In situ hybridization (ISH) may be employed to localize cells containing messenger RNA (mRNA), for a particular gene of interest, thereby identifying those cells capable of synthesizing a protein of interest. The present chapter presents a simple method for performing double-label ISH that has been validated for use in studies identifying the co-localization of cannabinoid CB1

From: Methods in Molecular Medicine: Marijuana and Cannabinoid Research: Methods and Protocols Edited by: E. S. Onaivi © Humana Press Inc., Totowa, NJ

Fig. 1. Film images show distribution of CBi (A, C) and preproenkephalin (B) mRNA in sagittal sections of rat brain detected using radioisotopic cRNA probes. CB1 mRNA was detected using an [35S]-labeled riboprobe for CB1 mRNA in the (A) absence or (C) presence of a digoxigenin-labeled probe for preproenkephalin mRNA. The distribution of preproenkephalin mRNA (B) was mapped radioisotopically using an [35S]-labeled riboprobe for preproenkephalin mRNA. Note the correspondence of the pattern of labeling produced by the radiolabeled probe for CB1 in single- (A) and double-labeled (C) sections. Note that the striatum shows high levels of expression of both CB1 and preproenkephalin mRNA. Hybridized slides were apposed to film for 3 d. The scale bar equals 200 |im.

receptor mRNA with other distinct species of peptide mRNAs (1,2). This method permits simultaneous detection of two distinct species of mRNA within the same tissue section. Double-label ISH may be accomplished by hybridizing tissue sections with a cocktail of radiolabeled and digoxigenin-labeled cRNA probes. Single-label ISH may be accomplished by following the procedures detailed for use with radioisotopic probes (here [35S]-labeled) only. Silver grains derived from conventional emulsion autoradiography are used to detect the radiolabeled cRNA probe (Fig. 1). An alkaline phosphatase (AP)-dependent chromagen reaction product is used to detect the nonisotopic (here, digoxigenin-labeled) cRNA probe. Thus, a co-localization of silver grains over cells marked by the colored reaction products identifies the double-labeled cell (Fig. 2). The specificity of the methods may be monitored by evaluating the correspondence of the radioisotopic and digoxigenin-labeled riboprobe for the same species of mRNA (see Note 1). These methods should be able to demonstrate identical patterns of mRNA co-localization when radioisotopic and digoxigenin-labeled mRNAs are substituted for each other in the same experiment. As an additional control, it is necessary to compare the pattern of mRNA expression produced by [35S]-labeling of each riboprobe using single probes hybridized separately on adjacent tissue sections.

The distribution of cannabinoid CB1 RNA in brain was first evaluated using oligonucleotide probes for CB1 (3-5). The development of methods for ISH employing highly sensitive RNA probes, complementary to the mRNA of interest, has facilitated identification of neurons containing even low levels of expression of a target mRNA. The methods described here have been used to specifically evaluate co-localization of cannabinoid CB1 receptor mRNA with other species of mRNA in rat striatum (2) and dorsal root ganglia (1) using highly sensitive cRNA probes. These methods were used to provide a direct demonstration of co-localization of CB1 mRNA with mRNA markers of stria-tonigral (marked by prodynorphin or preprotachykinin A mRNAs) and stri-

atopallidal (marked by proenkephalin mRNAs) projection neurons (2). CB1 mRNA was also localized to putative GABAergic interneurons that express high levels of GAD67 mRNA but not to cholinergic interneurons (expressing mRNAs for choline acetyltransferase or the vesicular acetylcholine transporter)

Fig. 2. Photomicrographs show co-localization of CB1 mRNA with mRNAs for (A) preproenkephalin and (B) prodynorphin in rat striatum. CB1 mRNA was detected radioisotopically using an [35S]-labeled riboprobe. Preproenkephalin and prodynorphin mRNAs were detected nonisotopically using digoxigenin-labeled riboprobes for each species of mRNA. In all panels, the silver grains mark cells expressing CB1 mRNA, whereas the colored reaction products mark cells expressing mRNAs for pre-proenkephalin and prodynorphin in A and B, respectively. The filled arrow shows a double-labeled cell whereas the open arrow shows a cell expressing CB1 mRNA only. (C) Hybridization of the same tissue section with an [35S]-labeled riboprobe for CB1 and a combination of digoxigenin-labeled riboprobes for preproenkephalin and prodynorphin mRNAs revealed co-localization of CB1 mRNA in striatal projection neurons. Striatopallidal neurons expressing preproenkephalin mRNA and striatonigral neurons expressing prodynorphin mRNA were detected using digoxigenin-labeled riboprobes for each respective species of mRNA. In all panels, the accumulation of silver grains over cells marked by the colored reaction product mark the double-labeled cells. Virtually all striatal projection neurons expressed CB1 mRNA. The scale bar equals 20 |im. (Adapted from ref. 2.)

or interneurons expressing preprosomatostatin mRNA (2). In dorsal root ganglia, these methods were used to demonstrate co-localization of CB1 mRNA in small subpopulations of neurons expressing mRNA for preprotachykinin A (a substance P precursor) and a-calcitonin gene related peptide (1). The present methods were adapted from procedures for double-label ISH described previously (6,7).

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