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Regulation of Synapse Development by Activity Dependent Transcription in Inhibitory Neurons Regulation of Synapse Development by Activity Dependent Transcription in Inhibitory Neurons The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Mardinly, Alan Robert. 2013. Regulation of Synapse Development by Activity Dependent Transcription in Inhibitory Neurons. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:11151534 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA 2013 Alan Robert Mardinly All rights reserved Dissertation Advisor: Professor Michael E. Greenberg Alan Robert Mardinly Regulation of Synapse Development by Activity Dependent Transcription in Inhibitory Neurons Abstract Neuronal activity and subsequent calcium influx activates a signaling cascade that causes transcription factors in the nucleus to rapidly induce an early-response program of gene expression. This early-response program is composed of transcriptional regulators that in turn induce transcription of late-response genes, which are enriched for regulators of synaptic development and plasticity that act locally at the synapse. The main focus of this thesis is to describe the activity-induced transcriptional program in inhibitory neurons and identify its functional roles. We find strong similarity of the early-induced transcription factors induced by activity in excitatory and inhibitory neurons, but significant differences in the late-response genes. This finding suggests that commonly induced transcription factors may regulate distinct sets of target genes to perform cell-type specific functional roles. To test this hypothesis we focused on the immediate early transcription factor Npas4, which in excitatory neurons transcribes Bdnf to promote increased inhibition. While Npas4 is highly induced in response to activity in inhibitory neurons, Bdnf is not expressed, suggesting that Npas4 may perform a unique function in inhibitory neurons. Conditional deletion of Npas4 in somatostatin (SST) expressing inhibitory neurons causes a decrease in excitatory – but not inhibitory – synapses both in vitro and in vivo, showing that Npas4 functions to promote development of excitation onto SST neurons. This function is the reciprocal if its function in promoting inhibition to excitatory neurons, demonstrating that the functional role of activity- iii dependent transcriptional programs is uniquely adapted to the requirements of a specific cell-type within a circuit. To investigate the mechanisms by which Npas4 controls development of excitatory input to SST neurons, we identified Npas4 target genes in inhibitory neurons and found that Npas4 regulates a unique transcriptional program of activity-induced late-response genes distinct from the program that it regulates in excitatory neurons. Finally, we demonstrate that SST neurons in vivo induce expression of a subset of Npas4 target genes. These genes include Nptx2, which has a well-characterized role in clustering AMPA receptors specifically to non-spiny excitatory synapses, suggesting a possible mechanism by which activity-dependent transcription in inhibitory neurons mediated by Npas4 controls the development of excitation. vi TABLE OF CONTENTS Abstract………………………………………………………………………………………………………….iii Table of Contents…………………………………………………………………………………………….v List of Figures…………………………………………………………………………………………………vi Acknowledgements……………………………………………………………………………………….vii Collaborator Contributions……………………………………………………………………………..x Chapter 1: Activity-Dependent Transcription and GABAergic Neurons in the Nervous System……………………………………………………………………………………………….1 Chapter 2: Npas4 regulates excitatory input onto somatostatin positive inhibitory neurons through a unique activity-dependent transcriptional program……………………………………………………………………………………………………..…43 Chapter 3: General discussion…………………………………………………………………..…113 Chapter 4: In vivo analysis of synaptic puncta using Array Tomography….…124 Chapter 5: References………………………………………………………………………………...164 Appendix 1: Loss of inhibitory interneurons in the dorsal spinal cord and elevated itch in Bhlhb5 mutant mice…………………………………………………………..198 Appendix 2: Protocol for Generating Sphere Models in Imaris and Using AT Analyzer……………………………………………………………………………………………………...212 Appendix 3: AT Analzyer Code and Comments…………………………………………...216 Appendix 4: Technical Notes to Supplement Published Array Tomography Protocols……………………………………………………………………………………………………..242 v List of Figures Figure 2.1: Neuronal cultures enriched for inhibitory and excitatory neurons………47 Figure 2.2: Neuronal culture systems to assay cell-type specific inducible transcriptional programs……………………………………………………………………………………51 Figure 2.3: The transcriptional program induced by activity in inhibitory neurons…………………….