Corticotropin-Releasing Factor Overexpression in the CentralAmygdala: Gene Expression, HPA Axis Function, and Behavior Público
Martin, Elizabeth (2009)
Abstract
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
Major Depressive Disorder Anxiety Disorders Pharmacotherapy for Depression and Anxiety SymptomsPage 6- Anatomy of Emotion
The Limbic System Limbic System Disruptions in Mood and Anxiety DisordersPage 12- Neurotransmission in Emotion
Amino acid Neurotransmitters Monoamines NeuropeptidesPage 18- CRF Mediates the Endocrine, Autonomic, and Behavioral Response to Stress
CRF and the Endocrine Response to Stress CRF and the Autonomic Response to Stress CRF and the Behavioral Response to StressPage 24- CRF in Psychopathology and Response to Antidepressant Drugs
CRF in the Psychopathology of Mood and Anxiety Disorders CRF in the Mechanism of Action of Antidepressant DrugsPage 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 SSRIsChapter 2: Viral Vector and Transgenic Tools to Manipulate Gene Expression within CRF-Expressing Cells
Page 41- IntroductionPage 44- Materials and Methods
Designing and Creating CRF-Cre Vectors Producing CRFp-Cre Virus Animal Subjects Histological Analysis Production and Testing of CRFp3.0Cre Transgenic Electrophysiological Analysis of Putative CeA CRF Neurons Page 54- Results Assessment of Promoter Activity In Vitro Assessment of Promoter Selectivity In Vivo Generating a Transgenic Mouse with the CRFp3.0Cre Construct Crossing CRFp3.0Cre with Cre-Recombinase Reporter Strains Fluorescence-guided Electrophysiological Recordings Page 58- Discussion Design of CRF-Cre Vectors Assessment of Promoter Selectivity In Vivo Fluorescence-guided Electrophysiological RecordingPage 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 transcriptionChapter 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- IntroductionPage 73- Materials and Methods
Design and Creation of the LVCRFp3.0CRF Construct Determining Virus Titer In vivo Functional Analysis of LVCRFp3.0CRF Animal Subjects Surgery and Injection of Virus Experiment 1 Behavior Experiment 2 Behavior Statistical Analysis Page 81- Results In Vitro and In Vivo Functional Assay of LVCRFp3.0CRF 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 TestChapter 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 Animal Subjects Experiment 1 Endocrine Analysis Experiment 2 Endocrine Analysis ACTH and Corticosterone Radioimmunoassay Statistical Analysis Page 103- Results Experiment 1 Endocrine Analysis Experiment 2 Endocrine Analysis Dexamethasone-Suppression Test CRF-Stimulation Test Dex/CRF Test Adrenal Gland and Body Weight Page 106- Discussion Experiment 1 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 WeightChapter 5: Chronic CeA CRF-OE Alters Expression of Genes Involved in HPA Axis Regulation
Page 118- Introduction Page 120- Materials and Methods Animal Subjects Histological Processing Page 123- Results Experiment 1 Gene Expression Experiment 2 Gene Expression Page 125- Discussion Experiment 1 Gene Expression 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 Summary and Conclusions Limitations and Methodological Considerations Continuing Progress Future DirectionsPage 143- LVCRFp3.0CRF Lentiviral Vector
Summary and Conclusions Limitations and Methodological Considerations Continuing Progress Future Directions Page 148- ConclusionPage 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 AKeen-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- AbstractPage 158- Introduction
Page 159- Materials and Methods
Production and testing of recombinant lentiviral vectors Animals and Housing Surgical Procedures Monitoring Estrous Cycles Dexamethasone (DEX) suppression test Behavioral Tests Immunohistochemistry In situ hybridizationPage 166- Results
Design of the Lenti-CMV-CRF vector and verification of CRF constitutive expression. Dysregulation of HPA axis Effects on emotionality Adverse consequences on reproductive parameters and sexual behaviorPage 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 CMartin 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 DElizabeth 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
Sample Procurement Microarray Data AnalysisPage 225- Summary and Conclusions
Page 227- Appendix D Tables and Figures
Table 1: Number of Genes DetectedTable 2: Signal log ratio (SLR) of comparisons between AM and PM samples
Hippocampus Hypothalamus Septum Prefrontal Cortex Amygdala Page 232- Works CitedAbout this Dissertation
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