DOI: 10.5061/dryad.gmsbcc2ws
title: Abnormal cytoskeletal remodeling but normal neuronal excitability in a mouse model of the recurrent developmental and epileptic encephalopathy-susceptibility KCNB1-p.R312H variant

description: Integrin_K+ Channel_Complexes (IKCs), are implicated in neurodevelopment
 and cause developmental and epileptic encephalopathy (DEE) through
 mechanisms that were poorly understood. Here, we investigate the function
 of neocortical IKCs formed by voltage-gated potassium (Kv) channels KCNB1
 and a5b5 integrin dimers in wild type (WT) and homozygous knockin (KI)
 Kcnb1R312H(+/+) mouse model  of DEE. Kcnb1R312H(+/+) mice suffer
 from severe cognitive deficit and compulsive behavior. Their brains show
 neuronal damage in multiple areas and disrupted cortico-cortical and
 cortico-thalamic connectivity along with aberrant glutamatergic vesicular
 transport. Surprisingly, the electrical properties of Kcnb1R312H(+/+)
 pyramidal neurons are similar to those of WT neurons, indicating that the
 arginine to histidine replacement does not affect the conducting
 properties of the mutant channel. In contrast, fluorescence recovery after
 photobleaching, biochemistry and immunofluorescence, reveal marked
 differences in the way WT and Kcnb1R312H(+/+) neurons modulate the
 remodeling of the actin cytoskeleton, a key player in the processes
 underlying neurodevelopment. Together these results demonstrate that Kv
 channels can cause multiple conditions, including epileptic seizures,
 through mechanisms that do not involve their conducting functions and put
 forward the idea that the etiology of DEE may be primarily non-ionic.
description: Original data used in the study entitled: "Abnormal cytoskeletal
 remodeling but normal neuronal excitability in a mouse model of the
 recurrent developmental and epileptic encephalopathy-susceptibility
 KCNB1-p.R312H variant" by Alessandro Bortolami, Elena
 Forzisi-Kathera-Ibarra, Anastasia Balatsky, Mansi Dubey, Rusheel Amin,
 Srinidi Venkateswaran, Stefania Dutto, Ishan Seth, Adam Ashor, Angel
 Nwandiko, Ping-Yue Pan, David P. Crockett, Federico Sesti.  Data are
 organized in folders named after each figure of the related paper. For
 each figure of the paper, the corresponding original data are in folders
 named after the figure's experimental procedures. For example the
 folder FIGURE2.zip contains the zipped sub-folders Dead reckoning WT and
 R312H, Flipping grid, Marble burying WT and R312H, Nest WT and R312H,
 Startle reflex and looming WT and R312H, Visual Cliff WT and R312H, along
 with the statistical analysis saved in the prism file Behavior WT and
 R312H. Folders can contain sub-folders, sub-sub-folders, etc.
 corresponding to a specific experimental condition. Please note: 1.       
 The genotype "Kcnb1R312H" is often called "R312H" or
 "R". 2.        The genotype Wild type is often called
 "WT" 3.        Some data may contain the genotype “F614L” which
 was not used in the related paper. A) Western blots (WBs) are organized in
 the PDF file uncropped_WBs. Each slide is named with the  primary antibody
 (i.e. VGluT1, VGluT2, Actin, etc.). The genotype is indicated in each lane
 of the blot. For example, a blot in the slide named VGluT1, may show an
 experiment with genotypes such as WT, R312H and NULL. VGluT1 was the
 antibody used in the procedure. Some may contain samples from the genotype
 called F416L which was not used in the related paper B) Images are placed
 into folders named after the antibody used. subfolders are named following
 the genotype of the animal analyzed. some folders may contain "20x,
 100x" subfolders. These folders specify the magnification of the
 objective used to acquire the image. Images are labeled as follows
 4[1]-[X]-DAPI.jpg = Image 4, DAPI visualization\ 4[0]-[X]-GFP.jpg = Image
 4, green channel\ 4[2]-[X]-CY3.jpg = Image 4, far-red channel\
 4[3]-[X]-CY5.jpg = Image 4, red channel\ 4.jpg = Composite Therefore, the
 composite image is indicated with the number alone, thus 4.jpg in the
 example. A different set of images would therefore be named as:
 5[1]-[X]-DAPI.jpg\ 5[0]-[X]-GFP.jpg\ 5[2]-[X]-CY3.jpg\ 5[3]-[X]-CY5.jpg\
 5.jpg + Composite\ etc. **Usage notes for files formats:** Confocal images
 in Nikon .ND2 format or Olympus .iob or Leica .lif format  can be imported
 in ImageJ/Fiji or read with the freely available software NIS Elements.
