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. 2021 Feb 20;24(3):102213.
doi: 10.1016/j.isci.2021.102213. eCollection 2021 Mar 19.

Reduction of prefrontal purinergic signaling is necessary for the analgesic effect of morphine

Yeting Zeng  1 , Huoqing Luo  2 , Zilong Gao  3   4 , Xiaona Zhu  2 , Yinbo Shen  1 , Yulong Li  4   5   6   7 , Ji Hu  2   8   9   10 , Jiajun Yang  1
Affiliations

Reduction of prefrontal purinergic signaling is necessary for the analgesic effect of morphine

Yeting Zeng et al. iScience. .

Abstract

Morphine is commonly used to relieve moderate to severe pain, but repeated doses cause opioid tolerance. Here, we used ATP sensor and fiber photometry to detect prefrontal ATP level. It showed that prefrontal ATP level decreased after morphine injection and the event amplitude tended to decrease with continuous morphine exposure. Morphine had little effect on prefrontal ATP due to its tolerance. Therefore, we hypothesized that the analgesic effect of morphine might be related to ATP in the medial prefrontal cortex (mPFC). Moreover, local infusion of ATP partially antagonized morphine analgesia. Then we found that inhibiting P2X7R in the mPFC mimicked morphine analgesia. In morphine-tolerant mice, pretreatment with P2X4R or P2X7R antagonists in the mPFC enhanced analgesic effect. Our findings suggest that reduction of prefrontal purinergic signaling is necessary for the morphine analgesia, which help elucidate the mechanism of morphine analgesia and may lead to the development of new clinical treatments for neuropathic pain.

