Princeton RAtlas: A Common Coordinate Framework for Fully cleared, Whole Rattus norvegicus Brains

Abstract

Whole-brain clearing and imaging methods are becoming more common in mice but have yet to become standard in rats, at least partially due to inadequate clearing from most available protocols. Here, we build on recent mouse-tissue clearing and light-sheet imaging methods and develop and adapt them to rats. We first used cleared rat brains to create an open-source, 3D rat atlas at 25 μm resolution. We then registered and imported other existing labeled volumes and made all of the code and data available for the community (https://github.com/emilyjanedennis/PRA) to further enable modern, whole-brain neuroscience in the rat. Key features • This protocol adapts iDISCO (Renier et al., 2014) and uDISCO (Pan et al., 2016) tissue-clearing techniques to consistently clear rat brains. • This protocol also decreases the number of working hours per day to fit in an 8 h workday. Graphical overview.

Keywords: Image registration; Light-sheet imaging; Neuroscience; Rat; Rattus norvegicus; Tissue clearing; iDISCO; uDISCO.

Figure 1.. Modifications to iDISCO and uDISCO protocols allow for fully cleared rat brains.

A. Schematics of iDISCO protocol and the modified rat iDISCO and uDISCO protocols (abbreviations DCM, BABB, and DBE all refer to solutions in Recipes). B. Example images of brains before clearing and after clearing with each protocol from the schematic. The uncleared brain is approximately 2.5 cm long and 1.7 cm wide. All images were taken on the same piece of paper for easy comparison. C. Computational slices through each cleared brain in B. Labels (a253, k320, z268) reflect the animal’s name. D. Drawing showing the orientation of the brain (left) and the FRC-QE scores (right) for each brain in B along the coronal axes in slice units. E. Quantification of the values in (D), with a Bonferroni-corrected t-test. Horizontal white bars are median values, and the violin limits are the 10% (bottom) and 90% (top) of the data. Asterisks indicate significance p < 0.05 compared with iDISCO a253.

Figure 2.. Creation of the Princeton RAtlas (PRA).

A. Schematics showing the paired alignments to create the seed brain (left, middle) and the multilevel refinement resulting in the PRA (right). B. Summed axial projection of the PRA. mBrains and fBrains are male and female brains respectively, defined by external genitalia.

Figure 3.. Quantification of computational alignment efficacy compared with humans.

A. Slices from the Waxholm Space Atlas of the Adult Rat Brain demonstrating the location (white arrow, +) of identifiable points of interest. Abbreviations: d/v dorsal/ventral, c/r caudal/rostral, L/R left/right. B. Drawing of the location of each slice from A shown in sagittal and axial spaces. C. Sagittal (left) and axial (right) examples of four human annotators’ points for location VM1 (ventricle middle 1) in a single brain compared with imported and aligned ground truth data from the Waxholm atlas. D. Sagittal (left) and axial (right) examples of four human annotators’ points for location VM1 made in Waxholm atlas space, compared with the ground truth data from the atlas. E. Simplified diagram demonstrating how to calculate the distance metric used in F. F. Quantification of distances between user-annotated points in distinct stages of alignment and the imported annotations (WHS). G. Quantification of distances demonstrating the accuracy (left) and precision (right) of human annotators for all four annotated points.