Rola receptorów neurotrofin TrkB i TrkC w rozwoju mózgu oposa Monodelphis domestica : praca doktorska

Tepper, Beata; Tepper, Beata

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Rola receptorów neurotrofin TrkB i TrkC w rozwoju mózgu oposa Monodelphis domestica : praca doktorska

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Title: Rola receptorów neurotrofin TrkB i TrkC w rozwoju mózgu oposa Monodelphis domestica : praca doktorska

Abstract:

The opossum, Monodelphis domestica is a small, omnivorous marsupial species from Brazil. Like other marsupials, opossums are born at an earlier stage of development than eutherians. Newborn opossums weighing only 100-120 mg, are at a developmental stage comparable to that of mice at day 12 after conception, and at 6 weeks of embryonic development in humans. Almost all brain structures, including the neocortex, develop within three weeks after birth, which is slower than in mice or rats. The slow and protracted postnatal development of the opossum brain allows to study early stages of the development in the mammalian brain structures.Neurotrophins and their receptors play an important role in processes involved in shaping the nervous system. They have been shown to be essential for division, migration, survival and differentiation of progenitor cells, as well as in synaptic plasticity which is crucial for learning and memory.The aim of the current study was to investigate the role of neurotrophin receptors, mainly TrkB, a specific receptor for brain-derived neurotrophic factor (BDNF) and TrkC, a specific receptor for neurotrophin 3 (NT-3) in the development of two brain structures, the neocortex and the cerebellum. The in vivo electroporation technique was used. The shRNA construct for the trkB or trkC gene was injected into the lateral ventricles of the opossum brain at postnatal day 7 (P), when deep layers of the cerebral cortex begin to form. The effect of injections on the development of neocortex in opossums was analyzed 2 or 5 days after electroporation. The inhibition of TrkB or TrkC receptors activity in the neocortex of opossums at P7, resulted in a reduction of the number of proliferating progenitor cells in vivo, which was not due to an increase in apoptosis. Moreover, the lack of TrkB or TrkC receptors activity stopped the migration and caused the arrest of newborn neurons in the intermediate zone, therefore, they could not penetrate the subplate zone.In order to study the role of neurotrophin receptors in development of the opossum cerebellum, first, developmental sequences and timing of different types of cerebellar cells generation were examined using BrdU injections into opossums at different ages (from newborn to adult). After a month or three, the immunohistochemical staining was performed to label BrdU cells of the cerebellum and detect specific cellular markers in various cell types. The presented study showed that Purkinje neurons and deep cerebellar nuclei cells are formed between P1-P5. Approximately three weeks later, the proliferation of granule cells started. Cerebellar cell cultures from P3 or P22 opossums were used to study the influence of the TrkC receptor on the development of Purkinje or granule cells, respectively. The plasmid shRNA targeting trkC was used to reduce TrkC receptors activity in cells that were cultured for the next 8 days after electroporation. Analysis of dendritic arbor structures showed that TrkC downregulation resulted in an increase in the number of dendrites and branching of Purkinje cells but had no effect on cerebellar granule cells. The presented results show that the selected neurotrophin receptors have a significant impact on the proliferation and migration of neocortical progenitor cells and development of the dendritic tree in Purkinje cells of the opossum cerebellum, demonstrating region-specific and cell-specific effects