Localization of cells expressing AChE mRNA in rat striatum using nonradioactive in situ hybridization

A better understanding of the role of AChE in mammalian brain requires know ledge of the distribution of AChE synthesizing cells in this tissue. The ai m of the present study is to test a nonradioactive approach for the localiz ation of AChE mRNA positive cells in rat striatum. Nonradioactive in situ h ybridization has not been used before for the localization of this mRNA in mammalian brain. In order to find optimal conditions for localization, we e mployed both RNA and oligonucleotide probes. We also examined various prehy bridization protocols and approaches. The total number of cells in brain se ctions was determined by subsequent fluorescent staining of the nuclei. Opt imal AChE mRNA localization was obtained with a digoxigenine-labeled RNA pr obe. We were not able to localize AChE mRNA with nonradioactively 3' end-la beled oligonucleotides. An acetylation step prior to hybridization was foun d to be essential for optimal signal/background ratios; high nonspecific st aining was observed, if this step was omitted. In accordance with reports o f other authors, who used radioactive in situ hybridization, we found very low percentages of AChE mRNA-positive cells in striatum, although this area exhibits very high AChE staining. In comparison to radioactive techniques, the nonradioactive approach avoids the risks of radioactivity, and is much less time consuming. In our experiments AChE mRNA localization in striatum was practically the same as that demonstrated previously by radioactive ap proaches. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.