 Confocal images in .XCF format can be imported in Image J or read with the
 Olympus Fluoview software Images are typically in JPEG format but some can
 be in other readable formats such as, PNG and TIFF. Files in HEIF format
 (Apple high efficiency image file) can be read using Apple OS or ImageJ.
 Data files are in Excel format or .cvs (comma separated values) format or
 more commonly in Prism format. These data can be read using Microsoft
 office or numbers or Prism software. Electrophysiology files are in .abf
 (Axon binary file) format and can be read with Axon pClamp software. Usage
 notes are also indicated in the various figure folders when opportune. 
 **Figure folders:** **FIGURE 1** was generated from DVC tracking that is
 stored in the DVC metadata repository of Tecniplast. The folder contains
 original data of tracking distances in the cage and tracking distances in
 the running wheel (Figures 1C, D). The other graphs in the Figure 1A and 
 B of the related paper were generated and directly downloaded from the
 Tecniplast site/software. The folder contains the sub-folders: “Female
 Tracking distance WT and R312H”,  “Male Tracking distance WT and R312H”,
 “Running wheel tracking distance R312H” and “Running wheel tracking
 distance WT”. Each sub-folder contains a file with the measurements of
 each animal along with identification number, sex and genotype. The files
 are in .cvs (comma separated values) format that can be read by many
 software including Excel, Numbers etc. The data of the cvs files are
 summarized in the Excel file “DVC analysis” that contains also the
 calculated cumulative distances. The Excel file includes empty cells not
 meant for analysis. The latter data are in the Prism file “DVC Analysis WT
 vs R312H” that was used to generate the figures and compute statistics.
 **FIGURE 2** was generated from video recordings, pictures, and analysis
 of animal behavior. Data are organized into subfolders named after the
 behavioral task. Each subfolder contains sub-subfolders named after the
 genotype of the mouse analyzed for the specific task (WT or R312H). If a
 subfolder is not present, the genotype is evidenced in the title of the
 image/video. Images and videos were analyzed by multiple observers with
 the methodologies explained in the method section of the related paper.
 The dead reckoning task was analyzed using Ethovision. A circular shape
 was used to evidence the arena and to calculate the tracking of the mouse.
 The "house" was evidenced by a smaller circle to quantify the
 number of house comebacks. The data used for Figure 2 are stored in the
 folders: Dead reckoning WT and R312H, Flipping grid, Marble burying WT and
 R312H, Nest WT and R312H, Startle reflex and looming WT and R312H, Visual
 Cliff WT and R312H, along with the statistical analysis saved in prism
 file Behavior WT and R312H. **FIGURE 3** was constructed using raw images
 of the neuroanatomy of WT and R312H animals. Images are classified into
 folders naming the genotype and the animal replicate. For example, WT1
 means WT animal was used, and it is the replicate number 1. Images are
 then organized into subfolders named after the brain region analyzed. For
 example: Nucleus Habenular, etc. images are then named as follows:
 5[1]-[X]-DAPI.jpg\ 5[0]-[X]-GFP.jpg\ 5[2]-[X]-CY3.jpg\ in which:\
 5[1]-[X]-DAPI.jpg - Nuclei\ 5[0]-[X]-GFP.jpg - NF-M\ 5[2]-[X]-CY3.jpg -
 L1-CAM Images can be opened on Image J. For the analysis, pictures were
 converted to grayscale and the thickness/fluorescence intensity of a
 specific brain region was calculated. The value of the background was
 measured to be removed in each replicate. The data used for Figure 3 are
 stored in the folders: Analysis L1 and NFM neuroanatomical WT and R312H,
 NFM L1 WT and R312H, along with the statistical analysis saved in prism
 format. **FIGURES 4 - 5 and S1** were generated from patch clamp
 recordings. The original recordings used for Figures 4, 5 and S1 are
 stored in the folders named electrophysiology (WT and R312H) and Ephys
 Null (Null). Within these two sub-folders data are segregated in
 sub-sub-folders named after the date of the experiment and the person who
 took the recordings (ex: Ale 010224= Alessandro Bortolami, 01/02/2024).
