Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice

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.

Methods
This dataset was generated using a combination of advanced techniques in molecular biology, neurophysiology, and microscopy:

Genetic Engineering: Mouse neurons were genetically modified to express specific fluorescent markers or proteins of interest to facilitate visualization and functional analysis.

Organotypic Slice Cultures: Brain slices from mice were cultured in vitro, preserving the three-dimensional structure and connectivity of the tissue for extended experimental manipulation and imaging.

Dissociated Cell Cultures: Neurons were enzymatically dissociated from mouse brains and plated in vitro, allowing detailed single-cell analyses.

Acute Brain Slices: Freshly prepared brain slices from adult mice were used for high-resolution electrophysiological and imaging studies.

Patch Clamp Technique: Electrophysiological recordings were conducted on individual neurons to measure their ionic currents and membrane properties.

Microscopy Techniques:

Immunofluorescence Microscopy: Specific proteins were visualized using antibodies conjugated to fluorescent dyes.

Confocal Microscopy: High-resolution images were acquired to examine subcellular structures and protein localization.

STED Microscopy: Super-resolution imaging was employed to resolve structures below the diffraction limit of light microscopy.

In Vivo Two-Photon Microscopy: Structural information was imaged in living mice.

In Vivo GRIN Lens Microscopy: Deep brain imaging was performed using gradient-index lenses in vivo.

Image Analysis:

Imaris: 3D image reconstruction and quantitative analyses of confocal and STED datasets.

Fiji/ImageJ: Additional image processing and quantitative analysis to refine and validate findings.