Corticotropin-Releasing Factor Overexpression in the Central Amygdala: Gene Expression, HPA Axis Function, and Behavior Open Access

Martin, Elizabeth (2009)

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Mood and anxiety disorders including major depressive disorder and post-traumatic stress disorder have been associated with a disrupted hypothalamic-pituitary-adrenal (HPA) axis response to stress, attributed to corticotropin-releasing factor (CRF) overexpression in the paraventricular nucleus of the hypothalamus (PVN). However, PVN output is determined by summation of signals from limbic and brainstem sources; disruption in one of these regions may result in increased PVN CRF and thus HPA axis hyperactivity. Long-term gene expression changes which confer the chronic nature of these disorders may take place primarily in the PVN or may take place primarily in limbic structures, which then modulate the PVN. The utility of CRFergic circuits as pharmaceutical targets for the treatment of mood and anxiety disorders could be improved with greater knowledge of distinct, regionally-specific CRF expression patterns. The goal of this research is to develop tools to manipulate gene expression within CRF-producing cells. Here we describe a transgenic mouse in which 3.0Kb of the CRF promoter reliably targets transgene expression to CRF-producing neurons. The cell-type specificity of this promoter was also employed in a lentiviral vector to overexpress CRF from CRFergic cells. Because the CeA is known to influence the behavioral stress-response and hypothesized to play a role in HPA axis regulation, this virus was injected bilaterally into the CeA of adult male rats. Chronic CRF overexpression in the CeA increased expression of CRF and vasopressin in the PVN, leading to increased HPA axis activation, and decreased expression of MR in the hippocampus, resulting in HPA axis disinhibition. These gene-expression changes and HPA axis hyperactivity also resulted in an increase in anxiety-like behavior. These data suggest that HPA axis hyperactivity in human patients may be secondary to altered signals from CRF neurons within the CeA. This and future work elucidating the precise mechanisms through which overexpression of CRF precipitates psychopathology may provide useful preventative and therapeutic tools for mood and anxiety disorders.

Table of Contents

Chapter 1: General Introduction and Background

Page 1- Symptoms and Epidemiology Mood and Anxiety Disorders

  1. Major Depressive Disorder
  2. Anxiety Disorders
  3. Pharmacotherapy for Depression and Anxiety Symptoms

Page 6- Anatomy of Emotion

  1. The Limbic System
  2. Limbic System Disruptions in Mood and Anxiety Disorders

Page 12- Neurotransmission in Emotion

  1. Amino acid Neurotransmitters
  2. Monoamines
  3. Neuropeptides

Page 18- CRF Mediates the Endocrine, Autonomic, and Behavioral Response to Stress

  1. CRF and the Endocrine Response to Stress
  2. CRF and the Autonomic Response to Stress
  3. CRF and the Behavioral Response to Stress

Page 24- CRF in Psychopathology and Response to Antidepressant Drugs

  1. CRF in the Psychopathology of Mood and Anxiety Disorders
  2. CRF in the Mechanism of Action of Antidepressant Drugs

Page 31- Chapter 1 Tables and Figures

  • Figure 1-1: Papez's Limbic Circuit
  • Figure 1-2: The Limbic System in the Human Brain
  • Figure 1-3: The limbic system in the rat brain
  • Figure 1-4: Efferent Projections from the CeA
  • Table 1-1: Anatomy of Mood and Anxiety Disorders
  • Figure 1-5: Gs-mediated Signal Transduction Cascades
  • Table 1-2: Neuropeptides in Stress and Interactions with CRF
  • Figure 1-6: CRF Peptide Distribution in the Rat Brain
  • Figure 1-7: CRF Receptor Distribution in the Rat Brain
  • Figure 1-8: CRF in a Hypothesized Mechanism of Action of SSRIs

Chapter 2: Viral Vector and Transgenic Tools to Manipulate Gene Expression within CRF-Expressing Cells

