Sex Steroid Hormone Regulation of Drug Reinforcement

作为学生、博士后和导师，我研究了G蛋白偶联受体(GPCRs)的分子生物学和信号转导机制。在过去的四年里，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方面的神经生物学作用。我在这两个领域的经验激发了我对了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，药物滥用责任的性别差异已经有了很好的记录，越来越多的证据表明雌激素增强了药物的强化作用。我最近观察到，新发现的雌激素受体GPR30是一种GPCR，在投射到伏隔核的腹侧被盖区(VTA)的多巴胺细胞中特异表达，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生涯的主题。在指导获奖期间，我将学习几种新的方法，以支持我作为一名药物滥用研究人员的发展。我将接受玛丽·克里策博士和马克·怀特曼博士的培训，前者是一位高技能的神经解剖学家，对与动机行为相关的大脑区域中性类固醇受体的表达进行了开创性的观察，后者以开发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世界。我将在我的主要导师萨利姆·尼古拉博士的帮助下，正确地设计和进行可卡因自我给药实验。我的合作导师Anne Etgen博士将继续就雌激素在大脑中的作用机制向我提供建议。这将使我有能力开发关于性类固醇激素在大脑中的作用的独立研究项目，这些项目不会与我两位导师的实验室正在进行的研究项目重叠。这项培训将在机构环境中进行，拥有出色的设施和研究人员之间长期合作互动的历史。这些经历将有助于我实现长期的职业目标，作为大学或学术医学中心的教员，在我自己的实验室进行研究。我的长期研究目标是利用行为、电化学、分子和药理学技术相结合的方法，确定药物滥用易感性性别差异的分子基础和神经回路。 拟议的研究计划测试了这样的假设，即雌二醇(E2)通过提高伏隔核中突触多巴胺的水平来增强可卡因的增强，而GPR30介导了E2的这些作用。临床和临床前证据表明，卵巢类固醇激素，特别是雌激素，调节多巴胺的神经传递，这可能与药物强化方面的性别差异有关，女性比男性更容易吸毒和复发。众所周知，雌激素和相关雌激素在大脑中具有快速的作用，可以在几秒钟到几分钟内观察到，这表明表达在质膜上的雌激素结合分子参与了细胞对雌二醇的反应。GPR30是一种GPR，在体外可介导多种细胞信号通路中雌二醇的激活，是一种潜在的介导雌二醇对脑功能的快速调节的候选基因。我的初步免疫组织化学研究表明，GPR30在中脑多巴胺神经元中高度表达。令人惊讶的是，人们对卵巢类固醇对中脑边缘药物奖励回路的影响知之甚少，特别是雌二醇快速调节伏核中突触多巴胺供应的可能性。因此，所提出的特定目标测试了雌激素通过GPR30作用于伏隔核内通过提高突触多巴胺水平来增强可卡因强化的假说。该研究将采用免疫组织化学和示踪相结合的方法，体内注射雌二醇或GPR30的特异性激动剂(G1)，以及在体内敲除VTA内的GPR30，以探讨GPR30在介导雌激素调节可卡因强化和伏隔核多巴胺释放中的作用。目的1利用GPR30和多巴胺神经元的标志物酪氨酸羟化酶双标记免疫细胞化学，结合逆行追踪，确定GPR30表达的神经元在皮质-中脑边缘多巴胺系统中的神经解剖分布。目的2将确定GPR30激动剂G1是否模仿雌激素增强可卡因的能力，通过促进可卡因的自我给药来衡量，并在递增比率时间表下增加服用可卡因的动机。然后，我们将使用针对VTA的体内RNA干扰来评估GPR30基因敲除动物中可卡因增强的E2/G1调节。目的3将结合体内注射E2和G1、快速扫描循环伏安法测量伏隔核多巴胺释放和体内RNA干扰敲除VTA中的GPR30来验证GPR30激活调节伏隔核壳中的强张性和相性多巴胺释放的假设。这些研究将为我未来在毒瘾方面的研究提供严格的培训。他们还将深入了解雌二醇对伏隔核多巴胺神经传递的突触前调节的一般分子机制，从而也可能揭示药物滥用易感性的性别差异。

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.