Mapping functional neuronal ensembles in premotor cortex during complex, goal-directed behaviour

The coordinated recruitment of neural ensembles is thought to underlie behaviourally relevant information processing in the brain. These ensembles are temporally dynamic with individual neurons flexibly recruited into functional groups. In premotor cortex, intermingled neurons are activated during movement planning and execution but also in response to contextual cues, and reward. However, probing the behavioural role of these ensembles and understanding the circuit mechanisms underlying dynamic ensemble formation has been challenging due to the difficulty of flexibly manipulating individual cells. Here, we present an approach to understand the role of dynamic ensembles during goal directed behaviour in premotor cortex of mice by combining a virtual reality (VR) context-dependent motor task with holographic optogenetics and 2-photon population imaging. The behavioural task includes distinct epochs of running, contextual cues, directional licking and reward which gives us a rich spectrum of behavioural epochs to investigate dynamic ensemble formation. The custom-built VR setup is controlled and displayed via Bonsai/Bonvision and communicates with an Arduino that moves the lick spouts, delivers acoustic cues, monitors the spouts for touch, and delivers rewards. For targeted intervention and functional mapping of neuronal ensembles we use an all-optical approach with co-expression of ChRmine and GCaMP6m for targeted holographic stimulation of individual neurons. We established mapping of optogenetic responses and functional connectivity as well as an all-optical protocol to estimate crosstalk activation of the opsin by the imaging laser. Taken together, the combination of VR task and holographic optogenetics now allows mapping and interfering with functional ensembles during behaviour.