In situ proximity ligation assay to study and understand the distribution and balance of GPCR homo- and heteroreceptor complexes in the brain

The existence of homo- and heteroreceptor complexes with allosteric receptor-receptor interactions increases the diversity of receptor function including recognition, traffi cking, and signaling. This phenomenon increases our understanding of how brain function is altered through molecular integration of receptor signals. An alteration in specifi c heteroreceptor complexes or their balance/equilibrium with the corresponding homoreceptors is considered to have a role in the pathogenic mechanisms that lead to mental and neurological diseases, including drug addiction, depression, Parkinson’s disease, and schizophrenia. However, despite extensive experimental work supporting the formation of these receptor complexes in cellular models, their detection and visualization in the brain remained largely unknown until recent years, when a well-characterized in situ proximity ligation assay (in situ PLA) was adapted to validate the existence of GPCR homo- and heteroreceptor complexes in their native environment. In this chapter we will describe the in situ PLA procedure as a high selectivity and sensitivity assay to detect and characterize GPCR homo- and heteroreceptor complexes and their balance and distribution ex vivo in the brain by confocal laser microscopy. Herein, we outlined in detail the in situ PLA assay and how to use it in an optimal way on work with formalin-fixed free-floating rat brain sections