Hippocampal AMPARs involve the central sensitization of rats with irritable bowel syndrome

Abstract Objective The roles of hippocampal AMPARs were investigated in irritable bowel syndrome (IBS)‐like rats to clarify the central sensitization mechanisms. Methods IBS model was induced by neonatal maternal separation. The effects of AMPARs on visceral hypersensitivity were examined by the responses of abdominal muscle to colorectal distension after the bilateral intrahippocampal injections of CNQX (an AMPAR inhibitor). The expressions of hippocampal AMPARs (GluR1 and GluR2) were determined by Western blot. Results The IBS‐like rats showed visceral hypersensitivity when compared with controls. Bilateral intrahippocampal injections of CNQX alleviated the visceral pain in IBS‐like rats. The maximal effect appeared at the time point of 30 min, and the duration lasted for 90 min after CNQX application, under 40 and 60 mmHg CRD. The expressions of hippocampal GluR2 significantly increased in IBS‐like rats when compared with controls (p < .05). However, the levels of hippocampal GluR1 had no significant differences in rats. Hippocampal LTP induced by HFS was significantly enhanced when compared with controls (p < .05). The expressions of GluR2 significantly increased in the control and IBS‐like rats after 60 min LTP of recordings (p < .05), but not GluR1. Conclusion Neonatal maternal separation enhances the expression of GluR2 and facilitates the LTP in the hippocampus, which could lead to the formation of visceral hypersensitivity when grown up.


| INTRODUCTION
Irritable bowel syndrome (IBS) influences around 11% of the population globally (Canavan, West, & Card, 2014). It manifests as abdominal hypersensitivity and abnormal gastrointestinal function. The IBS patients suffer huge psychological burdens and usually need more medications. IBS turns out to be a great social and economic burden (Plavsic, Hauser, Tkalcic, Pletikosic, & Salkic, 2015). Thus, greater efforts have been made to explore the mechanism of IBS and also in search of new therapies for it.
It is widely accepted that visceral hypersensitivity is the main factor causing visceral pain and bowel motor disorders in IBS patients (Agrawal et al., 2008). Visceral hypersensitivity results from peripheral sensitization and central sensitization (Price, Zhou, Moshiree, Robinson, & Verne, 2006): the former refers to sensitization of primary noxious sensor, and the latter indicates a persistent central synaptic transmission change during injury or after injury. Previously, most researches about the mechanism of visceral hypersensitivity focused on peripheral sensitization and the synaptic plasticity change in cornu dorsal medullae spinalis. Little attention has been paid to higher central synaptic plasticity.
Pain is conducted by a complex neural network in the central nervous system ). Chronic pain not only manifests hyperalgesia, allodynia, and spontaneous pain but also leads to various mood diseases, such as anxiety and depression ).
Thus, studies concerning pain are no longer restricted to the spinal cord, anterior cingulate, and almond, which have long been accepted as pain centers. The hippocampus also participates in pain and painful memories (Currie & Wang, 2004;Minami, 2012). Chronic pain lasts after the damage has cured. It is because of the functional or structural changes in the brain like memory processes. Therefore, chronic pain has been considered to be "a persistence of pain memory and the inability to extinguish the memory of pain evoked by an initial inciting injury" (Apkarian, Baliki, & Geha, 2009). A greater number of researchers believe that chronic pain shares a similar mechanism with memory, i.e., long-term potentiation. The hippocampus plays a key role in memory; thus, we choose it as the target of our study.
A huge number of experimental results have revealed that glutamate is an excitatory neurotransmitter that mediates painful information transfer. The a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors (AMPARs) are glutamate receptors widespread in the brain.
Our previous results suggest that spinal AMPARs may participate in the process of central hypersensitivity (Lin & Al-Chaer, 2005), but the roles of hippocampal AMPARs in chronic visceral pain remain unclear.
In this study, an IBS model was induced by neonatal maternal separation as in our previous studies. The effects of intrahippocampal injections of CNQX (an AMPAR inhibitor) on visceral hypersensitivity were examined using electromyogram (EMG), i.e., the responses of abdominal muscle to colorectal distension (CRD). The expressions of hippocampal AMPARs (GluR1 and GluR2) were evaluated by Western blot. In addition, the field potentials of CA1 region were recorded in the slices to figure out the hippocampal LTP. Finally, the expressions of GluR1 and GluR2 in the slices were evaluated by Western blot 60 min after high-frequency stimulation (HFS). A plausible mechanism of central hypersensitivity of chronic visceral pain in IBS was raised.