 Electrophysiology files are in Axon .abf format and can be opened in Clamp
 Fit. Notes about each experiment are illustrated in the MOV file ephys lab
 notebook. All analyses are contained in the Prism files current clamp and
 voltage clamp. I/V curves, the Rheobase, Cell Capacitance, Input
 resistance, Resting membrane potential, AP peak height, and AP half-width
 were calculated with Clamp fit. Resting membrane potential was measured
 using both Clamp fit and the reading on the amplifier during the
 experiment. **FIGURE 6 and S3** were generated from raw images of WT and
 R312H animal brains. Images are classified into folders naming the
 genotype and the animal replicate. For example, WT1 means WT animal was
 used, and it is the replicate number 1. Images are then organized into
 subfolders named after the brain region analyzed. for example: Nucleus
 Habenular, etc. images are then named as follows: 5[1]-[X]-DAPI.jpg\
 5[0]-[X]-GFP.jpg\ 5[2]-[X]-CY5.jpg\ in which:\ 5[1]-[X]-DAPI.jpg - Nuclei\
 5[0]-[X]-GFP.jpg - VGluT1\ 5[2]-[X]-CY5.jpg - VGluT2 Heatmaps were
 generated using Image J. Briefly, VGluT1, and VGluT2 images were converted
 to 16bit. images were then colored using "Fire" which highlights
 areas of low and high expression. Analysis was done using Image J and
 measuring on grayscale images the fluorescence intensity of specific brain
 regions, using the atlas mask to define the brain region of interest. The
 brain area to analyze was evidenced using the polygon tool and the
 fluorescence intensity was obtained. The data used for Figures 6 and S3
 are stored in the folders named Analysis VGlut1 and VGlut2 brain
 localization, VGluT IHC WT and R312H along with statistical analyses in
 prism format. **FIGURE 7** was constructed  using tracing data. The data
 are organized in folders named after the animal genotype and paired with
 the unique animal ID used for the procedure. For example: WT W. means WT
 genotype and animal ID: W. The subfolder contains the Z-stack images and
 the single images. Images can be opened into Image J. Images are named in
 sequential order from rostral to caudal. for example, 1 means it is the
 first image of the series and the most rostral image acquired. 2, 3, and 4
 are progressively more caudal compared to image 1. Images can be opened in
 Image j, ordered using the function images to stack. once the stack has
 been created, the z-stack can be obtained using the Z-project plug-in. For
 the quantification, after creating Z-stack images, we converted the images
 into grayscale and we analyzed the fluorescence intensity of specific
 brain areas. The value of the background was measured to be removed in
 each replicate. High-magnification images are included in each
 genotype-specific folder. The subfolders "WT series" and
 "R312H series" contain the heat maps created on the atlas
 evidencing the areas of connectivity". To create these heatmaps,
 singular brain slices were analyzed and if connectivity was present, the
 result was reported onto the atlas. Colors were assigned following the
 scheme: 1/3=0-33% red\ 2/3=34-66% yellow\ 3/3=67-100% green These files
 can be opened in Image J. The data used for Figure 7 are stored in folders
 Analysis BDA CX Th 488, BDA MW3000 488, MAP R312H BDA and MAP WT BDA,
 along with the statistical analyses in prism format. **FIGURE 8 and 9**
 were constructed using raw FRAP data. The data are organized into folders
 indicating the date of the experiment and the genotype used on that
 specific day. Data are in oib (Olympus image files) format that can be
 opened with Fiji. Within each folder, data are organized in subfolders
 indicating the genotype "WT" and "R312H". Data are
 further organized in sub-subfolders indicating the treatment used. For
 example, CytoD, Jasp, K+, etc.. Once opened, files are prepared as series,
 and each frame (total of 20) is 0.5 seconds of exposure. the 4th frame is
 the photobleaching. Images were analyzed evidencing the area of the bleach
 with a square and the fluorescent intensity was acquired. Results were
 normalized to the initial fluorescent intensity of each replicate. To
 obtain the initial florescence we plotted the raw data of the first frame
 of each replicate. To measure the Koff, acquired fluorescent data over
 time were plotted and fitted using Prism. Fit R results were used to
 obtain Koff results. The initial fluorescence drop was calculated on the
 fluorescence immediately after the photobleaching (4th frame). The mobile
 fraction was calculated based on the following formula: mobile=b-a in
 which b is the final recovery (Frame 20) and a is the initial bleach
 (Frame 4). The data used to construct Figures 8 and 9 are stored in the
 folders FRAP analysis, WT FRAP and R312H FRAP along with the statistical
 analyses in prism format. **FIGURES 10 and S10** were constructed using
 images of primary neuron cultures (PNC). The folders are organized with
 the date of the experiment and the genotype of the E13-derived embryo
 analyzed. Each folder contains the replicates in the folder named
 "WT1" etc. which means WT animal was used, and it is the
 replicate number 1. Within each folder, a sub-folder indicating the
 magnification can be found "20x, 100x". this indicates the
 magnification used to acquire the image. images are named as follows, and
 each color indicates: 5[1]-[X]-DAPI.jpg - Nuclei\ 5[0]-[X]-GFP.jpg -
 Actin\ 5[2]-[X]-CY5.jpg - MAP2 For the Sholl analysis of a single neuron,
 data were converted into 16-bit images using Image J. The image threshold
 was adjusted using the threshold function. Neurons were highlighted in
 black while the background resulted in white. after the neuron of interest
 was selected (pyramidal neuron), the other neurons were removed with the
 brush tool. Once only one neuron was present in the image, the pointing
 tool was used to select the soma of the neuron. After selecting the soma,
 the neuroanatomy Sholl plugin was used. Using Prism, the radius was
 plotted on the X axis and the number of interactions on the Y axis. For
 the Sholl area, data were converted into 16-bit images using Image J. The
 image threshold was adjusted using the threshold function. Neurons were
 highlighted in black while the background resulted in white. After the
 neuron of interest was selected (pyramidal neuron), the pointing tool was
 used to set the center of the Sholl radius on the soma of the neuron.
