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)

Permanent URL: https://etd.library.emory.edu/concern/etds/7s75dd098?locale=en
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Abstract

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

2.3.7.1 Small Animal Species................................................................... 20

2.3.7.2 Large Animal Species.................................................................. 20

2.3.7.3 Unique Species............................................................................. 22

2.3.7.4 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

Appendix

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

References...................................................................................................................... 186

Figure Index

Figure 2.3-1. Preliminary Ex Vivo Scans of Rat Brains................................................. 27

Figure 2.3-2. Subsequent Ex Vivo Scans of Rat Brains with Refinements................. 28

Figure 2.3-3. Spectrum of Susceptibility Artifact on a 3 x 3 Mouse Brain Grid.......... 29

Figure 2.3-4. High-Throughput MRI Imaging of Mouse and Rat Brains................... 30

Figure 2.3-5. Refinement of the Embedding Container and Mouse Tumor Brains... 31

Figure 2.3-6. Application of Method to Large Animal Brains....................................... 32

Figure 2.3-7. Human Brain Sections and Rare and Unique Brains............................ 33

Figure 3.2-1. Pubmed Count, by year, of "Fear Conditioning" Literature.................... 55

Figure 3.2-2. Neurobiological Circuit of Fear Conditioning and Extinction................ 56

Figure 3.4-1. Neurobiological Circuit of Olfactory Fear Conditioning.......................... 57

Figure 3.7-1. Neurobiological IL/PL Circuit of Fear Conditioning and Extinction...... 58

Figure 4.4-1. MEMRI of Olfactory System Following Mn2+ Infusion into the Nose... 77

Figure 4.4-2. Imaging of Mn2+ Enhanced Signal throughout the Olfactory System... 78

Figure 4.4-3. Quantification of Mn2+ Enhanced Signal Across Brain Regions........... 79

Figure 4.4-2. DTI Probabilistic Tractography with Unilateral Olfactory Bulb Seed.... 80

Figure 4.4-3. Comparison of Results from MEMRI and Probabilistic Tractography.. 81

Figure 5.3-1. High-Throughput Layout Utilized to Scan 6 Brains Ex vivo................. 106

Figure 5.3-2. IL and PL MRI Seeds for DTI Tractography and BDA Injection Sites. 107

Figure 5.3-3. Tractography Data for the IL and PL Compared to Vertes (2004)...... 108

Figure 5.3-4. Relative IL and PL Connectivity............................................................. 110

Figure 5.4-1. Combined Results for IL to Amygdala and PL to Striatum................... 111

Figure 5.4-2. Comparison of the BDA Projections of IL/PL........................................ 113

Figure 5.4-3. Comparison of the BDA Projections of IL/PL........................................ 114

Table 5.4-1. Compilation of the Tractography Data for IL and PL............................... 115

Figure 6.3-1. Probabilistic Connections of the Visual Cortex and Auditory Cortex... 136

Figure 6.4-1. The Probabilistic Connections of the Human MGN/S.......................... 138

Figure 6.4-2. The Probabilistic Connections of the Mouse MGN/S........................... 140

Figure 6.4-3. The Anterograde Classical Tracing Results of the Mouse MGN/S..... 142

Table 6.4-1. Comparison of MGN/S Connectivity in Mice and Humans................... 144

Figure 7.3-1. Comparison of Thy1-YFP Signal between MR Imaged and Naïve Mouse.. 165

Figure 7.3-2. Auditory Fear Conditioning/High-Throughput Ex Vivo Structural MRI.. 166

Figure 7.3-3. VBM Analysis of Auditory Fear Conditioning........................................ 168

Figure 7.3-4. Dendritic Spine Density in the Auditory Cortex.................................... 170

Figure 7.3-5. Dendritic Spine Width, Length, and their Ratio in the Auditory Cortex... 171

Figure 7.3-6. Nuclei Density and Width Measurements in the Auditory Cortex...... 173

Figure 7.3-7. Correlations of VBM and Dendritic Spine/Nuclei Density Analysis.... 175

Figure 7.3-8. Schematic of Volume Changes with Dendritic Spine Changes........... 176

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