| Opioid analgesics represented by morphine are the most commonly used and most effective in clinical application,which plays an important role in the medical treatment of pain.However,long-term use of opioid analgesics produces adverse effects that include the development of tolerance and dependence,limiting their clinical utility.The molecular basis of opioid dependence is associated with the compensatory adaptation process in vivo after chronic exposure to opioid agonists.It exists not only in the opioid receptor system,but also in non-opioid receptor systems,such as dopamine receptors and glutamate receptors,as well as other modulators that affecting the functions of opioid receptor.The imidazoline receptor antisera-selected protein（IRAS）was cloned from the cDNA library of human brain hippocampus by Pilez et al.IRAS,a functional I1 imidazoline receptor,is widely distributed in the central nervous system.It was reported to regulate cell migration and proliferation,participate in neuroprotection and neuroinflammation.IRAS is involved in the regulation of several molecules,and associated with various diseases such as cancer,tumor,hypertension,diabetes,and depression.In the previous study,we found that IRAS could interact withμopioid receptor（MOR）and modulate it’s trafficking and desensitization,indicating IRAS might regulate opioid tolerance and dependence vitro.Opioid agonists,such asβ-endorphin or methadone,promotes MOR trafficking and desensitization.In contrast,morphine was shown to drive poor internalization and desensitization in vitro,which underlines the strong tolerance and dependence in vivo.However,whether IRAS could regulate the tolerance and dependence of morphine in vivo and the underlying mechanism is remained to know.In the present study,we relied on the IRAS knockout（KO）mice which generated by our research group,to investigate the role of IRAS in morphine tolerance,dependence and cognitive impairment,and the possible underlying mechanisms.1.IRAS KO enhances morphine tolerance and dependence1.1 IRAS KO enhances morphine toleranceFirst,there was no significant difference in baseline pain threshold and spontaneous activity between the two genotypes.The acute analgesic effect and hyperactivity caused by morphine were not affected in IRAS KO mice.After chronic morphine treatment（5 mg/kg or10 mg/kg,s.c.）,the MPE%of KO group was significantly lower than WT group on day 7,9and 11 assessed by tail flick test.The paw withdrawal latency of the KO group was significantly lower than that WT group on day 3,4,5,and 6 assessed by hot plate test.Following morphine chronic treatment（10 mg/kg,s.c.）,mice were challenged with naloxone（1 mg/kg,i.p.）.The results showed that the KO group displayed stronger systemic withdrawal symptoms than the WT group,including jumping,face wiping,paw tremor,head shaking and wet-dog shaking.These results suggested that IRAS KO accelerated the morphine tolerance and the physical dependence induced by naloxone precipitated.The possible molecular mechanism of opioid tolerance is related to the compensatory adaptation of MOR and post-receptor signaling transduction.We first examined the expression of MOR and cAMP activity in the spinal cord and the pericardial gray（PAG）,which area associated with analgesia and tolerance of morphine.Western blot results showed that there was no significant change in the expression of MOR on the membrane of WT morphine-tolerant mice in PAG and spinal cord,but the expression of MOR in KO group was significantly decreased.The levels and activity of cAMP showed no significant difference between WT and KO morphine tolerance group in the brain region of PAG,while cAMP levels in spinal cord were significantly higher in IRAS KO mice than WT group.These results suggested that IRAS knockout alters MOR expression levels after long-term morphine treatment and promotes compensatory increases in cAMP,but may not affect G protein activation.We further examined changes in ERK（extracellular regulated protein kinases）and CaMKII-ɑ（calmodulin-dependent protein kinase II）activity in spinal cord which is the downstream signaling to MOR.The results showed that IRAS knockdown had no significant effect on phosphorylation of ERK and CaMKII-ɑafter morphine physical dependence.It is speculated that IRAS may regulate morphine tolerance and dependence in some other way.Given that morphine tolerance and dependence are highly correlated with glutamate synaptic plasticity,we further examined changes in glutamate receptor expression in related brain regions following morphine treatment.The results showed that IRAS knockout up-regulated the phosphorylation of GluR1-S845 in the spinal cord compared with WT group,but had no significant effect on the total GluR1 and NMDA NR2A/2B expression.1.2 IRAS KO enhances morphine psychological dependenceIn the morphine CPP test,the results showed that morphine at 3 mg/kg or 10 mg/kg can produce CPP acquisition both in WT and KO mice,and the KO mice showed higher preference scores compared with WT mice at the dose of 3 mg/kg.After 30 days of spontaneous morphine（3 mg/kg）withdrawal,morphine（3 mg/kg,s.c.）significantly induced CPP restatement in KO mice,but had no significant effect on WT mice.These results suggested that IRAS knockout can enhance the psychological dependence of morphine.The neural pathways associated with drug addiction in the central nervous system are complex,and the mesolimbic dopamine system plays an important role in this process.Ventral tegmental area(VTA and Nucleus accumbens（NAc）constitute the key brain regions of the mesolimbic dopamine system.Therefore,we examined the expression of some signaling molecules and glutamate receptors that associated with opioid dependence in the VTA and NAc after CPP test.The results indicate that IRAS knockout did not affect the expression of ERK,AKT and CaMKII-ɑphosphorylation which downstream to MOR after morphine treatment.In morphine dependence mice,IRAS KO enhanced phosphorylation of AMPA receptor GluR1-S845 in VTA,and increased AMPA GluR1/R2 subunit and NMDA NR2A/2B subunit receptor expression in cell surface.IRAS KO reduced expression of the AMPA receptor GluR1/2subunit and the NMDA receptor NR2A/2B subunit after morphine dependence,whereas these subunits were significantly elevated in WT mice.The results suggest that the effect of IRAS on morphine dependence might be involved in the regulation of AMPA/NMDA receptors.1.3 