 After selecting the center, the neuroanatomy Sholl plugin was used. Using
 Prism, the radius was plotted on the X axis and the number of interactions
 on the Y axis. The maximum dendrite length was calculated using the
 segmented line tool. The longest dendrite of a single neuron was used for
 the analysis. The data used to construct Figures 10 and S10 are stored in
 the folders Analysis Sholl's Final Actin, Analysis Sholls area and
 PNC actin toxins, along with the prism files containing the statistical
 analyses. **FIGURE S2** was constructed using Western blots of WT or R312H
 brain lysates stained for VGluT1, VGlut2, SNAP25, and GAD65 antibodies.
 The files are contained in the sub-folder "VGluT1 WB" and are
 named with the date of experiment, the primary antibody (i.e. VGluT1,
 VGlut2, etc.), and the genotype of each lane of the blot moving from left
 to right. Additional conditions may be indicated such for example the date
 of the experiment. Raw images of the blots were imported in Image J. The
 rectangular blot area of a single replicate was evidenced with a rectangle
 and the intensity was calculated using the intensity measuring tool. The
 rectangle was maintained constant for all the replicates. For each WB an
 actin control is present on the same membrane. Actin fluorescence
 intensity was calculated and used to normalize the results.\ The data used
 to construct Figure S2 are stored in the folders Analysis GAD65 VGluT1
 VGlut2 SNAP25 and Analysis VGluT Cortex only, along with statistical
 analyses in prism format. **FIGURE S4** was constructed using tracing
 data. The data are organized in folders named after the animal genotype
 and paired with the unique animal ID used for the procedure. for example:
 WT W. means WT genotype and animal ID: W. The subfolder contains the
 Z-stack images and the single images. images can be opened into Image J.
 Images are named in sequential order from rostral to caudal. for example:
 1 means it is the first image of the series and the most rostral image
 acquired. 2, 3, and 4 are progressively more caudal compared to image 1.
 Images can be opened into Image J. Images are named in sequential order
 from rostral to caudal. for example: 1 means it is the first image of the
 series and the most rostral image acquired. 2, 3, and 4 are progressively
 more caudal compared to image 1. Images can be opened in Image j, ordered
 using the function images to stack. once the stack has been created, the
 z-stack can be obtained using the Z-project plug-in. For the
 quantification, after creating Z-stack images, we converted the images
 into grayscale and we analyzed the fluorescence intensity of specific
 brain areas. The data used to construct Figure S4 are stored in the
 folders  Analysis Fluorogold and Fluorogold along with statistical
 analyses in prism format. **FIGURE S5** was constructed using tracing
 data. The data are organized in  folders named after the animal genotype
 and paired with the unique animal ID used for the procedure. For example:
 WT W. means WT genotype and animal ID: W. images can be opened into Image
 J. Each folder contains two high-magnification images of different layers
 of the cortex. Images were opened on Image J, converted in grayscale and
 the fluorescence intensity was measured using the fluorescence intensity
 tool. The value of the background was measured to be removed in each
 replicate. The data used to construct Figure S5 are stored in the folders
 Analysis BDA 594 Tracing and BDA 594 Injection thalamus along with
 statistical analyses in prism format. **FIGURE S6** was constructed
 using raw images of the corpus callosum of WT and R312H animals. Images
 are classified into folders naming the genotype and the animal replicate.
 For example, WT1 means WT animal was used, and it is the replicate number.
 Due to the high size volume, the images are divided into two different zip
 files named Pack One and Pack 2.\ Images are then named as follows:
 5[1]-[X]-DAPI.jpg\ 5[0]-[X]-GFP.jpg\ 5[2]-[X]-CY5.jpg\ 5[3]-[X]-CY3.jpg\
 in which: 5[1]-[X]-DAPI.jpg - Nuclei\ 5[0]-[X]-GFP.jpg - NF-L\
 5[2]-[X]-CY5.jpg - HDAC-1\ 5[3]-[X]-CY3.jpg - SMI-32 Images are Z-stacks
 at 60x magnifications that can be opened on Image J. Images were processed
 in Z-project and converted in grayscale. The resulting images were
 analyzed using the fluorescence intensity. For the analysis of HDAC-1, the
 image of HDAC-1 and DAPI were opened simultaneously. The tool
 colocalization was used to calculate the overlap between the two markers.
