Gaining insights into brain function by identifying neuronal circuits connecting different regions of the brain is of prime interest in cognitive neuroscience. For visualization of anatomical connections, the tract-tracing technique has been proved to be an extremely useful tool which provides valuable information on afferent and efferent connectivity of the brain. A variety of neuroanatomical tracers exists, and numerous investigations using classical tracers have contributed valuable descriptions of connectivity in the mammalian brain (1). These studies, however, require fixed, histologically processed tissue for data analysis and therefore cannot be applied for noninvasive/longitudinal investigations. Manganese-enhanced Magnetic Resonance Imaging is a recently introduced non-invasive technique that represents the first effort in the direction of studying neuronal connectivity in vivo by means of MRI (2). It is based on the paramagnetic properties of Mn2+ ion and on the fact that, once taken up by neurons, Mn2+ is transported anterogradely along the axon. However, the technique presents several drawbacks that can challenge its applicability, the most important being the potential toxicity of the ion in the tissue. In this study, we have developed an entirely new tracing technique where we have synthesized stable and bifunctional neuronal tracers that allow both, in vivo brain connectivity studies by means of MRI and postmortem microscopic investigation in fixed tissue, in the same experimental animal. Existing tracer molecules with excellent tracing properties could be covalently connected to organic macrocyclic moiety caging gadolinium as MR reporter. We started with biocytin and biotinylated-dextranamine (BDA) as model molecules which are well known anterograde and also retrograde (as opposed to Mn2+) neuronal tracer. Due to high interaction of biotin-avidin, both tracers can be visualized in postmortem tissue by using a host of avidin conjugated markers at the light- and electron microscope level. We have performed in vitro cell studies in neuroblastoma cell lines to prove their uptake competence and in vivo MR/histological visualization to prove their in vivo tracing efficiency as potential neuroanatomical tract-tracers. Our results reveal that these new tracers allow for a new strategy of neuronal tract-tracing, combining the powerful spatial resolution of the conventional microscopic techniques and in vivo visualization by MRI. References. (1) Prog Neurobiol (2000), 62, 327 and (2) Neuroimage (2008), 40, 458.