The primary motor cortex (M1) is known to be a critical site for movement initiation and motor learning. Surprisingly, it has also been shown to possess reward-related activity, presumably to facilitate reward-based learning of new movements. However, whether reward-related signals are represented among different cell types in M1, and whether their response properties change after cue-reward conditioning remains unclear. Here, we performed longitudinal in vivo two-photon Ca²⁺ imaging to monitor the activity of different neuronal cell types in M1 while mice engaged in a classical conditioning task. Our results demonstrate that most of the major neuronal cell types in M1 showed robust but differential responses to both cue and reward stimuli, and their response properties undergo cell-type specific modifications after associative learning. PV-INs’ responses became more reliable to the cue stimulus, while VIP-INs’ responses became more reliable to the reward stimulus. PNs only showed robust response to the novel reward stimulus, and they habituated to it after associative learning. Lastly, SOM-IN responses emerged and became more reliable to both conditioned cue and reward stimuli after conditioning. These observations suggest that cue- and reward-related signals are represented among different neuronal cell types in M1, and the distinct modifications they undergo during associative learning could be essential in triggering different aspects of local circuit reorganization in M1 during reward-based motor skill learning.

Using two-photon microscopy, we imaged subpopulations of neurons in awake and behaving mice on day 1 and day 7 of a head-fixed classical conditioning task where an auditory tone was paired with water rewards. We imaged pyramidal neurons, PV-INs, SOM-INs and VIP-INs in M1 to understand how M1 represents reward and reward-related cues. 

dF/F files show the change in GCaMP6f fluorescence and are saved as .mat files and behavioural data (acquired using wavesurfer) was saved as H5 files. H5 files log cue, reward and mouse lick timing. Imaging was performed at 30 fps.