Abnormal cytoskeletal remodeling but normal neuronal excitability in a mouse model of the recurrent developmental and epileptic encephalopathy-susceptibility KCNB1-p.R312H variant
Data files
Feb 03, 2025 version files 53.30 GB
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FIGURE_1.zip
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FIGURE_2.zip
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FIGURE_3.zip
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FIGURE_7.zip
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FIGURE_S2.zip
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FIGURE_S4.zip
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FIGURE_S5.zip
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FIGURE_S6.zip
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FIGURE_S7.zip
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FIGURE_S8.zip
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FIGURES_10-S10.zip
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FIGURES_4-5-S1.zip
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FIGURES_6-S3.zip
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FIGURES_8-9.zip
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README.md
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Uncropped_WBs.pdf
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Abstract
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.
README
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