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.