HIGH-RESOLUTION 2-PHOTON MICROSCOPY-BASED RAT BRAIN ATLAS INTEGRATED INTO THE BRAINGLOBE ECOSYSTEM

Rats are widely used model organisms in neuroscience, yet available brain atlases remain limited in resolution, dimensionality, and biological diversity. Existing three-dimensional rat atlases are primarily derived from magnetic resonance imaging (MRI), which enables whole-brain coverage but constrains fine anatomical detail and is largely based on adult, male animals.

Here, we present a high-resolution three-dimensional brain atlas of female Lister Hooded rats aged 5–8 weeks, generated using automated serial two-photon tomography. This approach enables the capture of fine anatomical detail not accessible in MRI-derived atlases. To generate a population-based anatomical template, we used the symmetric group-wise normalisation (SyGN) framework implemented in Advanced Normalisation Tools (ANTs), together with brainglobe-template-builder, an open-source tool within the BrainGlobe ecosystem for standardising neuroanatomical template construction.

To ensure broad accessibility, the template and curated annotations derived from the Waxholm Space rat brain atlas (an MRI-based reference for rat neuroanatomy) were packaged in a standard format and distributed via the modernised BrainGlobe Atlas API. The BrainGlobe ecosystem provides a unified framework for atlas-based image registration, coordinate transformation, and visualisation across species and atlas modalities, enabling direct comparison and reuse of data across studies. This integration allows seamless use of the atlas alongside existing resources.

By introducing a microscopy-derived three-dimensional atlas for female rats, this work fills a gap in available neuroanatomical resources and provides a foundation for anatomical alignment, circuit mapping, and brain-wide analyses in systems and comparative neuroscience. The modular pipeline enables extension to additional sexes and future integration of cell-type– or projection-specific information.