Mechanisms of sudden onset of malignant MDMA neurotoxicity

描述（由申请人提供）：虽然娱乐性使用3,4-亚甲基二氧甲基苯丙胺（MDMA；“摇头丸”）通常耐受性良好，但一些使用者偶尔会受到严重和恶性神经毒性的影响，特别是在身体活动和温暖环境温度等外部因素的影响下。这种突然发生的恶性神经毒性反应不能简单地解释为药物过量的结果。由于身体活动和温暖的环境温度通常与恶性反应同时发生，因此假设这两个外部因素加剧了mdma诱导的神经毒性。尽管多年来这一概念一直是非常具有挑战性的，以调查这两个因素对MDMA的神经毒性作用的作用机制。在目前的建议中，我们开发了一种新的大鼠模型，使我们能够概括这两个外部因素的机制作用。为了更好地描述这个模型并理解机制，我们提出了几个假设，重点关注我们将测试的两个具体目标。目的1：探讨各外部因素及其联合作用在mdma神经毒性中的作用。具体来说，我们假设这两个外部因素的组合在加剧mdma诱导的神经毒性方面比单独一个更严重。动物将在身体活动、温暖的环境温度或两者结合的影响下接受检查。体温和脑电图（EEG）的变化将被确定为对娱乐相关剂量的MDMA的反应。我们预测，体力活动或温暖的环境温度本身对娱乐性相关剂量的MDMA诱导的神经毒性的严重程度几乎没有加剧作用。然而，这两种情况的结合会促使娱乐相关剂量的MDMA产生恶性神经毒性的突然发作，表现为高热、癫痫样脑电图活动甚至死亡。我们将测试5HT2AR对这些反应至关重要，并进一步测试外部因素导致5HT2AR活性的改变或适应，然后在这些条件下服用MDMA时促进神经毒性作用。目的2：表征5HT外排的变化，并测试神经毒性严重程度与继发性5HT外排和5ht2ar介导的信号转导的病理生理学相关性。我们将检验mdma产生的过量5HT外排在理论上可以被描述为两种成分的组成：分别与良性和恶性神经毒性相关的原发性和继发性5HT外排。为了验证这一假设，将在不同的实验条件下对动物进行检查，同时使用体内微透析测定前额皮质5HT。在初步结果的支持下，我们将检验三个相关的假设。1) MDMA使CNS内5HT的外源性外源性和依赖性增加。假设，外部因素无关的5HT外排主要是由于含有5HT的囊泡的耗竭（定义为初级外排），而外部因素依赖的5HT外排源于5ht2ar促进的神经回路（作为次级外排）。2)二次外排量与mdma诱导神经毒性的严重程度成正比。我们的假设预测了二次流出量的等级顺序为：暖环境温度下的体力活动>体力活动=仅暖环境温度>标准实验条件。3)我们将测试是否会突然发生恶性MDMA神经毒性，同时假设继发性原发性外排。为了进一步了解潜在的分子机制，我们将分析5HT2AR蛋白/mRNA表达和PLC活性在温暖环境温度下对身体活动的响应。总之，本提案描述了环境和MDMA神经毒性在娱乐相关剂量下的神经毒性之间相互作用的局部和电路机制。

DESCRIPTION (provided by applicant): Although recreational uses of 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy") are usually well- tolerated, some users are occasionally struck by severe and malignant neurotoxicity, particularly under the influence of external factors such as physical activity and a warm ambient temperature. Such a sudden onset of malignant neurotoxic responses cannot be explained simply as a result of drug overdose. Motivated by the fact that physical activity and warm ambient temperature are usually coincidental with malignant reactions, it is hypothesized that these two external factors exacerbate MDMA-induced neurotoxicity. Despite this notion for many years it has been very challenging to investigate the mechanism underlying the action of these two factors on neurotoxic effects of MDMA. In the current proposal, we have developed a novel rat model that allows us to recapitulate the mechanistic role of these two external factors. To better characterize this model and understand the mechanisms, we propose several hypotheses focused on two specific aims that we will test. Aim 1: To investigate the role of each external factor and their combination in the MDMA-induced neurotoxicity. Specifically, we hypothesize that the combination of these two external factors is more serious than each one alone in exacerbating the MDMA-induced neurotoxicity. Animals will be examined under the influence of physical activity, warm ambient temperature or their combination. Changes in body temperature and electroencephalography (EEG) will be determined in response to MDMA at recreationally relevant doses. We predict that physical activity or warm ambient temperature alone has little exacerbating effect on the severity of neurotoxicity induced by MDMA at recreationally relevant doses. However, the combined condition will promote recreationally relevant doses of MDMA to produce a sudden onset of malignant neurotoxicity, showing hyperthermia, seizure-like EEG activity and even death. We will test that 5HT2ARs are crucial for such reactions and further test that external factors cause an alteration or adaptation of 5HT2AR activity, which then promotes neurotoxic effects when MDMA is taken under these conditions. Aim 2: To characterize changes in 5HT efflux and test that the neurotoxic severity is pathophysiologically associated with secondary 5HT efflux and 5HT2AR-mediated signal transduction. We will test that MDMA-produced excessive 5HT efflux can be theoretically described as a composition of two components: primary and secondary 5HT effluxes in correlation with benign and malignant neurotoxicity, respectively. To test this hypothesis, animals will be examined under different experimental conditions while the prefrontal cortical 5HT will be determined using in vivo microdialysis. Supported by our preliminary results, three related hypotheses will be tested. 1) MDMA produces external factor-independent and - dependent increases in 5HT efflux in the CNS. Hypothetically, external factor-independent 5HT efflux is primarily due to depletion of 5HT-containing vesicles (defined as primary efflux), whereas external factor-dependent 5HT efflux is derived from a 5HT2AR-facilitated neural circuit (as secondary efflux). 2) The amount of secondary efflux is positively proportional to the severity of MDMA-induced neurotoxicity. Our hypothesis predicts that the rank order of the amount of secondary efflux will be: physical activity in the warm ambient temperature >physical activity =warm ambient temperature alone >standard experimental condition. 3) We will test that there will be a sudden onset of malignant MDMA neurotoxicity while hypothetical secondary primary efflux. To further understand the underlying molecular mechanisms, we will analyze changes in 5HT2AR protein/mRNA expression and PLC activity in response to physical activity in the warm ambient temperature. In summary, this proposal describes both local and circuitry mechanisms underlying the neurotoxic interaction between environments and MDMA neurotoxicity at recreationally relevant doses. PUBLIC HEALTH RELEVANCE: 3,4-Methylenedioxymethamphetamine (MDMA "ecstasy") neurotoxicity associated with serotonin (5HT) efflux is primarily due to depleting 5HT-containing vesicles. This effect depends on doses. However, a sudden onset of malignant MDMA neurotoxicity depends not only on doses but also physical activity and ambient temperature. How these two external factors influence MDMA-evoked neurotoxicity is not well understood. Using a rat model, this application proposes to test the hypothesis that increased physical activity in combination with warm ambient temperatures markedly enhances the responsivity of 5HT2ARs and subsequently a 5HT2AR-facilitated neural circuit. As a result, there is a great amount of secondary increase in 5HT efflux in the CNS due to the sensitized 5HT2AR- facilitated circuit and thereby promoting the neurotoxic effect of MDMA, which may be the mechanism underlying the sudden onset of malignant neurotoxicity at recreationally relevant doses that usually produce only a benign response.