Vesicular monoamine transporter 2: a modulator of outcomes associated with posttraumatic stress disorder 公开
Cliburn Branco, Rachel (Summer 2018)
Abstract
Vesicular monoamine transporter 2 (VMAT2, SLC18A2) is a transmembrane transporter protein that packages monoamine neurotransmitters into vesicles in preparation for release from the presynaptic neuron. In this way, VMAT2 is a critical modulator of monoaminergic neurotransmission in the central nervous system. Our laboratory has generated a suite of tools to study VMAT2 function. We have generated transgenic strains of mice that express either 5% of wild type (VMAT2-LO mice) or 200% of wild type levels (VMAT2-HI mice) of VMAT2 protein. We developed a novel antibody to the VMAT2 protein and demonstrate that the antibody specifically recognizes VMAT2 and localizes to synaptic vesicles. We show the distribution of VMAT2 in monoaminergic regions of mouse brain, notably the midbrain, striatum, olfactory tubercle, dopaminergic paraventricular nuclei, tuberomammillary nucleus, raphe nucleus, and locus coeruleus. VMAT2 protein is also expressed in areas critical to the expression of fear behavior, the hippocampus and amygdala. Furthermore, immunohistochemical analysis confirmed that variation in VMAT2 gene dose results in variation in VMAT2 protein expression in these areas. We performed radioactive uptake to measure vesicular storage capacity, high performance liquid chromatography to measure monoamine metabolite content, and fast-scan cyclic voltammetry to measure stimulated dopamine release. We found that VMAT2-LO mice have reduced monoaminergic vesicular storage capacity in both the striatum and frontal cortex, show a pattern of reduced monoamine metabolites, and greatly reduced capacity to release dopamine upon stimulation. VMAT2-HI animals showed improved storage capacity, a pattern of increased monoamine metabolites, increased capacity to release dopamine upon stimulation, and altered dopamine release patterns following pharmacological blockade of the dopamine transporter. Since variation in SLC18A2, the gene that encodes VMAT2, confers risk for the development of PTSD, we used the VMAT2-transgenic mice to probe whether VMAT2 causally impacts fear behavior. VMAT2-LO mice showed increased cued and contextual fear expression, altered fear extinction, inability to discriminate threat from safety cues, and altered startle response compared to wild-type mice. Alternately, VMAT2-HI did not show changes in respect to wild-type mice in these behavioral assays. To test whether VMAT2 protein amount plays a causal role in deleterious non-fear symptoms associated with PTSD, we tested transgenic mice that represent a continuum of VMAT2 gene dose in a variety of sensory, affective, social, and appetitive assays. We found that VMAT2-LO mice tend to show a high-anxiety phenotype, but no deficits in sensory or social function. By contrast, VMAT2-HI mice are similar to wild-type mice in most assays, with some evidence of a reduced anxiety phenotype. These behavioral characterizations of VMAT2 transgenic mice may help characterize subsets of highly heterogeneous psychiatric disorders in humans. Together, these data corroborate and enrich the human genetic data showing that reduced VMAT2 expression impacts monoamine function and fear behavior. Since presynaptic monoamine function mediates PTSD-like outcomes in our mouse model, targeting this system may be an exciting strategy for future pharmacotherapies for disorders like PTSD.
Table of Contents
Abstract…………………………………...……………………….……2
Acknowledgements……………………...…………………….……….4
List of Figures....……………………………………………….………..9
I. Chapter 1. Introduction: Posttraumatic stress disorder, vesicular function, and the evidence for VMAT2 as a potential mediator of fear behavior ... 12
Background…………………………………………………….. 13
Rationale & Hypothesis………………………………………..33
II. Chapter 2. Immunological analysis of the expression of VMAT2 in VMAT2 transgenic mice…………37
Abstract…………………38
Introduction……………………39
Materials & Methods…………41
Results……………………………46
Discussion…………………………51
Figures………………………………55
III. Chapter 3. VMAT2 transgenic mice represent a continuum of monoaminergic function……………65
Abstract……………………………66
Introduction………………………67
Materials & Methods……………70
Results………………………………74
Discussion…………………………76
Figures………………………………81
IV. Chapter 4. VMAT2 gene dose mediates behavioral response to fear…………………86
Abstract……………………………87
Introduction………………………88
Materials & Methods……………91
Results………………………………95
Discussion…………………………97
Figures………………………………102
V. Chapter 5. The effect of VMAT2 gene dose on sensory, affective, social, and appetitive assays ……………......108
Abstract………………………………109
Introduction…………………………110
Materials & Methods………………113
Results…………………………………121
Discussion……………………………125
Figures…………………………………131
VI. Chapter 6: Discussion, Future Directions, and Concluding Remarks……………………139
VII. Appendix 1: The effect of D2 and D3 receptor blockade on cocaine-induced increase in dopamine release ……148
Abstract…………………149
Introduction……………150
Materials & Methods..152
Results…………………..153
Discussion……………..154
Figures…………………156
VIII. Appendix 2: Levodopa and dopamine dynamics in Parkinson’s disease metabolomics…..………161
Abstract………………162
Introduction…………163
Materials & Methods…………165
Results……………………168
Discussion………………171
Figures……………………179
IX. References…………197
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