 ] 570 nM, indicating
faster on-responses at physiological concentrations. No improvements in rise responses were detected in GCaMP5 variants7 (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 CaMRS20 interface are rate-limiting in both on- and off-responses. For monitoring in vivo Ca2 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): rst, rise responses at all values of D[Ca2 ] had at least two exponential components; second, rise kinetics were not saturated at concentrations for which equilibrium uorescence was near-maximal and third, the rst data point after the mixing dead time
(B1 ms) was increasingly elevated from baseline with increasing values of D[Ca2 ]. For example, for GCaMP3, within the dead time the uorescence change was 10% complete at D[Ca2]
KD 250 nM, 50% at D[Ca2 ] 1 mM and 65% at D[Ca2 ] 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-afnity binding step that can drive transition to a high-uorescence state.
Imaging sensory-evoked Ca2 activity in Drosophila. As our rst 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 (Johnstons organ neurons, JONs; Fig. 4a,b). JON population activity as assessed by eld potential recording is highly reproducible between stimulus trials29. 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-afnity variants would generate larger signals to the same courtship song. For Fast-GCaMP-EF05 and another high-afnity variant, Fast-GCaMPRS06 (Fig. 4d), more ROIs showed measurable responses than GCaMP3 and the overall distribution of responses was shifted to larger values of peak DF/F0 (Fig. 4e; Fast-GCaMP-RS06,
P 0.0025; Fast-GCaMP-EF05, Po10 6, Kolmogorov
Smirnov test). Low-afnity variants (Fast-GCaMP-RS09, -EF15 and -EF18) generated little to no uorescence increase, whereas the highest-afnity 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 uorescence change.
To test the song response speeds for two variants (Fast-GCaMP-EF05 and Fast-GCaMP-RS06), we estimated the rising and falling t1/2 in ROIs with a response signal-to-noise ratio (SNR) of at least 2. Compared to GCaMP3 and GCaMP5G, Fast-GCaMP-RS06 performed 34 times faster, whereas Fast-GCaMPEF05 performed only 1.41.6-fold faster (Table 2 and Fig. 4g). Rise and decay times for the new variants tended to be less variable (Fast-GCaMP-RS06: CVrise 0.36, CV
decay
0.40; Fast-
GCaMP-EF05: CVrise 0.23, CV
decay
0.35) than for GCaMP3
(CVrise 1.02, CV
decay
0.45) and GCaMP5G (CVrise 0.31,
CVdecay 0.68).
4 NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170 ARTICLE
1
EF25 EF19 RS06 RS08
OGB-1
Syringe B
GCaMP3
Normalized
response
0.5
0
Excitation
Loading ports
Syringe A
10
Observation cell
[Ca2+ ]
(M)
0
0
50 100 Time (ms)
150
0.6
1.6
0.5
EF RS
GCaMP3 EF05 RS06
0.4
GCaMP3
OGB-1
1.2
Decay t 1/2
0.3
Rise t 1/2(s)
0.8
0.2
0.1
0.4
0
0
0.2 1
0 2 4 6 8 KD (M)
[Ca2+] step (M)
Dead time (~1 ms) Post-mixing
Post-mixing
1
GCaMP3 EF06 RS09 EF09
Dead time
8
2
Fraction of signal
0.8
F/F 0
6
4
0.6
0.4
0
[Ca2+ ]
(M)
2
0.2
0
0 Time (s) Time (ms)
0.5 1
0 10 20
0 0.1 [Ca2+] (M)
1 10
Figure 3 | Stopped-ow measurement of calcium off- and on-responses from Fast-GCaMPs. (a) Stopped-ow uorimeter. (b) The uorescence decay response of selected variants at 37 C to a step in [Ca2 ]free from 10 mM to o10 nM. Traces are scaled to the baseline by bi-exponential t and to
the maximum uorescence intensity at [Ca2 ]free 10 mM. (c) Relationship between off-response t1/2 and KD (at 25 C) for variants with mutations
at the loop domain (EF variants) or at the RS20CaM interface (RS variants). The solid line represents a reciprocal curve through the GCaMP3 data point. (d) Calcium-dependence of the rise responses (t1/2) 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 [Ca2 ] steps from 0. Data are shown at 1-s (left) and 20-ms (right) timescales. The rst data point of each DF/F0 trace represents the magnitude of uorescence change during the dead time. The magnitude of DF/F0 during the post-mixing phase is the difference between A0 (estimated through bi-exponential tting) and the rst 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.
