Figure 7

Summary of experimental strategies and approaches. Gfap-hM3Dq mice express hM3Dq, a Gq-coupled engineered GPCR in GFAP⁺ glial cells throughout the nervous system. The ligand of hM3Dq, CNO effectively induces hind paw mechanical analgesia (Fig. 1) and decreases thermal sensitivity (Fig. 2) in GFAP-hM3Dq mice. To distinguish the potential analgesic effect of CNS astrocytes vs PNS GFAP⁺ glia following their Gq-GPCR activation, a peripheral acting blocker of muscarinic receptors, Trospium chloride, was administrated to block CNO-induced and hM3Dq-mediated Gq-GPCR activation in peripheral glia. Astrocytic Gq-GPCR activation alone did not lead to significant changes in hind paw mechanical sensitivity (Fig. 3), which suggested that peripheral GFAP⁺ glial Gq-GPCR activation is responsible for CNO-induced hind paw analgesia. The glial-induced analgesic effect was independent from inflammation and not affected by peripheral sympathectomy (Fig. 4), suggesting that peripheral glial Gq-GPCR activation directly modulate sensory neuronal activity and/or afferent excitability. To further test this hypothesis, peripheral acting and selective adenosine receptor A₁ adenosine receptor antagonists were administrated prior to CNO in GFAP-hM3Dq mice. Blockade of peripheral A1R activation completely abolished peripheral GFAP⁺ glial activation induced mechanical analgesia (Fig. 5); similar results were repeated using A1R KO mice that also express hM3Dq in GFAP⁺ glia (Fig. 6). These experiments strongly suggested that peripheral glial Gq-GPCR activation decreases hind paw mechanical sensitivity via peripheral activation of A1R.