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Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain
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Research Report
Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain Yu Chen1, Ai-qin Chen1, Xiao-qing Luo1, Li-xia Guo, Ying Tang, Cheng-jia Bao, Ling Lin, Chun Linn Fujian Medical University, Department of Physiology and Pathophysiology, Laboratory of Pain Research, Key Laboratory of Brain Aging and Neurodegenerative Diseases, Neuroscience Research Center, Fuzhou City, Fujian Province 350108, PR China
art i cle i nfo
ab st rac t
Article history:
Pain and learning memory have striking similarities in synaptic plasticity. Activation of the
Accepted 2 May 2014
N-methyl-D-aspartic acid receptors 2B subunits (NR2B-NMDAs) is responsible for the hippocampal LTP in memory formation. In our previous study, we found the significant
Keywords:
enhancement of CA1 hippocampal long-term potentiation (LTP) induced by high-frequency
Chronic visceral pain
stimulation (HFS) in rats with chronic visceral pain. However, it is unclear whether the
Irritable bowel syndrome
NR2B-NMDAs are required for the LTP in chronic visceral pain. In this study, a rat model
Hippocampus
with irritable bowel syndrome (IBS) was established by colorectal distention (CRD). The
Long-term potentiation
sensitivity of visceral pain and HFS-induced LTP at SC-CA1 synapses were significantly
N-methyl-D-aspartic acid receptor
enhanced in IBS-like rats (po0.05). In addition, hippocampal NR2B protein levels signifi-
2B subunit
cantly increased in IBS-like rats (po0.05). To test whether NR2B-NMDAs are responsible for
Tyrosine kinase
the LTP, effects of Ro 25-6981, a selective antagonist of NR2B-NMDAs, on field potential in CA1 region were investigated in vitro. Our results demonstrated that Ro 25-6981 dosedependently inhibited the facilitation of CA1 LTP in IBS-like rats. The plausible activation mechanism of hippocampal NR2B-NMDAs in the LTP enhancement was further explored. Western blot data indicated that expression of tyrosine phosphorylated NR2B protein in hippocampus significantly enhanced in IBS-like rats. Accordingly, genistein, a specific inhibitor of tyrosine kinases, dose-dependently blocked the facilitation of hippocampal LTP in IBS-like rats. Furthermore, EMG data revealed that intra-hippocampal injection of Ro 25-6981 dose-dependently attenuated the visceral hypersensitivity. In conclusion, hippocampal NR2B-NMDAs are responsible for the facilitation of CA1 LTP via tyrosine phosphorylation, which leads to visceral hypersensitivity. & 2014 Published by Elsevier B.V.
n Correspondence to: Department of Physiology and Pathophysiology, Laboratory of Pain Research, Neuroscience Research Center, Fujian Medical University, No.1 Xueyuan Road, Shangjie Zhen, Minhou County, Fuzhou City, Fujian Province 350108, PR China. Fax: þ86 591 2286 2439. E-mail address: [email protected] (C. Lin). 1 These three authors contributed equally to this work.
http://dx.doi.org/10.1016/j.brainres.2014.05.001 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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1.
Introduction
Irritable bowel syndrome (IBS), characterized by recurrent abdominal pain and altered bowel habit, is a common somatoform disorder without apparent organic changes (Matheis et al., 2007; Li et al., 2012). Although the pathogenesis is unknown, it involves intestinal hypersensitivity and emotional stress (Wesselmann et al., 2009; Li et al., 2012). Currently, patients with IBS are clinically treated to alleviate pain with anti-spasm medications, probiotics, or acupuncture treatments (Wesselmann et al., 2009; MacPherson et al., 2012; Chiba et al., 2013). However, clinical treatments for IBS patients remain unsatisfactory, mainly due to the difficulty in suppressing the repeated visceral pain (Asano et al., 2012). Previous researches related to chronic visceral pain have mainly focused on the level of periphery and spinal cord, whereas few have addressed the role of limbic system in visceral chronic pain. A growing body of clinical evidence has indicated that nociceptive pain and its emotional components result in the development of a “chronic pain memory” (Choi et al., 2007; Sanchez, 2011; Noel et al., 2012). Nociceptive stimulation can cause not only pain, but pain memory persisting for many years (Choi et al., 2007; Noel et al., 2012). In addition, Ji et al. (2003) proposed that pain and learning memory share striking similarities in synaptic plasticity. Long-term potentiation (LTP), as a cellular model for synaptic plasticity, is proposed as a fundamental mechanism of learning and memory (Bliss and Collingridge, 1993). Chronic pain, namely pathological learning, is due to long-term plastic changes taking place in the painprocessing pathway (Wu and Zhuo, 2009; Li et al., 2010). Our previous study demonstrated that LTP was enhanced in hippocampal CA1 region evoked by high-frequency stimulation (HFS) in IBS-like rats (Zhu et al., 2012). However, plausible molecular mechanisms of the enhanced synaptic responses in chronic visceral pain remain largely unclear. Subunits composed of N-methyl-D-aspartate receptors (NMDARs) are heteromeric complexes including the essential NR1 subunit and some regulatory NR2A-D subunits, and the amount of NR2 expression is developmentally related (Monyer et al., 1992; Zhuo, 2009). LTP is induced by the transient activation of NMDARs, particularly by NR2B subunit, in learning and memory formation (Berberich et al., 2005; Clayton et al., 2002; Oh-Nishi et al., 2009). It is accepted that the phosphorylation of NR2B-NMDAs may play a key role in the modulation of NMDA receptor functions (Rostas et al., 1996; Xu et al., 2012). Nakazawa et al. (2001) provided an additional evidence that the phosphorylated NR2B protein increased in the hippocampus after LTP induction. Nakazawa et al. (2001) also concluded that the Tyr-1472 of NR2B subunit is a major phosphorylation site which may modulate hippocampal synaptic plasticity. Wu and Zhuo (2009) mentioned “N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity plays roles not only in physiological functions such as learning and memory, but also in unwanted pathological conditions such as chronic pain”. However, it is still unclear whether tyrosine phosphorylated NR2B-NMDARs are required for hippocampal LTP and visceral hypersensitivity. In this study, IBS-like animal model was established by colorectal distention (CRD) on post-natal day 8–14 according
to our previous studies (Luo et al., 2014). Firstly, visceral hypersensitivity was evaluated by recording amplitude of EMG to CRD. Then, expressions of hippocampal NR2B and its tyrosine phosphorylation were analyzed by western blot method. Secondly, effects of Ro 25-6981, a selective antagonist of NR2B-NMDAs, and genistein, a specific inhibitor of tyrosine kinase, on CA1 hippocampal LTP were tested by electrophysiological recordings in vitro. Finally, effects of intra-hippocampal injection of Ro 25-6981 on visceral hypersensitivity were evaluated in IBS-like rats.
2.
Results
2.1. Hypersensitivity of visceral pain and facilitation of hippocampal LTP in IBS-like rats The sensitivity of visceral pain was assessed by recording response of EMG to CRD pressure in rats. In IBS-like rats, the visceral pain sensitivity significantly increased from 228.427 19.90% to 440.327132.19% under 40 mm Hg CRD and from 295.92723.77 to 757.537119.95% under 60 mm Hg CRD (Bonferroni t-test, po0.05, Fig. 1B). To examine whether hippocampal synaptic plasticity was facilitated in the condition of chronic visceral pain, the field excitatory postsynaptic potential (fEPSP)s in the hippocampal CA1 region was recorded. The fEPSPs were recorded 10 min pre-HFS and 60 min post-HFS in control and IBS-like rats
Fig. 1 – Neonatal CRD resulted in visceral pain hypersensitivity in adult rats. (A) A original typical recording of EMG in control and IBS-like rats under 40–60 mmHg CRD pressure. (B) The statistical chart of the percentage of EMG amplitude over baseline under 40–60 mmHg CRD in control and IBS-like rats. n¼ 6 for each group; npo0.05, vs controls. EMG: electromyography; CRD: colorectal distension; IBS: irritable bowel syndrome.
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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(Fig. 2A and B). The slope of fEPSPs increased by 99.1710.8% of the baseline mean control values at 60 min post-HFS in IBS-like rats, while 58.476.8% in the controls (Student's t-test, po0.05, Fig. 2A).The amplitude of fEPSPs increased by 105.1713.0% and 48.6711.5%, respectively, of the baseline mean control values at 60 min post-HFS in IBS-like rats and controls (Student's t-test, po0.05, Fig. 2B). The enhancement lasted for more than 1 h until the end of experiment. These results suggested that the CA1 hippocampal LTP was facilitated in IBS-like rats. These results were consistent with our previous reports (Zhu et al., 2012).
2.2. Upregulation of hippocampal both NR2B protein and its phosphorylation in IBS-like rats Western blot results indicated that expression of hippocampal NR2B protein significantly enhanced by 34% (from 0.8670.09 to 1.1570.09) in IBS-like rats as compared with that in controls (unpaired t-test, t¼ 3.737, po0.05, Fig. 3B), implying that the
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NR2B protein may be implicated in the facilitation of hippocampal LTP in chronic visceral pain. Expression of tyrosine phosphorylation of hippocampal NR2B protein increased by 80% (from 0.2670.02 to 0.4670.04) in IBS-like rats as compared with that in controls (unpaired t-test, t¼ 8.374, po0.05, Fig. 3B), suggesting that tyrosine phosphorylation may be a activation mechanism of hippocampal NR2B-NMDAs in chronic visceral hypersensitivity. Furthermore, the ratio (0.2970.03) of normalized pho-NR2B of normalized NR2B in IBS-like rats was significantly enhanced as compared with that (0.3970.08) in controls (unpaired t-test, t¼2.791, po0.05, Fig. 3C).
