Downregulation of TrkC Receptors Increases Dendritic Arborization of Purkinje Cells in the Developing Cerebellum of the Opossum, Monodelphis domestica.

Beata Tepper,Ruzanna Djavadian,Sonia Ngati,Magdalena Braszak,Katarzyna Bartkowska,Krzysztof Turlejski,Malgorzata Okrasa

doi:10.3389/fnana.2020.00056

Abstract

In therian mammals, the cerebellum is one of the late developing structures in the brain. Specifically, the proliferation of cerebellar granule cells occurs after birth, and even in humans, the generation of these cells continues during the first year of life. The main difference between marsupials and eutherians is that the majority of the brain structures in marsupials develop after birth. Herein, we report that in the newborn laboratory opossum (Monodelphis domestica), the cerebellar primordium is distinguishable in Nissl-stained sections. Additionally, bromodeoxyuridine birthdating experiments revealed that the first neurons form the deep cerebellar nuclei (DCN) and Purkinje cells, and are generated within postnatal days (P) 1 and 5. Three weeks after birth, progenitors of granule cells in the external germinal layer (EGL) proliferate, producing granule cells. These progenitor cells persist for a long time, approximately 5 months. Furthermore, to study the effects of neurotrophic tropomyosin receptor kinase C (TrkC) during cerebellar development, cells were obtained from P3 opossums and cultured for 8 days. We found that TrkC downregulation stimulates dendritic branching of Purkinje neurons, which was surprising. The number of dendritic branches was higher in Purkinje cells transfected with the shRNA TrkC plasmid. However, there was no morphological change in the number of dendritic branches of granule cells transfected with either control or shRNA TrkC plasmids. We suggest that inhibition of TrkC activity enables NT3 binding to the neurotrophic receptor p75NTR that promotes dendritic arborization of Purkinje cells. This effect of TrkC receptors on dendritic branching is cell type specific, which could be explained by the strong expression of TrkC in Purkinje cells but not in granule cells. The data indicate a new role for TrkC receptors in Monodelphis opossum.

Highlights

In all mammalian species including marsupials, the cerebellum contains two hemispheres connected with a medially located structure, the vermis, and the laterally located flocculus and paraflocculus
The vast majority of BrdU-positive cells that were generated on P1 migrated and were located in both the deep cerebellar nuclei (DCN) of the cerebellum (Figures 2E,F) and the Purkinje cell layer (Figures 2E,G)
We report that molecular markers for different cerebellar cell types are similar in marsupials and eutherian mammals; the lack of tropomyosin receptor kinase C (TrkC) signaling leads to unexpected effects in opossums

Summary

Introduction

In all mammalian species including marsupials, the cerebellum contains two hemispheres connected with a medially located structure, the vermis, and the laterally located flocculus and paraflocculus. The Purkinje cell is one of the largest neurons in the brain. Neurons of the deep nuclei and Purkinje cells are generated in the ventricular zone on embryonic day (E) 14 and E15, and granule cells proliferate from the caudal part of the ventricular zone known as the rhombic lip (Altman and Bayer, 1985a,b,c; Hausmann et al, 1985). Progenitor cells that proliferate in the rhombic lip migrate and create the second germinal zone called the external germinal layer (EGL). The EGL produces progenitor cells that migrate in the inner granular layer (IGL) and differentiate into neurons (Rakic and Sidman, 1970; Hatten and Heintz, 1995; Alder et al, 1996; Weisheit et al, 2006). The proliferation of cerebellar interneurons and granule cells that arise from the EGL lasts for over 3 weeks after birth

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