https://doi.org/10.5061/dryad.0k6djhb9p

Description of the data and file structure

The behavioral data involves the use of viral-mediated gene transfer to overexpress or knockdown a particular gene/protein (Arhgef11/PDZ-RhoGEF) in the medial prefrontal cortex of young adult male mice. This same viral approach was used to overexpress PDZ_RhoGEF into the medial prefrontal cortex of young adult mice and assess the effects on the density and morphology of dendritic spines. In addition, in cultured neurons, the spatial localization of PDZ-RhoGEF within dendritic spine neck and head regions was assessed. Finally, the spatial overlap between PDZ-RhoGEF and overexpressed DISC1 protein was also assessed in dendritic spines.

Files and variables

File: AAV-arhgef11_shrna_object_in_place.zip

Description: AAV-arhgef11 shrRNA-GFP or AAV-scrambled control shRNA-GFP infused into the medial prefrontal cortex of young adult male mice. Five days post-infusion, mice were test in an object-in-place task. For trial 1, mice are allowed to explore four unique non-identical objects. Mice are then returned to their home cage, then allowed to explore these same objects; however, the location of two of the objects are swapped between trials and the other two objects remain in a fixed location between trials. When opening this zip file, there is a folder named 'AAV-arhgef11 shRNA object in place. Inside this folder there are AVI video files for trial 1 and trial 2 for the object in place task for each mouse. Abbreviations in the file names: Control KD (control knockdown condition -- AAV-scrambled control shRNA-GFP); PRG KD (PDZ-RhoGEF knockdown condition -- AAV-arhgef11 shRNA-GFP). The number after KD is the mouse number, and each file also indicates if it is trial 1 or trial 2

Control KD1 trial 1: Control Mouse 1, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD1 trial 2: Control Mouse 1, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD2 trial 1: Control Mouse 2, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD2 trial 2: Control Mouse 2, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD3 trial 1: Control Mouse 3, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD3 trial 2: Control Mouse 3, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD4 trial 1: Control Mouse 4, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD4 trial 2: Control Mouse 4, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD5 trial 1: Control Mouse 5, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD5 trial 2: Control Mouse 5, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD6 trial 1: Control Mouse 6, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD6 trial 2: Control Mouse 6, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD7 trial 1: Control Mouse 7, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD7 trial 2: Control Mouse 7, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD8 trial 1: Control Mouse 8, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD8 trial 2: Control Mouse 8, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD9 trial 1: Control Mouse 9, trial 1 of object-in-place task; AAV-scrambled control shRNA-GFP

Control KD9 trial 2: Control Mouse 9, trial 2 of object-in-place task; AAV-scrambled control shRNA-GFP

PRG KD1 trial 1: PDZ-RhoGEF knockdown Mouse 1, trial 1 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD1 trial 2: PDZ-RhoGEF knockdown Mouse 1, trial 2 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD2 trial 1: PDZ-RhoGEF knockdown Mouse 2, trial 1 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD2 trial 2: PDZ-RhoGEF knockdown Mouse 2, trial 2 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD3 trial 1: PDZ-RhoGEF knockdown Mouse 3, trial 1 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD3 trial 2: PDZ-RhoGEF knockdown Mouse 3, trial 2 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD4 trial 1: PDZ-RhoGEF knockdown Mouse 4, trial 1 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD4 trial 2: PDZ-RhoGEF knockdown Mouse 4, trial 2 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD5 trial 1: PDZ-RhoGEF knockdown Mouse 5, trial 1 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD5 trial 2: PDZ-RhoGEF knockdown Mouse 5, trial 2 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD6 trial 1: PDZ-RhoGEF knockdown Mouse 6, trial 1 of object-in-place task; AAV-Arhgef11 shRNA-GFP

PRG KD6 trial 2: PDZ-RhoGEF knockdown Mouse 6, trial 2 of object-in-place task; AAV-Arhgef11 shRNA-GFP

File: HSV-PDZRhoGEF_forced_swim.zip

Description: HSV-PDZ-RhoGEF-GFP or HSV-GFP was infused into the medial prefrontal cortex of young adult mice. Five days later, mice were tested in the forced swim task. Upon opening the zip file, there is a folder named 'HSV-PDZRhoGEF forced swim.' When this folder is opened there are AVI files of the forced swim task for each individual mouse. Abbreviations on the AVI file names GFP (mouse infused with HSV-GFP; control group), PRG (mouse infused with HSV-PDZ-RhoGEF-GFP). Some of the videos show the behavior for two mice at once and the file indicates which mouse in on which side of the video.