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/F0 was 3.81.3% for GCaMP3 (means.d., n 2 responding out of
45 ROIs), whereas the peak of Fast-GCaMP-EF05 and Fast-GCaMP-RS06 responses were 15.96.0% (n 28 responding out
of 35 ROIs) and 14.03.7% (n 12 responding out of 34 ROIs)
respectively, comparable to GCaMP5G (15.45.8%, n 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 afnity 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, 3516%; Fast-GCaMP-RS06, 3922%; and GCaMP5G, 3724% versus GCaMP3, 115%; means.d.; Fig. 4i), with Fast-GCaMP-RS06 showing an
average off-response time of t1/2 0.250.01 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 afnity (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 914 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).
NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 5
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
2-P
GCaMP5G
Normalized F
0
RS06 EF05
GCaMP3
1
Speaker
5 m
1 s
Courtship song
Song response
Response time (s)
1
*
1
RS06
GCaMP3
5
5
0
{
2
EF05
0.8
GCaMP5G
2
Responding ROIs
0.2
0
Cumulative fraction
2
EF05
Peak F/F 0
0.6
0.5
1
RS06
Rise
1
Decay
GCaMP5G
GCaMP3
0.4
1
0.5
0.5
RS08
EF15
0
0 0 1 2 3
0
0 0.5 1 1.5
GCaMP3
EF05
RS06
EF05
RS06
EF05
RS06
GCaMP5G
Peak F/F0
Cuvet KD (M)
GCaMP3
GCaMP5G
GCaMP3
GCaMP5G
Pulse trains
Peak signal (F/F0)
0.4
1
20% F/F
10% F/F
1 s 1 s
30 ms IPI
0.2
240 ms IPI
0.5
20 pulses at 30 ms
10 pulses at 240 ms
GCaMP3
0
0
GCaMP5G
EF05
RS06
GCaMP3
GCaMP5G
EF05
RS06
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/F0 range of GCaMP3 70%, GCaMP5G 60%, EF05 100%, and RS06 50%. (d) Fraction of responding ROIs (* represents Po0.005 by
Fishers exact test, GCaMP3, n 95 ROIs, four animals; GCaMP5G, n 92 ROIs, seven animals; EF05, n 83 ROIs, three animals; RS06, n 56 ROIs, 3
animals; error bars, s.e.m.). (e) Cumulative distribution of peak uorescence amplitudes (peak DF/F0). (f) Peak DF/F0. The dependence of peak DF/F0 on KD (black line) is calculated using nH 3 (error bar: s.e.m.; RS08: lled black circle; EF15: open black circle). (g) Rise (left) and decay (right) times (t1/2) of
song-responsive ROIs for GCaMP3 (rise, n 53 ROIs; decay n 39 ROIs), GCaMP5G (rise, n 72 ROIs; decay, n 69 ROIs), EF05 (rise, n 63 ROIs;
decay, n 54 ROIs) and RS06 (rise, n 46 ROIs; decay, n 34 ROIs). (h) Example uorescence responses to trains of sound pulses (black). (i) Responses
(DF/F0) 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).
