Measurement of Chloride Movement in Neuronal Preparations

Rochelle D. Schwartz‐Bloom1, A. Christine Engblom2, Karl E. O. Åkerman3, Jon R. Inglefield4

1 Duke University Medical Center, Durham, North Carolina, 2 Royal Danish School of Pharmacy, Copenhagen, Denmark, 3 Uppsala University, Uppsala, Sweden, 4 U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.10
DOI:  10.1002/0471142301.ns0710s04
Online Posting Date:  May, 2001
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Abstract

In this unit, protocols are described for biochemical and optical techniques that have been used by investigators to measure ligand‐gated chloride movement in vesicular structures called synaptoneurosomes (also referred to as microsacs), in cultured neurons, and in the acute brain slice. These techniques can be applied to other ions as well. The measurement of uptake and efflux of radioisotopic chloride in synaptoneurosomes is used to study the responses of gamma‐aminobutyric acid (GABA) receptors, which are coupled to chloride channels. Similar chloride flux assays for primary neuronal cultures are also presented. Alternatively, the efflux of chloride from synaptoneurosomes and primary neuronal cultures can be studied using fluorescent dyes and photometry. Finally, the measurement of chloride uptake can be studied in individual neurons in brain slices using fluorescent dyes and optical imaging by nonconfocal and confocal microscopy. Several support protocols are provided as well, outlining the preparation of synaptoneurosomes from specific brain regions, and the preparation, loading, and calibration of chloride‐sensitive fluorescent dyes.

     
 
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Table of Contents

  • Basic Protocol 1: Uptake/Efflux of Radioisotopic Cl− in Synaptoneurosomes
  • Basic Protocol 2: Uptake of Radioisotopic Cl− in Primary Cultures
  • Basic Protocol 3: Efflux of Cl− in Synaptoneurosomes Measured with Fluorescent Dyes and Photometry
  • Support Protocol 1: Preparation of Synaptoneurosomes
  • Basic Protocol 4: Efflux of Cl− in Primary Cultures Measured with Fluorescent Dyes and Photometry
  • Basic Protocol 5: Uptake of Cl− in the Acute Brain Slice Measured with Fluorescent Dye and Epifluorescence Imaging
  • Alternate Protocol 1: Uptake of Cl− in the Acute Brain Slice Measured with Fluorescent Dye and Confocal Imaging
  • Support Protocol 2: Preparation of MEQ‐Loaded Acute Brain Slices
  • Support Protocol 3: Calibration of Fluorescent Dye in Synaptoneurosomes and Cultured Cells
  • Support Protocol 4: Calibration of Fluorescent Dye In Brain Slices
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Uptake/Efflux of Radioisotopic Cl− in Synaptoneurosomes

  Materials
  • PEI assay buffer: 36Cl flux assay buffer containing 0.05% (v/v) polyethyleneimine
  • 36Cl flux assay buffer (see recipe)
  • 36Cl (∼10 to 20 mCi/g, in HCl form; NEN Life Sciences)
  • 10× γ‐aminobutyric acid (GABA) or other agonist in 36Cl flux assay buffer
  • Synaptoneurosomes from individual brain regions (see protocol 4)
  • Picrotoxin assay buffer, ice cold: 36Cl flux assay buffer containing 100 µM picrotoxin
  • Water‐miscible scintillation fluid
  • 2.4‐cm glass fiber filters (Whatman GF/C or Schleicher & Schuell no. 30)
  • 1‐liter vacuum flask with Tygon tubing and vacuum pump (do not use a vacuum line)
  • Single‐place stainless steel filter holder (Hoefer Pharmacia Biotech)
  • Gas drying jar (containing Drierite or equivalent)
  • 7‐ml polyethylene scintillation vials
  • 12 × 75–mm glass test tubes
  • Shaking water bath, 30°C (uptake) or room temperature (efflux)
  • Filter forceps (not pointed)
  • Repeat pipettor with 1‐liter total volume and 1‐ to 10‐ml dispensing range
  • Additional reagents and equipment for determination of protein concentration (Lovrien and Matulis, )
NOTE: Prior to starting this assay, have all solutions and 36Cl mixtures prepared and have the filter apparatus ready, because tissue and 36Cl are added to the tubes in a timed fashion.

Basic Protocol 2: Uptake of Radioisotopic Cl− in Primary Cultures

  Materials
  • 36Cl (∼10 to 20 mCi/g in HCl form; NEN Life Sciences)
  • 36Cl flux assay buffer (see recipe)
  • γ‐Aminobutyric acid (GABA) or agonist
  • Picrotoxin assay buffer, ice cold: recipe36Cl flux assay buffer containing 100 µM picrotoxin
  • Cultured neurons plated on coverslips, grown in modified essential medium (MEM) or equivalent
  • 0.2 N NaOH
  • 0.2 M HCl
  • Water‐miscible scintillation fluid
  • 35‐mm plastic petri dishes
  • Forceps
  • Blotting or tissue paper
  • 20‐ml liquid scintillation vials
NOTE: Prior to assay, cultured cells should be kept at 37°C until needed. Keep the 36Cl flux assay buffer at room temperature and the wash buffers ice cold. Prepare all solutions ahead of time.

