Despite the widespread use of mice in neuroscience, rats offer unique benefits for translational research. However, comprehensive methodologies like tissue clearing and quantitative light-sheet microscopy, common in mouse studies, are scarce for rats. This gap is partly due to a lack of robust computational tools, limiting the use of rats in large-scale, unbiased experiments. Our study leverages recent advancements, including a detailed MRI-based rat brain atlas with 222 regions and an average light sheet template, to conduct an extensive analysis. Despite its comprehensiveness, this atlas from Waxholm lacks certain areas, notably the pedunculopontine nucleus (PPN), crucial for our research. We developed custom computational tools to integrate the PPN into the atlas and conducted a whole-brain evaluation of virally labeled cells expressing Choline acetyltransferase (ChAT). By aligning our data with the MRI Waxholm space and the Princeton RAtlas, we delineated the PPN’s 3D boundaries and expanded the atlas's ontology. This allowed us to unbiasedly examine and quantify ChAT-positive virally infected cells in the brain, with a focus on the PPN. This approach not only fills a significant gap in rat-based neuroscience research tools but also enhances our understanding of brain-wide distribution of specific neuronal populations.