Project Summary To determine the anatomical basis of complex neural behavior, it is critical to have the ability to trace more than one circuit simultaneously in the same animal. That’s because complex animal behaviors or neural computation should be understood through the interaction of more than one circuit – cooperative, antagonistic, or else. In addition, it is necessary to rapidly capture the connectivity information in the dynamically changing brains during development and learning. Engineered G-deleted rabies is a current state-of-art method to retrogradely trace the presynaptic input neurons of a defined cell type. However, it remains unfeasible to trace more than one neural circuit simultaneously. In addition, the current approach using AAV helpers and rabies requires several weeks for tracing. In this proposed research, we will overcome these disadvantages by developing two novel trans-synaptic viral tracer systems: SWORD: Sendai with Orthogonal Rabies Duplex Tracing (Aim 1) and a rapid TRIO/cTRIO: cell-type specific tracing the relationship between input and output (Aim 2). This research is significant because these new methods will allow more comprehensive analysis of neural connectivity in more than one circuit and in more diverse context such as the developing brain where distinct synaptic networks emerge and neural plasticity such as learning across many model species. The proposed research is innovative, because we are developing and validating technically innovative solutions, SWORD and rapid TRIO/cTRIO, to overcome the limitations of the current state-of-the art tracing method. These viral-genetic tools will have a positive and broad impact on the neuroscience field as it will enhance our understanding of neural circuit organization for the complex behaviors and help to identify the circuit-specific therapeutic targets to cure brain disorders.