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Activation of mu opioid receptor inhibits the excitatory glutamatergic transmission in the anterior cingulate cortex of the rats with peripheral inflammation
European Journal of Pharmacology 628 (2010) 91–95
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Activation of mu opioid receptor inhibits the excitatory glutamatergic transmission in the anterior cingulate cortex of the rats with peripheral inflammation Weihong Zheng ⁎ Department of Pharmacology, Medical Science College, China Three Gorges University, Yichang, Hubei 443002, PR China
a r t i c l e
i n f o
Article history: Received 29 August 2009 Received in revised form 9 November 2009 Accepted 17 November 2009 Available online 26 November 2009 Keywords: Excitatory postsynaptic current Opioid receptor Anterior cingulate cortex Complete Freund′s adjuvant
a b s t r a c t Emerging evidence recently indicates that the anterior cingulate cortex is critically involved in the central processing and modulation of noxious stimulus, although the neuroadaptation in the anterior cingulate cortex has not been well documented in the conditions of chronic pain. Meanwhile, the cellular mechanism underlying opiate analgesia in the anterior cingulate cortex remains unclear. To address these issues, the present study was undertaken to explore the adaptation of excitatory glutamatergic transmission and mu opioid receptor-mediated modulation of glutamatergic transmission in the anterior cingulate cortex slices from the complete Freund′s adjuvant (CFA)-inflamed rats. The results demonstrated that glutamatergic paired-pulse facilitation was decreased in the anterior cingulate cortex neurons from the CFA-inflamed rats, indicating an enhanced presynaptic glutamate release. In addition, activation of mu opioid receptor significantly inhibited the glutamatergic excitatory postsynaptic currents (EPSCs) in the anterior cingulate cortex neurons, which was attained through the suppression of presynaptic glutamate release. Taken together, these findings provided the evidence for the functional adaptation of central glutamatergic transmission induced by peripheral inflammation, and elucidated the cellular mechanism underlying opiate analgesia in the anterior cingulate cortex. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Anterior cingulate cortex is well documented as a critical brain region processing the cognitive and emotional functions of the brain (Frankland et al., 2004). Recently, increasing evidence demonstrates that the forebrain anterior cingulate cortex is critically involved in the supraspinal perception of noxious stimulus and pain modulation (Zhuo, 2008). Peripheral noxious stimuli significantly increase the excitability of anterior cingulate cortex neurons (Sikes et al., 2008; Vogt and Sikes, 2000), whereas local lesions of the medial frontal cortex, including the anterior cingulate cortex, reduce acute nociceptive responses, injury-related aversive behaviors, and chronic pain in patients (Johansen et al., 2001; Rios et al., 1999; Zhuo, 2006). As the principal excitatory neurotransmitter in neuronal circuits, glutamatergic transmission is essentially involved in a variety of brain functions, including pain modulation. Although emerging studies have provided some implicative hints to indicate the critical involvement of anterior cingulate cortex glutamatergic transmission in pain modulation in mice (Toyada et al., 2009; Wu et al., 2008), additional substantive evidence is required to further clarify the
adaptation of excitatory synaptic transmission in the anterior cingulate cortex in the condition of chronic pain. Currently opiates, especially the agents acting on mu opioid receptor, serve as the most effective analgesics for the clinical patients with moderate and severe pain. Although there exist 3 types of opioid receptors, mu, kappa and delta, in the neurons, most opioid actions, rewarding, addiction and analgesia, are mediated through the mu opioid receptor. Activation of mu opioid in the spinal cord and some supraspinal regions, including periaqueductal gray and nucleus raphe magnus, significantly inhibits the excitability of pain-related neurons, thus mediating the analgesia induced by opiates in these brain regions critically involved in the pain modulation. Although microinjection of mu agonist into the anterior cingulate cortex produces a robust analgesia effect in the rats (LaGraize et al., 2006), the cellular mechanism remains unclear. Hence, the present study is also designed to address this issue by investigating the effect of activation of mu opioid receptors on the glutamatergic transmission in the anterior cingulate cortex from the inflamed rats, by which the underlying central mechanisms for opioid analgesia are explored. 2. Materials and methods
⁎ Department of Pharmacology, Medical Science College, China Three Gorges University, 8 University Avenue, Yichang, Hubei 443002, PR China. Tel.: +86 717 6397466; fax: +86 717 6397328. E-mail address: [email protected]. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.11.041
2.1. Animals and behavioral test All experimental protocols and animal handling procedures were approved by the Institutional Animal Care and Use Committee at
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W. Zheng / European Journal of Pharmacology 628 (2010) 91–95
