Mechanisms of Olfactory Fear Learning and Memory in Adult Mice Open Access

Abstract

A significant amount is known regarding the molecular mechanisms underlying the processing of emotional stimuli in the central nervous system; however, fewer studies have investigated the mechanisms accompanying emotional learning at the level of specific sensory modalities. This dissertation utilizes the olfactory system, whose primary sensory receptive field maps are exquisitely organized, respond dynamically to cues in the environment, and remain plastic from development through adulthood, to understand the mechanisms underlying fear learning and memory at the primary sensory system level. Using a transgenic mouse in which olfactory sensory neurons (OSNs) expressing the M71 odorant receptor (OR) may be visualized by LacZ histochemistry (M71-LacZ mouse line), we have previously demonstrated that olfactory fear conditioning leads to increased odorant-specific receptor representation in the main olfactory epithelium (MOE) and in glomeruli within the olfactory bulb (OB). This dissertation reports on the effects of cue-specific extinction, as well as on the mechanisms underlying the behavioral and structural plasticity of the olfactory system in mice following the acquisition of cued olfactory fear. We first demonstrate that olfactory extinction training specific to the conditioned odor stimulus reverses the conditioning-associated freezing behavior and odor learning-induced structural changes in the olfactory epithelium and olfactory bulb in an odorant ligand-specific manner. These data suggest that learning-induced freezing behavior, structural alterations, enhanced neural sensory representation, and histone modifications at the M71 locus can be reversed in adult mice following extinction training. Second, we used 5-Ethynyl-2'-deoxyuridine (EdU) labeling of MOE OSNs to understand the dynamics of OSN cell turnover as a function of fear learning. Finally, using intranasal or intraperitoneal (I.P.) administration of Tropomysin receptor kinase B (TrkB) agonists and antagonists prior to olfactory fear conditioning, we demonstrate a role for brain-derived neurotrophic factor (BDNF) signaling through its receptor, TrkB in mediating the structural plasticity accompanying olfactory fear learning. The studies contained in this dissertation provide novel evidence of the mechanisms underlying the behavioral and structural plasticity of the olfactory system in mice following the acquisition and extinction of cued olfactory fear, and contribute to a growing body of literature suggesting a critical role for primary sensory systems, particularly the olfactory system, in emotional learning and memory.

