Abstract
Superimposed on the vast and complex synaptic network is a largely invisible set of chemical inputs, such as neurotransmitters and neuromodulators, that exert a profound influence on brain function across many structures and temporal scales. Thus, the determination of the spatiotemporal relationships between these chemical signals with synaptic resolution in the intact brain is essential to decipher the codes for transferring information across circuitry and systems. Recent advances in imaging technology have been employed to determine the extent of spatial and temporal neurotransmitter dynamics in the brain, especially glutamate, the most abundant excitatory neurotransmitter. Here, we discuss recent imaging approaches, particularly with a focus on the design and application of genetically encoded indicator iGluSnFR, in analyzing glutamate transients in vitro, ex vivo, and in vivo.
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References
Attwell D, Laughlin SB (2001) An energy budget for signaling in the grey matter of the brain. J Cereb Blood Flow Metab 21:1133–1145. doi:10.1097/00004647-200110000-00001
Kwon H-B, Sabatini BL (2011) Glutamate induces de novo growth of functional spines in developing cortex. Nature 474:100–104. doi:10.1038/nature09986
Lüscher C, Malenka RC (2012) NMDA receptor-dependent long-term potentiation and long-term depression (LTP/LTD). Cold Spring Harb Perspect Biol. doi:10.1101/cshperspect.a005710
Volterra A, Liaudet N, Savtchouk I (2014) Astrocyte Ca2+ signalling: an unexpected complexity. Nat Rev Neurosci 15:327–335. doi:10.1038/nrn3725
Arundine M, Tymianski M (2004) Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury. Cell Mol Life Sci 61:657–668. doi:10.1007/s00018-003-3319-x
Woodroofe N, Amor S (2014) Neuroinflammation and CNS disorders. John Wiley & Sons, West Sussex, UK
Clements JD (1996) Transmitter timecourse in the synaptic cleft: its role in central synaptic function. Trends Neurosci 19:163–171. doi:10.1016/S0166-2236(96)10024-2
Marcaggi P, Attwell D (2004) Role of glial amino acid transporters in synaptic transmission and brain energetics. Glia 47:217–225. doi:10.1002/glia.20027
Ventura R, Harris KM (1999) Three-dimensional relationships between hippocampal synapses and astrocytes. J Neurosci 19:6897–6906
Veruki ML, Mørkve SH, Hartveit E (2006) Activation of a presynaptic glutamate transporter regulates synaptic transmission through electrical signaling. Nat Neurosci 9:1388–1396. doi:10.1038/nn1793
Moussawi K, Riegel A, Nair S, Kalivas PW (2011) Extracellular glutamate: functional compartments operate in different concentration ranges. Front Syst Neurosci. doi:10.3389/fnsys.2011.00094
Ferraguti F, Shigemoto R (2006) Metabotropic glutamate receptors. Cell Tissue Res 326:483–504. doi:10.1007/s00441-006-0266-5
Hollmann M, Heinemann S (1994) Cloned glutamate receptors. Annu Rev Neurosci 17:31–108. doi:10.1146/annurev.ne.17.030194.000335
Zito K, Scheuss V (2009) NMDA receptor function and physiological modulation. Encyclopedia Neurosci. doi:10.1016/b978-008045046-9.01225-0
Conn PJ, Pin JP (1997) Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 37:205–237. doi:10.1146/annurev.pharmtox.37.1.205
Marcaggi P, Mutoh H, Dimitrov D et al (2009) Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization. Proc Natl Acad Sci U S A 106:11388–11393. doi:10.1073/pnas.0901290106
Vafabakhsh R, Levitz J, Isacoff EY (2015) Conformational dynamics of a class C G-protein-coupled receptor. Nature 524:497–501. doi:10.1038/nature14679
Masugi-Tokita M, Shigemoto R (2007) High-resolution quantitative visualization of glutamate and GABA receptors at central synapses. Curr Opin Neurobiol 17:387–393. doi:10.1016/j.conb.2007.04.012
