In Vivo Calcium Imaging of Freely Behaving Mice

For time-lapse imaging in freely behaving mice using an integrated miniature fluorescence microscope (nVistaHD, Inscopix), we followed a previously established protocol^{18 (link),38 (link)}. Briefly, at least 3 weeks after the surgical implantation procedure, we examined the expression of a Ca²⁺ indicator and tissue health by imaging mice under isoflurane anesthesia using a two-photon microscope (Ultima IV, Bruker, Germany) equipped with a tunable Ti:Sapphire laser (Insight, Spectra Physics, Santa Clara, CA). For the CA1-implanted mice, we inserted into the guide tube a ‘microendoscope’ consisting of a single gradient refractive index lens (0.44 pitch length, 0.47 NA, GRINtech GmbH, Germany). We selected for further imaging only those mice that exhibited homogenous GCaMP6 expression and appeared to have healthy tissue. For the selected CA1-implanted mice, we affixed the microendoscope within the guide tube using an ultraviolet-curing adhesive (Norland, NOA81, Edmund Optics, Barrington, NJ). Next, we attached the microscope’s base plate to the dental acrylic cap using light cured acrylic (Flow-It ALC, Pentron, Orange, CA). All mice were returned to their home cages for several days following the aforementioned procedure.