Sex Steroid Hormone Regulation of Drug Reinforcement

作为学生、博士后和讲师，我研究了G蛋白偶联受体（gpcr）的分子生物学和信号转导机制。在过去的4年里，我研究了雌激素在女性生殖功能控制和脑损伤后神经保护中的神经生物学作用。我在这两个领域的经历激发了我对理解成瘾障碍和精神疾病的性别差异和激素控制的分子基础的兴趣。事实上，滥用药物的性别差异是有据可查的，而且越来越多的证据表明，雌激素会增强药物的效力。我最近观察到，新发现的雌激素受体GPR30是一种GPCR，它在投射到伏隔核的腹侧被盖区（VTA）的多巴胺细胞中特异性表达，这促使我研究这种GPCR在药物成瘾的激素调节中发挥作用的可能性。药物强化和成瘾的神经生物学研究是我职业生涯的全新方向，也是我将全身心投入科研生涯的课题。在指导奖励期间，我将学习一些新的方法，以支持我作为药物滥用研究人员的发展。我将接受Mary Kritzer博士和Mark Wightman博士的培训，Mary Kritzer博士是一位非常熟练的神经解剖学家，她对涉及动机行为的大脑区域的性类固醇受体的表达进行了开创性的观察，Mark Wightman博士因开发了通过快速扫描循环伏安法实时测量清醒动物突触多巴胺水平的方法而闻名于世。我的主要导师Saleem Nicola博士将协助我正确设计并进行可卡因自我给药实验。我的共同导师安妮·埃特根博士将继续就雌激素在大脑中的作用机制向我提供建议。这将使我能够开展独立的研究项目，研究性类固醇激素在大脑中的作用，而不会与我的任何一位导师的实验室正在进行的研究项目重叠。该培训将在一个拥有优秀设施和研究人员之间长期合作互动历史的机构环境中进行。这些经历将有助于实现我的长期职业目标，在我自己的实验室作为大学或学术医学中心的教员进行研究。我的长期研究目标是利用行为学、电化学、分子和药理学技术的结合，确定药物滥用倾向中性别差异的分子基础和神经回路。

As a student, postdoctoral fellow and instructor, I have studied the molecular biology and signal transduction mechanisms of G protein coupled receptors (GPCRs). Over the past 4 years, I investigated the neurobiology of estrogen action in the control of female reproductive function and in neuroprotection following brain insult. My experience in these two fields stimulated my interest in understanding the molecular basis of sex differences in and hormonal control of addictive disorders and psychiatric diseases. Indeed, sex differences in drug abuse liability are well documented, and there is growing evidence that estrogens potentiate drug reinforcement. My recent observation that the newly discovered estrogen receptor GPR30, which is a GPCR, is specifically expressed in dopamine cells of the ventral tegmental area (VTA) that project to the nucleus accumbens prompted me to examine the possibility that this GPCR plays a role in the hormonal regulation of drug addiction. The study of drug reinforcement and the neurobiology of addiction is an entirely new orientation for my career, and the topic to which I will devote my scientific career. During the mentored award period, I will learn several new methods needed to support my development as a drug abuse researcher. I will receive training from Dr. Mary Kritzer, a highly skilled neuroanatomist who has made seminal observations on the expression of sex steroid receptors in brain regions involved in motivated behaviors, and Dr. Mark Wightman, who is world renowned for the development of methods for real time measurement of synaptic dopamine levels by fast scan cyclic voltammetry in awake animals. I will be assisted by my primary mentor Dr. Saleem Nicola to properly design and carry out cocaine self administration experiments. My co- mentor, Dr. Anne Etgen will continue to advise me on mechanisms of estrogen action in the brain. This will equip me to develop independent research projects on the role of sex steroid hormones in the brain that do not overlap with the research projects going on in the lab of either of my mentors. This training will take place at an institutional environment with outstanding facilities and a long history of collaborative interactions among the research faculty. These experiences will facilitate achievement of my long term career objective, to conduct research in my own laboratory as a faculty member at a university or academic medical center. My long-term research goal is to determine the molecular basis and neural circuits that underlie sex differences in drug abuse liability using a combination of behavioral, electrochemical, molecular and pharmacological techniques. The proposed research plan tests the hypothesis that estradiol ( E2) enhances cocaine reinforcement by elevating synaptic dopamine levels in the nucleus accumbens, and that GPR30 mediates these actions of E2. Clinical and preclinical evidence indicates that ovarian steroid hormones, particularly estrogens, modulate dopamine neurotransmission, and that this may be relevant to sex differences in drug reinforcement, with females being more vulnerable than males to drug addiction and relapse. It is well documented that estradiol and related estrogens have rapid actions in the brain that can be observed within seconds to minutes, suggesting that estrogen binding molecules expressed at the plasma membrane participate in mediating cellular responses to estradiol. GPR30, a GPCR which was recently shown to mediate estradiol activation of several cell signaling pathways in vitro, is a potential candidate for mediating rapid estradiol regulation of brain functions. My preliminary immunohistochemistry studies show that GPR30 is highly expressed in midbrain dopamine neurons. Surprisingly little is known about the influence of ovarian steroids on the mesolimbic drug reward circuits, especially the possibility that estradiol rapidly modulates synaptic dopamine availability in the nucleus accumbens. Therefore, the proposed specific aims test the hypothesis that estradiol acts via GPR30 to enhance cocaine reinforcement by elevating synaptic dopamine levels in the nucleus accumbens. The proposed studies will employ a combination of immunohistochemistry and tract tracing, in vivo administration of estradiol or a specific agonist for GPR30 (G1) and in vivo knockdown of GPR30 in the VTA to investigate the role of GPR30 in mediating estrogen regulation of cocaine reinforcement and dopamine release in the nucleus accumbens. Aim 1 will use double-label immunocytochemistry for GPR30 and tyrosine hydroxylase, a marker of dopamine neurons, combined with retrograde tract tracing to identify the neuroanatomical distribution of GPR30-expressing neurons in the cortico-mesolimbic dopamine system. Aim 2 will determine whether the GPR30 agonist G1 mimics the ability of estradiol to potentiate cocaine reinforcement as measured by facilitation of the acquisition of cocaine self administration and to increase motivation to take cocaine under a progressive ratio schedule. We will then assess E2/G1 modulation of cocaine reinforcement in animals subjected to GPR30 knockdown using in vivo RNA interference targeted to the VTA. Aim 3 will employ a combination of in vivo infusions of E2 and G1, fast scan cyclic voltammetry measurement of dopamine release in the nucleus accumbens and in vivo RNA interference to knock down GPR30 in the VTA to test the hypothesis that activation of GPR30 modulates both tonic and phasic dopamine release specifically in the nucleus accumbens shell in response to cocaine. These studies will provide rigorous training for my future research in drug addiction. They will also provide insight into the general molecular mechanisms underlying presynaptic regulation of dopamine neurotransmission in the nucleus accumbens by estradiol and thus may also shed light on the basis of sex differences in drug abuse liability.