Monocular deprivation (MD), the temporary closure of one eye, shifts neuronal responses in the visual cortex towards the open eye. This functional change is accompanied by formation of new dendritic spines. However, the presynaptic partners of these new spines have not yet been identified. Here we introduce a novel approach, two-timeframe monosynaptic rabies virus tracing, to survey the whole brain for new inputs associated with ocular dominance (OD) plasticity.First, in a tdTomato reporter mouse we infect starter cells (expressing a helper construct) with a rabies virus carrying eGFP and inducible Cre recombinase, thus tracing existing inputs to these cells. Next, injecting 4OHT induces expression of tdTomato in rabies-infected neurons. Neurons infected after 4OHT metabolism express only eGFP, allowing read-out of two sets of input cells: those infected before 4OHT injection (timeframe 1), expressing eGFP and tdTomato, and those infected after 4OHT is metabolized (timeframe 2). The latter ones are putative new inputs.To identify new connections formed during OD plasticity in adult mice, the experiment was arranged such that timeframe 1 inputs reflected pre-MD inputs to neurons in binocular V1, while timeframe 2 captured peri-MD inputs. Preliminary results show that MD increases the formation of new inputs to binocular V1 neurons brain-wide. Crucially, a substantial fraction of timeframe 2 inputs originated in the opposite visual cortex (MD: 48.6%; Control: 26.7%), which largely represents the eye that had been left open during MD. These results lay the foundation for using two-timeframe tracing as a tool for investigating novel neuronal connectivity.