Dual anterograde and retrograde viral tracing of reciprocal connectivity

Abstract

Current large-scale approaches in neuroscience aim to unravel the complete connectivity map of specific neuronal circuits, or even the entire brain. This emerging research discipline has been termed connectomics. Recombinant glycoprotein-deleted rabies virus (RABV ΔG) has become an important tool for the investigation of neuronal connectivity in the brains of a variety of species. Neuronal infection with even a single RABV ΔG particle results in high-level transgene expression, revealing the fine-detailed morphology of all neuronal features—including dendritic spines, axonal processes, and boutons—on a brain-wide scale. This labeling is eminently suitable for subsequent post-hoc morphological analysis, such as semiautomated reconstruction in 3D. Here we describe the use of a recently developed anterograde RABV ΔG variant together with a retrograde RABV ΔG for the investigation of projections both to, and from, a particular brain region. In addition to the automated reconstruction of a dendritic tree, we also give as an example the volume measurements of axonal boutons following RABV ΔG-mediated fluorescent marker expression. In conclusion RABV ΔG variants expressing a combination of markers and/or tools for stimulating/ monitoring neuronal activity, used together with genetic or behavioral animal models, promise important insights in the structure–function relationship of neural circuits.