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

Catherine Le Moine¹

¹Université 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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Abstract

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

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Materials

Basic Protocol 1: In Situ Hybridization

Materials

Cryostat sections of fresh-frozen tissue or 1% paraformaldehyde–perfused tissue (e.g., rat brain sections) onto gelatin-coated slides (unit 1.1)
4× SSC/0.1% Denhardt
4× 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
Hybridization buffers appropriate for oligonucleotide or cRNA probes (see recipes)
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

Materials

X-ray film (Fig. 1.10.1; see Basic 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: Generation of Radioactive Brain Paste Standards

Materials

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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Figures

Figure 1.10.1

X-ray film showing autoradiograms of the hybridized sections from control (left) and treated (right) animals and radioactive standards (top). Measurement of optical densities is done for each standard and a calibration curve is generated (see Fig. 1.10.2A). The entire range of the 16 radioactive “spots” corresponds to 1:25, 1:50, 1:100, 1:150, 1:200, 1:300, 1:400, 1:600, 1:800, 1:1200, 1:1600, 1:2400, 1:3200, 1:4800, 1:9600, and 1:19,200 of the original³⁵S-nucleotide dilutions. Note that the seven lowest dilutions (bottom row of the standards) show a saturated signal that cannot be used for accurate calibration with the present experimental conditions. After obtaining a correct calibration, the areas of interest are then delineated on each section, and the optical densities are measured and converted into concentration of radioactivity using the calibration curve.

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Figure 1.10.2

(A) Relation between the arbitrary units of radioactivity and the optical density measured on X-ray film. The same standard section was successively exposed 1, 2, 4, and 6 days. The upper limits of the linear portions of the curves with respect to exposure time are indicated by the vertical dashed lines on each curve. (B) Relation between the arbitrary units of radioactivity and the silver grain densities measured on radioactive standards after micro-autoradiography. Exposure times for the same standards are 2 and 3 weeks.

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Figure 1.10.3

Protocol for measurement of silver grain densities at the cellular level after in situ hybridization with a ³⁵S-labeled probe. (A) Bright-field image showing different types of cells containing the same mRNA: medium-sized and large-sized neurons. (B) The same image after combined illumination (normal bright-field plus fluorescent epi-illumination), where labeled cells are delineated on the video monitor. For a given microscope field, once the cells are delineated, the bright field is turned off so that only silver grains are visible (C). The number and density of silver grains are then automatically measured for each cell by an image analysis system after an appropriate calibration with measurements of the reflected light in a given square area for 0, 1, and 2 grains (C; see Support Protocol).

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Figure 1.10.4

Optical densities measured by densitometry on X-ray film for a given mRNA as a function of the thickness of the cryostat sections. The densitometric signal is stronger with increasing thickness of the sections, with a better homogeneity of the signal, starting from 10 µm, compared to 8- and 9-µm sections.

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Figure 1.10.5

Dark-field photomicrographs showing the expression ofc-fos mRNA in the striatum (ST) following treatment with saline (A), the full D1 receptor agonist SKF-82958 alone (B), and in combination with the D2-receptor agonist quinelorane (C). Note the heterogeneous “patchy” induction ofc-fos mRNA in the striatum following combined treatment with SKF-82958 plus quinelorane in C. Reproduced from Svenningsson et al. (2000) with permission from Elsevier Science.

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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.
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	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.