Role Of Amygdala Calcium/calmodulin Dependent Protein Kinase ?? In Opioid-induced Hyperalgesia In Rats

ObjectiveTo explore the role of amygdala calcium/calmodulin dependent protein kinase ?? in fentanyl-induced hyperalgesia in rats.Methods1.SPF Sprague-Dawley male rats(60-100 g)were randomly assigned to receive fentanyl or saline(Fentanyl group and Control group,n = 4 each).Fentanyl was injected four times(s.c.,60 ?g/kg per injection)at 15min intervals,resulting in total doses of 240 ?g/kg to induce OIH in Fentanyl group.Control group received saline injections.The PWMT and PWTL were tested for the nociceptive thresholds evaluated at different time points including D-1,DO(baseline),1 h,5 h,6 h,6.5 h,7 h,8 h,and D1,D2,D3,D4,D5 after the last saline or fentanyl injection.2.SPF Sprague-Dawley male rats(60-100 g)were randomly assigned to receive fentanyl or saline(Fentanyl group and Saline group,n=6 each).The level of p-CaMKII ? in the right laterocapcular division of central nucleus of amygdala(CeLC)was examined 6.5 h after the last injection of fentanyl or saline by Western blotting.3.SPF Sprague-Dawley male rats(60-100 g)were randomly assigned to receive 50%DMSO,KN92 10 nmol,KN935 nmol,KN93 7.5 nmol or KN93 10 nmol 6.5 h after the last fentanyl injection(n = 10 each).One week after cannulation in the right CeLC,Vehicle group,KN92 group,KN93(5 nmol)group,KN93(7.5 nmol)group and KN93(10 nmol)group received equal volume(0.5 ?l)of 50%DMSO,KN92 10 nmol,KN93 5 nmol,KN93 7.5 nmol and KN93 10 nmol respectively after mechanical allodynia and thermal hyperalgesia were developed(6.5 h after the last fentanyl injection).The PWMT and PWTL were tested at before OIH induction(Baseline),before drug infusion and 0.5 h after drug infusion.Then the level of p-CaMKII ? in the right CeLC tissues from each group was analyzed by Western blotting.4.SPF Sprague-Dawley male rats(60-100 g)were randomly divided into Saline group and Fentanyl group(n = 6 each).Recordings of miniature excitatory postsynaptic currents(mEPSCs)were performed using whole-cell voltage-clamp method in the right CeLC brain slices 12h after the last saline or fentanyl injection.Then KN93(10 ?M),a CaMKII inhibitor,was added into the brain slices to test its effect on the mEPSCs on right CeLC neurons.Results1.Compared with saline treated rats,fentanyl administration initially increased mechanical and thermal threshold.And this analgesic effect disappeared 5 h later.Mechanical allodynia and thermal hyperalgesia were observed from 5h after the last injection,peaked at 6.5 h,and lasted for 4 days.2.Compared with Saline group,the level of p-CaMKII ? in right CeLC in Fentanyl group was increased(P<0.05).3.PWMT and PWTL were significantly decreased 6.5 h after the last fentanyl administration in Vehicle group,KN92 group,KN93(5 nmol)group,KN93(7.5 nmol)group and KN93(10 nmol)group(P<0.05).30 min after drugs injection,Opioid-induced hyperalgesia was found to be attenuated by KN93 in a dose-dependent fashion.At lower doses,KN93(7.5 nmol)partially suppressed allodynia and hyperalgesia,at the highest dose,KN93(10 nmol)completely reversed allodynia and hyperalgesia(P<0.05).While neither KN92(10 nmol)nor vehicle altered the pain threshold(P>0.05);KN93,at the highest dose we used(10 nmol),significantly reversed the fentanyl-induced activation of p-CaMKII? in right CeLC(p<0.05).In contrast,neither KN92(10 nmol)nor vehicle changed the increased level of p-CaMKIIa in right CeLC induced by fentanyl(P>0.05).4.Compared with Saline group,the amplitude and frequency of mEPSCs recorded from the right CeLC neurons in Fentanyl group were significantly increased(P<0.05).In slices(12 h post induction),KN93(10?M)obviously reversed the increased amplitude and frequency of mEPSCs recorded from the CeLC neurons in OIH rats,but not in Saline rats(P<0.05).Conclusion1.Fentanyl exposure induced mechanical allodynia and thermal hyperalgesia in rats in a time-dependent manner.2.Fentanyl exposure increased the level of p-CaMK?? in right late roc apcular division of central nucleus of amygdala(CeLC).3.At CeLC,injection of KN93,a CaMK?? inhibitor,reversed fentanyl-induced behavioral hyperalgesia in a dose-dependent manner.4.CaMK?? inhibitor can reverse the enhanced synaptic transmission induced by fentanyl in right CeLC neurons.