A framework to determine active neurons and networks within the mouse brain reveals how brain activity changes over the course of the day.

The mouse brain's activity changes drastically over a day despite being generated from the same neurons and physical connectivity. To better understand this, we develop an experimental-computational pipeline to determine which neurons and networks are most active at different times of the day. We genetically mark active neurons of freely behaving mice at four times of the day with a c-Fos activity-dependent TRAP2 system. Neurons are imaged and digitized in 3D, and their molecular properties are inferred from the latest brain spatial transcriptomic dataset. We then develop a new computational method to analyze the network formed by the identified active neurons. Applying this pipeline, we observe region and layer-specific activation of neurons in the cortex, especially activation of layer five neurons at the end of the dark (wake) period. We also observe a shift in the balance of excitatory (glutamatergic) neurons versus inhibitory (GABAergic) neurons across the whole brain, especially in the thalamus. Moreover, as the dark (wake) period progresses, the network formed by the active neurons becomes less modular, and the hubs switch from subcortical regions, such as the posterior hypothalamic nucleus, to cortical regions in the default mode network. Taken together, we present a pipeline to understand which neurons and networks may be most activated in the mouse brain during an experimental protocol, and use this pipeline to understand how brain activity changes over the course of a day.