Rui Tao, Ph.D. - US grants

DESCRIPTION (provided by applicant): The overall objective of this project is to understand the physiological and pathological mechanisms of opioid-mediated plasticity in the neural circuitry controlling serotonin (5-HT) release in the mammalian forebrain, and thereby provide a sound basis for understanding and treatment of psychiatric disorders such as depression and drug addiction. The experimental approach combines behavioral measures with microdialysis in the CNS of freely behaving rats. We propose that mu, delta, kappa and ORL-1 opioids have different roles in regulation of 5-HT release. By reverse microdialysis infusion of selective opioid receptor agonists into the dorsal raphe nucleus (DRN) we will test the specific hypothesis that mu- and delta-opioids produce increases, and kappa- and ORL-1 decreases in 5-HT release. We further hypothesize that opioids have regionally selective effects on 5-HT. Preliminary data indicate that mu- and delta- opioids act in the DRN to increase 5-HT release in specific forebrain projection sites. In contrast, kappa-opioids may inhibit 5-HT release by acting on terminals in widespread areas of the brain including the DRN, median raphe nucleus, nucleus accumbens and dorsal hippocampus. The hypothesis that the increase in 5-HT elicited by mu-opioids is mediated indirectly by neural circuitry will also be examined. Specifically, mu-opioids may inhibit both GABAergic and glutamatergic inputs to 5-HT neurons in the DRN. This hypothesis will be investigated by using selective GABA and glutamate receptor antagonists to characterize the tonic activity of inhibitory and excitatory influences on 5-HT neurons. Preliminary data indicate a strong tonic influence of GABA compared with a relatively weak effect of glutamate in the DRN. We propose experiments to further examine the hypothesis that the effect of mu-opioids on 5-HT can be attributed to direct inhibition of both GABA and glutamate neurons. Because, GABA tone is predominant, the net effect of inhibiting both inputs is an increase in 5-HT release. In contrast, kappa- and ORL-1-opioids may directly inhibit 5-HT neurons. Prolonged opioid treatment may produce plasticity in the strength of synaptic connections in the raphe and thus affect the regulation of 5-HT release. To examine this hypothesis, opioid dependence will be induced by either direct infusion of opioids into the DRN for 4 days or by subcutaneous implantation of morphine pellets. Based on our previous observations, we will test the hypothesis that mu-opioids cause adaptations in 5-HT release by enhancement of GABA inputs. Similarly, the possibility of enhanced glutamate synaptic transmission will be tested. By determining changes in 5-HT during withdrawal, we will test the specific hypothesis that the enhancement in GABA exceeds the change in glutamate. This may result in a more pronounced net inhibitory effect of afferent inputs, thus decreased 5-HT release after tolerance to opioids develops. Further experiments will test the hypothesis that the excitability of 5-HT neurons is increased after prolonged treatment with ORL-1 opioids. Finally, we will test the hypothesis that 5-HT release is enhanced after regular exercise and this may ameliorate the adverse consequences of prolonged opioid treatment. Recent data suggest that the increase in 5-HT release associated with treadmill locomotion is enhanced after regular exercise. To examine the role of exercise in alleviating behavioral depression, opioid-dependent rats will be exercised regularly on a treadmill. We hypothesize that regular exercise results in an increase in the strength of glutamate relative to GABA connections with 5-HT neurons in the DRN, and that this may normalize the enhancement in net inhibitory associated with prolonged exposure to opioids.

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.