The Development and Application of Ex Vivo Magnetic Resonance Imaging Techniques to Understand the Neural Basis of Pavlovian Fear Conditioning and Extinction. Open Access

Keifer Jr, Orion Paul (2014)

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Pavlovian fear conditioning is the pairing of a neutral stimulus (conditioned stimulus) with an aversive stimulus (unconditioned stimulus) until the presentation of the neutral stimulus results in fear behaviors (conditioned response). Conversely, extinction is the process of presenting the conditioned stimulus until it no longer elicits fear behaviors. The power of these paradigms is the simplicity of execution and the translational validity with human physiology (emotional memory) and pathophysiology (fear and anxiety disorders). In fact, decades of cat, rat, and monkey fear conditioning studies have led to an anatomically constrained hypothetical fear circuit. Concomitantly, magnetic resonance imaging (MRI) allows for unparalleled study of the structure and function of the brain. While most MRI work is human focused, it is notable that MRI is one of a few research tools that can be directly translated between humans and experimental animals. This dissertation reports on the development, application, and validation of ex vivo MRI methods to study brain structure, especially in the context of the fear circuit in mice. First, connections of the olfactory bulb and the amygdala were evaluated using ex vivo diffusion weighted imaging (DWI) and were compared to the results from in vivo manganese enhanced MRI. Second, the projections of the infralimbic and prelimbic cortices were compared and contrasted using a combination of ex vivo DWI and anterograde tracing with biotinylated dextran amine (BDA). Third, the projections of the posterior thalamic group (medial geniculate, paraintralaminar, and suprageniculate nucleus) were analyzed with ex vivo DWI, and those results were validated using BDA tracing and were also compared to in vivo DWI results in humans. Fourth, ex vivo voxel-based morphometry analysis (VBM) was performed on structural scans of Thy1-YFP mice after auditory fear conditioning with followup confocal analysis of the dendritic density and morphology in areas of VBM significance. The results across all four approaches are broadly impactful as they validate the use of ex vivo MRI, refine underlying mechanisms of DWI and VBM signals, are a proof of concept for the translational utility of MRI, and provide much needed anatomical and structural evidence for the fear circuit in mice.

Table of Contents

Chapter 1: General Overview and Framework

1.1. An Overall Perspective on the Dissertation 2

1.1.1. The Particulars of the Technical Development 3

1.1.2. The Particulars of the Scientific Application 5

Chapter 2: The Development and Refinement of Ex vivo Magnetic Resonance Imaging

2.1. Context, Author Contribution's, and Acknowledgement of Reproduction 8

2.2. Introduction 8

2.3. Methods and Results 12

2.3.1. Practical Considerations 12

2.3.2. Brain Perfusions, Fixation, and Extraction 14

2.3.3. Evolution of the Embedding Procedure 15

2.3.4. Embedding Iteration 1 16

2.3.5. Embedding Iteration 2 17

2.3.6. Embedding Iteration 3 18

2.3.7. Application to Other Species and Modifications 20 Small Animal Species 20 Large Animal Species 20 Unique Species 22 Post-Mortem Human Brain Sections 22

2.4. Discussion 12

Chapter 3: A Review of the Pertinent Literature for the Relevant Neurobiology of Fear Conditioning and Extinction

3.1. Context, Author Contribution, and Acknowledgement of Reproduction 35

3.2. Introduction 35

3.3. A Brief Discussion of the Amygdala 38

3.4. Olfactory Fear Conditioning - Main Olfactory Bulb to the Amygdala 40

3.5. Auditory Fear Conditioning - Medial Geniculate Nucleus (and closely adjacent suprageniculate and posterior intralaminar nucleus) to the Amygdala 41

3.6. Auditory Fear Conditioning - The Auditory Cortex to the Amygdala 45

3.7. Fear Expression and Extinction - The Infralimbic and Prelimbic Cortex to the Amygdala 48

3.8. Discussion 52

Chapter 4: Mapping of the Mouse Olfactory System with Manganese-Enhanced Magnetic Resonance Imaging and Diffusion Tensor Imaging

