Labeling Dual Presynaptic Inputs using cFork Anterograde Tracing System

Understanding brain function-related neural circuit connectivity is essential for investigating how cognitive functions are decoded in neural circuits. Trans-synaptic viral vectors are useful for identifying neural synaptic connectivity because of their ability to be transferred from transduced cells to synaptically connected cells. However, concurrent labeling of multisynaptic inputs to postsynaptic neurons is impossible with currently available trans-synaptic viral vectors. Here, we report a neural circuit tracing system that can simultaneously label postsynaptic neurons with two different markers, the expression of which is defined by presynaptic input connectivity. This system, called "cFork (see fork)", includes delivering serotype 1-packaged AAV vectors (AAV1s) containing Cre or flippase recombinase (FlpO) into two different presynaptic brain areas, and AAV5 with a dual gene expression cassette in postsynaptic neurons. Our in vitro and in vivo tests showed that selective expression of two different fluorescence proteins, EGFP and mScarlet, in postsynaptic neurons could be achieved by AAV1-mediated anterograde trans-synaptic transfer of Cre or FlpO constructs. When this tracing system was applied to the somatosensory barrel field cortex (S1BF) or striatum innervated by multiple presynaptic inputs, postsynaptic neurons defined by presynaptic inputs were simultaneously labeled with EGFP or mScarlet. Our new anterograde tracing tool may be useful for elucidating the complex multisynaptic connectivity of postsynaptic neurons regulating diverse brain functions.