Araque A, Carmignoto G, Haydon PG (2001) Dynamic signaling between astrocytes and neurons. Annu Rev Physiol 63:795–813. doi:10.1146/annurev.physiol.63.1.795
Chefer VI, Thompson AC, Zapata A, Shippenberg TS (2009) Overview of brain microdialysis. Curr Protoc Neurosci. Chapter 7:Unit7.1. doi: 10.1002/0471142301.ns0701s47
McLamore ES, Mohanty S, Shi J et al (2010) A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux. J Neurosci Methods 189:14–22. doi:10.1016/j.jneumeth.2010.03.001
Namiki S, Sakamoto H, Iinuma S et al (2007) Optical glutamate sensor for spatiotemporal analysis of synaptic transmission. Eur J Neurosci 25:2249–2259. doi:10.1111/j.1460-9568.2007.05511.x
Brun MA, Tan K-T, Griss R et al (2012) A semisynthetic fluorescent sensor protein for glutamate. J Am Chem Soc 134:7676–7678. doi:10.1021/ja3002277
Takikawa K, Asanuma D, Namiki S et al (2014) High-throughput development of a hybrid-type fluorescent glutamate sensor for analysis of synaptic transmission. Angew Chem Int Ed 53:13439–13443. doi:10.1002/anie.201407181
Oldenziel WH, Beukema W, Westerink BHC (2004) Improving the reproducibility of hydrogel-coated glutamate microsensors by using an automated dipcoater. J Neurosci Methods 140:117–126. doi:10.1016/j.jneumeth.2004.04.038
Rahman MA, Kwon N-H, Won M-S et al (2005) Functionalized conducting polymer as an enzyme-immobilizing substrate: an amperometric glutamate microbiosensor for in vivo measurements. Anal Chem 77:4854–4860. doi:10.1021/ac050558v
Hu Y, Mitchell KM, Albahadily FN et al (1994) Direct measurement of glutamate release in the brain using a dual enzyme-based electrochemical sensor. Brain Res 659:117–125
Broussard GJ, Liang R, Tian L (2014) Monitoring activity in neural circuits with genetically encoded indicators. Front Mol Neurosci. doi:10.3389/fnmol.2014.00097
Lin MZ, Schnitzer MJ (2016) Genetically encoded indicators of neuronal activity. Nat Neurosci 19:1142–1153. doi:10.1038/nn.4359
Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat Biotechnol 19:137–141. doi:10.1038/84397
Tallini YN, Ohkura M, Choi B-R et al (2006) Imaging cellular signals in the heart in vivo: cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proc Natl Acad Sci U S A 103:4753–4758. doi:10.1073/pnas.0509378103
Tian L, Hires SA, Mao T et al (2009) Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6:875–881. doi:10.1038/nmeth.1398
Akerboom J, Chen T-W, Wardill TJ et al (2012) Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci 32:13819–13840. doi:10.1523/JNEUROSCI.2601-12.2012
Chen T-W, Wardill TJ, Sun Y et al (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499:295–300. doi:10.1038/nature12354
Baird GS, Zacharias DA, Tsien RY (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci U S A 96:11241–11246. doi:10.1073/pnas.96.20.11241
Wang Q, Shui B, Kotlikoff MI, Sondermann H (2008) Structural basis for calcium sensing by GCaMP2. Structure 16:1817–1827. doi:10.1016/j.str.2008.10.008
Petreanu L, Gutnisky DA, Huber D et al (2012) Activity in motor-sensory projections reveals distributed coding in somatosensation. Nature 489:299–303. doi:10.1038/nature11321
Shigetomi E, Bushong EA, Haustein MD et al (2013) Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses. J Gen Physiol 141:633–647. doi:10.1085/jgp.201210949
Issa JB, Haeffele BD, Agarwal A et al (2014) Multiscale optical Ca2+ imaging of tonal organization in mouse auditory cortex. Neuron 83:944–959. doi:10.1016/j.neuron.2014.07.009
Vanni MP, Murphy TH (2014) Mesoscale transcranial spontaneous activity mapping in GCaMP3 transgenic mice reveals extensive reciprocal connections between areas of somatomotor cortex. J Neurosci 34:15931–15946. doi:10.1523/JNEUROSCI.1818-14.2014
