Imaging Nervous System Activity

R. Douglas Fields1, Neil Shneider2, George Z. Mentis2, Michael J. O'Donovan2

1 Section on Nervous System Development and Plasticity, National Institute of Child Health and Human Development (NICHD), NIH, Bethesda, Maryland, 2 Section on Developmental Neurobiology, National Institute of Neurological Disorders and Stroke (NINDS), NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.3
DOI:  10.1002/0471142301.ns0203s49
Online Posting Date:  October, 2009
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Abstract

This unit describes methods for loading ion‐ and voltage‐sensitive dyes into neurons, with a particular focus on the spinal cord as a model system. In addition, we describe the use of these dyes to visualize neural activity. Although the protocols described here concern spinal networks in culture or an intact in vitro preparation, they can be, and have been, widely used in other parts of the nervous system. Curr. Protoc. Neurosci. 49:2.3.1‐2.3.14. © 2009 by John Wiley & Sons, Inc.

Keywords: ion‐sensitive dyes; voltage‐sensitive dyes; electroporation; inverse Pericam; retrograde loading

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Bath Application of Membrane‐Permeant Calcium‐Sensitive Fluorescent Indicators (Acetoxymethyl Ester Dyes) to Cultured Neurons
  • Alternate Protocol 1: Bath Application of Membrane‐Permeant Calcium‐Sensitive Dyes to En‐Bloc Nervous System Preparations or Tissue Slices
  • Basic Protocol 2: Retrograde Loading of Calcium‐Sensitive Dyes into En‐Bloc or Slice Preparations by Microinjection
  • Alternate Protocol 2: Retrograde Loading of Calcium‐Sensitive Dyes into En‐Bloc or Slice Preparations Using a Suction Electrode
  • Basic Protocol 3: Retrograde Loading of Voltage‐Sensitive Dyes into En‐Bloc or Slice Preparations by Microinjection
  • Basic Protocol 4: Loading of Ion‐Sensitive Dyes into En‐Bloc Preparations or In Vivo by Direct Bolus Injection
  • Basic Protocol 5: Loading of Calcium‐Sensitive Dyes into En‐Bloc Preparations by Electroporation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Bath Application of Membrane‐Permeant Calcium‐Sensitive Fluorescent Indicators (Acetoxymethyl Ester Dyes) to Cultured Neurons

  Materials
  • Cultured neurons (see Chapter 3)
  • Bathing solution: e.g., Tyrode's solution (unit 4.24) at room temperature for frog or chick neurons; Ringers solution (e.g., Invitrogen) prewarmed to 37°C for rat or mouse neurons
  • Calcium‐sensitive membrane‐permeant indicator dye solution (fura‐2 AM, indo‐1 AM, fluo‐3, or calcium green 1; see recipe)
  • Balanced salt solution lacking serum, such as artificial cerebrospinal fluid (aCSF; see recipe)
  • BSA (electrophoresis grade)
  • 35‐mm tissue culture dish
  • Bath sonicator
  • Epifluorescence microscope equipped with excitation and emission filters that are appropriate for the specific fluorescent probes (see unit 2.1 and Table 2.3.1)
    Table 2.3.1   MaterialsCharacteristics of Calcium‐Sensitive Indicators Used to Monitor Neuronal Activity

    Excitation λ (nm) Emission λ (nm)
    [Ca2+] [Ca2+] K d Comments
    Indicator Low High Low High
    Fura‐2 360 335 510 505 145 May be used as a ratiometric quantitative indicator or as a single‐wavelength indicator for nonquantitative measurements. AM form is fluorescent.
    Indo‐1 345 330 475 400 230 Emission ratiometric indicator for quantitative measurements. Can be excited by argon ion laser for confocal microscopy. AM form is fluorescent.
    Fluo‐3 500 500 525 525 390 Single‐wavelength indicator for nonquantitative calcium measurements. Large signal of nearly 100‐fold increase in fluorescence upon binding calcium (5‐ to 10‐fold increase from the normal cytosolic calcium concentration of neurons). AM form is not fluorescent.
    Calcium green 1 488 488 525 525 190 Single‐wavelength indicator that is more fluorescent than fluo‐3 at resting calcium levels. Can be coupled to dextran for retrograde tracing studies.
    Mag‐Fura‐2 370 330 510 510 25,000 Useful for measuring high levels of intracellular calcium concentration, which would be beyond the sensitivity of indicators with lower K d values (e.g., Fura‐2). AM form is fluorescent.

