Jan 27, 2026
Miniscope calcium signal recording of cortical activity in freely moving mice
- Hong-yuan Chu1,2
- 1Georgetown University;
- 2ASAP
Protocol Citation: Hong-yuan Chu 2026. Miniscope calcium signal recording of cortical activity in freely moving mice. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm1w5nv3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 22, 2026
Last Modified: January 30, 2026
Protocol Integer ID: 241636
Keywords: ASAPCRN, miniscope calcium signal recording of cortical activity, miniscope calcium signal recording, motor cortical activity in mice, cortical activity, mice this protocol, moving mice, task training protocol, mouse reach, mice, prism grin lens implantation
Funders Acknowledgements:
ASAP
Grant ID: ASAP 025187
ASAP
Grant ID: ASAP-020572
Abstract
This protocol was used to record motor cortical activity in mice that had received AAV-GCaMP6f injections and Prism GRIN Lens Implantation (dx.doi.org/10.17504/protocols.io.4r3l21rrqg1y/v1) and were trained on the Mouse reach-to-grab task training protocol (dx.doi.org/10.17504/protocols.io.ewov11bb2vr2/v1).
Troubleshooting
Habituation
5d
Following post-op recovery, bring the mouse to the behavior room for a 1-hour habituation.
Gently handle the mouse by the scruff of the neck.
Slightly loosen the side screw on the baseplate and remove the baseplate cover with blunt forceps.
Attach the nVue 2.0 miniscope to the baseplate and slightly tighten the side screw.
Note
You must hear a click to make sure the miniscope is securely attached to the baseplate.
Place the mouse back in its home cage and allow it to walk freely to habituate to the weight of the miniscope for about 30 min, 2 days in a row.
At the end of the habituation session, take the mouse out of the home cage.
Slightly loosen the side screw on the baseplate, remove the miniscope, and replace it with the baseplate cover.
Return the mouse to its home cage.
Repeat the same process on the next 3 days, but place the mouse in the reaching box for habituation.
Recording
20m
The day before the recording session, remove all food from the mouse’s home cage at the end of the light phase, but keep water.
On the day of the recording session, bring the mouse to the behavior room and allow it to habituate again to the room for about 30 min.
Then, gently handle the mouse by the scruff of the neck.
Slightly loosen the side screw on the baseplate and remove the baseplate cover with blunt forceps.
Attach the miniscope to the baseplate and slightly tighten the side screw.
Note
you must hear a click to make sure the miniscope is securely attached to the baseplate.
Place the mouse back in its home cage.
Start Inscopix data acquisition software, and turn on the green channel LED to check parameters for the best signal quality.
Note
Green channel parameters: for GCaMP6f imaging with the prism GRIN lens in M1, we use a frame rate of 20 Hz, LED power between 0.2 and 0.4 mW/mm2, exposure time 50 ms, and sensor gain of 2 to 4. Digital focus is manually chosen such as to have as many cells as possible in focus, typically between 0 and 1000 Inscopix units (i.e., ~ 0-300 μm).
Once your parameters are set, turn off the LED and start the recording on the green channel.
Gently place the mouse in the reaching box and start the behavior video recording.
Record mouse reaching behavior with concomitant calcium imaging for 20 minutes, then stop both recordings and leave the mouse in the testing box.
On Inscopix data acquisition software, switch to the red channel.
Record multiple videos at different digital focal planes, about 5 seconds each.
Note
Red channel parameters: for tdtomato imaging with the prism GRIN lens in M1, we use a frame rate of 10Hz, LED power between 0.2 and 0.4 mW/mm2, exposure time 50-100 ms, and sensor gain of 2 to 4. Digital focus is manually chosen such as to have as many cells as possible in focus, typically between 0 and 1000 Inscopix units (i.e., ~ 0-300 μm), with steps of 25 or 50 Inscopix units. The number of videos depends on the quality of the signal.
Remove the mouse from the test box.
Slightly loosen the side screw on the baseplate, remove the miniscope, and replace it with the baseplate cover.
Return the mouse to its home cage and provide food ad libitum.