Basic Protocol 3: Efflux of Cl− in Synaptoneurosomes Measured with Fluorescent Dyes and Photometry

  Materials
  • Synaptoneurosomes (see protocol 4)
  • SPQ/MQAE loading buffer (see recipe)
  • 2 M 6‐methoxy‐N‐(sulfopropyl)quinolinium (SPQ) or 1 M N‐(6‐methoxyquinolyl)acetoethyl ester (MQAE; both from Molecular Probes; store in small aliquots at −20°C)
  • SPQ/MQAE wash buffer (see recipe), ice cold
  • Low‐Cl buffer (see recipe), room temperature and 37°C
  • 2.5 mM nigericin in ethanol
  • 3.5 mM tributyltin acetate in ethanol
  • 10.5 M KSCN
  • 1.75 mM valinomycin in ethanol
  • Fluorescence spectrophotometer (Hitachi F 2000 or F 4000 or equivalent) equipped with thermosetting, magnetic stirring, and a 1‐ml quartz cuvette

Support Protocol 1: Preparation of Synaptoneurosomes

  Materials
  • Synaptoneurosome preparation buffer (see recipe)
  • Animal for sacrifice
  • Nylon monofilament mesh with 147‐µm pore size (Small Parts)
  • Swinnex filter holders (Millipore)
  • 10‐µm Mitex filters (Millipore)
  • 10‐ and 60‐ml plastic syringes
  • 15‐ml glass tissue grinder (e.g., Duall, Kontes)
  • 12 × 75–mm glass test tubes
NOTE: Keep tissue and buffer ice cold throughout the entire preparation.

Basic Protocol 4: Efflux of Cl− in Primary Cultures Measured with Fluorescent Dyes and Photometry

  • Rat cerebellar granule cells cultured on poly‐L‐lysine‐coated glass coverslips ( appendix 2A)
  • 10 mM GABA
  • 10 mM pentobarbitone
  • 4‐ml quartz spectrophotometer cuvette with coverslip holder

Basic Protocol 5: Uptake of Cl− in the Acute Brain Slice Measured with Fluorescent Dye and Epifluorescence Imaging

  Materials
  • MEQ‐loaded acute brain slice (see protocol 8)
  • Oxygenated Ringers solution (see recipe)
  • 50 to 1000 µM γ‐aminobutyric acid (GABA) or other agonist in recipeRingers solution
  • Plexiglas imaging chamber (Fig. ) with outflow and inflow tubes (∼1.7‐mm‐o.d.; ∼1.2‐mm i.d.), flow rate restrictor (in line with tubing), and switchable valve (chamber can be constructed by any instrument shop to fit stage)
  • Upright epifluorescence microscope (Nikon Optiphot or equivalent) equipped with:
  •   10× objective
  •   UV water‐immersible 40× objective (Olympus or equivalent)
  •   Mercury lamp
  •   335‐nm excitation filter
  •   360‐nm dichroic beam splitter
  •   440‐nm emission filter (100‐nm band pass)
  •   Silicon‐intensified target (SIT) camera or charge‐coupled device (CCD)
  •    intensified camera
  • Platinum‐wire holder containing threads of nylon stocking glued with Superglue (“harp”)
  • Image analysis computer system and video monitor
NOTE: Protect eyes from UV light. Use the shield that attaches to the microscope and do not look directly at the blue light beam.

Alternate Protocol 1: Uptake of Cl− in the Acute Brain Slice Measured with Fluorescent Dye and Confocal Imaging

  • Confocal microscope attached to an upright microscope (Nikon Optiphot or equivalent) equipped with:
  •   10× objective
  •   UV water‐immersible 40× objective (Olympus or equivalent)
  •   Air table and air tank
  •   UV laser with a 364‐nm excitation line
  •   400‐nm barrier filter
  •   Optical disk recorder (Panasonic OMDR LQ 3031 or equivalent)
NOTE: Protect eyes from the UV light. Use the shield that attaches to the microscope and do not look directly at the blue light beam.

Support Protocol 2: Preparation of MEQ‐Loaded Acute Brain Slices

  Materials
  • 6‐methoxy‐N‐ethylquinolinium chloride (MEQ; Molecular Probes)
  • N 2 tank
  • Sodium borohydride
  • Ethyl acetate
  • Anhydrous MgSO 4
  • Oxygenated Ringers solution (see recipe), ice cold and room temperature
  • Brain tissue for slicing (fresh)
  • 12 × 75–mm glass test tubes
  • 0.5‐ to 1.0‐ml glass microvial with conical insert
  • Mesh supports (glue a piece of crinoline, obtained at any fabric store, to a plastic ring to make a floatable support that fits an 80‐ml beaker)
  • Vibrating tissue slicer (Vibratome or equivalent)
  • Additional reagents and equipment for preparation of brain slices (unit 6.4)
NOTE: Use distilled, deionized water throughout.

Support Protocol 3: Calibration of Fluorescent Dye in Synaptoneurosomes and Cultured Cells

  Materials
  • 2.5 mM nigericin in ethanol
  • 3.5 mM tributyltin acetate in ethanol
  • SPQ‐ or MQAE‐loaded synaptoneurosomes (in 1‐ml quartz cuvette; see protocol 3, step ) or cultured cells (in 4‐ml quartz cuvette; see protocol 5, step )
  • Low‐Cl buffer supplemented with varying NaCl and gluconate (see recipe)
  • 2 M NaCl
  • 10.5 M KSCN
  • 1.75 mM valinomycin in ethanol

Support Protocol 4: Calibration of Fluorescent Dye In Brain Slices

  • MEQ‐loaded brain slice (see protocol 6, step , or see protocol 7, step )
  • 200 µM nigericin/100 µM tributyltin acetate/low‐Cl Ringers solution (see recipe)
  • 200 µM nigericin/100 µM tributyltin acetate/low‐Cl Ringers solution with varying NaCl and gluconate concentrations (see recipe)
  • 150 mM KSCN/25 µM valinomycin/low‐Cl Ringers solution (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
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