Japanese scientists have discovered structure-function relationships between aldolase C/zebrin II expression and complex spike synchrony in the cerebellum. Previous separate studies had observed intricate parasagittal organization of Purkinje cells (PCs) along the expanse of the cerebellar cortex, along with synchronous complex spike activities among these PCs corresponding with proposed "microzones" that are activated depending on sensory stimuli patterns. The scientists conducted this study to define the relationship between these rows of PCs and accompanying "microzone" spike activity, using aldolase C/zebrin II compartments as markers for PC groups, and calcium indicators to track neural spike activity in climbing fibers to PCs.

To visually confirm a relationship between aldolase C compartments and complex spike synchrony at single cell resolution, the scientists performed in-vivo two-photon calcium imaging upon the cerebellar cortexes of transgenic mice expressing Aldoc-tdTomato, a gene controlling the aldolase C gene promoter and enabling aldolase C to be detected through red fluorescent protein tdTomato. After confirming that tdTomato was specific to aldolase C in the mice, the scientists injected the prepared calcium dyes of OGB-1 and Cal-520^® into the Crus IIa cerebellar areas of interest. Through acquiring two-photon images of these stained areas, the scientists were able to observe how the boundary of high-synchrony areas precisely matched boundaries of aldolase C compartments, as well as how extensive this synchrony across the mouse cerebellar cortex could be (>100 µm). By applying stimuli through air puffs upon the mice's ipsilateral perioral surfaces, the scientists could also observe the "microzones" activated within aldolase C boundaries.

Observation of PC activity in these aldolase C compartments/microzones was implemented through two-photon calcium detection of the OGB-1 and Cal-520^® signals emitted from the cerebellar cortex layer. The materials and methods section describes OGB-1 or Cal-520^® as green calcium dyes suitable for imaging of neural activity based on previous studies (Hashizume et al., 2013; Tada et al., 2014). Successful use of both OGB-1 and Cal-520^® interchangably within this study to acquire definitive images and results confirms that Cal-520^® is a novel calcium indicator that is as reliable as the classic OGB-1, if not better.