4.1. Context, Author's Contribution, and Acknowledgement of Reproduction 60

4.2. Introduction 60

4.3. Methods 65

4.3.1. Animal Preparation and Imaging 65

4.3.2. Imaging Processing 67

4.4. Results 70

4.4.1. MEMRI Results 70

4.4.2. Probabilistic Tractography 71

4.4.3. Overlap of DTI and MEMRI 71

4.5. Discussion 73

4.6. Conclusion 75

Chapter 5: A DTI Tractography analysis of Infralimbic and Prelimbic Connectivity in the Mouse using High-throughput MRI

5.1. Context, Author's Contribution, and Acknowledgement of Reproduction 83

5.2. Introduction 83

5.3. Methods 88

5.3.1. Animals 88

5.3.2. Fixation Procedure 88

5.3.3. Mounting Procedure 88

5.3.4. Imaging Parameters 90

5.3.5. ROI Selection 91

5.3.6. DTI Registration 91

5.3.7. Probabilistic Tractography 93

5.3.8. Determining Connectivity of the IL/PL 93

5.3.9. Intra-mice Comparison of IL/PL Connectivity 94

5.4. Results 95

5.4.1. Probabilistic Tractography (PT) 95

5.4.2. IL Connectivity via PT 96

5.4.3. PL Connectivity via PT 97

5.4.4. Direct Contrast of IL and PL Connectivity-Preferential IL Connectivity 98

5.4.5. Direct Contrast of IL and PL Connectivity-Preferential PL Connectivity 99

5.4.6. BDA Anterograde Tracing of IL/PL Connectivity 100

5.5. Discussion 101

Chapter 6: A Comparative Analysis of Mouse and Human Medial Geniculate Nucleus Connectivity: A DTI and Classical Tracing Study

6.1. Context, Author Contribution's, and Acknowledgement of Reproduction 119

6.2. Introduction 119

6.3. Materials & Methods 122

6.3.1. Human Participants 122

6.3.2. Animal Studies 123

6.3.3. Magnetic Resonance Imaging - Human 124

6.3.4. Magnetic Resonance Imaging - Mouse 124

6.3.5. Diffusion Tensor Imaging Analysis 125

6.4. Results 127

6.4.1. Isolation of Human MGN/S 127

6.4.2. MGN/S Probabilistic Connectivity in Humans 127

6.4.3. MGN/S Probabilistic Connectivity in Mice 128

6.4.4. Mouse Anterograde Tracing Results 129

6.5. Discussion 129

Chapter 7: Parallel Macroscopic and Microscopic Increase in Brain Structure After Learning: Voxel-Based Morphometry Predicts Shifts in Dendritic Spine Density and Morphology with Auditory Fear Conditioning

7.1. Context, Author's Contribution, and Acknowledgement of Reproduction 146

7.2. Introduction 146

7.3. Results 149

7.3.1. Auditory Fear Conditioning 149

7.3.2. Magnetic Resonance Imaging - Voxel Based Morphometry Analysis 150

7.3.3. Confocal Microscopy - Dendritic Spine Density, Width, and Length 151

7.3.4. Confocal Microscopy - Nuclei/Cell Density 152

7.3.5. Correlational Metrics between VBM and Confocal Analysis 155

7.4. Discussion 156

7.5. Methods 161

7.5.1. Mice 161

7.5.2. Auditory Fear Conditioning 161

7.5.3. Magnetic Resonance Imaging 162

7.5.4. Voxel-Based Morphometry Analysis 162

7.5.5. Dendritic Spine Density and Morphology Analyses 163

7.5.6. Nuclei Density/Width Analysis 164

Chapter 8: Overall Discussion and Future Directions

8.1. General Discussion 179

8.2. Future Directions 181

8.2.1. Researching Mechanisms of MRI Techniques 181

8.2.2. Further Scientific Exploration of the Role of the Auditory Cortex 182

8.2.3. Further Comparative Work between Humans and Mice 182

8.2.4. Work with Collaborators to Expand the Use of Ex vivo Imaging 183


A.1 Scientific Publications Outside of the Scope of the Dissertation 185

References 186

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