Stereological Assessment of Locus Coeruleus in the Mouse: A Methodological Study in Pups and Adult Animals

Unbiased stereology represents the most accurate approach for estimating the total number of neurons of specific brain regions; however, its reliability critically depends on the use of rigorously defined and anatomically appropriate sampling parameters. The brain nucleus Locus Coeruleus (LC) plays a key role in several brain functions. LC impairment has been associated with a range of disorders affecting individuals across the lifespan, from infancy to adulthood. In animal models of these conditions, precise estimation of LC neuronal number is essential. The LC analysis poses specific methodological challenges due to its small size, indistinct anatomical boundaries, and age-dependent changes in neuronal density. In this study, we present a detailed and reproducible stereological workflow for the quantification of LC neurons in the mouse brain across the lifespan. Using C57BL/6J mice at postnatal, adult, and aged stages, we optimized all key components of the Optical Fractionator method, LC neurons were identified by immunoperoxidase staining for tyrosine hydroxylase (TH) and quantified using systematic-random sampling implemented in Stereo Investigator® software. We show that age-specific adjustment of stereological parameters is necessary to obtain reliable estimates, particularly at early postnatal stages characterized by high neuronal packing density. With the optimized protocols described here, TH+ LC neuron counts consistently met accepted precision criteria, as assessed by the Gundersen coefficient of error.