The role of glycine receptors during cerebral cortical development

This study focuses on glycine receptors (GlyR) and explores their possible physiological functions on the control of the migration of cortical interneurons and the generation of projection neurons during brain development. Chapter one is the introduction and exposes the context for the experimental work presented in the subsequent chapters. This chapter begins with a description of the characteristics of the brain in terms of its anatomy, morphology, cellular structure and functionality. Then, it focuses on general events that occur during brain development and explains, in general terms, how the main characteristics of the brain emerge starting from cycling progenitors to functional neurons. Subsequently, it separately explains the development of the two major types of neurons present in the cortex, the projection neurons and the interneurons. Following that, there is an introduction to GlyR by describing their properties and physiological roles in the functioning of the central and peripheral nervous system. Finally, the most important studies that link GlyR to a possible role on the brain development are presented and discussed. Chapter two describes the materials and methods that were used during the experimental work. Chapter three presents the results of the study in relation to the effect of GlyR and interneuron development. Based on the literature we hypothesized that activation of GlyR expressed by interneurons can trigger a sequence of molecular events that ultimately contribute to their tangential migration in the forebrain. To test this hypothesis, we performed patch-clamp recordings on interneurons migrating in brain slice culture and demonstrated the functional expression of alpha two homomeric GlyR. Complementary electrophoretic analyses (western blot) and immunolabelings were performed to: 1) confirm the results and unravel the possible dynamic changes that occurred during development; 2) get an insight in the cellular distribution of the receptors. We further explored the possible effects of GlyR activation on cell migration by using time lapse experiments in slices or explant cultures in combination with ex-vivo electroporation. These experiments demonstrated that GlyR activation can change the speed of cell migration by affecting nucleokinesis (translocation of the nucleus towards the centrosome). Therefore, we assessed the effect of GlyR activation on two processes that have been associated with the regulation of nuclear translocation, the spontaneous calcium activity and the phosphorylation of myosin II. Altogether, our results support a mechanism by which GlyR activation depolarizes cell membrane activating voltage gated calcium channels that further contribute to increase intracellular calcium concentration. This increase in intracellular calcium contributes to myosin contraction at the rear of the cell which ultimately controls the frequency of nuclear translocations. Chapter four presents experimental results obtained during the study of dorsal progenitors and radially migrating projection neurons. Electrophysiological experiments were carried out to identify and characterize GlyR expressed by progenitors and radially migrating neurons at embryonic day 13 (E13). In addition, calcium imaging was performed to get some insight into the cellular and molecular mechanisms downstream GlyR activation. To understand the physiological relevance of GlyR on these cells, we assessed cell proliferation using pharmacological tools and shRNA-mediated knockdown. Finally, the last two chapters are devoted to a general discussion, the conclusions of the study, and perspectives.

Publication year:2013

Accessibility:Open