Genetically Encoded Glutamate Indicators with Altered Color and

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Genetically Encoded Glutamate Indicators with Altered Color and Topology Jiahui Wu, Ahmed S Abdelfattah, Hang Zhou, Araya Ruangkittisakul, Yong Qian, Klaus Ballanyi, and Robert E. Campbell ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b01085 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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Genetically Encoded Glutamate Indicators with Altered Color and Topology Jiahui Wu†,§, Ahmed S. Abdelfattah†,#, Hang Zhou†, Araya Ruangkittisakul‡, Yong Qian†, Klaus Ballanyi‡ and Robert E. Campbell*,† †

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2

‡

Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

§

Present address: Department of Pharmacology, Weill-Cornell Medical College, Cornell University, New York, New York, USA 10065 #

Present address: Howard Hughes Medical Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, Virginia, USA 20147

Supporting Information Placeholder ABSTRACT: Glutamate is one of the 20 common amino acids, and of utmost importance for chemically mediated synaptic transmission in nervous systems. To expand the color palette of genetically encoded indicators for glutamate, we used protein engineering to develop a red intensity-based glutamate-sensing fluorescent reporter (R-iGluSnFR1). Manipulating the topology of R-iGluSnFR1, and a previously reported green fluorescent indicator, led to the development ncp of non-circularly permutated (ncp) variants. R- and R iGluSnFR1 display glutamate affinities of 11 µM and 0.9 µM, respectively. We demonstrate that these glutamate indicators are functional when targeted to the surface of ncp HEK-293 cells. Furthermore, we show that G -iGluSnFR enabled reliable visualization of extrasynaptic glutamate in organotypic hippocampal slice cultures, while R-iGluSnFR can reliably resolve action potential-evoked glutamate transients by electrical field stimuli in cultures of dissociated hippocampal neurons.

Glutamate plays a pivotal role in chemical communication in 1,2 the nervous system of both vertebrates and invertebrates. In the mammalian brain, glutamate actions have most extensively been studied at synapses, where it is released at the presynaptic terminal of one neuron to activate mostly excitatory receptors on the membrane of a postsynaptic 2–4 neuron. Moreover, glutamate actions on neurons are controled by its uptake into both neurons and neighboring 2–4 glial cells who themselves express glutamate receptors. 4,5 Finally, glutamate also acts on extrasynaptic receptors. To gain a better understanding of the various actions of glutamate, methods to visualize its release and dynamics in neuronal tissues are required.

Fluorescent protein (FP)-based genetically encoded neurotransmitter indicators (GENIs) convert “invisible” inter-neural signaling events into optical signals that can be visualized using fluorescence microscopy. The earliest GENIs for glutamate were based on the modulation of intramolecular Förster resonance energy transfer (FRET) efficiency between a cyan FP and a yellow FP due to the clamshell-like conformational change of Escherichia coli 6 protein GltI upon binding to glutamate. Examples of such indicators include fluorescent indicator protein for glutamate 7 (FLIPE), glutamate-sensing fluorescent reporter (GluSnFR) 8 and the further optimized SuperGluSnFR variant. While these FRET-based indicators all benefit from an inherently ratiometric response, their signal changes in response to glutamate are relatively small (i.e., ∆Rmax = 44% for SuperGluSnFR). To develop a GENI for glutamate with larger signal changes and increased utility for in vivo imaging, Marvin et al. engineered an intensity-based GluSnFR (iGluSnFR, hereafter 9 designated green (G)-iGluSnFR). G-iGluSnFR was constructed by insertion of a circularly permutated (cp) green FP (GFP), with termini in close proximity to the chromophore, into the hinge region of GltI where a substantial conformational change upon glutamate binding is expected. G-iGluSnFR exhibits fast kinetics of response and a (∆F/F)max of 4.5, enabling high signal-to-noise detection of neural glutamate release. The effectiveness of using bindingdependent conformational changes to modulate the fluorescence of a cpGFP has been most clearly illustrated by 2+ 10 the success of the GCaMP-series of Ca indicators. A drawback of green intensity-based indicators is that they require excitation with light in the blue to cyan region of the spectrum. Relative to more red-shifted excitation light, light in this range is associated with higher levels of

