Head-fixation of awake rodents is a method that allows for sophisticated investigation and manipulation of neural circuits in vivo, that would otherwise be impossible in completely freely moving animals. However, while it is known that head-fixation induces stress, its scale and habituation dynamics remain unclear. Thus, interpretation of physiological and behavioral experiments would greatly benefit from the characterization of the stress response. In our study, we used the Mobile HomeCage system (Neurotar, Finland) where animals are head-fixed to an aluminum frame, but otherwise freely moving in an ultralight carbon container floating above an air-dispensing base. To better understand this experimental environment, we analyzed locomotion and stress during an extended habituation protocol. For 25 consecutive days, mice were prepared as they would be for recording experiments, i.e. head-fixed while standing on the air-lifted platform for 2 hours per day. Throughout 25 days, blood samples were taken periodically from the tail vein to measure variation in the stress-related hormone, corticosterone. These data were compared and contrasted with behavioral data including locomotion during the 2-hours head-fixed habituation sessions and several classical behavioral measurements known to be affected by chronic mild stress.

Stress and behavioral correlates in the head-fixed method