Habituation and monitoring of mice for awake imaging

Habituation of the mouse pre-imaging

1. Following surgery, wait 5 days for the mouse to fully recover.

2. Habituation sessions should be performed by the same person and in the same room as the imaging experiments.

3.  On day 1 of habituation, take the mouse out of the cage and let it walk on the experimenter’s loosely folded arms and hands for 10 minutes or longer. If the cage was kept in a different room beforehand, it should be allowed to rest for at least 15 minutes after transportation.

4. On day 2, first handle the mouse for 5 minutes, then head fix it on the head fixation device (a suspended bed with a head fixation bar). Leave it fixed for 5 minutes. Do not produce loud noises in the room which could increase mouse stress. Using a plastic pipette, offer a drop of sweetened condensed milk within sniffing and licking distance for the head-fixed mouse. Release the fixation and bring the mouse back to its cage.

5. On days 3-5, repeat step 4. With each session, increase the time period over which the mouse is head fixed, from 5 to 20, 50, and 120 minutes. Place the fixation apparatus with the head-fixed mouse under the imaging objective with the lights off, mimicking the conditions for imaging. Provide a drop of sweetened condensed milk (can be replaced by water with sugar) every 15 minutes.

6. On day 6, begin imaging sessions.

The time frame proposed in this protocol was based on our personal experience and was not based on any measurement of mouse stress indicators. We believe that the duration of each session, time interval between sessions and number of sessions could be adapted based on observed mouse behavior during the sessions as well as, to some extent, external schedule constraints. 

Monitoring of the mouse during imaging

During imaging and habituation under the microscope (sessions 3-5), the mouse was monitored continuously using a video camera.

Setup: The commercially bought camera ((Lifecam Studio, Microsoft) was modified: the IR filter initially inside was removed manually after opening the front of the camera with a screwdriver. A NIR longpass filter (Midwest Optical LP920-25.5) was added in front of the camera. An IR illumination (M940L3 - IR (940 nm) LED, Thorlabs) was used to make the mouse visible to the modified camera in the otherwise dark room; this IR illumination was invisible for the PMT photodetectors.

For both habituation and imaging sessions, visible signs of stress in the mouse included: eyes partially closed; accumulation of secretions in the eyes or nose; excessive alertness (unnaturally stiff position); extended (>10s) or frequent (>33% of the time approximately) bursts of movement other than grooming behavior (i.e. struggling). When stress signs were observed, the mouse was released from fixation and returned to its cage. Normal (non-stressed) signs included: grooming behavior; accepting the reward (sweet milk); and a natural-looking position.