A rapid workflow for neuron counting in combined light sheet microscopy and magnetic resonance histology

Information on regional variation in cell numbers and densities in the CNS provides critical insight into structure, function, and the progression of CNS diseases. However, variability can be real or a consequence of methods that do not account for technical biases, including morphologic deformations, errors in the application of cell type labels and boundaries of regions, errors of counting rules and sampling sites. We address these issues in a mouse model by introducing a workflow that consists of the following steps: 1. Magnetic resonance histology (MRH) to establish the size, shape, and regional morphology of the mouse brain in situ. 2. Light-sheet microscopy (LSM) to selectively label neurons or other cells in the entire brain without sectioning artifacts. 3. Register LSM volumes to MRH volumes to correct for dissection errors and both global and regional deformations. 4. Implement stereological protocols for automated sampling and counting of cells in 3D LSM volumes. This workflow can analyze the cell densities of one brain region in less than 1 min and is highly replicable in cortical and subcortical gray matter regions and structures throughout the brain. This method demonstrates the advantage of not requiring an extensive amount of training data, achieving a F1 score of approximately 0.9 with just 20 training nuclei. We report deformation-corrected neuron (NeuN) counts and neuronal density in 13 representative regions in 5 C57BL/6J cases and 2 BXD strains. The data represent the variability among specimens for the same brain region and across regions within the specimen. Neuronal densities estimated with our workflow are within the range of values in previous classical stereological studies. We demonstrate the application of our workflow to a mouse model of aging. This workflow improves the accuracy of neuron counting and the assessment of neuronal density on a region-by-region basis, with broad applications for studies of how genetics, environment, and development across the lifespan impact cell numbers in the CNS.