Fiber photometry recording

Four to five weeks following stereotactic surgery, mice injected with AAV2/9-hSyn-DIO-GCaMP6s underwent unilateral implantation of a chronically implanted optical fiber (200 μm diameter, 0.37 numerical aperture; Shanghai Zhuen Bio-Technology Co., Ltd, China). Targeted implantation sites were the vLGN at −2.35 mm bregma, 2.5 mm lateral, −3.6 mm ventral, and the l/vlPAG at −4.25 mm bregma, 0.5 mm lateral, −2.9 mm ventral. Fibers were secured to the skull using miniature screws and dental cement (Shanghai Yuyan Bio-Technology Co., Ltd, China). Animals were allowed to recover on a heating pad post-surgery. An accurate implantation site was confirmed post-experimentally via coronal brain section analysis.

Calcium transients were captured using a fiber photometry system (R810, RWD Life Science Co., Ltd). GCaMP6s fluorescence was excited by a 470 nm LED (30 μW at fiber tip), and calcium-independent signals by a 405 nm LED (20 μW at fiber tip). LEDs alternated at 20 Hz, with emissions recorded by an sCMOS camera (Photo-metrics Prime) at a matching frequency.

In vivo recordings were performed in mice during exposure to open spaces at both ground level and elevated heights. To acclimate them, the mice were handled for 10 min each day across three days. For the tests, each mouse was placed in a transparent cylindrical tube (8.9 cm inner diameter, 20 cm height) set on a round acrylic platform (9.5 cm diameter), located in the center of a 20 × 20 × 20 cm open-field arena with transparent walls. The animal was then exposed to the open space by swiftly removing the tube. Each trial consisted of 30-s recording sessions, separated by 2-min intervals, and was repeated 4–5 times per mouse. The moment of tube removal marked time 0 for each trial. For height exposure trials, the same mice were tested on a similar platform elevated 30 cm above the ground. Signal quantification involved subtracting the calibrated 410 nm signal from the 470 nm signal to correct for both movement and bleaching. ΔF/F ratio was calculated as (470 nm signal - calibrated 410 nm signal) (calibrated 410 nm signal)⁻¹, then further normalized using the -5 to 0 s pre-exposure window as the baseline period. The area under the curve (AUC) for the response to the exposure to height or open space was then calculated over a 0 to 10 s period post-exposure.