2.3. The inhibitory effects of Ro 25-6981 on CA1 hippocampal LTP in IBS-like rats in vitro To examine whether the NR2B-NMDARs were required for the facilitation of CA1 hippocampal LTP, the effect of Ro 25-6981 on hippocampal LTP was tested in IBS-like rats. Bath application of Ro 25-6981 (0.25, 0.5, and 1.0 mg/L or 0.67, 1.33, 2.67 μM) into the brain slices had no significant effects on basal synaptic responses in IBS-like rats (ANOVA, p40.05, Fig. 4A and B). After HFS, 0.25 mg/L (or o.67 μM) Ro 25-6981 had no significant influence on fEPSPs in IBS-like rats (ANOVA/SNK-q-test, p40.05, Fig. 4). However, 0.5 mg/L (or 1.33 μM) Ro 25-6981 reduced the amplitude and slope of fEPSPs by 59% (from 100.1717.9% to 41.4712.9%) and 38% (from 96.2710.8% to 59.8714.1%), respectively, at 60 min post-HFS (ANOVA/SNK-q-test, po0.05, Fig. 4A and B). 1.0 mg/L (or 2.67 μM) Ro 25-6981 inhibited the amplitude and slope of fEPSPs by 72% (from 100.1717.9% to 28.0710.7%) and 54% (from 96.2710.8% to 44.3713.4%), respectively, at 60 min post-HFS (ANOVA/SNK-q-test, po0.05, Fig. 4A and B). The suppression of the CA1 hippocampal LTP by Ro 25-6981 presented a dose-dependent manner (ANOVA/SNK-q-test, po0.05, Fig. 4A and B). These results suggested that NR2BNMDAs played an essential role in the enhancement of LTP in hippocampal CA1 region of IBS-like rats.
2.4. Tyrosine kinase inhibitor suppressed the facilitation of CA1 hippocampal LTP in IBS-like rats in vitro
Fig. 2 – The enhanced LTP in hippocampal CA1 region evoked by HFS in IBS-like rats. The standardized fEPSPs slope (A) and (B) amplitude in control and IBS-like rats. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with inter-train interval of 10 s). Examples of representative fEPSPs traces obtained from control and IBS slices at 5 min pre-HFS and at 60 min post-HFS, respectively. Neonatal CRD resulted in the facilitation of hippocampal LTP in adult rats. n ¼8 slices from four rats for each group. po0.05, vs controls. HFS: high frequency stimulation; IBS: irritable bowel syndrome; fEPSP: field excitatory postsynaptic potential.
The expression of hippocampal tyrosine phosphorylation of NR2B protein significantly increased in IBS-like rats as compared with that in controls (unpaired t-test, po0.05, Fig. 3B). To further explore the potential activation mechanism of NR2B-NMDAs in enhancement of hippocampal LTP, the effects of genistein, a specific inhibitor of tyrosine kinases, on the induction and maintenance of LTP were estimated in IBS-like rats. Before HFS, bath application of genistein (2.5, 5, and 10 mg/L or 9.25, 18.5, 37 μM) had no significant effects on basic fEPSPs in IBS-like rats (ANOVA, p40.05, Fig. 5A and B). After HFS, 2.5 mg/L (or 9.25 μM) genistein had no significant influence on the fEPSPs in IBS-like rats (ANOVA/SNK-q-test, p40.05, Fig. 5A and B). However, 5 mg/L (or 18.5 μM) genistein could significantly inhibit the amplitude of fEPSPs by 41% (from 100.1717.9% to 59.5716.9%), and the slope of fEPSPs by 31% (from 96.2710.8% to 66.1721.7%) at 60 min post-HFS in IBS-like rats (ANOVA/SNK-q-test, po0.05, Fig. 5A and B). Moreover, 10 mg/L (or 37 μM) genistein could significantly block the amplitude of fEPSPs by 72% (from 100.1717.9% to 28.4710.2%), and the slope by 70% (from 96.2710.8% to
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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Fig. 3 – Expression of hippocampal NR2B-containing NMDA receptors in rats. (A) Western blot results showed that the protein expression of hippocampal NR2B-NMDAs and tyrosine phosphorylation of NR2B-NMDAs in rats. (B) Statistical chart showed that expression of NR2B-NMDAs and tyrosine phosphorylation of NR2B-NMDAs significantly increased in the IBS-like rats, n¼ 3, for each group. (C) The ratio of normalized pho-NR2B of normalized NR2B in hippocampus of rats. The ratio was enhanced in IBS-like rats. npo0.05, vs controls. NR2B: N-methyl-D-aspartic acid receptor 2B subunit; IBS: irritable bowel syndrome.
Fig. 4 – Inhibitory effects of Ro 25-6981 on enhanced LTP at SC-CA1 synapses in IBS-like rats. The effect of Ro 25-6981 on the standardized fEPSPs amplitude (A) and slope (B) in IBS-like rats. Bath application of Ro 25-6981 (0.5-1.0 mg/L or 1.33- 2.67 μM) dose-dependently blocked the LTP in IBS-like rats. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with intertrain interval of 10 s). N¼ 8 slices from four rats for each group. npo0.05, vs IBSþDMSO rats. Ro: Ro 25-6981; L: low dosage (0.25 mg/L or 0.67 μM); M: medial dosage (0.5 mg/L or 1.33 μM); H: high dosage (1.0 mg/L or 2.67 μM); IBS: irritable bowel syndrome; HFS: high frequency stimulation; LTP: long-term potentiation; fEPSP: field excitatory postsynaptic potential.
28.9712.0%) at 60 min post-HFS in IBS-like rats (ANOVA/SNKq-test, po0.05, Fig. 5A and B). These results suggested that tyrosine phosphorylation of CA1 hippocampal NR2B-NMDAs was involved in the facilitation of hippocampal LTP in IBSlike rats.
To further examine the correlation of tyrosine kinase activity with the maintenance of hippocampal LTP in IBS-like rats, genistein was applied at 15 min post-LTP induction. After LTP induction, 2.5 mg/L (or 9.25 μM) genistein had no significant influence on the fEPSPs in IBS-like rats (ANOVA/SNK-q-test,
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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Fig. 5 – Suppression effects of genistein on enhanced LTP in IBS-like rats. The effect of genistein on the standardized fEPSPs amplitude (A) and slope (B) in IBS-like rats. Genistein (5–10 mg/L or 18.5–37 μM) dose-dependently blocked the LTP in IBS-like rats. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with inter-train interval of 10 s). N¼ 8 slices from four rats for each group.npo0.05, vs IBSþDMSO rats. G: genistein; L: low dosage (2.5 mg/L or 9.25 μM); M: medial dosage (5 mg/L or 18.5 μM); H: high dosage (10 mg/L or 37 μM); IBS: irritable bowel syndrome; HFS: high frequency stimulation; LTP: long-term potentiation; fEPSP: field excitatory postsynaptic potential.
Fig. 6 – Tyrosine phosphorylation of the NR2B subunit contributes. to the maintenance of LTP. The effect of genistein on the fEPSPs amplitude (A) and slope (B) in IBS-like rats. Genistein (5–10 mg/L or 18.5–37 μM) dose-dependently blocked the maintenance of LTP 30 min and 60 min post-HFS in IBS-like rats. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with inter-train interval of 10 s). N¼6 slices from three rats for each group. npo0.05, vs IBSþDMSO rats. G: genistein; L: low dosage (2.5 mg/L or 9.25 μM); M: medial dosage (5 mg/L or 18.5 μM); H: high dosage (10 mg/L or 37 μM); IBS: irritable bowel syndrome; HFS: high frequency stimulation; LTP: long-term potentiation; fEPSP: field excitatory postsynaptic p40.05, Fig. 6A and B). However, 5 mg/L (or 18.5 μM) genistein could significantly inhibit the slope of fEPSPs by 31% (from 105.6778.37% to 72.9074.02%) and the amplitude of fEPSPs by 47% (from 144.46711.79% to 76.0177.77%), slope of fEPSPs by 44% (from 119.8879.70% to 67.60710.65%) and the amplitude of fEPSPs by 47% (from 148.83713.67% to 79.5077.62%), respectively, at 30 and 60 min post-HFS in IBS-like rats (ANOVA/SNKq-test, po0.05, Fig. 6A and B). Moreover, 10 mg/L (or 37 μM) genistein could significantly block the slope of fEPSPs by 64% (from 105.6778.37% to 38.2374.88%) and the amplitude of fEPSPs by 68% (from 144.46711.79% to 46.85714.30%), slope of fEPSPs by 71% (from 119.8879.70% to 34.9674.94%) and the amplitude of fEPSPs by 65% (from 148.83713.67% to 51.61717.17%), respectively, at 30 and 60 min post-HFS in IBS-like rats (ANOVA/SNK-q-test, po0.05, Fig. 6A and B). The results showed that genistein significantly inhibited CA1 hippocampal LTP at 30 min after LTP induction in IBS-like rats, and the inhibitory effect sustained for at least 60 min. These results provide new evidence to demonstrate that tyrosine phosphorylation of CA1 hippocampal NR2B-NMDAs was involved in the maintenance of hippocampal LTP in IBSlike rats.
2.5. Ro 25-6981 treatment attenuated visceral pain hypersensitivity in IBS-like rats Whether NR2B-dependent facilitation of hippocampal LTP was involved in visceral hypersensitivity was studied in IBS-like rats. Effects of NR2B-NMDAs on visceral hypersensitivity were examined in vivo. Bilateral intra-hippocampal injection of Ro 25-6981 (1, 2, 4 μg/μl, 2 μl each side) had no effect on the response of EMG to 40–60 mmHg CRD in controls (Bonferroni t-test, p40.05, Fig. 7A and C). There was no obvious change in responses of EMG to 40–60 mmHg CRD between 1 μg/μl Ro 25-6981 and vehicle in IBS-like rats (Bonferroni t-test, p40.05, Fig. 7B and D). Responses of EMG to 40–60 mmHg CRD were decreased by 39% (from 413.89723.29% to 253.44726.17%) and 35% (from 593751.64% to 382.60723.17%), respectively, after injection of 2 μg/μl Ro 25-6981 as compared with vehicle in IBSlike rats (Bonferroni t-test, po0.05, Fig. 7B and D). Responses of EMG were decreased by 75% (from 554.25765.38% to 141.16710.10%) under 40 mmHg CRD, and decreased by 61% (from 704.567139.73% to 278.27739.76%) under 60 mmHg CRD after injection of 4 μg/μl Ro 25-6981 as compared with vehicle in IBS-like rats (Bonferroni t-test, po0.05, Fig. 7B and D).
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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Fig. 7 – Bilateral intra-hippocampal injection of Ro 25-6981 decreased chronic visceral pain in IBS-like rats. The percentage of EMG amplitude over baseline under 40 (A) and 60 mmHg CRD (C) at 30 min after injection in controls; under 40 (B) and 60 mmHg CRD (D) at 30 min after injection in IBS-like rats. Bilateral intra-hippocampal injection of Ro 25-6981 (2–4 μg/μl, 2 μl per side) significantly inhibited chronic visceral pain in IBS-like rats. The time course of EMG were under 40 (E) and 60 mmHg CRD (F) in IBS-like rats. The maximal inhibition was observed at 30 min after Ro 25-6981 administrated in IBS-like rats (n ¼6 for each group); npo0.05. vs before the injection of Ro 25-6981; EMG: electromyography; CRD: colorectal distension; IBS: irritable bowel syndrome; Ro: Ro 25-6981.
