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

Summary 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.


INTRODUCTION
Pain management is a major public health burden (Loeser, 2012). Morphine is an opioid drug, commonly used for the relief of moderate to severe pain (Galanie et al., 2015;Kalso et al., 2004;. Anti-nociceptive tolerance is one of the most common adverse effects and is defined as the inability to exert analgesic effect following repeated opioid exposure (Fields and Margolis, 2015). Morphine tolerance is caused by neuroinflammation (Eidson et al., 2017;Zhang et al., 2017) and changes in some important receptors, including the m-opioid receptor (MOR), N-methyl-D-aspartic acid receptor (NMDAR), and g-aminobutyric acid receptor (Corder et al., 2017;Dang and Christie, 2012;Martini and Whistler, 2007;Williams et al., 2013). Here we focused on the relationship between morphine and a classical neurotransmitter, adenosine 5 0 -triphosphate (ATP), which has been widely investigated in recent years (Illes et al., 2019;Kato et al., 2017).
Purinergic signaling plays an important role in the central nervous system (CNS) (Burnstock, 2017) and participates in pain management by mediating the activation of various signal molecules (Jiang et al., 2013;Kasuya et al., 2017;Zhang et al., 2020b). When peripheral or central nerve injury occurs, ATP increased release from damaged neurons. Extracellular ATP subsequently activates purinergic receptors to enhance neuroinflammation by mediating neuronal inflammatory signaling pathways (Chen et al., 2018;Kopp et al., 2019). The critical functions of ATP are mediated via purinergic receptors including P1 receptors and P2 receptors (Burnstock and Kennedy, 1985;Jacobson and Gao, 2006). There are two families of P2 receptors, namely, the P2X family and the P2Y family. The P2X family that is a ligand-gated ion channel consists of P2X (1-7) (Brake et al., 1994;Jarvis and Khakh, 2009). These receptors are widely distributed in the CNS (Burnstock et al., 2011). Given the important role of ATP in pain, inhibiting the release of ATP or affecting the function of the receptors can relieve neuronal inflammation and, thus, the pain response.
The P2X4 receptor (P2X4R) and P2X7 receptor (P2X7R) have been extensively studied in the chronic neuropathic and inflammatory pain (Burnstock and Kennedy, 2011;Duarte et al., 2007;Zhang et al., 2020a). Although immunohistochemical evidences suggested that purinergic receptors were widely distributed in key parts of the brain for pain processing, most studies on the interaction between purinergic receptors and morphine mainly concentrated on the peripheral nerves or spinal cord. Few studies have investigated the role of purinergic signaling in pain in the medial prefrontal cortex (mPFC). P2X4R and P2X7R in the mPFC changed significantly during the formation of anti-nociceptive tolerance to morphine (Metryka et al., 2019). Owing to the development of optical fiber recording, scientists have detected the dynamic changes of dopamine during morphine addiction and drug resistance (Calipari et al., 2016;. At present, the detection of dynamic changes of ATP in the formation of morphine tolerance has not been reported. In this study, an ATP sensor (Wu et al., 2021) is used to explore whether the purinergic signaling in the mPFC is involved in the regulation of morphine analgesia and drug resistance. Our results illustrate that (1) prefrontal purinergic signaling is involved in the analgesic effect of morphine, (2) blocking the prefrontal P2X7R mimics morphine's analgesic effect, and (3) antagonizing the prefrontal P2X4R and P2X7R enhances the analgesic effect of morphine after the development of drug resistance. Our results add a new perspective to the current knowledge on morphine analgesia and tolerance.

