Korean J Pain 2023; 36(2): 163-172
Published online April 1, 2023 https://doi.org/10.3344/kjp.22372
Copyright © The Korean Pain Society.
Kai Wen Xi1,2,3 , De Duo Chen1 , Xin Geng2 , Yan Bian4 , Min Xin Wang1 , Hui Bian1
1Department of Physiology, Faculty of Basic Medical Science, Kunming Medical University, Kunming, Yunnan, China
2Second Department of Neurosurgery, The First Affiliated Hospital, Kunming Medical University, Kunming, Yunnan, China
3Department of Cerebrovascular Surgery, Xinyu People’s Hospital, Xinyu, Jiangxi, China
4Department of Oncology, The Second Affiliated Hospital, Kunming Medical University, Kunming, Yunnan, China
Correspondence to:Hui Bian
Department of Physiology, Faculty of Basic Medical Science, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming 650500, Yunnan, China
Tel: +86-871-65922852, Fax: +86-871-65922865, E-mail: firstname.lastname@example.org
Handling Editor: Jong Yeon Park
Received: November 4, 2022; Revised: February 16, 2023; Accepted: February 19, 2023
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Synaptic plasticity contributes to nociceptive signal transmission and modulation, with calcium/calmodulin-dependent protein kinase II (CaMK II) playing a fundamental role in neural plasticity. This research was conducted to investigate the role of CaMK II in the transmission and regulation of nociceptive information within the nucleus accumbens (NAc) of naïve and morphine-tolerant rats.
Methods: Randall Selitto and hot-plate tests were utilized to measure the hindpaw withdrawal latencies (HWLs) in response to noxious mechanical and thermal stimuli. To induce chronic morphine tolerance, rats received intraperitoneal morphine injection twice per day for seven days. CaMK II expression and activity were assessed using western blotting.
Results: Intra-NAc microinjection of autocamtide-2-related inhibitory peptide (AIP) induced an increase in HWLs in naïve rats in response to noxious thermal and mechanical stimuli. Moreover, the expression of the phosphorylated CaMK II (p-CaMK II) was significantly decreased as determined by western blotting. Chronic intraperitoneal injection of morphine resulted in significant morphine tolerance in rats on Day 7, and an increase of p-CaMK II expression in NAc in morphine-tolerant rats was observed. Furthermore, intra-NAc administration of AIP elicited significant antinociceptive responses in morphine-tolerant rats. In addition, compared with naïve rats, AIP induced stronger thermal antinociceptive effects of the same dose in rats exhibiting morphine tolerance.
Conclusions: This study shows that CaMK II in the NAc is involved in the transmission and regulation of nociception in naïve and morphine-tolerant rats.
Keywords: Analgesics, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Morphine, Neuronal Plasticity, Nociception, Nucleus Accumbens, Pain, Peptides, Rats.
Pain is a predominant problem in modern healthcare . In addition to diminishing patient quality of life, pain also exerts immense financial costs [2,3]. For example, nearly 50–100 million Americans live in constant pain, with an estimated annual cost of $635 billion . Opioid analgesics such as morphine remain the major treatment modality for moderate to severe pain; however, most chronic pain patients develop opioid tolerance after long-term opioid use . Although many studies have focused on opioid analgesic tolerance, the underlying mechanisms of the initiation and development in opioid tolerance remain unclear because of their complexity.
Synaptic plasticity participates in the transmission and regulation of pain signals [6,7]. Opioids largely influence the synaptic plasticity of learning and memory within the neural system that are crucial to analgesic tolerance development . Calcium/calmodulin-dependent protein kinase II (CaMK II) plays a fundamental role in neural plasticity, long-term potentiation, learning, and memory [9,10]. Acute or chronic morphine exposure enhances CaMK II expression in the prefrontal cortex or spinal cord [11,12]. In rodent models of morphine tolerance, administration of CaMK II inhibitors into the spinal cord, hippocampus, or prefrontal cortex not only prevented, but also reversed morphine tolerance. This occurs either directly or through learning/memory pathways [10,11,13,14]. The above results imply that CaMK II is essential to the development and retention of morphine tolerance.
