Quantitative In Situ Hybridization for the Study of Gene Expression at the Regional and Cellular Levels

Catherine Le Moine1

1 Université Victor Segalen Bordeaux 2, Bordeaux Cedex, France
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.10
DOI:  10.1002/0471142301.ns0110s23
Online Posting Date:  August, 2003
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Quantitative in situ hybridization allows measurement of mRNA level modifications in a variety of experimental conditions. This analysis may be performed both at the regional anatomical and cellular levels by densitometry, neuronal counting and silver grain measurements.

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Table of Contents

  • Basic Protocol 1: In Situ Hybridization
  • Basic Protocol 2: Measurement of Gene Expression and mRNA Quantification
  • Support Protocol 1: Generation of Radioactive Brain Paste Standards
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: In Situ Hybridization

  • Cryostat sections of fresh‐frozen tissue or 1% paraformaldehyde–perfused tissue (e.g., rat brain sections) onto gelatin‐coated slides (unit 1.1)
  • recipe4× SSC/0.1% Denhardt
  • recipe4× SSC (see recipe for 8× stock)
  • 4× SSC/1.33% triethanolamine/0.25% acetic anhydride, pH 8
  • 70%, 80%, 90%, and 100% ethanol
  • 35S‐ or 33P‐labeled probes (synthetic oligonucleotides, cRNA; unit 1.3), sp. act., ∼0.4 µCi/ng
  • recipeHybridization buffers appropriate for oligonucleotide or recipecRNA probes (see reciperecipes)
  • 1:3 (v/v) Ilford K5 emulsion/1× SSC
  • Toluidine blue (or Mayer's hematoxylin‐eosin)
  • Eukitt cytological mounting medium (Electron Microscopy Sciences)
  • X‐ray films (Kodak BIOMAX) and photographic emulsion (Ilford K5)
  • Developer and fixative for autoradiography, and material for staining (toluidine blue or Mayer's hematoxylin and eosin; unit 1.3)
  • Additional reagents and equipment for standard in situ hybridization protocols (unit 1.3)

Basic Protocol 2: Measurement of Gene Expression and mRNA Quantification

  • X‐ray film (Fig. ; see protocol 1)
  • Optical table
  • CCD video camera (e.g., Sony or Panasonic)
  • Microscope equipped with fluorescent epi‐polarization
  • Image analyzer system (Visioscan and DensiRag from Biocom or Mercator from Explora Nova) allowing densitometric analysis and silver grain counting
  • Statistical analysis software (GraphPad Prism, Statview)
To analyze macroscopically

Support Protocol 1: Generation of Radioactive Brain Paste Standards

  • Rat brains perfused with 0.9% NaCl (unit 1.1)
  • 0.1 M phosphate buffer, pH 7.2 ( appendix 2A)
  • 1000 Ci/mmol35S‐labeled nucleotide
  • Liquid nitrogen
  • Scintillation cocktail
  • Photographic emulsion
  • Sonicator
  • Glass pipets
  • Straws or PCR‐type microcentrifuge tubes
  • Gelatin‐coated slides
  • Cryostat
  • Scintillation counter vials
  • Scintillation counter
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Literature Cited

