Application of Single‐Cell Microfluorimetry to Neurotoxicology Assays

Tobi L. Limke1, William D. Atchison2

1 Millipore Corporation, Billerica, Massachusetts, 2 Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.15
DOI:  10.1002/0471140856.tx1215s42
Online Posting Date:  November, 2009
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Abstract

Intracellular signaling events play fundamental roles in regulating physiological function. In neurons, these include inducing growth and differentiation, secretion, gene expression, and controlling processes associated with learning and memory. All of these processes have in common the vital dependence on changes in intracellular Ca2+ [Ca2+]i. Numerous toxicants, including metals, polychlorinated biphenyls, and biological neurotoxins, can disrupt [Ca2+]i. Understanding how toxicants disrupt Ca2+‐dependent neuronal signaling, and thus induce neuronal death or dysfunction, requires the ability to monitor [Ca2+]i at the level of individual cells. A series of fluorophores that can report on changes in [Ca2+]i has been pivotal in this process. This section describes how to use these fluorophores to study effects of neurotoxicants on two types of processes: changes in [Ca2+]i in individual cells and changes in mitochondrial membrane potential. Similar techniques using distinct fluorophores can be applied to other physiological processes. Curr. Protoc. Toxicol. 42:12.15.1‐12.15.13. © 2009 by John Wiley & Sons, Inc.

Keywords: fluorescence; calcium; fluorophores; mitochondria

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring Changes in [Ca2+]
  • Support Protocol 1: Measuring the Viability of Dye‐Loaded Cells
  • Basic Protocol 2: Measuring Changes in Mitochondrial Membrane Potential
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring Changes in [Ca2+]

  Materials
  • HEPES‐buffered saline solution (HBS; see recipe)
  • Fura‐2 acetoxymethylester (Fura‐2 AM; see recipe)
  • Cell culture of interest grown on poly‐L‐lysine‐coated glass coverslips
  • Ethanol
  • EGTA/HBS (see recipe)
  • 37°C water bath
  • Glass or plastic foil‐covered scintillation vials
  • Sharp forceps
  • O‐ring chamber or other secure chamber to hold coverslips
  • Cotton swabs
  • Kimwipes
  • Dark box to hold cell chamber
  • Inverted fluorescence microscope system coupled to an excitation light source (such as a xenon lamp in a covered housing) with fluorescence filters for 340 and 380 nm excitation (preferably mounted on a rotating filter wheel for rapid switching between excitation wavelengths) and a 510‐nm emission barrier
  • Perfusion system: consisting of perfusion pump, tubing leading from bottle of buffer to microscope and out to waste container
  • Temperature control system integrated into microscope stage, optional

Support Protocol 1: Measuring the Viability of Dye‐Loaded Cells

  Materials
  • HBS balanced salt solution (see recipe)
  • Tetramethylrhodamine ethyl ester (TMRE; see recipe)
  • Cells of interest cultured on poly‐L‐lysine‐coated glass coverslips
  • Ethanol
  • 37°C water bath
  • Sharp forceps
  • O‐ring chamber
  • Cotton swabs
  • Kimwipes
  • Dark box to hold cells
  • Inverted fluorescence microscope system coupled to excitation light source (such as a xenon lamp in a covered housing) with fluorescent filter for 540‐nm excitation (preferably mounted on a rotating filter wheel for rapid periods of excitation), and 590‐nm emission barrier filter
  • Perfusion system consisting of perfusion pump, tubing to lead from buffer bottle to microscope and out to waste container
  • Temperature control system integrated into microscope stage, optional
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Figures