Responses of EMG were significantly decreased by 30% (from 360.88731.30% to 253.44726.17%) under 40 mmHg CRD after injection of 2 μg/μl Ro 25-6981 as compared with 1 μg/μl Ro 25-6981 in IBS-like rats, and significantly decreased by 28% (from 502.54733.87% to 382.60723.17%) under 60 mmHg CRD (Bonferroni t-test, po0.05, Fig. 7B and D). Responses of EMG were significantly decreased by 61% (from 360.88731.30% to 141.16710.10%) under 40 mmHg CRD after injection of 4 μg/μl Ro 25-6981 as compared with 1 μg/μl Ro 25-6981 in IBS-like rats, and significantly decreased by 45% (from 502.54733.86% to 278.27739.76%) under 60 mmHg CRD (Bonferroni t-test, po0.05, Fig. 7B and D). These EMG data showed that intrahippocampal injection of the NR2B-NMDAs antagonist had a
dose-dependent inhibitory effect on visceral hypersensitivity in IBS-like rats. These results suggested that NR2B-NMDAs-dependent facilitation of hippocampal LTP may a key mechanism underlying the visceral hypersensitivity.
2.6. Time course of inhibitory effect of Ro 25-6981 on visceral hypersensitivity in IBS-like rats To further observe time profiles of Ro 25-6981, responses of EMG to 40–60 mmHg CRD were recorded every half an hour within three hours after intra-hippocampal injection of 4 μg/μl Ro 25-6981 in IBS-like rats. The lowest percentage of EMG
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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amplitude over baseline was observed at 30 min after injection of 4 μg/μl Ro 25-6981 (141.16710.10% under 40 mmHg CRD; 278.27739.76% under 60 mmHg CRD). Responses of EMG to 40 mmHg CRD were lowered by 69%, 52%, 37%, and 32% respectively at 30, 60, 90, and 120 min after injection of 4 μg/μl Ro 25-6981 in IBS-like rats. Responses of EMG to 60 mmHg CRD were lowered by 65%, 60%, 52%, 45% and 33% respectively at 30, 60, 90, and 120 min after injection of 4 μg/μl Ro 25-6981 in IBS-like rats. These results suggested that the maximal inhibition was observed at 30 min after administration of Ro 25-6981 under either 40 or 60 mmHg CRD in IBS-like rats (Bonferroni t-test, po0.05, Fig. 7E and F).
3.
Discussion
We previously observed that LTP induced by HFS at SC-CA1 synapses significantly increased in IBS-like rats (Zhu et al., 2012). In the present study, we provide further evidence to demonstrate that hippocampal NR2B receptor plays an important role in the enhanced LTP and visceral hypersensitivity in IBS-like rats and that activities of NR2B-NMDARs are mainly regulated by tyrosine kinase. Firstly, expressions of the hippocampal NR2B protein and tyrosine phosphorylated NR2B protein significantly increased in IBS-like rats. Secondly, the application of Ro 25-6981could dose-dependently block the facilitation of LTP in hippocampal CA1 region. Thirdly, genistein could also dose-dependently inhibit the induction and maintenance of hippocampal LTP in IBS-like rats in vitro. Finally, bilateral intra-hippocampal injection of Ro 25-6981 significantly attenuated visceral hypersensitivity in IBS-like rats.
3.1.
Facilitation of hippocampal LTP in IBS-like rats
It is claimed that synaptic plasticity is a key mechanism for chronic pain and plastic changes contributes to pain perception, fear and memory in Science (Li et al., 2010). It is concluded that LTP is a well-characterized form of synaptic Q3 plasticity (Ji et al., 2003; Wu et al., 2009; Li et al., 2010). In this study, the amplitude and slope of fEPSPs were ca. 49% and 57%, respectively, in controls, which is in agreement with data reported by Li et al. (2011). Although there are differences between the synaptic plasticity contributing to learning memory and pain memory, there are also striking similarities (Ji et al., 2003). In addition, LTP induced by HFS in the hippocampal CA1 region significantly enhanced and maintained for more than 1 h in IBS-like rats. These results indicate that CRD in neonatal rats could result in chronic visceral pain-related memory when they grew up. Thus, LTP is a key mechanism of chronic visceral pain in agreement with memory and other pains (Kodama et al., 2007; Li et al., 2010; Ohnami et al., 2012). Our results demonstrated that LTP induced by HFS at SCCA1 synapses significantly enhanced in IBS-like rats. This viewpoint is conflict with that in neuropathic pain reported by Kodama et al. (2007). They reported that hippocampal synaptic transmission in CA1 region was impaired in a murine model of neuropathic pain prepared by partial ligation of the sciatic nerve. Some reasonable explanations will
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be needed. Firstly, types of stimulus inducing LTP in neuropathic and visceral pain are different. Their LTP was induced by tetanus in the neuropathic pain, while ours was induced by HFS at SC-CA1 synapses. Secondly, there are some fundamental differences between the mechanisms responsible in visceral and somatic pain (Wesselmann et al., 2009). The neuropathic pain model reported by Kodama et al. is one of somatic pain. Thus, differences of pain models may result in the variation in synaptic responses. Finally, the model reported by Kodama et al. was prepared by partial ligation of the sciatic nerve, which is an acute pain, while the visceral pain established in this study is a chronic pain (up to 8 weeks). However, the interpretation of mechanisms responsible to different pains will need further studies in the future. Compared to other limbic structures such as the anterior cingulate cortex (ACC) and amygdala, the role of the hippocampus in the formation of chronic pain is not fully elucidated (Zhuo, 2007; Li et al., 2010; Kim et al., 2010; Crock et al., 2012). Our results reveal that hippocampal formation plays an important role in chronic visceral pain. There are several observations to support our results. Firstly, pain is a complex phenomenon resulting from a three-dimensional integration of sensory-discriminative, affective-motivational and cognitive-evaluative axes (Liu and Chen, 2009). Pain memory may persist for many years, which may be a key role for chronicity and repeatability of IBS (Choi et al., 2007; Noel et al., 2012). Secondly, hippocampal synaptic plasticity is a main cellular model for memory (Bliss and Collingridge, 1993), which contributes to the formation of pain-related memory and emotional responses (Chou et al., 2011). Thirdly, CA1 hippocampal synaptic transmission could be influenced by neuropathic and inflammatory pain (Kodama et al., 2007; Zhao et al., 2009). Furthermore, recent studies have demonstrated that CA1 plays a critical role in the learning and memory (Porte et al., 2011; Place et al., 2012). Khanna and Sinclair proposed that noxious stimuli produce prolonged changes in the CA1 region (Khanna and Sinclair, 1989). Finally, induction of persistent peripheral neuropathic pain could affect basic hippocampal processing such as the spatial encoding performed by CA1 region cells (Cardoso-Cruz et al., 2011). Thus, hippocampal formation plays an integral role in chronic visceral pain.
3.2. Role of NR2B in facilitation of CA1 hippocampal LTP and formation of visceral hypersensitivity in IBS-like rats Subunits composed of NMDA receptors are classified as families of NR1, NR2, and NR3 based on genotype (Monyer et al., 1992). As mentioned, NR2 subunit is composed of NR2A-D, which has a strong effect on the biophysical and pharmacological properties of NMDAR assemblies (Monyer et al., 1992; Zhuo, 2009). NR2B-NMDAs are widespread in areas related to pain transmission, such as the dorsal horn of spine and hippocampus (Oh-Nishi et al., 2009; Luo et al., 2014). Some experiment in mutant mice suggested that LTP is dependent on the NR2A subunit important for LTP (Clayton et al., 2002; Oh-Nishi et al., 2009; Zhuo, 2009). Ro 25-6981 is 5000 folds more selective for the heteromeric NR1/NR2B receptors than the NR1/NR2A receptors (Fischer et al., 1997). In our study, expression of hippocampal NR2B protein
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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increased in IBS-like rats, which was similar with the formalin-induced chronic pain (Wei et al., 2001). Together with, Ro 25-6981, a selective antagonist of NR2B-NMDAs, dose-dependently inhibited the facilitation of hippocampal LTP in vitro in IBS-like rats. Thus, it is hypothesized that NR2B subunit plays a critical role in hippocampal LTP enhancement in chronic visceral pain, which is in consistent with the study of genetic overexpression of NR2B subunit indicating the involvement of NR2B receptor in LTP (Tang et al., 1999). A study in vivo indicated intra-hippocampal injection of nonselective NMDA antagonists, AP5 and MK801, significantly reduced pain behavior in both the acute and tonic phases of the formalin test (Chen et al., 2009). However, the therapeutic potential of NMDA antagonists has been restricted in the treatment of chronic pain due to their serious side effects including hallucinations, learning and memory impairments, and sensorimotor disturbances (Soleimannejad et al., 2007; Qu et al., 2009). Our present study indicated that the blockade of NR2B receptors in the hippocampal CA1 region significantly inhibited neonatal CRD – induced chronic visceral hypersensitivity without significant side effects. In our studies, Ro 25-6981 attenuated visceral pain by ca. 45% and 70%, respectively, 30 min after intrathecal (75 μg/10 μl) and intra-hippocampal (4 μg/2 μl) injection (Luo et al., 2014). Explanations for the findings are that hippocampal NR2B subunit may play much more important role in chronic visceral pain. In other studies, the application dosage of Ro 25-6981 was 0.3–2 mg/L (Bartlett et al., 2007; Li and Han, 2008), and in this study, the effective dosages of Ro 25-6981 were between 0.5 and 1.0 mg/L. In our study, intra-hippocampal injection of Ro 25-6981 did not completely attenuate visceral pain hypersensitivity. The reason is likely because of other signaling molecule in hippocampus formation and other brain regions mediating chronic pain (Zhuo, 2007; Crock et al., 2012; Ikeda et al., 2013). Therefore, further studies will be required to the comprehensive understanding of mechanisms of chronic visceral pain.
