DOI: 10.5061/dryad.n02v6wx76
title: Data from: Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice

description: The axon initial segment (AIS) constitutes not only the site of action
 potential initiation, but also a hub for activity-dependent modulation of
 output generation. Recent studies shedding light on AIS function used
 predominantly post-hoc approaches since no robust murine in vivo
 live reporters exist. Here, we introduce a reporter line in which the AIS
 is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by
 Cre recombinase, tagging the native Ank3 gene. Using confocal,
 superresolution, and two-photon microscopy as well as whole-cell
 patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the
 subcellular scaffold of the AIS and electrophysiological parameters of
 labeled cells remain unchanged. We further uncover rapid AIS remodeling
 following increased network activity in this model system, as well as
 highly reproducible in vivo labeling of AIS over weeks. This novel
 reporter line allows longitudinal studies of AIS modulation and plasticity
 in vivo in real-time and thus provides a unique approach to study
 subcellular plasticity in a broad range of applications.
description: This dataset was generated using a combination of advanced
 techniques in molecular biology, neurophysiology, and
 microscopy: <strong>Genetic
 Engineering</strong>: Mouse neurons were genetically modified to
 express specific fluorescent markers or proteins of interest to facilitate
 visualization and functional analysis. 
 <strong>Organotypic Slice Cultures</strong>: Brain slices from
 mice were cultured in vitro, preserving the three-dimensional structure
 and connectivity of the tissue for extended experimental manipulation and
 imaging. <strong>Dissociated Cell
 Cultures</strong>: Neurons were enzymatically dissociated from mouse
 brains and plated in vitro, allowing detailed single-cell
 analyses. <strong>Acute Brain
 Slices</strong>: Freshly prepared brain slices from adult mice were
 used for high-resolution electrophysiological and imaging
 studies. <strong>Patch Clamp
 Technique</strong>: Electrophysiological recordings were conducted
 on individual neurons to measure their ionic currents and membrane
 properties. <strong>Microscopy
 Techniques</strong>: 
 <strong>Immunofluorescence Microscopy</strong>: Specific
 proteins were visualized using antibodies conjugated to fluorescent
 dyes. <strong>Confocal
 Microscopy</strong>: High-resolution images were acquired to examine
 subcellular structures and protein localization. 
 <strong>STED Microscopy</strong>: Super-resolution imaging was
 employed to resolve structures below the diffraction limit of light
 microscopy. <strong>In Vivo Two-Photon
 Microscopy</strong>: Structural information was imaged in living
 mice. <strong>In Vivo GRIN Lens
 Microscopy</strong>: Deep brain imaging was performed using
 gradient-index lenses in vivo. 
 <strong>Image Analysis</strong>: 
 <strong>Imaris</strong>: 3D image reconstruction
 and quantitative analyses of confocal and STED datasets.
 <strong>Fiji/ImageJ</strong>: Additional image
 processing and quantitative analysis to refine and validate
 findings.
description: # Ankyrin-G-GFP data respository
 [https://doi.org/10.5061/dryad.n02v6wx76](https://doi.org/10.5061/dryad.n02v6wx76) ## Description of the data and file structure 1\. General Information Dataset Title: AnkG-GFP Data Repository – Raw Data Authors: Christian Thome, Jan Maximilian Janssen, Seda Karabulut, Claudio Acuna, Elisa D’Este, Stella J. Soyka, Konrad Baum, Michael Bock, Nadja Lehmann, Johannes Roos, Nikolas A. Stevens, Masashi Hasegawa, Dan A. Ganea, Chloé M. Benoit, Jan Gründemann, Lia Min, Kalynn M. Bird, Christian Schultz, Vann Bennett, Paul M. Jenkins, Maren Engelhardt Date of Data Collection: 2019–2023 Associated Publication: “Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice” in eLife Description: This dataset includes raw experimental data used in the study. It contains imaging files, processed data, and statistical outputs. ### Files and variables 2\. Dataset Structure This dataset corresponds to the scientific publication on the characterization of the Ankyrin-G-GFP (Ank-G-GFP) mouse model. It contains raw and processed image files, electrophysiology data, and statistical analyses related to the figures presented in the paper. The dataset is structured in folders named after the figures and supplementary data in the publication. Each folder corresponds to a specific figure or panel in the publication, containing the original data used for analysis and