 Following, to calculate the amount of translocated HDCA-1, the HDAC-1
 image was opened and converted to 16-bit, and the threshold tool was used
 to highlight neurons in black eliminating the areas of high-fluorescence
 (nuclei). The fluorescence intensity was then calculated using the
 fluorescence intensity tool. The data used to construct Figure S6 are
 stored in the folders Analysis HDAC1 SMI32 DAPI and HDAC1 SMI32 DAPI,
 along with statistical analyses in prism format. **FIGURE S7** was
 constructed with raw images of the neocortex of WT and R312H animals.
 Images are classified into folders naming the genotype and the animal
 replicate. For example, WT1 means WT animal was used, and it is the
 replicate number 1. Images are then named as follows: 5[1]-[X]-DAPI.jpg\
 5[0]-[X]-GFP.jpg\ 5[2]-[X]-CY5.jpg in which: 5[1]-[X]-DAPI.jpg - Nuclei\
 5[0]-[X]-GFP.jpg - Ankyrin-G\ 5[2]-[X]-CY3.jpg - Map2 Images were opened
 on Image J, converted in grayscale and the fluorescence intensity was
 measured using the fluorescence intensity tool. The value of the
 background was measured to be removed in each replicate.\ The data used to
 construct Figure S7 are stored in the folders Analysis Ankyrin G and
 Ankyrin G along with statistical analyses in prism format. **FIGURE S8**
 was constructed from Western blots of WT or R312H brain lysates stained
 for Actin G and F. The files are contained in the sub-folder "Actin
 WB" and are named with the date of the experiment, the primary
 antibody (i.e. Actin), and the genotype and the type of actin (G or F) of
 each lane of the blot moving from left to right. Each replicate Actin is
 one next to the other one, and the organization is specified in the title
 of the blot image. Additional conditions may be indicated such for example
 the date of the experiment. Raw images of the blots were imported in Image
 J. The rectangular blot area of a single replicate was evidenced with a
 rectangle and the intensity was calculated using the intensity measuring
 tool. The rectangle was kept constant for all the replicates. The value of
 the background was measured to be removed in each replicate. The amount of
 G and F actin was calculated and plotted in different graphs.\ The data
 used to construct Figure S8 are stored in the folders Analysis Actin F/G.
 **FIGURE S9** was constructed using proliferation data using CHO cells.
 The proliferation data were generated by our Infinite M+ Nano (Tecan)
 reader as Excel files and then converted into the PRISM files contained in
 the folder. Each trial is listed on the Prism \ file specifying the day of
 the experiment, the plasmid transfected, and the treatment used. Data are
 normalized from the day 0 fluorescence. Day 2 fluorescence results have
 been used to measure the proliferation rate on day 2. The figure was also
 constructed using raw images of N2a cells. The data are in the N2a actin
 folder which is organized in subfolders indicating the day of the
 experiment. Sub-subfolders indicate the plasmid transfected.
 Sub-sub-subfolders indicate the treatment used. Images were opened in
 Image J, and cells positive for GFP fluorescence were used for the
 analysis. Using the segmented line tool, the length of the longest neurite
 was measured and plotted. The data used to construct Figure S9 are stored
 in folders Cho proliferation, N2a actin and Analysis N2a actin along with
 the statistical analyses in prism format. \** ** **Keys:** \** ** **DVC
 analysis** Tracking Distance: on the Y-axis is indicated the distance run
 in meters during a day. On the X-axis is indicated the day number.
 Cumulative distance (Figure 1C): the tracking distance run starting from
 day 1. Genotype are indicated as follow: WT: C57BL6/J control group R312H:
 homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse
 Running wheel Distance (Figure 1D): the Cumulative distance run on the
 running wheel by each cohort of animals. On the X-axis is indicated the
 day number. WT: C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse **Behavior** 1.     Nesting:
 nestlet building score was attributed according to Deacon, R. M. Measuring
 motor coordination in mice. In summary, the Y-axis score was attributed
 from 1 (No nest) to 5, where mice could construct a perfect nest. WT:
 C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse \ 2.     Material Torn:
 nestlet were individually weighted before the start of the trial.
 Following the given time, nestlet was let to dry, and weighed once again.