Page 41- Introduction

Page 44- Materials and Methods

  1. Designing and Creating CRF-Cre Vectors
  2. Producing CRFp-Cre Virus
  3. Animal Subjects
  4. Histological Analysis
  5. Production and Testing of CRFp3.0Cre Transgenic
  6. Electrophysiological Analysis of Putative CeA CRF Neurons
Page 54- Results
  1. Assessment of Promoter Activity In Vitro
  2. Assessment of Promoter Selectivity In Vivo
  3. Generating a Transgenic Mouse with the CRFp3.0Cre Construct
  4. Crossing CRFp3.0Cre with Cre-Recombinase Reporter Strains
  5. Fluorescence-guided Electrophysiological Recordings
Page 58- Discussion
  1. Design of CRF-Cre Vectors
  2. Assessment of Promoter Selectivity In Vivo
  3. Fluorescence-guided Electrophysiological Recording

Page 62- Chapter 2 Figures and Tables

  • Figure 2-1: Design of LVCRFp3.0CRF
  • Figure 2-2: Cre-recombinase mechanism of action
  • Figure 2-3: In Vitro Functional Assay
  • Figure 2-4: In Vivo Functional Assay
  • Figure 2-5: Cre-Recombinase Expression in CRFp3.0Cre Transgenic Mice
  • Figure 2-6: Functional Cre-recombinase expression in CRFp3.0Cre-Rosa26 Mice
  • Figure 2-7: CRFp3.0Cre-Td/EGFP Reporter Transgenic Mice
  • Figure 2-8: Physiological properties of CRF-containing neurons in the CeA
  • Figure 2-9: Cis and Trans elements regulate CRF gene transcription

Chapter 3: Behavioral effects of lentiviral-vector mediated region and cell-type specific overexpression of CRF within CRF-producing cells of the central amygdala.

Page 71- Introduction

Page 73- Materials and Methods

  1. Design and Creation of the LVCRFp3.0CRF Construct
  2. Determining Virus Titer
  3. In vivo Functional Analysis of LVCRFp3.0CRF
  4. Animal Subjects
  5. Surgery and Injection of Virus
  6. Experiment 1 Behavior
  7. Experiment 2 Behavior
  8. Statistical Analysis
Page 81- Results
  1. In Vitro and In Vivo Functional Assay of LVCRFp3.0CRF
  2. Behavioral Effects of Chronic CRF-OE from the CeA
Page 84- Discussion Page 88 Chapter 3 Figures and Tables
  • Figure 3-1: LVCRFp3.0CRF Design
  • Figure 3-2: Timeline for Experiment 1
  • Figure 3-3: Timeline for Experiment 2
  • Table 3-1: Open Field Test in Experiment 1
  • Figure 3-4: Elevated Plus Maze in Experiment 1
  • Table 3-2: Additional measures of behavioral activity in the EPM
  • Figure 3-5: Defensive Withdrawal in Experiment 1
  • Table 3-3: Additional measures of behavior in the DW Test
  • Table 3-4; Forced Swim Test in Experiment 1
  • Table 3-5: Experiment 1 Sucrose Preference Tests
  • Table 3-6: Experiment 2 Sucrose Preference Test

Chapter 4: Effects of Region and Cell-Type Specific Overexpression of CRF within CeA CRF cells on HPA Axis Activity

Page 98- Introduction Page 101- Materials and Methods
  1. Animal Subjects
  2. Experiment 1 Endocrine Analysis
  3. Experiment 2 Endocrine Analysis
  4. ACTH and Corticosterone Radioimmunoassay
  5. Statistical Analysis
Page 103- Results
  1. Experiment 1 Endocrine Analysis
  2. Experiment 2 Endocrine Analysis
    1. Dexamethasone-Suppression Test
    2. CRF-Stimulation Test
    3. Dex/CRF Test
    4. Adrenal Gland and Body Weight
Page 106- Discussion
  1. Experiment 1
  2. Experiment 2
Page 110- Chapter 4 Figures and Tables
  • Figure 4-1: Experiment 1 Timeline and Experimental Design
  • Figure 4-2: Experiment 2 Timeline and Experimental Design
  • Figure 4-3: Dex/CRF Test in Experiment 1
  • Figure 4-4: Dex-Suppression Test in Experiment 2
  • Figure 4-5: CRF-Stimulation Test in Experiment 2
  • Figure 4-6: Dex/CRF Test in Experiment 2
  • Table 4-1: Experiment 2 Additional Endocrine Results and Outliers
  • Table 4-2: Experiment 2 Adrenal Gland and Body Weight

