Resolving differences between clinical opioids at single neurons

项目总结 MU阿片受体激动剂(MOR)是目前最有效的止痛药，但阿片类药物 滥用和过量用药继续困扰着美国。阿片类药物临床使用的长期历史提供了证据 这些药物在潜在的生物学上有所不同。例如，缺乏完全的交叉容差 镇痛剂和特定的副作用对一种药物的反应比另一种药物更强烈(史密斯 和Peppin，2014)表明，它们在神经回路水平上的功能并不相同。更进一步，最近 动物研究表明，吗啡和芬太尼之间缺乏交叉耐受性，当这些药物 直接微量注射到疼痛调制电路(Bobeck等人，2012,2019)。配基偏置是电流， 关于这可能如何发生的主要假设，然而这个想法充其量也是不完整的(奥斯汀·扎马利帕et 等人，2018年；科尼贝尔和凯利，2019年；尤丁和罗哈克斯，2019年)。这项建议的首要目标是 利用神经元的反应为理解这些化合物奠定了新的基础 导致阿片类药物在体内的不同作用。我之前已经用全细胞录音演示过 从脑片上看，腹侧被盖区(VTA)的神经元对阿片类药物表现出独立的反应 缺乏体内交叉耐受性并以不同方式影响酒精消费的受体激动剂(酱等人， 1991年；米切尔等人，2014年；马戈利斯等人，2017年)。这项研究提供了概念观察的证据 电生理学可以用来检测药理上的差异。启用性能分析和比较 更多的分子，在这里我建议使用多电极细胞外记录急性脑 切片极大地增加了我们可以记录的神经元的数量，从而增加了 这种方法。我们将研究4种临床化合物(吗啡、芬太尼、 羟考酮和丁丙诺啡)，并使用DAMGO，一种高选择性的MOR激动剂，广泛用于临床前 研究，作为额外的参照物。奖赏回路(VTA)中大脑区域的大多数神经元， 急性发作时痛觉厌恶环路(缰核)和呼吸环路(前Bötzinger复合波)自发放电 脑片，这些脑区也高度表达MOR。因此，我们可以利用这个更高的 通过通过量方法测量不同MOR激动剂在这些大脑中的效力和疗效 区域，以及比较在单个神经元中顺序应用每个配体的反应 每个大脑区域。此外，我们将比较幼稚的动物和制造吗啡的动物的结果。 依附的。通过提高我们对特定回路中单个神经元的阿片类药物反应的理解 由于体内阿片类药物的影响，这项工作将(1)证明使用这种方法的可行性 快速表征化合物和(2)确定MOR功能的生物变异，这将使更多 涉及阿片信号的临床问题的有效治疗进展，包括阿片类药物的使用 精神障碍、酒精使用障碍和对止痛药的耐受性。

PROJECT SUMMARY Agonists of the mu opioid receptor (MOR) are currently the most effective pain-relieving drugs, but opioid abuse and overdose continues to plague the USA. The long history of clinical opioid use provides evidence that these drugs differ in underlying biology. For instance, a lack of complete cross tolerance between analgesics and that specific side effects are more intense in response to one drug compared to another (Smith and Peppin, 2014) suggests that they do not function identically at the level of neural circuits. Further, recent animal studies demonstrate a lack of cross tolerance between morphine and fentanyl when these drugs are microinjected directly into the pain modulation circuit (Bobeck et al., 2012, 2019). Ligand bias is the current, dominant hypothesis for how this might happen, however this idea is at best incomplete (Austin Zamarripa et al., 2018; Conibear and Kelly, 2019; Yudin and Rohacs, 2019). The overarching objective of this proposal is to lay a new groundwork for understanding these compounds using responses in neurons from the circuits that contribute to the different in vivo effects of opioids. I have previously demonstrated with whole cell recordings from brain slices, neurons in the ventral tegmental area (VTA) show independent responding to delta opioid receptor agonists that lack in vivo cross tolerance and differentially affect alcohol consumption (Jiang et al., 1991; Mitchell et al., 2014; Margolis et al., 2017). This study provides proof of concept observations that electrophysiology can be used to detect differences in pharmacologies. To enable profiling and comparison of a larger number of molecules, here I propose to use multielectrode extracellular recording from acute brain slices to greatly increase the number of neurons we can record from, thereby increasing the statistical power of the approach. We will investigate the neuronal responses to 4 clinical compounds (morphine, fentanyl, oxycodone, and buprenorphine) and use DAMGO, a highly selective MOR agonist used widely in preclinical studies, as an additional comparator. The majority of neurons in brain regions in the reward circuit (VTA), pain-aversion circuit (habenula), and respiration circuit (Pre-Bötzinger Complex) fire spontaneously in acute brain slices, and these brain regions also highly express the MOR. Therefore, we can use this higher throughput approach to measure potency and efficacy of different MOR agonists in each of these brain regions, as well as compare the responses to sequential application of each ligand in individual neurons in each brain region. Further, we will compare results between naïve animals and those made morphine dependent. By improving our understanding of opioid responses of individual neurons in the specific circuits that underly in vivo opioid effects, this work will (1) demonstrate the viability of using this approach to characterize compounds quickly and (2) identify biological variation of MOR function that will enable more effective therapeutic development for clinical problems that involve opioid signaling, including opioid use disorder, alcohol use disorder, and tolerance to pain medications.