 The percentage of the nestlet was obtained considering the % pre- and
 post-trial. On the Y-axis 100% intends that all nesting material was torn
 and 0% no material was torn. WT: C57BL6/J control group R312H: homozygote
 knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse \ 3.     Marble
 Burying: 8 marbles were positioned in the cage. After the trial, the
 marbles were counted. Y-axis goes from 0, no marble buried, to 8, all
 marble buried. WT: C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse 4.     House Comebacks: On
 the Y-axis is indicated the number of house comebacks each mouse performed
 during the dead reckoning behavior. WT: C57BL6/J control group R312H:
 homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse
 5.     Speed: On the Y-axis is indicated the number of average speed of
 each mouse during the duration of the dead reckoning behavior in
 centimeters/second (cm/s). WT: C57BL6/J control group R312H: homozygote
 knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse 6.     Distance
 Moved: on the Y-axis is indicated the space traveled by each mouse during
 the dead reckoning behavior in centimeters (cm). WT: C57BL6/J control
 group R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse 7.     Hanging test: Measurement of the time in seconds
 from the start of the trial until the tested animal loses its grip.
 Maximum time was set at 120 seconds. Y-axis goes from 0 seconds, no grip
 maintained, to 120 seconds, grip maintained for 120 seconds. WT: C57BL6/J
 control group R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse **IHC** L1 and NFM: 1.     Heights: on the Y-axis is
 indicated the height of a specific brain region in Pixels (Px). The brain
 region of interest is stated alongside the Y-axis. WT: C57BL6/J control
 group R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse 2.     Fluorescence intensity: on the Y-axis is
 indicated the fluorescence intensity of a specific brain region in A.U.
 The brain region of interest is stated alongside the Y-axis. WT: C57BL6/J
 control group R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse VGluTs: 1.     Fluorescence intensity: on the Y-axis is
 indicated the fluorescence intensity of a specific brain region in A.U.
 The brain region of interest is stated alongside the Y-axis. WT: C57BL6/J
 control group R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse Ankyrin G: 1.     Fluorescence intensity: on the Y-axis
 is indicated the fluorescence intensity of the Cortex in A.U. WT: C57BL6/J
 control group R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse HDAC1 SMI32: 1.     SMI32 Immunofluorescence: on the
 Y-axis is indicated the fluorescence intensity of SMI32 in the CC as A.U.
 WT: C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse 2.     % of translocated
 HDAC1: on the Y-axis is indicated the % of translocated HDAC1 in the
 axonal projections. Total HDAC1 and axonal HDAC1 intensity were calculated
 and the % was plotted. WT: C57BL6/J control group R312H: homozygote
 knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse **FRAP** 1.    
 Pre-Bleach: on the Y-axis is indicated the initial fluorescence intensity
 in A.U. of the region of interest of the neurons transfected
 pre-bleaching. WT: C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse 2.     Fluorescence
 recovery: on the Y-axis is indicated the % of recovery. The initial three
 points starting from coordinates X=0 and Y=0 refer to the pre-bleach. On
 the X-axis is indicated the time in seconds. WT: C57BL6/J control group
 R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H*
 mouse Cyto D, Jasp, Phall, FN, GxTx, Cilengitide, PND1186, and [K], refers
 to the presence of a specific treatment in the concentration stated in the
 paper. 3.    Mobile Fraction: on the Y-axis is indicated the % of mobile
 fraction is indicated with the formula stated in the method section of the
 paper. WT: C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse Cyto D, Jasp, Phall, FN,
 GxTx, Cilengitide, PND1186, and [K], refers to the presence of a specific
 treatment in the concentration stated in the paper. 4.     Koff: the Koff
 plotted on the Y-axis refers to the dissociation rate constant of globular
 actin from binding to another monomer of actin to form a Filamentous actin
 polymer. WT: C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse. **Electrophysiology
 (EPHYS)** 1.     Representative traces: in the representative traces, on
 the Y-axis is indicated the current in nA, on the X-axis the time in
 seconds. The total duration of the trace is 0.5 s. WT derived PNC: PNC
 from C57BL6/J control group R312H derived PNC: PNC from homozygote knockin
 (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse NULL derived PNC: from
 homozygote C57BL/6J-*Kcnb1em2Sesf/*J, *Kcnb1NULL* mouse.\ 2.     IV curve:
 I/V curve (current-voltage curve) is a graphical representation of the
 relationship between the electrical current (I) flowing through a system
 and the applied voltage (V) across it. on the Y-axis is indicated current
 in nA, and on the X-axis is indicated the voltage in mV. WT derived PNC:
 PNC from the C57BL6/J control group R312H derived PNC: PNC from homozygote
 knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse. NULL derived
 PNC: from homozygote C57BL/6J-*Kcnb1em2Sesf/*J, *Kcnb1NULL* mouse. 3.    
