| Pain is a complex experience encompassing sensory-discriminative, affective-motivational and cognitive-emotional components mediated by different mechanisms and is processed in a neural network. The persistence of pain is highly related to the strengthening of synaptic transmission on the pain conduction pathway. After various painful stimuli, the enhancement of the excitatory synaptic transmission was observed in the spinal cord, the amygdala, the hippocampal formation, the anterior cingulate cortex (ACC) and other pain-related brain formations. Furthermore, besides the changes of excitatory synaptic transmission, it was reported that the change of the inhibitory synaptic transmission was also involved in the persistence of chronic pain. Previous studies in our laboratory showed that the disruption in the balance of excitatory and inhibitory amino acids in the spinal cord and an imbalance between excitatory and inhibitory synaptic transmission in the ACC played an important role in peripheral inflammatory pain.Recent studies have revealed that an extensive cortical network is associated with pain processing and plays a major role in the representation and modulation of pain. Substantial evidence indicates that the primary somatosensory (S1) cortex, one of the most important structures in the cortical network, is involved mainly in processing the sensory-discriminative aspects of pain. With the use of magnetic resonance imaging and positron emission tomography in humans, it has been demonstrated that noxious stimulation caused significant activation of the S1cortex. Our previous observations showed that c-Fos-labeled neurons became densely increased in the superficial layers (Ⅱ-Ⅲ) of the S1 cortex and reached a peak at 2 h after BV treatment, which indicated that BV-induced peripheral persistent nociception could evoke increased neuronal activities in the S1 area with predominant localization in layerⅡ-Ⅲ. However, compared with pain-related formations mentioned above, the changes of synaptic transmission in the S1 cortex have been largely neglected in the painful situation. Moreover, in the case of inflammatory pain, it is unknown what the molecular and cellular mechanisms are responsible for the changes in the S1 cortex.In the present study, we address the above questions based on bee venom (BV)-induced peripheral inflammatory pain model, which has been well studied in our laboratory. Male Sprague-Dawley albino rats were randomly divided into two groups: saline group and BV group. The detailed experimental design and procedures are as follows:(1) Whole-cell patch-clamp recordings. The whole recordings were acquired on the pyramidal cells in layerⅡ/Ⅲof S1 brain slices. Then the changes of excitatory postsynaptic currents (EPSC) and inhibitory postsynaptic currents (IPSC) caused by peripheral persistent pain were studied. Following these, the whole synaptic plasticity was analyzed.(2) Immunohistochemistry (IHC). Two hours after BV injection (0.2 mg/50μl), the expression of AMPA receptor subunits GluR1, GluR2, GluR3, and GABAA subunit GABAAα1 in S1 were observed with IHC methods, which mainly aimed to identify the change of total protein in S1 under peripheral persistent pain.(3) Western blotting. The expression of total protein and subcellular fraction of AMPA receptor subunits GluR1, GluR2, GluR3, and GABAA subunit GABAAα1 in S1 were observed with western blotting methods, which mainly aimed to investigate the subcelluar distribution of AMPA receptor and GABAA under peripheral persistent pain.Results1. Changes in excitatory synaptic transmission induced by peripheral persistent nociception in the S1 cortexIn the continuous presence of bicuculline, sEPSC were recorded from pyramidal neurons in the layerⅡ/Ⅲof the contralateral S1 cortex. In the slices of the BV group, the frequency of sEPSC in layerⅡ/Ⅲneurons averaged 11.23±0.81 Hz (n = 7 neurons/7 rats), which was significantly increased compared with that of the saline group (4.93±0.40 Hz, n = 9 neurons/9 rats) (P < 0.01). The amplitude of sEPSC in the BV group (16.59±1.15 pA, n = 7 neurons/7 rats) was also much higher than that in the saline group (10.13±1.47 pA, n = 9 neurons/9 rats) (P < 0.05). However, there were no significant differences in the half-width between the two groups. These data suggested an overall enhancement of presynaptic transmitter release probability and postsynaptic AMPA receptor function in the layerⅡ/Ⅲof the S1 cortex. Next, we recorded AMPA receptor-mediated mEPSC in the layerⅡ/Ⅲof the contralateral S1 cortex neurons in the presence of 1μM TTX. Compared with the saline group, there was an obvious increase in mEPSC frequency in the BV group (saline: 4.88±0.23 Hz, n = 5 neurons/5 rats; BV: 5.85±0.21 Hz, n = 7 neurons/7 rats; P < 0.01). Furthermore, there was a significant difference in the amplitude of mEPSC between the two groups (saline: 12.52±1.11 pA, n = 5 neurons/5 rats; BV: 16.83±0.95 pA, n = 7 neurons/7 rats; P < 0.05). However, no significant differences were observed in the half-width between the two groups. Moreover, to test the upregulation of postsynaptic AMPA receptor function by BV, glutamate was infused to observe the changes of mEPSC. We found that both the frequency and amplitude of mEPSC in the saline and BV groups were increased in response to