The nucleus accumbens (NAc) is a limbic structure that is associated with reward, addiction, and pleasure. Recent pain studies have shown that the NAc also plays a major role in pain modulation and antinociceptive tolerance and acts as a source of analgesia [1,15,16]. The authors’ earlier report showed that the microinjection of the CaMK II inhibitor autocamtide-2-related inhibitory peptide (AIP) or KN93 into the NAc alleviates pain that is related to behaviors in the peripheral neuropathic pain model [17,18]. However, the role of CaMK II in the NAc in naïve and morphine-tolerant rats has yet to be the subject of in-depth examination. Therefore, the current research was conducted to determine whether CaMK II participates in the transmission of nociception in the NAc of naïve rats and to examine the role of CaMK II in rats exhibiting morphine tolerance.
The solutions for NAc microinjection were dissolved in 0.9% sterilized saline, and the drug concentration was 4, 8 or 12 µg of AIP (a selective inhibitor of CaMK II; GL Biochem) per milliliter. The experimental group was injected with 1 µL of 0.9% sterilized saline.
In this study, adult male Sprague–Dawley rats with weights within the range of 200–250 g (Experiment Animal Center of Kunming Medical University) were used. These were housed in cages, provided with
Rats received intraperitoneal morphine injection (10 mg/kg, morphine hydrochloride injection, Shenyang First Pharmaceutical Factory) twice a day (09:00 and 21:00) for a total of seven days for induction of chronic morphine tolerance. To determine antinociceptive tolerance, the authors assessed the effect of the antinociception after 30 minutes of the first morphine injection every day, which was also compared with that of the first day. The increase in hindpaw withdrawal latencies (HWLs) in response to noxious thermal and mechanical stimulation due to morphine markedly decreased after repeated use of morphine. After seven days of repeated injection, the chronic morphine tolerance rat model was established.
Before the experiment, the rats were subjected to behavioral test training using noxious thermal and mechanical stimuli for three to five days. The HWLs, in response to noxious thermal and mechanical stimuli, were examined as described elsewhere [18–20].
In the experiment, HWLs, in response to nociceptive thermal stimulation were assessed by a hot-plate (Type YSL-6B Intelligent Heat Panel Instrument) that was set to a temperature of 52 ± 0.2°C. Radiant heat stimulation was applied to the plantar surface of the hind paw of the rats, and latency was measured in seconds and then recorded as the HWL of each rat to thermal stimulation.
Rat HWLs to nociceptive mechanical stimulation were assessed using the Randall Selitto Test (Ugo Basile, Type 37215). The push rod was applied at a loading rate of 30 g/s onto the dorsal surface of its hind paw, then recorded the latency necessary to induce a withdrawal reaction as HWL in response to mechanical stimulation. Each rat was examined using thermal and mechanical stimuli. To prevent tissue damage due to thermal and mechanical stimulation, a cut-off limit of pain detection was set at 15 seconds.
Before microinjection into the NAc, the HWLs were detected thrice to obtain an average value that was designated as the basal HWLs. The HWLs examined in subsequent experiments were then recorded as the percentage change at the basal level (% changes of HWL) in every rat examined.
A 60 mg/kg intraperitoneal injection of sodium pentobarbital was used to anesthetize the rats. Then, the rats were placed on a brain stereotaxic instrument. A stainless steel guide cannula with an external diameter of 0.8 mm was directed into the bilateral NAc (1.6 mm anterior to Bregma, 1.6 mm lateral to midline, and 7.0 mm ventral to the skull surface) following Paxinos and Watson , which was fixed using dental acrylic to the skull. After the surgery, the rats received daily injections of penicillin (80,000 units/one rat) for three days. Three days of recuperation were allowed before the following behavioral experiment was performed.
During the experiment, a syringe needle (0.4 mm external diameter) was inserted into the guide cannula, which was 1.5 mm longer than the cannula tip. Microinjected 0.5 µL of solution into each lateral NAc at a rate of 0.5 µL/min. The syringe needle was left in place for 1 minute after every microinjection.
At the end of the experiment, the rats’ brains were removed, and microinjection sites were confirmed according to the brain atlas. Only the results from rats whose injection sites were within the NAc were included in the statistical analysis.
