To visualize the microglia, we utilized biotinylated anti-mouse FcgR secondary antibody by addition of high concentrations of Triton X-100 (1%) and revealed secondary antibody binding by the avidin-biotin-immunoperoxidase (ABC) method (25, 28). Application of this method revealed the non-neuronal cells with branches similar to the microglia in the cerebellar cortex and raises the speculation that FcgRs are localized on microglia (Fig. 1A, D). To confirm that, the ionized calcium binding adaptor molecule 1 (Iba1) marker was used to demonstrate the morphological features of microglial cells (29–31). A transverse section of the cerebellum immunostained with Iba1 shows that microglia are the only immuno-reactive cells with this marker and the morphology closely resemble the FcgRs+ cells. (Fig. 1B, C). Double immunostaining confirmed that FcgRs are co-labeled with the Iba1 (Fig. 1F). The detail of co-localization of FcgR and Iba1 immunostaining is demonstrated in higher magnification (Fig. 1D-F). Positive microglial cell appeared to contain FcgR-like immunoreactivity.
To determine whether FcgR is expressed in other non-neuronal cells, the wt cerebellar sections were stained by double-fluorescence immunohistochemistry, FcgRs -like immunoreactivity was not colocalized in GFAP-positive astrocytes (Fig. 2A–C) and in MBP-positive oligodendrocytes (Fig. 2D–F).
To understand the expression of FcgRs during the development of the microglia, the cerebellar sections were used during perinatal development. FcgRs immunoreactivity is distinguishable at prenatal stages of development and by E17, FcgR immunoreactivity is weak at the core of the cerebellar sections in developing white matter (Fig. 3A, B). The FcgR immunoreactivity is strong in microglial precursor cells which are located in developing white matter at around P1 (Fig. 3C) and P3 (Fig. 3D). By P10, developing microglial cells with their extended process are clearly detectable by FcgRs which are highly populated in the white matter and indicated stream migratory pathway to cerebellar cortex (Fig. 3E, F).
To determine whether FcgRs expression profile is altered in the cerebellum with PCs migration disorders, we used nax mutant cerebellum which shows an excessive PC migration and compared it with other Reelin mutant mice cerebellum with prominent ectopic PCs in white matter and lack of PCs cluster dispersal.
The cerebellar cortex of nax mouse is abnormal and three layer of the cortex is indistinguishable with PCs invading the molecular layer and significantly reduce in the number of granule cells ((18) and Fig. 4). In a P17 wt mouse, the PCs bodies almost form a monolayer while their dendrite extension is apparent in developing molecular layer and directed toward the pial surface (Fig. 4A) while, in nax cerebellum PC bodies with less dendritic arborization and form a multilayer of the cells instead of a monolayer in cortex (Fig. 4D). Multilayer PCs occupy all molecular layer and their dendrites are branched in multidirectional pattern in nax cerebellum (Fig. 4D) in comparison to the wt (Fig. 4A). Recently we have shown the distribution of both ramified and activated microglial cells in nax cerebellum compared to the wt siblings cerebellum (32). In spite of the activated microglia, the FcgR expression in microglia of the nax cerebellum with excessive PCs migration which were detected by Iba1 at P17 were colocalized, which is similar to the wt cerebellum (Fig. 4B, E; higher magnification in Fig. 4C,F). To determine the FcgR expression profile in cerebellar with ectopic Pcs in white matter, FcgR and Iba1 immunopeoxidase staining performed using the scm cerebellum (Fig. 5). Surprisingly, no FcgR immunoractivity was observed in scm cerebellum (Fig. 5D, E) compare to scm wt cerebellum (Fig. 5A, B, C). Iba1 immunostaining was utilized to determine whether the lack of immunoractivity in FcgRs is due to the lack of microglia in cerebellum. Results revealed the existence of ramified microglia and they are not activated in respond to ectopic neuronal environment (Fig. 5F, G). To test the hypotheis that FcgR expression is absent in scm pathway, we have pursued in Reeler and Apoer−/− mutants. Double immunoflouresent staining using anti- FcgR and Iba1 antibodies (Fig. 6) in reeler wt confirmed the colocalizrtion of the FcgR and Iba1 (Fig. 6A-C). However, double immunoflouresent staining using anti- FcgR and Iba1 antibodies in reeler (Fig. 6D-F), Apoer−/− (Fig. 6G-I), and scm (Fig. 6J-L) mutants revealed lack of FcgR expression on their microglia despite their well-organized Iba1 immunopositve microglia when compared with control wt (Fig. 6A-C).