glutamate infusion. However, only the increase in the amplitude of mEPSC in the BV group was more significant than that in the saline group (saline: 132.35±7.73%, n = 5 neurons/5 rats; BV: 154.18±8.29%, n = 7 neurons/7 rats; P < 0.05). These data further proofed the results obtained from sEPSC.2. Effects of peripheral inflammation on the expression and subcellular distribution of GluR1, 2, 3 proteins in the S1 cortexIHC and Western blot results showed that, in the contralateral layerⅡ/Ⅲof the S1 cortex, total GluR1, 2, 3 protein levels of BV-injected rats were not significantly different from those of saline rats at 2 h post-saline injection (P > 0.05). These results indicate that the BV-induced peripheral inflammation has not altered the total protein expression of AMPA receptor subunits, suggesting that the changed synaptic transmission in the S1 cortex may not be caused by the changes in the total proteins of AMPA receptors.Meanwhile, quantification of the protein levels revealed that, the amount of GluR1 was decreased by 45.06% in the cytosolic fraction (n = 4, P < 0.05) and correspondingly increased by 21.38% in the membrane fraction at 2 h post-BV injection compared with the values of the saline group (n = 4). Conversely, compared with the saline group, the amount of GluR2 in the BV-treated group was 42.01% higher in the cytosolic fraction (n = 4, P < 0.05) and correspondingly 29.53% lower in the membrane fraction (n = 4, P < 0.05). However, no significant differences were observed in GluR3 between the saline and BV groups in either the cytosolic or membrane fractions (n = 4).3. Changes in inhibitory synaptic transmission induced by peripheral persistent nociception in the S1 cortexsIPSC were recorded in the continuous presence of CNQX. It was found that the amplitude of sIPSC was significantly lowered in the slices from the BV group (21.40±3.15 pA, n = 8 neurons/8 rats) compared with that from the saline group (35.63±4.01, n = 7 neurons/7 rats) (P < 0.05). However, further analysis of frequency or half-width of sIPSC did not reveal any significant differences between the two groups. These data indicated a decrease of postsynaptic GABAA function in the layerⅡ/Ⅲof the S1 cortex in BV-induced inflammation.Meanwhile, in order to further compare the changes in excitatory and inhibitory synaptic transmission, we examined GABAA-induced mIPSC from the layerⅡ/Ⅲof the contralateral S1 cortex neurons in the two groups in the presence of CNQX and TTX. Analysis of pooled data showed that the amplitude of mIPSC was significantly reduced in the BV group (12.70±1.01 pA, n = 8 neurons/8 rats), compared with that in the saline group (16.24±1.11 pA, n = 7 neurons/7 rats) (P < 0.05). Like the results from sIPSC, BV injection did not cause any significant changes in the frequency and half-width of mIPSC. Besides, to test the downregulation of postsynaptic GABAA function after BV injection, GABA was added to the ACSF. After analyzing the indices of the mIPSC, we detected that both the frequency and amplitude in the two groups were increased after the infusion of GABAA. Especially, the increase of amplitude in the BV grouP was significantly less than that in the saline grouP (saline: 142.58±8.23%, n = 7 neurons/7 rats; BV: 118.33±7.82%, n = 8 neurons/8 rats; P < 0.05). These data were consistent with the results from sIPSC.4. Effects of peripheral inflammation on the expression and subcellular distribution of GABAAα1 proteins in the S1 cortex IHC and Western blot results showed that, in the contralateral layerⅡ/Ⅲof the S1 cortex, total GABAAα1 protein levels of BV-injected rats were not significantly different from those of saline rats at 2 h post-saline injection (P < 0.05). These results indicate that the BV-induced peripheral inflammation has not altered the total protein expression of GABAA, suggesting that the changed synaptic transmission in the S1 cortex may not be caused by the changes in the total proteins of GABAA.Meanwhile, the amount of GABAAα1 protein in the BV-treated group was 58.42% higher in the cytosolic fraction (n = 4, P < 0.05) and correspondingly 29.30% lower in the membrane fraction (n = 4, P < 0.05) than that in the saline group. These data showed that GABAA was redistributed in the layerⅡ/Ⅲof the contralateral S1 cortex neurons as a result of BV-induced peripheral inflammation.Conclusion:(1) One change of synaptic plasticity in layerⅡ/Ⅲof S1 cortex under peripheral persistent pain is the enhancement of excitatory synaptic transmission, which includes increased presynaptic excitatory transmitter release and the up-regulation of postsynaptic AMPA receptors.(2) The trafficking of AMPA receptors, including insertion into plasma membrane of GluR1-containing AMPA receptors and internalization of GluR2-containing AMPA receptors contributed to the changes of excitatory synaptic plasticity in layerⅡ/Ⅲof S1 cortex under peripheral persistent pain.(3) The other change of synaptic plasticity in layerⅡ/Ⅲof S1 cortex under peripheral persistent pain is the impairment of inhibitory synaptic transmission, which includes down-regulation of postsynaptic GABAA.(4) The trafficking of GABAA (internalization/endocytosis) contributed to the change of inhibitory synaptic plasticity in layerⅡ/Ⅲof S1 cortex under peripheral persistent pain. |
PDF Full Text Request