Murakami T, Yoshida T, Matsui T, Ohki K (2015) Wide-field Ca2+ imaging reveals visually evoked activity in the retrosplenial area. Front Mol Neurosci. doi:10.3389/fnmol.2015.00020
Sun XR, Badura A, Pacheco DA et al (2013) Fast GCaMPs for improved tracking of neuronal activity. Nat Commun 4:2170. doi:10.1038/ncomms3170
Helassa N, Zhang X, Conte I et al (2015) Fast-response calmodulin-based fluorescent indicators reveal rapid intracellular calcium dynamics. Sci Rep 5:15978. doi:10.1038/srep15978
Huber D, Gutnisky DA, Peron S et al (2012) Multiple dynamic representations in the motor cortex during sensorimotor learning. Nature 484:473–478. doi:10.1038/nature11039
Ziv Y, Burns LD, Cocker ED et al (2013) Long-term dynamics of CA1 hippocampal place codes. Nat Neurosci 16:264–266. doi:10.1038/nn.3329
Vogt N (2015) Voltage sensors: challenging, but with potential. Nat Methods 12:921–924. doi:10.1038/nmeth.3591
Fioravante D, Regehr WG (2011) Short-term forms of presynaptic plasticity. Curr Opin Neurobiol 21:269–274. doi:10.1016/j.conb.2011.02.003
Xie Y, Chan AW, McGirr A et al (2016) Resolution of high-frequency mesoscale intracortical maps using the genetically encoded glutamate sensor iGluSnFR. J Neurosci 36:1261–1272. doi:10.1523/JNEUROSCI.2744-15.2016
Vyleta NP, Smith SM (2011) Spontaneous glutamate release is independent of calcium influx and tonically activated by the calcium-sensing receptor. J Neurosci 31:4593–4606. doi:10.1523/JNEUROSCI.6398-10.2011
Schellenberg GD, Furlong CE (1977) Resolution of the multiplicity of the glutamate and aspartate transport systems of Escherichia coli. J Biol Chem 252:9055–9064
Dwyer MA, Hellinga HW (2004) Periplasmic binding proteins: a versatile superfamily for protein engineering. Curr Opin Struct Biol 14:495–504. doi:10.1016/j.sbi.2004.07.004
Okumoto S, Looger LL, Micheva KD et al (2005) Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc Natl Acad Sci U S A 102:8740–8745. doi:10.1073/pnas.0503274102
Hires SA, Zhu Y, Tsien RY (2008) Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters. Proc Natl Acad Sci U S A 105:4411–4416. doi:10.1073/pnas.0712008105
Marvin JS, Borghuis BG, Tian L et al (2013) An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods 10:162–170. doi:10.1038/nmeth.2333
Marvin JS, Schreiter ER, EchevarrÃa IM, Looger LL (2011) A genetically encoded, high-signal-to-noise maltose sensor. Proteins 79:3025–3036. doi:10.1002/prot.23118
Brunert D, Tsuno Y, Rothermel M et al (2016) Cell-type-specific modulation of sensory responses in olfactory bulb circuits by serotonergic projections from the raphe nuclei. J Neurosci 36:6820–6835. doi:10.1523/JNEUROSCI.3667-15.2016
Borghuis BG, Marvin JS, Looger LL, Demb JB (2013) Two-photon imaging of nonlinear glutamate release dynamics at bipolar cell synapses in the mouse retina. J Neurosci 33:10972–10985. doi:10.1523/JNEUROSCI.1241-13.2013
Borghuis BG, Looger LL, Tomita S, Demb JB (2014) Kainate receptors mediate signaling in both transient and sustained OFF bipolar cell pathways in mouse retina. J Neurosci 34:6128–6139. doi:10.1523/JNEUROSCI.4941-13.2014
Yonehara K, Farrow K, Ghanem A et al (2013) The first stage of cardinal direction selectivity is localized to the dendrites of retinal ganglion cells. Neuron 79:1078–1085. doi:10.1016/j.neuron.2013.08.005
Baxter PS, Bell KFS, Hasel P et al (2015) Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system. Nat Commun 6:6761. doi:10.1038/ncomms7761
O’Herron P, Chhatbar PY, Levy M et al (2016) Neural correlates of single-vessel haemodynamic responses in vivo. Nature. doi:10.1038/nature17965