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autofluorescence, higher levels of phototoxicity and decreased penetration into tissue. One solution to these problems is to use laser scanning two-photon imaging, in which high intensities of near-infrared light are used for excitation. A second solution is to replace the GFP portion of 11–13 the indicator with a yellow-light excitable red FP (RFP). We have previously used such an approach to create the 13 2+ cpRFP-based (specifically, cpmApple) Ca indicator R15 GECO1 (Ref. 14), and the voltage indicator FlicR1. The X-ray crystal structure of GCaMP2 revealed that an 2+ arginine side chain on the Ca -binding domain is key to 2+ manipulating the Ca -dependent protonation state of the 16 chromophore, and thereby its fluorescence intensity. The 17 X-ray crystal structure of R-GECO1 (PDB ID 4I2Y) revealed a similar interaction between Lys270 (Figure S1) of the cpRFP and the chromophore (Figure S2). We speculated that this interaction stabilizes the chromophore in its brightly 2+ fluorescent phenolate form in the Ca -bound state. Further, 2+ we presume that in the absence of Ca , this interaction is absent and the chromophore is in its dimly fluorescent phenol form. The fact that Lys270 of R-GECO1 is a residue of cpRFP (as opposed to a residue of the binding module, as in GCaMP), suggested that this cpRFP has a ‘built in’ fluorescence modulation mechanism and could be particularly amenable for construction of other types of indicators. We reasoned that it should be possible to couple the glutamate-dependent conformational change of GltI into a modulation of the chromophore protonation state, and fluorescence intensity, of the R-GECO1-derived cpRFP. To test this idea, we replaced the cpGFP domain (residues 248 to 496) of G-iGluSnFR with the cpRFP (Figure S1) domain of R-GECO1. Codons encoding linker residues Pro248 and Ala494 were fully randomized using an NNK codon (N = A, G, T, and C and K = G and T; Figure S1). This library of variants was expressed in E. coli in the context of bacterial colonies on solid media. Picking of clones that exhibited the brightest red fluorescence led to the identification of a variant with 54 ± 5% decrease in fluorescence intensity (dynamic range = (Fmax-Fmin)/Fmin = 1.2 ± 0.2) upon binding to glutamate. This variant, designated red (R)-iGluSnFR0.1, carries mutations Pro248Glu and Ala494Pro at the randomized linker positions. To further increase the relatively low fluorescent brightness and dynamic range of R-iGluSnFR0.1, we performed 7 iterative rounds of directed evolution by library creation and fluorescence-based screening of E. coli colonies. In each round we picked colonies that exhibited the highest fluorescence intensity, and then tested the glutamate– dependent response of soluble protein extracted from an overnight E. coli culture (see Supporting Information). Genes encoding the most promising variants were used as the template for the subsequent round of library creation. These efforts ultimately led to the identification of R-iGluSnFR1, which exhibits a dynamic range of 4.9 ± 0.2 (Figure 1), and a glutamate affinity (Kd) of 11 ± 2 µM. R-iGluSnFR1 has 12 mutations relative to R-iGluSnFR0.1 (Figure S1; Table S1; Table S2). Of these 12 mutations, 5 are in the GltI-derived domain and 7 are in the cpRFP (Figure 1; Figure S1).

