Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots

Misa Arizono1, Hiroko Bannai2, Katsuhiko Mikoshiba3

1 Interdisciplinary Institute for Neuroscience (IINS), Université Bordeaux Segalen, Bordeaux, 2 Division of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya, 3 Laboratory for Developmental Neurobiology, Brain Science Institute, Saitama
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.21
DOI:  10.1002/0471142301.ns0221s66
Online Posting Date:  January, 2014
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Abstract

This unit describes the method that we have developed to clarify endogenous mGluR5 (etabotropic tamate eceptors 5) dynamics in astrocytes by single‐particle tracking using quantum dots (QD‐SPT). QD‐SPT has been a powerful tool to examine the contribution of neurotransmitter receptor dynamics to synaptic plasticity. Neurotransmitter receptors are also expressed in astrocytes, the most abundant form of glial cell in the brain. mGluR5s, which evoke intracellular Ca2+ signals upon receiving glutamate, contribute to the modulation of synaptic transmission efficacy and local blood flow by astrocytes. QD‐SPT has previously revealed that the regulation of the lateral diffusion of mGluR5 on the plasma membrane is important for local Ca2+ signaling in astrocytes. Determining how mGluR5 dynamics are regulated in response to neuronal input would enable a better understanding of neuron‐astrocyte communication in future studies. Curr. Protoc. Neurosci. 66:2.21.1‐2.21.18. © 2014 by John Wiley & Sons, Inc.

Keywords: astrocyte; mGluR5; single particle tracking; quantum dot

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Primary Culture of Hippocampal Neurons and Astrocytes
  • Basic Protocol 2: Transfection of Plasmid Encoding Fluorescent Protein into Astrocytes
  • Basic Protocol 3: Labeling mGluR5s with QDs
  • Basic Protocol 4: Recording and Analyzing mGluR5 Dynamics to Calculate Diffusion Parameters
  • Basic Protocol 5: Visualizing and Quantifying Diffusion Hindrances
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Primary Culture of Hippocampal Neurons and Astrocytes

  Materials
  • 4% (w/v) polyethyleneimine stock solution: dilute 50% (w/v) polyethyleneimine solution (Sigma‐Aldrich, cat. no. P3143) with sterile water, then filter sterilize (store up to 1 year or longer at –20°C)
  • Hanks’ balanced salt solution (HBSS; e.g., Life Technologies, cat. no. 14170‐112)
  • 1 M HEPES, pH 7.3, filter‐sterilized (prepare in lab or purchase from Life Technologies, cat. no. 15630‐080).
  • Minimum essential medium (MEM; e.g., Life Technologies, cat. no. 11090‐081)
  • B27 supplement (e.g., Life Technologies, cat. no. 17504‐044, or Miltenyi Biotec, cat. no. 130‐093‐566)
  • 200 mM L‐glutamine, filter‐sterilized (prepared in lab or purchase from Life Technologies, cat. no. 25030‐081)
  • 100 mM sodium pyruvate (e.g., Life Technologies, cat. no. 11360‐070)
  • Penicillin‐streptomycin solution (penicillin, 10,000 U/ml/streptomycin, 10,000 µg/ml; e.g., Life Technologies, cat. no. 15140‐122)
  • 18‐ to 20‐day pregnant Wistar or Sprague Dawley rat
  • 2.5% trypsin solution (Sigma‐Aldrich, cat. no. T4674; prepare 150‐µl aliquots and store at −20°C)
  • 0.5% (w/v) DNase I stock solution (e.g., Roche Diagnostics, cat. no. 10104159001) in HBSS supplemented with 120 mM MgSO 4 (prepare 150‐µl aliquots and store at −20°C)
  • Neurobasal A medium (Life Technologies, cat. no. 10888‐022)
  • 18‐mm‐diameter round coverslips (thickness no. 1, e.g., Hecht Assistent, cat. no. 1001/18; http://www.hecht‐assistent.de/)
  • 12‐well culture plates (12‐well multiwell plate, BD Falcon, cat. no. 353043)
  • Sterile hood with UV light source
  • Improved Neubauer cell‐counting chamber
  • Additional reagents and equipment for dissection of rat embryos to isolate hippocampi (unit 3.2)

