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

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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.