High‐Affinity Choline Uptake (HACU) and Choline Acetyltransferase (ChAT) Activity in Neuronal Cultures for Mechanistic and Drug Discovery Studies

Balmiki Ray1, Jason A. Bailey1, Jay R. Simon1, Debomoy K. Lahiri2

1 Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, 2 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.23
DOI:  10.1002/0471142301.ns0723s60
Online Posting Date:  July, 2012
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Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory‐related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High‐affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate‐limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well‐documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide‐based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK‐N‐SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease. Curr. Protoc. Neurosci. 60:7.23.1‐7.23.16. © 2012 by John Wiley & Sons, Inc.

Keywords: brain enzyme; cholinesterase; CNS; synapse; neuronal differentiation; enzymatic activity; cell culture

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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Affinity Choline Uptake (HACU) Assay
  • Basic Protocol 2: CellTiter‐Glo (CTG) Assay
  • Basic Protocol 3: ChAT Activity Assay
  • Support Protocol 1: Cholinergic Differentiation of Human SK‐N‐SH Cells
  • Alternate Protocol 1: HACU and ChAT Assays in Synaptosomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: High‐Affinity Choline Uptake (HACU) Assay

  • HACU buffer (see recipe), freshly prepared
  • [3H]Choline chloride (66.7 Ci/mmol; 1 mCi/ml; Perkin Elmer, cat. no. NET109250UC)
  • 10 mM hemicholinium‐3 (HC‐3; Sigma, cat. no. H108) stock solution prepared in sterile distilled water (store up to 7 days at −20°C)
  • Differentiated cholinergic SK‐N‐SH cells in a 24‐well plate (see protocol 4)
  • 1× Dulbecco's phosphate‐buffered saline (DPBS; Cellgro, cat. no. 20‐031‐CV)
  • Mammalian protein extraction buffer (M‐PER; Pierce, product no. 78505)
  • Liquid biodegradable counting cocktail
  • Water bath with heater and shaker (Precision Scientific Company, cat. no. 66802)
  • Inverted phase‐contrast microscope (Leica DMIL HC; Leica Microsystems)
  • Plastic liquid scintillation counting vials (Research Products International, cat. no. 125501)
  • Liquid scintillation spectrometer (Beckman model LS 3801)

Basic Protocol 2: CellTiter‐Glo (CTG) Assay

  • Cell lysates from HACU assay (see protocol 1)
  • CellTiter‐Glo (CTG) reagent (Promega, cat. no. G7572)
  • 96‐well flat‐bottom white polystyrene plates (Corning, cat. no. 3688)
  • Glowmax luminometer (Turner Biosystems)

Basic Protocol 3: ChAT Activity Assay

  • Lysates from HACU assay (see protocol 1)
  • Mammalian protein extraction buffer (M‐PER; Pierce, product no. 78505)
  • ChAT buffer (see recipe)
  • 50 mM HCl (Sigma, cat. no. 84435)
  • 30 ng/ml sodium tetraphenylboron (Strem Chemicals, cat. no. 93‐057) in 3‐heptanone (MP Biomedicals, cat. no. 195217)
  • Biodegradable counting cocktail (Econo‐Safe; Research Products International, cat. no. 111175)
  • 1.5‐ml microcentrifuge tubes, prechilled
  • Plastic liquid scintillation counting vials (Research Products International, cat. no. 125501)
  • Liquid scintillation spectrometer (Beckman model LS 3801)
CAUTION: 3‐Heptanone is an eye and skin irritant and should be handled with caution. Wear gloves and work in a well‐ventilated fume hood.

Support Protocol 1: Cholinergic Differentiation of Human SK‐N‐SH Cells

  • Human SK‐N‐SH cells (ATCC)
  • MEM‐complete (see recipe)
  • MEM‐complete/ATRA (see recipe)
  • Trypsin‐EDTA (Cellgro/Mediatech, cat. no. 25‐051‐Cl)
  • Trypan blue (Sigma, cat. no. T8154)
  • 100‐mm tissue culture plates (Corning, cat. no. 430167)
  • 15‐ml polyethylene tubes (Corning, cat. no. 430052)
  • Fire‐polished Pasteur pipets
  • Improved Neubauer hemocytometer
  • 24‐well tissue culture plates (Corning, cat. no. 3526)
  • Standard cell culture equipment (humidified 37°C, 5% CO 2 incubator; laminar flow hood)
NOTE: Unless otherwise noted, all media, solutions, and reagents added to cells should be sterile and prewarmed to 37°C prior to use.

Alternate Protocol 1: HACU and ChAT Assays in Synaptosomes

  • Brain tissue
  • 0.32 M sucrose solution, cold
  • Mammalian protein extraction buffer (M‐PER; Pierce, cat. no. 78505)
  • Protease inhibitors (Roche)
  • Potter Elvehjem homogenizer with a Teflon‐coated pestle
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  •   FigureFigure 7.23.1 Schematic of overall procedure for measurement of HACU and ChAT activity. Human neuroblastoma cells are cultured and differentiated with all‐ trans retinoic acid (ATRA). Approximately 100,000 cells are plated in 24‐well cell culture dishes and subjected to HACU and ChAT assays as outlined.
  •   FigureFigure 7.23.2 Layout of a 24‐well plate for the HACU assay. Four replicate sets can be assayed, with three columns allocated to total uptake and three to hemicholinium (HC‐3) blanks (nonspecific uptake). Thus, for each set, n = 3. The number of replicate sets can be decreased or increased by using 12‐ or 48‐well plates.
  •   FigureFigure 7.23.3 Phase‐contrast images of 7‐day‐differentiated human SK‐N‐SH cells before and after HACU experiment. A representative area of a well was imaged at 10× magnification. Cell morphology and number both remain unchanged after [3H]choline uptake.
  •   FigureFigure 7.23.4 Schematic diagram of calculations for net HACU activity in neuronal cells. Raw [3H]choline uptake (in dpm) of both the total uptake and blank wells is adjusted by the relative CTG value to obtain adjusted dpm. The relative CTG value is determined by dividing the raw CTG value (in relative luminescence units or RLU) of all wells by the lowest CTG value. Net HACU is calculated by subtracting the adjusted dpm values from each total uptake well by the average of the adjusted dpm value of the three blank wells.
  •   FigureFigure 7.23.5 Separation of [14C]ACh from the ChAT assay reaction mixture by extraction with sodium tetraphenylboron/3‐heptanone followed by centrifugation yields a biphasic liquid. The clear organic layer contains [14C]ACh.


Literature Cited

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