Basic Protocol 2: Transfection of Plasmid Encoding Fluorescent Protein into Astrocytes

  Materials
  • Cultured cells on circular glass coverslips (DIV 7 to 9; see protocol 1)
  • Purified plasmid (1 mg/ml DNA concentration) of fluorescent protein (CFP) subcloned into a mammalian expression vector (0.5 µg/well required)
  • Opti‐MEM I reduced‐serum medium (Life Technologies, cat. no. 31985‐070)
  • Lipofectamine 2000 reagent (Life Technologies, cat. no. 11668‐027)
  • 1.5‐ml microcentrifuge tubes (autoclaved)

Basic Protocol 3: Labeling mGluR5s with QDs

  Materials
  • Cultured cells on circular glass coverslips (DIV 8 to 10, >24 hr after transfection; see protocol 2)
  • Imaging medium (see recipe)
  • Primary antibody (rabbit) against extracellular part of mGluR5 (Alomone Labs, cat. no. AGC‐007): 10‐µl aliquots of antibody reconstituted according to the manufacturer's instructions should be kept at −20°C and at 4°C after thawing (avoid freeze‐thaw cycles)
  • Secondary antibody: biotinylated Fab fragment of goat anti–rabbit IgG (Jackson Immunoresearch, cat. no. 111‐066‐047)
  • 1 µM Qdot 605 or 625 streptavidin conjugate (Life Technologies, cat. no. Q10101MP or A10196)
  • 1× QD binding buffer (see recipe)
  • Sucrose
  • Heating block
  • 10‐cm‐diameter dish (e.g., Cell Culture Dishes, 100 × 20 mm style; BD Falcon, cat. no. 353003)
  • Vacuum pump

Basic Protocol 4: Recording and Analyzing mGluR5 Dynamics to Calculate Diffusion Parameters

  Materials
  • Cells on coverslips labeled with QDs ( protocol 3)
  • Imaging medium (see recipe)
  • Recording chamber that allows recording in imaging medium (e.g., Ludin chamber; Life Imaging Service, http://www.lis.ch/)
  • Inverted fluorescent microscope (e.g., IX70, Olympus) equipped with a Plan Apo 60× objective lens (NA 1.42) (e.g., Olympus)
  • Cooled charge‐coupled device (CCD) camera (e.g., ORCA II‐ER, Hamamatsu Photonics) or EM‐CCD camera (e.g., ImagEM, Hamamatsu Photonics)
  • Appropriate filter sets (e.g., excitation: 470 to 490 nm, emission: 515 to 550 nm for CFP or GFP signal and excitation: 420 to 490 nm, emission: 595 to 615 nm for QD signal)
  • Light‐emitting diode (LED) illumination system (e.g., precisExcite, CoolLED 490 nm for CFP or GFP signal and 440 nm for QD signal) or mercury/xenon lamp (e.g., Olympus)
  • PC and software for image acquisition (e.g., MetaMorph, Molecular Devices)

Basic Protocol 5: Visualizing and Quantifying Diffusion Hindrances

  Materials
  • Cells on coverslips labeled with QDs ( protocol 3)
  • Imaging medium (see recipe)
  • Recording chamber that allows recording in imaging medium (e.g., Ludin chamber; Life Imaging Service, http://www.lis.ch/)
  • Inverted fluorescent microscope (e.g., IX70, Olympus) equipped with a Plan Apo 60× objective lens (NA 1.42) (e.g., Olympus)
  • Cooled charge‐coupled device (CCD) camera (e.g., ORCA II‐ER, Hamamatsu Photonics) or EM‐CCD camera (e.g., ImagEM, Hamamatsu Photonics)
  • Appropriate filter sets (e.g., excitation: 470 to 490 nm, emission: 515 to 550 nm for CFP or GFP signal and excitation: 420 to 490 nm, emission: 595 to 615 nm for QD signal)
  • Light‐emitting diode (LED) illumination system (e.g., precisExcite, CoolLED 490 nm for CFP or GFP signal and 440 nm for QD signal) or mercury/xenon lamp (e.g., Olympus)
  • PC and software for image acquisition (e.g., MetaMorph, Molecular Devices)
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Figures