China Three Gorges University, and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Young male adult Sprague–Dawley rats (6–8 weeks old) were maintained on a 12 h light/dark cycle with food and water provided ad libitum. To induce inflammatory pain, 30 µl of 5% complete Freund′s adjuvant (CFA) was injected subcutaneously into the dorsal surface of the left hindpaw in the rats anesthetized with halothane (Guo et al., 2005). Meanwhile, saline was injected in the same site in another group of rats as control. Thermal nociceptive thresholds were assessed using radiant heat (Hargreaves et al., 1988). Briefly, for this test, the rat was placed in a clear Plexiglas box resting on an elevated glass plate maintained at 30 °C. After a 1 h acclimation, a radiant beam of light was positioned under the hindpaw and the time for the rat to remove the paw from the thermal stimulus was electronically recorded as the paw withdrawal latency (PWL). The intensity of the beam was adjusted to produce basal PWLs of ∼ 10 s. A maximal PWL of 16 s was set to prevent excessive tissue damage due to the repeated application of a noxious thermal stimulus. 2.2. Brain slice preparation The rats were deeply anesthetized with halothane inhalation and then decapitated. The whole brain was quickly removed and placed into the cold (2–4 °C) Krebs solution containing (in mM) NaCl, 126; KCl, 2.5; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.4; glucose, 11; and NaHCO3, 25 with pH 7.35. Coronal brain slices (300 μm) containing the anterior cingulate cortex were cut on a Vibratome (series 1000, Technical Products International, Inc., St. Louis, MO, USA) in cold (2–4 °C) Krebs solution. The slices were then transferred to a chamber filled with Krebs solution saturated with 95% O2 and 5% CO2 and allowed to rest at 35 °C for at least 1 h. 2.3. Whole-cell patch clamp recordings in adult anterior cingulate cortex slices Visualization of whole-cell patch clamp recordings was performed on the anterior cingulate cortex slices after one-hour recovery. Excitatory postsynaptic currents (EPSCs) were recorded from layer II/III neurons in the anterior cingulate cortex with an Axon 200B amplifier (Molecular Devices, CA) and stimulations (0.25 ms, 0.05–0.3 mA) were delivered by a bipolar tungsten stimulating electrode placed in layer V of the anterior cingulate cortex. Recording electrodes (2–5 MΩ) contained a pipette solution composed of (mM): K-gluconate, 125; NaCl, 5; MgCl2 1; EGTA, 0.5; Mg-ATP, 2; Na3GTP, 0.1; HEPES, 10; pH 7.2; and 280–300 mOsmol. For paradigm of paired-pulse ratios (PPR), a pair of EPSCs was evoked by two stimuli with an interval of 40, 70 and 100 ms, and the PPR at each interval was calculated by dividing the second EPSC amplitude by the first one. Four PPRs were averaged to obtain a mean PPR to minimum the variances among consecutive events. Miniature EPSCs (mEPSCs) were recorded in 60-s epochs with TTX (1 μM) in the bath Krebs solution. mEPSCs were detected and analyzed using an event detection program (Mini Analysis Program; Synaptosoft, Inc., Decatur, GA). A seal resistance of ≥2 GΩ and an access resistance of 15–20 MΩ were considered acceptable. The series resistance was optimally compensated by ≥70% and constantly monitored throughout the experiments. The membrane potential was held at −70 mV throughout the experiment.
identify significant difference. All data are expressed as mean± S.E.M. P b 0.05 was considered statistically significant. 3. Result 3.1. Subcutaneous injection of CFA induced ipsilateral thermal hyperalgesia Firstly we observed the thermal hyperalgesia development in the rats with CFA injection. Consistent with the previous studies (Guo, et al., 2005; Zhao et al., 2006), local swelling and thermal hyperalgesia gradually occurred in the ipsilateral hindpaw following unilateral injection of CFA into the plantar. The inflammation in the ipsilateral plantar reached a maximal plateau after 24 h and lasted more than 4 days. As shown in Fig. 1, PWLs to noxious thermal stimulus were significantly shortened in the ipsilateral (two-way ANOVA, P b 0.01), but not the contralateral, hindpaws through the 4 days after injection. Meanwhile, no any detectable change occurred in the hindpaws of the rats with saline injection (data not shown). These results suggested that thermal hyperalgesia occurred in CFA-inflamed rats. 3.2. CFA-induced peripheral inflammation enhanced glutamatergic transmission in the anterior cingulate cortex To detect the functional adaptation of excitatory glutamatergic transmission in the anterior cingulate cortex during chronic pain, we measured the paired-pulses facilitation of evoked EPSCs in the anterior cingulate cortex neurons from the rats receiving unilateral saline or CFA 4 days previously. EPSCs were evoked by electrical stimulation within the anterior cingulate cortex in the presence of bicuculline (30 μM), and these evoked EPSCs can be completely blocked by the application of CNQX (10 μM) and AP-5 (10 μM), confirming its components mediated by the excitatory transmitter glutamate. In order to identify the glutamatergic adaptation in the anterior cingulate cortex neurons induced by peripheral inflammation, the EPSC PPR paradigm was employed to detect any potential alteration of the probability of presynaptic transmitter release (Zhao et al., 2006; Zucker and Regehr, 2002). In the present study, evoked EPSC PPR was significantly decreased in anterior cingulate cortex neurons from CFA-inflamed rats when compared with saline controls (CFA: 1.38 ± 0.07, n = 11; saline: 1.68 ± 0.08, n = 11; P b 0.01) (Fig. 2A,B), indicating an increased probability of presynaptic transmitter release in the glutamatergic synapses of CFA-inflamed rats. To exclude the potential influence of inter-pulse intervals, three different stimulus intervals (40, 70, and 100 ms) were applied (Zhao et al., 2006). In neurons from CFA-treated
2.4. Drug, data analysis and statistics 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (AP-5), [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), CTAP (H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) and all other drugs were purchased from Sigma Aldrich, and were applied through the perfusion solution. Results were analyzed by paired or unpaired t-test, or two-way ANOVA followed by post-hoc test to
Fig. 1. Subcutaneous injection of CFA induced thermal hyperalgesia in the ipsilateral hindpaw of the rats. Intra-plantar injection of CFA (30 μl, n = 7) induced progressive PWL reduction of the ipsilateral paw but not the uninjected contralateral paw. ⁎⁎P b 0.01 vs. the uninjected contralateral hindpaw.