 G/Gmax: The ratio G/Gmax is commonly used in electrophysiology to describe
 the relative conductance of an ion channel under different conditions. On
 the Y-axis is plotted the G/Gmax ratio. On the X-axis is plotted the
 voltage in mV. WT derived PNC: PNC from the C57BL6/J control group R312H
 derived PNC: PNC from homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse. NULL derived PNC: from homozygote
 C57BL/6J-*Kcnb1em2Sesf/*J, *Kcnb1NULL* mouse. 4.     Number AP/Sec (HZ):
 on the Y-axis is indicated the number of Action potentials per second
 (Hz). On the X-axis is indicated the input current in nA. The graph shows
 the number of Action Potential/second (HZ) at a given input current. WT
 derived PNC: PNC from the C57BL6/J control group R312H derived PNC: PNC
 from homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H*
 mouse. NULL derived PNC: from homozygote C57BL/6J-*Kcnb1em2Sesf/*J,
 *Kcnb1NULL* mouse. 5.     Rheobase: On the Y-axis, the value of the
 Rheobase is represented in millivolts (mV), which corresponds to the
 minimum current amplitude required to elicit an action potential in a
 neuron when the stimulus is applied for a long duration. This measure is
 an indicator of neuronal excitability and helps in assessing the threshold
 at which a neuron becomes activated. WT derived PNC: PNC from C57BL6/J
 control group R312H derived PNC: PNC from homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse. NULL derived PNC: from
 homozygote C57BL/6J-*Kcnb1em2Sesf/*J, *Kcnb1NULL* mouse. 6.     Cell
 Capacitance: On the Y-axis, the value of the cell capacitance is
 represented in picofarads (pF), which quantifies the cell's ability
 to store and separate electrical charge across its membrane. WT derived
 PNC: PNC from C57BL6/J control group R312H derived PNC: PNC from
 homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse.
 NULL derived PNC: from homozygote C57BL/6J-*Kcnb1em2Sesf/*J, *Kcnb1NULL*
 mouse. 7.     Resistance GΩ (Input resistance): On the Y-axis, the value
 of the input resistance is represented in giga-ohms (GΩ), a measure of how
 much a cell resists the flow of electrical current when a voltage is
 applied across its membrane. WT derived PNC: PNC from C57BL6/J control
 group R312H derived PNC: PNC from homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse. NULL derived PNC: from
 homozygote C57BL/6J-*Kcnb1em2Sesf/*J, *Kcnb1NULL* mouse. 8.     I=0 (RMP):
 On the Y-axis, the resting membrane potential is represented in millivolts
 (mV), which denotes the baseline electrical potential difference across
 the cell membrane when the cell is at rest and not actively firing. WT
 derived PNC: PNC from the C57BL6/J control group R312H derived PNC: PNC
 from homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H*
 mouse. NULL derived PNC: from homozygote C57BL/6J-*Kcnb1em2Sesf/*J,
 *Kcnb1NULL* mouse. 9.     Current: on the Y-axis is indicated the current
 amplitudes at +80 mV. WT derived PNC: PNC from the C57BL6/J control group
 R312H derived PNC: PNC from homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse. NULL derived PNC: from
 homozygote C57BL/6J-*Kcnb1em2Sesf/*J, *Kcnb1NULL* mouse. GxTx – or +
 denotes the presence or absence of GxTx at the concentration stated in the
 paper. 10.  Spikes at 0.05: on the Y-axis is indicated the number of
 Action potentials per second (Hz) at 50 pA. WT derived PNC: PNC from the
 C57BL6/J control group R312H derived PNC: PNC from homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse. NULL derived PNC: from
 homozygote C57BL/6J-*Kcnb1em2Sesf/*J, *Kcnb1NULL* mouse. GxTx – or +
 denotes the presence or absence of GxTx at the concentration stated in the
 paper. \# all the recording files are provided and the lab notebook
 uploaded can be used to retrieve the individual recording used. **N2a
 cells** 1.     WT: neurite length of WT transfected N2a cells. the Y-axis
 indicates the neurite length in Px. Cyto D, Jasp, and Lat A refer to the
 presence of a specific treatment in the concentration stated in the paper.
 Cnt indicates the control condition. 2.     R312H: neurite length of R312H
 transfected N2a cells. the Y-axis indicates the neurite length in Px. Cyto
 D, Jasp, and Lat A refer to the presence of a specific treatment in the
 concentration stated in the paper. Cnt indicates the control condition.
 3.     Mock: neurite length of Mock transfected N2a cells. the Y-axis
 indicates the neurite length in Px. Cyto D, Jasp, and Lat A refer to the
 presence of a specific treatment in the concentration stated in the paper.
