Tracking Quantum Dot–Tagged Calcium Channels at Vertebrate Photoreceptor Synapses: Retinal Slices and Dissociated Cells

Aaron J. Mercer1, Wallace B. Thoreson2

1 Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University Of Michigan, Ann Arbor, Michigan, 2 Departments of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.18
DOI:  10.1002/0471142301.ns0218s62
Online Posting Date:  January, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

At synapses in the central nervous system, precisely localized assemblies of presynaptic proteins, neurotransmitter‐filled vesicles, and postsynaptic receptors are required to communicate messages between neurons. Our understanding of synaptic function has been significantly advanced using electrophysiological methods, but the dynamic spatial behavior and real‐time organization of synapses remains poorly understood. In this unit, we describe a method for labeling individual presynaptic calcium channels with photostable quantum dots for single‐particle tracking analysis. We have used this technique to examine the mobility of L‐type calcium channels in the presynaptic membrane of rod and cone photoreceptors in the retina. These channels control release of glutamate‐filled synaptic vesicles at the ribbon synapses in photoreceptor terminals. This technique offers the advantage of providing a real‐time biophysical readout of ion channel mobility and can be manipulated by pharmacological or electrophysiological methods. For example, the combination of electrophysiological and single‐particle tracking experiments has revealed that fusion of nearby vesicles influences calcium channel mobility and changes in channel mobility can influence release. These approaches can also be readily adapted to examine membrane proteins in other systems. Curr. Protoc. Neurosci. 62:2.18.1‐2.18.23. © 2013 by John Wiley & Sons, Inc.

Keywords: single particle tracking; L‐type calcium channels; ribbon synapse; membrane diffusion

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of the Ambystoma tigrinum Retinal Model
  • Basic Protocol 2: Immunohistochemical Attachment of QDs to L‐Type CaV Channels
  • Basic Protocol 3: Imaging and Analysis of Individual QDs on Retinal Slices
  • Support Protocol 1: Construction of the Retinal Slice Perfusion Chamber
  • Support Protocol 2: Dissociated Ambystoma tigrinum Retinal Preparation
  • Support Protocol 3: Controls
  • Support Protocol 4: Fixed Tissue Immunohistochemistry of Ambystoma tigrinum Eyes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of the Ambystoma tigrinum Retinal Model

  Materials
  • Dow Corning vacuum grease
  • HEPES‐buffered amphibian extracellular saline solution, pH 7.8 (HAESS; see recipe), 4°C
  • Adult aquatic tiger salamanders, male or female, 18 to 25 cm in length (Kons Scientific or Charles D. Sullivan Co., http://www.researchamphibians.com)
  • Plastic perfusion chamber ( protocol 4)
  • 25 × 75–mm microscope slides
  • Filter paper (type AAWP, 0.8 µm pores; Millipore)
  • Linoleum tissue‐dissecting block
  • Cotton balls
  • Heavy shears or small animal guillotine
  • Binocular dissecting microscope
  • Microsurgical tools (e.g., Word Precision Instruments)
    • 2× 12 cm‐long forceps with 0.08 × 0.04 mm tips
    • 10.5 cm‐long fine‐tip spring Vannas scissors, 3 mm blades
    • 10.5 cm‐long curved fine‐tip spring Vannas scissors
    • Microscalpel
  • Razor blade tissue chopper (e.g., Stoelting Tissue Slicer 51425)
  • Razor blades (Ted Pella, cat. no. 121‐6)

Basic Protocol 2: Immunohistochemical Attachment of QDs to L‐Type CaV Channels

  Materials
  • Plastic perfusion chamber with retinal tissue ( protocol 1) or slide with dissociated retinal tissue
  • HEPES‐buffered amphibian extracellular saline solution (HAESS), 4°C
  • Bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A9418)
  • Primary antibody: rabbit anti‐α 2δ 4 antibody (Qin et al., )
  • Secondary antibody: goat anti‐rabbit biotinylated antibody (Jackson ImmunoResearch, cat. no. 111‐065‐003)
  • Qdot 525 streptavidin conjugate (1 µM; Invitrogen, cat. no. Q10141MP)
  • Humidified chamber: large petri dish with the perfusion chamber placed at the center and damp paper towels at the periphery