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Fig. 3. Activation of mu opioid receptor inhibited the evoked EPSCs in the anterior cingulate cortex neurons. Representative EPSCs in an anterior cingulate cortex neuron from the CFA-inflamed rat, under the conditions of control, the presence of selective mu receptor agonist DAMGO, and DAMGO plus CTAP, a selective mu receptor antagonist.
Fig. 2. Peripheral inflammation increased presynaptic glutamate release in the anterior cingulate cortex neurons. A, Representative pairs of EPSCs evoked by two consecutive stimuli at an interval of 40 ms in an anterior cingulate cortex neuron from a salineinjected or CFA-inflamed rat. B, Paired-pulse ratios at three between stimuli intervals as indicated in the anterior cingulate cortex neurons of the control group (n = 13) and of the inflamed group (n = 10 at each interval). ⁎P b 0.05, ⁎⁎P b 0.01.
using synaptic analyses of both PPR and miniature EPSCs. DAMGO (1 μM) significantly increased the ratio of the paired EPSCs in the anterior cingulate cortex neurons from the CFA-injected rats (1.33 ± 0.07 vs. 1.72 ± 0.13, n = 7, P b 0.01, Fig. 4), which indicating a presynaptic action site of mu receptor-mediated inhibition of evoked EPSCs. This was further confirmed by the following investigation of the effects of DAMGO on the miniature spontaneous EPSCs in the anterior cingulate cortex neurons from the inflamed rats. Consistently, DAMGO (1 μM) significantly decreased the mEPSCs frequency (13.21 ± 1.76 Hz, vs. 7.27±1.17 Hz; n = 6, P b 0.05) without significantly altering the mEPSCs amplitude (−23.3 ±1.45 pA vs. −22.3 ± 1.58 pA, n = 6, P N 0.05) (Fig. 5). Taken together, these observations consolidated the notion that the mu opioid receptor-mediated inhibition of glutamatergic transmission was fulfilled through the suppression of presynaptic glutamate release. 4. Discussion
rats, the values of PPRs were significantly reduced at all three stimulus intervals (Fig. 2B). This suggested a stimulus interval-independent reduction in the PPR of glutamate EPSCs, and confirmed the enhanced excitatory glutamatergic synaptic transmission in the anterior cingulate cortex neurons induced by chronic peripheral inflammation. 3.3. Activation of mu opioid receptor inhibited EPSC in the anterior cingulate cortex To address the cellular mechanisms underlying the opiates analgesia, the effect of activation of mu opioid receptor on the enhanced glutamatergic transmission was examined in the anterior cingulate cortex slices from the CFA-inflamed rats. In the brain slices from the inflamed rats, bath application of DAMGO (1 μM), a selective mu opioid receptor agonist, significantly decreased the amplitude of evoked EPSCs by 53.1 ± 3.0% (− 264.4 ± 38.5 pA vs. − 119.9 ± 38.5 pA, n = 10, P b 0.01), while this inhibition was completely abolished by the co-application of selective mu antagonist CTAP (1 μM) (control, −252.7 ± 26.1 pA; DAMGO, −127.8 ± 20.3 pA, P b 0.01 vs. control; DAMGO+CTAP, −245.7 ± 22.6 pA, n = 6, P N 0.05 vs. control, Fig. 3), suggesting that DAMGO-mediated inhibition was specifically mediated through mu opioid receptor. Meanwhile, perfusion of DAMGO (1 μM) induced similar inhibition of evoked EPSC (− 189.8 ± 9.8 pA, vs. −111.1 ± 8.5 pA, n = 7, P b 0.01) in the anterior cingulate cortex neurons from the saline-treated rats.
In the present study, we identified the enhanced glutamatergic transmission and mu opioid receptor-mediated inhibition of presynaptic glutamate release in the anterior cingulate cortex neurons from the rats with the chronic inflammatory pain. To our knowledge, this is the first investigation to combine these two issues together to elucidate the underlying mechanism for opioid analgesia in the anterior cingulate cortex, the key brain region for pain and emotion modulation. 4.1. Functions of the anterior cingulate cortex in pain Series of evidence have demonstrated the critical involvement of the forebrain anterior cingulate cortex in the perception of noxious
3.4. Activation of mu opioid receptor decreased the presynaptic release of glutamate We next determined whether the EPSC inhibition by activation of mu opioid receptor involved a pre- or post-synaptic site, or both,
Fig. 4. Mu-opioid receptor-mediated inhibition of EPSCs involved a presynaptic site. Representative EPSC pairs at 40 ms interval before and after addition of DAMGO in an anterior cingulate cortex neuron from the CFA-inflamed rats.
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AMP-PKA signaling pathway was postulated to be responsible for the enhanced glutamtergic transmission in the anterior cingulate cortex neurons from the rats with chronic pain. 4.3. Mu-inhibition of EPSC and functional significance
Fig. 5. Activation of mu opioid receptor reduced presynaptic release of glutamate. A, Current traces with spontaneous events of miniature EPSCs in the absence and presence of DAMGO from an anterior cingulate cortex neuron of the inflamed rat. B, Group data of the DAMGO effect on miniature EPSC frequency and amplitude in anterior cingulate cortex neurons from the inflamed rats. ⁎P b 0.05.
stimulus and analgesia (Zhuo, 2006), while it is usually established as a critical brain region processing the cognitive and emotional functions. Previous electrophysiological evidence showed that the anterior cingulate cortex neurons responded to peripheral noxious stimulus in the rodents (Sikes et al., 2008; Vogt and Sikes, 2000; Yang et al., 2006), and neuroimaging studies in humans further confirmed these observations and showed that the anterior cingulate cortex, together with other cortical structures, were activated by acute noxious stimuli, psychological pain, and social pain (Eisenberger et al., 2003; Hofbauer et al., 2001; Koyama et al., 2000). Therefore, the anterior cingulate cortex was served as a potential target for pain modulation and analgesia.
