Retrograde tract-tracing "Plus": Adding extra value to retrogradely traced neurons

Abstract

Classical neuroanatomical tract-tracing methods have formed the basis for most of our current understanding of brain circuits. However, to obtain a deeper knowledge of the main operational principles of the brain, the simple delineation of brain connectivity is not suffi cient. This particularly holds true in regard to the analysis of connections within the diseased brain, for instance, the study of a number of major neurological disorders through the use of animal models. In other words, the information gathered from tract- tracing techniques is often too static, and recent fi ndings in the fi elds of neurophysiology, receptor mapping, and neuroimaging (among others) need to be integrated within the context of structural data of brain connectivity as seen with neuroanatomical tracing techniques. During the past few years, our laboratory has pioneered a number of combinations of retrograde tracers with in situ hybridization, analyzing the changes in mRNA expression levels within brain circuits of interest. More recently, we have succeeded in combining a number of tract-tracing methods with a newly introduced technique known as in situ proximity ligation assay (PLA). The PLA technique is particularly well suited for the analysis of protein-protein interactions. This combination of methods enabled us to elucidate unequivocally the presence of GPCR heteromers within identifi ed brain circuits and we strongly believe that this will soon become a popular approach in the fi eld. Here we provide readers with a landscape view of these approaches, together with step-by-step protocols so that these methods may be easily reproduced even by inexperienced users.