Reduction of ATP in the mPFC is involved in the analgesic effect of morphine
We used a genetically encoded G protein-coupled receptor (GPCR)-activation-based (GRAB) sensor for ATP designed by the Li Yulong Lab (Wu et al., 2021) to record extracellular ATP dynamics in the mPFC. The extracellular ATP level is expressed by the fluorescence intensity of green fluorescent protein (Figures 1A and 1B). To quantify changes in ATP dynamics, we used peak analysis to identify transient fluorescent events. By using fiber photometry recordings, we found that intraplantar injection of formalin caused a slow and sustained increase in ATP level ( Figure 1C). Meanwhile, morphine (intraperitoneally [i.p.]) significantly decreased the ATP concentrations in the mPFC compared with saline (i.p.) ( Figures 1D and 1E).
Event amplitude of ATP in the mPFC tends to decrease with continuous morphine exposure A single i.p. injection of morphine can cause a decrease of ATP in the mPFC. We used fiber photometry and an ATP sensor to conduct longitudinal measurements of the ATP dynamics in the mPFC during the morphine tolerance developed. The experimental procedures are shown in the Figure 2A. Five mice were repeatedly i.p. injected with morphine (10 mg/kg) for 1 week. Moreover, the signal changes in the mPFC were recorded four times from day 1 and recorded 1 day apart (Days 1, 3, 5, and 7). Interestingly, with repeated morphine administration for 7 days, the inhibition amplitude of ATP in the mPFC tended to decrease. On the last day, the downward trend nearly leveled off ( Figures 2B-2F).
Long-term administration of morphine induces tolerance to its analgesic effect. The experimental group received repeated i.p. injection of morphine (10 mg/kg), and the control group was given saline (0.25 mL per mouse) for 7 consecutive days. Both groups underwent formalin tests on Days 1 and 7 (first injection and the final injection) 30 minutes after morphine or saline administration. In the experimental group, the analgesic efficacy of morphine was significantly suppressed at the final injection compared with the first day, indicating the formation of opioid analgesic tolerance ( Figure 2G). In comparison, the control group showed no differences in the time of licking and biting of the injected paw between Day 1 and Day 7 (see Figure S1A).

Local infusion of ATP weakens the analgesic effect of morphine
As morphine and pain caused different changes in the ATP levels in the mPFC and morphine had little effects on the prefrontal ATP when the morphine tolerance developed, we hypothesized that the analgesic effect of morphine might be related to ATP. We further explored the relationship between analgesia and the purinergic system by changing the ATP level or affecting its function in the mPFC. We first tested whether the analgesic effect of morphine was abolished by compensating for the decrease of ATP. We directly infused ATP intracerebrally (i.c.) into the mPFC, whereas morphine was infused i.p. (Figures 3A and 3B). In the group injected with morphine plus ATP (0.5 mg per mouse), the anti-nociceptive effect in the formalin test was partially reversed compared with that of the group injected with morphine plus vehicle ( Figures 3C and 3D). Infusion of ATP into the mPFC alone neither altered the pain threshold nor exacerbated the pain caused by formalin, so the reduced analgesia of morphine was not caused by the pain increased by ATP injection (see Figures S2A-S2C). However, the analgesic effect of morphine was not completely blocked by the local infusion of ATP. Group morphine plus ATP only achieved 55.6% of the analgesic effect of group morphine alone in the first phase and achieved 72.1% in the second phase. In other words, the analgesic efficiency of morphine in the first phase of the formalin test decreased by 44.4% with ATP infusion and decreased by 27.9% in the second phase. Even if the concentration of ATP iScience Article is increased, the blocking efficiency cannot reach 100% (see Figures S3A and S3B). These results suggested that the acute analgesic efficacy of morphine at least partially depends on ATP or purinergic receptors in the mPFC.