| Animals
Sprague-Dawley (SD) rats were obtained from the Experimental Animal Center of Fujian Medical University. Male rats (about 250 g) were used in the following study when they were 8 weeks old. IBS-like rats were induced by neonatal maternal separation (NMS) for three hours a day during postnatal days 3-21 Xiao et al., 2016). The controls were treated just like the IBS-like rats except for NMS. Rats have free access to food and water. They were maintained on a 12-hours light/dark cycle. All procedures were conducted during the light cycle. The rats were monitored routinely at least once daily by researchers. Rats were gently handled before experiments to alleviate stress and anesthetized during recording. Euthanasia was performed by administering an intraperitoneal injection of a lethal dose of pentobarbital sodium (150-200 mg/kg) at the end of the experiments.
All animal procedures were approved by the Committee for Care and Use of Laboratory Animals at Fujian Medical University.

| Measurement of visceral sensitivity
The visceral hypersensitivity was assessed as described before  by recording the spikes of the abdominal muscle to CRD (40 mmHg, 60 mmHg) when they were 8 weeks old. Distention balloons were placed in the descending colons under isoflurane anesthesia (VMR, Matrix, USA). Then, silver bipolar electrodes were inserted into the abdominal muscle. Distention was produced by rapidly inflating the balloon (10 s every 4 min) to the desired pressure.
The average of three recordings is taken as the amplitude of EMG.
The EMG recordings were collected and analyzed using RM6240BD (Chengyi, China).

| Surgery and intrahippocampal injection
After anesthesia, rats were placed in a stereotaxic instrument (Narishige, Japan). Through a midline incision along the skull, a stainless steel guide cannula was put stereotaxically and directed into the CA1 region of the hippocampus . Dental resin was used to fix two cannulae on the skull with two little screws. After a 3-day recovery period, 2 μl CNQX was injected into the hippocampal CA1 region within 5 min. DMSO was injected to the controls in the same way.

| Hippocampal electrophysiological recordings
The Schaffer collateral-Commissural pathway was stimulated and fEP-SPs were recorded from the dendritic layer of the CA1 pyramidal cells as reported by Kleppisch (Chen et al., 2014;Kleppisch et al., 1999). In the experiments, the amplitudes of the fEPSPs of the downward peak were measured. The test stimulating frequency was 0.1 Hz. The intensity was adjusted to produce about 50% of the maximal field potential amplitude. The baseline field potential was recorded at least 10 min before LTP induction or administration of agents. LTP was induced by HFS and recorded 60 min after HFS . The fEPSPs were amplified and analyzed using the RM6240BD.

| Statistical analysis
SPSS 10.0 was used to perform the analysis. A one-way repeated measures analysis of variance (ANOVA) was used to compare the EMG between control and IBS-like rats. One or two-way repeated measures ANOVA and Bonferroni post hoc test were adopted for multiple comparisons after the intrahippocampal injection of compounds. Western blot results were examined using unpaired t-test.
The 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. All data were expressed as mean ± SEM. p < .05 was considered to be statistically significant.

