Measurement of Chloride Movement in Neuronal Preparations

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Abstract
Table of Contents
Materials
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Literature Cited

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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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: ³⁶ Cl⁻ flux assay buffer containing 0.05% (v/v) polyethyleneimine
³⁶ Cl⁻ flux assay buffer (see recipe )
³⁶ Cl⁻ (∼10 to 20 mCi/g, in HCl form; NEN Life Sciences)
10× γ‐aminobutyric acid (GABA) or other agonist in ³⁶ Cl⁻ flux assay buffer
Synaptoneurosomes from individual brain regions (see protocol 4 )
Picrotoxin assay buffer, ice cold: ³⁶ Cl⁻ 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 ³⁶ Cl⁻ mixtures prepared and have the filter apparatus ready, because tissue and ³⁶ Cl⁻ are added to the tubes in a timed fashion.

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

Materials

³⁶ Cl⁻ (∼10 to 20 mCi/g in HCl form; NEN Life Sciences)
³⁶ Cl⁻ flux assay buffer (see recipe )
γ‐Aminobutyric acid (GABA) or agonist
Picrotoxin assay buffer, ice cold: recipe³⁶ Cl⁻ 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 ³⁶ Cl⁻ 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)
N₂ tank
Sodium borohydride
Ethyl acetate
Anhydrous MgSO₄
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

Figure 7.10.1 A typical filtration apparatus. The stainless steel filter holder has two parts; the glass fiber filter is placed on the mesh support and then wet with buffer to keep it in place. The entire unit is placed onto the vacuum flask for filtration.

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Figure 7.10.2 A typical concentration‐response experiment for muscimol‐induced ³⁶ Cl⁻ uptake in cerebral cortical synaptoneurosomes is shown (see ). Values obtained in dpm (average of triplicates) have been converted to nmol of ³⁶ Cl⁻ taken up per mg of synaptoneurosomal protein in a 5‐sec period.

View Image

Figure 7.10.3 A model experiment showing responses of synaptoneurosomes loaded with Cl⁻ ‐sensitive fluorescent dye (see ) upon addition of substances that change the intrasynaptoneurosomal Cl⁻ concentration. This is also applicable to dye‐loaded cultured cells (see ). GABA and barbiturate cause increases in the fluorescence in response to the efflux of Cl⁻ through GABA_A receptor–coupled Cl⁻ channels. The intracellular Cl⁻ concentration is estimated (see 3) by adding nigericin and tributyltin (which equalize the Cl⁻ gradient and give maximal fluorescence), and valinomycin and KSCN (which cause quenching of the dye and give minimal fluorescence).

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Figure 7.10.4 A typical brain slice superfusion (imaging) chamber. The chamber is made of Plexiglas with two wells for filling and removing buffer from the chamber. The wells are connected to the central chamber by a thin tunnel to allow the buffer to fill the central chamber. The slice is placed in the central chamber and the harp is placed on top of the slice to keep it from moving. A single inflow tube is shown here. For treatment of dye‐loaded slices with GABA or other agonists, a separate reservoir is used, and a switch valve is used to alternate between perfusion media.

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Figure 7.10.5 Pseudocolor‐enhanced video images from a typical confocal imaging experiment in an acute brain slice. The response of individual pyramidal neurons in area CA1 of hippocampus, loaded with MEQ, to 100 µM GABA (see ) is shown. The left panel shows fluorescence before the addition of GABA (basal) and the right panel shows the fluorescence 20 min after the addition of a maximally effective concentration of GABA (100 µM) to the perfusion medium. At 20 min after application, GABA produced an average Δ F of 22%. Note that one cell was relatively insensitive to GABA.

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Videos

Literature Cited

Literature Cited
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Measurement of Chloride Movement in Neuronal Preparations

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited