Prism probe implantation

One week post virus-injection, a 1 mm diameter gradient refractive index lens (GRIN) attached to a prism (Inscopix) was lowered stereotaxically into the craniotomy at a rate of 10 µm/s while the tissue was treated constantly with saline to minimize desiccation. The prism lens was positioned 1.2–1.3 mm deep and 0.15–0.2 mm away from the injection site. Lens implants were secured to the skull with a thin layer of Kwik-Sil silicone elastomer, followed by a thick layer of adhesive cement (super-bond C&B, Sun Medical). The lens cuff was filled with Kwik-Cast (WPI) for protection during a 1–2 week interval to allow for viral expression. A custom-made head bar was cemented to the skull with dental acrylic for head fixation in behavioural experiments.

Once viral expression was confirmed, mice underwent anaesthesia to secure a baseplate (Inscopix), which was cemented on the prism probe to support the connection of the miniaturized microscope during in vivo imaging under freely moving conditions. During the procedure, a baseplate was attached to the miniature epifluorescence microscope (nVista HD, Inscopix) and stereotaxically positioned to a desired focal plane with the help of visible landmarks (GCaMP6m-expressing neurons and blood vessels) using 20–30% LED power, a frame rate of 5 Hz and digital gain of 4. Once the focal plane was identified, the microscope and baseplate were raised by ~50 µm to compensate for shrinkage of the adhesive cement and were subsequently fixed in place using the same compound, followed by a thin layer of dental acrylic mixed with black carbon spherical powder (Sigma Aldrich) to minimize the light interference of the imaging field. The baseplate was covered with a protective cap (Inscopix), and imaging began within 1–2 days.