Histomorphometrical Analyses of the Mouse Suprachiasmatic Nucleus

The mammalian central circadian clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus in the brain and is responsible for coordinating daily rhythms of biological processes spanning from gene expression to behavior. Light, the primary environmental zeitgeber, entrains the SCN via melanopsin-expressing intrinsically photosensitive retinal ganglion cells that project through the retino-hypothalamic tract. Altered circadian rhythms are common in individuals diagnosed with neurodevelopmental and neurodegenerative disorders, and often, associated with structural alterations of the SCN and impaired retinal input; importantly, these anomalies can be recapitulated in animal models. Here, we describe step-by-step protocols for quantitative histomorphometrical analysis of the SCN and the assessment of retinal-SCN connectivity, previously used in mouse models of neurodevelopmental and neurodegenerative disorders. These include measurement of the SCN area, perimeter, height and width using Nissl- or DAPI-stained coronal sections, as well as densitometric and plot profile analyses of cholera toxin β-subunit-labeled retinal projections using Axiovision or Fiji/ImageJ. The protocols incorporate standardized region-of-interest, measurements by masked observers, and consistent scaling procedures to enhance reproducibility. These methods provide a rigorous framework for detecting structural anomalies and connectivity defects in the circadian system and can be broadly applied to other experimental models of circadian dysfunction. Key features • Histomorphometrical analyses of the SCN can provide anatomical bases to understand altered sleep and circadian rhythms in animal models of disease. • Exploration of retinal-SCN connectivity to facilitate the identification of the underlying causes of deficits in the response to photic cues in animal models of disease. • The protocols described here employ widely used and accessible software and provide rigorous but easy-to-follow instructions. • These analyses do not require expensive staining procedures and can be easily implemented in any laboratory. • Strengths for reproducibility: usage of fixed region-of-interest (ROI), measurements averaged from multiple sections per animal, masked observers thoroughly trained.