Minicube

The fiber photometry system used LEDs to deliver 405 nm and 473 nm light. These light streams were sinusoidally modulated at 211 Hz and 531 Hz respectively, combined into a minicube (Doric Lenses, Quebec, Canada), and coupled to an optical patch cord (400 μm; Doric Lenses). The optical patch cord was connected to the animal using a zirconia sleeve (2.5 mm; Doric Lenses). Emitted fluorescence was received through the same optical patch cord and collected by a femtowatt photodetector (New Focus 2151, Newport Corporation, Irvine, CA). This signal was processed using Doric Photometry Software. Photometry data analysis and statistical tests were performed using custom code in MATLAB (MathWorks, Natick, MA). Data were collected at 12 kHz, and low-pass filtered at 12 Hz. The calcium activity-independent 405 nm reference channel was fit to the calcium activity-dependent 473 nm channel using linear least squares. Relative fluorescence changes, reported as ΔF/F, were calculated using the following equation:
$$\frac{\Delta \text{F}}{\text{F}}=\frac{473\text{ nm signal}-\text{fitted }405\text{ nm signal}}{\text{mean }473\text{ nm signal}}\times 100$$
Mean ΔF/F before and after an event or behavior was calculated by calculating the mean signal in a 5 s window pre- and post-timepoint.

Fiber optic cables (1m, 400μm core, 0.48 NA; Doric Lenses) were coupled to implanted optic fibers with zirconia or bronze sleeves (Doric Lenses). Excitation and emission light was passed through a fluorescence minicube (FMC6_AE(405)_E1(465–480)_F1(500–540)_E2(555–570)_F2(580–680)_S, Doric Lenses). Excitation light (~100 μW) was provided by 405nm and 465nm LEDs (Doric) modulated at 531 or 211 Hz using an RZ5P fiber photometry processor (Tucker-Davis Technologies, TDT) running Synapse, and emission light collected via fluorescence photodetector heads integrated into the minicube (Doric Lenses). Signals were demodulated, digitized at 1 kHz, and acquired via the RZ5P processor. Transistor–transistor logic (TTL) time stamps were used to align the signal, video frames, and experimental stimuli. Photometry recordings were done between 4 and 8 weeks following viral vector injection.