Local infusion of BBG into the mPFC mimics morphine's analgesic effect
The supplement of exogenous ATP or the event amplitude of ATP in the mPFC decreases with repeated administration of morphine; it can be proved that the reduction of purinergic signaling molecule is necessary for morphine analgesia. Then we infused purinergic receptor antagonists to block the purinergic function in the mPFC to determine whether it could produce an analgesic effect like morphine. By infusing different types of purinergic receptor antagonists into the mPFC, such as PPADS, TNP-ATP, or BBG, we can not only determine whether purinergic antagonists produce an analgesic effect but also distinguish which receptor is involved in pain transduction. We infused these antagonists 10 minutes before the formalin injection ( Figure 4A). The results showed that only BBG caused a reduction in the licking and biting time of the injected paw in response to formalin. BBG could achieve 63.3% of the analgesic efficiency of iScience Article morphine in the first phase and showed no significant difference in the analgesic effect in the second phase. In comparison, neither PPADS nor TNP-ATP showed an effect in the two-phase pain induced by formalin ( Figure 4B). Overall, we concluded that P2X7R, but not P2X4R, was involved in acute morphine analgesia. As a P2X7R antagonist, BBG exerted an anti-nociceptive effect in the formalin test.   (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, t 12 = 9.156, p < 0.0001, two-tailed, Student's paired t test) and the second phase (right) (n = 7, t 12 = 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 G SEM. Previous experiments have shown that P2XR antagonists can produce an analgesic effect ( Figure 4B). To confirm whether these antagonists can be used to enhance the analgesic efficacy after the development of morphine tolerance or not we divided the morphine-tolerant mice into four groups (n = 7-8) that received morphine combined with vehicle or three P2XR antagonists (i.p. or i.c.) on the seventh day. Antagonists of P2XRs or vehicle were administered to the mPFC (i.c.) 10 minutes before the i.p. injection of morphine (i.p.) and 40 minutes before the formalin test (see Figure 5A).
The results demonstrated that TNP-ATP and BBG enhanced the analgesic effect of morphine in two phases of the nociceptive response in the formalin pain assay compared with the vehicle. In comparison, PPADS had no effect on morphine analgesic efficacy in the morphine-tolerant mice ( Figure 5B). Thus, antagonists of P2X4R and P2X7R can enhance anti-nociceptive effect in morphine-tolerant mice. . Theoretically, affecting the function of P2X7R in the mPFC can also alter the pain response. Our experiment confirmed this conclusion for the first time ( Figure 4B). Drug resistance is a major limitation in the clinic. The mPFC is involved in morphine tolerance by sending messages through neuronal connections with the striatum and hippocampus (Delgado et al., 2003;Esmaeili et al., 2012). These two regions are required to regulate the rewarding effects of morphine and the formation of morphine tolerance through the dopamine system (Jordan et al., 2019;. It is necessary to carry out a series of studies on the mPFC to investigate morphine analgesia and tolerance.