| DISCUSSION
In the present study, we found that hippocampal GluR2 may con-

| The effect of AMPARs on IBS-like rats
AMPARs are important ionotropic glutamate receptors, mediating most fast excited nervous transmissions. They participate both in physiological and pathophysiological sensations (Triller & Choquet, 2005). In our study, hippocampal injection of high dose CNQX (an AMPARs antagonist) could significantly attenuate visceral hypersensitivity in rats, and the inhibitory rates of IBS-like rats were higher than that of control rats. Medial dose CNQX only showed alleviatory effects in IBS-like rats but not in controls. Our results are in accordance with those of Lin (Lin & Al-Chaer, 2005): low, medial, and high doses of CNQX all significantly decreased the neuronal responses to CRD in IBS-like rats. However, medial and high doses of CNQX decreased the neuronal responses to CRD in controls. Wu et al. (2008) claimed AMPARs were responsible for anterior cingulate cortex (ACC) nociceptive transmissions in control rats. These results suggest that AMPARs play a more important role not only in a visceral physiological sense but also in the formation of chronic visceral hyperalgesia.  (Advokat & Rutherford, 1995) or hot plate (Nishiyama, Yaksh, & Weber, 1998).
Injection of AMPARs antagonist in amygdala did not change pain reactions in acute pain rats (Ghalandari-Shamami, Hassanpour-Ezatti, & Haghparast, 2011). Therefore, the roles of AMPARs in different pains are not always the same.
The analgesia effect of high dose CNQX in IBS-like rats reach a peak at 30 min after injection, then decreased gradually, lasted about 90 min, and pain reactions returned to normal 120 min after injection.
There might be several reasons for this degeneration in analgesia effect. First, brain CNQX concentration decreased as time went on.
Second, pain perceptions from other brain areas exceeded the inhibition of CNQX. Third, CNQX only selectively blocks AMPARs, the functions of other glutamate receptors still exist. Fourth, visceral pain sensation may be recovered through other mechanisms apart from AMPARs. Park announced an intrathecal administration of CNQX could significantly alleviate the CFA-induced mechanical pain and thermal pain up to 24 hr (Park et al., 2008). A plausible reason for the time difference might be that our study was of visceral pain, and theirs was of somatic pain. The analgesia effects of CNQX on different types of pain might be different.
GluR1 and GluR2 are considered to be closely related to pain sensation among the AMPAR subunits. Choi and Pezet have revealed that GluR1 expression in the spinal dorsal horn neuron membrane upregulated formalin-induced inflammation pain in animals, but the expression of GluR2 was unchanged (Choi, Svensson, Koehrn, Bhuskute, & Sorkin, 2010;Pezet et al., 2008). Park et al. (2008) observed GluR1 and GluR2 changed in CFA-induced chronic pain animal models, i.e., membrane GluR1 increased, and GluR2 decreased. With neuropathic pain, F I G U R E 4 The time curve of CNQX effects on visceral hypersensitivity in rats. The time curve of the inhibitory effects of 40 nmol CNQX on EMG under 40 and 60 mmHg CRD in control (a, b) and IBS-like rats (c, d). The maximal inhibition was observed at 30 min after CNQX application in control and IBS-like rats. The inhibitory effects of CNQX lasted for 90 min in IBS-like rats, but 30/60 min in control rats. n = 7, *:p < .05, vs pre-drug. EMG, electromyography; CRD, colorectal distention; IBS, irritable bowel syndrome; CNQX, an AMPAR inhibitor   Gray, Fink, Sarinana, Vissel, & O'Dell (2007) demonstrated that LTP in the hippocampal CA1 region does not require insertion and activation of GluR2-lacking AMPA receptors. In this study, GluR2 expression increased after high-frequency stimulation in the hippocampus slides of IBS-like rats, which was in accordance with the previous studies, indicating that GluR2 plays an important role in LTP maintenance and synapse plasticity in IBS-like rats.

| Hippocampus LTP, memory, and pain
It is well known that the hippocampus is involved in many functions of the brain, such as learning and memory, mood and emotion, feelings and motivation.However, there are always controversies concerning whether the hippocampus participates in pain procession. Shih reported formalin-induced nociceptive processing increased blood oxygenation level-dependent signals in the hippocampus . The reorganizations of signal processing in the hippocampus and between hippocampus and cortex seem to contribute to the transition from subacute to chronic pain (Mutso et al., 2014). Pain behaviors were significantly reduced by the injection of NMDA receptor antagonists in the hippocampus (Soleimannejad, Naghdi, Semnanian, Fathollahi, & Kazemnejad, 2007). Our results showed that the hippocampus AMPA receptor antagonist administration significantly alleviated the visceral pain in IBS-like rats. These results suggest that the hippocampus is involved in pain-related processing.
A great number of studies have proved that sensory-related longterm synaptic plasticity is a crucial mechanism of chronic pain (Woolf & Salter, 2000;Zhuo, 2008). Wei, Xu, Qu, Milbrandt, & Zhuo (2000) first recorded a hippocampal reaction to peripheral noxious stimulations in adult rats. Zhao reported that in a bee venom-induced persistent pain animal model, hippocampus LTP was remarkably enhanced (Zhao et al., 2009). In this study, we observed that LTP induced by HFS in the hippocampus CA1 region significantly increased in IBS-like rats, which is in accordance with our previous data . Our data suggest that the hippocampus LTP might be the cellular mode of pain.
However, further investigations are still needed to provide direct evidence for the relationship that exist between hippocampus LTP and pain.
The formation of chronic pain is closely similar to those of learning and memory. Moreover, chronic pain can lead to various changes in the nervous system like those in memory process and these changes last after the cure of the injuries. From functional modification to structural recombination, these different types of memories were also occurring in chronic visceral pain (Yi & Zhang, 2011). Central sensitization has long been considered as a critical mechanism in chronic pain, and LTP is the cellular basis of memory. Ji found astonishing similarities between central sensitization and LTP after comparing the production and sustaining of them, particularly in the regulations and transportations of NMDARs and AMPARs (Ji, Kohno, Moore, & Woolf, 2003).
Among the over 100 kinds of LTP-related factors, many of them participate in spinal central sensitization and lead to hyperalgesia. From this, we can deduce that there might be similar molecular mechanisms underlying chronic pain and memory. Chronic pain could be called a kind of noxious memory, but more researches should be carried out to understand and distinguish the roles of hippocampal LTP in pain and memory.