Basic Protocol 3: Imaging and Analysis of Individual QDs on Retinal Slices

  Materials
  • Antibody‐incubated retinal tissue ( protocol 2)
  • TMC vibration isolation table
  • Nikon E600FN upright microscope with the following components:
    • 60×, 1.2‐ or 1.0‐NA water‐immersion objective
    • FITC filter cube (Chroma Technologies, cat. no. 41001)
    • Hg/Xe epifluorescent light source (OptiQuip, http://optiquipsurgical.com/)
    • Lambda 10‐2 shutter (Sutter Instruments)
  • Photometrics Ds‐Qi1 EMCCD camera
  • Nikon NIS‐Elements Imaging Software
  • Computer running Microsoft Excel
NOTE: The specific components of a single imaging setup vary among laboratories. We describe the setup used for our studies in retinal slices as a foundation for others to follow.

Support Protocol 1: Construction of the Retinal Slice Perfusion Chamber

  Materials
  • Machine shop tools
  • 2 mm‐thick acrylic plastic
  • 20‐G plastic tubing
  • Ag/AgCl reference electrode
  • Reference lead wire
  • 25 × 75–mm glass slide
  • Spinal needles, 20‐GA, 3.5‐in. (BD Medical Systems)
  • Small screws

Support Protocol 2: Dissociated Ambystoma tigrinum Retinal Preparation

  Materials
  • Concanavalin A (Sigma‐Aldrich, cat. no. C7275) or CellTak (BD Biosciences)
  • Sylgard (optional)
  • 0.5 mM Ca2+ HAESS (low Ca2+ version of HAESS; see recipe)
  • Bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A9418)
  • Cysteine (Sigma‐Aldrich, cat. no. 168149)
  • Papain (Sigma‐Aldrich, cat. no. 76220)
  • DNase (Worthington Biochemicals, cat. no. LS0066331)
  • 18‐mm‐diameter glass coverslips
  • Imaging chamber for 18‐mm coverslips (Warner Instruments, cat. no. RC‐41LP)
  • 35‐mm petri dishes
  • Rocking platform
  • Pasteur pipet with a fire‐polished tip, ∼1 mm O.D., and attached rubber bulb
  • Pasteur pipet with large (∼5 mm) fire‐polished opening to use as a transfer pipet
  • Additional reagents and equipment for isolation, enucleation, and quartering of salamander eyecup ( protocol 1, steps 4 to 19)

Support Protocol 3: Controls

  Materials
  • HEPES‐buffered extracellular amphibian saline solution (HAESS; see recipe)
  • Agar
  • Quantum dots (QDs; Invitrogen, cat. no. Q10141MP; 1 µM solution)
  • Dow Corning vacuum grease
  • Plastic perfusion chamber ( protocol 4)
  • Additional reagents and equipment for antibody‐based QD attachment ( protocol 2) to fixed retinal tissue ( protocol 7)

