Cell-Type Specific Behavioral and Molecular Characterization of Fear Controlling Amygdala Sub-Populations Público
McCullough, Kenneth (2017)
Abstract
Behavioral and molecular characterization of cell-type specific populations governing fear learning and related behaviors is a promising avenue for the identification of more targeted therapeutics for the treatment of fear-related disorders such as Posttraumatic Stress Disorder. In the amygdala, a number neuronal of sub-populations within previously identified nuclei have been identified by their distinct mRNA and protein expression profiles. These sub-populations appear to differentially regulate fear behaviors by supporting the learning and expression of fear or by inhibiting fear expression and supporting fear extinction. Here, we approach the identification, behavioral characterization and molecular characterization of potentially behaviorally relevant amygdala sub-populations in three ways. First, we perform an in-depth analysis of the expression and co-expression of six mRNA markers that may relate to behaviorally relevant functional sub-populations within the central amygdala (CeA). Second, we perform an in-depth behavioral and molecular characterization of one such CeA population, the dopamine receptor 2 (Drd2) expressing population. Third, we behaviorally and molecularly characterize a fear inhibiting population, the Thy-1 population, found within the basolateral amygdala (BLA).
In our characterization of CeA sub-populations, we find that within the lateral compartment of the central amygdala (CeL), Somatostatin (Sst), Tachykinin (Tac2) Neurotensin (Nts) and Corticotropin releasing factor (Crf) mRNAs mark a single convergent population; however, within the medial compartment of the central amygdala (CeM) these RNAs mark independent populations. Additionally, protein kinase C delta (Prkcd) and Drd2 mRNAs mark non-overlapping sub-populations within the capsular compartment of the central amygdala (CeC) and CeL. Further characterization of the CeA Drd2 population identifies this population as a fear-supporting population whose activity is sufficient to enhance fear expression and block fear extinction. Cell-type specific characterization of actively translating RNAs using translating ribosome affinity purification (TRAP) reveals that Sst5r, Npy5r, Fgf3, ErbB4, Fkbp14, Dlk1, Ssh3 and Adora2a are each differentially regulated within Drd2 neurons following fear conditioning. Pharmacological manipulation of Drd2 neurons through D2R and A2AR recapitulate fear-supporting profile observed with direct chemogenetic activation. Finally, using optogenetics and chemogenetics we identify the Thy-1 population of the BLA as a fear-inhibiting population. Isolation and sequencing of RNA from Thy-1 neurons reveals Ntsr2, Dkk3, Rspo2, and Wnt7a as being upregulated in Thy-1 neurons compared to other amygdala neurons. Pharmacological activation of NTSR2 is sufficient to recapitulate fear-suppression profile observed using direct manipulations of this population. Our efforts have clarified the identities of sub-populations of the CeA and provided important molecular characterizations of crucial fear-controlling populations of the CeA and BLA.Table of Contents
Chapter Index
Chapter 1: Introduction: Bridging the Gap: Towards a Cell-Type Specific Understanding of Neural Circuits Underlying Fear Behaviors. 16
1.1 Context, Author's Contribution and Acknowledgement of Reproduction. 17
1.2 Abstract. 17
1.3 Introduction.. 18
1.4 Background on Circuitry and Fear. 20
Pavlovian Conditioning. 20
Fear learning: Basic Circuitry and Key Players. 21
1.5 Optogenetic Tracing of Fear Circuitry. 24
Inputs to Lateral Amygdala. 26
Studies focused on Basolateral Amygdala. 27
Studies Focused on Medial Prefrontal Cortex. 29
The Central Nucleus of the Amygdala. 30