GFP1 on left GFP2 on right: HSV-GFP infused mouse 1 (GFP1) is on the left-hand side of the video and HSV-GFP infused mouse 2 (GFP2) is on the right-hand side of the video.

GFP8 on right PRG7 on left: HSV-GFP infused mouse 8 (GFP8) is on the right-hand side of the video and HSV-PDZ-RhoGEF-GFP infused mouse 7 (PRG7) is on the left-hand side of the video.

GFP 10 on right PRG9 on left: HSV-GFP infused mouse 10 (GFP10) is on the right-hand side of the video and HSV-PDZ-RhoGEF-GFP infused mouse 9 (PRG9) is on the left-hand side of the video.

GFP 11 on right PRG10 on left: HSV-GFP infused mouse 11 (GFP11) is on the right-hand side of the video and HSV-PDZ-RhoGEF-GFP infused mouse 10 (PRG10) is on the left-hand side of the video.

PRG1: HSV-PDZ-RhoGEF-GFP infused mouse 1 (PRG1) in the only mouse in this video

PRG2 on left GFP3 on right: HSV-PDZ-RhoGEF-GFP infused mouse 2 (PRG2) is on the left-hand side of the video and HSV-GFP mouse 3 (GFP3) is on the right-hand side of the video.

PRG3 on left GFP4 on right: HSV-PDZ-RhoGEF-GFP infused mouse 3 (PRG3) is on the left-hand side of the video and HSV-GFP mouse 4 (GFP4) is on the right-hand side of the video.

PRG4 on left GFP5 on right: HSV-PDZ-RhoGEF-GFP infused mouse 4 (PRG4) is on the left-hand side of the video and HSV-GFP mouse 5 (GFP5) is on the right-hand side of the video.

PRG5 on left GFP6 on right: HSV-PDZ-RhoGEF-GFP infused mouse 5 (PRG5) is on the left-hand side of the video and HSV-GFP mouse 6 (GFP6) is on the right-hand side of the video.

PRG6 on left GFP7 on right: HSV-PDZ-RhoGEF-GFP infused mouse 6 (PRG6) is on the left-hand side of the video and HSV-GFP mouse 7 (GFP7) is on the right-hand side of the video.

PRG8 on left GFP9 on right: HSV-PDZ-RhoGEF-GFP infused mouse 8 (PRG8) is on the left-hand side of the video and HSV-GFP mouse 9 (GFP9) is on the right-hand side of the video.

File: HSV-PDZRhoGEF_Social_Approach.zip

Description: HSV-PDZ-RhoGEF-GFP or HSV-GFP was infused into the medial prefrontal cortex of young adult mice. Five days later, mice were tested in the three chamber social approach task. Each end chamber of the apparatus contains a mesh cylinder. One mesh cylinder contains and age and sex matched stimulus mouse, whereas the other mesh cylinder is empty -- in the file descriptions below, which cylinder contains the stimulus mouse is indicated. Upon opening the zip file, there is a folder named 'HSV-PDZRhoGEF social approach.' When this folder is opened there are AVI files of the social approach task for each individual mouse. Abbreviations on the AVI file names GFP (mouse infused with HSV-GFP; control group), PRG (mouse infused with HSV-PDZ-RhoGEF-GFP). The number after GFP or PRG is the mouse number.

GFP1: HSV-GFP infused mouse 1; stimulus mouse is in the left-hand side end chamber cylinder; right-hand end chamber cylinder is empty

GFP2: HSV-GFP infused mouse 2; stimulus mouse is in the right-hand side end chamber cylinder; left-hand end chamber cylinder is empty

GFP3: HSV-GFP infused mouse 3; stimulus mouse is in the right-hand side end chamber cylinder; left-hand end chamber cylinder is empty

GFP4: HSV-GFP infused mouse 4; stimulus mouse is in the right-hand side end chamber cylinder; left-hand end chamber cylinder is empty

PRG1: HSV-PDZ-RhoGEF-GFP infused mouse 1; stimulus mouse is in the left-hand side end chamber cylinder; right-hand end chamber cylinder is empty

PRG2: HSV-PDZ-RhoGEF-GFP infused mouse 2; stimulus mouse is in the left-hand side end chamber cylinder; right-hand end chamber cylinder is empty

