Imaris stitcher

The 12 months old BAC-CAG/CamK2a-CreERT2/WT/MORF3 and CamK2a-CreERT2/WT/MORF3 mice were induced by one dose of Tamoxifen (20mg/kg body weight, i.p.). Mice were perfused, sectioned, and stained as previously described (Veldman et al., 2020 (link)). The striatum of each brain sections was imaged by the Andor DragonFly spinning disk confocal (Belfast, UK) with 60x silicon lens. The images were stitched by Imaris stitcher (Oxford Instruments). The spines on the terminal 30 μm of the dendrites, which has clear dendrite terminals in the top 50 μm sections, were reconstructed and analyzed using Aivia (Bellevue,US). 5~6 medium spiny neurons were analyzed per mouse. For each neuron, the spine density of the terminal 30 μm in 3~5 dendrites were analyzed. Unpaired t-test were used to compare the average spine density per neuron in BAC-CAG and WT mice.

Multiple images of each section were captured using a Zeiss LSM700 laser scanning confocal microscope at 20x (excitation/emissions: 510/510 nm (blue), 639/660 (red), 594/594 nm (green), 647/647 nm (magenta) and stitched together using the Imaris Stitcher (Oxford Instruments, Abingdon, Oxfordshire, UK). The VMHvl was delineated by a contour, and cells outside of the contour were masked using Imaris (version 9.5, Oxford Instruments). Nuclei were classified based on gene expression into the following seven categories: 1) total Esr1 expressing cells, 2) total Tac1 cells 3) total Rprm cells, 4) total Pdyn cells, 5) Tac1/Esr1 co-expressing cells, 6) Rprm/Esr1 co-expressing cells, and 7) Pdyn/Esr1 co-expressing cells. Thus, Esr1 was labeled in two separate series. Because these series yielded nearly identical results when examined separately for effects of age, sex, and T treatment, the mean Esr1 count for the first and second series was calculated for each animal and used in statistical analyses. Cells were counted in one or two matched sections containing the largest cross-sectional areas of the caudo-lateral VMHvl; if two sections were counted, an average was calculated and used in statistical analyses. Total cell number was also recorded by counting the number of DAPI-stained cells with a neuronal morphology (large, regularly shaped, and uniformly stained cell nuclei).