Bao H, Goldschen-Ohm M, Jeggle P et al (2016) Exocytotic fusion pores are composed of both lipids and proteins. Nat Struct Mol Biol 23:67–73. doi:10.1038/nsmb.3141
Poleg-Polsky A, Diamond JS (2016) Retinal circuitry balances contrast tuning of excitation and inhibition to enable reliable computation of direction selectivity. J Neurosci 36:5861–5876. doi:10.1523/JNEUROSCI.4013-15.2016
Zhang R, Li X, Kawakami K, Du J (2016) Stereotyped initiation of retinal waves by bipolar cells via presynaptic NMDA autoreceptors. Nat Commun 7:12650. doi:10.1038/ncomms12650
Haustein MD, Kracun S, X-H L et al (2014) Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway. Neuron 82:413–429. doi:10.1016/j.neuron.2014.02.041
Rosa JM, Bos R, Sack GS et al (2015) Neuron-glia signaling in developing retina mediated by neurotransmitter spillover. eLife 4:e09590. doi:10.7554/eLife.09590
Stork T, Sheehan A, Tasdemir-Yilmaz OE, Freeman MR (2014) Neuron-glia interactions through the heartless FGF receptor signaling pathway mediate morphogenesis of drosophila astrocytes. Neuron 83:388–403. doi:10.1016/j.neuron.2014.06.026
Parsons MP, Vanni MP, Woodard CL et al (2016) Real-time imaging of glutamate clearance reveals normal striatal uptake in Huntington disease mouse models. Nat Commun 7:11251. doi:10.1038/ncomms11251
Poskanzer KE, Yuste R (2016) Astrocytes regulate cortical state switching in vivo. Proc Natl Acad Sci U S A 113:E2675–E2684. doi:10.1073/pnas.1520759113
Looger LL, Griesbeck O (2012) Genetically encoded neural activity indicators. Curr Opin Neurobiol 22:18–23. doi:10.1016/j.conb.2011.10.024
Akerboom J, Tian L, Marvin J, Looger L (2012) Engineering and application of genetically encoded calcium indicators. In: Martin J-R (ed) Genetically encoded functional indicators. Humana Press, New York, pp 125–147
Moretti R, Bender BJ, Allison B, Meiler J (2016) Rosetta and the design of ligand binding sites. Methods Mol Biol 1414:47–62. doi:10.1007/978-1-4939-3569-7_4
Pettersen EF, Goddard TD, Huang CC et al (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. doi:10.1002/jcc.20084
Bender BJ, Cisneros A, Duran AM et al (2016) Protocols for molecular modeling with Rosetta3 and RosettaScripts. Biochemistry 55:4748–4763. doi:10.1021/acs.biochem.6b00444
Hughes MD, Nagel DA, Santos AF et al (2003) Removing the redundancy from randomised gene libraries. J Mol Biol 331:973–979
Edelheit O, Hanukoglu A, Hanukoglu I (2009) Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol 9:61. doi:10.1186/1472-6750-9-61
Liu H, Naismith JH (2008) An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol 8:91. doi:10.1186/1472-6750-8-91
Quan J, Tian J (2011) Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc 6:242–251. doi:10.1038/nprot.2010.181
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492
Huang R, Fang P, Kay BK (2012) Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries. Methods 58:10–17. doi:10.1016/j.ymeth.2012.08.008
Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. Genome Res 2:28–33. doi:10.1101/gr.2.1.28
Gruet A, Longhi S, Bignon C (2012) One-step generation of error-prone PCR libraries using Gateway® technology. Microb Cell Factories 11:14. doi:10.1186/1475-2859-11-14
Drobizhev M, Makarov NS, Tillo SE et al (2011) Two-photon absorption properties of fluorescent proteins. Nat Methods 8:393–399. doi:10.1038/nmeth.1596
Makarov NS, Drobizhev M, Rebane A (2008) Two-photon absorption standards in the 550–1600 nm excitation wavelength range. Opt Express 16:4029–4047. doi:10.1364/OE.16.004029
Vandenberghe LH, Xiao R, Lock M et al (2010) Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing. Hum Gene Ther 21:1251–1257. doi:10.1089/hum.2010.107
