Fiber photometry

Photometry recording setup is as follows: 473-nm laser (Omicron) was delivered via 400-μm, core NA 0.48 patch cords connected via rotary joint (Doric) to fiber optic cannula implanted in mice, and the light power at the fiber tip (200-μm diameter) was calibrated to be 1 to 2 μW/mm² at 0.5-mm tissue depth. Light stimulation frequency was determined by an optical chopper (Thorlabs). GCaMPs or GFP fluorescence emission was received by a femtowatt photoreceiver (NewPort), and signals were phase-lock amplified (Stanford Research Systems) and digitized (LabJack), then recorded by a customized Python program based on Fiberkontrol (https://github.com/logang/Fiberkontrol) (56).

Acute electric shocks experiments were conducted in sound-attenuated fear-conditioning chambers (Med Associates Inc.). Fiber optic–implanted mouse was attached to patch cords and habituated in the chamber for 1 hour. Random interspersed electric shocks of specific intensity and duration were delivered to the metal grid flooring under Video Freeze software command (Med Associates Inc.), and video recording was done using a high-speed firewire monochrome NIR video camera with NIR in-house lighting. TTL signal outputs were sent from Video Freeze to fiber photometry recording program to indicate timings of electric shocks.

Shock-tone conditioning was conducted in a fear-conditioning chamber with black acrylic A-roof and steel grid flooring, and surfaces were cleaned with detergent with mild lemon scent. Mice were habituated in this chamber for 1 hour 1 day before conditioning and were further habituated for 30 min on conditioning day. Five 20-s pure tones (90 dB, 75 kHz) (CS) that ended with 1-s electric shocks (0.7 mA; US) were delivered to the fear-conditioning chamber over a duration of 10 min. Mice were then given 10-min rest before return to the home cage. Tone extinction was performed the following day in a separate fear-conditioning chamber consisting of white acrylic curved walls and flooring, and surfaces were cleaned with 2% acetic acid solution. Each mouse was attached to patch cords and habituated in the tone extinction chamber for 30 min. Thirty tone extinction trials consisting of 10-s pure tones (90 dB, 75 kHz) were then delivered to the tone extinction chamber. Photometric recordings were conducted at tone extinction trials 1, 2, 9, 10, 19, 20, 29, and 30, and TTL signal outputs were sent from Video Freeze to fiber photometry recording program to indicate tone timings. Mouse freezing behavior was recorded by the NIR video camera and analyzed by Video Freeze.

Acute pinches were conducted on mice lightly anesthetized with 1.25% isoflurane and placed on a platform housed in a fear-conditioning chamber. The experimenter was cued to pinch mice upon hearing a 5-s sound cue commanded by Video Freeze, and pinches were performed by lightly squeezing tail or paws with a blunt tip forceps for 5 s or until a reflex behavior response was triggered.

Tail dips were conducted on mice lightly anesthetized with 1.25% isoflurane and placed on a platform housed in a fear-conditioning chamber. The experimenter was cued to lower the mouse tail into a temperature-controlled water bath for 20 s upon hearing a sound cue commanded by Video Freeze. TTL signal outputs were sent from Video Freeze to fiber photometry recording program to indicate timings of sound cues.