Mouse layer 2/3 (L2/3) pyramidal neurons were imaged in brain slices from 1421-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/F0 scaled inversely with in vitro KD (Fig. 5d), with the largest responses for the highest-afnity variant (Fast-GCaMP-EF05, n 10 SCGN and n 7 L2/3
pyramidal neurons) and no detectable response for the EF13 variant (n 6 neurons), similar to performance in Drosophila
(Fig. 5d, curve). The peak DF/F0 for GCaMP5G (n 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 uorescence (Fig. 5c).
To quantify response times in both SCGN (Fig. 5e) and L2/3 neurons (Fig. 5f), we estimated time to rst response (tresponse)
and decay t1/2 (Table 2). We calculated tresponse 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
6 NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170 ARTICLE
Table 2 | In vivo response times of Fast-GCaMPs.
Variant Protein Drosophila Mammaliant1/2(off) SCGN L2/3
25 C 37 C trise tdecay tresponse tdecay tresponse tdecay
GCaMP3 344 150 946133 77055 13636 795162 14610 31613 GCaMP5G 351 154 63123 1,07087 20242 29117
Fast-GCaMP-EF05 357 183 59418w 55927w 917* 36530* 14315 25413z
Fast-GCaMP-RS05 42 27 759* 24878* Fast-GCaMP-RS06 63 34 23126w 26920w 816w 17340z
Fast-GCaMP-RS09 51 25 8211 21019*
Measurements on puried protein were performed using stopped-ow uorometry. Drosophila measurements show the t at the start and after the termination of courtship song at 25 C. Mammalian measurements show the time to rst response (t ) and decay time (t ) to trains of 510 impulses or action potentials at 35 C. All values are in ms and are expressed as means.e.m. *Po0.05, wPo0.01, zPo0.002, smaller values compared with GCaMP3 by paired t-test; for SCGN: GCaMP3 n 7, EF05 n 10, RS05 n 7, RS09 n 5; for L2/3: GCaMP3 n 3, GCaMP5G n 5, EF05
n 7 and RS06 n 5.
GCaMP3 Alexa 594
SCG neuron
L2/3 neuron
1
EF05
GCaMP3
GCaMP5G
300
GCaMP5G
Peak F/F 0(%)
20 m
20 m
20 m
Normalized F
0
200
RS06
100
GCaMP3
Number of spikes
EF05
RS06
120 mV
0.25 s
0
0 25
10
15
5 20
30
SCG neurons
L2/3 neurons
GCaMP3
2
0.55
3
L2/3 SCG
1
0.4
Peak F/F 0
EF05 GCaMP5G
GCaMP3
RS05
RS06
RS09
EF13
Response time (s)
1
0.3
0.2
0.1
0.5 0.8
0
Decay time (s)
Response time (s)
0.3
0.20.15
0.6
0.5
0.3
0.1
Decay time (s)
2
0.2
1
Drosophila
0.4
0.2
0.1
0.05
0
0
0
0
GCaMP3
EF05
RS05
0 0.5 1 1.5
RS09
GCaMP3
EF05
RS05
RS09
GCaMP3
EF05
RS06
EF05
RS06
Cuvet KD (M)
GCaMP5G
GCaMP5G
Figure 5 | Responses of fast GCaMPs in superior cervical ganglion and neocortical pyramidal neurons. (a) Top left: Epiuorescence image of layer 2/3 pyramidal neuron expressing GCaMP3 in acute brain slice form postnatal day 16 mouse. Top right: Same cell lled 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 uorescence 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/F0 range of GCaMP3 120%, GCaMP5G 500%, EF05 120% and RS06 50%. (c) In layer 2/3 pyramidal neurons, the peak DF/F0 was correlated with the number (4 or greater) of evoked spikes. Peak DF/F0 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/F0 and the afnity of the variant for both SCGN (open circles) and layer 2/3 pyramidal neurons (closed circles; for SCGN: GCaMP3, n 7; EF05, n 10; RS05,
n 7, RS09, n 5; for L2/3: GCaMP3, n 3; GCaMP5G, n 5; EF05, n 7; RS06, n 5). Error bars, s.e.m. (e) Kinetics of Ca2 -mediated uorescence
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.
variants are associated with improved reporting performance
when expressed in situ.