4.2. Functional significance of enhanced EPSC in the anterior cingulate cortex In the present study, an enhanced presynaptic glutamate release, although we cannot exclusively rule out the possible concurrence of adaptation of postsynaptic glutamate receptors (Wu et al., 2008), was observed in the anterior cingulate cortex neurons from the rats with chronic pain, which was clearly indicated by the significantly decreased paired-pulsed facilitation in glutamatergic synapses in the anterior cingulate cortex slices from the inflamed rats. Previous studies found the similar enhanced excitatory glutamatergic transmission in the anterior cingulate cortex of mice with persistent inflammatory pain (Toyada et al., 2009) and neuropathic pain (Xu et al., 2008). These findings suggested that increased presynaptic glutamate release was a fundamental feature of the long-term synaptic plasticity in the anterior cingulate cortex neurons during chronic pain, which would be responsible for the emotional response to chronic pain and the aversion to noxious stimulus. Previous studies reported an increased activity of intracellular cyclic AMP-PKA signaling pathway in the anterior cingulate cortex neurons from the animals with chronic pain (Wu et al., 2008; Liauw et al., 2005), and upregulation of this signaling pathway was proved to increase the neurotransmitters, both glutamate (Fu et al., 2008) and GABA (Bonci and Williams, 1997), release from the presynaptic terminals under various pathophysiological conditions. Therefore, an upregulated cyclic
To our knowledge, the present study is the first to report the mu opioid receptor-mediated inhibition of excitatory glutamatergic transmission in the anterior cingulate cortex. Activation of mu opioid receptor suppressed the presynaptic glutamate release in the spinal cord (Terman et al., 2001), the central amygdala (Zhu and Pan, 2005) and some other brain regions (Ostermeier et al., 2000; Sbrenna et al., 1999; Yang et al., 2004), which was consistent with the present finding. In general, this mu-mediated action was realized through the inhibition of the intracellular PLA2 signaling pathway and, subsequently, the 4-AP sensitive potassium channels in the presynaptic terminals (Zhu and Pan, 2005). Mu opioid receptor was well documented to underlie the most analgesic effect of opiates. The existence of mu opioid receptors in the anterior cingulate cortex was previously well demonstrated by the binding assay with the radioactive-labeled selective agonists (Vogt et al., 1995, 2001) and immunostaining studies (Chen et al., 2008; Zubieta et al., 2001). It was reported that activation of endogenous mu opioid receptor activity in the anterior cingulate cortex largely underlay the central mechanisms for placebo-mediated analgesia (Wager et al., 2007), and microinjection of morphine into the anterior cingulate cortex produced a selective naloxone reversible analgesia and alleviated the fear memory of the noxious stimulus in nerve-damaged animals (LaGraize et al., 2006; Zubieta et al., 2001), although the underlying mechanisms remained unclear. In the present study, we observed that activation of mu opioid receptor significantly suppressed the enhanced presynaptic glutamate release in the anterior cingulate cortex neurons in the inflamed rats. As discussed above, the enhanced glutamatergic transmission in the anterior cingulate cortex was supposed to be critical for the negative emotion and aversion during chronic pain (Zhuo, 2008), thus mu opioid receptor-mediated inhibition of EPSC in this brain region would underlie the mechanism for the alleviation of stress and aversion during opiates-mediated analgesia. 5. Conclusion In conclusion, our experiment demonstrated that chronic peripheral inflammation significantly enhanced the presynaptic glutamate release in the anterior cingulate cortex, which was significantly suppressed by the activation of mu opioid receptor. This investigation may clarify, at least partially, the underlying supraspinal mechanism for opiatemediated analgesia and suppression of an aversion to noxious stimulus (LaGraize et al., 2006). References Bonci, A., Williams, J.T., 1997. Increased probability of GABA release during withdrawal from morphine. J. Neurosci. 17, 796–803. Chen, T.C., Cheng, Y.Y., Sun, W.Z., Shyu, B.C., 2008. Differential regulation of morphine antinociceptive effects by endogenous enkephalinergic system in the forebrain of mice. Mol. Pain 4, 41. Eisenberger, N.I., Lieberman, M.D., Williams, K.D., 2003. Does rejection hurt? An FMRI study of social exclusion. Science 302, 290–292. Frankland, P.W., Bontempi, B., Talton, L.E., Kaczmarek, L., Silva, A.J., 2004. The involvement of the anterior cingulate cortex in remote contextual fear memory. Science 304, 881–883. Fu, Y., Han, J., Ishola, T., Scerbo, M., Adwanikar, H., Ramsey, C., Neugebauer, V., 2008. PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior. Mol. Pain 4, 26. Guo, J.D., Wang, H., Zhang, Y.Q., Zhao, Z.Q., 2005. Alterations of membrane properties and effects of D-serine on NMDA-induced current in rat anterior cingulate cortex neurons after monoarthritis. Neurosci. Lett. 384, 245–249. Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77–88. Hofbauer, R.K., Rainville, P., Duncan, G.H., Bushnell, M.C., 2001. Cortical representation of the sensory dimension of pain. J. Neurophysiol. 86, 402–411.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Activation of mu opioid receptor inhibits the excitatory glutamatergic transmission in the anterior cingulate cortex of the rats with peripheral inflammation Weihong Zheng ⁎ Department of Pharmacology, Medical Science College, China Three Gorges University, Yichang, Hubei 443002, PR China