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Figure 1. Modeled structure of red glutamate indicators and responses to glutamate. (A) Location of substitutions in RiGluSnFR1. The linker to connect the normal termini of the mApple RFP is represented as a green line. (B) Topology of ncp R-iGluSnFR1 and R -iGluSnFR1. The N- and C-terminus of ncp each indicator is labeled with N and C. In R -iGluSnFR1, the linker included to connect the former termini is represented as a blue line. (C,D) Normalized excitation and emission ncp spectra of R-iGluSnFR1 (C) and R -iGluSnFR1 (D). Topologically, R-iGluSnFR1 is composed of a circularly permuted FP inserted into GltI. An alternate topology for this protein, that would have an identical connection between the FP domain and the GltI domain, would be to 18 insert GltI into the FP. A protein with an altered topology could, in principle, exhibit differences in rate of folding, overall stability, rate of degradation, or other properties, that could change the steady state concentration or function when expressed in mammalian cells. To modify the topology of R-iGluSnFR1, we used a linker with 37 amino acids to genetically connect the GltI-derived N- and C-termini, and reinstated the normal termini of the mApple RFP (Figure S3). We designated this non-cp (ncp) variant as ncp ncp R -iGluSnFR1 (Figure 1, Figure S3). R -iGluSnFR1 exhibits a glutamate affinity of 0.9 ± 0.1 µM, while maintaining a 4.8 ± 0.2-fold dynamic range (Figure 1, Table S2). This preservation of function and dynamic range suggested that this same 9 strategy should also be applicable to G-iGluSnFR. ncp Accordingly, we created G -iGluSnFR, which similarly exhibits an increased glutamate affinity of 22 ± 2 µM (33.0 ± 0.7 µM for G-iGluSnFR), and a 4.4 ± 0.2-fold dynamic range (Figure S3). We conclude that this ncp topology, in which the glutamate-binding domain is inserted within the FP (i.e., a 18 camgaroo-like topology), retains the functionally important features of the cp topology. Systematic in vitro characterization revealed that RiGluSnFR1 has excitation and emission peaks at 562 nm and 588 nm, respectively, in the glutamate-free state. These peaks shift to 564 nm and 592 nm, respectively, in the ncp glutamate-bound state (Figure 1, Table S2). R -iGluSnFR1 has very similar spectral properties, but with a slightly redshifted emission (Figure 1, Table S2). Both red indicators exhibit a decrease in both extinction coefficient and quantum yield upon binding to glutamate (Table S2). Absorbance spectra reveal an increase in the phenol form in the glutamate-bound state (Figure S4). pH titrations were ncp used to determine that R-iGluSnFR1 and R -iGluSnFR1 have a pKa of 5.1 in the glutamate-free state, and pKas of 8.5 and 8.3, respectively, in the glutamate-bound state (Figure S4). These results indicate that, in contrast to R-GECO1, Lys270 is positioned to stabilize the phenolate in the unbound form. The conformational change upon binding to glutamate must cause Lys270 to move further from the chromophore, which shifts the equilibrium towards the phenol form. To determine the specificity of these indicators, we titrated ncp purified R-iGluSnFR1 and R -iGluSnFR1 proteins against the structurally similar amino acids aspartate, glutamine and asparagine. The results show that R-iGluSnFR1 has a Kd of 39 ± 4 µM for aspartate, 1.3 ± 0.3 mM for asparagine, and no fluorescence response for glutamine at concentrations up to 1


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ncp

mM. R -iGluSnFR1 has a Kd of 4.9 ± 0.8 µM for aspartate, 2 ± 2 mM for asparagine, and 400 ± 100 µM for glutamine ncp (Figure S5). Overall, the specificity of R- and R -iGluSnFR1 for glutamate have decreased relative to G-iGluSnFR, which was reported to have no detectable affinity for any amino 9 acids other than glutamate and aspartate. As previously 9 ncp reported for G-iGluSnFR, R- and R -iGluSnFR exhibited rise-times faster than could be monitored by our stoppedflow fluorometer (Figure S6). To explore the utility of these new indicators as possible ncp GENIs, we expressed R-iGluSnFR1, R -iGluSnFR1, Gncp iGluSnFR, and G -iGluSnFR, on the surface of HEK-293 9 cells and tested their responses to glutamate. Buffers with various concentrations of glutamate were perfused over transfected HEK-293 cells, and green or red fluorescence was visualized by fluorescence microscopy. The relationship between fluorescence intensity and glutamate concentration was in good qualitative agreement with in vitro titrations ncp (Figure 2). When R-iGluSnFR1 or R -iGluSnFR1 were concp expressed with G-iGluSnFR or G -iGluSnFR, addition of glutamate gave the expected decrease of red fluorescence and increase of green fluorescence (Figure 3). These results ncp indicate that R-iGluSnFR1 and R -iGluSnFR1 retain their function on the cell surface and enable multicolor imaging in combination with spectrally distinct GFP-based indicators. ncp

Figure 2. In situ glutamate titration of R-iGluSnFR1 (R), R ncp ncp ncp iGluSnFR1 (R ), G-iGluSnFR (G), and G -iGluSnFR (G ) on the surface of HEK-293 cells. Values in brackets represent the apparent dissociation constant (Kd,apparent) of each glutamate indicator.