Paxinos G, Franklin KBJ (2012) Paxinos and Franklin’s the mouse brain in stereotaxic coordinates, 4th edn. Academic Press, Amsterdam
Deverman BE, Pravdo PL, Simpson BP et al (2016) Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34:204–209. doi:10.1038/nbt.3440
Chia TH, Levene MJ (2009) Microprisms for in vivo multilayer cortical imaging. J Neurophysiol 102:1310–1314. doi:10.1152/jn.91208.2008
Low RJ, Gu Y, Tank DW (2014) Cellular resolution optical access to brain regions in fissures: imaging medial prefrontal cortex and grid cells in entorhinal cortex. Proc Natl Acad Sci U S A 111:18739–18744. doi:10.1073/pnas.1421753111
Guo ZV, Hires SA, Li N, O'Connor DH, Komiyama T, Ophir E, Huber D, Bonardi C, Morandell K, Gutnisky D, Peron S, Xu N-l, Cox J, Svoboda K (2014) Procedures for behavioral experiments in head-fixed mice. PLoS One 9(2). doi:10.1371/journal.pone.0088678
Kislin M, Mugantseva E, Molotkov D, Kulesskaya N, Khirug S, Kirilkin I, Pryazhnikov E, Kolikova J, Toptunov D, Yuryev M, Giniatullin R, Voikar V, Rivera C, Rauvala H, Khiroug L (2014) Flat-floored air-lifted platform: a new method for combining behavior with microscopy or electrophysiology on awake freely moving rodents. J Vis Exp 88:–e51869. doi:10.3791/51869
Urbain N, Gervasoni D, Soulière F et al (2000) Unrelated course of subthalamic nucleus and globus pallidus neuronal activities across vigilance states in the rat. Eur J Neurosci 12:3361–3374
Urbain N, Salin PA, Libourel P-A et al (2015) Whisking-related changes in neuronal firing and membrane potential dynamics in the somatosensory thalamus of awake mice. Cell Rep 13:647–656. doi:10.1016/j.celrep.2015.09.029
Guizar-Sicairos M, Thurman ST, Fienup JR (2008) Efficient subpixel image registration algorithms. Opt Lett 33(2):156–158
Wu M, Chen R, Soh J, Shen Y, Jiao L, Wu J, Chen X, Ji R, Hong M (2016) Super-focusing of center-covered engineered microsphere. Sci Rep 6:31637. doi:10.1038/srep31637
Granseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51:773–786. doi:10.1016/j.neuron.2006.08.029
Shen Y, Lai T, Campbell RE (2015) Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications. Neurophotonics 2:031203. doi:10.1117/1.NPh.2.3.031203
Dana H, Mohar B, Sun Y et al (2016) Sensitive red protein calcium indicators for imaging neural activity. Elife. doi:10.7554/eLife.12727
Lin JY, Knutsen PM, Muller A et al (2013) ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat Neurosci 16:1499–1508. doi:10.1038/nn.3502
Drobizhev M, Tillo S, Makarov NS, Hughes TE, Rebane A (2009) Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. J Phys Chem B 113(4):855–859. doi:10.1021/jp8087379
Acknowledgements
This work is supported by NIH DP2 MH107059 (L.T.), Brain Initiative U01NS090604 (L.T., E.K.U., G.J.B.) and U01NS09058 (R.L.), Rita Allen Foundation (R.L.), Human Frontier Research Program (G.J.B.), and NIH R21NS095325 (B.P.M.). We are grateful for the contributions of Douglas Unger in generating the rotation matrix. We are grateful to Loren Looger, Jonathan Marvin and Philip Borden for their pioneering work in engineering iGluSnFR and critical comments. We also thank Lisa Makhoul for careful reading and discussion of this book chapter.
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Broussard, G.J., Unger, E.K., Liang, R., McGrew, B.P., Tian, L. (2018). Imaging Glutamate with Genetically Encoded Fluorescent Sensors. In: Parrot, S., Denoroy, L. (eds) Biochemical Approaches for Glutamatergic Neurotransmission. Neuromethods, vol 130. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7228-9_5
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