Table of Contents

Table of Contents CHAPTER 1: INTRODUCTION.......................................................................................... 1 Preamble ................................................................................................................................................1 Context, Author's Contribution, and Acknowledgement of Reproduction.................................1 Introduction...........................................................................................................................................2 PTSD as a disorder of fear dysregulation ........................................................................................3 Factors and stages underlying the development of PTSD.............................................................6 How we model fear responses in animals: classical Pavlovian fear conditioning ....................9 The fear response is a hardwired process involving the amygdala ........................................... 11 Odors can be potent and long lasting emotional and trauma memory cues............................ 14 The value of the olfactory system as a model to study learning and plasticity ........................ 16 Anatomy and circuitry of the olfactory system ............................................................................. 18 Olfactory signal transduction and upstream stages of olfactory processing: From the main olfactory epithelium to the olfactory bulb..............................................................................................18 Downstream olfactory processing: From the olfactory bulb to higher brain regions..............20 Adult neurogenesis in the olfactory system...........................................................................................21 Associative plasticity in the rodent olfactory system .................................................................... 22 Olfactory plasticity across development.................................................................................................22 Olfactory plasticity in adulthood...............................................................................................................24 Transgenerational and in utero olfactory plasticity ............................................................................ 27 Overview of Dissertation Goals:...................................................................................................... 30 CHAPTER 2: EXTINCTION REVERSES OLFACTORY FEAR CONDITIONED INCREASES IN NEURON NUMBER AND GLOMERULAR SIZE ........................ 39 Context, Author's Contribution, and Acknowledgement of Reproduction.............................. 39 Abstract ............................................................................................................................................... 39 Significance Statement ..................................................................................................................... 40 Introduction........................................................................................................................................ 40 Methods ............................................................................................................................................... 42 Animals .............................................................................................................................................................. 42 Olfactory fear conditioning, extinction and testing.............................................................................43 Freezing behavior data analysis.................................................................................................................46 Beta-galactosidase staining of the MOE OSNs and OB glomeruli ............................................... 46 Quantitation of M71-positive OSNs in the MOE ................................................................................ 46 Measurement of glomerular area in the olfactory bulb......................................................................47 Native Chromatin Immunoprecipitation (N-ChIP) on the MOE....................................................48 Statistics............................................................................................................................................................. 48 Results ................................................................................................................................................. 49 Behavioral responses following olfactory fear acquisition and extinction ................................. 49 Cue-specific olfactory extinction 3 weeks following acquisition reverses M71-specific neuroanatomical enhancements.................................................................................................................50 Cue-specific olfactory extinction immediately following acquisition blocks M71-specific neuroanatomical enhancements.................................................................................................................52 Olfactory fear acquisition and extinction are accompanied by a dynamic regulation of histone marks around the M71 locus ....................................................................................................... 53 Discussion........................................................................................................................................... 54 CHAPTER 3: REGULATION OF CELL SURVIVAL IN THE STRUCTURAL PLASTICITY ACCOMPANYING OLFACTORY FEAR CONDITIONING.........80 Context, Author's Contribution, and Acknowledgement of Reproduction.............................. 80 Introduction........................................................................................................................................ 80 Methods ............................................................................................................................................... 83 Animals .............................................................................................................................................................. 83 Olfactory fear conditioning, extinction and testing.............................................................................84 Freezing behavior data analysis.................................................................................................................84 Zinc Sulfate administration and experimental timelines...................................................................85 EdU administration and experimental timelines..................................................................................88 Perfusion............................................................................................................................................................ 89 Beta-galactosidase staining of the MOE OSNs and OB glomeruli ............................................... 90 Quantitation of whole mount M71-positive X-gal-labeled OSNs in the MOE ......................... 90 RNAscope investigation of the MOE......................................................................................................91 GFP, EdU, TUNEL and Caspase-3 immunohistochemical staining of the MOE ....................91 Quantitation of M71-positive and EdU-positive OSNs in the MOE.............................................92 Measurement of glomerular area in the olfactory bulb......................................................................92 Statistics............................................................................................................................................................. 93 Results ................................................................................................................................................. 93 RNAscope in the mouse MOE. ................................................................................................................. 93 Experiment 3.1: Time course of epithelial recovery following zinc sulfate (ZnSO4) induced ablation of the MOE......................................................................................................................................94 Experiment 3.2: Investigation of the OB glomeruli 2 days following zinc sulfate (ZnSO4) induced ablation of the MOE. .................................................................................................................... 94 Experiment 3.3: Olfactory fear conditioning 3 days post- zinc sulfate (ZnSO4) induced ablation of the MOE......................................................................................................................................95 Experiment 3.4: Olfactory fear conditioning followed 1 week later by zinc sulfate (ZnSO4) induced ablation of the MOE. .................................................................................................................... 96 EdU and TUNEL labeling of the mouse MOE.....................................................................................96 Experiment 3.5: Baseline labeling of M71-expressing OSNs using EdU...................................97 Experiment 3.6: Examining the proliferation of M71-expressing OSNs following olfactory fear conditioning.............................................................................................................................................98 Experiment 3.7: Examining the survival of M71-expressing OSNs following olfactory fear conditioning. .................................................................................................................................................... 99 Discussion.........................................................................................................................................101 CHAPTER 4: BDNF-TRKB SIGNALING IN OLFACTORY FEAR LEARNING AND MEMORY ................................................................................................................... 126 Context, Author's Contribution, and Acknowledgement of Reproduction............................126 Introduction......................................................................................................................................126 Brain-derived neurotrophic factor (BDNF) signaling mechanisms in PTSD and fear learning ........................................................................................................................................................................... 127 BDNF-TrkB signaling in the olfactory system.................................................................................. 129 Methods .............................................................................................................................................130 Animals........................................................................................................................................................... 130 Drug administration .................................................................................................................................... 131 Olfactory fear conditioning and testing ............................................................................................... 131 Freezing behavior data analysis.............................................................................................................. 135 Beta-galactosidase staining of the MOE OSNs and OB glomeruli ............................................ 135 Quantitation of M71-positive OSNs in the MOE ............................................................................. 135 Measurement of glomerular area in the olfactory bulb................................................................... 136 Statistics.......................................................................................................................................................... 136 Results ...............................................................................................................................................137 Experiment 4.1: Both I.P. and intranasal administration of the TrkB agonist 7,8-DHF prior to olfactory fear conditioning lead to increases in M71 structure............................................... 137 Experiment 4.2: Intranasal administration of the TrkB agonist 7,8-DHF prior to olfactory fear conditioning leads to increases in M71 structure, but no differences in freezing behavior, compared to vehicle-administered controls. ................................................................... 138 Experiment 4.3: Intranasal administration of the broad spectrum TrkB antagonist K252a prior to olfactory fear conditioning leads to decreases in M71 structure, but no differences in freezing behavior, compared to vehicle-administered controls. .................................................. 139 Experiment 4.4: I.P. administration of the TrkB antagonist ANA-12 prior to olfactory fear conditioning leads to decreases in M71 structure compared to vehicle-administered controls. ........................................................................................................................................................................... 140 Experiment 4.5: Intranasal administration of the TrkB antagonist ANA-12 prior to olfactory fear conditioning leads to decreases in M71 structure, but no differences in freezing behavior, compared to vehicle administered controls..................................................................... 141 Discussion.........................................................................................................................................142 CHAPTER 5: DISCUSSION............................................................................................. 161 Summary of results .........................................................................................................................161 Integration of findings....................................................................................................................164 Implications and future directions ...............................................................................................167 Associative plasticity in the human olfactory sensory system ..................................................... 168 Olfactory cues potent and salient trauma reminders and triggers in individuals with posttraumatic stress disorder (PTSD) ................................................................................................... 170 Clinical relevance of the olfactory system and its potential for inclusion in therapeutic treatment ......................................................................................................................................................... 173 Conclusion ........................................................................................................................................175 REFERENCES ..................................................................................................................... 176

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	Extinction Fear learning Olfaction
Committee Chair / Thesis Advisor	Ressler, Kerry, Emory University
Committee Members	Gourley, Shannon L, Emory University Zheng, James, Emory University Scott, John W, Emory University Jovanovic, Tanja , Emory University

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