The Roles Of ERK1/2 And CRF System In Cocaine Addiction

BackgroundCocaine is one of commonly abused drugs due to its easy-getting and strongly addictive effects. Last several decades, scientists did a lot of work to investigate the mechanisms of cocaine addiction. Exposure to cocaine causes plasticity in neural circuits related to reward and motivation, supporting the idea that addiction is a physiological and psychological disorder.Till now, most studies focus on the role of dopaminergic system in cocaine addiction. Mesolimbic pathway (originating from Ventral Tegmental Area to limbic area), mesocortical pathway and substantia nigra-striatum pathway have been regarded as key neural targets for mediating neuroadaptation induced by cocaine abuse. Notably, striatum is the essential bridge among the three pathways. By binding to the dopamine transporter (DAT), cocaine blocks the reuptake of released dopamine (DA), causing a long-lasting rise of synaptic DA concentrations. Accordingly, DA receptors can be activated for a long time during and after cocaine administration. The molecular mechanism of the neuroadaptations in neural circuits caused by cocaine abuse remains largely unknown. Recently, the roles of extracellular signal-regulated protein kinase (ERK), expecially ERK1/2, in cocaine addiction attract much attention. ERK1/2 is one of important kinases that is widely distributed in the brain, and plays an important role in cell migration and matures, and is involved in mediating the physiological changes in biological disorder. Exposure to cocaine has been reported to induce ERK1/2 activation in the dopaminergic system. Importantly, blockade of ERK1/2 pathway has been reported to attenuate abnormal behaviors associated with cocaine treatment, including hyperlocomotor activity and conditioned place preference (CPP), suggesting that ERK1/2 pathways may play an important role in incubation of cocaine addiction. Notably, most previous studies focused on the role of ERK1/2 in long-term changes induced by chronic drug exposure in the brain. ERK1/2 was significantly activated only at the early time point after acute cocaine treatment, and it returned to normal level after repeated cocaine administration, indicating that early ERK1/2 activation may be critical for the subsequent long-term changes after cocaine exposure. However, how ERK activation leads to long-term changes remains unclear. Thus, it is essential to identify the mechanisms of ERK1/2 activation after acute cocaine treatment. It has been reported that acute cocaine treatment upregulates the level of p-ERK1/2 in ventral straitum (nucleus accumbence, NAc). However, NAc is divided into shell and core. The core and shell regions might mediate distinct processes associated with cocaine-induced behavioral abnormalities, thus it is important to investigate the function of cocaine-induced p-ERK1/2 in the core and shell of NAc, respectively. In addition, the dorsal striatum attracts more and more attention about its roles in cocaine addiction. However, the studies about the function and mechanisms of ERK1/2 in dorsal striatum need systematic investigation.Similar to other abused drugs, main characteristics of cocaine addiction are compulsive drug use despite adverse consequences and high rates of relapse during periods of abstinence. Environment changes or emotional problem easily lead to cocaine relapse after long term withdrawal, indicating that a pathological process of learning and memory is involved in formation of cocaine addiction. Long term potentiation (LTP) in hippocampus has been an important and useful cellular model of synaptic plasticity and is considered as one of the cellular substrates of learning and memory. Previous studies showed that LTP in hippocampus is significantly enhanced after cocaine self-administration, and this enhancement even persists after extinction of cocaine administration. Cocaine withdrawal is reported to impair the habit learning and memory process by attenuating the depotentiation in corticostriatal synapses. However, the mechanisms by which cocaine abuse leads to the electrophysiological changes in hippocampus and corticostriatal synapses remain unclear. Stress is a well-known risk factor for drug abuse and relapse. On the other hand, stress can be a result of cocaine abuse, especially during cocaine withdrawal. More recent efforts have begun to identify the relationships between neural activity during stress and drug relapse outcomes. Corticotrophin-releasing factor (CRF)-related peptides are primary regulator of the stress response. Till now several CRF-related peptides have been identified: CRF, urocortin 1 (UCN1), UCN2 and UCN3. These CRF-related peptides actions are mediated through two different G-protein-coupled receptors, CRF₁ and CRF₂. Last decades, abundant evidences showed that the CRF related peptides and their receptors in HPA play an important role in cocaine addiction. However, recent studies find that CRF related peptides and their receptors are widely distributed outside the thalamus, and this extra-thalumal CRF system carry out important regulating roles under both physiological and pathological conditions. Till now, no data have been reported about the roles of CRF related peptides and their receptors in cocaine-induced changes of learning and memory.Aim1. The first aim of this study is to investigate the expression, time course and location of p-ERK1/2 in dorsal striatum after acute cocaine treatment and identify the effects of ERK1/2 on cocaine-induced hyperlocomotor behavior. Furthermore, it is to investigate the upstream and downstream of cocaine-induced p-ERK1/2.2. The second aim of this study is to determine the respective effect of ERK1/2 in the shell and core of NAc on cocaine-induced changes in c-Fos expression.3. The third aim