Support Protocol 4: Fixed Tissue Immunohistochemistry of Ambystoma tigrinum Eyes

  Materials
  • 4% paraformaldehyde (see recipe)
  • 30% (w/v) sucrose in deionized H 2O
  • 0.1 M phosphate‐buffered saline, pH 7.4 (PBS; appendix 2A)
  • OCT Compound (Sakura FineTek, cat. no. 4853)
  • Pulverized dry ice
  • Blocking solution (see recipe)
  • Normal serum
  • Primary antibody: rabbit anti‐α 2δ 4 antibody (Qin et al., )
  • Secondary antibody: goat anti‐rabbit fluorophore‐conjugated secondary antibody (e.g., FITC‐conjugated anti‐rabbit IgG; Sigma‐Aldrich, cat. no. F0382)
  • VectaShield Hard Mount with DAPI (Vector Labs, cat. no. H‐1200)
  • Tissue‐Tek cryomolds (10 mm × 10 mm × 5 mm)
  • Leica CM1800 cryostat
  • Fisher SuperFrost glass slides
  • PAP pen (or colored nail polish)
  • Humidified chamber: large petri dish with the slide containing the section placed at the center and damp paper towels at the periphery
  • Razor blades
  • 24 mm × 60 mm coverslips
  • Additional reagents and equipment for removing the eyes from a tiger salamander ( protocol 1, steps 4 to 13) and cryostat sectioning (unit 1.1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Alcor, D., Gouzer, G., and Triller, A. 2009. Single‐particle tracking methods for the study of membrane receptors dynamics. Eur. J. Neurosci. 30:987‐997.
   Babai, N. and Thoreson, W.B. 2009. Horizontal cell feedback regulates calcium currents and intracellular calcium levels in rod photoreceptors of salamander and mouse retina. J. Physiol. 587:2353‐2364.
   Babai, N., Morgans, C.W., and Thoreson, W.B. 2010. Calcium‐induced calcium release contributes to synaptic release from mouse rod photoreceptors. Neuroscience 165:1447‐1456.
   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.
   Bates, I.R., Hebert, B., Luo, Y., Liao, J., Bachir, A.I., Kolin, D.L., Wiseman, P.W., and Hanrahan, J.W. 2006. Membrane lateral diffusion and capture of CFTR within transient confinement zones. Biophys. J. 91:1046‐1058.
   Bauer, C.S., Tran‐Van‐Minh, A., Kadurin, I., and Dolphin, A.C. 2010. A new look at calcium channel alpha2delta subunits. Curr. Opin. Neurobiol. 20:563‐571.
   Bruchez, M. Jr., Moronne, M., Gin, P., Weiss, S., and Alivisatos, A.P. 1998. Semiconductor nanocrystals as fluorescent biological labels. Science 281:2013‐2016.
   Catterall, W.A. 2000. Structure and regulation of voltage‐gated Ca2+ channels. Annu. Rev. Cell Dev. Biol. 16:521‐555.
   Chan, W.C. and Nie, S. 1998. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281:2016‐2018.
   Charrier, C., Ehrensperger, M.V., Dahan, M., Levi, S., and Triller, A. 2006. Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane. J. Neurosci. 26:8502‐8511.
   Choquet, D. 2010. Fast AMPAR trafficking for a high‐frequency synaptic transmission. Eur. J. Neurosci. 32:250‐260.
   Cooper, N.G. and McLaughlin, B.J. 1984. The distribution of filipin‐sterol complexes in photoreceptor synaptic membranes. J. Comp. Neurol. 230:437‐443.
   Cristofanilli, M. and Akopian, A. 2006. Calcium channel and glutamate receptor activities regulate actin organization in salamander retinal neurons. J. Physiol. 575:543‐554.
   Cristofanilli, M., Mizuno, F., and Akopian, A. 2007. Disruption of actin cytoskeleton causes internalization of Ca(v)1.3 (alpha 1D) L‐type calcium channels in salamander retinal neurons. Mol. Vis. 13:1496‐1507.
   Dahan, M., Levi, S., Luccardini, C., Rostaing, P., Riveau, B., and Triller, A. 2003. Diffusion dynamics of glycine receptors revealed by single‐quantum dot tracking. Science 302:442‐445.
   Davies, A., Kadurin, I., Alvarez‐Laviada, A., Douglas, L., Nieto‐Rostro, M., Bauer, C.S., Pratt, W.S., and Dolphin, A.C. 2010. The alpha2delta subunits of voltage‐gated calcium channels form GPI‐anchored proteins, a posttranslational modification essential for function. Proc. Natl. Acad. Sci. U.S.A. 107:1654‐1659.
   Eggermann, E., Bucurenciu, I., Goswami, S.P., and Jonas, P. 2012. Nanodomain coupling between Ca(2) channels and sensors of exocytosis at fast mammalian synapses. Nat. Rev. Neurosci. 13:7‐21.