CaMK II expression levels were assessed using western blotting, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference. Upon completion of the experiment, the rats were anesthetized using 4% isoflurane, and the brains were immediately collected. The NAc was dissected on ice and they were flash frozen at –80°C. The NAc tissue samples were then homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology) on ice. After centrifugation at 14,000 rpm for 10 minutes at 4°C, the supernatant was collected. The protein concentrations were then measured using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). An equal amount of 20 µg of total protein was loaded into each lane, which were then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Later, the proteins were transferred onto PVDF membranes (MilliporeSigma), then blocked with 5% nonfat dry milk in Tris-buffered saline Tween (TBST) for 1 hour at room temperature, followed by incubation in blocking solution containing primary antibody (rabbit anti-CaMK II antibody 1:5,000; Abcam, rabbit anti-CaMK II (phospho T286) antibody 1:1,000; Abcam); or horse radish peroxidase (HRP)-conjugated GAPDH (1:10,000; Kangcheng) overnight at 4°C. The membranes were later washed with TBST thrice, with 10 minutes for each wash and then incubated for 1 hour at room temperature with an HRP-conjugated anti-rabbit secondary antibody (1:5,000; ZhongShan GoldenBridge Technology) or TBST. After another batch of 3 × 10 minutes washes using TBST, the bands were visualized using enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology). The relative band densities were evaluated using ImageJ. The ratios of the optical densities of total CaMK II (t-CaMK II) or phosphorylated CaMK II (p-CaMK II) relative to those of GAPDH were determined for each sample.
The data collected from the experiment were expressed as mean ± standard error of the mean and analyzed with GraphPad Prism 5. Differences between groups were evaluated by two-way ANOVA for repeated measures or the Student’s
To examine the role of CaMK II in pain regulation in the NAc, four groups of naïve rats were subjected to intra-NAc microinjection of 4 µg (n = 8), 8 µg (n = 9) or 12 µg (n = 7) of AIP, with 1 µL of 0.9% sterilized saline as a control (n = 10). Relative to the control group, HWLs in response to noxious thermal and mechanical stimuli in the rat group treated with AIP significantly increased followed intra-NAc administration of 4 µg of AIP (hot-plate test: Fleft/left = 194.60,
To study the effect of AIP on CaMK II expression, rats received an intra-NAc microinjection of 12 µg of AIP (n = 3) or 1 µL of 0.9% sterilized saline as a control (n = 3). CaMK II expression and activity were assessed based on the degree of autophosphorylation at Thr 286 using western blotting. P-CaMK II/t-CaMK II ratio (n = 3; t = 3.57;
To elicit chronic tolerance to morphine antinociception, the rats (n = 10) intraperitoneally received morphine (10 mg/kg) twice a day for a total of 7 days. As shown in Fig. 3, the antinociceptive effects caused by morphine at 30 minutes after the first injection significantly decreased, and the rats exhibited morphine tolerance on day 7, relative to that of the first day (hot-plate test: tleft = 7.03,
The above results revealed the involvement of CaMK II in rat NAc chronic morphine tolerance. Thus, experiments were conducted to correlate the biochemical changes with the behavioral effects. Four groups of rats exhibiting chronic morphine tolerance received intra-NAc microinjections,
The above results showed that AIP induced antinociceptive effects both in naïve and morphine-tolerant rats. So, are there some changes in the antinociceptive effect of AIP in these two different states? In order to compare the changes of antinociceptive effect induced by AIP under normal and morphine tolerance conditions, Fig. 6 was used to depict the alteration in antinociception caused by AIP microinjection into the NAc of rats with morphine tolerance, relative to that in naïve rats. These results showed a significant increase in HWLs in response to noxious thermal stimulation post administration of 4 µg of AIP (hot-plate test: tleft/left = –1.60,
The results of this study showed that the naïve rat HWLs, in response to both thermal and mechanical stimuli, significantly increased after intra-NAc microinjection of AIP. These findings imply that AIP imparts an antinociceptive effect within the NAc. To further detect the antinociceptive effect of AIP in chronic morphine tolerance, the authors also observed that microinjection of AIP into the NAc resulted in prolonged HWLs in rats with chronic morphine tolerance. Interestingly, the thermal antinociceptive effect caused by AIP was stronger in rats exhibiting morphine tolerance relative to naïve rats using the same dose in the NAc. These results suggest that CaMK II participates in the modulation of nociception in naïve rats as well as in chronic morphine-induced antinociceptive tolerance in the NAc. Therefore, targeting CaMK II may not only help in elucidating the mechanism underlying pain modulation, but also provide a potential target of pharmacological intervention for morphine tolerance management.