Figure 3. Red and green glutamate indicators on the extracellular plasmalemmal surface of HEK-293 cells. HEKncp 293 cells coexpressing R -iGluSnFR and G-iGluSnFR (A and ncp B), R-iGluSnFR and G -iGluSnFR (C and D), and RiGluSnFR and G-iGluSnFR (E and F) were treated with 2 mM glutamate. Normalized fluorescence intensity (F.I.) of region of interest (ROI) in (A, C, and E) was plotted against time (B, D, and F). Scale bar represents 10 µm. To further explore the utility of these glutamate indicators, we inserted their genes into the pCMV(MinDis) plasmid 19,20 under control of the human synapsin I promoter, and used this plasmid to transfect dissociated rat hippocampal neurons or organotypic rat hippocampal slices. Confocal imaging revealed all indicators correctly localized to the plasma membrane (Figure S7). As previously reported for 15 ncp mApple-derived indicators, R- and R -iGluSnFR1 exhibit lysosomal accumulation when expressed in neurons. We ncp found that G -iGluSnFR performed similarly to G-iGluSnFR and enabled reliable and robust imaging of both spontaneous and theophylline-induced glutamate release in both hippocampal neurons and glial cells (Figure 4) in organotypic rat hippocampal slices.

ncp

Figure 4. G -iGluSnFR expressed on the extracellular surface of a hippocampal neuron (A-C) and glial cells (D-F) ncp in organotypic rat hippocampal slices. G -iGluSnFR exhibits responses to both spontaneous (B, E) and theophyllineinduced (C, F) glutamate changes. For (C) and (F), artificial cerebrospinal fluid (ACSF) with 10 mM theophylline was added one minute before start of image acquisition. Intensities in ROI 1, 2, and 3 in (A, D) were plotted against time in blue, red, and black, respectively. ROI intensities from (A) are plotted in (B, C), and ROI intensities from (D) are plotted in (E, F). Scale bar represents 30 µm. ncp

To characterize the performance of R- and R -iGluSnFR1 on the extracellular surface of dissociated rat hippocampal neurons, we imaged their fluorescence during electrical field stimulations that could evoke action potentials (APs). We found that both red indicators could reliably resolve glutamate transients induced by electrical field stimuli ncp (Figure 5). Furthermore, R- and R -iGluSnFR1's fluorescence response plateaued at higher numbers of field stimuli (50 to 160) (Figure 5) than G-iGluSnFR, which 9 plateaued at ~5 field stimuli as previously reported. We were unable to achieve reliable and robust imaging of spontaneous ncp or theophylline-induced glutamate release with R- or R iGluSnFR1 expressed in organotypic rat hippocampal slices. We speculate that this is due to the low response of these indicators to a single field stimulus (-1.1 ± 0.2%) (Figure 5D). In contrast, G-iGluSnFR has a response of 14 ± 2% to 1 field 9 stimulus. The combined use of a green indicator that increases fluorescence in response to glutamate, and a red indicator that decreases fluorescence in response to glutamate, should enable highly sensitive ratiometric imaging.

ncp

Figure 5. R- and R -iGluSnFR1 characterization in cultured rat hippocampal neurons. (A) Fluorescence image of cultured rat hippocampal neurons expressing R-iGluSnFR1 under the control of synapsin promotor. Scale bar represents 80 µm. (B) Fluorescence response of R-iGluSnFR1 to electric field stimulation-induced glutamate release. Vertical black marks indicate field stimulus application (50V, 80 Hz, 1 ms, 20 stimuli). (C) Representative traces of R-iGluSnFR1 fluorescence responses to field stimulation in cultured rat hippocampal neurons. (D) Average fluorescence change of RiGluSnFR1 in response to field stimulation (n = 30-70 neurons ncp for each field stimulus train; error bars are s.e.m). (E,F) R iGluSnFR1 characterized as in (C,D) For panel F, n = 36-86 neurons for each field stimulation train and error bars are s.e.m. In summary, inspired by the ‘built-in’ fluorescence modulation mechanism of R-GECO1, we have engineered a single RFP-based glutamate indicator, R-iGluSnFR1, and a ncp topological variant, R -iGluSnFR1. These new red glutamate indicators will create new opportunities for multi-color and multi-analyte imaging in combination with green fluorescent indicators.



