This article describes a method for real-time imaging of neuronal activity in the medial prefrontal cortex of anesthetized mice using a gradient-index lens and a miniscope. The technique enables the correlation of calcium transients with specific behaviors during imaging sessions.
Take an anesthetized transduced mouse implanted with a gradient-index lens targeting the medial prefrontal cortex, where neurons express genetically encoded calcium indicators.
Remove the lens cap and clean its surface for clear light transmission.
Secure the miniscope to the lens holder.
Adjust the miniscope position to identify the optimal focal plane. This adjustment ensures clear visualization of neuronal cell bodies.
Detach the cable and allow the mouse to recover.
Connect the cable to the miniscope and transfer the mouse to a chamber with a behavioral recording setup.
During imaging, the miniscope emits light to excite the calcium indicators, which fluoresce upon intracellular calcium influx.
The gradient-index lens relays these optical signals from deep brain tissues to the miniscope, reducing light scattering and enabling real-time visualization of neuronal activity.
Process the recorded calcium transient activity with behavioral observations to correlate neuronal activity to specific behaviors.
After briefly anesthetizing the mouse with isoflurane, loosen the locking screw in the base with a small screwdriver before removing the protective cap, and clean the surface of the GRIN lens with an acetone-soaked cotton swab. Connect the miniscope to the cable and start the NewView software to begin identifying the best focal plane with adjusting the position of the miniscope relative to the base by slightly tightening or loosening with the help of blunt forceps. Once the best focal plane is determined, tighten the locking screw before disconnecting the cable and placing the mouse back in its home cage.
Turn on the behavior camera software to view the mouse behavior arena through a live stream function. Manually adjust the focus of the top camera. Select Trigger Strobe and check Enable/disable trigger followed by clicking Record button. Then, use browse to select the location where behavior recordings will be saved. Select the desired image format.
Bring the mouse close to the arena and connect the miniscope to the cable linked to the data acquisition system. Then, place the mouse in the center of the arena. In the behavior camera software, click Start Recording.
In NewView, check LED1, click on Capture and then trigger buttons to start recording. This allows simultaneous recordings of calcium imaging and mouse behavior. Hit Stop after 3,000 frames and save the files.
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