Postnatal development of cerebellar zones revealed by neurofilament heavy chain protein expression

Abstract

The cerebellum is organized into parasagittal zones that control sensory-motor behavior. Although the architecture of adult zones is well understood, very little is known about how zones emerge during development. Understanding the process of zone formation is an essential step toward unraveling how circuits are constructed to support specific behaviors. Therefore, we focused this study on postnatal development to determine the spatial and temporal changes that establish zonal patterns during circuit formation. We used a combination of wholemount and tissue section immunohistochemistry in mice to show that the cytoskeletal protein neurofilament heavy chain (NFH) is a robust marker for postnatal cerebellar zonal patterning. The patterned expression of NFH is initiated shortly after birth, and compared to the domains of several known zonal markers such as zebrin II, HSP25, neurogranin, and phospholipase Cβ4 (PLCβ4), NFH does not exhibit transient expression patterns that are typically remodeled between stages, and the adult zones do not emerge after a period of uniform expression in all lobules. Instead, we found that throughout postnatal development NFH gradually reveals distinct zones in each cerebellar lobule. The boundaries of individual NFH zones sharpen over time, as zones are refined during the second and third weeks after birth. Double labeling with neurogranin and PLCβ4 further revealed that although the postnatal expression of NFH is spatially and temporally unique, its pattern of zones respects a fundamental and well-known molecular topography in the cerebellum. The dynamics of NFH expression support the hypothesis that adult circuits are derived from an embryonic map that is refined into zones during the first 3-weeks of life.

Pathogenesis of severe ataxia and tremor without the typical signs of neurodegeneration

“…4B; n = 6). Wholemount analysis of an early zonal marker, the cystoskeletal protein neurofilament heavy chain (NFH; White and Sillitoe, 2013), further revealed that the boundaries in Car8 wdl are not sharply delineated because of poor sculpting (Fig. S4; n = 4).…”

In vivo analysis of Purkinje cell firing properties during postnatal mouse development

“…Immunohistochemistry was carried out as described previously (Reeber et al 2011;Sillitoe et al 2003Sillitoe et al , 2010White and Sillitoe 2013b). Briefly, tissue sections were thoroughly washed, blocked with 10% normal goat serum (NGS; Sigma, St. Louis, MO) for 1 h at room temperature, and then incubated in 0.1M PBS containing 10% NGS, 0.1% Tween-20, and the anti-CAR8 primary antibodies (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) for 16 -18 h at room temperature, shaking gently.…”

Mossy Fibers Terminate Directly Within Purkinje Cell Zones During Mouse Development

“…1b). To test if the labeled neurons reflected the known zonal organization of the cerebellum, and to determine whether they were Purkinje cells, I co-labeled the tissue with neurofilament heavy chain (NFH), a marker of postnatal Purkinje cell zones [17]. NFH expression revealed a pattern of zones that was complementary to a subset of traced Purkinje cells (Fig.…”

“…We used an anti-SMI-32 primary antibody (1:1500; Covance, Princeton, NJ,) to reveal Purkinje cell zones. See White and Sillitoe [17] for details about the NFH staining pattern.…”