ASSOCIATED CONTENT Supporting Information Experimental methods; Supporting figures S1-7; Supporting tables S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank the University of Alberta Molecular Biology Services Unit, W. Zhang for technical support, and C.W. Cairo for providing access to instrumentation. We thank the Janelia Research Campus Histology group for help with preparing cultured neurons. A.S.A. was supported by a Vanier Canada Graduate Scholarship and an Alberta Innovates Health Solutions (AIHS) Studentship. This work was supported by grants from CIHR (MOP-123514), NSERC (RGPIN 288338-2010), and Brain Canada to R.E.C and NSERC (RGPIN 06484-2014) and University Hospital Foundation (Hochhausen Fund) to K.B..

REFERENCES (1) Takeuchi, A. (1987) The transmitter role of glutamate in nervous systems. Jpn. J. Physiol. 37, 559-572. (2) Zhou, Y., and Danbolt, N. C. (2014) Glutamate as a neurotransmitter in the healthy brain. J Neural Transm 121, 799-817. (3) Attwell, D., and Laughlin, S. B. (2001) An energy budget for signaling in the grey matter of the brain. J. Cereb. Blood Flow Metab. 21, 1133-1145. (4) Okubo, Y., and Iino, M. (2011) Visualization of glutamate as a volume transmitter. J. Physiol. 589, 481-488. (5) Rodriguez, M., Sabate, M., Rodriguez-Sabate, C., and Morales, I. (2013) The role of non-synaptic extracellular glutamate. Brain Res. Bull. 93, 17-26. (6) Barash, H., and Halpern, Y. S. (1975) Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12. Biochim. Biophys. Acta 386, 168-180. (7) Okumoto, S., Looger, L. L., Micheva, K. D., Reimer, R. J., Smith, S. J., and Frommer, W. B. (2005) Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc. Natl. Acad. Sci. U.S.A. 102, 8740-8745. (8) Hires, S. A., Zhu, Y., and Tsien, R. Y. (2008) Optical measurement of synaptic glutamate spillover and reuptake by linker optimized

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glutamate-sensitive fluorescent reporters. Proc. Natl. Acad. Sci. U.S.A. 105, 4411-4416. (9) Marvin, J. S., Borghuis, B. G., Tian, L., Cichon, J., Harnett, M. T., Akerboom, J., Gordus, A., Renninger, S. L., Chen, T.-W., Bargmann, C. I., Orger, M. B., Schreiter, E. R., Demb, J. B., Gan, W.-B., Hires, S. A., and Looger, L. L. (2013) An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat. Methods 10, 162-170. (10) Chen, T.-W., Wardill, T. J., Sun, Y., Pulver, S. R., Renninger, S. L., Baohan, A., Schreiter, E. R., Kerr, R. A., Orger, M. B., Jayaraman, V., Looger, L. L., Svoboda, K., and Kim, D. S. (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295300. (11) Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L., and Lukyanov, S. A. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969-973. (12) Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., and Tsien, R. Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567-1572. (13) Shaner, N. C., Lin, M. Z., McKeown, M. R., Steinbach, P. A., Hazelwood, K. L., Davidson, M. W., and Tsien, R. Y. (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545-551. (14) Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y.-F., Nakano, M., Abdelfattah, A. S., Fujiwara, M., Ishihara, T., Nagai, T., and Campbell, R. E. (2011) An expanded palette of genetically encoded Ca²⁺ indicators. Science 333, 1888-1891. (15) Abdelfattah, A. S., Farhi, S. L., Zhao, Y., Brinks, D., Zou, P., Ruangkittisakul, A., Platisa, J., Pieribone, V. A., Ballanyi, K., Cohen, A. E., and Campbell, R. E. (2016) A bright and fast red fluorescent protein voltage indicator that reports neuronal activity in organotypic brain slices. J. Neurosci. 36, 2458-2472. (16) Wang, Q., Shui, B., Kotlikoff, M. I., and Sondermann, H. (2008) Structural basis for calcium sensing by GCaMP2. Structure 16, 18171827. (17) Akerboom, J., Carreras Calderón, N., Tian, L., Wabnig, S., Prigge, M., Tolö, J., Gordus, A., Orger, M. B., Severi, K. E., Macklin, J. J., Patel, R., Pulver, S. R., Wardill, T. J., Fischer, E., Schüler, C., Chen, T.W., Sarkisyan, K. S., Marvin, J. S., Bargmann, C. I., Kim, D. S., Kügler, S., Lagnado, L., Hegemann, P., Gottschalk, A., Schreiter, E. R., and Looger, L. L. (2013) Genetically encoded calcium indicators for multicolor neural activity imaging and combination with optogenetics. Front Mol Neurosci 6, 2. (18) Baird, G. S., Zacharias, D. A., and Tsien, R. Y. (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. U.S.A. 96, 11241-11246. (19) Glover, C. P., Bienemann, A. S., Heywood, D. J., Cosgrave, A. S., and Uney, J. B. (2002) Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity. Mol. Ther. 5, 509-516. (20) Kügler, S., Kilic, E., and Bähr, M. (2003) Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10, 337-347.


