Targeting GPCRs in amygdalar and cortical neural ensembles to treat pain aversion

项目总结 疼痛是一种具有感觉和情感成分的多维体验。痛苦的厌恶性，即 它与生俱来的不愉快，导致大多数慢性疼痛患者的痛苦，并经常导致共病 焦虑症和抑郁症等疾病。尽管阿片类镇痛剂有成瘾的特性，但它仍在临床上使用 很有用，因为它们可以深刻地抑制疼痛的影响。因此，发现可以改变神经活动的目标 选择性地在产生疼痛厌恶的神经回路中，而不是在阿片类药物的奖赏或呼吸回路中 此外，开发新的、更安全的止痛药也是一个有吸引力的策略。 最近，通过将体内成像和化学发生操作相结合，在基础神经动力学 我们的合作发现，自由行为的小鼠受到有害刺激后的杏仁外侧核(BLA) BLA中编码疼痛的负性情感配价的独特神经集合(Corder等人，Science， 2019年)。利用Gi/o-蛋白偶联DREADDS抑制这一伤害性编码集成的化学发生 在不改变戒断反应、焦虑或奖励的情况下，减轻疼痛情感行为。而且，我们的 对这种伤害性感受器的功能研究揭示了它在痛觉异常现象中的因果作用。 基于这些令人兴奋的发现，我们现在寻求通过确定疼痛治疗的新靶点 用原位杂交和单细胞RNA测序对这些BLA伤害性感受细胞进行分子鉴定 (scRNA-seq)。我们对BLA伤害性感受细胞的初步scRNA-seq研究表明，它们表达数十个GI/O。 蛋白偶联受体(Gi/o-GPCRs)，可作为抗痛觉影响的靶点。此外， 我们的追踪研究显示，在前扣带回皮质(ACC)有一组V层锥体细胞投射 与扣带回切开术可用于治疗顽固性慢性疼痛的事实一致。 解析这些ACC伤害性感受细胞的分子特征也可能揭示治疗疼痛影响的新靶点。 因此，在这里，我们建议在BLA和ACC(目标1，发现)、测试中编目候选Gi/o-GPCR靶标 它们治疗疼痛的效用(目标2，验证)，并验证这些新靶点对大脑的奖励和 呼吸回路(目标3，安全性和可译性)。在目标1中，我们将识别疼痛影响中的Gi/o-GPCR靶点 使用小鼠遗传学、病毒示踪剂、scRNA-seq和生物信息学分析来研究BLA和ACC的电路。在……里面 目的2，我们将验证这些新靶点的神经生理效应和止痛特性，使用 活体脑组织切片、急、慢性疼痛动物模型和钙离子的电生理记录 行为小鼠BLA和ACC神经活动的影像研究。在目标3中，我们将验证安全和 新型抗伤害性药物靶点的可译性。我们将评估每个目标的滥用可能性，并 使用奖励处理和全身体积描记的行为分析对呼吸的影响， 分别进行了分析。为了评估我们在啮齿动物身上的结果是否有可能转化为临床，我们还将分析 用原位杂交法研究药物靶点在人体组织中的表达模式。

PROJECT SUMMARY Pain is a multidimensional experience with sensory and affective components. The aversive quality of pain, i.e. its inherent unpleasantness, causes a majority of chronic pain patients’ suffering and often leads to comorbid disorders such as anxiety and depression. Despite their addictive qualities, opioid analgesics remain clinically useful since they can profoundly dampen pain affect. Thus, discovering targets that could alter neural activity selectively in neural circuits that generate pain aversion, but not in the reward or breathing circuits that opioids also alter, is an attractive strategy to develop novel, safer analgesics. Recently, by combining in vivo imaging and chemogenetic manipulations of neural dynamics in the basal and lateral amygdala (BLA) of freely behaving mice encountering noxious stimuli, our collaboration discovered a distinct neural ensemble in the BLA that encodes the negative affective valence of pain (Corder et al., Science, 2019). Chemogenetic inhibition of this nociceptive coding ensemble using Gi/o-protein-coupled-DREADDs alleviated pain affective behaviors without altering withdrawal reflexes, anxiety or reward. Moreover, our functional studies of this nociceptive ensemble revealed its causal role in the phenomenon of allodynia. Based on these exciting findings, we now seek to identify novel targets to treat pain by determining the molecular identity of these BLA nociceptive cells via in situ hybridization and single cell RNA-sequencing (scRNA-seq). Our preliminary scRNA-seq studies of BLA nociceptive cells suggest they express dozens of Gi/o protein-coupled receptors (Gi/o-GPCRs) that could be targeted for anti-nociception against pain affect. Further, our tracing studies have revealed a set of layer V pyramidal cells in anterior cingulate cortex (ACC) that project onto BLA nociceptive neurons, consistent with the fact cingulotomy can be used to treat intractable chronic pain. Resolving the molecular identity of these ACC nociceptive cells could also reveal new targets to treat pain affect. Thus, here we propose to catalog candidate Gi/o-GPCR targets in BLA and ACC (Aim 1, Discovery), test their utility to treat pain (Aim 2, Validation), and verify these new targets have no effect in the brain’s reward and breathing circuitry (Aim 3, Safety & Translatability). In Aim 1 we will identify Gi/o-GPCR targets in pain affect circuits of the BLA and ACC using mouse genetics, viral tracers, scRNA-seq and bioinformatics analyses. In Aim 2, we will validate the neurophysiological effects and analgesic properties of these new targets, using electrophysiological recordings in live brain tissue slices, animal models of acute and chronic pain, and Ca2+ imaging studies in behaving mice of BLA and ACC neural activity. In Aim 3, we will verify the safety and translatability of the novel antinociceptive drug targets. We will evaluate each target for abuse potential and effects on breathing by using behavioral assays for reward processing and whole-body plethysmography, respectively. To evaluate whether our results in rodents are likely to translate clinically, we will also analyze expression patterns of the drug targets in human tissue using in situ hybridization.