Ultima 4 two photon microscope

Male and female Thy1-GFP-M line mice^{41 (link)} (n = 5 per condition) were purchased from The Jackson Laboratory (JAX #007788) and maintained in UCSC animal facilities according to an IACUC approved protocol. In vivo transcranial two-photon imaging and data analysis were performed as previously described.^{42 (link)} Briefly, mice were anesthetized with an intraperitoneal (i.p.) injection of a mixture of ketamine (87 mg/kg) and xylazine (8.7 mg/kg). A small region of the exposed skull was manually thinned down to 20–30 μm for optical access. Spines on apical dendrites in mouse primary sensory cortices were imaged using a Bruker Ultima IV two-photon microscope equipped with an Olympus water-immersion objective (40x, NA = 0.8) and a Ti:Sapphire laser (Spectra-Physics Mai Tai, excitation wavelength 920 nm). Images were taken at a zoom of 4.0 (pixel size 0.143 × 0.143 μm) and Z-step size of 0.7 μm. The mice received an i.p. injection of DOI (10 mg/kg) or TBG (50 mg/kg) immediately after they recovered from the anesthesia of the first imaging session. The mice were re-imaged 24 h after drug administration. Dendritic spine dynamics were analyzed using ImageJ. Spine formation and elimination were quantified as percentages of spine numbers on day 0.