DiscussionBy making targeted changes in Ca2-sensing components of
GCaMP3, we have generated a series of variants termed Fast-GCaMPs. Fast GCaMPs respond to Ca2 with up to 20-fold improved kinetics and have afnities spanning the range of intracellular neuronal Ca2 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 uorescence5,7. Another effort has led to similar brightness improvements and modest kinetic improvements7. Although a combinatorial approach can be effective at maximizing one parameter at a time, parameters such as KD,
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 benecial features, and can lead to kinetic
NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 7
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
improvements beyond previous fast-responding GECIs including TN-XL and GCaMP1.6 (decay t 240260 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 uorescence signals is limited by both calcium dynamics and probe response kinetics. As an illustration of why these factors matter, a recent GCaMP optimization effort12 showed an improvement in physiological off-responses from t1/2 0.6 s using GCaMP3 to t1/2 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, t1/2 0.10.2 s for RS06 and RS09, and we
found that stopped-ow measurements on puried protein were faster still, with t1/2 730 ms. In addition, the variant of
GCaMP6 produced by Ohkura et al.12 and GCaMP8 did not have shorter rising t1/2 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-ow measurements or an expression system in which calcium signals are extremely rapid (for example, single spikes in unbuffered dendritic spines8).
Our nding of a submillisecond response for calcium steps 4KD is consistent with previous observations on GCaMP1.6 (ref. 32) and calmodulin itself33. GCaMP may therefore have a low-afnity binding state capable of rapid transition to a high-uorescence state. A likely rapid-binding candidate is the lowafnity pair of sites at the N-domain33, 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 Ca2 binding, CaM must interact with
RS20 to allow a conformational change to a high-uorescence state. Ca2 dissociation from the high-uorescence state is energetically unfavored because RS20 binding increases Ca2 afnity22. Consistent with this concept is the recent observation that alterations in a linker domain led to both strongly increased afnity and considerable slowing of off-responses, indicating that bound Ca2 is effectively trapped6. The relatively bright GECI
YC-Nano15 has high afnity, making it useful for detecting single action potentials34; however, high afnity is accompanied by extremely slow off-kinetics, precluding the tracking of successive spikes occurring at high frequency. The same difculty is apparent for the faster GCaMP5 family of GECIs7. Our ndings demonstrate the converse point: perturbations to CaMRS20 interactions decreased afnity and led to considerable speeding of off-responses.
We constructed a molecular dynamics model based on our observations. Several conditions had to be satised: (1) based on our results, the elimination of Ca2 binding in any EF-hand loop resulted in reduced Ca2 afnity, 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 uorescence and high Ca2 afnity. (3) Elimination of either loop I or loop II leads to a signicant reduction in Rf (Fig. 2c), indicating a necessity for Ca2 binding to both sites of the
N-lobe to achieve protection of the chromophore and conformational changes that lead to high uorescence. (4) The elimination of Ca2 binding to loop III or loop IV led to reduced Ca2 afnity and left the Rf intact (Fig. 2b), indicating that the C-lobe is
required for high-afnity Ca2 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 Ca2 to the N-lobe occurs on a submillisecond timescale, with lower afnity than the slower-binding C-lobe (Figs 4a and 5b).
We propose a kinetic model in which GCaMP has two pathways to a high-uorescence state (Fig. 6). Loops I and II (N-lobe) begin in a low-afnity (1 mM) state, while loops III and IV (C-lobe) begin in a high-afnity (250 nM) state. In C-like activation for small Ca2 transients, Ca2 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 afnity of the N-lobe22. After the N-lobe binds to Ca2, the entire CaM
RS20 complex shifts in conformation20, leading to reduced chromophore-solvent access, leading to a high-uorescence state.
