Sensory satellite glial Gq-GPCR activation alleviates inflammatory pain via peripheral adenosine 1 receptor activation

Abstract

Glial fibrillary acidic protein expressing (GFAP⁺) glia modulate nociceptive neuronal activity in both the peripheral nervous system (PNS) and the central nervous system (CNS). Resident GFAP⁺ glia in dorsal root ganglia (DRG) known as satellite glial cells (SGCs) potentiate neuronal activity by releasing pro-inflammatory cytokines and neuroactive compounds. In this study, we tested the hypothesis that SGC Gq-coupled receptor (Gq-GPCR) signaling modulates pain sensitivity in vivo using Gfap-hM3Dq mice. Complete Freund's adjuvant (CFA) was used to induce inflammatory pain, and mechanical sensitivity and thermal sensitivity were used to assess the neuromodulatory effect of glial Gq-GPCR activation in awake mice. Pharmacogenetic activation of Gq-GPCR signaling in sensory SGCs decreased heat-induced nociceptive responses and reversed inflammation-induced mechanical allodynia via peripheral adenosine A1 receptor activation. These data reveal a previously unexplored role of sensory SGCs in decreasing afferent excitability. The identified molecular mechanism underlying the analgesic role of SGCs offers new approaches for reversing peripheral nociceptive sensitization.

Figure 1

CNO-induced GFAP⁺ glial Gq-GPCR activation alleviated CFA-induced mechanical allodynia in vivo. (A) Schematic timeline of CFA-induced inflammatory pain animal model; (B) Von Frey apparatus for assessing mechanically induced pain thresholds. (C) CFA injection into hind paw induced comparable levels of mechanical allodynia in both Gfap-hM3Dq mice and WT littermate controls (N = 3 per genotype). (D) 0.25 mg/kg CNO i. p. administration largely reversed CFA-induced mechanical allodynia in CFA-injected paws in Gfap-hM3Dq mice, but not in WT littermate controls (N = 5 per genotype); **p < 0.01; ***p < 0.001 compared to pre-CNO. (B) was taken by Alison Xie; (C) and (D) were prepared using Graphpad Prism 7.04.

Figure 2

CNO-induced GFAP⁺ glial Gq-GPCR activation decreased heat sensitivity in the hind paws of naive mice. (A) Baseline heat sensitivity without any drug administration of animals used in (B). Gfap-hM3Dq mice exhibited similar heat sensitivity compared to their WT littermate controls in Hargreaves test (N = 9 control, N = 10 Gfap-hM3Dq). (B) 0.25 mg/kg CNO i. p. administration significantly increased paw withdrawal frequency at 30 min after CNO delivery (N = 9 control, N = 10 Gfap-hM3Dq). (C) Baseline heat sensitivity without any drug administration of animals used in (D). Gfap-hM3Dq mice exhibited similar heat sensitivity compared to their WT littermate controls (N = 10 control, N = 17 Gfap-hM3Dq). (D) Intrathecal administration (i. t.) of 0.5 mg/kg CNO significantly increased paw withdrawal latency up to 2 h (N = 10 control, N = 17 Gfap-hM3Dq). In both (B) and (D), *p < 0.05; **p < 0.01; ***p < 0.001 compared to pre-CNO. Figures were prepared using Graphpad Prism 7.04.

Figure 3

CNO-induced analgesia in Gfap-hM3Dq mice was due to Gq-GPCR activation in peripheral GFAP⁺ glia and not due to CNO-induced decreases in motor activity. (A) Schematic timeline of peripheral hM3Dq blockade experiments Gfap-hM3Dq mice were subjected to CFA-induced inflammatory pain model as in Fig. 1. Von Frey tests were used to assess both baseline and inflammation induced mechanical pain threshold in both CFA-injected and saline-injected hind paws (N = 21 mice). (B) A summary of peripheral Gq-GPCR activation blockade in Gfap-hM3Dq mice with saline or Trospium pretreatment. (C,D) Trospium pretreatment prevented CNO-induced increases of pain threshold in both CFA-injected (C) and saline-injected (D) hind paws in Gfap-hM3Dq mice (N = 11 saline pretreated, N = 10 trospium pretreated). (E) 0.25 mg/kg CNO i. p. administration had no effect on rotarod performance (three trials, T1, T2, and T3) in WT littermate control mice, nor did pretreatment with trospium (N = 6 per group). (F) CNO led to decreased rotarod performance in Gfap-hM3Dq mice. Trospium pretreatment did not alter CNO-induced decreases in rotarod performance (N = 5 per group). ⁺p < 0.01 compared to baseline in saline-treated mice; *p < 0.01, **p < 0.001 compared to saline-treated mice. (C–F) Were prepared using Graphpad Prism 7.04.

Figure 4

Peripheral chemical sympathectomy reversed inflammation induced mechanical allodynia in CFA-injected hind paws in Gfap-hM3Dq mice, while CNO induced additional analgesia effects in both CFA-injected and saline-injected hind paws. (A) Experimental timeline of 6-OHDA induced peripheral chemical sympathectomy and CFA-induced inflammatory pain in Gfap-hM3Dq mice (N = 6 vehicle injection, N = 5 6-OHDA injection). (B) Peripheral sympathectomy blocked CFA-induced mechanical allodynia in Gfap-hM3Dq mice. CNO-induced analgesia was observed in CFA-injected hind paws in both the vehicle- and 6-OHDA-injected groups. (C) Sympathectomy does not affect either the baseline pain threshold or CNO-induced analgesia in saline-injected hind paw. ⁺p < 0.01 compared to baseline in vehicle treated mice; ^#p < 0.01 compared to baseline in sympathectomized mice. (B,C) Were prepared using Graphpad Prism 7.04.

Figure 5

Pharmacological blockade of peripheral A₁ Adenosine receptors prevented CNO-induced analgesia in Gfap-hM3Dq mice. Peripheral administration of SPT, a blood brain barrier impermeable inhibitor of adenosine receptors ablated CNO-induced analgesia in CFA-injected (A) and saline-injected (B) hind paws (N = 13 saline treated, N = 5 SPT treated). A₁ adenosine receptor blocker CPX also prevented CNO-induced analgesia in CFA-injected (C) and saline-injected (D) hind paws (N = 13 saline treated, N = 12 CPX treated). ⁺p < 0.01 compared to baseline in saline-treated animals; *p < 0.05, **p < 0.01, ***p < 0.001 compared to saline-treated animals. Figures were prepared using Graphpad Prism 7.04.

Figure 6

Gfap-hM3Dq mice lacking A₁R, but not those lacking A_2AR failed to exhibit CNO-induced analgesia. CNO-induced analgesia was observed in A₁ heterozygous (Het) but not in A₁ knockout (KO) mice in both CFA-injected (A) and saline-injected (B) hind paws (N = 9 A₁ Het mice, and N = 13 A₁ KO mice). CNO-induced analgesia remained intact in both A_2A heterozygous and in A_2A knockout mice in both the CFA-injected (C) and saline-injected (D) hind paws (N = 7 per group). ⁺p < 0.01 compared to baseline in heterozygous mice; ^#p < 0.01 compared to baseline in knockout mice. Figures were prepared using Graphpad Prism 7.04.

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.