representation. Below is an outline of the dataset structure along with a description of the content of each figure. Figure 1: ‘Genetic strategy and creation of the ankyrin-G-GFP line’ illustrates the strategy used to generate the Ank-G-GFP mouse line, including genetic targeting, Cre-dependent recombination, and the sequence of steps leading to the successful integration of GFP into the Ank3 gene. The folder ‘Fig1‘ contains sketches and diagrams of the genetic targeting strategy (.pptx) as well as vector and genomic sequences in .gb and .docx formats. Figure 2: ‘Ank-G-GFP activation and expression in different experimental models’ shows images of Ank-G-GFP expression in different neuron types and brain regions after Cre-mediated recombination. Data are split by the respective subplots into folders Fig2A, Fig2B, Fig2C, Fig2D, Fig2E, Fig2F, Fig2G, each containing the original imaging data (.nd2, .tif, .oir, .jpg). Figure 3: ‘Ank-G-GFP activation and expression in nodes of Ranvier’ investigates whether Ank-G-GFP expression affects the morphology of nodes of Ranvier. The folder ‘Fig3AC+suppl’ contains the original images of the ankyrin-G-GFP labeling as .nd2 files and quantitative analysis as .xls and .pzfx files. The folder ‘Fig3B’ contains files of paranodal protein visualization and node reconstructions (.png, .tif, .ics, .ids, .h5). Figure 4: ‘Ank-G-GFP expression and molecular AIS composition’ examines whether the molecular organization of the axon initial segment (AIS) remains unchanged after ankyrin-G-GFP expression. The folder ‘Fig4+suppl’ contains immunohistochemical verification images (.nd2, .tif, .jpg) of ankyrin-G-GFP, Nav1.6, betaIV-spectrin, Kv2.1, NF-186, TRIM46, and FGF14 at the axon initial segment as well as intensity gradient measurements (.xls). Figure 5: ‘Ank-G-GFP expression preserves axonal characteristics’ evaluates axonal integrity and the presence of structural components such as synaptopodin clusters, GABAergic synapses, and microglia interactions in the AIS. The folders Fig5A, Fig5B, and Fig5C contain raw and processed imaging data (.nd2, .tif, .jpg). Figure 6: ‘The AIS nanostructure is maintained after GFP fusion to ankyrin-G’ uses super-resolution imaging (STED) to analyze the nanoscale structure of the AIS after Ank-G-GFP expression. The folder ‘Fig6’ contains STED images from the Abberior microscope (.msr, .tif). Figure 7: ‘Neuronal excitability is not affected by GFP fusion to ankyrin-G’ investigates electrophysiological properties of neurons expressing Ank-G-GFP. The folder 'Fig7CD' contains raw cell images (.nd2), electrophysiology recordings (.cfs), and a spreadsheet of genotypes and corresponding electrophysiology data (.xlsx). Figure 8: ‘AIS structural plasticity in response to network activity changes’ demonstrates that ankyrin-G-GFP-expressing AIS undergo structural modifications under different network activity conditions. The folder 'Fig8A-Dexamples' contains raw and processed images (.nd2, .jpg) as well as ROI traces (.h5). The folder 'Fig8E' contains raw images as .avi and .tif as well as as ROI traces as .traces and .csv. Figure 9: ‘Live imaging of ank-G-GFP+-AIS in vivo’ presents in vivo two-photon microscopy imaging of AIS over time. The folders 'Fig9A-C' and 'Fig9D' contain live imaging files, processed images, ROI traces and statistics (.traces, .csv, .xlsx, .tif). \################################################################# 3\. Software Recommendations & Requirements Imaging data: .nd2 (Nikon Elements), .oir (Olympus), .msr (Abberior STED), .ics (meta-data), .ids (pixel data), .tif, .png, .jpg. Use Fiji (ImageJ) or ImSpector software (Abberior) for .msr. Electrophysiology data: .cfs. Use Clampfit (Axon pCLAMP) or Signal (Cambridge Electronic Design). Morphometric traces: Use (Fiji) for .traces and AISuite for .h5. Spreadsheet and statistical analysis: .csv, .xls, .xlsx. Use Microsoft Excel, LibreOffice, or Python (pandas library). Vector and genomic data: .gb, .docx. Use Microsoft Word and Benchling. Figure sketches and diagrams: .pptx. Use Microsoft PowerPoint. ## Access information 4\. Data Usage Citation: While not required, users are encouraged to cite this data repository as well as the associated publication when using this dataset. \################################################################# 5\. Contact Information Corresponding Author: Prof. Maren Engelhardt Email: [maren.engelhardt@jku.at](mailto:maren.engelhardt@jku.at) First Author: Dr. Christian Thome Email: [christian.thome@jku.at](mailto:christian.thome@jku.at) Institution: Johannes Kepler Universität Linz