PRG3: HSV-PDZ-RhoGEF-GFP infused mouse 3; stimulus mouse is in the right-hand side end chamber cylinder; left-hand end chamber cylinder is empty

File: HSV-PDZRhoGEF_object_in_place.zip

Description: HSV-PDZ-RhoGEF-GFP or HSV-GFP infused into the medial prefrontal cortex of young adult male mice. Five days post-infusion, mice were test in an object-in-place task. For trial 1, mice are allowed to explore four unique non-identical objects. Mice are then returned to their home cage, then allowed to explore these same objects; however, the location of two of the objects are swapped between trials and the other two objects remain in a fixed location between trials. When opening this zip file, there is a folder named 'HSV-PDZRhoGEF object in place.' Inside this folder there are AVI video files for trial 1 and trial 2 for the object in place task for each mouse. Abbreviations in the file names: GFP (HSV-GFP infused mouse); PRG (HSV-PDZ-RhoGEF-GFP infused mouse). The number after GFP or PRG is the mouse number, and each file also indicates if it is trial 1 or trial 2

GFP1 trial 1: HSV-GFP infused mouse 1; trial 1 of object in place task

GFP1 trial 2: HSV-GFP infused mouse 1; trial 2 of object in place task

GFP2 trial 1: HSV-GFP infused mouse 2; trial 1 of object in place task

GFP2 trial 2: HSV-GFP infused mouse 2; trial 2 of object in place task

GFP3 trial 1: HSV-GFP infused mouse 3; trial 1 of object in place task

GFP3 trial 2: HSV-GFP infused mouse 3; trial 2 of object in place task

GFP4 trial 1: HSV-GFP infused mouse 4; trial 1 of object in place task

GFP4 trial 2: HSV-GFP infused mouse 4; trial 2 of object in place task

GFP5 trial 1: HSV-GFP infused mouse 5; trial 1 of object in place task

GFP5 trial 2: HSV-GFP infused mouse 5; trial 2 of object in place task

GFP6 trial 1: HSV-GFP infused mouse 6; trial 1 of object in place task

GFP6 trial 2: HSV-GFP infused mouse 6; trial 2 of object in place task

GFP7 trial 1: HSV-GFP infused mouse 7; trial 1 of object in place task

GFP7 trial 2: HSV-GFP infused mouse 7; trial 2 of object in place task

GFP8 trial 1: HSV-GFP infused mouse 8; trial 1 of object in place task

GFP8 trial 2: HSV-GFP infused mouse 8; trial 2 of object in place task

GFP9 trial 1: HSV-GFP infused mouse 9; trial 1 of object in place task

GFP9 trial 2: HSV-GFP infused mouse 9; trial 2 of object in place task

GFP10 trial 1: HSV-GFP infused mouse 10; trial 1 of object in place task

GFP10 trial 2: HSV-GFP infused mouse 10; trial 2 of object in place task

GFP11 trial 1: HSV-GFP infused mouse 11; trial 1 of object in place task

GFP11 trial 2: HSV-GFP infused mouse 11; trial 2 of object in place task

GFP12 trial 1: HSV-GFP infused mouse 12; trial 1 of object in place task

GFP12 trial 2: HSV-GFP infused mouse 12; trial 2 of object in place task

PRG1 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 1; trial 1 of object in place task

PRG1 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 1; trial 2 of object in place task

PRG2 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 2; trial 1 of object in place task

PRG2 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 2; trial 2 of object in place task

PRG3 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 3; trial 1 of object in place task

PRG3 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 3; trial 2 of object in place task

PRG4 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 4; trial 1 of object in place task

PRG4 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 4; trial 2 of object in place task

PRG5 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 5; trial 1 of object in place task

PRG5 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 5; trial 2 of object in place task

PRG6 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 6; trial 1 of object in place task

PRG6 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 6; trial 2 of object in place task

PRG7 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 7; trial 1 of object in place task

PRG7 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 7; trial 2 of object in place task

PRG8 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 8; trial 1 of object in place task

PRG8 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 8; trial 2 of object in place task

PRG9 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 9; trial 1 of object in place task

PRG9 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 9; trial 2 of object in place task

PRG10 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 10; trial 1 of object in place task

PRG10 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 10; trial 2 of object in place task

PRG11 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 11; trial 1 of object in place task

PRG11 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 11; trial 2 of object in place task

