Computational protocol: Activity-Dependent Regulation of Dendritic Growth and Maintenance by Glycogen Synthase Kinase 3β

Similar protocols

Protocol publication

[…] Hippocampal slice cultures were prepared from postnatal day 6 (P6) Sprague Dawley rat pups (mixed sex) as previously described . Briefly, rat pups were anesthetized and decapitated according to institutional guidelines, and then hippocampi were removed and transversely sliced (400 µm thick) using a McIlwain Tissue Chopper (Ted Pella, Inc). Slices were transferred onto 0.4 µm Millicell cell culture membrane inserts (Millipore) and cultured for 2 weeks. Biolistic transfection of neurons in hippocampal slices was then performed using a Helios gene gun (Bio-Rad) . Cell imaging was performed using a two-photon FV1000 laser scanning confocal (Olympus) 48 hours after transfection, and again 10 days after transfection. CA3 pyramidal neurons expressing EGFP were randomly chosen by location and morphology. Maximal intensity projections of z-stacks were used to generate 2D images for analysis. Tracing and quantification of dendrites were done using the Simple Neurite Tracer ImageJ plugin . [...] We used the maximal intensity projection of z-stacks of images to generate the 2D images for analysis. Tracing and quantification of dendrites were done using ImageJ with the NeuronJ plugin . Only neurons showing intact dendritic arborization were analyzed and only branches over 10 µm in length were quantified. Sholl analysis was performed using ImageJ with the Sholl analysis plugin developed by the Ghosh Lab (http://biology.ucsd.edu/labs/ghosh/software) with a 10 µm interval in radius between concentric circles. […]

Pipeline specifications