The Roles Of Spinal ERK Signaling Pathway In Morphine Dependence And Withdrawal

ObjectiveThe development of dependence to opiates continues to be a significant clinical and social problem. Opioid dependence involves psychological dependence characterized by compulsive, out - of - control drug use despite serious negative consequences and relapse, while physical dependence is characterized by producing withdrawal symptoms after discontinuation or reduction in the use of a substance. Psychological dependence is the key point in the study of dependence and the center of most of experimental studies. However, physical dependence is one of the most important causes of compulsive drug - taking behavior and short term relapse. Thus, elucidation of the various mechanisms involved in physical dependence is requisite to the development of treatment strategies to attenuate or circumvent opioid dependence.Accumulating evidence has suggested that the spinal cord neurons sensitiza-tion participated in mediating morphine withdrawal. Sensitization is considered as a form of synaptic plasticity, which was considered as a model of learning and memory. Drug dependence can be considered as a kind of learning and memory process regarding the effect of addictive drugs for the specific neurons in the brain. More and more studies have demonstrated that synaptic plasticity, a possible substrate for learning and memory, was involved in initiation and development of drug dependence and may contribute to the learning of addictive behaviors. Moreover, the development of drug dependence shares common cellular and molecular mechanisms with traditional learning models. Extracellular signal - regulated kinase ( ERK) , one member of the mitogen - activated protein ki-nases ( MAPK) family, transduces a broad range of extracellular stimuli into di-verse intracellular responses. It is now well established that ERK signaling pathway is a critical player in synaptic and neuronal plasticity. In the present study, we investigated whether and how activation of the spinal ERK pathway contributes to morphine withdrawal and withdrawal - induced spinal cord neurons sensi-tization.Materials and MethodsThe following procedures were taken to set up a chronic morphine dependence model. Rats were subcutaneously injected with morphine (bid!, for 5d). The dose for each injection was 10 mg/kg on d 1, and increased by 10 mg/kg each day. On d 6, 4 h after injection of morphine 50 mg/kg, morphine withdrawal syndrome was precipitated by administration of naloxone (4 mg/kg, i. p. ). Following injection of naloxone, signs of withdrawal were scored for a total of 60 min at 15 min intervals. Ten previously identified behavioral characteristics of the rat opiate abstinence syndrome were assessed. The absolute frequency of five episodic behaviors was recorded and scored based on multiples of five incidents (0 = no incidents;1 = 1 ~ 5 incidents;2=6-10 incidents;and 3 = > 10 incidents). Behaviors scored in this manner included: jumping, teeth chatter, writhing, wet -dog shakes, and rearing. Five withdrawal behaviors could not be defined in discrete episodes ( ptosis, lacrimation, piloerectioni, irritability, and diarrhea) , and the severity of these behaviors was assessed Using a four -point scale;0 = absent;1 = mild;2 = moderate;3 = severe. The scores for each time period were then added together. To examine and quantify the response of rats to nonnoxious tactile stimulation [ a probe drawn lightly across the flank - touched - evoked agitation ( TE ) ] (allodynia ) , a three - point rating scale was employed: 0 = no change/no effect of stroking;1 = mild squeaking, occasional scratching or biting at the flank;2 = vigorous squeaking, gnawing at flank leading to depilation, vigorous squeaking evoked by stroking probe, bites and chews on the probe. The presence or absence of TE was assessed in each animal during each 5 min intervals, for the 60 min period after injection of naloxone. To observe the role of spinal intraceDular signaling pathway in morphinewithdrawal, some chemicals were intrathecally injected 30 min prior to injection of naloxone.Immunohistochemical technique and Western blotting assay were used to detect the expression of spinal ERK, pERK, CREB, pCREB, Fos, iNOS and nNOS.ResultsFirstly, we study the time course of the spinal pERK expression in morphine dependent and withdrawal rats. We found that both cytosolic and nuclear pERK, not total ERK, were increased in the spinal cord of morphine dependence and withdrawal rats. The expression of pERK, especially in the nuclear fraction, reached at its peak level 30 min after morphine withdrawal. One hour after morphine withdrawal, the overexpression of pERK decreased ( but it was still higher than that in the control group) . The time course of changes of pERK expression, at least in part, correlated significantly with morphine withdrawal symptoms. Next, we found that intrathecal (i. t. ) injection of U0126, a MEK inhibitor or ERK antisense ODN, not DMSO or missense ODN, significantly attenuated naloxone - precipitated withdrawal symptoms and withdrawal - induced allodynia. The immunohistochemical studies indicated that morphine dependence and withdrawal increased the expression of pERK, which mainly distributed in the superficial laminae of the spinal cord. The increased expression of pERK was inhibited by i. t. U0126 or ERK antisense ODN. Western blqtfting assay further revealed that i. t. U0126 or ERK antisense - ODN, not DMSO or mis-sense ODN, reduced the cytosolic and nuclear pERK expression in the spinal cord of morphine withdrawal rats.Fos protein, the product of c -fos immediate early gene (IEG) , has been used as a maker for neuronal activation in the central nervous systeta ( CNS). Unlike the expression of pCREB, few Fos positive neurons expressed in the spinal cord of morphine dependence rats. Morphine withdrawal markedly increased Fos expression, which distributed in all the laminae of the spinal cord. These results suggested that the latent sensitization of the spinal cord neurons had de-veloped in morphine dependence rats, and naloxone - precipitated withdrawal could unmask the latent sensitization and therefore lead to hypersensitization in the spinal cord neurons. Intrathecal injection of U0126 or ERK antisense ODN also significantly reduced the Fos expression in the spinal cord in morphine withdrawal rats. In accordance with of the immunohistochemical results, western blot studies further showed that the nuclear Fos highly expressed in the spinal cord of morphine withdrawal group rats. Intrathecal injection of UO12J6 or ERK antisense ODN significantly inhibited the increased expression of the nuclear Fos.It is well established that nuclear translocation of ERK is essential for its action on gene induction and transcription. The transcription factor CJREB is an important effector of ERK. Several lines of evidence, in vitro and in Vivo, have indicated that ERK activation and nuclear translocation contributed to CREB phosphorylation and CREB - dependent gene expression is required for long -term changes in synaptic plasticity induced by chronic morphine administration in supraspinal level. In this study, morphine dependence and withdrawal significantly increased the spinal pCREB expression. Intrathecal injection of U0126 or ERK antisense ODN markedly inhibited pCREB expression in the spinal cord of morphine withdrawal rats. These results suggest that activation of ERK[ may contribute to the increase of pCREB expression in the spinal cord in morphine withdrawal and the function of pERK is partly accomplished via the CREB - dependent gene expression.Many studies have showed that the activation of NMD A receptor and intra-cellular PKC was involved in the development of morphine dependence and withdrawal. Furthermore, NMDA receptor and PKC are known to activate the ERK pathway in the CNS. The present study was to assess the roles of NM)DA receptor and PKC on morphine withdrawal - induced the activation of the spinal ERK pathway. Intrathecally injection of NMDA receptor antagonist MK801 or PKC inhibitor CHE attenuated naloxone - precipitated withdrawal response and withdrawal - induced allodynia. Correlated with behavior results, western blot results showed that intrathecal injection of MK801 or CHE markedly inhibited the increase of cytosolic and nuclear pERK, not total ERK, in the spinal cord ofmorphine withdrawal rats.Nitric oxide ( NO) is an important intra - and inter - cellular messenger and plays a crucial role in a number of physiological and pathological conditions within the nervous system. A growing body of evidence suggests that the spinal NO participates in the development of morphine tolerance, dependence and withdrawal. In vivo and in vitro studies in various cell types have shown that there is a cross talk between NO and MAPK signaling pathway. Herein, we examine the hypothesis that the cross - talk between NO and ERK pathway in the spinal cord level may mediate morphine withdrawal and withdrawal — induced spinal neuronal sensitization in morphine - dependent rats. The present study has shown the following findings (1) i. t. pretreatment with various NOS inhibitors reduced morphine withdrawal - induced spinal ERK activation;( 2 ) spinal ERK inhibition by U0126 also reduced the increased expression of spinal nNOS and iNOS induced by morphine withdrawal;and (3 ) Inhibitory expression of both pERK and NOS in the spinal cord was accompanied by suppressed naloxone - precipitation withdrawal and decreased spinal neuronal sensitization.Conclusions1. Activation of spinal ERK signaling pathway implicates in the development of morphine dependence and withdrawal.2. NMD A receptor and PKC involve the activation of spinal ERK signaling pathway induced by morphine dependence and withdrawal, and the function of pERK is partly accomplished via the CREB - dependent gene expression.3. Cross talk between NO and ERK in the spinal cord may mediate morphine withdrawal and withdrawal - induced spinal neuronal sensitization.4. A model for the role of the spinal ERK signaling pathway in morphine dependence and withdrawal:Chronic morphine treatment induces release of Get and G^7 - subunit from Gi/o protein, which in turn activates ERK pathway in a ras - dependent manner. Up - regulation of the cAMP/PKA pathway also facilitates MEK1/2, and then activates ERK pathway. Activated ERK pathway increases the expression ofmorphine dependence - related proteins including ion channels or receptors and intracellular signal molecular (for example, NOS, PKC et al) , which result in spinal neuronal latent sensitization. Postsynaptic NMDA receptor is activated by increased release of neuronal transmitters during morphine withdrawal. The Ca+ + influx is initiated by activation of NMDA receptor and triggers a large increase in intracellular Ca+ + concentration ( [ Ca+ + ] i). This [Ca++]i elevation in turn activates ERK pathway. Morphine withdrawal - induced overshoot of cAMP/PKA system also activates ERK pathway. Ca+ + combines with CaM and forms Ca - CaM complex, which in turn activates NOS and results in NO production. NO activates ERK through cGMP/PKG - dependent and - independent mechanisms. On the other hand, NO diffuses into presynapse and increases the neuronal transmitters release, which further regulates and maintains NMDA receptor function. Activated ERK further increases the expression of morphine dependence - related proteins and also translocates onto membrane an