a r t i c l e
i n f o
Article history: Received 29 August 2009 Received in revised form 9 November 2009 Accepted 17 November 2009 Available online 26 November 2009 Keywords: Excitatory postsynaptic current Opioid receptor Anterior cingulate cortex Complete Freund′s adjuvant
a b s t r a c t Emerging evidence recently indicates that the anterior cingulate cortex is critically involved in the central processing and modulation of noxious stimulus, although the neuroadaptation in the anterior cingulate cortex has not been well documented in the conditions of chronic pain. Meanwhile, the cellular mechanism underlying opiate analgesia in the anterior cingulate cortex remains unclear. To address these issues, the present study was undertaken to explore the adaptation of excitatory glutamatergic transmission and mu opioid receptor-mediated modulation of glutamatergic transmission in the anterior cingulate cortex slices from the complete Freund′s adjuvant (CFA)-inflamed rats. The results demonstrated that glutamatergic paired-pulse facilitation was decreased in the anterior cingulate cortex neurons from the CFA-inflamed rats, indicating an enhanced presynaptic glutamate release. In addition, activation of mu opioid receptor significantly inhibited the glutamatergic excitatory postsynaptic currents (EPSCs) in the anterior cingulate cortex neurons, which was attained through the suppression of presynaptic glutamate release. Taken together, these findings provided the evidence for the functional adaptation of central glutamatergic transmission induced by peripheral inflammation, and elucidated the cellular mechanism underlying opiate analgesia in the anterior cingulate cortex. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Anterior cingulate cortex is well documented as a critical brain region processing the cognitive and emotional functions of the brain (Frankland et al., 2004). Recently, increasing evidence demonstrates that the forebrain anterior cingulate cortex is critically involved in the supraspinal perception of noxious stimulus and pain modulation (Zhuo, 2008). Peripheral noxious stimuli significantly increase the excitability of anterior cingulate cortex neurons (Sikes et al., 2008; Vogt and Sikes, 2000), whereas local lesions of the medial frontal cortex, including the anterior cingulate cortex, reduce acute nociceptive responses, injury-related aversive behaviors, and chronic pain in patients (Johansen et al., 2001; Rios et al., 1999; Zhuo, 2006). As the principal excitatory neurotransmitter in neuronal circuits, glutamatergic transmission is essentially involved in a variety of brain functions, including pain modulation. Although emerging studies have provided some implicative hints to indicate the critical involvement of anterior cingulate cortex glutamatergic transmission in pain modulation in mice (Toyada et al., 2009; Wu et al., 2008), additional substantive evidence is required to further clarify the
adaptation of excitatory synaptic transmission in the anterior cingulate cortex in the condition of chronic pain. Currently opiates, especially the agents acting on mu opioid receptor, serve as the most effective analgesics for the clinical patients with moderate and severe pain. Although there exist 3 types of opioid receptors, mu, kappa and delta, in the neurons, most opioid actions, rewarding, addiction and analgesia, are mediated through the mu opioid receptor. Activation of mu opioid in the spinal cord and some supraspinal regions, including periaqueductal gray and nucleus raphe magnus, significantly inhibits the excitability of pain-related neurons, thus mediating the analgesia induced by opiates in these brain regions critically involved in the pain modulation. Although microinjection of mu agonist into the anterior cingulate cortex produces a robust analgesia effect in the rats (LaGraize et al., 2006), the cellular mechanism remains unclear. Hence, the present study is also designed to address this issue by investigating the effect of activation of mu opioid receptors on the glutamatergic transmission in the anterior cingulate cortex from the inflamed rats, by which the underlying central mechanisms for opioid analgesia are explored. 2. Materials and methods
⁎ Department of Pharmacology, Medical Science College, China Three Gorges University, 8 University Avenue, Yichang, Hubei 443002, PR China. Tel.: +86 717 6397466; fax: +86 717 6397328. E-mail address: [email protected]. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.11.041
2.1. Animals and behavioral test All experimental protocols and animal handling procedures were approved by the Institutional Animal Care and Use Committee at
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China Three Gorges University, and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Young male adult Sprague–Dawley rats (6–8 weeks old) were maintained on a 12 h light/dark cycle with food and water provided ad libitum. To induce inflammatory pain, 30 µl of 5% complete Freund′s adjuvant (CFA) was injected subcutaneously into the dorsal surface of the left hindpaw in the rats anesthetized with halothane (Guo et al., 2005). Meanwhile, saline was injected in the same site in another group of rats as control. Thermal nociceptive thresholds were assessed using radiant heat (Hargreaves et al., 1988). Briefly, for this test, the rat was placed in a clear Plexiglas box resting on an elevated glass plate maintained at 30 °C. After a 1 h acclimation, a radiant beam of light was positioned under the hindpaw and the time for the rat to remove the paw from the thermal stimulus was electronically recorded as the paw withdrawal latency (PWL). The intensity of the beam was adjusted to produce basal PWLs of ∼ 10 s. A maximal PWL of 16 s was set to prevent excessive tissue damage due to the repeated application of a noxious thermal stimulus. 