Glucocorticoid Effects on Cerebellar Development in a Chicken Embryo Model: Exploring Changes in PAX6 and Metalloproteinase‐9 After Exposure to Dexamethasone

Abstract

The developing cerebellum is vulnerable to effects of glucocorticoids and cerebellar dysfunction is associated with neurodevelopmental disorders (e.g. autism). Transcription factor PAX6 and matrix metalloproteinase-9 (MMP-9) are critical for normal cerebellar development and are highly expressed in migrating neurones. Alterations in MMP-9 and PAX6 are associated with altered cerebellar development. In the present study, we characterised the growth rate and development of the cortical layers, and further investigated how the levels of PAX6 and MMP-9, as well as glucocorticoid receptor (GR) and proliferating cell nuclear antigen (PCNA), change in the cerebellum during the foetal period [embryonic day (E)12-21] in chicken, which corresponds to the human perinatal period. Dexamethasone (DEX) was administered in ovo at E13 and E16, aiming to investigate how prenatal exposure to glucocorticoids interferes with normal development. DEX reduced foetal and cerebellar weight at E17 in a dose-dependent manner linked to a reduced level of PCNA and, over time, down-regulation of GR. We report that promoter activity of PAX6 and MMP-9 increased as a result of GR-stimulation in vitro. Prenatal DEX increased the protein level of PAX6 in a transient manner. PAX6 is reduced in mature granule neurones, and this occurred earlier in embryos exposed to DEX than in non-exposed controls. DEX exposure also led to a slow-onset down-regulation of MMP-9. Taken together, these findings indicate that excess prenatal glucocorticoid stimulation disturbs normal development of the cerebellum through mechanisms associated with reduced proliferation and accelerated maturation where PAX6 and MMP-9 play important roles.

Opioid receptor‐mediated changes in the NMDA receptor in developing rat and chicken

“…The expression of GluN2B in the chicken cerebellum displayed two peaks, one at E15 (not significant) and one at E18. Interestingly, this expression pattern is closely correlated with chicken cerebellar Pax6 expression (Austdal et al, 2016), an essential regulator of granule neurone differentiation and migration (Yamasaki et al, 2001). The expression of GluN2B also matches the cerebellar growth rate observed from E13-E18 (Austdal et al, 2016).…”

“…Interestingly, this expression pattern is closely correlated with chicken cerebellar Pax6 expression (Austdal et al, 2016), an essential regulator of granule neurone differentiation and migration (Yamasaki et al, 2001). The expression of GluN2B also matches the cerebellar growth rate observed from E13-E18 (Austdal et al, 2016). These features closely resemble rat cerebellar development (Zhong et al, 1995), but occurs in ovo in the chicken as opposed to postnatally in the rat.…”

“…Cerebella were harvested for western blotting as described by Austdal et al (Austdal et al, 2016). The membranes were probed with primary antibodies against GluN2B (1:1000), MOR (1:250) and β‐actin (1:10000) followed by a HRP‐conjugated secondary antibody (1:10000).…”

“…Comparative anatomy studies have shown that the cerebellum is a well‐conserved structure between species (Sultan and Glickstein, 2007) and the ontogeny of the opioid system in both developing chicken and rat brain, including the cerebellum, has been well characterised (Vernadakis et al, 1990; Spain et al, 1985). The migration of granule neurones from the external granule layer (EGL) to the internal granule layer (IGL) occurs before hatching in the chicken (Austdal et al, 2016), while it is completed by two weeks after birth in the rat (Hager et al, 1995). The migration is guided by N‐methyl‐D‐aspartate receptor (NMDAR)‐mediated calcium ion (Ca 2+ ) influx, among many other signals (Komuro and Rakic, 1993).…”

Exploring the overlapping binding sites of ifenprodil and EVT‐101 in GluN2B‐containing NMDA receptors using novel chicken embryo forebrain cultures and molecular modeling

“…Isolated tissues from chicken forebrain (E7‐18) and mouse cerebellum (postnatal day 21) were frozen in N 2 (−196°C) before long‐term storage at −20°C. To prepare for western blotting analysis, tissue was homogenized as previously described . In short, samples were kept on ice, added tris‐EDTA (TE) buffer containing the same protease inhibitors as described above, and homogenized using a motorized pellet pestle.…”