Videos

Literature Cited

Literature Cited
   Aras, M.A., Hartnett, K.A., and Aizenman, E. 2008. Assessment of cell viability in primary neuronal cultures. Curr. Protoc. Neurosci. 44:7.18.1‐7.18.15.
   Choi, J., Sawant, S.G., Couch, D.B., Ho, I.K., and Farley, J.M. 1995. Continuous measurement of changes in intracellular calcium concentration in mouse splenic T cells attached to a glass substrate. J. Biomed. Sci. 2:379‐383.
   Denny, M.F. and Atchison, W.D. 1994. Methylmercury‐induced elevations in intrasynaptosomal zinc concentrations: An 19F‐NMR study. J. Neurochem. 63:383‐386.
   Denny, M.F. and Atchison, W.D. 1996. Mercurial‐induced alterations in neuronal divalent cation homeostasis. Neurotoxicol. 17:47‐61.
   Denny, M.F., Hare, M.F., and Atchison, W.D. 1993. Methylmercury alters intrasynaptosomal concentrations of endogenous polyvalent cations. Toxicol. Appl. Pharmacol. 122:222‐232.
   Edwards, J.R., Marty, M.S., and Atchison, W.D. 2005. Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury. Toxicol. Appl. Pharmacol. 208:222‐232.
   Ehrich, M. and Sharova, L. 2000. In vitro methods for detecting cytotoxicity. Curr. Protoc. Toxicol. 3:2.6.1‐2.6.27.
   Friedman, P.A. and Gesek, F.A. 1995. Stimulation of calcium transport by amiloride in mouse distal convoluted tubule cells. Kidney Int. 48:1427‐1434.
   Grynkiewicz, G., Poenie, M., and Tsien, R.Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440‐3450.
   Hare, M.F., McGinnis, K.M., and Atchison, W.D. 1993. Methylmercury increases intracellular concentrations of Ca2+ and heavy metals in NG108‐15 cells. J. Pharmacol. Exp. Ther. 266:1626‐1635.
   Kowaltowski, A.J., Smaili, S.S., Russell, J.T., and Fiskum, G. 2000. Elevation of resting mitochondrial membrane potential of neural cells by cyclosporine A, BAPTA‐AM, and bcl. Am. J. Physiol. Cell Physiol. 279:C852.
   Limke, T.L. and Atchison, W.D. 2002. Acute exposure to methylmercury opens the mitochondrial transition pore in rat cerebellar granule cells. Toxicol. Appl. Pharmacol. 178:52‐61.
   Marty, M.S. and Atchison, W.D. 1997. Pathways mediating Ca2+ entry in rat cerebellar granule cells following in vitro exposure to methyl mercury. Toxicol. Appl. Pharmacol. 147:319‐330.
   Neve, E.P., Boyer, C.S., and Moldeus, P. 1995. N‐ethyl maleimide stimulates arachidonic acid release through activation of the signal‐responsive phospholipase A2 in endothelial cells. Biochem. Pharmacol. 49:57‐63.
   Peng, S.Q., Hajela, R.K., and Atchison, W.D. 2005. Fluid flow‐induced increase in inward Ba2+ current expressed in HEK293 cells transiently transfected with human neuronal L‐type Ca2+ channels. Brain Res. 1045:116‐123.
   Scaduto, R.C. Jr. and Grotyohann, L.W. 1999. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys. J. 76:469‐477.
   Sutachan, J.J., Montoya, G.J.V., Xu, F., Chen, D., Blanck, T.J., and Recio‐Pinto, E. 2006. Pluronic F‐127 affects the regulation of cytoplasmic Ca2+ in neuronal cells. Brain Res. 1068:131‐137.
   Ward, C.A. and Moffat, M.P. 1992. Positive and negative inotropic effects of phorbol 12‐myristate 13‐acetate: Relationship to PKC‐dependence and changes in [Ca2+]i. J. Mol. Cell Cardiol. 24:937‐948.
   Xu, Y.J., Shao, Q., and Dhalla, N.S. 1997. Fura‐2 fluorescent technique for the assessment of Ca2+ homeostasis in cardiomyocytes. Mol. Cell Biochem. 172:149‐157.
   Yuan, Y. and Atchison, W.D. 2007. Methylmercury induced increase of intracellular Ca2+ increases spontaneous synaptic current frequency in rat cerebellar slices. Mol. Pharmacol. 71:1109‐1121.
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