A neural circuit targeting technique for investigating functional input-output organization in the nervous system

Neurons communicate information across circuits and the function of cells in these circuits is determined by both the afferent inputs they receive and the efferent outputs they send to other brain regions^1,2. To study the activity and function of specific neuronal populations, transneuronal anterograde³and retrograde^4–6viral approaches have been employed to define neural circuit elements by inputs or outputs, respectively. However, what is missing is a way to study the function of neurons based on both their inputs and outputs. Applying a combination of multiple recombinases and transneuronal anterograde/retrograde viruses, we developed a technique called input-output<underline>P</underline>rojection-based<underline>IN</underline>tersectional<underline>C</underline>ircuit-tagging<underline>E</underline>nabled by<underline>R</underline>ecombinases (PINCER) to target specific neuronal cell types and investigate functional input-output organization in neural circuits. We show the logic and application of this technique within vivocalcium imaging and optogenetic approaches to reveal the distinct functions and neural dynamics of connectivity defined neuronal populations in the amygdala for emotional processing. Specifically, PINCER allowed the parsing of valence and salience functions of the amygdala to reveal an input-output cell type selectively mediating aversive memory formation. This technique allows neuroscientists to identify novel subclasses of cells based on their combinatorial input-output anatomical connectivity, providing a tool for fine dissection of the functional properties of neural circuits.