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Figure 1. Modeled structure of red glutamate indicators and responses to glutamate. (A) Location of substitutions in R-iGluSnFR1. The linker to connect the normal termini of the mApple RFP is represented as a green line. (B) Topology of R-iGluSnFR1 and Rncp-iGluSnFR1. The N- and C-terminus of each indicator is labeled with N and C. In Rncp-iGluSnFR1, the linker included to connect the former termini is represented as a blue line. (C,D) Normalized excitation and emission spectra of R-iGluSnFR1 (C) and Rncp-iGluSnFR1 (D). 153x92mm (300 x 300 DPI)



Figure 2. In situ glutamate titration of R-iGluSnFR1 (R), Rncp-iGluSnFR1 (Rncp), G-iGluSnFR (G), and GncpiGluSnFR (Gncp) on the surface of HEK-293 cells. Values in brackets represent the apparent dissociation constant (Kd,apparent) of each glutamate indicator.

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Figure 3. Red and green glutamate indicators on the extracellular plasmalemmal surface of HEK-293 cells. HEK-293 cells coexpressing Rncp-iGluSnFR and G-iGluSnFR (A and B), R-iGluSnFR and Gncp-iGluSnFR (C and D), and R-iGluSnFR and G-iGluSnFR (E and F) were treated with 2 mM glutamate. Normalized fluorescence intensity (F.I.) of region of interest (ROI) in (A, C, and E) was plotted against time (B, D, and F). Scale bar represents 10 µm.

371x249mm (300 x 300 DPI)



Figure 4. Gncp-iGluSnFR expressed on the extracellular surface of a hippocampal neuron (A-C) and glial cells (D-F) in organotypic rat hippocampal slices. Gncp-iGluSnFR exhibits responses to both spontaneous (B, E) and theophylline-induced (C, F) glutamate changes. For (C) and (F), artificial cerebrospinal fluid (ACSF) with 10 mM theophylline was added one minute before start of image acquisition. Intensities in ROI 1, 2, and 3 in (A, D) were plotted against time in blue, red, and black, respectively. ROI intensities from (A) are plotted in (B, C), and ROI intensities from (D) are plotted in (E, F). Scale bar represents 30 µm. 207x117mm (300 x 300 DPI)


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Figure 5. R- and Rncp-iGluSnFR1 characterization in cultured rat hippocampal neurons. (A) Fluorescence image of cultured rat hippocampal neurons expressing R-iGluSnFR1 under the control of synapsin promotor. Scale bar represents 80 µm. (B) Fluorescence response of R-iGluSnFR1 to electric field stimulation-induced glutamate release. Vertical black marks indicate field stimulus application (50V, 80 Hz, 1 ms, 20 stimuli). (C) Representative traces of R-iGluSnFR1 fluorescence responses to field stimulation in cultured rat hippocampal neurons. (D) Average fluorescence change of R-iGluSnFR1 in response to field stimulation (n = 30-70 neurons for each field stimulus train; error bars are s.e.m). (E,F) Rncp-iGluSnFR1 characterized as in (C,D) For panel F, n = 36-86 neurons for each field stimulation train and error bars are s.e.m. 175x230mm (300 x 300 DPI)



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Genetically Encoded Glutamate Indicators with Altered Color and

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