Chapter 5: Chronic CeA CRF-OE Alters Expression of Genes Involved in HPA Axis Regulation

Page 118- Introduction Page 120- Materials and Methods
  1. Animal Subjects
  2. Histological Processing
Page 123- Results
  1. Experiment 1 Gene Expression
  2. Experiment 2 Gene Expression
Page 125- Discussion
  1. Experiment 1 Gene Expression
  2. Experiment 2 Gene Expression
Page 130- Figure 5 Figures and Tables
  • Figure 5-1: Timeline and Experimental Design for Experiment 1 and 2
  • Figure 5-2: CeA CRF Transcript in Experiment 1 Subjects
  • Figure 5-3: PVN CRF Transcript in Experiment 1 Subjects
  • Figure 5-4: PVN AVP Transcript in Experiment 1 Subjects
  • Figure 5-5: Hippocampal GR Transcript in Experiment 1 Subjects
  • Figure 5-6: Hippocampal MR Transcript in Experiment 1 Subjects
  • Table 5-1: Experiment 1 Additional In Situ Hybridization Data
  • Figure 5-7: CeA CRF Transcript in Experiment 2 Subjects
  • Figure 5-8: PVN CRF Expression in Experiment 2 Subjects
  • Figure 5-9: PVN AVP Transcript in Experiment 2 Subjects
  • Table 5-2: Experiment 2 Additional In Situ Hybridization Data
  • Table 5-3: Experiment 2 Grubbs Test for Outliers

Chapter 6: General Discussion Page 141- CRFp3.0Cre Transgenic Mouse
  1. Summary and Conclusions
  2. Limitations and Methodological Considerations
  3. Continuing Progress
  4. Future Directions

Page 143- LVCRFp3.0CRF Lentiviral Vector

  1. Summary and Conclusions
  2. Limitations and Methodological Considerations
  3. Continuing Progress
  4. Future Directions
Page 148- Conclusion

Page 153- Chapter 6 Figures

  • Figure 6-1: Experimental Design for LVCRFp3.0CRF Experiment #3
  • Table 6-1: Hypothalamic Regulation
  • Figure 6-2: Limbic Networks Facilitate and Suppress HPA-Axis Activity

Appendix A

Keen-Rinehart, E, Michopoulos, V, Toufexis, DJ, Martin, EI, Nair H, Ressler KJ, Davis M, Owens MJ, Nemeroff CB, Wilson ME (2008). Continuous expression of corticotropin-releasing factor in the central nucleus of the amygdala emulates the dysregulation of the stress and reproductive axes. Molecular Psychiatry.

Page 156- Title Page Page 157- Abstract

Page 158- Introduction

Page 159- Materials and Methods

  1. Production and testing of recombinant lentiviral vectors
  2. Animals and Housing
  3. Surgical Procedures
  4. Monitoring Estrous Cycles
  5. Dexamethasone (DEX) suppression test
  6. Behavioral Tests
  7. Immunohistochemistry
  8. In situ hybridization

Page 166- Results

  1. Design of the Lenti-CMV-CRF vector and verification of CRF constitutive expression.
  2. Dysregulation of HPA axis
  3. Effects on emotionality
  4. Adverse consequences on reproductive parameters and sexual behavior

Page 168- Discussion

Page 174- Acknowledgements and Author Contributions

Page 175- Appendix A Figures and Tables

§Figure 1: Constitutive CRF Overexpression

§ Figure 2: Lenti-CMV-CRF injection into the CeA increased CRF protein

§ Figure 3: Lenti-CMV-CRF increased CRF and AVP protein expression

§ Figure 4: Lenti-CMV-CRF on the dexamethasone suppression test.