3.3. Contribution of tyrosine phosphorylated NR2BNMDAs to CA1 hippocampal LTP in IBS-like rats The phosphorylation of NR2B-NMDAs plays a key role in modulation of NMDA receptor functions (Rostas et al., 1996; Nakazawa et al., 2001; Xu et al., 2012). The intracellular C-terminal cytoplasmic region of the NR2B-NMDAs is a functionally important region targeted by numerous protein kinases, including Fyn-mediated tyrosine kinases (Nakazawa et al., 2001). NR2B-NMDAs have seven phosphorylation sites for tyrosine kinase, with its main phosphorylation sites including S1303, S1323, S1480, Y1252, Y1336, and Y1472 (Nakazawa et al., 2001; Qiu et al., 2011). It is addressed that Tyr-1472 of NR2B is a major phosphorylation site, which may modulate hippocampal synaptic plasticity (Nakazawa et al., 2001). In this study, western blot data showed that expression of Tyr-1472 of NR2B protein in hippocampus significantly enhanced in IBS-like rats, in agreement with the paper reported by Xu et al. in inflammatory pain (Xu et al., 2012).We reasonably infer hippocampal NR2B phosphorylation at tyrosine 1472 may contribute to central sensitization of chronic visceral pain.
Since the tyrosine phosphorylation of NR2B subunit has already increased in IBS-like rats, these results do not exclude the possibility that there may be the constitutive activity of tyrosine kinase in maintaining the level of phosphorylation. As mentioned, the level of the phosphorylated NR2B protein increased in the hippocampus after induction of LTP (Rostas et al., 1996). In addition, tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP. Our results suggested that tyrosine phosphorylation of the NR2B subunit contributed to the maintenance of LTP in IBS-like rats, which is similar with the findings reported by Rostas et al. (1996). Thus, tyrosine phosphorylation activity was enhanced in the maintenance of CA1 hippocampal LTP in IBS-like rats. Rostas et al. (1996) mentioned “the increase in tyrosine phosphorylation of NR2B required LTP induction but was not involved in the induction process itself since the increase in tyrosine phosphorylation was not observed until between 5 and 15 min after LTP induction”. In this study, the tyrosine kinase inhibitor restrains the induction of LTP, which inhibits the tyrosine phosphorylation of NR2B and results in the inhibitory effect on LTP maintenance. Thus, it is speculated that tyrosine phosphorylation is one of the activating mechanisms of the hippocampal NR2BNMDAs in the facilitation of LTP in IBS-like rats.
4.
Experimental procedures
4.1.
Animals
Male Sprague-Dawley neonatal rats were provided by the Department of Experimental Animal Center, Fujian Medical University. The animals were housed with a nursing adult female rat until they were 25 days old. After separation from the adult female rats, the weaned rats were raised for two months before surgery and housed six in a cage with access to food and water ad libitum. The animal experimental procedures were conducted in accordance with the guidelines of the International Association for the Study of Pain (Zimmermann, 1983). The experiments were approved by the Animal Care and Use Committee of Fujian Medical University.
4.2.
Animal model of visceral hypersensitivity
In order to study mechanisms of chronic visceral pain, we developed a modified rat model of IBS as described by Al-Chaer et al., (2000). Hypersensitive SD rats were daily generated by 60 mmHg CRD during postnatal days 8–14. The controls received the same procedure except CRD. Above experiments were done by the same investigator. No treatment, procedures or further interventions were performed by the investigator until the testing date (8 weeks old).
4.3.
Assessment of visceral hyperalgesia
Visceral hypersensitivity was assessed by electromyographic (EMG) measurements of visceromoter response to CRD as described previously (Luo et al., 2014). Briefly, under anesthesia with isofluorane by anesthesia machine (VMR, Matrix,
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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USA), two silver bipolar electrodes were inserted the external oblique muscle of the abdomen. After the balloon was advanced into the colon, it was secured by taping the attached tubing to the rat's tail. The balloon was inflated to 40 and 60 mmHg for 10 s followed by 4 min rest. Responses of EMG were recorded three times under the same CRD pressure in each rat. The average of three recordings is taken as the magnitude of EMG activity. The magnitude of EMG activity was measured by a RM6240BD multi-channel physiological signal acquisition and processing system (the instrument, made in Chendu, China). The EMG data of 10 s before CRD (baseline) and 10 s during CRD were recorded for calculation. The responses were taken as the percentage of EMG amplitude during CRD over baseline using RM6420BD program. In order to avoid the interference of other factors, a single blind operation was adopted in the experiment. Furthermore, the rats were kept in a quiet environment, with relative humidity maintained at 40–70% and room temperature at 23–27 1C.
4.4.
Surgery and intra-hippocampal injection
The rats were anesthetized by intraperitoneal chloral hydrate (300 mg/kg) and were placed on a stereotaxic instrument (Narishige, Japan). The skin was cut along the midline of the skull. A cotton swab soaked with 3% H2O2 was used to abrade the tissue until the skull was exposed. A stainless steel guide cannula of 0.65 mm outer-diameter was placed stereotaxically and directed into the CA1 region of the hippocampus (4.0 mm posterior to the bregma, 2.8 mm lateral to the midline and 2.5 mm from the surface of the skull) according to Paxinos and Watson (1998). The two cannulas were fixed on the skull with two small screws by dental resin. The rats were given 3 days to recover from the operation. On the day of experiment, a stainless steel injection tube with 0.4 mm diameter was directly inserted into the guide cannula with 0.5 mm beyond the tip of the latter. Then 2 μl of drug solution was injected into the CA1 region of the hippocampus within 5 min. Control group was injected with the vehicle. The injection needle was left in place for an additional 5 min to allow diffusion.
4.5.
Hippocampal slice preparation
Rats were anesthetized with intraperitoneal chloral hydrate (300 mg/kg), and immediately after decapitation the brains were placed in ice-cold ACSF bubbled with 95% O2–5% CO2 (pH 7.4). The hippocampal formation was cut into 500-μm-thick transverse slices using a vibratome (ZQP-86, Zhixing Company, Shanghai, China) after fixed using 502 glue. The slices were incubated in an interface recording chamber maintained at a constant temperature (2871 1C), and were allowed to equilibrate for at least 1 h. The upper surface of the slices was in contact with a humidified, oxygenated (95% O2–5% CO2) stream.
4.6.
Electrophysiological recordings
The Schaffer collateral-Commissural pathway was stimulated and fEPSPs recorded from the dendritic layer of the CA1 pyramidal cells as reported by Kleppisch et al. (1999). The amplitude of the fEPSPs was measured for the downward
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peak. The intensity of the test stimulus, delivered at 0.1 Hz, was adjusted for each slice so that the fEPSPs were equal to approximately 50% of the maximal amplitude of the fEPSPs. The slices were stimulated for 10 min (0.01 Hz) so as to generate baseline values before LTP induction. Baseline values were recorded for at least 10 min before application of drugs. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with inter-train interval of 10 s). fEPSPs were amplified and analyzed using a RM6240BD multi-channel physiological signal acquisition and processing system.
4.7.
Western blotting
Equal amounts of protein (50 μg) from the hippocampus of rats were separated and electro-transferred onto PVDF membranes (Invitrogen, USA), which were probed with mouse anti-NR2B monoclonal antibody (1:500, Abcam, USA), Rabbit anti-NR2B (pTyr1472) polyclonal antibody (1:1000, Abcam, USA) and mouse anti-β-actin primary antibody (1:1000, Abcam, USA). Blots were washed and incubated in peroxidase-conjugated goat anti-rabbit Ig G (1:5000, Abcam, USA) or goat anti-mouse Ig G (1:5000, Abcam, USA). Bands were visualized using an ECL system (Made in Xiamen, China).
4.8.
Statistical analysis
All data were expressed as mean7S.E.M. A one-way repeated measures analysis of variance (ANOVA) was used to determine whether there was a significant difference in the response to CRD pressure between control and IBS-like rats. One or two way repeated measures analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for multiple comparison after intra-hippocampal injection of compounds. Western blot results were examined by unpaired t-test. LTP was measured 60 min after HFS and reported as mean7SEM of baseline fEPSPs amplitude or slope. Student's t-test was used to compare the hippocampal field potential in control and IBS-like rats. One way ANOVA with Student– Newman–Keuls (SNK) multiple comparisons post-hoc analysis was used to compare the different groups of hippocampal slices of rats in vitro (SPSS 10.0). po0.05 was considered to be statistically significant.
Conflict of interest statement The authors declare that they have no competing interests.
Author contributions YC: Participated in the design of the research and data analysis. AQC: Participated in the research and data analysis. XQL: Participated in the research and manuscript writing. LXG: Carried out molecular techniques and participated in the analysis of results. YT: Participated animal models and the analysis of results. CJB: Participated in the research and interpreted results. LL: Participated in the research.
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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CL: Designed, supervised studies, interpreted results, and prepare the manuscript.
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Uncited reference Sprengel et al. (1998).
Acknowledgments The present work was supported by grants from National Natural Science Foundation of China (81100998), Natural Science Foundation of Fujian Province (2012J05053), Doctoral Fund of Fujian Medical University (2010BS008), and the Major Fund of Fujian Medical University (09ZD009).
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hippocampal CA1 and dentate gyrus injection of MK801 and AP5 in the formalin test in adult male rats. Eur. J. Pharmacol. 562 (1-2), 39–46. Sprengel, R., Suchanek, B., Amico, C., Brusa, R., Burnashev, N., Rozov, A., Hvalby, O., Jensen, V., Paulsen, O., Andersen, P., Kim, J.J., Thompson, R.F., Sun, W., Webster, L.C., Grant, S.G., Eilers, J., Konnerth, A., Li, J., McNamara, J.O., Seeburg, P.H., 1998. Importance of the intracellular domain of NR2 subunits for NMDA receptor function in Vivo. Cell 92 (2), 279–289. Tang, Y.P., Shimizu, E., Dube, G.R., Rampon, C., Kerchner, G.A., Zhuo, M., Liu, G., Tsien, J.Z., 1999. Genetic enhancement of learning and memory in mice. Nature 401 (6748), 63–69. Wei, F., Wang, G.D., Kerchner, G.A., Kim, S.J., Xu, H.M., Chen, Z.F., Zhuo, M., 2001. Genetic enhancement of inflammatory pain by forebrain NR2B overexpression. Nat. Neurosci. 4 (2), 164–169. Wesselmann, U., Baranowski, A.P., Bo¨rjesson, M., Curran, N.C., Czakanski, P.P., Giamberardino, M.A., Ness, T.J., Robbins, M.T., Traub, R.J., 2009. Emerging therapies and novel approaches to visceral pain. Drug Discov. Today Ther. Strategy 6 (3), 89–95. Wu, L.J., Zhuo, M., 2009. Targeting the NMDA receptor subunit NR2B for the treatment of neuropathic pain. Neurotherapeutics 6 (4), 693–702. Xu, L., Pan, Y., Zhu, Q., Gong, S., Tao, J., Xu, G.Y., Jiang, X., 2012. Arcuate Src activation-induced phosphorylation of NR2B NMDA subunit contributes to inflammatory pain in rats. J. Neurophysiol. 108 (11), 3024–3033. Zhao, X.Y., Liu, M.G., Yuan, D.L., Wang, Y., He, Y., Wang, D.D., Chen, X.F., Zhang, F.K., Li, H., He, X.S., Chen, J., 2009. Nociception-induced spatial and temporal plasticity of synaptic connection and function in the hippocampal formation of rats: a multi-electrode array recording. Mol. Pain 5, 55. Zhu, F.C., Chen, Y., Lin, C., Cai, Q.Y., Chen, A.Q., 2012. Inhibition of niflumic acid on synaptic long-term potentiation in hippocampus CA1 region in rats with chronic visceral pain. Chin. J. Neuroanat. 28 (2), 151–156. Zhuo, M., 2007. A synaptic model for pain: long-term potentiation in the anterior cingulate cortex. Mol. Cells 23 (3), 259–271. Zhuo, M., 2009. Plasticity of NMDA receptor NR2B-NMDAs in memory and chronic pain. Mol. Brain 3 (2), 4. Zimmermann, M., 1983. Ethical guidelines for investigations of Q4 experimental pain in conscious animals 16 (2) 109–110.
