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Abstract
Chronic pain pathologies plague society. While opioids effectively relieve pain, significant side effects, abuse liability, and development of analgesic tolerance outweigh their benefits. Furthermore, increasing levels of opioid prescription and repeated subsequent use has led to the current opioid epidemic, requiring substantive research efforts, as evidenced by the NIH’s Helping to End Addiction Long-term Initiative (HEAL) initiative. Whether other endogenous neuromodulators and their receptors can be harnessed to relieve pain with efficacy similar to opioids has not been thoroughly explored. The role of the neuromodulator Acetylcholine (ACh) in CNS function has been enigmatic. In this thesis, we explored the role of ACh in the ventrolateral periaqueductal gray (vlPAG), a key nucleus of the descending pain modulatory pathway. We found that nocifensive behaviors decreased ACh release in the vlPAG. Additionally, reversing this decrease in ACh by selectively activating cholinergic projections from Pedunculopontine Tegmental Nucleus (PPTg) to the vlPAG decreased the somatic and affective components of acute and chronic pain in opioid naïve and tolerant mice.
We determined that the antinociceptive effects of this cholinergic circuit were mediated through ⍺7 nicotinic acetylcholine receptors (nAChR), which are co-expressed on μ-opioid receptor-expressing (Oprm1+) GABAergic vlPAG neurons. Parallel in vivo experiments revealed an unexpected physiological impact of ɑ7 nAChRs activation: these non-selective cation channels inhibited neuronal activity in a cell-autonomous manner multiple minutes after receptor activation. Using molecular biology and slice electrophysiology, we identified the intracellular signaling mechanism underlying this counter-intuitive decrease in activity: activation of ɑ7 nAChRs decreased neuronal excitability by phosphorylating voltage-gated potassium channels, a mechanism similar to the mechanism proposed for opioid receptors in the vlPAG.
Finally, we explored the manifestation and progression of chronic pain state and opioid tolerance in the vlPAG in vivo. The value of obtaining insights in awake-behaving animals is perhaps of utmost importance in the descending pain modulatory pathways, given that the central dogma comes from work conducted in lightly anesthetized animals, which has been questioned. In this thesis, we use 2-photon imaging to track the progression of aberrant neuronal ensemble dynamics. This work leads to unexpected insights into the recruitment of non-pain-responsive neurons into the pain-encoding ensemble. We also observe that ⍺7 nAChRs agonists effectively relieve pain by inhibiting the Oprm1+ pain-sensitive neuronal ensembles in the vlPAG, even in opioid-tolerant animals. Finally, unlike opioids, ⍺7 nAChRs agonists did not show the development of tolerance, reward profile, or withdrawal symptoms.
To obtain these insights, we have employed methods that allow for rigorous cross-verification at multiple levels. Cross-disciplinary approaches include mRNA measurements, cell-type specific anatomical tracing, ex-vivo electrophysiology to assay receptor function and neuronal excitability, and in-vivo 2-photon imaging to track the cellular basis for the onset and progression of chronic pain and opioid tolerance. These assays are supplemented by cell-type and circuit-specific manipulations to assay the impact on sensory and affective-motivational aspects of pain behaviors. Together, these investigations identify a novel circuit that modulates pain signaling and non-opioid therapeutic targets for the management of chronic pain.