of this study is to investigate the changes of the LTP in hippocampal CA1 slices from chronic cocaine treatment rats and cocaine withdrawal rats. Furthermore, it is to investigate the roles of CRF related peptides and their receptors in the process.4. The last aim of this study is to investigate the changes of LTP in corticostriatal slices from acute cocaine treatment rats, chronic treatment rats and cocaine withdrawal rats. Furthermore, it is to identify the roles of CRF related peptides and their receptors in the process.Methods1. Setting up different cocaine treatment models, including acute cocaine treatment (one single injection), chronic cocaine treatment (consective 7d or 14d), short term cocaine withdrawal treatment (3d withdrawal after chronic cocaine treatment) and long term cocaine withdrawal treatment (14d withdrawal after chronic cocaine treatment) models.2. Immunostaining: to observe the distribution and location of p-ERK1/2 in striatum.3. Western blots: to observe the expression of p-ERK1/2, t-ERK1/2, c-Fos, p-CREB, p-Elk-1.4. RT-PCR: to observe the level of c-fos mRNA in ventral striatum (nucleus accubence, NAc)5. Whole-cell patch clamp: to observe the resting membrane potaentials (RMP) and input resistance of pyramid neuron in hippocampus and medium spiny neurons (MSNs) in striatum. 6. Field EPSP recordings: to observe the LTP of hippocampal CA1 slices and corticostriatal slices.Results1. Acute cocaine injection results in up-regulation of p-ERK1/2 in dorsal striatum, but has no effects on t-ERK1/2.2. The level of acute cocaine-induced p-ERK1/2 is time-dependent; the level of p-ERK1/2 is increased at 5 min, and reaches to a peak at 10 min, then returns to basal level at 20 min after acute cocaine injection.3. The p-ERK1/2 positive staining is located in the cytosotic part of medium spiny neurons (MSNs) at 5 min after acute cocaine injection, and is mainly detected in the nucleus of MSNs at 10 min after cocaine treatment.4. Specific blockade of ERK1/2 activation partially attenuates acute cocaine-induced hyperlocomotor behavior.5. Acute cocaine-induced p-ERK1/2 is mainly located in dopamine D1 receptor (D1R)-positive MSNs, and little is collocated with D2R in dorsal striatum.6. Specific blockade of D1R significantly inhibits acute cocaine-induced p-ERK1/2 in dorsal striatum, and the similar results occur in the presence of specific antagonist for D2R.7. Specific blockade of ERK1/2 activation significantly attenuates acute cocaine-enhanced c-Fos, p-CREB, and p-Elk-1 in dorsal striatum.8. Acute cocaine injection increases the level of c-Fos protein and c-fos mRNA in nucleas accubence (NAc); specific blockade of ERK1/2 activation significantly attenuates acute cocaine-induced c-fos mRNA in NAc, and partially reduces cocaine-induced c-Fos protein in the shell, but completely reduces cocaine-induced c-Fos protein in the core of NAc. 9. There is no effect of chronic cocaine treatment and cocaine withdrawal on the basic properties of hippocampal CA1 pyramid neurons (RMP and input resistance).10. The magnitude of LTP in hippocampal CA1 is significantly enhanced by cocaine withdrawal, but not affected by chronic cocaine treatment.11. Specific blockade of CRF₁ in vitro significantly attenuates the magnitude of LTP in hippocampal slices from controls and cocaine withdrawals; In the presence of CRF₁ antagonist, the magnitude of LTP in hippocampal slices from cocaine withdrawal groups is still greater than that from controls.12. Specific blockade of CRF₂ in vitro significantly attenuates the magnitude of LTP in hippocampal slices from cocaine withdrawals.13. The basic properties of MSNs in dorsal striatum (RMP and input resistance) are not affected by acute or chronic cocaine treatment or cocaine withdrawal.14. The magnitude of LTP in corticostriatal slices is not affected by acute or chronic cocaine treatment or cocaine withdrawal.15. Application of CRF (20-80 nM) in vitro significantly increases the magnitude of LTP in corticostriatal slices from controls and cocaine withdrawal.16. Specific blockade of CRF₁ in vitro significantly attenuates CRF-enhanced magnitude of LTP in corticostriatal slices from controls and cocaine withdrawal groups.17. Specific blockade CRF₂ in vitro significantly attenuates CRF-enhanced magnitude of LTP in corticostriatal slices from cocaine withdrawal groups.18. Application of UCN2 in vitro significantly increases the magnitude of LTP in corticostriatal slices from cocaine withdrawal groups.19. Specific blockade of CRF₂ significantly attenuates UCN2-enhanced magnitude of LTP in corticostriatal slices from cocaine withdrawal groups. Conclusions1. Acute cocaine-induced p-ERK1/2 in dorsal striatum is a major mediating factor for cocaine abuse-caused neuroadaptations, also contributes to acute cocaine-induced abnormal behavior. In dorsal striatum, D1R is one main regulator to mediate p-ERK1/2 activation induced by acute cocaine treatment. In addition, the increased p-ERK1/2 carrys out its subsequent mediating roles via changing the expression of transcripts, including c-Fos, CREB and Elk-1.2. ERK1/2 plays an important mediating role in acute cocaine-caused hyperactivity in ventral striatum (NAc). ERK1/2 carrys out its mediating function mainly in the core part of NAc.3. Cocaine withdrawal enhances the magnitude of LTP in hippocampal slice. CRF₁ and CRF₂ both are involved in the process. CRF₁ contributes to the formation of LTP in hippocampal slices both under physiological and pathological conditions. Cocaine withdrawal partially induces the changes of hippocampal synaptic plasticity via changing the CRF₂ activity, which leads to the change of learning and memory.4. CRF₂ is the main receptor type involved in the CRF and UCN2-induced enhancement of the magnitude of LTP in corticostriatal slices in cocaine withdrawal.