The Intercalated Cell Masses. 31
Bed Nucleus of the Stria Terminalis. 32
1.6 Search for the Memory Engram... 33
1.7 Cell Type Specific Targeting of Behavioral Processes. 36
Differential Molecular Markers of Central Amygdala Cell Types: PKCd, Sst, and Tac2 39
The Parabrachial Nucleus and Calcitonin Gene-Related Peptide. 42
BLA Inhibitory Neuronal Sub-Populations: PV and SOM... 43
Thy1-Population of Pyramidal BA Neurons. 44
Hypothalamic Sub-Populations: OT, ESR1, SF1. 45
Alternative Targets. 47
1.8 Cell-Type Specific Transcriptome Sequencing. 48
1.9 Summary. 51
Chapter 2: Co-Expression Analysis of Prkcd, Sst, Tac2, Crf, Nts and Drd2 Sub-Populations Within the Central Amygdala. 57
2.1 Context, Author's Contribution and Acknowledgement of Reproduction. 58
2.2 Abstract. 58
2.3 Introduction.. 59
2.4 Results. 62
Distribution of Labeled Cells. 63
Prevalence of Labeled Cells. 64
Co-localization of CeA Markers. 64
2.5 Discussion.. 67
2.6 Methods. 70
RNA Scope Staining. 70
Image Acquisition.. 71
Data Analysis. 71
Statistical Analysis. 71
2.7 Figures. 73
Chapter 3: Behavioral and Molecular Characterization of Central Amygdala Dopamine Receptor 2 Expressing Neurons' Role in Fear Behavior. 84
3.1 Context, Author's Contribution and Acknowledgement of Reproduction.. 85
3.2 Abstract. 85
3.3 Introduction.. 86
3.4 Results. 89
Examination and Cell-Type Specific Manipulation of CeA Drd2 Population. 89
Characterization of Dynamic mRNA Changes in Drd2 Cells After Fear Conditioning. 91
Manipulation of A2A Receptor During Fear Behavior. 92
Examination of Role of D2R in Fear Learning and Extinction. 93
Dynamic Regulation of Drd2 After Fear Extinction. 94
Examination of Role of D2R in Consolidation of Extinction Learning. 95
3.5 Discussion.. 96
3.6 Methods. 99
Animals. 99
Surgical Procedures. 100
Drug Administration.. 100
Behavioral Assays. 101
Auditory Cue-Dependent Fear Conditioning. 101
Auditory Cue-Dependent Fear and Extinction.. 101
Open Field. 101
Brain Collection Following Behavior. 101
Real Time PCR.. 102
RNA-Seq Library Preparation.. 102
Analysis of RNA Sequencing Data. 103
Translating Ribosome Affinity Purification.. 103
Statistics. 103
RNA Scope Staining. 104
Image Acquisition.. 104
3.7 Figures. 105
Chapter 4: Molecular Characterization of a ‘Fear-Off' Neuronal Population within the Basolateral Amygdala. 116
4.1 Context, Author's Contribution and Acknowledgement of Reproduction. 117
4.2 Abstract. 117
4.3 Introduction.. 118
4.4 Results. 120
Thy1 Marks Consistent Population of BLA Neurons. 120
Separate Neuron Populations Active During Fear Processes. 122
Electrophysiological Characterization of Thy1-eNpHR Neurons. 123
Optogenetic Silencing of Thy1 Neurons. 123
Chemogenetic Activation of Thy1 Neurons. 125
Isolation and Molecular Characterization of Thy1 Neurons. 126
Pharmacological Manipulation of Neurotensin Receptor 2. 129
Examination of Projection Patterns of BLA Thy1-Cre Neurons. 131
4.5 Discussion.. 133
4.6 Methods. 136
Animals. 136
Surgical Procedures. 136
Laser Delivery. 137
Drug Administration.. 138
Behavioral Assays. 138
Auditory Cue-Dependent Fear Conditioning. 138
Auditory Cue-Dependent Fear Expression and Extinction.. 138
Behavioral Tests For c-fos Expression Experiments. 139
Open Field. 139
Dissociation of Amygdala Tissue for FACS. 139
Immunolabeling Cell Suspension for FACS. 140
Flow Cytometry. 140
Real Time PCR.. 141
Immunohistochemistry. 141
RNA-Seq Library Preparation.. 142
Analysis of RNA Sequencing Data. 142
Statistics. 143
Analysis of BLA Thy1-Cre Projections. 143
4.7 Figures. 144
4.8 Supplemental Discussion.. 155
4.9 Supplemental Figures. 161
Chapter 5: Conclusion, Discussion and Future Directions. 176
5.1 Summary of Results. 177
5.2 Integration of Findings. 179
5.3 Future Directions. 180
References. 185
Figure Index