§ Figure 5: Anxiety- and depression-related behaviors.

§ Figure 6: Increased CRF produces disturbances in the rat estrous cycle

§ Figure 7: Increased CRF in the CeA decreases the number of GnRH cells.

§ Figure 8: Lenti-CMV-CRF on sexual behavior in ovariectomized rats.

Appendix B:

Martin EI and Nemeroff CB. 2008. The Role of Corticotropin-Releasing Factor in the Pathophysiology of Depression: Implications for Antidepressant Mechanisms of Action. Psychiatric Annals April 2008

Page 183- Title Page

Page 184- Text

Page 193- Appendix B Figures and Tables

  • Figure 1: ACTH (A) and cortisol (B) response to the combined Dex/CRF test
  • Figure 2: Rat endogenous CRF expression
  • Figure 3: Dose- related change in cerebrospinal fluid levels of CRF

Appendix C

Martin EI and Nemeroff CB. 2007. The Biology of Generalized Anxiety Disorder and Major Depressive Disorder: Commonalities and Distinguishing Features. In The Future of Psychiatric Diagnosis: Refining the Research Agenda Depression and Generalized Anxiety Disorder, American Psychiatric Institute Press

Page 196- Title Page

Page 197- Anatomy of GAD and MDD

1.Cortical activity is differentially disrupted in GAD and MDD in a subregion-specific manner.

2.Limbic and Paralimbic Regions are differentially disrupted in GAD and MDD

3.Distinct Cortical-Limbic Neural Networks mediate GAD and MDD.

Page 202- Neuroendocrinology

1.HPA axis hyperactivity characterizes MDD but not GAD.

2.The hypothalamic pituitary-gonadal axis in MDD and GAD.

3.HPT axis alterations are common in MDD but not in GAD.

Page 206- Neuropeptides

1.NPY is decreased in MDD and may be a neural correlate of resiliency to mood and anxiety disorders.

2.Cholecystokinin provokes panic and anxiety but not depression.

3.Galanin has depressogenic effects and may modulate anxiety.

Page 208- Neurotransmitters

Page 212- GAD and MDD are characterized by distinct somatic symptoms

Page 213- Summary and Implications for Research

Page 216- Acknowledgements Financial Disclosures

Page 217- Appendix C Figures and Tables

  • Table1: Neuroanatomical and neuroimaging studies in MDD and GAD
  • Table 2: Endocrine and NP disruptions in MDD and GAD
  • Table 3: Neurotransmitter abnormalities in MDD and GAD
  • Figure 1: Cortical Regions involved in GAD and MDD
  • Figure 2: Limbic and Paralimbic Regions involved in GAD and MDD
  • Figure 3: Hypothesized mechanism of action of SSRIs

Appendix D

Elizabeth I. Martin, Terrell E. Brotherton, Becky Kinkead, K.V. Thrivikraman, Paul M. Plotsky, Charles B. Nemeroff, Michael J. Owens. Diurnal Regulation of Gene Expression in Select Brain Regions of Adult Male Sprague Dawley Rats Society for Neuroscience Annual meeting, November, 2007

Page 223- Title Page

Page 224- Introduction

Page 224- Materials and Methods

  1. Sample Procurement
  2. Microarray
  3. Data Analysis

Page 225- Summary and Conclusions

Page 227- Appendix D Tables and Figures

Table 1: Number of Genes Detected

Table 2: Signal log ratio (SLR) of comparisons between AM and PM samples

Hippocampus Hypothalamus Septum Prefrontal Cortex Amygdala Page 232- Works Cited

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