Alternate Protocol 1: Bath Application of Membrane‐Permeant Calcium‐Sensitive Dyes to En‐Bloc Nervous System Preparations or Tissue Slices

  • Neural tissue: brain slice or spinal cord preparation
  • DMSO (anhydrous) containing 2.5 mg/ml Pluronic F‐127 detergent
  • Artificial cerebrospinal fluid (aCSF)
  • Superfusion system, including:
    • Pump
    • 95% O 2/5% CO 2
    • Regulators
    • Perfusion chamber with a Sylgard base and an inlet and outlet for the perfusion fluid (e.g., aCSF)
    • Heater (if necessary)
  • Fine‐tipped microdissection scissors, vibrating blade mounted on a micromanipulator, or Vibratome (see unit 1.1)

Basic Protocol 2: Retrograde Loading of Calcium‐Sensitive Dyes into En‐Bloc or Slice Preparations by Microinjection

  Materials
  • En‐bloc or slice preparation of neural tissue
  • Calcium‐sensitive membrane‐impermeant indicator dye solutions (calcium green or fura‐2 coupled to dextran or the membrane‐impermeant salts; see recipe) in aCSF
  • Glass microelectrodes pulled on a standard puller from 1‐ to 1.5‐mm filament glass to have ∼50‐MΩ impedance when filled with KCl (i.e., suitable for intracellular recording; see Chapter 6)
  • Dissecting microscope
  • Micromanipulator (see Chapter 6)
  • Microscope equipped with excitation and emission filters that are appropriate for the specific fluorescent probes (see unit 2.1 and Table 2.3.1)
  • Additional reagents and equipment for perfusion ( protocol 2)

Alternate Protocol 2: Retrograde Loading of Calcium‐Sensitive Dyes into En‐Bloc or Slice Preparations Using a Suction Electrode

  • aCSF (see recipe)
  • Suction electrode: 1‐ to 3‐in. (2.5‐ to 7.5‐cm) length of PE 50‐90 tubing pulled over a flame to provide a taper with a 100‐ to 200‐um tip diameter (also see Chapter 6)
  • PE 50‐160 polyethylene tubing
  • Tygon tubing, 1‐ to 1.5‐mm i.d.

Basic Protocol 3: Retrograde Loading of Voltage‐Sensitive Dyes into En‐Bloc or Slice Preparations by Microinjection

  Materials
  • En‐bloc or slice preparation of neural tissue
  • 10 to 20 mg/ml voltage‐sensitive dye (di‐8‐ANEPPQ or di‐12‐ANEPEQ; Molecular Probes) in ethanol, chloroform, or DMSO
  • Glass microelectrodes pulled on a standard puller from 1‐ to 1.5‐mm filament glass to have ∼50‐MΩ impedance when filled with KCl (i.e., suitable for intracellular recording; see Chapter 6)
  • Micromanipulator (see Chapter 6)
  • Epifluorescence microscope equipped with excitation and emission filters that are appropriate for the specific fluorescent probes (see unit 2.1 and Table 2.3.1)
  • Additional reagents and equipment for pressure‐injection of dye ( protocol 3 and perfusion ( protocol 2)

Basic Protocol 4: Loading of Ion‐Sensitive Dyes into En‐Bloc Preparations or In Vivo by Direct Bolus Injection

  Materials
  • Calcium‐sensitive membrane‐permeant indicator dye solutions (Calcium green AM; Oregon Green 488 Bapta AM; Fluo‐4 AM see recipe)
  • 20% (w/v) Pluronic F‐127 in DMSO
  • En‐bloc preparation of neural tissue
  • 0.2‐ to 0.4‐µm filter
  • Standard patch electrode: glass pipet pulled to a tip diameter of ∼2 to 3 µm (unit 6.3)
  • Micromanipulator
  • Pressure ejection device (e.g., Picospritzer)
  • Epifluorescence microscope equipped with excitation and emission filters that are appropriate for the specific fluorescent probes (see unit 2.1 and Table 2.3.1)
  • Additional reagents and equipment for superfusion ( protocol 2)

Basic Protocol 5: Loading of Calcium‐Sensitive Dyes into En‐Bloc Preparations by Electroporation

  Materials
  • En‐bloc or slice preparation of neural tissue
  • aCSF (see recipe): normal and low calcium/high magnesium formulations
  • Calcium‐sensitive membrane‐impermeant indicator dye solutions (calcium green or fura‐2 coupled to dextran or their membrane‐impermeant salts; see recipe) in aCSF
  • Electroporation equipment:
    • Electroporator and gold‐plated electrodes electroporation electrodes, 3‐ to 5‐mm in length (e.g., ECM 830 electroporation system, manufactured by BTX Harvard Apparatus)
    • Broken glass microelectrode pulled on a standard puller from 1 to 1.5 mm filament glass micromanipulator (see Chapter 6)
    • Power source
  • Glass microelectrode mounted on a micromanipulator for dye injection
  • Epifluorescence microscope equipped with excitation and emission filters that are appropriate for the specific fluorescent probes (see unit 2.1 and Table 2.3.1)
  • Additional reagents and equipment for perfusion ( protocol 2)
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Literature Cited

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