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Dendritic Development of Gabaergic Cortical Interneurons Revealed by Biolistic Transfection with GFP

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Dendritic Development of Gabaergic Cortical Interneurons Revealed by Biolistic Transfection with GFP Graduate Theses, Dissertations, and Problem Reports 2002 Dendritic development of GABAergic cortical interneurons revealed by biolistic transfection with GFP Xiaoming Jin West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Jin, Xiaoming, "Dendritic development of GABAergic cortical interneurons revealed by biolistic transfection with GFP" (2002). Graduate Theses, Dissertations, and Problem Reports. 1708. https://researchrepository.wvu.edu/etd/1708 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Dendritic Development of GABAergic Cortical Interneurons Revealed by Biolistic Transfection with GFP Xiaoming Jin Dissertation submitted to the School of Medicine at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Anatomy Ariel Agmon, Ph.D., Chair Albert Berrebi, Ph.D. Richard Dey, Ph.D. Peter Mathers, Ph.D. Adrienne Salm, Ph.D. Department of Neurobiology and Anatomy Morgantown, West Virginia 2002 Keywords: neocortex, GAD, gene gun, dendrite, growth, BDNF, neuronal activity, organotypic, brain slice, culture. ABSTRACT Dendritic Development of GABAergic Cortical Interneurons Revealed by Biolistic Transfection with GFP Xiaoming Jin Neurons using γ-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. Acknowledgments I would like to express my sincere gratitude to my graduate advisor Dr. Ariel Agmon for providing constant guidance, support and encouragement, and for promoting tremendous professional growth through the years of my graduate studies. His diligence and motivation have made a deep impression on me and will continue to motivate me in my future career. I would like to thank each of my committee members for always making themselves available, and for their kind support, guidance and encouragement. This work could not have been completed without their insight and help. In particular, I thank Dr. Peter Mathers for his collaboration on the whole research project, for generating the DNA constructs, and for his critical comments and suggestions on molecular biology and on the manuscripts. I gratefully acknowledge the help of Dr. Albert Berrebi with the immunocytochemistry method and for his critical comments on the data and the manuscripts. Many thanks go to Dr. Richard Dey and Dr. Adrienne Salm, for their help as graduate program coordinators during my graduate studies, for allowing me to use their lab facilities, and for their invaluable suggestions and comments on the research project. I would also like to thank Dr. Gabor Szabo for providing the GAD67 promoter. Appreciation is extended to Cary Johnson for excellent lab support, to Jeffery Altemus for technical support in confocal imaging, to Colette Ramsburg for preparing the DNA constructs, and to Jim Porter for help in patch-clamp recording. I would also like to thank Jason Wells, Yunyong Ma and Jen Leasa for their help and friendship in the lab. I thank my parents, my sister and brothers, for their understanding, support and encouragement. And most importantly, I’d like to thank my wife Jianhua Gao, for her ongoing understanding, contribution, love and support. iii Table of Contents Abstract………….……...…………………………...…………………………...……....ii Acknowledgments……...…………………………………………………………..……iii Table of contents………………….……………………………………………..………iv List of figures…………..………………………………………………………..………vi List of abbreviations…………….….…………………………………………..………vii Chapter 1: Introduction (Literature Review) A. GABAergic interneurons in the cerebral cortex……………………………………...02 1. Physiological functions of GABAergic cortical interneurons………………………...02 2. Pyramidal neurons and interneurons in the cerebral cortex…………………………...09 3. GAD67 and GAD65…….…………………………………………………………….11 4. Classification of GABAergic cortical interneurons…………………………………...13 5. Development of GABAergic cortical interneurons…………………………………...22 B. BDNF involvement in dendritic growth…………………………………………….26 1. BDNF as a neurotrophic factor………………………………………………………..26 2. Developmental and activity-dependent regulation of BDNF and trkB receptors……..29 3. Physiological roles of BDNF………………………………………………………….34 4. BDNF involvement in activity-dependent development of GABAergic interneurons..41 References…………………………………………………………………….………….50 Chapter 2: Biolistic Transfection with GAD67-GFP Reveals Functional GABAergic Interneurons in Organotypic Mouse Brain Slice Culture Abstract…………………………………………………………………….…………….82 Introduction………………………………………………………………….…………...83 Materials and Methods…………………………………………………………………...84 Results…………………………………………………………………….……………...88 Discussion…………………………………………………………………….………….91 References………………………………………………………………….……..…….101 Chapter 3: Vertical Bias in Dendritic Trees of Non-pyramidal Neocortical Neurons Expressing GAD67–GFP In Vitro Abstract………………………………………………………………………………....106 Introduction……………………………………………………..………….…………...107 Materials and Methods……………………………………………….…………………109 Results………………………………………………………….……………………….112 Discussion………………………………………………………….…………………...119 References…………………………………………………………………….………...140 Chapter 4: BDNF Mediates Activity-Dependent Dendritic Growth in Developing Non-pyramidal Neocortical Interneurons in Vitro Abstract………………………………………………………….……………………...152 Introduction……………………………………………………………….…………….153 Materials and Methods………………………………………………………………….154 Results………………………………………………………………….……………….160 iv Discussion…………………………………………………….………………………...167 References………………………………………………………………………………185 Chapter 5: General Discussion Discussion.………………………………………………..…………….…….…….…..196 References…………………………………………………………………….………...204 Appendix: Detailed Methods………………… ……………………………………...208 Curriculum Vitae……………………………………….……………………………..217 v List of Figures Chapter 1 Figure 1.1 Classification of GABAergic cortical neurons………………………………48 Figure 1.2 Synaptic contacts of GABAergic cortical interneurons……………………..49 Figure 1.3 Interneuron migration and neocortical development …..……………………50 Chapter 2 Figure 2.1 GAD67-GFP transfection revealed GABAergic neurons…………………...96 Figure 2.2 Action potential firing patterns of GAD67-GFP expressing neurons……….97 Figure 2.3 Spontaneous postsynaptic currents from GFP expressing neurons………….99 Figure 2.4 Putative unitary IPSCs generated by GFP expressing neurons…………….100 Chapter 3 Figure 3.1
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