Persistent pain is a devastating state that often destroys the quality of life. To understand the molecular and cellular mechanisms underlying persistent pain, we studied the signal transduction pathway for pain-related synaptic plasticity in the spinal dorsal horn. Using two animal models of persistent pain, i.t. NMDA and i.d. capsaicin, our study showed that the phosphorylation and cell-surface localization of GluA1 are significantly increased after NMDA-R activation and that these increases were blocked by the removal of ROS. ROS-dependent increase of GluA2-S880 phosphorylation and thus decreased cell-surface localization of GluA2 were also parts of the signaling pathway. Thus, our study suggests a signal transduction pathway that consists of NMDA-R activation, ROS production, AMPA-R phosphorylation, and AMPA-R trafficking to and from the cell surface, plays a critical role in pain-related synaptic plasticity in DHNs.
In this study, we used i.t. NMDA injection to induce pain-related behavior of mice for the following reasons: 1) the i.t. NMDA injection produces pain-related behavior in animals [1
], 2) i.t. NMDA induces pain-related behavior in animals without the involvement of other factors such as inflammation, 3) i.t. NMDA model allows us to study central mechanism with minimal peripheral involvement, and 4) the i.t. NMDA injection could activate a large population of DHNs through immediate, direct, strong, and homogenous stimulation. Thus the changes in the signaling pathway could be large enough to be detected by biochemical and optical imaging analyses, as shown in our study.
Since NMDA is not an endogenous neurotransmitter, the AMPA-R phosphorylation was also examined in i.d. capsaicin-injected mice, and then compared to that of the i.t. NMDA mice. Capsaicin will activate peripheral nerve endings of nociceptors and will cause glutamate release from nociceptor terminals in the spinal cord and then activate NMDA-Rs, as well as, other glutamate receptors on DHNs [48
]. Furthermore, blocking NMDAR activation by an NMDAR inhibitor, AP7, prevents the development of the sensitization of spinothalamic tract neurons in the spinal cord after i.d. capsaicin [8
]. Thus the activation of NMDA-Rs in the spinal cord by i.d. capsaicin is closer to a physiological condition compared to that produced by i.t. NMDA. Despite these differences, the results are similar: ROS-dependent increases in phosphorylation of GluA1 and GluA2 are in concert with pain-related behavior. Therefore, this parallel study suggests that the ROS-dependent AMPA-R phosphorylation is a common component for the pain-related signaling pathway at least in these two pain models.
We do not believe that the DHN sensitization after i.t. NMDA is the same as central sensitization induced by a nerve or tissue injury or inflammation. The reasons include that NMDA is not an endogenous neurotransmitter although it is specific for NMDA-Rs, and it activates non-synaptic NMDA-Rs as well as synaptic NMDA-Rs. However, our present data and previous studies strongly suggest that a similar mechanism is involved at least for the initial phase of DHN sensitization regardless of animal models of pain [23
] in vivo
or in vitro
To analyze pain behavior, we performed open-field exploratory activity tests in addition to Von Frey tests. Exploratory activities require brain functions and do not entirely rely on spinal reflexes. Furthermore, the spontaneous activity test can also be used for the detection of spontaneous pain, which is difficult to measure in most animal models of pain. Consistent results from two types of pain-related behavior tests provided us a solid foundation for our analyses of pain-related signaling pathway.
A main finding from our study is the link from ROS to AMPA-R phosphorylation and cell-surface localization in the L4/5 spinal cord, as a part of the signaling pathway for the induction of pain-related synaptic plasticity. ROS is known as a critical component for the signaling pathway [23
]. However, molecules downstream of ROS are not known. It has been well known that increases of PKC, CaMKII, and PKA activities are essential for the induction of pain-related behavior [9
]. Since all these kinases have target sites on AMPA-Rs [2
] and AMPA-Rs play a role in pain [12
], we analyzed the changes of AMPA-R phosphorylation (S818, S831, and S845 of GluA1 and S880 of GluA2) in two animal models of pain. Our data demonstrated that phosphorylation on the all four examined sites was significantly increased by NMDA-R activation in concert with pain-related behavior. Importantly, the changes in AMPA-R phosphorylation after NMDA-R activation were dependent on ROS levels. Furthermore, the changes of AMPA-R localization on the cell surface in the L4/5 spinal cord were also dependent on ROS. It has been shown that the cell-surface localization of AMPA-Rs is regulated by phosphorylation of AMPA-R in the brain [6
] and spinal cord [40
]. AMPA-R trafficking to the cell surface has the priming effect on AMPA-R trafficking to the synapse [30
]. Taken together, it is likely that the ROS-dependent changes of AMPA-R phosphorylation and subsequent AMPA-R trafficking in and out of the cell surface are essential parts of signaling pathways for pain-related synaptic plasticity.