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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Research Report
Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain Yu Chen1, Ai-qin Chen1, Xiao-qing Luo1, Li-xia Guo, Ying Tang, Cheng-jia Bao, Ling Lin, Chun Linn Fujian Medical University, Department of Physiology and Pathophysiology, Laboratory of Pain Research, Key Laboratory of Brain Aging and Neurodegenerative Diseases, Neuroscience Research Center, Fuzhou City, Fujian Province 350108, PR China
art i cle i nfo
ab st rac t
Article history:
Pain and learning memory have striking similarities in synaptic plasticity. Activation of the
Accepted 2 May 2014
N-methyl-D-aspartic acid receptors 2B subunits (NR2B-NMDAs) is responsible for the hippocampal LTP in memory formation. In our previous study, we found the significant
Keywords:
enhancement of CA1 hippocampal long-term potentiation (LTP) induced by high-frequency
Chronic visceral pain
stimulation (HFS) in rats with chronic visceral pain. However, it is unclear whether the
Irritable bowel syndrome
NR2B-NMDAs are required for the LTP in chronic visceral pain. In this study, a rat model
Hippocampus
with irritable bowel syndrome (IBS) was established by colorectal distention (CRD). The
Long-term potentiation
sensitivity of visceral pain and HFS-induced LTP at SC-CA1 synapses were significantly
N-methyl-D-aspartic acid receptor
enhanced in IBS-like rats (po0.05). In addition, hippocampal NR2B protein levels signifi-
2B subunit
cantly increased in IBS-like rats (po0.05). To test whether NR2B-NMDAs are responsible for
Tyrosine kinase
the LTP, effects of Ro 25-6981, a selective antagonist of NR2B-NMDAs, on field potential in CA1 region were investigated in vitro. Our results demonstrated that Ro 25-6981 dosedependently inhibited the facilitation of CA1 LTP in IBS-like rats. The plausible activation mechanism of hippocampal NR2B-NMDAs in the LTP enhancement was further explored. Western blot data indicated that expression of tyrosine phosphorylated NR2B protein in hippocampus significantly enhanced in IBS-like rats. Accordingly, genistein, a specific inhibitor of tyrosine kinases, dose-dependently blocked the facilitation of hippocampal LTP in IBS-like rats. Furthermore, EMG data revealed that intra-hippocampal injection of Ro 25-6981 dose-dependently attenuated the visceral hypersensitivity. In conclusion, hippocampal NR2B-NMDAs are responsible for the facilitation of CA1 LTP via tyrosine phosphorylation, which leads to visceral hypersensitivity. & 2014 Published by Elsevier B.V.
n Correspondence to: Department of Physiology and Pathophysiology, Laboratory of Pain Research, Neuroscience Research Center, Fujian Medical University, No.1 Xueyuan Road, Shangjie Zhen, Minhou County, Fuzhou City, Fujian Province 350108, PR China. Fax: þ86 591 2286 2439. E-mail address: [email protected] (C. Lin). 1 These three authors contributed equally to this work.
http://dx.doi.org/10.1016/j.brainres.2014.05.001 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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1.
Introduction
Irritable bowel syndrome (IBS), characterized by recurrent abdominal pain and altered bowel habit, is a common somatoform disorder without apparent organic changes (Matheis et al., 2007; Li et al., 2012). Although the pathogenesis is unknown, it involves intestinal hypersensitivity and emotional stress (Wesselmann et al., 2009; Li et al., 2012). Currently, patients with IBS are clinically treated to alleviate pain with anti-spasm medications, probiotics, or acupuncture treatments (Wesselmann et al., 2009; MacPherson et al., 2012; Chiba et al., 2013). However, clinical treatments for IBS patients remain unsatisfactory, mainly due to the difficulty in suppressing the repeated visceral pain (Asano et al., 2012). Previous researches related to chronic visceral pain have mainly focused on the level of periphery and spinal cord, whereas few have addressed the role of limbic system in visceral chronic pain. A growing body of clinical evidence has indicated that nociceptive pain and its emotional components result in the development of a “chronic pain memory” (Choi et al., 2007; Sanchez, 2011; Noel et al., 2012). Nociceptive stimulation can cause not only pain, but pain memory persisting for many years (Choi et al., 2007; Noel et al., 2012). In addition, Ji et al. (2003) proposed that pain and learning memory share striking similarities in synaptic plasticity. Long-term potentiation (LTP), as a cellular model for synaptic plasticity, is proposed as a fundamental mechanism of learning and memory (Bliss and Collingridge, 1993). Chronic pain, namely pathological learning, is due to long-term plastic changes taking place in the painprocessing pathway (Wu and Zhuo, 2009; Li et al., 2010). Our previous study demonstrated that LTP was enhanced in hippocampal CA1 region evoked by high-frequency stimulation (HFS) in IBS-like rats (Zhu et al., 2012). However, plausible molecular mechanisms of the enhanced synaptic responses in chronic visceral pain remain largely unclear. Subunits composed of N-methyl-D-aspartate receptors (NMDARs) are heteromeric complexes including the essential NR1 subunit and some regulatory NR2A-D subunits, and the amount of NR2 expression is developmentally related (Monyer et al., 1992; Zhuo, 2009). LTP is induced by the transient activation of NMDARs, particularly by NR2B subunit, in learning and memory formation (Berberich et al., 2005; Clayton et al., 2002; Oh-Nishi et al., 2009). It is accepted that the phosphorylation of NR2B-NMDAs may play a key role in the modulation of NMDA receptor functions (Rostas et al., 1996; Xu et al., 2012). Nakazawa et al. (2001) provided an additional evidence that the phosphorylated NR2B protein increased in the hippocampus after LTP induction. Nakazawa et al. (2001) also concluded that the Tyr-1472 of NR2B subunit is a major phosphorylation site which may modulate hippocampal synaptic plasticity. Wu and Zhuo (2009) mentioned “N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity plays roles not only in physiological functions such as learning and memory, but also in unwanted pathological conditions such as chronic pain”. However, it is still unclear whether tyrosine phosphorylated NR2B-NMDARs are required for hippocampal LTP and visceral hypersensitivity. In this study, IBS-like animal model was established by colorectal distention (CRD) on post-natal day 8–14 according
to our previous studies (Luo et al., 2014). Firstly, visceral hypersensitivity was evaluated by recording amplitude of EMG to CRD. Then, expressions of hippocampal NR2B and its tyrosine phosphorylation were analyzed by western blot method. Secondly, effects of Ro 25-6981, a selective antagonist of NR2B-NMDAs, and genistein, a specific inhibitor of tyrosine kinase, on CA1 hippocampal LTP were tested by electrophysiological recordings in vitro. Finally, effects of intra-hippocampal injection of Ro 25-6981 on visceral hypersensitivity were evaluated in IBS-like rats.
2.
Results
2.1. Hypersensitivity of visceral pain and facilitation of hippocampal LTP in IBS-like rats The sensitivity of visceral pain was assessed by recording response of EMG to CRD pressure in rats. In IBS-like rats, the visceral pain sensitivity significantly increased from 228.427 19.90% to 440.327132.19% under 40 mm Hg CRD and from 295.92723.77 to 757.537119.95% under 60 mm Hg CRD (Bonferroni t-test, po0.05, Fig. 1B). To examine whether hippocampal synaptic plasticity was facilitated in the condition of chronic visceral pain, the field excitatory postsynaptic potential (fEPSP)s in the hippocampal CA1 region was recorded. The fEPSPs were recorded 10 min pre-HFS and 60 min post-HFS in control and IBS-like rats
Fig. 1 – Neonatal CRD resulted in visceral pain hypersensitivity in adult rats. (A) A original typical recording of EMG in control and IBS-like rats under 40–60 mmHg CRD pressure. (B) The statistical chart of the percentage of EMG amplitude over baseline under 40–60 mmHg CRD in control and IBS-like rats. n¼ 6 for each group; npo0.05, vs controls. EMG: electromyography; CRD: colorectal distension; IBS: irritable bowel syndrome.
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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(Fig. 2A and B). The slope of fEPSPs increased by 99.1710.8% of the baseline mean control values at 60 min post-HFS in IBS-like rats, while 58.476.8% in the controls (Student's t-test, po0.05, Fig. 2A).The amplitude of fEPSPs increased by 105.1713.0% and 48.6711.5%, respectively, of the baseline mean control values at 60 min post-HFS in IBS-like rats and controls (Student's t-test, po0.05, Fig. 2B). The enhancement lasted for more than 1 h until the end of experiment. These results suggested that the CA1 hippocampal LTP was facilitated in IBS-like rats. These results were consistent with our previous reports (Zhu et al., 2012).