DISCUSSION
One major challenge in the development of a purinergic sensor is that the component of extracellular nucleotide signals are at low concentrations, at hundreds of nanomolar to low micromolar levels (Jacobson et al., 2015). Thus, it is difficult to identify the actual ATP level in the brain tissue in vivo, both spatially and temporally. This is the first study to conduct longitudinal measurements of ATP dynamics in the mPFC during the development of morphine tolerance. ATP is an energy information substance that is involved in pain (Jiang et al., 2013;Kobayashi et al., 2013). Extracellular ATP binds to P2X or P2Y to regulate neuropathic and inflammatory pain via changes in the phosphorylation of signal molecules (  iScience Article electrical activity of neurons (Khakh and North, 2012). ATP is a danger signal in the CNS and seems to be a mainly slow transmitter/modulator (Illes et al., 2019). It shows a slow and persistent upregulation during sustained damage (Cotrina and Nedergaard, 2009;Rodrigues et al., 2015). Extracellular ATP changes dramatically only if a severe injury occurs, such as a spinal cord injury (Peng et al., 2009). Our results were consistent with this conclusion. With the prolongation of the time, extracellular ATP in the mPFC showed a gradual and slow increase (Figures 1C and S5B) in mice with intraplantar injection of formalin. In recent years, extensive studies have confirmed the pivotal role of ATP in pain. Many chemicals have been used to reduce the neuroinflammatory response and subsequently alleviate neuropathic pain by decreasing ATP release, for example, the inhibitor of vesicular nucleotide transporter (Kato et al., 2017;Yin et al., 2019) and the inhibitor of mammalian target of rapamycin (Cui et al., 2014). The decrease of ATP level in the mPFC may counter nociceptive input and transmission in the CNS. Our results clearly showed that prefrontal ATP participated in pain management. When morphine does not change ATP level in the CNS, the analgesic effect is greatly reduced ( Figures 2F, 2G, 3C, and 3D). However, the analgesic effect was not completely blocked. Further research is needed to confirm the key role of the purinergic receptors in the analgesic effect.
P2X7Rs are highly expressed in the mPFC, mostly in glial cells Scholz and Woolf, 2007). The finding that antagonists of P2X7R exert an analgesic effect has been verified in extensive experiments (  iScience Article Microglia cells with high expression of P2X7Rs are activated first, and then, astrocytes with a lower level of P2X7Rs are promoted by inflammatory cytokine interleukin (IL)-1 release from microglia (Narcisse et al., 2005). Therefore, P2X7R not only binds to microglia but also acts on astrocytes. The released IL-1 from microglia directly mediates pain sensation through the p-38MAPK pathway (Clark et al., 2010a) or an indirect release of cathepsin (Clark et al., 2010b). By activating P2X7R, extracellular ATP can immediately open potassium channels to increase potassium influx (Ferrari et al., 2006). The increase in potassium ions leads to enhance the release of IL1-b and caspase-1 from macrophages and microglia (Hayashi et al., 2016;Kahlenberg and Dubyak, 2004;Manfredini et al., 2019). These two inflammatory factors, in turn, cause neuroinflammatory and pain responses (Mingam et al., 2008). Chen et al. (2017) found that Pannexin-1, which mediated pore formation, was activated when P2X7R bound to ATP. When P2X7R in the spinal cord is antagonized, it disrupts P2X7R's connection with Pannexin-1 and then reduces the transmission of pain (Bravo et al., 2015). Our finding that the infusion of BBG to the mPFC exerted an analgesic effect ( Figure 4B) demonstrated the important role of P2X7R in pain management. In addition to the aforementioned factors, activation of the P2X7R also induces other downstream inflammatory factors associated with the occurrence and maintenance of pain, including COX-2, NADPH oxidase, MIP-1a, TNF-a, and iNOS. These inflammatory factors not only influence the analgesic effect of P2X7R antagonist but also inhibit the analgesic effect of morphine (Puchałowicz et al., 2014;Samad et al., 2001;Takenouchi et al., 2009;Woolf et al., 1997). Experimentally, we also found that infusion of BBG into the mPFC could directly induce the analgesic effect. Therefore, the analgesic effect induced by pretreatment of P2X7R antagonist may be the analgesic effect of BBG itself, or it may be that BBG enhances the sensitivity of mice to morphine ( Figures 5B and S4A). More researches are needed to further confirm this conclusion.
