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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Abstract

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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Materials

Basic Protocol 1: High‐Affinity Choline Uptake (HACU) Assay

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
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   Biedler, J.L., Helson, L., and Spengler, B.A. 1973. Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res. 33:2643‐2652.
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   Crouch, S.P., Kozlowski, R., Slater, K.J., and Fletcher, J. 1993. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J. Immunol. Methods 160:81‐88.
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