2.2. Brain slice preparation The rats were deeply anesthetized with halothane inhalation and then decapitated. The whole brain was quickly removed and placed into the cold (2–4 °C) Krebs solution containing (in mM) NaCl, 126; KCl, 2.5; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.4; glucose, 11; and NaHCO3, 25 with pH 7.35. Coronal brain slices (300 μm) containing the anterior cingulate cortex were cut on a Vibratome (series 1000, Technical Products International, Inc., St. Louis, MO, USA) in cold (2–4 °C) Krebs solution. The slices were then transferred to a chamber filled with Krebs solution saturated with 95% O2 and 5% CO2 and allowed to rest at 35 °C for at least 1 h. 2.3. Whole-cell patch clamp recordings in adult anterior cingulate cortex slices Visualization of whole-cell patch clamp recordings was performed on the anterior cingulate cortex slices after one-hour recovery. Excitatory postsynaptic currents (EPSCs) were recorded from layer II/III neurons in the anterior cingulate cortex with an Axon 200B amplifier (Molecular Devices, CA) and stimulations (0.25 ms, 0.05–0.3 mA) were delivered by a bipolar tungsten stimulating electrode placed in layer V of the anterior cingulate cortex. Recording electrodes (2–5 MΩ) contained a pipette solution composed of (mM): K-gluconate, 125; NaCl, 5; MgCl2 1; EGTA, 0.5; Mg-ATP, 2; Na3GTP, 0.1; HEPES, 10; pH 7.2; and 280–300 mOsmol. For paradigm of paired-pulse ratios (PPR), a pair of EPSCs was evoked by two stimuli with an interval of 40, 70 and 100 ms, and the PPR at each interval was calculated by dividing the second EPSC amplitude by the first one. Four PPRs were averaged to obtain a mean PPR to minimum the variances among consecutive events. Miniature EPSCs (mEPSCs) were recorded in 60-s epochs with TTX (1 μM) in the bath Krebs solution. mEPSCs were detected and analyzed using an event detection program (Mini Analysis Program; Synaptosoft, Inc., Decatur, GA). A seal resistance of ≥2 GΩ and an access resistance of 15–20 MΩ were considered acceptable. The series resistance was optimally compensated by ≥70% and constantly monitored throughout the experiments. The membrane potential was held at −70 mV throughout the experiment.
identify significant difference. All data are expressed as mean± S.E.M. P b 0.05 was considered statistically significant. 3. Result 3.1. Subcutaneous injection of CFA induced ipsilateral thermal hyperalgesia Firstly we observed the thermal hyperalgesia development in the rats with CFA injection. Consistent with the previous studies (Guo, et al., 2005; Zhao et al., 2006), local swelling and thermal hyperalgesia gradually occurred in the ipsilateral hindpaw following unilateral injection of CFA into the plantar. The inflammation in the ipsilateral plantar reached a maximal plateau after 24 h and lasted more than 4 days. As shown in Fig. 1, PWLs to noxious thermal stimulus were significantly shortened in the ipsilateral (two-way ANOVA, P b 0.01), but not the contralateral, hindpaws through the 4 days after injection. Meanwhile, no any detectable change occurred in the hindpaws of the rats with saline injection (data not shown). These results suggested that thermal hyperalgesia occurred in CFA-inflamed rats. 3.2. CFA-induced peripheral inflammation enhanced glutamatergic transmission in the anterior cingulate cortex To detect the functional adaptation of excitatory glutamatergic transmission in the anterior cingulate cortex during chronic pain, we measured the paired-pulses facilitation of evoked EPSCs in the anterior cingulate cortex neurons from the rats receiving unilateral saline or CFA 4 days previously. EPSCs were evoked by electrical stimulation within the anterior cingulate cortex in the presence of bicuculline (30 μM), and these evoked EPSCs can be completely blocked by the application of CNQX (10 μM) and AP-5 (10 μM), confirming its components mediated by the excitatory transmitter glutamate. In order to identify the glutamatergic adaptation in the anterior cingulate cortex neurons induced by peripheral inflammation, the EPSC PPR paradigm was employed to detect any potential alteration of the probability of presynaptic transmitter release (Zhao et al., 2006; Zucker and Regehr, 2002). In the present study, evoked EPSC PPR was significantly decreased in anterior cingulate cortex neurons from CFA-inflamed rats when compared with saline controls (CFA: 1.38 ± 0.07, n = 11; saline: 1.68 ± 0.08, n = 11; P b 0.01) (Fig. 2A,B), indicating an increased probability of presynaptic transmitter release in the glutamatergic synapses of CFA-inflamed rats. To exclude the potential influence of inter-pulse intervals, three different stimulus intervals (40, 70, and 100 ms) were applied (Zhao et al., 2006). In neurons from CFA-treated
2.4. Drug, data analysis and statistics 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (AP-5), [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), CTAP (H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) and all other drugs were purchased from Sigma Aldrich, and were applied through the perfusion solution. Results were analyzed by paired or unpaired t-test, or two-way ANOVA followed by post-hoc test to
Fig. 1. Subcutaneous injection of CFA induced thermal hyperalgesia in the ipsilateral hindpaw of the rats. Intra-plantar injection of CFA (30 μl, n = 7) induced progressive PWL reduction of the ipsilateral paw but not the uninjected contralateral paw. ⁎⁎P b 0.01 vs. the uninjected contralateral hindpaw.
