Transmission Electron Microscopy as the Visualization Technique for Analysis of Circadian Synaptic Plasticity in the...

Examining circadian synaptic plasticity requires housing mice under different lighting conditions (light/dark cycle, LD 12:12, and constant darkness, DD), providing access to running wheels, and sacrificing them at four defined time points within 24 h-at the beginning and middle of the day/subjective day and at the beginning and middle of the night/subjective night. Brains are then properly fixed for transmission electron microscopy (TEM). The barrel cortex, with its precise somatotopic organization, provides an ideal model for such analysis. To obtain the required brain area, the brains are tangentially cut with a vibratome, and then, sections containing the barrel cortex are selected and embedded in Polybed resin. From the prepared blocks containing the selected barrels, consecutive ultrathin sections are cut. Synaptic density, excitatory and inhibitory, is analysed from electron micrographs using the stereological dissector method. Additionally, TEM images are used for 3D reconstructions of dendritic spines. Changes in the shape of dendritic spines indicate remodeling of neurons during the day. The number of excitatory synapses peaks during sleep (day) in mice, while inhibitory synapses peak during their activity phase (in the middle of the night).