The submillisecond responses we observe suggest a second possible kinetic pathway, in which high calcium levels can drive rapid binding to the low-afnity state of the N-lobe, which then would be sufcient to drive the CaMRS20 conformational shift and chromophore protection. In this N-like mode, C-lobe binding to Ca2 is not required, as evidenced by the fact that elimination of loop III or IV Ca2 binding sites leaves a functional (albeit low-afnity) probe. Finally, after removal of calcium, off-responses are limited in part by dissociation of the CaMRS20 interface domain followed by Ca2 unbinding.
cpEGFP
N
RS20
C
Large calcium step
Small calcium step
Fast binding to N-lobe
Slow binding to C-lobe
Fast binding of Ca2+ to N-lobe
cpEGFP
N
C
cpEGFP N
C
Slow binding of Ca2+ to C-lobe
cpEGFP C
N
N
C
cpEGFP
N-like activation C-like activation
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 afnity for Ca2 is indicated by deeper sockets in the N- and C-lobes.
8 NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170 ARTICLE
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-Rf variants7. The residues altered for high response and high per-molecule uorescence reside in different probe domains7, 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 afnities. Coexpression can expand detection range35. 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 amplied 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 hexahistidine 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 1.0 at 600 nm, the
temperature reduced to 25 C, and protein expression induced with 1 mM IPTG for 1216 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 uoride, pH 8.0) and lysed by two passes through the Emulsiex C3 homogenizer (Avestin, Ottawa, ON, Canada). Cell debris were removed by 40 000 g centrifugation for 1 h at 4 C. Clear lysates were puried by loading onto nickel-nitrilotriacetic acid Superow resin columns (Qiagen) followed by 40 ml wash buffer. Protein was eluted with 500 mM imidazole and concentrated with 10 kDa Amicon Ultra centrifugal lters (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 puried protein. To measure the properties of puried Fast-GCaMP proteins, the Ca2 buffer used was 10 mM complexometry grade
EGTA (Sigma-Aldrich) for steady-state measurements and 2 mM BAPTA (Molecular Probes) for kinetic measurements. Various free Ca2 concentrations were generated by mixing of high-Ca2 (Ca2 plus EGTA or BAPTA) and zero-Ca2 (EGTA or BAPTA alone) solutions36 obtained from a commercial source (Invitrogen, Grand Island, NY) or made according to the method of
Neher42. Free Ca2 concentrations were veried through titration of Fura-2 and Fura-4F (Molecular Probes). Free Ca2 concentrations were calculated using MaxChelator (C. Patton, maxchelator.stanford.edu) assuming an ionic strength of 0.15 N for 10 mM K2H2EGTA, 100 mM KCl and 30 mM KMOPS (pH 7.20).
Excitation and emission spectra were measured on a FluoroLog 3 spectrouorometer (Horiba Jobin Yvon Inc., Edison, NJ, USA). For calcium-dependence, steady-state measurements were made using an F-2500 uorescence spectrophotometer (488 nm excitation, 509 nm emission) running FL Solutions version4.1 software (Hitachi, Japan) at 23 C using 0.52 mM of puried protein suspended in either zero-Ca2 buffer or high-Ca2 buffer, followed by reciprocal dilution with the other buffer to reach free Ca2 concentrations between 0.01 and 10 mM. Calcium/magnesium selectivity was measured by competition assay in which 0.01100 mM MgCl2 was added to a solution containing 0.160.27 mM free Ca2 .
Extinction coefcients (e490nm) were estimated using the absorption coefcient A490nm at saturating [Ca2]free 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 (A280nm); in the
cases listed in Table 1, concentrations were further conrmed using A447nm after
alkali denaturation with 0.1 M NaOH for 3 minutes to eliminate uorescence and generate an absorption band with e447nm 44 000 M 1 cm 1 (ref. 38). Quantum
yield (QY) was determined by scaling Fmax/e490nm proportionally using QY 0.65
for GCaMP3 (ref. 7) as a benchmark.
