During this early period of development, GABA- or glycine-mediated opening of Cl− channels causes membrane depolarization, as Cl− floods out of the cell. Although GABA and glycine are known to be important for nervous system development, researchers had not been able to confirm that early Cl−-mediated depolarization was essential for their effects. The authors therefore overexpressed human KCC2 in zebrafish embryos from the onset of development, to set up a 'reversed' Cl− gradient across the neuronal membranes. They examined the embryos between 26 and 32 hours after fertilization, before endogenous expression of KCC2 would normally begin. Overexpression of KCC2 resulted in gross morphological changes in the nervous system, including the absence of a number of major axonal tracts in the brain and spinal cord, accompanied by abnormal locomotor (swimming) behaviour.
The authors examined the expression of markers for newly differentiated neurons and determined that the defects in axonal growth were due to abnormalities in neurogenesis. There were fewer neurons overall and, in particular, fewer motor neurons and interneurons. Next the authors considered whether the reduced numbers of neurons resulted from altered proliferation, differentiation or cell death. Staining with acridine orange indicated that levels of apoptosis were normal, and levels of proliferation, as assessed using two mitotic markers, were also unaffected. Curiously, there was also no dramatic change in the glial population, indicating that the progenitors had not simply switched fate. Thus, the overexpression of KCC2 seems to have stalled the progenitors at some point before their final differentiation into neurons.
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