Molecular identification of bulbospinal ON neurons by GPER, which drives pain and morphine tolerance
Yingfu Jiao, Po Gao, Li Dong, Xiaowei Ding, Youqiang Meng, Jiahong Qian, Ting Gao, Ruoxi Wang, Tao Jiang, Yunchun Zhang, Dexu Kong, Yi Wu, Sihan Chen, Saihong Xu, Dan Tang, Ping Luo, Meimei Wu, Li Meng, Daxiang Wen, Changhao Wu, Guohua Zhang, Xueyin Shi, Weifeng Yu, Weifang Rong
Yingfu Jiao, Po Gao, Li Dong, Xiaowei Ding, Youqiang Meng, Jiahong Qian, Ting Gao, Ruoxi Wang, Tao Jiang, Yunchun Zhang, Dexu Kong, Yi Wu, Sihan Chen, Saihong Xu, Dan Tang, Ping Luo, Meimei Wu, Li Meng, Daxiang Wen, Changhao Wu, Guohua Zhang, Xueyin Shi, Weifeng Yu, Weifang Rong
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Research Article Neuroscience

Molecular identification of bulbospinal ON neurons by GPER, which drives pain and morphine tolerance

Abstract

The rostral ventromedial medulla (RVM) exerts bidirectional descending modulation of pain attributable to the activity of electrophysiologically identified pronociceptive ON and antinociceptive OFF neurons. Here, we report that GABAergic ON neurons specifically express G protein–coupled estrogen receptor (GPER). GPER+ neurons exhibited characteristic ON-like responses upon peripheral nociceptive stimulation. Optogenetic activation of GPER+ neurons facilitated, but their ablation abrogated, pain. Furthermore, activation of GPER caused depolarization of ON cells, potentiated pain, and ameliorated morphine analgesia through desensitizing μ-type opioid receptor–mediated (MOR-mediated) activation of potassium currents. In contrast, genetic ablation or pharmacological blockade of GPER attenuated pain, enhanced morphine analgesia, and delayed the development of morphine tolerance in diverse preclinical pain models. Our data strongly indicate that GPER is a marker for GABAergic ON cells and illuminate the mechanisms underlying hormonal regulation of pain and analgesia, thus highlighting GPER as a promising target for the treatment of pain and opioid tolerance.

Authors

Yingfu Jiao, Po Gao, Li Dong, Xiaowei Ding, Youqiang Meng, Jiahong Qian, Ting Gao, Ruoxi Wang, Tao Jiang, Yunchun Zhang, Dexu Kong, Yi Wu, Sihan Chen, Saihong Xu, Dan Tang, Ping Luo, Meimei Wu, Li Meng, Daxiang Wen, Changhao Wu, Guohua Zhang, Xueyin Shi, Weifeng Yu, Weifang Rong

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Figure 2

ON cells exclusively express GPER in the RVM.

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ON cells exclusively express GPER in the RVM. (A) Schematic of the exper...
(A) Schematic of the experimental model. (B and C) Iontophoretic application of E2 caused dose-dependent (current-dependent) excitation of RVM ON cells without affecting OFF cells. n = 7–11 cells/group. (D and E) Intracellular recording, functional identification, and immunolabeling revealed positive GPER expression for the great majority (16 of 18) of RVM ON cells. The ON cells tested (n = 16) were not immunoreactive for TPH (see Supplemental Figure 4F for representative TPH immunofluorescence). Scale bar: 100 μm. (F and G) Intracellular recording, functional identification, and immunolabeling showing an absence of GPER immunoreactivity in RVM OFF neurons (n = 7). One of the 7 OFF neurons tested was immunoreactive to TPH. Scale bar: 40 μm. (H) ON cells recorded using the piggyback electrodes showed membrane potential depolarization and accelerated firing rates following iontophoretic application of E2. n = 6–7 cells/group. (I) Schematic showing in vivo 2-photon calcium imaging of RVM GPER+ neurons through surgery exposure from the ventral side in mice. GperCre mice were injected with pAAV-EF1a-DIO-CGaMP6(F)-P2A-NLS-tdTomato into the RVM to induce Cre-driven expression of GCaMP6 in GPER+ neurons. The image shows tdTomato+ fluorescence in RVM neurons infected with virus. Scale bar: 100 μm. (J) TdTomato+ (GPER+) neurons exhibited a transient rise in calcium in response to a noxious pinch applied to the hind paw. Scale bar: 100 μm. (K) Averaged calcium signal of 63 neurons from 5 mice during a noxious pinch applied to the hind paw. Data are representative of at least 3 independent experiments and are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 2-way ANOVA followed by Bonferroni’s post hoc test (C) and paired, 2-tailed Student’s t test (H).