In Situ Hybridization of Adrenergic Receptor mRNA in Brain

Abstract

The advent of transgenic mice has ushered in an explosion of new knowledge regarding key roles of adrenergic receptors (ARs) in central nervous system- (CNS) mediated blood pressure control, states of alertness/sedation, analgesic pathways, and temperature control; these findings have renewed interest in examining human CNS tissue for expression of adrenergic receptor subtype mRNA in specific brain regions. In situ hybridization enables detection of rare mRNA species in specific cells within the human CNS. However, identification of adrenergic receptor mRNA in human tissues is difficult owing to restricted access to tissue for harvest immediately after death. Therefore, in situ hybridization methods need to be carefully optimized to preserve remaining mRNA signal in human studies. Although [α-³⁵S]uridine triphosphate (UTP) isotopic in situ hybridization is our preferred method, a summary of nonisotopic in situ hybridization is also included (in Subheading 4.). [α-³⁵S]-UTP isotopic in situ hybridization is highly sensitive, enabling identification of rare RNA species in human tissues (1). We have had best results in human tissues when the actions of tissue RNases on mRNA signal can be arrested within 2 h of death. We have published identification of α₂-AR mRNA in human spinal cord using this technique (2). This chapter is designed to facilitate the experienced researcher switching from nonhuman models to human tissues, as well as providing the investigator unfamiliar with in situ hybridization with a step-by-step approach.