We also investigated the role of other purinergic receptors in morphine-tolerant mice. TNP-ATP and PPADS are two types of non-selective P2XR antagonists that activate multiple targets. TNP-ATP blocks the P2X1-7 receptors, whereas PPADS inhibits the P2X1-3 and P2X5-7 receptors (Horvath and DeLeo, 2009). Although PPADS and TNP-ATP can block P2X7R, the blocking efficiency is not high (Donnelly-Roberts and Jarvis, 2007). Besides, other P2X1, 3, 5, 6 receptors are less distributed in the mPFC, so they may not play a major role in the mPFC (Yao et al., 2000). As TNP-ATP has an antagonistic effect on P2X4R, whereas PPADS does not, this difference is often used to explore the role of P2X4R ( Raghavendra et al., 2003). This finding indicates that dysfunction of glial cells can abolish morphine analgesia. Other researchers (Tai et al., 2010) found that TNP-ATP alleviated morphine tolerance by regulating the expression of NMDAR subunits and excitatory amino acids, so they concluded that TNP-ATP and NMDAR antagonists acted on the same pathway to influence the opioid analgesic tolerance (Tai et al., 2010). Horvath et al. (2010) found that intracerebral injection of P2X4 receptor antisense oligonucleotide (selectively inhibits the expression of P2X4 receptors) had no effect on the baseline response to mechanical and thermal stimulation, whereas it could inhibit the expression of ED2 to block analgesic tolerance to morphine (Horvath et al., 2010). Furthermore, researchers knocked out the P2X4 gene in mice and found that the acute toxicity and tissue damage caused by intraplantar injection of formalin in P2X4 À/À mice was not significantly different from that in normal mice . These results indicate that only with the participation of morphine can P2X4R antagonists maintain the anti-inflammatory and anti-nociceptive effects of morphine in tolerant mice, which corresponded with our results. In our research, two non-selective receptor antagonists, PPADS or TNP-ATP, given alone did not change the licking and biting time of the injected paw compared with the vehicle ( Figure 4B). After the formation of morphine tolerance, TNP-ATP restored the analgesic effect of morphine on the seventh day, whereas PPADS did not ( Figure 5B). This result showed the crucial role of P2X4R in morphine tolerance, consistent with previous research (Metryka et al., 2019). As the infusion of P2X4R antagonist into the mPFC alone did not induce analgesic effects in morphine-tolerant mice (see Figure S4A), the analgesia of P2X4R antagonist (i.c.) plus morphine may be due to the effect of P2X4R antagonists on morphine sensitivity. In summary, antagonists of P2X4R significantly reduced formalinevoked nociceptive behaviors, suggesting facilitatory or permissive role for P2X4Rs in the mPFC in inflammatory pain. iScience Article In conclusion, our results demonstrated that the reduction of prefrontal ATP was involved in morphine's analgesic effect. The prefrontal ATP level was decreased after morphine administration, and the event amplitude tended to decrease during continuous morphine exposure. The inhibition amplitude nearly disappeared after the development of morphine tolerance. Moreover, the analgesic effect of acute morphine administration was partially revised by local infusion of ATP in the mPFC. So the conclusion was that the acute analgesic effect of morphine mainly depended on P2X7R for the result that selective antagonisting P2X7R mimicked morphine's analgesic effect. Furthermore, inhibitors of P2X4R or P2X7R could increase anti-nociceptive to morphine. Overall, our study thus provides a new perspective for the mechanism of the analgesic effect of morphine and opioid analgesic tolerance.