   Gomez‐Varela, D., Kohl, T., Schmidt, M., Rubio, M.E., Kawabe, H., Nehring, R.B., Schafer, S., Stuhmer, W., and Pardo, L.A. 2010. Characterization of Eag1 channel lateral mobility in rat hippocampal cultures by single‐particle‐tracking with quantum dots. PLoS One 5:e8858.
   Groc, L., Choquet, D., and Chaouloff, F. 2008. The stress hormone corticosterone conditions AMPAR surface trafficking and synaptic potentiation. Nat. Neurosci. 11:868‐870.
   Hall, D. 2008. Analysis and interpretation of two‐dimensional single‐particle tracking microscopy measurements: Effect of local surface roughness. Anal. Biochem. 377:24‐32.
   Heine, M., Thoumine, O., Mondin, M., Tessier, B., Giannone, G., and Choquet, D. 2008. Activity‐independent and subunit‐specific recruitment of functional AMPA receptors at neurexin/neuroligin contacts. Proc. Natl. Acad. Sci. U.S.A. 105:20947‐20952.
   Huang, B., Wang, W., Bates, M., and Zhuang, X. 2008. Three‐dimensional super‐resolution imaging by stochastic optical reconstruction microscopy. Science 319:810‐813.
   Jakway, J.S. and Riss, W. 1972. Retinal projections in the tiger salamander, Ambystoma tigrinum. Brain Behav. Evol. 5:401‐442.
   Jares‐Erijman, E.A. and Jovin, T.M. 2003. FRET imaging. Nat. Biotechnol. 21:1387‐1395.
   Job, C. and Lagnado, L. 1998. Calcium and protein kinase C regulate the actin cytoskeleton in the synaptic terminal of retinal bipolar cells. J. Cell Biol. 143:1661‐1672.
   Krizaj, D., Mercer, A.J., Thoreson, W.B., and Barabas, P. 2011. Intracellular pH modulates inner segment calcium homeostasis in vertebrate photoreceptors. Am. J. Physiol. Cell Physiol. 300:C187‐197.
   Kunz, W.S. and Kunz, W. 1985. Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria. Biochim. Biophys. Acta 841:237‐246.
   Lasansky, A. 1973. Organization of the outer synaptic layer in the retina of the larval tiger salamander. Philos. Trans. R. Soc. Lond. B Biol. Sci. 265:471‐489.
   Liu, S.L., Zhang, Z.L., Sun, E.Z., Peng, J., Xie, M., Tian, Z.Q., Lin, Y., and Pang, D.W. 2011. Visualizing the endocytic and exocytic processes of wheat germ agglutinin by quantum dot‐based single‐particle tracking. Biomaterials 32:7616‐7624.
   Mavrakis, M., Rikhy, R., Lilly, M., and Lippincott‐Schwartz, J. 2008. Fluorescence imaging techniques for studying Drosophila embryo development. Curr. Protoc. Cell Biol. 39:4.18.1‐4.18.43.
   Medintz, I.L., Uyeda, H.T., Goldman, E.R., and Mattoussi, H. 2005. Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Materials 4:435‐446.
   Mercer, A.J. and Thoreson, W.B. 2011. The dynamic architecture of photoreceptor ribbon synapses: Cytoskeletal, extracellular matrix, and intramembrane proteins. Vis. Neurosci. 28:453‐471.
   Mercer, A.J., Chen, M., and Thoreson, W.B. 2011a. Lateral mobility of presynaptic L‐type calcium channels at photoreceptor ribbon synapses. J. Neurosci. 31:4397‐4406.
   Mercer, A.J., Rabl, K., Riccardi, G.E., Brecha, N.C., Stella, S. L. Jr., and Thoreson, W.B. 2011b. Location of release sites and calcium‐activated chloride channels relative to calcium channels at the photoreceptor ribbon synapse. J. Neurophysiol. 105:321‐335.
   Mercer, A.J., Szalewski, R.J., Jackman, S.L., Van Hook, M.J., and Thoreson, W.B. 2012. Regulation of presynaptic strength by controlling Ca2+ channel mobility: Effects of cholesterol depletion on release at the cone ribbon synapse. J. Neurophysiol. 107:3468‐3478.
   Mikasova, L., Groc, L., Choquet, D., and Manzoni, O.J. 2008. Altered surface trafficking of presynaptic cannabinoid type 1 receptor in and out synaptic terminals parallels receptor desensitization. Proc. Natl. Acad. Sci. U.S.A. 105:18596‐18601.
   Nachman‐Clewner, M., St Jules, R., and Townes‐Anderson, E. 1999. L‐type calcium channels in the photoreceptor ribbon synapse: Localization and role in plasticity. J. Comp. Neurol. 415:1‐16.
   Neher, E. and Sakaba, T. 2008. Multiple roles of calcium ions in the regulation of neurotransmitter release. Neuron 59:861‐872.
   Pang, J.J., Gao, F., Barrow, A., Jacoby, R.A., and Wu, S.M. 2008. How do tonic glutamatergic synapses evade receptor desensitization? J. Physiol. 586:2889‐2902.