Videos

Literature Cited

  Arizono, M., Bannai, H., Nakamura, K., Niwa, F., Enomoto, M., Matsu‐Ura, T., Miyamoto, A., Sherwood, M.W., Nakamura, T., and Mikoshiba, K. 2012. Receptor‐selective diffusion barrier enhances sensitivity of astrocytic processes to metabotropic glutamate receptor stimulation. Sci. Signal. 5:ra27.
  Bannai, H., Levi, S., Schweizer, C., Dahan, M., and Triller, A. 2006. Imaging the lateral diffusion of membrane molecules with quantum dots. Nat. Protoc. 1:2628‐2634.
  Bannai, H., Levi, S., Schweizer, C., Inoue, T., Launey, T., Racine, V., Sibarita, J.B., Mikoshiba, K., and Triller, A. 2009. Activity‐dependent tuning of inhibitory neurotransmission based on GABAAR diffusion dynamics. Neuron 62:670‐682.
  Charles, A.C., Merrill, J.E., Dirksen, E.R., and Sanderson, M.J. 1991. Intercellular signaling in glial cells: Calcium waves and oscillations in response to mechanical stimulation and glutamate. Neuron 6:983‐992.
  Ding, S., Fellin, T., Zhu, Y., Lee, S.Y., Auberson, Y.P., Meaney, D.F., Coulter, D.A., Carmignoto, G., and Haydon, P.G. 2007. Enhanced astrocytic Ca2+ signals contribute to neuronal excitotoxicity after status epilepticus. J. Neurosci. 27:10674‐10684.
  Goslin, K., Asmussen, H., and Banker, G. 1998. Rat hippocampal neurons in low‐density culture. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.), pp. 339‐370. MIT Press, Cambridge, Mass.
  Kusumi, A., Sako, Y., and Yamamoto, M. 1993. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium‐induced differentiation in cultured epithelial cells. Biophys. J. 65:2021‐2040.
  Lidke, D.S., Nagy, P., and Arndt‐Jovin, D.J. 2007. In vivo imaging using quantum‐dot‐conjugated probes. Curr. Protoc. Cell Biol. 36:25.1.1‐25.1.18.
  Magistretti, P.J. 2006. Neuron‐glia metabolic coupling and plasticity. J. Exp. Biol. 209:2304‐2311.
  Muir, J., Arancibia‐Carcamo, I.L., MacAskill, A.F., Smith, K.R., Griffin, L.D., and Kittler, J.T. 2010. NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the gamma2 subunit. Proc. Natl. Acad. Sci. U.S.A. 107:16679‐16684.
  Nakada, C., Ritchie, K., Oba, Y., Nakamura, M., Hotta, Y., Iino, R., Kasai, R.S., Yamaguchi, K., Fujiwara, T., and Kusumi, A. 2003. Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization. Nat. Cell Biol. 5:626‐632.
  Newman, E.A. 2003. New roles for astrocytes: Regulation of synaptic transmission. Trends Neurosci. 26:536‐542.
  Niwa, F., Bannai, H., Arizono, M., Fukatsu, K., Triller, A., and Mikoshiba, K. 2012. Gephyrin‐independent GABA(A)R mobility and clustering during plasticity. PLoS One 7:e36148.
  Panatier, A., Vallee, J., Haber, M., Murai, K.K., Lacaille, J.C., and Robitaille, R. 2011. Astrocytes are endogenous regulators of basal transmission at central synapses. Cell 146:785‐798.
  Perea, G. and Araque, A. 2007. Astrocytes potentiate transmitter release at single hippocampal synapses. Science 317:1083‐1086.
  Saxton, M.J. and Jacobson, K. 1997. Single‐particle tracking: Applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26:373‐399.
  Simard, M. and Nedergaard, M. 2004. The neurobiology of glia in the context of water and ion homeostasis. Neuroscience 129:877‐896.
  Triller, A. and Choquet, D. 2008. New concepts in synaptic biology derived from single‐molecule imaging. Neuron 59:359‐374.
  Viviani, B. 2006. Preparation and coculture of neurons and glial cells. Curr. Protoc. Cell Biol. 32:2.7.1‐2.7.21.
Key References
  Arizono et al., 2012. See above.
  The protocols described in this unit were used in this paper.
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