Sex Steroid Hormone Regulation of Drug Reinforcement

描述（由申请人提供）：作为一名学生，博士后研究员和讲师，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，我研究了雌激素在控制女性生殖功能和脑损伤后神经保护作用的神经生物学。我在这两个领域的经历激发了我对理解成瘾性疾病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，药物滥用倾向的性别差异是有据可查的，越来越多的证据表明，雌激素增强药物强化。我最近观察到，新发现的雌激素受体GPR 30，这是一种GPCR，特异性地表达在腹侧被盖区（VTA）的多巴胺细胞中，该细胞投射到延髓核，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一个全新方向，也是我将致力于科学事业的主题。在指导奖期间，我将学习几种新的方法，以支持我作为一个药物滥用研究人员的发展。我将接受玛丽·克里泽博士和马克·怀特曼博士的培训。克里泽博士是一位技艺高超的神经解剖学家，她对大脑中与动机行为有关的区域的性类固醇受体的表达进行了开创性的观察。马克·怀特曼博士因开发了一种方法而闻名世界，该方法通过快速扫描循环伏安法对清醒动物的突触多巴胺水平进行真实的时间测量。我将在我的主要导师Saleem Nicola博士的协助下，正确设计和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。这将使我能够开发关于性类固醇激素在大脑中的作用的独立研究项目，这些项目不会与我的导师的实验室中进行的研究项目重叠。这项培训将在一个机构环境中进行，该环境具有出色的设施和研究人员之间合作互动的悠久历史。这些经验将有助于实现我的长期职业目标，即在大学或学术医学中心担任教员，在自己的实验室进行研究。我的长期研究目标是使用行为，电化学，分子和药理学技术相结合，以确定药物滥用倾向的性别差异的分子基础和神经回路。 拟议的研究计划测试的假设，即雌二醇（E2）通过提高突触多巴胺水平在丘脑核增强可卡因的强化，和GPR 30介导E2的这些行动。临床和临床前证据表明，卵巢类固醇激素，特别是雌激素，调节多巴胺神经传递，这可能与药物强化的性别差异有关，女性比男性更容易成瘾和复发。有充分证据表明，雌二醇和相关雌激素在大脑中具有快速作用，可以在几秒到几分钟内观察到，这表明在质膜上表达的雌激素结合分子参与介导细胞对雌二醇的反应。GPR 30是一种GPCR，最近在体外被证明可以介导雌二醇激活几种细胞信号通路，是介导雌二醇快速调节脑功能的潜在候选者。我的初步免疫组织化学研究表明，GPR 30是高表达的中脑多巴胺神经元。令人惊讶的是，卵巢类固醇对中脑边缘系统药物奖赏回路的影响知之甚少，特别是雌二醇快速调节突触多巴胺在丘脑核中的可用性的可能性。因此，提出的具体目标测试的假设，雌二醇通过GPR 30的行为，以提高突触多巴胺水平的突触在丘脑核可卡因的强化。拟开展的研究将采用免疫组织化学和束示踪相结合的方法，在体内给予雌二醇或GPR 30（G1）的特异性激动剂，并在体内敲低VTA中的GPR 30，以研究GPR 30在介导雌激素调节可卡因强化和多巴胺释放中的作用。目的1将使用GPR 30和酪氨酸羟化酶（多巴胺神经元的标记物）的双标记免疫细胞化学，结合逆行追踪来确定GPR 30表达神经元在皮质-中脑边缘多巴胺系统中的神经解剖分布。目的2将确定GPR 30激动剂G1是否模拟雌二醇增强可卡因强化的能力，如通过促进可卡因自我给药的获得所测量的，并增加在渐进比例时间表下服用可卡因的动机。然后，我们将评估E2/G1调制的可卡因在GPR 30基因敲低的动物使用体内RNA干扰靶向VTA。目的3将采用E2和G1的体内输注、延髓核中多巴胺释放的快速扫描循环伏安法测量和体内RNA干扰的组合来敲低VTA中的GPR 30，以测试GPR 30的激活响应于可卡因而特异性地调节延髓核壳中的紧张性和阶段性多巴胺释放的假设。这些研究将为我今后在药物成瘾方面的研究提供严格的训练。他们还将提供深入了解突触前调节多巴胺神经传递的一般分子机制，在神经核雌二醇，因此也可能揭示药物滥用倾向的性别差异的基础上。

DESCRIPTION (provided by applicant): 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.