The mechanisms by which RhoA activity and associated synaptic effects are controlled by the DISC1 scaffolding-like protein

This dataset contains the raw data for the manuscript entitled "The mechanisms by which RhoA activity and associated synaptic effects are controlled by the DISC1 scaffolding-like protein." The raw data corresponding to each main figure and supplementary figure of the article are shown in a spreadsheet. The spreadsheet tabs clearly indicate which figure of the paper the dataset corresponds to. Also, the microscopy dendritic spine images and dendrite tree images used for quantification are compiled and shared. The raw microscopy images are from primary neuronal cultures transfected with full-length PDZ-RhoGEF (i.e, wild-type PDZ-RhoGEF) or PDZ-RhoGEF containing deletions of the enzymatic domain of the PDZ domain. The quantification of the microscopy images demonstrates the effects of these PDZ-RhoGEF constructs on dendritic spine density, dendritic spine morphology, and the length and branching of the apical and basal dendrite trees.

Methods
Primary Neuronal Transfection:

Primary cortical rat neurons were transfected with cDNA between DIV21-DIV23 with Lipofectamine 2000. cDNA of Myc-tagged PDZ-RhoGEF constructs in combination with GFP were mixed with Lipofectamine in DMEM containing 10 % fetal bovine serum and incubated at 37 °C for 30 minutes, then 100 µl of this combination was added to the cells. 4 hours later, the cell media was removed, and 500 µl of old neurobasal media that was not exposed to Lipofectamine (removed just prior to the addition of the cDNA+Lipofectamine) and 500 µl of fresh neurobasal media supplemented with B27 (Gibco), DL-AP5 (Tocris), penicillin/streptomycin, and L-glutamine was added to each culture dish.

Cell culture fixation and immunocytochemistry for dendritic spine and Sholl analysis:

 Five days following transfection with cDNA, primary cortical neurons were fixed (between DIV26-DIV28) by the application of a combination of 3.7 % formaldehyde and 4 % sucrose for 10 minutes at room temperature. The fixative was rinsed off with PBS, followed by incubation for 10 minutes with pre-chilled methanol. Cells were then rinsed with PBS containing 0.3 % Triton X-100, and blocked in this PBS-Triton containing 3 % normal donkey serum for 1 hour at room temperature. A 1:300 dilution of chicken anti-GFP antibody was used to enhance the GFP signal and the labeling of PDZ-RhoGEF constructs was accomplished via a mouse anti-Myc-Tag antibody.

Cells were incubated in primary antibody overnight at room temperature. Cells were then washed with PBS, and incubated with Donkey anti-chicken AlexaFluor 488 in PBS containing donkey serum for 3 hours at room temperature. Cells were then rinsed three times with PBS, and coverslipped with VectaShield hardset anti-fade mounting media.

Dendrite and dendritic spine imaging and analysis

 A Keyence BZ-X700E scanning microscope was used to image dendrites and dendritic spines. For dendrite images of pyramidal neurons, a 20x objective lens was used with a 0.3 µm step size for Z-stack collection. For images of dendritic spines, a 100x objective lens was used with a 0.1 µm step size for Z-stack collection. Spine images were all obtained for apical tree secondary dendrites, with each dendrite segment at least 50 µm long. BZ analyzer software was used to collapse and deconvolve images.

NeuronStudio software was used to analyze dendritic length and branching and to analyze dendritic spine density and morphology. For spine quantification, all spines from a given dendrite were manually selected, upon which the program places a hollow ellipse around the head of the spine. The diameter of the ellipse is indicative of the diameter of the spine head. In instances in which the ellipse is greater or less than the true size of the spine head, manual adjustments are made. Spines were then classified as thin, stubby, or mushroom. Briefly, spine that lack a clear neck region were always classified as stubby. Spines with a neck region and spine diameter larger than 0.5 µm were classified as mushroom spines. Spines with a neck region and a head diameter less than 0.5 µm were classified as thin. All dendrite and spine imaging and analysis was done blinded to experimental conditions.