Two photon imaging system

For imaging in Figure 4, mice were imaged throughout training in 15 min sessions per day. Volumetric imaging was performed using a resonant galvanometer two-photon imaging system (Bruker), with a laser (Insight DS+, Spectra Physics) tuned to 920 nm to excite the calcium indicator, GCaMP6f, through a 16×/0.8 water immersion objective (Nikon) interfacing with an Gradient Refractive Index (GRIN) lens through a few drops of distilled water. Prior to each session, mice were headfixed and each GRIN lens was carefully cleaned with 70% ethanol. Fluorescence was detected through GaAs photomultiplier tubes using the Prairie View 5.4 acquisition software. Black dental cement was used to build a well around the implant to minimize light entry into the objective from the projector. High-speed z-stacks were collected in the green channel (using a 520/44 bandpass filter, Semrock) at 512 × 512 pixels covering each x–y plane of 800 × 800 mm over a depth of ~150 μm (30 μm apart) by coupling the 30 Hz rapid resonant scanning (x–y) to a z-piezo to achieve ~3.1 Hz per volume. Average beam power measured at the objective during imaging sessions was between 20–40 mW. An incoming TTL pulse from ViRMEn at the start of behaviour enabled time-locking of behavioural epochs to imaging frames with millisecond precision.