Limitations of the study
In this study, we used the formalin test as a pain experiment to verify the relationship between purinergic signaling and morphine analgesia. We lack more other pain models to further verify the experimental results. Second, the changes of ATP level in the formation of morphine tolerance also need to be further explored with more technologies and methods.

Resource availability Lead contact
Jiajun Yang yangjiajunfzy@sina.com

Material availability
This study did not generate any new material.

Data and code availability
The study does not use any unpublished custom code, software, or algorithm that is central to supporting the main claims of the paper.

METHODS
All methods can be found in the accompanying Transparent Methods supplemental file.      Co., Ltd. Beijing China) were used.

Viral microinjection and stereotactic surgery
GRABATP (1.89 × 10 13 genomic copies per ml) was designed in Li Yulong Lab (Wu et al.,2021) . Optical fiber and cannula were purchased from RWD Life Science Co., LTD (Shenzhen China). Stereotaxic surgeries were performed as previously described (Zhang et al., 2020;Li et al., 2020;Qiu et al., 2020). The mice were anesthetized with an intraperitoneal injection of pentobarbital (100 mg/kg) and kept warm (37°C) with an electric heating pad (BrainKing Biotech). Then, the mice were placed in a stereotaxic apparatus (RWD Instruments, Shenzhen, China) to keep their skulls parallel to the reference panel. Next, 300 nl of AAV virus was slowly microsyringe injected (60 nl /min) by a glass pipette from a small hole (15-25 µm in diameter) into the unilateral mPFC (coordinates from the bregma: -1.6 mm AP, -0.3 mm ML, 2.0 mm DV).
The pipette remained in place for at least ten minutes after the injection. The coordinate of the fiber was 0.15 mm above the viral sites. The optical fiber was then fixed with dental cement (RWD Life Science Co., Ltd.).
For the implantation of the microinjection cannula in the mPFC, the procedure was the same as described before but without AAV virus injection (coordinates from the bregma: -1.6 mm AP, ±0.3 mm ML, 1.8 mm DV).
The locations of the virus, fiber, or microinjection in the mPFC were defined by visualization of stained tissue under a microscope.

Fiber photometry recording
After 2 weeks for recovery, fiber photometry recordings were conducted in the open-field chambers described before Wang et al., 2020). Continuous fiber photometry recordings were conducted before and after substances administration. For chronic morphine treatment, continuous fiber photometry recordings were conducted before and after morphine administration on days 1, 3, 5, 7 and the spontaneous fluorescent transient events were detected.
Fluorescence signals were obtained with a fiber photometry system (488 nm excitation laser, 505-544 nm emission filter, and a photomultiplier tube). The analog voltage signals were digitalized at 100 Hz and recorded by a Power 1401 digitizer and Spike2 software (CED, Cambridge, UK). The laser power was adjusted to 20-40 μW to minimize bleaching.
The photometry data were exported to MATLAB R2020b mat files. We calculated the values of fluorescence change (ΔF/F) by (F−F0)/F0, where F0 was the baseline fluorescence signal averaged over a 400-seconds-long control time window. The ΔF/F values are presented with a blue or red average plot indicating the average of the several yellow curves that represented the signal variation through trial and error. We collected the mean ΔF/F values 400 seconds before agent injection and the mean ΔF/F values after agent injection as the control or the treatment values. For the implantation of the microinjection cannula into mPFC, the procedure was the same as described before but without AAV virus injection.

Drug exposure
The procedure for intracerebral injection follows the steps described previously (Tsuda et al., 1999b). For fiber photometry recording experiment (Figure 1), formalin (5% in 20 μl) or saline (0.9% in 20 μl) was injected into hind paw ( Figure 1C) and morphine (10 mg/kg) or saline (0.9% in 0.25 ml) was intraperitoneally ( Figure 1D, E). Intraperitoneal injection of morphine (10 mg/kg) or saline (0.9% in 0.25 ml) was followed by formalin test in thirty minutes. In the chronic morphine tolerance experiment, the mice were given morphine (10 mg/kg) once a day for seven consecutive days to develop anti-nociceptive tolerance as described previously Shu et al., 2006;Zhang et al., 2018). Five mice with ATP sensor and fiber in the mPFC were used for fiber photometry recording. Continuous fiber photometry recordings were conducted before and after morphine administration on days 1, 3, 5, and 7. Seven mice were performed the formalin test thirty minutes after morphine administration on days 1 and 7 to assess the formation of opioid tolerance. To testify the real relationship between the morphine and ATP, we intraperitoneally (i.p.) injected with morphine followed by infusion of ATP (0.5 μg per mouse) or vehicle (1.0 μl per mouse). The saline was injected intraperitoneally as control group (see Figure S1A). All groups performed formalin test after assays administration. In acute analgesic experiments ( Figure A, B), the formalin test was performed ten minutes after infusion of substances to mPFC via indwelling intracerebral catheter. Intracerebral dosing into mPFC consisted of intracerebral administration of vehicle (1 μl per mouse), PPADS (0.2 μg per mouse), TNP-ATP (0.2 μg per mouse), and BBG (42.7 μg per mouse). For the chronic morphine experiment, the morphine tolerant mice were divided into four groups pretreated with different antagonists into mPFC. The mPFC was infused with vehicle or purinergic antagonists ten minutes before morphine administration on day 7 ( Figure 5A). The formalin test was performed thirty minutes after morphine injection. The substances were infused slowly into the corresponding brain region, and the entire injection process lasted one minute. After injection, the needle tube remained for at least another minute.