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Fig. 3. Activation of mu opioid receptor inhibited the evoked EPSCs in the anterior cingulate cortex neurons. Representative EPSCs in an anterior cingulate cortex neuron from the CFA-inflamed rat, under the conditions of control, the presence of selective mu receptor agonist DAMGO, and DAMGO plus CTAP, a selective mu receptor antagonist.
Fig. 2. Peripheral inflammation increased presynaptic glutamate release in the anterior cingulate cortex neurons. A, Representative pairs of EPSCs evoked by two consecutive stimuli at an interval of 40 ms in an anterior cingulate cortex neuron from a salineinjected or CFA-inflamed rat. B, Paired-pulse ratios at three between stimuli intervals as indicated in the anterior cingulate cortex neurons of the control group (n = 13) and of the inflamed group (n = 10 at each interval). ⁎P b 0.05, ⁎⁎P b 0.01.
using synaptic analyses of both PPR and miniature EPSCs. DAMGO (1 μM) significantly increased the ratio of the paired EPSCs in the anterior cingulate cortex neurons from the CFA-injected rats (1.33 ± 0.07 vs. 1.72 ± 0.13, n = 7, P b 0.01, Fig. 4), which indicating a presynaptic action site of mu receptor-mediated inhibition of evoked EPSCs. This was further confirmed by the following investigation of the effects of DAMGO on the miniature spontaneous EPSCs in the anterior cingulate cortex neurons from the inflamed rats. Consistently, DAMGO (1 μM) significantly decreased the mEPSCs frequency (13.21 ± 1.76 Hz, vs. 7.27±1.17 Hz; n = 6, P b 0.05) without significantly altering the mEPSCs amplitude (−23.3 ±1.45 pA vs. −22.3 ± 1.58 pA, n = 6, P N 0.05) (Fig. 5). Taken together, these observations consolidated the notion that the mu opioid receptor-mediated inhibition of glutamatergic transmission was fulfilled through the suppression of presynaptic glutamate release. 4. Discussion
rats, the values of PPRs were significantly reduced at all three stimulus intervals (Fig. 2B). This suggested a stimulus interval-independent reduction in the PPR of glutamate EPSCs, and confirmed the enhanced excitatory glutamatergic synaptic transmission in the anterior cingulate cortex neurons induced by chronic peripheral inflammation. 3.3. Activation of mu opioid receptor inhibited EPSC in the anterior cingulate cortex To address the cellular mechanisms underlying the opiates analgesia, the effect of activation of mu opioid receptor on the enhanced glutamatergic transmission was examined in the anterior cingulate cortex slices from the CFA-inflamed rats. In the brain slices from the inflamed rats, bath application of DAMGO (1 μM), a selective mu opioid receptor agonist, significantly decreased the amplitude of evoked EPSCs by 53.1 ± 3.0% (− 264.4 ± 38.5 pA vs. − 119.9 ± 38.5 pA, n = 10, P b 0.01), while this inhibition was completely abolished by the co-application of selective mu antagonist CTAP (1 μM) (control, −252.7 ± 26.1 pA; DAMGO, −127.8 ± 20.3 pA, P b 0.01 vs. control; DAMGO+CTAP, −245.7 ± 22.6 pA, n = 6, P N 0.05 vs. control, Fig. 3), suggesting that DAMGO-mediated inhibition was specifically mediated through mu opioid receptor. Meanwhile, perfusion of DAMGO (1 μM) induced similar inhibition of evoked EPSC (− 189.8 ± 9.8 pA, vs. −111.1 ± 8.5 pA, n = 7, P b 0.01) in the anterior cingulate cortex neurons from the saline-treated rats.
In the present study, we identified the enhanced glutamatergic transmission and mu opioid receptor-mediated inhibition of presynaptic glutamate release in the anterior cingulate cortex neurons from the rats with the chronic inflammatory pain. To our knowledge, this is the first investigation to combine these two issues together to elucidate the underlying mechanism for opioid analgesia in the anterior cingulate cortex, the key brain region for pain and emotion modulation. 4.1. Functions of the anterior cingulate cortex in pain Series of evidence have demonstrated the critical involvement of the forebrain anterior cingulate cortex in the perception of noxious
3.4. Activation of mu opioid receptor decreased the presynaptic release of glutamate We next determined whether the EPSC inhibition by activation of mu opioid receptor involved a pre- or post-synaptic site, or both,
Fig. 4. Mu-opioid receptor-mediated inhibition of EPSCs involved a presynaptic site. Representative EPSC pairs at 40 ms interval before and after addition of DAMGO in an anterior cingulate cortex neuron from the CFA-inflamed rats.
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AMP-PKA signaling pathway was postulated to be responsible for the enhanced glutamtergic transmission in the anterior cingulate cortex neurons from the rats with chronic pain. 4.3. Mu-inhibition of EPSC and functional significance
Fig. 5. Activation of mu opioid receptor reduced presynaptic release of glutamate. A, Current traces with spontaneous events of miniature EPSCs in the absence and presence of DAMGO from an anterior cingulate cortex neuron of the inflamed rat. B, Group data of the DAMGO effect on miniature EPSC frequency and amplitude in anterior cingulate cortex neurons from the inflamed rats. ⁎P b 0.05.
stimulus and analgesia (Zhuo, 2006), while it is usually established as a critical brain region processing the cognitive and emotional functions. Previous electrophysiological evidence showed that the anterior cingulate cortex neurons responded to peripheral noxious stimulus in the rodents (Sikes et al., 2008; Vogt and Sikes, 2000; Yang et al., 2006), and neuroimaging studies in humans further confirmed these observations and showed that the anterior cingulate cortex, together with other cortical structures, were activated by acute noxious stimuli, psychological pain, and social pain (Eisenberger et al., 2003; Hofbauer et al., 2001; Koyama et al., 2000). Therefore, the anterior cingulate cortex was served as a potential target for pain modulation and analgesia.