   Park, H., Li, Y., and Tsien, R.W. 2012. Influence of synaptic vesicle position on release probability and exocytotic fusion mode. Science 335:1362‐1366.
   Probst, J., Dembski, S., Milde, M., and Rupp, S. 2012. Luminescent nanoparticles and their use for in vitro and in vivo diagnostics. Exp. Rev. Mol. Diagn. 12:49‐64.
   Qin, N., Yagel, S., Momplaisir, M.L., Codd, E. E., and D'Andrea, M.R. 2002. Molecular cloning and characterization of the human voltage‐gated calcium channel alpha(2)delta‐4 subunit. Mol. Pharmacol. 62:485‐496.
   Raviola, E. and Gilula, N.B. 1975. Intramembrane organization of specialized contacts in the outer plexiform layer of the retina: A freeze‐fracture study in monkeys and rabbits. J. Cell Biol. 65:192‐222.
   Reed, M.A., Randall, J.N., Aggarwal, R.J., Matyi, R.J., Moore, T.M., and Wetsel, A.E. 1988. Observation of discrete electronic states in a zero‐dimensional semiconductor nanostructure. Phys. Rev. Lett. 60:535‐537.
   Ribrault, C., Reingruber, J., Petkovic, M., Galli, T., Ziv, N.E., Holcman, D., and Triller, A. 2011. Syntaxin1A lateral diffusion reveals transient and local SNARE interactions. J. Neurosci. 31:17590‐17602.
   Saxton, M.J. 1997. Single‐particle tracking: The distribution of diffusion coefficients. Biophys. J. 72:1744‐1753.
   Saxton, M.J. and Jacobson, K. 1997. Single‐particle tracking: Applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26:373‐399.
   Schubert, T. and Akopian, A. 2004. Actin filaments regulate voltage‐gated ion channels in salamander retinal ganglion cells. Neuroscience 125:583‐590.
   Shtengel, G., Galbraith, J.A., Galbraith, C.G., Lippincott‐Schwartz, J., Gillette, J.M., Manley, S., Sougrat, R., Waterman, C.M., Kanchanawong, P., Davidson, M.W., Fetter, R.D., and Hess, H.F. 2009. Interferometric fluorescent super‐resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. U.S.A. 106:3125‐3130.
   Smith, A.M., Duan, H., Mohs, A.M., and Nie, S. 2008. Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv. Drug Deliv. Rev. 60:1226‐1240.
   Steele, E.C.,Jr., Chen, X., Iuvone, P.M., and MacLeish, P.R. 2005. Imaging of Ca2+ dynamics within the presynaptic terminals of salamander rod photoreceptors. J. Neurophysiol. 94:4544‐4553.
   Takagahara, T. 1987. Excitonic optical nonlinearity and exciton dynamics in semiconductor quantum dots. Phys. Rev. B Condens. Matter 36:9293‐9296.
   Thoreson, W.B. 2007. Kinetics of synaptic transmission at ribbon synapses of rods and cones. Mol. Neurobiol. 36:205‐223.
   Thoreson, W.B., Nitzan, R., and Miller, R.F. 1997. Reducing extracellular Cl‐ suppresses dihydropyridine‐sensitive Ca2+ currents and synaptic transmission in amphibian photoreceptors. J. Neurophysiol. 77:2175‐2190.
   tom Dieck, S., Altrock, W.D., Kessels, M.M., Qualmann, B., Regus, H., Brauner, D., Fejtova, A., Bracko, O., Gundelfinger, E.D., and Brandstatter, J.H. 2005. Molecular dissection of the photoreceptor ribbon synapse: Physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex. J. Cell Biol. 168:825‐836.
   Townes‐Anderson, E., MacLeish, P.R., and Raviola, E. 1985. Rod cells dissociated from mature salamander retina: Ultrastructure and uptake of horseradish peroxidase. J. Cell Biol. 100:175‐188.
   Van Hook, M.J. and Thoreson, W.B. 2012. Simultaneous whole cell recordings from photoreceptors and second‐order neurons in an amphibian retinal slice preparation. J. Vis. Exp. e50007. http://www.jove.com/video/50007/.
   Watanabe, T.M., Sato, T., Gonda, K., and Higuchi, H. 2007. Three‐dimensional nanometry of vesicle transport in living cells using dual‐focus imaging optics. Biochem. Biophys. Res. Commun. 359:1‐7.
   Wu, S.M. 1987. Synaptic connections between neurons in living slices of the larval tiger salamander retina. J. Neurosci. Methods 20:139‐149.
   Wycisk, K.A., Budde, B., Feil, S., Skosyrski, S., Buzzi, F., Neidhardt, J., Glaus, E., Nurnberg, P., Ruether, K., and Berger, W. 2006. Structural and functional abnormalities of retinal ribbon synapses due to Cacna2d4 mutation. Invest. Ophthalmol. Vis. Sci. 47:3523‐3530.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library