Behavioral assays
Behavioral tests were performed during the dark phase (9:00-21:00). Three days before the experiment, all mice were handled for at least five minutes per day. The mice were placed in the experimental room for three hours before the tests. Then, the mice were placed in an experimental container for half an hour before the experiment.

Formalin test
Mice received an intraplantar injection of 20 µl of formalin (5% diluted in 0.9% sterile saline) in the hind paw with a microliter syringe as described previously Hussey et al., 2007;Magnúsdóttir et al., 2018). For the group given morphine alone, the mice were given formalin thirty minutes after morphine injection. Following formalin injection, the mice were immediately returned to the observation chamber (10 cm x 10 cm x 15 cm) with opaque sides and transparent bottoms and the observation time started. In experiments with the antagonists, mice were given intracerebrally ten minutes before the formalin pain assay. The animal behaviors were recorded by a video camera located beneath the observation chamber for a period of at least thirty minutes. The videos were recorded for subsequent analysis. Formalin injection causes intense licking and biting of the injected paw in two phases. The first phase was defined as acute neurogenic pain occurring within the first five minutes after the intraplantar injection of formalin. The second phase is chronic peripheral tonic inflammation pain and occurs 15-30 minutes after formalin administration. The licking and biting of the injected paw in two phases is considered a sign of nociception.

Morphine treatment
Adult male mice (aged 8-12 weeks) were used for the morphine tolerance experiment. The protocol conducts as described previously Shu et al., 2006;Zhang et al., 2018). Mice were intraperitoneally injected with morphine (10 mg/kg) daily for one week. To assess morphine tolerance, formalin test was carried out 30 minutes after injection of morphine on the first and seventh days. When morphine tolerance developed, the analgesic effect of morphine on the pain caused by formalin was significantly reduced ( Figure 2G).

Von Frey Test
The procedure is the same as described before , Young et al., 2016.
Von Frey Filaments (von Frey hair, Stoelting, Wood Dale, USA) were used in this test. Mice were placed in a plastic chamber (4cm x 4cm x 7cm) with a metal mesh bottom for 20 minutes for behavioral accommodation. Mechanical sensitivity was performed on the hind paw plantar surface. The paw was touched by one hair for several seconds and take a break for more than 10 seconds. The up and down methods were used to determine the bending force causing the median 50% withdrawal (Chaplan et al., 1994).

Hot plate test
Heat sensitivity was determined with a hot plate (BIOSEB, German). The hot plate test was performed as described as before (McCoy et al., 2013). The latency to licking a hind paw, jump, or shake hind paw were measured with a 30 seconds cut-off time.

Histology
The procedure for immunofluorescence staining follows the steps described previously (Yuan et al., 2019). Animals were deeply anesthetized by intraperitoneal injection of pentobarbital (100 mg/kg) and perfused with phosphate-buffered solution (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA). Then, the head was removed and soaked in 4% PFA overnight. After dissection, the brain was cryoprotected in 20% sucrose in PBS at 4°C overnight. Coronal sections (40 μm thick) containing the mPFC were obtained by a cryostat microtome. The slides were washed with 0.1 M PBS buffer three times. Finally, 4',6-diamidino-2-phenylindole (DAPI) were used to seal the slides. For the fluorescent images, an Olympus VS120 microscope was used to take the photos. The mPFC brain section containing the GRABATP and fiber channel is shown in Figure 1B.

Data analysis
ImageJ software was used to process the brain slides. All statistical analyses were performed with GraphPad Prism 8.0 or MATLAB R2020b. Intergroup comparisons of different results were performed using Student's paired / unpaired t-test and one-way analysis of variance (ANOVA).