Loading Neurons with Dextran‐Conjugated Calcium Indicators in Intact Nervous Tissue

Kerry R. Delaney1

1 University of Victoria, Victoria, British Columbia, Canada
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.5
DOI:  10.1002/0471142301.ns0205s50
Online Posting Date:  January, 2010
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Abstract

Dextran‐conjugated Ca2+ indicators are retained well in neurons for many days following loading in intact or semi‐intact brain tissue. Methods for loading neurons, as well as discussion of the unique properties of dextran‐conjugated dyes which need to be considered for their use, are presented. Curr. Protoc. Neurosci. 50:2.5.1‐2.5.11. © 2010 by John Wiley & Sons, Inc.

Keywords: calcium imaging; dendrites; presynaptic terminals; in vivo; en bloc; electroporation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Manual Loading of Neuronal Structures with Dextran‐Conjugated Dyes
  • Alternate Protocol 1: Deep‐Tissue Loading of Neuronal Structures with Dextran‐Conjugated Dyes
  • Alternate Protocol 2: Injection Loading of Neuronal Structures with Dextran‐Conjugated Dyes
  • Alternate Protocol 3: Electroporation Loading of Neuronal Populations with Dextran‐Conjugated Dyes
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Manual Loading of Neuronal Structures with Dextran‐Conjugated Dyes

  Materials
  • Dextran‐conjugated Ca2+ indicator (e.g., Calcium Green‐1, Oregon Green 488 BAPTA‐1, fura‐2, indo‐1, and fluo‐4; Molecular Probes)
  • 1.5% (w/v) bovine serum albumin (BSA)
  • Brain tissue or slice (e.g., units 6.4& 6.11)
  • Ringer's solution
  • Glass microscope slide
  • Electrophysiological glass pipets (e.g., Warner Instruments, A‐M Systems, WPI; 1.0‐ to 1.5‐mm o.d.) or other narrow glass tubing or rod
  • Micropipet puller
  • Stereomicroscope
  • Micromanipulator (optional)

Alternate Protocol 1: Deep‐Tissue Loading of Neuronal Structures with Dextran‐Conjugated Dyes

  • Polyvinyl alcohol (Sigma)

Alternate Protocol 2: Injection Loading of Neuronal Structures with Dextran‐Conjugated Dyes

  • Mineral oil
  • 10% to 20% (w/v) dextran‐conjugated indicator prepared in distilled water
  • Dextran Texas Red (Molecular Probes)
  • Microsyringe (e.g., Hamilton model 7100; Harvard Apparatus) or microinjector (e.g., Nanoject II, Drummond Scientific)

Alternate Protocol 3: Electroporation Loading of Neuronal Populations with Dextran‐Conjugated Dyes

  • Pulse generator and isolated current source capable of passing 5 µA of current across 10 megOhms of resistance
  • Oscilloscope
  • 100 KOhm resistor
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Figures

Videos

Literature Cited

Literature Cited
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   Delaney, K.R. and Hall, B.J. 1996. An in vitro nose‐brain preparation of frog for the study of odor‐induced oscillations in olfactory bulb and cortex. J. Neurosci. Methods 68:193‐202.
   Delaney, K.R., Davison, I., and Denk, W. 2001. Odour‐evoked [Ca2+] transients in mitral cell dendrites of frog olfactory glomeruli. Eur. J. Neurosci. 13:1658‐1672.
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