Fiber photometry

Fiber photometry recordings were performed as previously described (24). Briefly, an optic fiber was attached to the implanted fiber by a ferrule sleeve, and then GCaMP6s was stimulated by two light-emitting diodes (LEDs), a 531-Hz sinusoidal light (Thorlabs, M470F3), band-pass filtered at 470 ± 20 nm, and a 211-Hz sinusoidal light (Thorlabs, M405FP1), band-pass filtered at 405 ± 10 nm. (Filter cube: Doric FMC4; LED driver: DC4104). The 470-nm signal evokes Ca²⁺-dependent emission, while the 405-nm signal evokes Ca²⁺-independent isosbestic control emission. Before recording, a 180-s period of GCaMP6s excitation with both light channels was used to remove the majority of baseline drift. Laser intensity at the optic fiber tip was adjusted to ~50 μW before each day of recording. GCaMP6s fluorescent signal was isolated by band-pass filtering (525 ± 25 nm), transduced by a femtowatt silicon photoreceiver (Newport, 2151), and recorded by a real-time processor (TDT RZ5P). The envelopes of 531- and 211-Hz signals were extracted in real time by the TDT program Synapse at a sampling rate of 1017.25 Hz.

Fiber recordings were analyzed using custom MATLAB scripts available on GitHub. Baseline drift due to slow photobleaching artifacts was corrected by fitting a double exponential curve to the raw trace, then the photometry trace was z-scored relative to the mean and SD of the signal. The mean z score during 10 s preceding and following an event was compared using paired t tests. Shuffled traces were generated by replacing event times with a random time between the beginning and end of the session.