Stopped-ow measurements were performed at 25 C or 37 C with the AutoSF-120 and Stopow version 1.0.1830 data acquisition software (KinTek, Austin, TX) using 488 nm xenon arclamp monochromator excitation and 525/40 nm lter (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 ve shots were averaged and analyzed for 12 exponential components. Traces were tted to a double exponential f(t) A0 A1 exp( k1t) A2 exp( k2t). To
estimate the decay half-life (t1/2), f(0) was used in compensating for the instrument dead time and A0 was used as the equilibrium uorescence 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/level37. Injected ies were crossed to a w1118 strain and the signicant transformants were identied. The FruP1 Gal4 driver38 was used to express GCaMP3, GCaMP5G, and all variants. Within the antennal nerve, therefore, both Fru 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 control37.
Optical recordings 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 platform23. The antennal nerve was imaged via dissection through the proboscis. To reduce motion, some muscles surrounding the brain were removed. Antennal nerve axons were imaged on a Zeiss LSM 710 two-photon laser scanning microscope at 1516 frames per second using a 20 water immersion objective (NA 1.0) at 256 256 resolution
using pulsed 920 nm excitation (Coherent) and for detection, GaAsP photomultiplier tubes (Hamamatsu, Hamamatsu City, Japan) with 500550 nm emission lters (Chroma Technologies, Brattleboro, VT). Laser intensity was maintained at o14 mW. Two types of sound stimuli were delivered via a calibrated sound delivery system29: 10 seconds of wild-type y song and synthetic song pulse trains of 1020 pulses separated by IPIs of 240, 120, 60 or 30 ms. Stimulus trials were separated by at least 20 s. Sound 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/F0 was calculated during the 5 s following stimulus onset. Decay phase t1/2 was estimated through mono-exponential tting for the rst 5 s after termination 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 recombination as previously described39. Proper uorescent protein expression was assessed by epiuorescence microscopy.
SCGNs were cultured from the lower mandible of embryonic rats40 for 914 days. At 37 C, SCGNs were either AM-loaded with 15 mM of uorescent Ca2 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 (108 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 69 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 froma 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 lled 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 viain 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 P1421, 250 mM-thick cortical brain slices were prepared at in ice-cold articial CSF (aCSF) containing (in mM) 126 NaCl, 3 KCl, 1 NaH2PO4, 20D-glucose, 25 NaHCO3, 2 CaCl2 and 1 MgCl2 and saturated with 95% O2/5% CO2. Slices were preincubated at 34 C for 4060 min and then kept at room temperature. For recording, slices were transferred to an immersion-type recording chamber perfused at 24 ml min 1 with aCSF solution saturated with 95% O2/5% CO2 at B35 C. L2/3 pyramidal neurons were shadowpatched with borosilicate patch recording electrodes(69 MO) lled with a solution containing (in mM, pH to 7.30 with KOH) 133 methanesulfonic acid, 7.4 KCl, 0.3 MgCl2, 3 Na2ATP and 0.3 Na3GTP, 290 mOsm. Electrophysiological signals were acquired with an Axopatch 200B amplier 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 1530 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 | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 9
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3170
References
1. Tsien, R. Y. in Calcium as a Cellular Regulator (eds Carafoli, E & Klee, C. B)
2854 (Oxford University Press, 1999).
2. Tsien, R. Y. Indicators based on uorescence resonance energy transfer (FRET). Cold Spring Harb. Protoc. 4, 17 (2009).
3. Looger, L. L. & Griesbeck, O. Genetically encoded neural activity indicators. Curr. Opin. Neurobiol. 22, 1823 (2012).
4. Hendel, T. et al. Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro. J. Neurosci. 28, 73997411 (2008).
5. Tian, L. et al. Imaging neural activity in worms, ies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875881 (2009).