2.2. Upregulation of hippocampal both NR2B protein and its phosphorylation in IBS-like rats Western blot results indicated that expression of hippocampal NR2B protein significantly enhanced by 34% (from 0.8670.09 to 1.1570.09) in IBS-like rats as compared with that in controls (unpaired t-test, t¼ 3.737, po0.05, Fig. 3B), implying that the
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NR2B protein may be implicated in the facilitation of hippocampal LTP in chronic visceral pain. Expression of tyrosine phosphorylation of hippocampal NR2B protein increased by 80% (from 0.2670.02 to 0.4670.04) in IBS-like rats as compared with that in controls (unpaired t-test, t¼ 8.374, po0.05, Fig. 3B), suggesting that tyrosine phosphorylation may be a activation mechanism of hippocampal NR2B-NMDAs in chronic visceral hypersensitivity. Furthermore, the ratio (0.2970.03) of normalized pho-NR2B of normalized NR2B in IBS-like rats was significantly enhanced as compared with that (0.3970.08) in controls (unpaired t-test, t¼2.791, po0.05, Fig. 3C).
2.3. The inhibitory effects of Ro 25-6981 on CA1 hippocampal LTP in IBS-like rats in vitro To examine whether the NR2B-NMDARs were required for the facilitation of CA1 hippocampal LTP, the effect of Ro 25-6981 on hippocampal LTP was tested in IBS-like rats. Bath application of Ro 25-6981 (0.25, 0.5, and 1.0 mg/L or 0.67, 1.33, 2.67 μM) into the brain slices had no significant effects on basal synaptic responses in IBS-like rats (ANOVA, p40.05, Fig. 4A and B). After HFS, 0.25 mg/L (or o.67 μM) Ro 25-6981 had no significant influence on fEPSPs in IBS-like rats (ANOVA/SNK-q-test, p40.05, Fig. 4). However, 0.5 mg/L (or 1.33 μM) Ro 25-6981 reduced the amplitude and slope of fEPSPs by 59% (from 100.1717.9% to 41.4712.9%) and 38% (from 96.2710.8% to 59.8714.1%), respectively, at 60 min post-HFS (ANOVA/SNK-q-test, po0.05, Fig. 4A and B). 1.0 mg/L (or 2.67 μM) Ro 25-6981 inhibited the amplitude and slope of fEPSPs by 72% (from 100.1717.9% to 28.0710.7%) and 54% (from 96.2710.8% to 44.3713.4%), respectively, at 60 min post-HFS (ANOVA/SNK-q-test, po0.05, Fig. 4A and B). The suppression of the CA1 hippocampal LTP by Ro 25-6981 presented a dose-dependent manner (ANOVA/SNK-q-test, po0.05, Fig. 4A and B). These results suggested that NR2BNMDAs played an essential role in the enhancement of LTP in hippocampal CA1 region of IBS-like rats.
2.4. Tyrosine kinase inhibitor suppressed the facilitation of CA1 hippocampal LTP in IBS-like rats in vitro
Fig. 2 – The enhanced LTP in hippocampal CA1 region evoked by HFS in IBS-like rats. The standardized fEPSPs slope (A) and (B) amplitude in control and IBS-like rats. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with inter-train interval of 10 s). Examples of representative fEPSPs traces obtained from control and IBS slices at 5 min pre-HFS and at 60 min post-HFS, respectively. Neonatal CRD resulted in the facilitation of hippocampal LTP in adult rats. n ¼8 slices from four rats for each group. po0.05, vs controls. HFS: high frequency stimulation; IBS: irritable bowel syndrome; fEPSP: field excitatory postsynaptic potential.
The expression of hippocampal tyrosine phosphorylation of NR2B protein significantly increased in IBS-like rats as compared with that in controls (unpaired t-test, po0.05, Fig. 3B). To further explore the potential activation mechanism of NR2B-NMDAs in enhancement of hippocampal LTP, the effects of genistein, a specific inhibitor of tyrosine kinases, on the induction and maintenance of LTP were estimated in IBS-like rats. Before HFS, bath application of genistein (2.5, 5, and 10 mg/L or 9.25, 18.5, 37 μM) had no significant effects on basic fEPSPs in IBS-like rats (ANOVA, p40.05, Fig. 5A and B). After HFS, 2.5 mg/L (or 9.25 μM) genistein had no significant influence on the fEPSPs in IBS-like rats (ANOVA/SNK-q-test, p40.05, Fig. 5A and B). However, 5 mg/L (or 18.5 μM) genistein could significantly inhibit the amplitude of fEPSPs by 41% (from 100.1717.9% to 59.5716.9%), and the slope of fEPSPs by 31% (from 96.2710.8% to 66.1721.7%) at 60 min post-HFS in IBS-like rats (ANOVA/SNK-q-test, po0.05, Fig. 5A and B). Moreover, 10 mg/L (or 37 μM) genistein could significantly block the amplitude of fEPSPs by 72% (from 100.1717.9% to 28.4710.2%), and the slope by 70% (from 96.2710.8% to
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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Fig. 3 – Expression of hippocampal NR2B-containing NMDA receptors in rats. (A) Western blot results showed that the protein expression of hippocampal NR2B-NMDAs and tyrosine phosphorylation of NR2B-NMDAs in rats. (B) Statistical chart showed that expression of NR2B-NMDAs and tyrosine phosphorylation of NR2B-NMDAs significantly increased in the IBS-like rats, n¼ 3, for each group. (C) The ratio of normalized pho-NR2B of normalized NR2B in hippocampus of rats. The ratio was enhanced in IBS-like rats. npo0.05, vs controls. NR2B: N-methyl-D-aspartic acid receptor 2B subunit; IBS: irritable bowel syndrome.
Fig. 4 – Inhibitory effects of Ro 25-6981 on enhanced LTP at SC-CA1 synapses in IBS-like rats. The effect of Ro 25-6981 on the standardized fEPSPs amplitude (A) and slope (B) in IBS-like rats. Bath application of Ro 25-6981 (0.5-1.0 mg/L or 1.33- 2.67 μM) dose-dependently blocked the LTP in IBS-like rats. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with intertrain interval of 10 s). N¼ 8 slices from four rats for each group. npo0.05, vs IBSþDMSO rats. Ro: Ro 25-6981; L: low dosage (0.25 mg/L or 0.67 μM); M: medial dosage (0.5 mg/L or 1.33 μM); H: high dosage (1.0 mg/L or 2.67 μM); IBS: irritable bowel syndrome; HFS: high frequency stimulation; LTP: long-term potentiation; fEPSP: field excitatory postsynaptic potential.
28.9712.0%) at 60 min post-HFS in IBS-like rats (ANOVA/SNKq-test, po0.05, Fig. 5A and B). These results suggested that tyrosine phosphorylation of CA1 hippocampal NR2B-NMDAs was involved in the facilitation of hippocampal LTP in IBSlike rats.
To further examine the correlation of tyrosine kinase activity with the maintenance of hippocampal LTP in IBS-like rats, genistein was applied at 15 min post-LTP induction. After LTP induction, 2.5 mg/L (or 9.25 μM) genistein had no significant influence on the fEPSPs in IBS-like rats (ANOVA/SNK-q-test,
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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Fig. 5 – Suppression effects of genistein on enhanced LTP in IBS-like rats. The effect of genistein on the standardized fEPSPs amplitude (A) and slope (B) in IBS-like rats. Genistein (5–10 mg/L or 18.5–37 μM) dose-dependently blocked the LTP in IBS-like rats. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with inter-train interval of 10 s). N¼ 8 slices from four rats for each group.npo0.05, vs IBSþDMSO rats. G: genistein; L: low dosage (2.5 mg/L or 9.25 μM); M: medial dosage (5 mg/L or 18.5 μM); H: high dosage (10 mg/L or 37 μM); IBS: irritable bowel syndrome; HFS: high frequency stimulation; LTP: long-term potentiation; fEPSP: field excitatory postsynaptic potential.
Fig. 6 – Tyrosine phosphorylation of the NR2B subunit contributes. to the maintenance of LTP. The effect of genistein on the fEPSPs amplitude (A) and slope (B) in IBS-like rats. Genistein (5–10 mg/L or 18.5–37 μM) dose-dependently blocked the maintenance of LTP 30 min and 60 min post-HFS in IBS-like rats. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with inter-train interval of 10 s). N¼6 slices from three rats for each group. npo0.05, vs IBSþDMSO rats. G: genistein; L: low dosage (2.5 mg/L or 9.25 μM); M: medial dosage (5 mg/L or 18.5 μM); H: high dosage (10 mg/L or 37 μM); IBS: irritable bowel syndrome; HFS: high frequency stimulation; LTP: long-term potentiation; fEPSP: field excitatory postsynaptic p40.05, Fig. 6A and B). However, 5 mg/L (or 18.5 μM) genistein could significantly inhibit the slope of fEPSPs by 31% (from 105.6778.37% to 72.9074.02%) and the amplitude of fEPSPs by 47% (from 144.46711.79% to 76.0177.77%), slope of fEPSPs by 44% (from 119.8879.70% to 67.60710.65%) and the amplitude of fEPSPs by 47% (from 148.83713.67% to 79.5077.62%), respectively, at 30 and 60 min post-HFS in IBS-like rats (ANOVA/SNKq-test, po0.05, Fig. 6A and B). Moreover, 10 mg/L (or 37 μM) genistein could significantly block the slope of fEPSPs by 64% (from 105.6778.37% to 38.2374.88%) and the amplitude of fEPSPs by 68% (from 144.46711.79% to 46.85714.30%), slope of fEPSPs by 71% (from 119.8879.70% to 34.9674.94%) and the amplitude of fEPSPs by 65% (from 148.83713.67% to 51.61717.17%), respectively, at 30 and 60 min post-HFS in IBS-like rats (ANOVA/SNK-q-test, po0.05, Fig. 6A and B). The results showed that genistein significantly inhibited CA1 hippocampal LTP at 30 min after LTP induction in IBS-like rats, and the inhibitory effect sustained for at least 60 min. These results provide new evidence to demonstrate that tyrosine phosphorylation of CA1 hippocampal NR2B-NMDAs was involved in the maintenance of hippocampal LTP in IBSlike rats.
