The Role Of HCN Channels Within The Periaqueductal Gray In Neuropathic Pain

Background:Neuropathic pain is a common and severely disabling disease, affecting millions ofpeople worldwide. Neuropathic pain is characterized by mechanical allodynia, thermalhyperalgesia, and spontaneous pain. Both peripheral and central neuronal sensitizationcontribute to the development of neuropathic pain. Increased numbers of discharges from theinjured primary neurons lead to the initiation of neuropathic pain. The subsequenthyperexcitability of central neurons contributes to pain maintenance. Ion channels are primarydeterminants of neuronal excitability, and alterations in the expression and function of variouschannels in pain have been identified.Hyperpolarization-activated cyclic nucleotide-gated-cation (HCN) channels are widelydistributed in the nervous system, and comprised of four isoforms, from HCN1to HCN4. Ihcurrent, carried by HCN channels, is important in driving the repetitive firing of neurons.Abnormal alterations in Ih currents are involved in many neural diseases, includingneuropathic pain. Neuropathic pain involves the ectopic discharge of neurons. Many studyhave confirmed an important role for HCN channels in both inflammatory and neuropathicpain. Interestingly, previous studies have identified the HCN channel protein located in theventral-lateral periaqueductal gray (vlPAG), a region that is considered to be important forpain modulation. However, it is unclear whether HCN channels in the vlPAG are involved inpain modulation. In the present study, we found changes in the expression of HCN1andHCN2channels within the vlPAG in neuropathic pain. Furthermore, we detected anti-nociceptive effects in vivo by blocking vlPAG HCN channels in CCI rats.Methods:1. Sciatic nerve chronic constriction injury (CCI) was established as a neuropathic painmodel, and the pain threshold of PWMT and PWTL was evaluated by (Hargreaves) andmechanical (von Frey) tests. 2. By employing Real-time PCR, imunostaining and Western blot analysis, theexpression of HCN1and HCN2channel at vlPAG14days post-CCI surgery were detected.3. The function of these upregulated HCN channels was verified by the intra-vlPAGinfusion of ZD7288, a specific HCN blocker, and the change of pain threshold was evaluatedsimultaneously.4. For patch-clamp studies, Sprague–Dawley rat pups(p21) were used for brain slicepreparation. Whole-cell recordings of HCN channels and neural activity (AP frequencey,frequency of sEPSC) were conducted in vlPAG neurons in sham and CCI groups14days postsurgery. Additionally, some cells were intracellularly labeled with biocytin (0.5%) to confirmmorphological identification.5. HCN blocker ZD7288were applied to investigated changes in HCN channels andtheir influence on vlPAG neuronal activity, and AC agonist FSK, whcih could elevateintracellular cAMP, was used to study the role of intracellular cAMP in mediating the impactof CCI surgery on regulating HCN channel and neuronal activity.Results:1. CCI surgery decreased the pain threshold of PWMT and PWTL, with a maximal effectat2weeks post-operation. Thus,14days post-surgery was chosen as the critical time point inthis study. Immunostaining and western blot analysis revealed higher expressions of HCN1and HCN2proteins in CCI animals than in the naive and sham groups at14days post-CCI.2. The HCN channel blocker ZD7288or the same volume of saline was injectedintra-vlPAG. The effect at20μg of ZD7288reached its peak within30min of injection, andthen gradually decreased. In addition, either injection of saline or20μg of ZD7288had noobvious effect on thermal hypersensitivity and mechanical allodynia behaviors of naive rats,which indicates that the motion of animals was not affected.3. vlPAG neurons display electrophysiological feature of HCN currents. CCI surgeryproduced significant augmentation of the Ih current amplitude, when normalized to cellcapacitance, the Ih current density of CCI neurons was significantly increased compared withthe Ih current density in control neurons over the voltage range-90to-120mV.Furthermore, The voltage dependence was shifted to more depolarized potentials in neuronsfrom the CCI group. 4. The mean rate of AP firing in CCI rats was higher than that in control rats.Additionally, compared with the sham-operated group, CCI surgery significantly increasedthe frequency of sEPSCs in recorded neurons. Bath application of ZD7288(50μM), a HCNblocker, decreased AP firing and sEPSCs in recorded cells to the normal level. This resultsuggests that the enhanced Ih currents observed after CCI surgery contribute to the increase inneuronal activity in the vlPAG region.5. FSK, which elevates cAMP by directly activating adenylate cyclase, was bath applied.Increases in the frequency of AP and sEPSCs induced by a50pA current injection wereobserved in normal rat vlPAG neurons after bath application of FSK, and these increasesmirrored the change observed in the CCI induced neuropathic pain. Moreover, the increaseswere blocked by HCN antagonist ZD7288(50μM).Conclusion:These results have shown an increase of HCN1and HCN2channels expression after CCIsurgery in vlAPG region. The electrophysiological data also revealed functional interactionbetween cAMP and HCN channels after CCI surgery. Intracellular cAMP, increased after CCIsurgery, thus enhance activation of HCN channels, and facilitated the hyperexcitability ofneurons in vlPAG region. Taken together, our findings suggest a key regulatory role of HCNchannels on vlPAG neural activity and impact the facilitation role of vlPAG in modulation ofpain during neuropathic pain.