Keywords: Clinical Neuroscience; Molecular Neuroscience; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Effects of different stimulants on the ATP concentration in the mPFC (A) Schematic diagram of the principle of the GRAB-based ATP sensor. Extracellular ATP induces a conformational change in the sensor when binding to the corresponding sites and then increases the fluorescence signal. (B) Schematic diagram of the optical fiber recording in fluorescence signal changes of the ATP sensor in the mPFC after substance administration in free-moving mice. The location of the adeno-associated virus (AAV) and optical fiber embedded in the mPFC, and a fluorescently stained image showing AAV expression and optical fiber position. Coronal section of the mouse habenula showing expression of ATP (green) and nuclear staining (DAPI, blue). Scale bar, 300 μm. (C) Changes in the ATP sensor fluorescent signal in the mPFC of the mice after intraplantar injection of saline (0.9% in 20 μL in the red line) or formalin (5% in 20 μL in the blue line). The fluorescence signal changes are denoted by ΔF/F. (D and E) Changes in the ATP sensor fluorescent signal in the mPFC of the mice after intraperitoneal administration of morphine (10 mg/kg in the blue line) or saline (0.9% in 0.25 mL in the red line). The fluorescent signal changes are denoted by ΔF/F (n = 5, t8 = 5.399, p = 0.0006, two-tailed, Student's paired t test). Definition of statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns indicates not significant. Values are reported as the mean ± SEM.
Figure 2
Figure 2
Longitudinal measurements of ATP dynamics in the mPFC during the formation of morphine tolerance (A) A brief schematic diagram of the steps of morphine tolerance experiment. (B–E) Changes in the transient fluorescent event in the mPFC when mice were given morphine for 7 consecutive days, including every interval from the first day (Day 1, Day 3, Day 5, and Day 7) (the red line shows the point at which morphine is injected intraperitoneally). (F) A statistical chart of the variation in the signal recorded for 4 days (n = 5, F3,16 = 11.25; p = 0.0012; one-way ANOVA with Tukey's post hoc test; Day 1 versus Day 3: p = 0.0488; Day 1 versus Day 5: p = 0.0025; Day 1 versus Day 7: p = 0.0001). (G) Changes in analgesic efficacy on Day 1 and Day 7 in the mice given morphine for 7 consecutive days in the first phase (left) (n = 7, t12 = 9.156, p < 0.0001, two-tailed, Student's paired t test) and the second phase (right) (n = 7, t12 = 7.678, p < 0.0001, two-tailed, Student's paired t test). Definition of statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns indicates not significant. Values are reported as the mean ± SEM.
Figure 3
Figure 3
Effects of local infusion of ATP into the mPFC on morphine analgesia (A) A diagram of the location and mode of administration. ATP was intracerebrally injected into mPFC via a microsyringe (i.c.), morphine was intraperitoneally injected with a common syringe (i.p.), and formalin was intraplantar into the hind paw with a microsyringe (intraplantar). (B) Schematic diagram of the location of the cannula embedded in the mPFC (left) and a brain slide of the location of cannula insertion in the mPFC (right). Coronal section of the mouse habenula showing nuclear staining (DAPI, blue). Scale bar, 500 μm. (C) The licking and biting time of the injected paw in formalin test after the administration of morphine (intraperitoneally [i.p.]) and ATP (intracerebrally [i.c.]) into the mPFC in the first phase (left) (n = 7, F2,18 = 42.29, p < 0.0001, one-way ANOVA with Tukey's post hoc test; saline versus morphine plus vehicle: p < 0.0001; morphine plus vehicle versus morphine plus ATP: p = 0.0014) and the second phase (right) (n = 7, F2,18 = 53.02, p < 0.0001, one-way ANOVA with Tukey's post hoc test; saline versus morphine plus vehicle: p < 0.0001; morphine plus vehicle versus morphine plus ATP: p = 0.0228). (D) Morphine (i.p.) plus ATP (i.c.) to mPFC achieved 55.6% of the analgesic effect of morphine (i.p.) alone in the first phase and 72.1% in the second phase. In other words, local infusion of ATP antagonized 44.4% of the morphine analgesia in the first phase and antagonized 27.9% in the second phase. Definition of statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns indicates not significant. Values are reported as the mean ± SEM.
Figure 4
Figure 4
Effects of antagonists of P2XR in formalin experiments (A) Simple diagram of the administration sequence. (B) The local infusion of vehicle or P2XR antagonists into the mPFC caused different reactions to intraplantar injection of formalin. The licking and biting time of the injected paw in formalin test after the administration of intracerebral P2XR antagonists into the mPFC in the first phase (left) (n = 6–8, F4,31 = 31.70, p < 0.0001, one-way ANOVA with Tukey's post hoc test; vehicle versus PPADS: p = 0.2128; vehicle versus TNP-ATP: p > 0.9999; vehicle versus BBG: p = 0.0003; vehicle versus morphine: p < 0.0001); (PPADS versus TNP-ATP: n = 8, t14 = 1.990, p = 0.0665, two-tailed, Student's paired t test); (BBG versus morphine: n = 6–7, t11 = 2.791, p = 0.0176, two-tailed, Student's unpaired t test). The analgesic effects of BBG (i.c.) into mPFC were 63.3% as effective as morphine (i.p.) in the first phase. The licking and biting time of the injected paw in the formalin test after the administration of intracerebral P2XR antagonists into the mPFC in the second phase (right) (n = 6–8; F4,31 = 13.02, p < 0.0001, one-way ANOVA with Tukey's post hoc test; vehicle vs. PPADS: p = 0.1397; vehicle vs. TNP-ATP: p = 0.2990; vehicle versus BBG: p = 0.0001; vehicle vs. morphine: p < 0.0001); (PPADS versus TNP-ATP: n = 8, t14 = 0.3436, p = 0.7363, two-tailed, Student's paired t test); (BBG versus morphine: n = 6–7, t11 = 1.845, p = 0.0921, two-tailed, Student's unpaired t test). The analgesic effects of BBG (i.c.) into mPFC were not significantly different from morphine administration (i.p.) in the second phase. Definition of statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns indicates not significant. Values are reported as the mean ± SEM.
Figure 5
Figure 5
Effects of P2XR antagonists on analgesia in morphine-tolerant mice (A) A brief schematic of the morphine-tolerant experiment and the schedule of the antagonists and morphine administration after the formation of morphine tolerance on the last day. (B) Effects of pretreatment with P2XR antagonists (i.c.) into mPFC on morphine analgesia in morphine-tolerant mice. The licking and biting time of the injected paw in formalin test after the pretreatment with P2XR antagonists (i.c.) on tolerant mice in the first phase (left) (n = 7–8, F3,25 = 18.98, p < 0.0001, one-way ANOVA with Tukey's post hoc test; vehicle plus morphine versus PPADS plus morphine: p = 0.5033; vehicle plus morphine versus TNP-ATP plus morphine: p = 0.0003; vehicle plus morphine versus BBG plus morphine: p < 0.0001). The licking and biting time of the injected paw in formalin test after the pretreatment with PPADS (i.c.) or TNP-ATP (i.c.) on tolerant mice in the first phase (left) (n = 7–8, t13 = 2.943, p = 0.0114, two-tailed, Student's unpaired t test). The licking and biting time of the injected paw in formalin test after the pretreatment with P2XR antagonists (i.c.) on tolerant mice in the second phase (right) (n = 7–8; F3, 25 = 16.60; p < 0.0001; one-way ANOVA with Tukey's post hoc test; vehicle plus morphine versus PPADS plus morphine: p = 0.3177; vehicle plus morphine versus TNP-ATP plus morphine: p < 0.0001; vehicle plus morphine versus BBG plus morphine: p < 0.0001). The licking and biting time of the injected paw in formalin test after the pretreatment with PPADS (i.c.) or TNP-ATP (i.c.) on tolerant mice in the second phase (right) (n = 7–8, t13 = 3.377, p = 0.0050, two-tailed, Student's unpaired t test). Definition of statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns indicates not significant. Values are reported as the mean ± SEM.
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