Effects of conditional IRAS KO in specific brain on morphine tolerance and dependenceTo further validate the functional specificity of IRAS in different brain regions,we used IRASfloxed/floxed mice to evaluate the actions of morphine.Conditional IRAS KO in specific brain regions was through stereotaxic injection of CRE virus into the indicated brain.The results showed that specific IRAS KO in VTA had no significant effect on morphine tolerance and psychological dependence,while specific IRAS KO in NAc enhanced morphine tolerance and psychological dependence.We also found the specific IRAS KO in PAG enhanced morphine tolerance.These results suggested that NAc may be a key brain region in which IRAS plays a role in regulating morphine tolerance and dependence.PAG is also an important brain region in which IRAS plays a role in regulating morphine tolerance.Taken together,we propose that IRAS KO enhanced morphine tolerance,physical dependence and psychological dependence,and NAc is a key brain area associate with this process.IRAS plays an essential role in the development of morphine tolerance and dependence which the underlying mechanisms involved in regulation of MOR expression and cAMP levels,as well as the membrane expression of AMPA GluR1-S845 phosphorylation and AMPA/NMDA receptors,and ultimately affecting the function of glutamate system.2.Effects of IRAS KO on natural reward and cognitive impairment caused by morphine withdrawalThe harm of drug addiction to the human also includes negative emotions,decreased natural rewards,and impairment of cognitive function during drug addiction withdrawal.We further applied different experiment models to evaluate the effects of IRAS on natural rewards and cognitive impairment caused by morphine treatment.The results of sucrose preference and novel object recognition experiments showed that there was no significant difference between WT and KO group in na?ve mice or during the morphine withdrawal.The results also showed that there was no significant difference in natural reward that tested by sucrose administration between WT mice and KO mice under the basal condition.However,during morphine withdrawal period,sucrose administration in WT mice was strongly destroyed,but was not alted in KO mice comparing with vehicle control.These results suggest that IRAS KO alleviated the effects of morphine withdrawal on natural rewards.The results of water maze test showed that there was no significant difference in the acquisition and extraction of spatial memory between WT mice and KO mice in the basal condition.Morphine withdrawal caused deficit of acquisition and extraction of spatial memory in WT mice but not in KO mice.In the reverse learning test,IRAS KO mice showed cognitive flexibility impairment in the period of morphine withdrawal.These results suggest that IRAS KO did not affect cognitive flexibility impairment caused by morphine withdrawal,but inhibited the deficits of natural reward and spatial memory caused by morphine withdrawal.The neurobiological basis of learning and memory is synaptic plasticity,and the glutamate system plays an important role in the synaptic transmission of the central nervous system.Whether IRAS affects the natural reward and recognition impairment caused by morphine withdrawal through regulating the glutamatergic system?In the brain of prefrontal cortex（PFC）and hippocampus（Hip）.The results showed that the expression of GluR1/2 subunit and NMDA receptor NR2A/2B subunit in WT mice was significantly up-regulated during the morphine withdrawal,while IRAS KO down-regulated the expression of these subunits.Therefore,it is suggested that the underlying mechanism of IRAS on cognitive impairment induced by morphine withdrawal may be related to the regulation of AMPA and NMDA receptor expression in PFC and Hip.3.Role of PI3K in IRAS regulation of morphine tolerance and dependencePhosphoinositide 3-kinase（PI3K）plays an important role in regulating glutamatergic plasticity.Previous study reported that IRAS regulates apoptosis through PI3K/AKT signaling pathway,and also suggested that it may have interactions between IRAS and PI3K-p85regulatory subunits.The the above study suggested that IRAS affects morphine tolerance and dependence and cognitive impairment caused by morphine withdrawal through regulating the glutamatergic system,but how IRAS modulates the expression of glutamate receptor remains unclear.Whether can IRAS interact with PI3K-p85 and participate in regulation of the effect of morphine and glutamate receptor expression.It was found by immunoprecipitation experiments that there was indeed an interaction between IRAS and PI3K-p85 in vivo and in vitro.A direct interaction between IRAS and PI3K-p85 was found by Pull-down assay.In order to study the role PI3K in the effect of IRAS on regulating morphine tolerance and dependence,we take advantage of two diffierent PI3K inhibitors,LY294002 and wortmannin.We found that the PI3K inhibitor LY294002 significantly reduced morphine tolerance and naloxone-induced physical dependent in WT mice,but had no significant effect on KO mice.PI3K inhibitors LY294002 and wortmannin specifically inhibited CPP acquisition in WT mice,but had no significant effect in KO mice.This suggests that the effect of PI3K in regulating morphine tolerance and dependence is dependent on IRAS.Western blot results showed that PI3K inhibitor decreased the up-regulation of AMPA/NMDA receptors in WT morphine tolerance mice,but had no effect on the expression of these receptors in KO group,suggesting that inhibition of PI3K reversed the changes of glutamate receptors in WT mice,and this effect wass dependent on the presence of IRAS.These results suggest that IRAS interacted with PI3K-p85,which maybe mediated the effect of IRAS on regulation of glutamate receptors and morphine actions.In summary,for the first time we found that IRAS can regulate morphine tolerance and dependent behavior in animal models.We initially verified the key brain regions associated with the effect of IRAS on morphine tolerance and dependence,and revealed that the possible underlying molecular mechanism is associated with the compensatory adaptation of opioid receptor and glutamatergic system by coupling with PI3K.The findings of this study are important for further elucidating the neurobiological mechanisms of opioid addiction and developing of new analgesics or anti-addiction drugs. |