2.5. Ro 25-6981 treatment attenuated visceral pain hypersensitivity in IBS-like rats Whether NR2B-dependent facilitation of hippocampal LTP was involved in visceral hypersensitivity was studied in IBS-like rats. Effects of NR2B-NMDAs on visceral hypersensitivity were examined in vivo. Bilateral intra-hippocampal injection of Ro 25-6981 (1, 2, 4 μg/μl, 2 μl each side) had no effect on the response of EMG to 40–60 mmHg CRD in controls (Bonferroni t-test, p40.05, Fig. 7A and C). There was no obvious change in responses of EMG to 40–60 mmHg CRD between 1 μg/μl Ro 25-6981 and vehicle in IBS-like rats (Bonferroni t-test, p40.05, Fig. 7B and D). Responses of EMG to 40–60 mmHg CRD were decreased by 39% (from 413.89723.29% to 253.44726.17%) and 35% (from 593751.64% to 382.60723.17%), respectively, after injection of 2 μg/μl Ro 25-6981 as compared with vehicle in IBSlike rats (Bonferroni t-test, po0.05, Fig. 7B and D). Responses of EMG were decreased by 75% (from 554.25765.38% to 141.16710.10%) under 40 mmHg CRD, and decreased by 61% (from 704.567139.73% to 278.27739.76%) under 60 mmHg CRD after injection of 4 μg/μl Ro 25-6981 as compared with vehicle in IBS-like rats (Bonferroni t-test, po0.05, Fig. 7B and D).
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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Fig. 7 – Bilateral intra-hippocampal injection of Ro 25-6981 decreased chronic visceral pain in IBS-like rats. The percentage of EMG amplitude over baseline under 40 (A) and 60 mmHg CRD (C) at 30 min after injection in controls; under 40 (B) and 60 mmHg CRD (D) at 30 min after injection in IBS-like rats. Bilateral intra-hippocampal injection of Ro 25-6981 (2–4 μg/μl, 2 μl per side) significantly inhibited chronic visceral pain in IBS-like rats. The time course of EMG were under 40 (E) and 60 mmHg CRD (F) in IBS-like rats. The maximal inhibition was observed at 30 min after Ro 25-6981 administrated in IBS-like rats (n ¼6 for each group); npo0.05. vs before the injection of Ro 25-6981; EMG: electromyography; CRD: colorectal distension; IBS: irritable bowel syndrome; Ro: Ro 25-6981.
Responses of EMG were significantly decreased by 30% (from 360.88731.30% to 253.44726.17%) under 40 mmHg CRD after injection of 2 μg/μl Ro 25-6981 as compared with 1 μg/μl Ro 25-6981 in IBS-like rats, and significantly decreased by 28% (from 502.54733.87% to 382.60723.17%) under 60 mmHg CRD (Bonferroni t-test, po0.05, Fig. 7B and D). Responses of EMG were significantly decreased by 61% (from 360.88731.30% to 141.16710.10%) under 40 mmHg CRD after injection of 4 μg/μl Ro 25-6981 as compared with 1 μg/μl Ro 25-6981 in IBS-like rats, and significantly decreased by 45% (from 502.54733.86% to 278.27739.76%) under 60 mmHg CRD (Bonferroni t-test, po0.05, Fig. 7B and D). These EMG data showed that intrahippocampal injection of the NR2B-NMDAs antagonist had a
dose-dependent inhibitory effect on visceral hypersensitivity in IBS-like rats. These results suggested that NR2B-NMDAs-dependent facilitation of hippocampal LTP may a key mechanism underlying the visceral hypersensitivity.
2.6. Time course of inhibitory effect of Ro 25-6981 on visceral hypersensitivity in IBS-like rats To further observe time profiles of Ro 25-6981, responses of EMG to 40–60 mmHg CRD were recorded every half an hour within three hours after intra-hippocampal injection of 4 μg/μl Ro 25-6981 in IBS-like rats. The lowest percentage of EMG
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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amplitude over baseline was observed at 30 min after injection of 4 μg/μl Ro 25-6981 (141.16710.10% under 40 mmHg CRD; 278.27739.76% under 60 mmHg CRD). Responses of EMG to 40 mmHg CRD were lowered by 69%, 52%, 37%, and 32% respectively at 30, 60, 90, and 120 min after injection of 4 μg/μl Ro 25-6981 in IBS-like rats. Responses of EMG to 60 mmHg CRD were lowered by 65%, 60%, 52%, 45% and 33% respectively at 30, 60, 90, and 120 min after injection of 4 μg/μl Ro 25-6981 in IBS-like rats. These results suggested that the maximal inhibition was observed at 30 min after administration of Ro 25-6981 under either 40 or 60 mmHg CRD in IBS-like rats (Bonferroni t-test, po0.05, Fig. 7E and F).
3.
Discussion
We previously observed that LTP induced by HFS at SC-CA1 synapses significantly increased in IBS-like rats (Zhu et al., 2012). In the present study, we provide further evidence to demonstrate that hippocampal NR2B receptor plays an important role in the enhanced LTP and visceral hypersensitivity in IBS-like rats and that activities of NR2B-NMDARs are mainly regulated by tyrosine kinase. Firstly, expressions of the hippocampal NR2B protein and tyrosine phosphorylated NR2B protein significantly increased in IBS-like rats. Secondly, the application of Ro 25-6981could dose-dependently block the facilitation of LTP in hippocampal CA1 region. Thirdly, genistein could also dose-dependently inhibit the induction and maintenance of hippocampal LTP in IBS-like rats in vitro. Finally, bilateral intra-hippocampal injection of Ro 25-6981 significantly attenuated visceral hypersensitivity in IBS-like rats.
3.1.
Facilitation of hippocampal LTP in IBS-like rats
It is claimed that synaptic plasticity is a key mechanism for chronic pain and plastic changes contributes to pain perception, fear and memory in Science (Li et al., 2010). It is concluded that LTP is a well-characterized form of synaptic Q3 plasticity (Ji et al., 2003; Wu et al., 2009; Li et al., 2010). In this study, the amplitude and slope of fEPSPs were ca. 49% and 57%, respectively, in controls, which is in agreement with data reported by Li et al. (2011). Although there are differences between the synaptic plasticity contributing to learning memory and pain memory, there are also striking similarities (Ji et al., 2003). In addition, LTP induced by HFS in the hippocampal CA1 region significantly enhanced and maintained for more than 1 h in IBS-like rats. These results indicate that CRD in neonatal rats could result in chronic visceral pain-related memory when they grew up. Thus, LTP is a key mechanism of chronic visceral pain in agreement with memory and other pains (Kodama et al., 2007; Li et al., 2010; Ohnami et al., 2012). Our results demonstrated that LTP induced by HFS at SCCA1 synapses significantly enhanced in IBS-like rats. This viewpoint is conflict with that in neuropathic pain reported by Kodama et al. (2007). They reported that hippocampal synaptic transmission in CA1 region was impaired in a murine model of neuropathic pain prepared by partial ligation of the sciatic nerve. Some reasonable explanations will
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be needed. Firstly, types of stimulus inducing LTP in neuropathic and visceral pain are different. Their LTP was induced by tetanus in the neuropathic pain, while ours was induced by HFS at SC-CA1 synapses. Secondly, there are some fundamental differences between the mechanisms responsible in visceral and somatic pain (Wesselmann et al., 2009). The neuropathic pain model reported by Kodama et al. is one of somatic pain. Thus, differences of pain models may result in the variation in synaptic responses. Finally, the model reported by Kodama et al. was prepared by partial ligation of the sciatic nerve, which is an acute pain, while the visceral pain established in this study is a chronic pain (up to 8 weeks). However, the interpretation of mechanisms responsible to different pains will need further studies in the future. Compared to other limbic structures such as the anterior cingulate cortex (ACC) and amygdala, the role of the hippocampus in the formation of chronic pain is not fully elucidated (Zhuo, 2007; Li et al., 2010; Kim et al., 2010; Crock et al., 2012). Our results reveal that hippocampal formation plays an important role in chronic visceral pain. There are several observations to support our results. Firstly, pain is a complex phenomenon resulting from a three-dimensional integration of sensory-discriminative, affective-motivational and cognitive-evaluative axes (Liu and Chen, 2009). Pain memory may persist for many years, which may be a key role for chronicity and repeatability of IBS (Choi et al., 2007; Noel et al., 2012). Secondly, hippocampal synaptic plasticity is a main cellular model for memory (Bliss and Collingridge, 1993), which contributes to the formation of pain-related memory and emotional responses (Chou et al., 2011). Thirdly, CA1 hippocampal synaptic transmission could be influenced by neuropathic and inflammatory pain (Kodama et al., 2007; Zhao et al., 2009). Furthermore, recent studies have demonstrated that CA1 plays a critical role in the learning and memory (Porte et al., 2011; Place et al., 2012). Khanna and Sinclair proposed that noxious stimuli produce prolonged changes in the CA1 region (Khanna and Sinclair, 1989). Finally, induction of persistent peripheral neuropathic pain could affect basic hippocampal processing such as the spatial encoding performed by CA1 region cells (Cardoso-Cruz et al., 2011). Thus, hippocampal formation plays an integral role in chronic visceral pain.
3.2. Role of NR2B in facilitation of CA1 hippocampal LTP and formation of visceral hypersensitivity in IBS-like rats Subunits composed of NMDA receptors are classified as families of NR1, NR2, and NR3 based on genotype (Monyer et al., 1992). As mentioned, NR2 subunit is composed of NR2A-D, which has a strong effect on the biophysical and pharmacological properties of NMDAR assemblies (Monyer et al., 1992; Zhuo, 2009). NR2B-NMDAs are widespread in areas related to pain transmission, such as the dorsal horn of spine and hippocampus (Oh-Nishi et al., 2009; Luo et al., 2014). Some experiment in mutant mice suggested that LTP is dependent on the NR2A subunit important for LTP (Clayton et al., 2002; Oh-Nishi et al., 2009; Zhuo, 2009). Ro 25-6981 is 5000 folds more selective for the heteromeric NR1/NR2B receptors than the NR1/NR2A receptors (Fischer et al., 1997). In our study, expression of hippocampal NR2B protein
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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increased in IBS-like rats, which was similar with the formalin-induced chronic pain (Wei et al., 2001). Together with, Ro 25-6981, a selective antagonist of NR2B-NMDAs, dose-dependently inhibited the facilitation of hippocampal LTP in vitro in IBS-like rats. Thus, it is hypothesized that NR2B subunit plays a critical role in hippocampal LTP enhancement in chronic visceral pain, which is in consistent with the study of genetic overexpression of NR2B subunit indicating the involvement of NR2B receptor in LTP (Tang et al., 1999). A study in vivo indicated intra-hippocampal injection of nonselective NMDA antagonists, AP5 and MK801, significantly reduced pain behavior in both the acute and tonic phases of the formalin test (Chen et al., 2009). However, the therapeutic potential of NMDA antagonists has been restricted in the treatment of chronic pain due to their serious side effects including hallucinations, learning and memory impairments, and sensorimotor disturbances (Soleimannejad et al., 2007; Qu et al., 2009). Our present study indicated that the blockade of NR2B receptors in the hippocampal CA1 region significantly inhibited neonatal CRD – induced chronic visceral hypersensitivity without significant side effects. In our studies, Ro 25-6981 attenuated visceral pain by ca. 45% and 70%, respectively, 30 min after intrathecal (75 μg/10 μl) and intra-hippocampal (4 μg/2 μl) injection (Luo et al., 2014). Explanations for the findings are that hippocampal NR2B subunit may play much more important role in chronic visceral pain. In other studies, the application dosage of Ro 25-6981 was 0.3–2 mg/L (Bartlett et al., 2007; Li and Han, 2008), and in this study, the effective dosages of Ro 25-6981 were between 0.5 and 1.0 mg/L. In our study, intra-hippocampal injection of Ro 25-6981 did not completely attenuate visceral pain hypersensitivity. The reason is likely because of other signaling molecule in hippocampus formation and other brain regions mediating chronic pain (Zhuo, 2007; Crock et al., 2012; Ikeda et al., 2013). Therefore, further studies will be required to the comprehensive understanding of mechanisms of chronic visceral pain.