4.2. Functional significance of enhanced EPSC in the anterior cingulate cortex In the present study, an enhanced presynaptic glutamate release, although we cannot exclusively rule out the possible concurrence of adaptation of postsynaptic glutamate receptors (Wu et al., 2008), was observed in the anterior cingulate cortex neurons from the rats with chronic pain, which was clearly indicated by the significantly decreased paired-pulsed facilitation in glutamatergic synapses in the anterior cingulate cortex slices from the inflamed rats. Previous studies found the similar enhanced excitatory glutamatergic transmission in the anterior cingulate cortex of mice with persistent inflammatory pain (Toyada et al., 2009) and neuropathic pain (Xu et al., 2008). These findings suggested that increased presynaptic glutamate release was a fundamental feature of the long-term synaptic plasticity in the anterior cingulate cortex neurons during chronic pain, which would be responsible for the emotional response to chronic pain and the aversion to noxious stimulus. Previous studies reported an increased activity of intracellular cyclic AMP-PKA signaling pathway in the anterior cingulate cortex neurons from the animals with chronic pain (Wu et al., 2008; Liauw et al., 2005), and upregulation of this signaling pathway was proved to increase the neurotransmitters, both glutamate (Fu et al., 2008) and GABA (Bonci and Williams, 1997), release from the presynaptic terminals under various pathophysiological conditions. Therefore, an upregulated cyclic
To our knowledge, the present study is the first to report the mu opioid receptor-mediated inhibition of excitatory glutamatergic transmission in the anterior cingulate cortex. Activation of mu opioid receptor suppressed the presynaptic glutamate release in the spinal cord (Terman et al., 2001), the central amygdala (Zhu and Pan, 2005) and some other brain regions (Ostermeier et al., 2000; Sbrenna et al., 1999; Yang et al., 2004), which was consistent with the present finding. In general, this mu-mediated action was realized through the inhibition of the intracellular PLA2 signaling pathway and, subsequently, the 4-AP sensitive potassium channels in the presynaptic terminals (Zhu and Pan, 2005). Mu opioid receptor was well documented to underlie the most analgesic effect of opiates. The existence of mu opioid receptors in the anterior cingulate cortex was previously well demonstrated by the binding assay with the radioactive-labeled selective agonists (Vogt et al., 1995, 2001) and immunostaining studies (Chen et al., 2008; Zubieta et al., 2001). It was reported that activation of endogenous mu opioid receptor activity in the anterior cingulate cortex largely underlay the central mechanisms for placebo-mediated analgesia (Wager et al., 2007), and microinjection of morphine into the anterior cingulate cortex produced a selective naloxone reversible analgesia and alleviated the fear memory of the noxious stimulus in nerve-damaged animals (LaGraize et al., 2006; Zubieta et al., 2001), although the underlying mechanisms remained unclear. In the present study, we observed that activation of mu opioid receptor significantly suppressed the enhanced presynaptic glutamate release in the anterior cingulate cortex neurons in the inflamed rats. As discussed above, the enhanced glutamatergic transmission in the anterior cingulate cortex was supposed to be critical for the negative emotion and aversion during chronic pain (Zhuo, 2008), thus mu opioid receptor-mediated inhibition of EPSC in this brain region would underlie the mechanism for the alleviation of stress and aversion during opiates-mediated analgesia. 5. Conclusion In conclusion, our experiment demonstrated that chronic peripheral inflammation significantly enhanced the presynaptic glutamate release in the anterior cingulate cortex, which was significantly suppressed by the activation of mu opioid receptor. This investigation may clarify, at least partially, the underlying supraspinal mechanism for opiatemediated analgesia and suppression of an aversion to noxious stimulus (LaGraize et al., 2006). References Bonci, A., Williams, J.T., 1997. Increased probability of GABA release during withdrawal from morphine. J. Neurosci. 17, 796–803. Chen, T.C., Cheng, Y.Y., Sun, W.Z., Shyu, B.C., 2008. Differential regulation of morphine antinociceptive effects by endogenous enkephalinergic system in the forebrain of mice. Mol. Pain 4, 41. Eisenberger, N.I., Lieberman, M.D., Williams, K.D., 2003. Does rejection hurt? An FMRI study of social exclusion. Science 302, 290–292. Frankland, P.W., Bontempi, B., Talton, L.E., Kaczmarek, L., Silva, A.J., 2004. The involvement of the anterior cingulate cortex in remote contextual fear memory. Science 304, 881–883. Fu, Y., Han, J., Ishola, T., Scerbo, M., Adwanikar, H., Ramsey, C., Neugebauer, V., 2008. PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior. Mol. Pain 4, 26. Guo, J.D., Wang, H., Zhang, Y.Q., Zhao, Z.Q., 2005. Alterations of membrane properties and effects of D-serine on NMDA-induced current in rat anterior cingulate cortex neurons after monoarthritis. Neurosci. Lett. 384, 245–249. Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77–88. Hofbauer, R.K., Rainville, P., Duncan, G.H., Bushnell, M.C., 2001. Cortical representation of the sensory dimension of pain. J. Neurophysiol. 86, 402–411.
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