Magnetic resonance imaging of neuronal connectivity in vivo opens up the possibility of performing longitudinal investigations on neuronal networks. This is one main reason for the attention that paramagnetic ion manganese (Mn2+) has attracted as a potential anterograde neuronal tracer for MRI experiments. However, the correct and possibly repeated use of this tracer--or of any tracer for that matter, including heavy metals--requires the development of an administration strategy that minimizes its toxic effects. Here we first investigated the conditions that maximize the tracing efficiency of Mn2+ and preserve viability and tissue architectonics in combined MRI and histology experiments in rats. We demonstrate that most common protocols for neuronal tract tracing using Mn2+ result in large neuronal and glial lesions. The toxicity of manganese is distinct during intracortical injections and blocks the transfer of the tracer. After optimizing the technique, we could show that extensive cortical connectivity maps can be generated, with no sign of neuronal damage. Importantly, preservation of tissue viability improves the efficiency of Mn2+ in tracing neuronal connections. We have successfully used this technique to trace corticofugal somatosensory and motor pathways in individual animals and describe a connectivity index (CnI) based on Mn2+ transport that quantitatively reveals cortical heterogeneities in interhemispheric communication. Finally, we have significantly improved the resolution of the technique by continuously infusing very low concentrations of Mn2+ into the target area using osmotic pumps coupled to chronically implanted brain cannulae. The specific, nontoxic and quantitative nature of the neuronal tracings described here indicates the value of this tracer for chronic studies of development and plasticity as well as for studies of brain pathology.