With the increase in the incidence rate of pain, the widespread use of opioid therapy has proliferated in the past decades . Opioid tolerance is the main reason for decreased pain management and increased doses . In the past 15 years, the death rates caused by opioid overdose in the United States have increased four-fold . For example, over 130 Americans die every day due to opioid overdose in the United States . Therefore, there is an urgent need to develop a new treatment strategy that can both enhance opioid analgesia, as well as alleviate tolerance to improve patient safety .
A lot of studies have discussed the involvment of CaMK II in morphine tolerance at the spinal cord level [10,25–28]. Inhibition of spinal CaMK II can disrupt morphine antinociceptive tolerance in rodent models. The acute intrathecally administration of CaMK II inhibitor KN93 attenuated chronic morphine tolerance in rats . Further investigations have demonstrated that chronic morphine treatment results in a significant increase in CaMK II mRNA and protein expression in the spinal cord of mice. Confocal microscopy showed that the increase in expression of CaMK II was mainly localized to the superficial laminae of the dorsal horn . Another study showed that intraperitoneal injection of haloperidol, a butyrophenone antipsychotic drug, inhibited CaMK II; it both prevented and reversed morphine tolerance in mice. Their western blotting experimental results showed that pretreatment or short-term application of haloperidol inhibited the increased CaMK II α activity in the spinal cord .
However, few studies have focused on the participation of CaMK II beyond the spinal levels in morphine tolerance [11,12,14]. Fan et al.  reported that hippocampal CaMK II may act through learning and memory to affect morphine tolerance and dependence. Intrahippocampal dentate gyrus administration of CaMK II inhibitor in rats prevented the development of morphine tolerance. CaMK II may also affect morphine tolerance independent of learning and memory. Tang et al.  reported that intracerebroventricular KN93 injection dose-dependently reversed the established antinociceptive tolerance to morphine. Pretreating mice using KN93 could also prevent the generation of morphine tolerance. Further studies by Hu et al.  showed that chronic morphine treatment significantly increased CaMK II expression and activity in the cortices or prefrontal cortex in mice. The present study showed that chronic morphine tolerance induced a significant increase of p-CaMK II in the NAc, and inhibiting CaMK II
The µ-opioid receptor (µOR) is considered the most relevant factor influencing opioid tolerance. The desensitization of µOR may play an important role in the development of morphine tolerance . Biochemical evidence supports a direct interaction effect between CaMK II and the µOR . The µOR contains consensus sites for phosphorylation by CaMK II . Constitutively active CaMK II promotes agonist-induced desensitization of the µOR expressed in cellular models, and this effect is abolished by mutation of the putative CaMK II phosphorylation site [5,29,30]. This was further supported by the observation that µOR and CaMK II colocalize to anatomic areas responsible for the transmission and processing of nociceptive signals, including the dorsal root ganglia and the superficial laminae of the spinal cord dorsal horn . However, intracellular Ca2+, CaM, and CaMK II can each be regulated by morphine . Indeed, the expression and activity of CaMK II both increase after morphine exposure [10,11,25,26].
Furthermore, CaMK II may also interact with N-methyl-D-aspartic acid (NMDA) receptors, leading to morphine tolerance . CaMK II and the NMDA receptor may interact in a feedforward manner during the initiation stage of morphine tolerance. Activated CaMK II was phosphorylated, which in turn activated the NMDA receptor, leading to Ca2+ influx through the channels. In addition, increased Ca2+ influx through the activated NMDA receptor results in CaMK II autophosphorylation at position Thr 286 that then activates the kinase [5,11,12].
In conclusion, the results of this study indicate that administration of AIP into the NAc induced antinociceptive effects in naïve rats, and p-CaMK II expression was decreased by microinjection of AIP into the NAc. Moreover, increased p-CaMK II expression in the NAc was observed after chronic morphine tolerance. In addition, the administration of AIP into the NAc imparted antinociceptive effects in morphine-tolerant rats. These results reveal that CaMK II in the NAc participates in the nociception transmission and modulation in naïve and morphine-tolerant rats.
The datasets supporting the findings of this study are available from the corresponding author upon reasonable request.
No potential conflict of interest relevant to this article was reported.
This study was supported by the National Natural Science Foundation of China (No: 81760212).
Kai Wen Xi: Investigation; De Duo Chen: Investigation; Xin Geng: Investigation; Yan Bian: Investigation; Min Xin Wang: Investigation; Hui Bian: Writing/manuscript preparation.