Figure 1‑1. Neural circuits involves in fear and anxiety-related behaviors in rodents. 52
Figure 1‑2. Microcircuits and specific neuronal populations in the amygdala, ventromedial hypothalamus (VMH) and parabrachial nucleus (PBN) involved in fear and anxiety-related behaviors. 53
Figure 2‑1. Distribution of examined mRNAs across CeA sub-compartments. 73
Figure 2‑2. Co-expression of Sst, Tac2 and Prkcd (A-P -1.5). 74
Figure 2‑3. Co-expression of Crf, Nts and Prkcd (A-P -1.5). 75
Figure 2‑4. Co-expression of Sst and Nts, and Crf and Tac2 (A-P -1.5). 77
Figure 2‑5. Co-expression of examined mRNA's in anterior CeA (A-P -.9, -.8, and -1.22). 80
Figure 3‑1. Examination and cell-type specific manipulation of CeA Drd2 population. 105
Figure 3‑2. Examination of cell-type specific mRNA changes after fear conditioning. 107
Figure 3‑3. Selective blockade of A2AR blunts fear expression and enhances extinction consolidation. 109
Figure 3‑4. Blockade of Drd2 during extinction with common psychosis and MDD treatment, Sulpiride, enhances fear expression and within session extinction. 110
Figure 3‑5. Dynamic role of Drd2 during fear extinction. 112
Figure 4‑1. Thy1 lines mark BLA population that is active during expression of fear extinction. 144
Figure 4‑2 Halorhodopsin inhibition of BLA Thy-1 neurons. 146
Figure 4‑3. In Vivo inhibition of Thy1 neurons. 148
Figure 4‑4. Enhancing excitability of BLA Thy1 neurons using DREADDs. 150
Figure 4‑5. Workflow describing FACS sorting and sequencing of RNA of Thy1-eYFP cell bodies Cell-type specific RNA sequencing and identification of differentially regulated gene transcripts. 151
Figure 4‑6. Molecular characterization of basolateral amygdala Thy1 neurons. 153
Figure 4‑7. Modulating Neurotensin Receptor 2 activity alters fear expression and consolidation. 155
Figure 5‑1. Summary of Findings. 179
Supplemental Figure Index
Supplemental Figure 2‑1. Comparison of mouse brain atlases. 83
Supplemental Figure 3‑1. Fear conditioning of mice for TRAP collection. 114
Supplemental Figure 3‑2. Validation of TRAP pull-down. 114
Supplemental Figure 3‑3. Sulpiride effects on fear extinction rate. 115
Supplemental Figure 4‑1. Schematic of fiber optic fiber tip placement. 161
Supplemental Figure 4‑2. Genetic effects are not responsible for changes in fear expression of Thy1-eNpHR mice. 162
Supplemental Figure 4‑3. Flow chart of strategy for analysis of RNA sequencing differential expression data. 163
Supplemental Figure 4‑4. Replication of RNA sequencing results with qPCR. 164
Supplemental Figure 4‑5. Co-localization of Thy1-eYFP with additional differentially expressed genes. 165
Supplemental Figure 4‑6. Quantification of co-localization between Thy1-eYFP and additional proteins of interest. 166
Supplemental Figure 4‑7. Regional similarities in Thy1-eYFP, NTSR2, and DKK3 expression. 167
Supplemental Figure 4‑8. Differences in fear behavior after drug delivery are not due to anxiety like behavior after drug administration. 168
Supplemental Figure 4‑9. Infusion of AAV-DIO-YFP into Anterior BLA Thy1-Cre mouse. 169
Supplemental Figure 4‑10. Infusion of AAV-DIO-YFP into BLA Thy1-Cre mouse. 171
Supplemental Figure 4‑11. Infusion of AAV-DIO-YFP into Posterior BLA Thy1-Cre mouse. 172
Supplemental Figure 4‑12. Infusion of BDA into Anterior BLA. 173
Supplemental Figure 4‑13. Infusion of BDA into Posterior BLA. 174
Supplemental Figure 4‑14. Regional specificity of cre-recombinase mediated mCherry expression. 174
Supplemental Figure 4‑15. Double transgenic Thy1-eYFP/ Thy1-Cre mice have red-shifted expression in Thy1-eYFP neurons. 175
Index of Tables
Table 1.1 Descriptions of publications using optogenetics to query basic fear-related circuitries 54
Table1.2 Descriptions of publications using cell-type specific methodologies to query fear related circuitry. 56
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