Literature Cited
   Baskin, D.G., Filuk, P.E., and Stahl, W.L. 1989. Standardization of tritium‐sensitive film for quantitative autoradiography. J. Histochem. Cytochem. 37:1337‐1344.
   Bernard, V., Le Moine, C., and Bloch, B. 1991. Striatal neurons express increased level of dopamine D2 receptor mRNA in response to haloperidol treatment: A quantitative in situ hybridization study. Neuroscience 45:117‐126.
   Bisconte, J.C., Fulcrand, J., and Marty, R. 1968. Analyse autoradiographique dans le système nerveux central par photométrie et cartographie combinées. C. r. Scéanc. Soc. Biol. 161:2178‐2182.
   Fauchey, V., Jaber, M., Caron, M.G., Bloch, B., and Le Moine, C. 2000. Differential regulation of the D1, D2 and D3 receptor gene expression and changes in the phenotype of the striatal neurons in mice lacking the dopamine transporter. Eur. J. Neurosci. 12:19‐26.
   Frenois, F., Cador, M., Caillé, S., Stinus, L., and Le Moine, C. 2002. Neural correlates of the motivational ans somatic components of naloxone‐precipitated morphine withdrawal. Eur. J. Neurosci. 16:1377‐1389.
   Gerfen, C.R. 1989. Quantification of in situ hybridization histochemistry for analysis of brain function. Meth. Neurosci. 1:79‐97.
   Gerfen, C.R., McGinty, J.F., and Young, W.S. III 1991. Dopamine differentially regulates dynorphin, substance P, and enkephalin expression in striatal neurons: In situ hybridization histochemical analysis. J. Neurosci. 11:1016‐1031.
   Gerfen, C.R., Keefe, K.A., and Gauda, E.B. 1995. D1‐ and D2‐dopamine receptor function in the striatum: Coactivation of D1‐ and D2‐dopamine receptors on separate populations of neurons results in potentiated immediate early gene response in D1‐containing neurons. J. Neurosci. 15:8167‐8176.
   Georges, F., Stinus, L., Bloch, B., and Le Moine, C. 1999. Chronic morphine exposure as well as spontaneous withdrawal are associated with modifications in dopamine receptor and neuropeptide gene expression in the rat striatum. Eur. J. Neurosci. 11:481‐490.
   Georges, F., Stinus, L., and Le Moine, C. 2000. Mapping of c‐fos gene expression in the brain during morphine dependence and precipitated withdrawal and phenotypic identification of the striatal neurons involved. Eur. J. Neurosci. 12:4475‐4486.
   Griffin, W.S.T. 1987. Methods for hybridization and quantification of mRNA in individual brain cells. In In Situ Hybridization, Applications to Neurobiology. (K.L. Valentino, J.H. Eberwise, and J.D. Barchas eds.) pp. 97‐110. Oxford University Press, New York.
   Jaber, M., Cador, M., Dumartin, B., Normand, E., Stinus, L., and Bloch, B. 1995. Acute and chronic amphetamine treatments differently regulate neuropeptide messenger RNA levels and Fos immunoreactivity in rat striatal neurons. Neuroscience 65:1041‐1050.
   Le Moine, C. 2000. Quantitative in situ hybridization using radioactive probes to study gene expression in heterocellular systems. In Methods in Molecular Biology, In Situ Hybridization Protocols. (I. Darby, ed.) pp. 143‐156. Humana Press, Totowa, NJ.
   Le Moine, C. and Bloch, B. 1995. D1 and D2 dopamine receptor gene expression in the rat striatum: Sensitive cRNA probes demonstrate prominant segregation of D1 and D2 mRNAs in distinct neuronal populations of the dorsal and ventral striatum. J. Comp. Neurol. 355:418‐426.
   Le Moine, C., Normand, E., Guitteny, A.F., Fouque, B., Teoule, R., and Bloch, B. 1990. Dopamine receptor gene expression by enkephalin neurons in the rat forebrain. Proc. Natl. Acad. Sci. U.S.A. 87:230‐234.
   Le Moine, C., Svenningsson, P., Fredholm, B., and Bloch, B. 1997. Dopamine‐adenosine interactions in the striatum and the globus pallidus: Inhibition of striato‐pallidal neurons through either D2 or A2A receptors enhances D1 receptor‐mediated effects on. c‐fos expression J. Neurosci. 17:8038‐8048.
   Miller, J.A. 1991. The calibration of 35S or 32P with 14C‐labeled brain paste or 14C‐plastic standards for quantitative autoradiography using LKB Ultrofilm or Amersham Hyperfilm. Neurosci. Lett. 121:211‐214.
   Normand, E., Popovici, T., Onteniente, B., Fellmann, D., Piatier‐Tonneau, D., Auffray, C., and Bloch, B. 1988. Dopaminergic neurons of the substantia nigra modulate preproenkephalin A gene expression in rat striatal neurons. Brain Res. 439:39‐46.
   Smolen, A.J. and Beaston‐Wimmer, P. 1990. Quantitative analysis of in situ hybridization using image analysis. In In Situ Hybridization Histochemistry (M.F. Chesselet ed.) pp. 75‐188. C.R.C. Press, Boca Raton Ann Arbor, Boston.
   Svenningsson, P., Fredholm, B.B., Bloch, B., and Le Moine, C. 2000. Co‐stimulation of D1/D5 and D2 dopamine receptors leads to an increase in c‐fos mRNA in cholinergic interneurons and a redistribution of c‐fos mRNA in striatal projection neurons. Neuroscience 98:749‐757.
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