 Cnt indicates the control condition. **Primary neuron culture (PNC)**
 1.     Total Dendrite Length: On the Y-axis is indicated the total
 dendrite length of dendrites in Pixels (Px). PNC from the C57BL6/J control
 group and PNC from homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse, were analyzed in two separate sheets with the
 appropriate genotype annotated in the sheet name. Cyto D, Jasp, Phall, and
 Lat A refer to the presence of a specific treatment in the concentration
 stated in the paper. Cnt indicates the control condition. 2.     Number of
 intersections: the analysis takes in account the interactions of a single
 neuron. On the Y-axis is indicated the number of intersections obtained
 from the Sholl analysis. On the X-axis is indicated the Sholl radius
 centered on the soma. The Sholl radius is expressed in mm. PNC from the
 C57BL6/J control group and PNC from homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse, were analyzed in two
 separate sheets with the appropriate genotype annotated in the sheet name.
 Cyto D, Jasp, Phall, and Lat A refer to the presence of a specific
 treatment in the concentration stated in the paper. Cnt indicates the
 control condition. 3.     WT all (Density of intersections): on the Y-axis
 is indicated the number of intersections normalized to the number of cells
 within an area of the Sholl radius r. On the X-axis is indicated the Sholl
 radius centered on the soma. The Sholl radius is expressed in mm. PNC from
 the C57BL6/J control group and PNC from homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse, were analyzed in two
 separate sheets with the appropriate genotype annotated in the sheet name.
 Cyto D, Jasp, Phall, and Lat A refer to the presence of a specific
 treatment in the concentration stated in the paper. Cnt indicates the
 control condition. 4.     Inter WT/R312H at Max (Density of intersections
 @200mm): on the Y-axis is indicated the density of the intersection in
 correspondence of a Sholl radius of 200mm. The Sholl radius is expressed
 in mm. PNC from the C57BL6/J control group and PNC from homozygote knockin
 (KI) C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse, were analyzed in two
 separate sheets with the appropriate genotype annotated in the sheet name.
 Cyto D, Jasp, Phall, and Lat A refer to the presence of a specific
 treatment in the concentration stated in the paper. Cnt indicates the
 control condition. **Proliferation** 1.     WT: proliferating cells of WT
 transfected CHO cells. the Y-axis indicates the number of proliferating
 cells x10000 Cyto D, Jasp, Phall, and Lat A refer to the presence of a
 specific treatment in the concentration stated in the paper. Cnt indicates
 the control condition. 2.     R312H: proliferating cells of R312H
 transfected CHO cells. the Y-axis indicates the number of proliferating
 cells x10000 Cyto D, Jasp, Phall, and Lat A refer to the presence of a
 specific treatment in the concentration stated in the paper. Cnt indicates
 the control condition. 3.     Mock: proliferating cells of Mock
 transfected CHO cells. the Y-axis indicates the number of proliferating
 cells x10000 Cyto D, Jasp, Phall, and Lat A refer to the presence of a
 specific treatment in the concentration stated in the paper. Cnt indicates
 the control condition. **Tracing** BDA 594 1.     Fluorescence intensity:
 on the Y-axis is indicated the fluorescence intensity of projecting fibers
 in cortical layer IV or VI was expressed in A.U. The brain region of
 interest is stated above the graph and on the sheet page name. WT:
 C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse BDA 488  1.     Fluorescence
 intensity: on the Y-axis is indicated the fluorescence intensity of
 projecting fibers in a specific brain region expressed in A.U. The brain
 region of interest is stated above the graph and on the sheet page name.
 WT: C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse  Fluorogold 1.    
 Fluorescence intensity: on the Y-axis is indicated the fluorescence
 intensity of projecting fibers in the cortex expressed in A.U. WT:
 C57BL6/J control group R312H: homozygote knockin (KI)
 C57BL/6J-*Kcnb1em1Sesf*/J, *Kcnb1R312H* mouse **Western blot (WB)** 1.    
 WB analysis: the antibody used to detect the protein of interest is stated
 in the sheet description and above the graph. On the Y-axis is indicated
 the colorimetric analysis in A.U. The individual values account for the
 background detected and were normalized to the WT control. The following
 antibodies were used: VGluT1, VGluT2, GAD65, SNAP25, Actin. WT: C57BL6/J
 control group R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse 2.     F/G Actin WB: On the Y-axis is indicated the
 colorimetric analysis in A.U. The antibody used was Actin, in the analysis
 are present WT and R312H for both F and G actin. WT: C57BL6/J control
 group R312H: homozygote knockin (KI) C57BL/6J-*Kcnb1em1Sesf*/J,
 *Kcnb1R312H* mouse