This is the first study to demonstrate the potential role of GluA1-pS818 in the pain-related synaptic plasticity in the spinal cord. Our data demonstrated co-variance of GluA1-pS818 with pain behavior. This agrees with the role of GluA1-pS818 in synaptic plasticity in the brain which was established recently [3
]. The ROS-dependent increase in GluA1 (S831 and S845) and GluA2 (S880) phosphorylation during the induction of pain is a novel finding in our study, although the increases of the phosphorylation have been reported in some pain models [5
Both GluA1-S818 and GluA2-S880 are substrates of PKC and they are essential for hippocampal LTP [3
] and inflammatory pain [40
], respectively. Our study with animal models of pain clearly demonstrated that the GluA1/GluA2 phosphorylation at the PKC sites is significantly increased during the induction of pain, and then almost completely reversed when ROS were removed. The data thus suggest that ROS are critical for activation of PKC during the induction of pain. Similarly, the phosphorylation of S845 and S831, the sites of PKA and CaMKII, respectively, was also significantly increased during the induction of pain and regulated by ROS levels. However, the ROS scavenger effect on reversing phosphorylation at S831 is smaller compared to that at S818, S880, and S845. Thus the data suggests that at least some part of CaMKII activity increase during the induction of pain is not dependent on ROS. The small differences between control and NMDA+PBN might have been caused by either an insufficient ROS scavenging by PBN and TEMPOL or an imbalance between scavenging and production of ROS.
Our multidisciplinary data consistently demonstrated a significant reversal of NMDA or capsaicin effects on pain behavior and AMPA-R cell-surface localization, as well as on AMPA-R phosphorylation, by ROS scavengers. These data strongly suggested that ROS is a necessary intermediate element for induction of pain behavior, which is also consistent with previous studies from our group [23
]. Our data thus suggest that ROS is working as an intermediate signaling molecule between NMDA-R activation and AMPA-R phosphorylation by regulating kinases (and/or phosphatases) activities during development of pain related synaptic plasticity. Previous studies support our idea. ROS-dependent activation of PKA increases excitability of amygdala neurons and pain behavior [29
]. ROS are also known as signaling molecules for various kinases [33
By labeling surface proteins with a membrane-impermeable cross-linking reagent BS3
, we clearly demonstrated the increase of GluA1 on the cell surface after NMDA-R activation. Furthermore, the finding of ROS-dependency of GluA1 cell-surface localization was also a novel finding in this study. The insertion of the GluA1 into the plasma membrane has been reported in some pain models [5
]. Most previous studies, however, have shown the increase of GluA1 in a membrane fraction that could include not only the plasma membrane but also the internal membrane, and thus they have a contamination problem. GluA1 trafficking to the cell surface has the priming effect on AMPA-R trafficking to the synapse during hippocampal LTP [30
]. Therefore, the increase of cell-surface localization of GluA1 in the spinal cord after i.t. NMDA injection would likely contribute to the increase of synaptic AMPA-Rs, thus resulting in the pain-related behavior observed in our study. The ROS-dependent decrease of GluA2 cell-surface localization is also novel and consistent with a previous study which showed GluA2 internalization during inflammatory pain [40
The opposite movement of GluA1 and GluA2 in and out of the DHN surface would change surface GluA1/GluA2 ratio. Considering that only GluA2 subunit can block Ca2+
influx through AMPA-Rs, the NMDA induced trafficking of GluA1 and GluA2 would increase Ca2+
-permeable AMPA-Rs on the cell surface, and thus at the synapse of DHNs. Previous studies have shown that Ca2+
-permeable AMPA-Rs contribute to DHN sensitization and pain-related behavior [5
]. The increase of Ca2+
influx through AMPA-Rs may activate some signaling pathways in the micro domain around the Ca2+
-permeable AMPA-Rs. However, molecular mechanisms downstream of the Ca2+
influx through the Ca2+
-permeable AMPA-Rs and their contribution in the pain-related signaling pathway are yet to be revealed. Furthermore, the relationship and relative contribution between Ca2+
influx through AMPA-Rs and Ca2+
influx through NMDA-Rs is not yet clear. Further studies are warranted for resolving these issues.