Date of Graduation
School of Medicine
Physiology, Pharmacology & Neuroscience
Neurons using gamma-aminobutyric acid (GABA) as a neurotransmitter are critically important as inhibitory neurons in the CNS. A major hindrance to further studies of this important and highly diverse class of neurons is their relative scarcity, which makes it difficult to identify and target them for morphological and physiological studies. To overcome this difficulty, we developed a novel protocol for visualizing detailed morphology of GABAergic neurons in living mouse brain slice cultures, using biolistic transfection with GAD67-GFP. The morphological, immunocytochemical and physiological characteristics of GAD67-GFP expressing neurons indicated that they were functional GABAergic neurons. Additionally, our data demonstrate that this technique allows patch-clamp recording of firing patterns and postsynaptic currents from GABAergic cortical interneurons, and unitary inhibitory postsynaptic currents from neurons postsynaptic to them. We imaged and reconstructed dendritic morphology of 150 GAD67-GFP expressing cortical interneurons in organotypic slice cultures. Using a novel computational metric of dendritic polarization, we demonstrated that dendrites of about two thirds of these GAD67-GFP expressing neurons were not randomly oriented, but instead showed a highly significant tendency to extend along the vertical axis of the cortex, indicating that GABAergic interneurons make an important contribution to the vertical bias of the neocortical neuropil. We further tested the hypothesis that brain derived neurotrophic factor (BDNF) is involved in activity-dependent dendritic growth of cortical GABAergic interneurons. By repeated confocal imaging of the same living GFP-expressing neurons at 5-day intervals, we demonstrated that both high KCl medium and BDNF enhance dendritic growth of GABAergic neurons, and that the enhancing effect of depolarization on dendritic growth is mediated by BDNF, while the BDNF effect is not dependent on neuronal activity. These results suggest that enhanced neuronal activity acts to promote dendritic growth of GABAergic interneurons by increasing expression and release of BDNF from neighboring neurons. Altogether, we demonstrated two modes of dendritic development in GABAergic cortical interneurons: vertically-oriented growth and activity-dependent growth, with each probably serving different functional requirements of the cerebral cortex. Our results also demonstrate that biolistic transfection with GAD67-GFP is an efficient, flexible and reliable technique for electrophysiological and morphological studies of living GABAergic interneurons.
Jin, Xiaoming, "Dendritic development of GABAergic cortical interneurons revealed by biolistic transfection with GFP" (2002). Graduate Theses, Dissertations, and Problem Reports. 1708.