……………………………………………………………………………………….53 Figure 2.4: Npas4 is induced in inhibitory neurons by neuronal activity………………59 Figure 2.5: Npas4 is expressed in multiple inhibitory neuron subtypes………………..61 Figure 2.6: Selective removal of Npas4 from SST neurons in dissociated E16.5 cortical cultures…………………………………………………………………………………………………65 Figure 2.7: Deletion of Npas4 in SST neurons in vitro results in decrease density of excitatory, but not inhibitory, synapses……………………………………………………………….67 Figure 2.8: Selective deletion of Npas4 from SST neurons in vivo………………………...70 Figure 2.9: Npas4 regulates the development of functional excitatory synapses onto SST positive inhibitory neurons………………………………………………………………………….72 Figure 2.10: Selective deletion of Npas4 from SST neurons does not affect mIPSCs or event kinetics…………………………………………………………………………………………………….75 Figure 2.11: Npas4 regulates a cell-type specific activity induced transcriptional program in inhibitory neurons……………………………………………………………………………82 Figure 2.12: Cell-type specificity of Npas4 MGE target genes……………………………….85 Figure 2.13: Npas4 target genes are induced by physiological stimulation in SST neurons in vivo…………………………………………………………………………………………………..88 Figure 2.14: Narp is induced in SST neurons in response to activity…………………….92 Figure 3.1: Npas4 induces a unique transcriptional program in MGE-derived cultures……………………………………………………………………………………………………………116 Figure 3.2: Selective deletion of Igf-1 in VIP positive inhibitory neurons increase mEPSC frequency……………………………………………………………………………………………..121 Figure 4.1: Alignment and validation of synaptic marker staining on serial array tomography ribbons…………………………………………………………………………………………128 Figure 4.2: Volume rendering of 3D Array Tomography data for quantification of synaptic puncta………………………………………………………………………………………………..135 Figure 4.3: AT Analyzer produces unbiased, automated analysis of volume-rendered 3D array tomography data……………………………………………………………………………..…138 Figure 4.4: AT Analyzer method detects large developmental changes in synapse density…………………………………………………………………………………………………………….142 Figure 4.5: Array Tomography analysis of Ube3a knockout mice……………………….146 Figure 4.6: Array Tomography analysis of Ube3a knockout CA3 reveals elevated PSD-95/Synapsin co-clusters……………………………………………………………………………149 Figure 4.7: Array Tomography analysis of Ephexin 5 knockout CA1 reveals elevated synapse density………………………………………………………………………………………………..152 Figure 4.8: Array Tomography reveals that CR3 and C3 knockout mice have elevated synapse density in the Lateral Geniculate Nucleus……………………………….156 vi ACKNOWLEDGEMENTS “We have to watch ourselves most carefully. The real preparation for education is a study of one's self. The training of the teacher who is to help life is something far more than a learning of ideas. It includes the training of character; it is a preparation of the spirit." Montessori, The Absorbent Mind This PhD is in a narrow sense a record, or catalogue, of the work that I have performed over the last six years, but the contents of no thesis are ever truly reflective of the nature of the labor that went into producing it. It is perhaps the curse of modern biology that the conceptual ideas are relatively simple compared to the long, laborious, and technically demanding experiments demanded to test them. Therefore doctoral training is only partially a purely intellectual challenge; the demands placed upon the trainee and the advisor are extended, to quote Montessori, to “the training of character...the preparation of the spirit.” It has been my good fortune to train with Dr. Michael Greenberg, who understands this aspect of mentorship more than anybody I have ever encountered. I stand in awe of Mike’s capacity for clarity and incisiveness in the face of complexity, and I would consider my training a success if I have made even a small fraction of this capacity my own. Equally as important is his capacity for patience, understanding, and to allow people to come to their own conclusions. Even more than his great capacity for teaching how to think, Mike has a gift for teaching how to live life within science, how to behave, to persevere, be generous, and learn the appropriate lessons from adversity. I owe any success in my future scientific pursuits to his mentorship. I have been very fortunate to be guided throughout my graduate school career by a supportive and generous group of faculty advisors. Chinfei Chen has been a great mentor, confidant, and friend. She generously took me into her lab at the start of graduate school, and
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