PRG12 trial 1: HSV-PDZ-RhoGEF-GFP infused mouse 12; trial 1 of object in place task

PRG12 trial 2: HSV-PDZ-RhoGEF-GFP infused mouse 12; trial 2 of object in place task

File: HSV-PDZRhoGEF_Y-maze.zip

Description: Mice were pre-tested in the Y-maze five days prior to viral infusion. Mice were allowed free exploration of the three arms of the Y-maze. Five days following the pre-test, mice were infused with either HSV-PDZ-RhoGEF-GFP or HSV-GFP into the medial prefrontal cortex. Five days post-surgery, mice were tested again in the Y-maze (post-test). Upon opening the zip file there is a folder 'HSV-PDZRhoGEF Y-maze.' Inside this folder are individual AVI files for the pretest and posttest for each mouse. GFP (HSV-GFP infused mouse), PRG (HSV-PDZ-RhoGEF-GFP infused mouse). The number after GFP or PRG is the mouse number and each file indicates whether it is the pretest or posttest.

GFP1 pretest: HSV-GFP infused mouse 1; Y-maze performed five days prior to viral infusion

GFP2 pretest: HSV-GFP infused mouse 2; Y-maze performed five days prior to viral infusion

GFP3 pretest: HSV-GFP infused mouse 3; Y-maze performed five days prior to viral infusion

GFP4 pretest: HSV-GFP infused mouse 4; Y-maze performed five days prior to viral infusion

GFP5 pretest: HSV-GFP infused mouse 5; Y-maze performed five days prior to viral infusion

GFP6 pretest: HSV-GFP infused mouse 6; Y-maze performed five days prior to viral infusion

PRG1 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 1; Y-maze performed five days prior to viral infusion

PRG2 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 2; Y-maze performed five days prior to viral infusion

PRG3 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 3; Y-maze performed five days prior to viral infusion

PRG4 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 4; Y-maze performed five days prior to viral infusion

PRG5 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 5; Y-maze performed five days prior to viral infusion

PRG6 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 6; Y-maze performed five days prior to viral infusion

PRG7 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 7; Y-maze performed five days prior to viral infusion

PRG8 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 8; Y-maze performed five days prior to viral infusion

PRG9 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 9; Y-maze performed five days prior to viral infusion

PRG10 pretest: HSV-PDZ-RhoGEF-GFP infused mouse 10; Y-maze performed five days prior to viral infusion

GFP1 posttest: HSV-GFP infused mouse 1; Y-maze performed five days after viral infusion

GFP2 posttest HSV-GFP infused mouse 2; Y-maze performed five days after viral infusion

GFP3 posttest: HSV-GFP infused mouse 3; Y-maze performed five days after viral infusion

GFP4 posttest: HSV-GFP infused mouse 4; Y-maze performed five days after viral infusion

GFP5 posttest: HSV-GFP infused mouse 5; Y-maze performed five days after viral infusion

GFP6 posttest: HSV-GFP infused mouse 6; Y-maze performed five days after viral infusion

PRG1 posttest: HSV-PDZ-RhoGEF-GFP infused mouse 1; Y-maze performed five days after viral infusion

PRG2 posttest: HSV-PDZ-RhoGEF-GFP infused mouse 2; Y-maze performed five days after viral infusion

PRG3 posttest: HSV-PDZ-RhoGEF-GFP infused mouse 3; Y-maze performed five days after viral infusion

PRG4 posttest: HSV-PDZ-RhoGEF-GFP infused mouse 4; Y-maze performed five days after viral infusion

PRG5 posttest: HSV-PDZ-RhoGEF-GFP infused mouse 5; Y-maze performed five days after viral infusion

PRG6 posttest HSV-PDZ-RhoGEF-GFP infused mouse 6; Y-maze performed five days after viral infusion

PRG7 posttest: HSV-PDZ-RhoGEF-GFP infused mouse 7; Y-maze performed five days after viral infusion

PRG8 posttest: HSV-PDZ-RhoGEF-GFP infused mouse 8; Y-maze performed five days after viral infusion

PRG9 posttest HSV-PDZ-RhoGEF-GFP infused mouse 9; Y-maze performed five days after viral infusion

PRG10 posttest: HSV-PDZ-RhoGEF-GFP infused mouse 10; Y-maze performed five after after viral infusion

File: SIM_images_Primary_Neuronal_Cultures__endogenous_PDZ-RhoGEF_555__GFP-488.zip

Description: GFP plasmid was overexpressed in primary cortical cultures. After fixation, immunocytochemistry was performed to label endogenous PDZ-RhoGEF (PRG) in 555nm and enhance GFP expression (488nm). Structured illumination microscopy (SIM) was used to capture z-stack images of dendrite segments.

**Sub Folders: TIF Image Files. **Folders are labeled by cell number and contain .tif images of each z-stack slice. C1 is the 555 channel and C0 is the 488 channel. Z### indicates the z-stack image number. Sub-folders include:

TIF Images, cell 1, endogenous PRG 555, GFP 488

TIF Images, cell 2, endogenous PRG 555, GFP 488

TIF Images, cell 3, endogenous PRG 555, GFP 488

TIF Images, cell 4, endogenous PRG 555, GFP 488

TIF Images, cell 5, endogenous PRG 555, GFP 488

**Sub Folder: .ND2 Images. **Folders are labeled by cell number and contain .ND2 files (LIM image type). These can be opened in ImageJ. Files in this folder includes:

cell 1, endogenous PRG 555, GFP 488.nd2

cell 2, endogenous PRG 555, GFP 488.nd2

cell 3, endogenous PRG 555, GFP 488.nd2

cell 4, endogenous PRG 555, GFP 488.nd2

cell 5, endogenous PRG 555, GFP 488.nd2

**Sub Folder: Quantification. **Z-stack images were segmented and measured using Imaris and the data was exported to Excel. This folder contains the following Excel documents:

**PDZ-RhoGEF nanodomain concentration.xlsx : **Data from structured illumination microscopy (SIM) images. Image files included in analysis: cell 1, endogenous PRG 555, GFP 488; cell 2, endogenous PRG 555, GFP 488, cell 3, endogenous PRG 555, GFP 488; cell 4, endogenous PRG 555, GFP 488.

Sheet 1- Spine volume and nanodomain #: Each dendritic spine that was analyzed is listed as a row. The cell number (corresponding to the image file) is listed in column 1. The unique spine number/ ID is listed in column 2. The spine type classification is listed in column 3. The spine volume (µm3) measured using Imaris is listed in column 4. The number of PDZ-RhoGEF nanodomains in each spine is listed in column 5.

Sheet 2: Compartment vol. & nanodomain #: Each thin and mushroom spine compartment (head or neck) is listed as a row. Column 1 indicates the cell number (corresponding to the image files). Column 2 indicates the spine number/ ID (There is one head and one neck row for each thin and mushroom type dendritic spine). The spine type classification is listed in column 3. Each compartment (either head or neck region of each spine) is denoted in column 4. The corresponding volume measurement of that compartment (in µm3 ) is listed in column 5. The number of PDZ-RhoGEF nanodomains in each compartment is listed in column 6).

**Influence of spine head volume on proportion of spine head occupied by PDZ-RhoGEF (PRG): **Data froms structured illumination microscopy (SIM) images. Image files included in analysis: cell 1, endogenous PRG 555, GFP 488; cell 2, endogenous PRG 555, GFP 488, cell 3, endogenous PRG 555, GFP 488; cell 4, endogenous PRG 555, GFP 488; cell 5, endogenous PRG 555, GFP 488. Each thin and mushroom spine (column 1: spine classification/ type) was divided into neck and head compartments. The volume of the neck and head compartments (µm3) are listed in columns 2 and 3, respectively. Columns 4 and 5 list the proportion/ percentage of the neck or head compartment that is occupied/covered by PDZ-RhoGEF (PRG), respectively.

File: in_vivo_dendritic_spine_analysis_HSV-PDZ-RhoGEF_or_HSV-GFP.zip

Description: The mPFC of mice was infused with either HSV-PDZ-RhoGEF-GFP or HSV-GFP. 4 days later mice were transcardially perfused. mPFC pyramidal neuron apical tree dendrite segments from HSV-GFP and HSV-PDZ-RhoGEF-GFP groups were imaged using a keyonce.

**Sub-Folders: TIF Image files. **Z-stack images were collapsed and deconvolved using BZ analyzer software. Image files in the folder are named by condition, mouse number and cell number.

Quantification: Dendritic spine data from HSV-GFP and HSV-PDZ-RhoGEF-GFP.xlsx:

Sheet 1- Dendritic Spine Density: The dendritic spines on dendrite segments of multiple cells were analyzed for each mouse using the NeuronStudio program. Column 1 indicates the condition, mouse number, and cell number analyzed. GFP indicated mice infused with HSV-GFP and PDZ-RhoGEF indicated mice infused with HSV-PDZ-RhoGEF-GFP. The total number of mushroom, thin, and stubby type spines on the dendrite section is indicated in columns 2,3, and 4, respectively followed by the total number of spines (column 5). The length of the dendrite segment (µm2) analyzed is indicated in column 6. Column 7,8,9, and 10 depict mushroom, thin, stubby, and total spine density, respectively.

Sheet 2-Diameter individual spine heads: The spine head diameter (µm2) of each dendritic spine analyzed is noted by row. Column 1 indicated the condition, mouse number and cell number of the spine. GFP indicated mice infused with HSV-GFP and PDZ-RhoGEF indicated mice infused with HSV-PDZ-RhoGEF-GFP. Column 2 indicates the spine type (thin, mushroom, or stubby). Column 3 indicates the spine head diameter (µm2).

File: SIM_Images_Primary_Neuronal_Cultures__exogenous_DISC1-647__endogenous_PDZ-RhoGEF555__GFP-488.zip

Description: GFP and HA tagged DISC1 plasmids were overexpressed in primary cortical cultures. After fixation, immunocytochemistry was performed to label endogenous PDZ-RhoGEF (PRG) in 555nm, the HA tag on DISC1 in 647, and enhance GFP expression (488nm). Structured illumination microscopy (SIM) was used to capture z-stack images of dendrite segments.

**Sub-Folders: TIF Image Files. **Folders are labeled by cell number and contain .tif images of each z-stack slice. C1 is the 555 channel, C2 is the 488 channel, C0 is the 647 channel. Z### indicates the z-stack image number. Sub-folders include:

TIF Images, cell 1, endogenous PRG 555, exogenous DISC1 647, GFP 488

TIF Images, cell 2, endogenous PRG 555, exogenous DISC1 647, GFP 488

TIF Images, cell 3, endogenous PRG 555, exogenous DISC1 647, GFP 488

TIF Images, cell 4, endogenous PRG 555, exogenous DISC1 647, GFP 488

TIF Images, cell 5, endogenous PRG 555, exogenous DISC1 647, GFP 488

**Sub Folder: .ND2 Images. **Folders are labeled by cell number and contain .ND2 files (LIM image type). These can be opened in ImageJ. Files in this folder include:

cell 1, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2

cell 2, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2

cell 3, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2

cell 4, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2

cell 5, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2

**Sub Folder: Quantification. **Z-stack images were segmented and measured using Imaris and the data was exported to Excel. This folder contains the following Excel documents:

**Influence of DISC1 on dendritic spine volume.xlsx: **Data from structured illumination microscopy (SIM) images. Image files included in analysis: cell 1, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2, cell 2, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2, cell 3, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2, cell 4, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2, cell 5, endogenous PRG (555), exogenous DISC1 (647), GFP (488).nd2.

Sheet 1- Spine volume and nanodomain #: Each dendritic spine that was analyzed is listed as a row. The cell number (corresponding to the image file) is listed in column 1. The unique spine number/ ID is listed in column 2. The spine type classification is listed in column 3. The spine volume (µm3) measured using Imaris is listed in column 4. The number of PDZ-RhoGEF nanodomains in each spine is listed in column 5. The number of DISC1 nanodomains in each spine is listed in column 6. The number of PDZ-RhoGEF nanodomains that spatially overlap with DISC1 in each spine is listed in column 7.

Sheet 2: Compartment vol. & nanodomain #: Each thin and mushroom spine compartment (head or neck) is listed as a row. Column 1 indicates the cell number (corresponding to the image files). Column 2 indicates the spine number/ ID (There is one head and one neck row for each thin and mushroom type dendritic spine). The spine type classification is listed in column 3. Each compartment (either head or neck region of each spine) is denoted in column 4. The corresponding volume measurement of that compartment (in µm3 ) is listed in column 5. The number of PDZ-RhoGEF nanodomains in each compartment is listed in column 6). The number of DISC1 nanodomains in each spine is listed in column 7. The number of PDZ-RhoGEF nanodomains that spatially overlap with DISC1 in each spine is listed in column 8.

Code/software

The video files are AVI files, which can be opened with any video software. Video files will open with Windows Media Player or QuickTime video. Tif files will open with most imaging programs. ND2 images can be opened with ImageJ.