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
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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

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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
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Basic Protocol 1: Preparation of the Ambystoma tigrinum Retinal Model

  • 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.,
  • 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

  • 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

  • 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,
    • 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

  • 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

  • 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

  • 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

  • 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)
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