T here is a general consensus in the neuroscientific field on the prime importance of understanding brain connectivity to advance our understanding of brain function and disease. 1,2 In particular, the profuse pattern of connections of the cerebral cortex plays an important role for cognitive functions. 3 Research over the past few years has added additional complexity to the connectivity problem, challenging the view of the brain as a rigid system and demonstrating that it is rather a plastic structure with connections adapting to the environment. For instance, connectivity can change in the course of learning, drug addiction, epilepsy, and other neurodegenerative diseases. 2,4À6 In order to better relate the anatomical structure of the brain to a particular functional outcome and to follow changes with development and aging, longitudinal studies on an individual basis are required. There are not many techniques that give us the opportunity of approaching this goal. For its broad spatial coverage and 3D noninvasive nature, magnetic resonance imaging (MRI) stands as an excellent candidate for dynamic investigations of brain connectivity.We report here a strategy to produce novel MR contrast agents to dynamically trace brain connectivity in vivo by functionalizing classical neuroanatomical tracer molecules. Classical tracers are taken up by neurons and transported from the soma and dendrites to the distant synaptic terminals (anterograde tracers) or toward the soma (retrograde tracers). 7 These compounds are visualized with conventional histological techniques in post-mortem samples. Thus, the outputs from and inputs to a particular brain area are delineated only after processing of the fixed brain tissue. Previous and successful attempts to investigate neuronal connectivity in vivo using MRI and the paramagnetic ion manganese (Mn 2+ ) have been reported. 8 In manganese-enhanced MRI (MEMRI), Mn 2+ is taken up by neurons and transported to the distant synaptic terminals where it accumulates and reveals the projection fields. 8À14 Combined anterograde and retrograde transport has also been reported using MEMRI. 15 Also reported is, however, the toxicity associated with high concentration of Mn 2+ in tissue that challenges the applicability of the MEMRI technique. 10,16,17 Although some strategies have been reported to decrease the impact of Mn 2+ toxicity on MEMRI experiments, 10,18À20 new nontoxic paramagnetic tracers are desirable.To develop new neuronal tracers for connectivity studies in living animals, we have focused, as a proof of concept, on the wellknown classical tracer biocytin. 21 The backbone of this structure was functionalized by covalently linking the gadolinium (Gd 3+ )Received: March 7, 2011 Accepted: August 3, 2011 ABSTRACT: To investigate the connectivity of brain networks noninvasively and dynamically, we have developed a new strategy to functionalize neuronal tracers and designed a biocompatible probe that can be visualized in vivo using magnetic resonance imaging (MRI). Furthermore, the multi...