6. Horikawa, K. et al. Spontaneous network activity visualized by ultrasensitive Ca2 indicators, yellow Cameleon-Nano. Nat. Methods 7, 729732
(2010).7. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 1381913840 (2012).
8. Higley, M. J. & Sabatini, B. L. Calcium signaling in dendrites and spines: practical and functional considerations. Neuron 59, 902913 (2008).
9. Yang, S.-N., Tang, Y.-G. & Zucker, R. S. Selective induction of LTP and LTD by postsynaptic [Ca2 ]i elevation. J. Neurophysiol. 81, 781787 (1999).
10. Shouval, H. Z., Wang, S. S.-H. & Wittenberg, G. M. Spike timing dependent plasticity: a consequence of more fundamental learning rules. Front. Comput. Neurosci. 4, pii: 19 (2010).
11. Li, H. et al. Concurrent imaging of synaptic vesicle recycling and calcium dynamics. Front. Mol. Neurosci. 4, 110 (2011).
12. Ohkura, M., Sasaki, T., Kobayashi, C., Ikegaya, Y. & Nakai, J. An improved genetically encoded red uorescent Ca2 indicator for detecting optically
evoked action potentials. PLoS One 7, e39933 (2012).13. Strynadka, N. C. & James, M. N. Crystal structures of the helix-loop-helix calcium-binding proteins. Annu. Rev. Biochem. 58, 951998 (1989).
14. Reid, R. E. & Hodges, R. S. Co-operativity and calcium/magnesium binding to troponin C and muscle calcium binding parvalbumin: an hypothesis. J. Theor. Biol. 84, 401444 (1980).
15. Procyshyn, R. M. & Reid, R. E. A structure/activity study of calcium afnity and selectivity using a synthetic peptide model of the helix-loop-helix calcium-binding motif. J. Biol. Chem. 269, 16411647 (1994).
16. Wu, X. & Reid, R. E. Structure/calcium afnity relationships of site III of calmodulin: testing the acid pair hypothesis using calmodulin mutants. Biochemistry 36, 86498656 (1997).
17. Wu, X. & Reid, R. E. Conservative D133E mutation of calmodulin site IV drastically alters calcium binding and phosphodiesterase regulation. Biochemistry 36, 36083616 (1997).
18. Black, D. J., Tikunova, S. B., Johnson, J. D. & Davis, J. P. Acid pairs increase the N-terminal Ca2 afnity of CaM by increasing the rate of Ca2 association.
Biochemistry 39, 1383113837 (2000).19. Wang, S., George, S. E., Davis, J. P. & Johnson, J. D. Structural determinants of Ca2 exchange and afnity in the C terminal of cardiac troponin C.
Biochemistry 37, 1453914544 (1998).20. Wang, Q., Shui, B., Kotlikoff, M. I. & Sondermann, H. Structural basis for calcium sensing by GCaMP2. Structure 16, 18171827 (2008).
21. Akerboom, J. et al. Crystal structures of the GCaMP calcium sensor reveal the mechanism of uorescence signal change and aid rational design. J. Biol. Chem. 284, 64556464 (2009).
22. Bayley, P. M., Findlay, W. A. & Martin, S. R. Target recognition by calmodulin: dissecting the kinetics and afnity of interaction using short peptide sequences. Protein Sci. 5, 12151228 (1996).
23. Bagchi, I. C., Huang, Q. H. & Means, A. R. Identication of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase. J. Biol. Chem. 267, 30243029 (1992).
24. Chin, D. & Means, A. R. Methionine to glutamine substitutions in the C-terminal domain of calmodulin impair the activation of three protein kinases. J. Biol. Chem. 271, 3046530471 (1996).
25. Chin, D., Sloan, D. J., Quiocho, F. A. & Means, A. R. Functional consequences of truncating amino acid side chains located at a calmodulin-peptide interface.J. Biol. Chem. 272, 55105513 (1997).26. Reid, R. E. Synthetic fragments of calmodulin calcium-binding site III. A test of the acid pair hypothesis. J. Biol. Chem. 265, 59715976 (1990).
