Animals were electroporated with DNA constructs at stage of 45–46 and screened for those with sparsely transfected and well-isolated cells. For imaging, animals were anesthetized with 0.01% MS-222 (Sigma) and were placed in a Sylgard chamber covered by a glass coverslip. Images were collected every 4 h before and after each visual experience session (dark or STVE). Two-photon z-series were collected at 1 μm steps with a 20× water immersion objective (Olympus XLUMPlanFL 0.95NA) at 3–4× scan zoom using a custom-built microscope modified from an Olympus FV300 system20 (link).
Complete dendritic arbors of each neuron were reconstructed using a semi-manual function in the Filament module of Imaris (Bitplane, US). Total dendritic length and TBTN were automatically calculated by the software. 3D Sholl analysis calculated the number of branches that intersect concentric circles at increasing distances from the cell soma, using a customized Matlab program with reconstructed filament data exported from Imaris. The dendritic structural data of the control group (Figs.3 –6 ) was a subset of a previously reported data set[20 (link)]. These neurons were collected and processed in parallel with the GluACTP-expressing experimental groups.
Complete dendritic arbors of each neuron were reconstructed using a semi-manual function in the Filament module of Imaris (Bitplane, US). Total dendritic length and TBTN were automatically calculated by the software. 3D Sholl analysis calculated the number of branches that intersect concentric circles at increasing distances from the cell soma, using a customized Matlab program with reconstructed filament data exported from Imaris. The dendritic structural data of the control group (Figs.
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