3.3. Contribution of tyrosine phosphorylated NR2BNMDAs to CA1 hippocampal LTP in IBS-like rats The phosphorylation of NR2B-NMDAs plays a key role in modulation of NMDA receptor functions (Rostas et al., 1996; Nakazawa et al., 2001; Xu et al., 2012). The intracellular C-terminal cytoplasmic region of the NR2B-NMDAs is a functionally important region targeted by numerous protein kinases, including Fyn-mediated tyrosine kinases (Nakazawa et al., 2001). NR2B-NMDAs have seven phosphorylation sites for tyrosine kinase, with its main phosphorylation sites including S1303, S1323, S1480, Y1252, Y1336, and Y1472 (Nakazawa et al., 2001; Qiu et al., 2011). It is addressed that Tyr-1472 of NR2B is a major phosphorylation site, which may modulate hippocampal synaptic plasticity (Nakazawa et al., 2001). In this study, western blot data showed that expression of Tyr-1472 of NR2B protein in hippocampus significantly enhanced in IBS-like rats, in agreement with the paper reported by Xu et al. in inflammatory pain (Xu et al., 2012).We reasonably infer hippocampal NR2B phosphorylation at tyrosine 1472 may contribute to central sensitization of chronic visceral pain.
Since the tyrosine phosphorylation of NR2B subunit has already increased in IBS-like rats, these results do not exclude the possibility that there may be the constitutive activity of tyrosine kinase in maintaining the level of phosphorylation. As mentioned, the level of the phosphorylated NR2B protein increased in the hippocampus after induction of LTP (Rostas et al., 1996). In addition, tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP. Our results suggested that tyrosine phosphorylation of the NR2B subunit contributed to the maintenance of LTP in IBS-like rats, which is similar with the findings reported by Rostas et al. (1996). Thus, tyrosine phosphorylation activity was enhanced in the maintenance of CA1 hippocampal LTP in IBS-like rats. Rostas et al. (1996) mentioned “the increase in tyrosine phosphorylation of NR2B required LTP induction but was not involved in the induction process itself since the increase in tyrosine phosphorylation was not observed until between 5 and 15 min after LTP induction”. In this study, the tyrosine kinase inhibitor restrains the induction of LTP, which inhibits the tyrosine phosphorylation of NR2B and results in the inhibitory effect on LTP maintenance. Thus, it is speculated that tyrosine phosphorylation is one of the activating mechanisms of the hippocampal NR2BNMDAs in the facilitation of LTP in IBS-like rats.
4.
Experimental procedures
4.1.
Animals
Male Sprague-Dawley neonatal rats were provided by the Department of Experimental Animal Center, Fujian Medical University. The animals were housed with a nursing adult female rat until they were 25 days old. After separation from the adult female rats, the weaned rats were raised for two months before surgery and housed six in a cage with access to food and water ad libitum. The animal experimental procedures were conducted in accordance with the guidelines of the International Association for the Study of Pain (Zimmermann, 1983). The experiments were approved by the Animal Care and Use Committee of Fujian Medical University.
4.2.
Animal model of visceral hypersensitivity
In order to study mechanisms of chronic visceral pain, we developed a modified rat model of IBS as described by Al-Chaer et al., (2000). Hypersensitive SD rats were daily generated by 60 mmHg CRD during postnatal days 8–14. The controls received the same procedure except CRD. Above experiments were done by the same investigator. No treatment, procedures or further interventions were performed by the investigator until the testing date (8 weeks old).
4.3.
Assessment of visceral hyperalgesia
Visceral hypersensitivity was assessed by electromyographic (EMG) measurements of visceromoter response to CRD as described previously (Luo et al., 2014). Briefly, under anesthesia with isofluorane by anesthesia machine (VMR, Matrix,
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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USA), two silver bipolar electrodes were inserted the external oblique muscle of the abdomen. After the balloon was advanced into the colon, it was secured by taping the attached tubing to the rat's tail. The balloon was inflated to 40 and 60 mmHg for 10 s followed by 4 min rest. Responses of EMG were recorded three times under the same CRD pressure in each rat. The average of three recordings is taken as the magnitude of EMG activity. The magnitude of EMG activity was measured by a RM6240BD multi-channel physiological signal acquisition and processing system (the instrument, made in Chendu, China). The EMG data of 10 s before CRD (baseline) and 10 s during CRD were recorded for calculation. The responses were taken as the percentage of EMG amplitude during CRD over baseline using RM6420BD program. In order to avoid the interference of other factors, a single blind operation was adopted in the experiment. Furthermore, the rats were kept in a quiet environment, with relative humidity maintained at 40–70% and room temperature at 23–27 1C.
4.4.
Surgery and intra-hippocampal injection
The rats were anesthetized by intraperitoneal chloral hydrate (300 mg/kg) and were placed on a stereotaxic instrument (Narishige, Japan). The skin was cut along the midline of the skull. A cotton swab soaked with 3% H2O2 was used to abrade the tissue until the skull was exposed. A stainless steel guide cannula of 0.65 mm outer-diameter was placed stereotaxically and directed into the CA1 region of the hippocampus (4.0 mm posterior to the bregma, 2.8 mm lateral to the midline and 2.5 mm from the surface of the skull) according to Paxinos and Watson (1998). The two cannulas were fixed on the skull with two small screws by dental resin. The rats were given 3 days to recover from the operation. On the day of experiment, a stainless steel injection tube with 0.4 mm diameter was directly inserted into the guide cannula with 0.5 mm beyond the tip of the latter. Then 2 μl of drug solution was injected into the CA1 region of the hippocampus within 5 min. Control group was injected with the vehicle. The injection needle was left in place for an additional 5 min to allow diffusion.
4.5.
Hippocampal slice preparation
Rats were anesthetized with intraperitoneal chloral hydrate (300 mg/kg), and immediately after decapitation the brains were placed in ice-cold ACSF bubbled with 95% O2–5% CO2 (pH 7.4). The hippocampal formation was cut into 500-μm-thick transverse slices using a vibratome (ZQP-86, Zhixing Company, Shanghai, China) after fixed using 502 glue. The slices were incubated in an interface recording chamber maintained at a constant temperature (2871 1C), and were allowed to equilibrate for at least 1 h. The upper surface of the slices was in contact with a humidified, oxygenated (95% O2–5% CO2) stream.
4.6.
Electrophysiological recordings
The Schaffer collateral-Commissural pathway was stimulated and fEPSPs recorded from the dendritic layer of the CA1 pyramidal cells as reported by Kleppisch et al. (1999). The amplitude of the fEPSPs was measured for the downward
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peak. The intensity of the test stimulus, delivered at 0.1 Hz, was adjusted for each slice so that the fEPSPs were equal to approximately 50% of the maximal amplitude of the fEPSPs. The slices were stimulated for 10 min (0.01 Hz) so as to generate baseline values before LTP induction. Baseline values were recorded for at least 10 min before application of drugs. LTP was induced by two trains of HFS (200 pulses at 200 Hz, with inter-train interval of 10 s). fEPSPs were amplified and analyzed using a RM6240BD multi-channel physiological signal acquisition and processing system.
4.7.
Western blotting
Equal amounts of protein (50 μg) from the hippocampus of rats were separated and electro-transferred onto PVDF membranes (Invitrogen, USA), which were probed with mouse anti-NR2B monoclonal antibody (1:500, Abcam, USA), Rabbit anti-NR2B (pTyr1472) polyclonal antibody (1:1000, Abcam, USA) and mouse anti-β-actin primary antibody (1:1000, Abcam, USA). Blots were washed and incubated in peroxidase-conjugated goat anti-rabbit Ig G (1:5000, Abcam, USA) or goat anti-mouse Ig G (1:5000, Abcam, USA). Bands were visualized using an ECL system (Made in Xiamen, China).
4.8.
Statistical analysis
All data were expressed as mean7S.E.M. A one-way repeated measures analysis of variance (ANOVA) was used to determine whether there was a significant difference in the response to CRD pressure between control and IBS-like rats. One or two way repeated measures analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for multiple comparison after intra-hippocampal injection of compounds. Western blot results were examined by unpaired t-test. LTP was measured 60 min after HFS and reported as mean7SEM of baseline fEPSPs amplitude or slope. Student's t-test was used to compare the hippocampal field potential in control and IBS-like rats. One way ANOVA with Student– Newman–Keuls (SNK) multiple comparisons post-hoc analysis was used to compare the different groups of hippocampal slices of rats in vitro (SPSS 10.0). po0.05 was considered to be statistically significant.
Conflict of interest statement The authors declare that they have no competing interests.
Author contributions YC: Participated in the design of the research and data analysis. AQC: Participated in the research and data analysis. XQL: Participated in the research and manuscript writing. LXG: Carried out molecular techniques and participated in the analysis of results. YT: Participated animal models and the analysis of results. CJB: Participated in the research and interpreted results. LL: Participated in the research.
Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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CL: Designed, supervised studies, interpreted results, and prepare the manuscript.
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Uncited reference Sprengel et al. (1998).
Acknowledgments The present work was supported by grants from National Natural Science Foundation of China (81100998), Natural Science Foundation of Fujian Province (2012J05053), Doctoral Fund of Fujian Medical University (2010BS008), and the Major Fund of Fujian Medical University (09ZD009).
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Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001
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Please cite this article as: Chen, Y., et al., Hippocampal NR2B-containing NMDA receptors enhance long-term potentiation in rats with chronic visceral pain. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.001