27. George, S. E., Su, Z., Fan, D. & Means, A. R. Calmodulin-cardiac troponin C chimeras. Effects of domain exchange on calcium binding and enzyme activation. J. Biol. Chem. 268, 2521325220 (1993).
28. Kao, J. P. & Tsien, R. Y. Ca2 binding kinetics of fura-2 and azo-1 from
temperature-jump relaxation measurements. Biophys. J. 53, 635639 (1988).
29. Tootoonian, S., Coen, P., Kawai, R. & Murthy, M. Neural representations of courtship song in the Drosophila brain. J. Neurosci. 32, 787798 (2012).
30. Mank, M. et al. A FRET-based calcium biosensor with fast signal kinetics and high uorescence change. Biophys. J. 90, 17901796 (2006).
31. Geiger, A. et al. Correlating calcium binding, Frster resonance energy transfer, and conformational change in the biosensor TN-XXL. Biophys. J. 102, 24012410 (2012).
32. Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca2 probe
composed of a single green uorescent protein. Nat. Biotechnol. 19, 137141 (2001).33. Faas, G. C., Raghavachari, S., Lisman, J. E. & Mody, I. Calmodulin as a direct detector of Ca2 signals. Nat. Neurosci. 14, 301304 (2011).
34. Yamada, Y. Quantitative comparison of genetically encoded Ca2 indicators
in cortical pyramidal cells and cerebellar Purkinje cells. Front. Cell Neurosci. 5, 110 (2011).35. Brenowitz, S. D. & Regehr, W. G. Calcium dependence of retrograde inhibition by endocannabinoids at synapses onto Purkinje cells. J. Neurosci. 23, 63736384 (2003).
36. Tsien, R. & Pozzan, T. Measurement of cytosolic free Ca2 with quin2. Meth.
Enzymol. 172, 230262 (1989).37. Pfeiffer, B. D. et al. Renement of tools for targeted gene expression in Drosophila. Genetics 186, 735755 (2010).
38. Stockinger, P., Kvitsiani, D., Rotkopf, S., Tirin, L. & Dickson, B. J. Neural circuitry that governs Drosophila male courtship behavior. Cell 121, 795807 (2005).
39. Baneld, B. W., Kaufman, J. D., Randall, J. A. & Pickard, G. E. Development of pseudorabies virus strains expressing red uorescent proteins: new tools for multisynaptic labeling applications. J. Virol. 77, 1010610112 (2003).
40. Chng, T. H., Flood, E. A. & Enquist, L. W. Culturing primary and transformed neuronal cells for studying pseudorabies virus infection. Methods Mol. Biol. 292, 299316 (2005).
41. Pologruto, T., Sabatini, B. & Svoboda, K. ScanImage: exible software for operating laser scanning microscopes. BioMed. Eng. OnLine 2, 13 (2003).
42. Neher, E. The inuence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum. J. Phys. 395, 193214 (1988).
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 Investigator 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-ow uorimetry, Fred Hughson for plasmids and protein purication 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 purication and equilibrium measurements; X.R.S. performed stopped-ow measurements; D.A.P. performed imaging in Drosophila; A.B. and E.R.S. performed brain slice imaging; M.P.T. andI.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
Web End =http://www.nature.com/ http://www.nature.com/naturecommunications
Web End =naturecommunications
Competing nancial interests: The authors declare no competing nancial interests.
Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/
Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions/
Web End =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).
10 NATURE COMMUNICATIONS | 4:2170 | DOI: 10.1038/ncomms3170 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Copyright Nature Publishing Group Jul 2013
Abstract
The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca2+ -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 Ca2+ -chelating residues. Second, we find that off-responses to Ca2+ 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 Ca2+ and by slow binding to the C-lobe at lower Ca2+ . 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 Ca2+ 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.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer