DOI: 10.5061/dryad.0k6djhb9p
title: Microscopy images of the effects of PDZ-RhoGEF manipulation on dendritic spines and videos of effects on PDZ-RhoGEF on mouse behavioral phenotypes

description: PDZ-RhoGEF is a RhoA-specific guanine nucleotide exchange factor (GEF) for
 the RhoA small GTPase. The purpose of these experiments is to determine
 the effects of PDZ-RhoGEF overexpression and knockdown on mouse behavioral
 phenotypes. Relevant behaviors including assessments of spatial working
 memory, recognition memory, social behavior, and stress coping. Herpes
 simplex viral vectors (HSV) were used to overexpress PDZ-RhoGEF. Knockdown
 of PDZ-RhoGEF was accomplished via packaging of short hairpin RNA (shRNA)
 into adeno-associated viral vectors (AAV) against Arhgef11 (the gene that
 encodes the PDZ-RhoGEF protein). Viral vectors were surgically infused
 directly into the medial prefrontal cortex (mPFC) of young adult male
 mice. Using the same HSV approach, the effects of PDZ-RhoGEF
 overexpression on the density and morphology of dendritic spines was also
 analyzed. Finally, using structured illumination microscopy (SIM), the
 spatial location of PDZ-RhoGEF and that of one of its direct interacting
 proteins, DISC1, was examined within individual dendritic spines.
description: Object in place behavior videos: Mice were infused with
 HSV-PDZ-RhoGEF-green fluorescence protein (HSV-PRG-GFP) or HSV-GFP into
 the medial prefrontal cortex of mice. 5 days post-infusion, mice were
 assessed in an object-in-place recognition task. During trial 1, mice were
 allowed to freely explore 4 unique, non-identical objects in an open
 arena. Mice were then returned to their home cages for 7 minutes and then
 placed back into the open field arena (trial 2) with the same objects as
 before, however, the location of two of the objects were swapped in
 location. This results in two objects whose location is fixed between
 trials and two objects whose location is altered between trials. The
 amount of time mice spent directly investigating the swapped and
 non-swapped objects was done manually using a scientific stopwatch with
 0.01 second accuracy. For the knockdown of PDZ-RhoGEF, these same
 procedures were followed except mice were infused with AAV-Arhgef11
 shRNA-GFP or AAV-scrambled control shRNA-GFP into the medial prefrontal
 cortex and assessed four weeks post-infusion. The videos of this behavior
 have been uploaded as part of this data set. The raw data obtained by
 scoring the behavior from these videos was manually logged into
 spreadsheets that are included as supplementary data in the source
 publication. Y-maze spontaneous alternation behavior
 videos: 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). The arm
 entries of each mouse were manually scored and the spontaneous alternation
 percentage calculated as the number of arm triplet entries divided by the
 number of total possible arm triplet entries. The videos of this behavior
 have been uploaded as part of this data set. The raw data obtained by
 scoring the behavior from these videos was manually logged into
 spreadsheets that are included as supplementary data in the source
 publication. Social approach behavior videos: Mice were
 infused with HSV-PDZ-RhoGEF-GFP or HSV-GFP into the medial prefrontal
 cortex. Five days post-infusion, mice were placed in the center of a three
 chamber social apparatus. Each end chamber of the apparatus contained a
 mesh cylinder. The cylinder in one end chamber was empty and that of the
 other end chamber contained an aged and sex matched stimulus mouse. The
 amount of time mice spent directly investigating each cylinder was
 determined manually using a scientific stopwatch. The videos of this
 behavior have been uploaded as part of this data set. The raw data
 obtained by scoring the behavior from these videos was manually logged
 into spreadsheets that are included as supplementary data in the source
 publication. Forced swim test behavior videos: Mice
 were infused with HSV-PDZ-RhoGEF-GFP or HSV-GFP into the medial prefrontal
 cortex. Five days post-infusion, mice were individually placed into a
 beaker of room temperature water. The amount of time mice spent actively
 swimming while in contact with the water was manually scored using a
 scientific stopwatch. The videos of this behavior have been uploaded as
 part of this data set. The raw data obtained by scoring the behavior from
 these videos was manually logged into spreadsheets that are included as
 supplementary data in the source publication. The
 effects of PDZ-RhoGEF on dendritic spine density and morphology: Mice were
 infused with HSV-PDZ-RhoGEF-GFP or HSV-GFP into the medial prefrontal
 cortex. Five days post-infusion, mice were transcardially perfused and the
 GFP signal from the viral label enhanced using immunohistochemistry.
 Brains were coronally sectioned at 100um. Neurons were imaged using a
 Keyence BZ-X700E scanning microscope. Within neurons, we imaged secondary
 dendrites from the apical tree. Imaging was done using at 100x using a 0.1
 µm step size for Z-stack collection. Images for analyses, which are
 included in this dataset, were collapsed and deconvolved using BZ analyzer
 software. Dendritic spine analysis, which is included in this dataset, was
 done using NeuronStudio software. The localization of
 endogenous PDZ-RhoGEF within dendritic spines: Day in vitro (DIV) 21-23
 (DIV 21-23) cultured neurons derived from embryonic day 18 Sprague-Dawley
 rat were transfected with GFP and fixed 2 days later (3.7% formaldehyde),
 and subsequently immunostained for GFP and endogenous PDZ-RhoGEF. Neurons
 were imaged using a Nikon Structured Illumination microscope using a 100×
 1.49 NA oil objective with a laser excitation of 488nm (for GFP), 561nm
 (for PDZ-RhoGEF). Z stack (z = 0.12μm) images of secondary dendrites on
 pyramidal neurons were processed and analyzed using Nikon Elements
 software. Imaging and reconstruction parameters were determined with the
 support of the Biochemistry Optical Core at the University of
 Wisconsin-Madison. Acquisition was set to 5MHz at 14bit with EM gain and
 no binning. Auto exposure was kept consistent between images and within
 250-300ms, the EM gain multiplier was kept below 300, and conversion gain
 was held at 1x. Reconstruction parameters (Illumination Modulation
 Contrast, High Resolution, Noise Suppression and Out of Focus Blur
 Suppression) were kept consistent across all images. Dendrites and spines
 were segmented in the channel of the cell fill (GFP-488nm) using the
 Imaris surface tool and manually corrected when needed (i.e. splitting two
 overlapping spines). Object-to object stats were applied, and local
 background subtraction was used. Thresholding values were kept consistent
 between images. Spine segmentation was done by manually separating each
 spine from the dendrite surface using the cut surface tool. This
 segmentation was duplicated, and the head and neck were manually split in
 the duplicated segmentation using the same method. Spine classification
 was done using spine morphology, namely head volume and neck length.
 Spines with no discernable neck region were assigned as stubby spines,
 spines with a head volume of 0.4µm<sup>3</sup> or larger as
 mushroom spines, and those with a head volume below this value classified
 as thin. Spatial overlap between endogenous PDZ-RhoGEF
 and overexpressed DISC1: The same procedures were followed as in the above
 section described for PDZ-RhoGEF. The only difference is that cultured
 neurons were transfected not only with GFP, but also with DISC1.
 Immunohistochemisty was then used to label the GFP, endogenous PDZ-RhoGEF,
 and overexpressed DISC1. The same imaging procedures were used as in the
 above sections for endogenous PDZ-RhoGEF, with laser excitations of 488nm
 (for GFP), 561nm (for PDZ-RhoGEF), and 640nm (for DISC1).
description: # Microscopy images of the effects of PDZ-RhoGEF manipulation on dendritic
 spines and videos of effects on PDZ-RhoGEF on mouse behavioral phenotypes
 [https://doi.org/10.5061/dryad.0k6djhb9p](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.