Study Of The Molecular Mechanism On The Actin Rearrangement In Different Brain Regions Of Morphine Withdrawal And Relapse Rats

Opiate addiction is a complex relapsing brain disease process that is characterized by the compulsive seeking and taking of an opiate, despite adverse consequences, and the emergence of a negative emotional state when access to the opiate is denied. Addicted subjects are liable to return to compulsive drug-taking long after experiencing acute withdrawal symptoms. The aberrant reorganization of reward and memory circuits, brought about by chronic drug abuse, is hypothesized to be crucial to the mounting of these responses. However, it is clear that more work is necessary to fully understand the involvement of synaptic and structural plasticity in addictive behaviors. This experiment is beginning from researching the skeleton actin, combining methods such as ethology and molecular cell biology, to study the reorganization of skeleton action during morphine withdrawal and relapse of rats. This work deepens the knowledge of understanding the molecular mechanism of opioid addition.Part 1 Study of the molecular mechanism on the actin rearrangement in different brain regions of morphine withdrawal and induced again ratsObjective: To study of the actin rearrangement in different brain regions of morphine withdrawal and induced again ratsMethods: 1 The establishment of models of morphine dependence and withdrawal: Rats were randomly divided into 6 groups with 8 in each. N2, N4, N5 and N6 groups were established with morphine hydrochloride for 7 days. By the way of increasing doses of morphine hydrochloride two times per day through intraperitoneal injection. N1 and N3 groups were injected saline in intraperitoneal. All groups were stopped injection at d8-d20. Day21, N6 andN5 groups were injected in ventricle by 1μL saline and 0.5μg/μL Latrunculin A, and injected morphine with 10mg/kg after 10 min. N3 and N4 groups were injected morphine with 10mg/kg-1. N1 group were injected saline. 1.1 Symptom score : The changes of behaviors in 20 minutes were daily observed at day8-21, including: wet dog shaking, standing, stretching, cleaning fur, swallowing, tooth fibrillation, cunnilingus and diarrhea. All groups were observed at day21. All groups of animals were decapitated and separated related brain areas after fully tested. The related brain areas included Hip, Pfc, Str and NAc. 2 Determination of protein levels in rat brain regions by Western-blot: Brain tissues were homogenized, then all the related proteins were detected. F-actin in cystoskeleton fraction was separated from G-actin in endochylema with different buffer, then detect the changes of expression of actin(G-actin and F-actin), actin binding protein(cofilin, drebrin A/E, cortactin, WAVE-2 、N-WASP、Pan-Calcinerurin A、Profilin-1、ARP2、ARP3、SSH1and β-actin) by western blot.Results: 1 Morphological changes of different brain regions under the electron microscope : The control group was observed that the presynaptic mitochondrion, a large number of synaptic vesicles, evenly thickened postsynaptic membrane compared with the presynaptic membrane in Hip, Pfc and NAc. It was found that the membrane of postsynaptic was thickening in Hip, Pfc and NAc in morphine dependence group. The membrane of postsynaptic in morphine withdrawal group was still thickening. 2 Symptom score : The Symptom score of morphine withdrawal 14 days was no significant changes.The Symptom score of rats injected morphine again was up-regulated by 45%(P<0.001). Latrunculin A group was no significant changes. 3 Determination of protein levels in rat brain regions by Western-blot: 3.1 W group compared with NS/NS group: In Hip, cortactin was up-regulated by 66%(P<0.01); ARP2 was up-regulated by 35%(P<0.05). In Pfc, profilin-1was down-regulated by 27%(P<0.01). In Str, N-WASP was down-regulated by 20%(P<0.05). In NAc, P-cofilin/cofilin ratio was down-regulated by 20%(P<0.01); WAVE-2 was down-regulated by 45%(P<0.01). 3.2 M/M group compared with W group: In Hip, F-actin/G-actin ratio was down-regulated by 13%(P<0.05); drebrin A/E binding rate on F-actin was down-regulated by 12%(P<0.05); cortactin binding rate on F-actin was up-regulated by 30%(P<0.05); SSH1 was up-regulated by 40%(P<0.05); P-cofilin/cofilin ratio was down-regulated by 60%(P<0.05); N-WASP was up-regulated by 42%(P<0.05); calcinurin A was up-regulated by 13%(P<0.05). In Pfc, drenbrin A/E was up-regulated by 58%(P<0.05); P-cofilin/cofilin ratio was down-regulated by 13%(P<0.05); calcinurin A was up-regulated by 50%(P<0.05). In Str, drenbrin A/E was up-regulated by 45%(P<0.05). In NAc, calcinurin A was up-regulated by 13%(P<0.05). 3.3 M/Lat-A/M group compared with M/M group: In Hip, drebrin A/E binding rate on F-actin was down-regulated by 20%(P<0.05); SSH1 was down-regulated by 64%(P<0.01); ARP2 was down-regulated by 45%(P<0.05); P-cofilin/cofilin ratio was up-regulated by 60%(P<0.05); WAVE-2 was down-regulated by 21%(P<0.01). In Pfc, drenbrin A/E was down-regulated by 89%(P<0.01); N-WASP was up-regulated by 45%(P<0.05); WAVE-2 was down-regulated by 21%(P<0.01). In Str, drenbrin A/E was down-regulated by 56%(P<0.01); cortactin was up-regulated by 28%(P<0.05); N-WASP was up-regulated by 39%(P<0.05). In NAc, cortactin was up-regulated by 84%(P<0.05); N-WASP was up-regulated by 38%(P<0.001); WAVE-2 was up-regulated by 29%(P<0.01); calcinurin A was up-regulated by 20%(P<0.05).Conclusions: Chronic morphine treatment can induce ultrastructural synaptic changes in reward brain regions of rats. The changes can exist even after symptoms of withdrawal disappearance. Morphine induced again can make behavioral changes, actin remodeling and show specific brain regions.Reconstruction of actin may be related to nucleation and depolymerization processes in Hip.Part 2 Study of the molecular mechanism on the actin rearrangement in Hip of morphine induced CPP ratsObjective: To study of the actin rearrangement in Hip of morphine induced CPP ratsMethods: 1 The establishment of CPP models: Rats were randomly divided into 4 groups with 8 in each. Including : N1:NS/NS group,N2:M/NS/M group, N3:M/Lat-A/M group, N4:M/FK-506/M group. Preconditioning: all groups of animals were recorded in each compartment(black and white) at d1-d3. Conditioning, 1 week, N2, N3 and N4 groups were injected morphine with 10mg/kg, and then confined for 40 min to the assigned compartment every morning; injected saline every afternoon to another one. N1 group was injected saline all day, processing method as above. After a week, all groups were tested as the preconditioning. Withdrawal period: all groups were tested every 5 days. The period of morphine acute treatment again: N2, N3 and N4 groups were respectively injected in CA1 by 1μL solvent 、 0.5μg/μL Latrunculin A and 5.0μg/μL FK-506,and injected morphine with 10mg/kg after 30 min. Then all groups were tested after 10 min. All groups of animals were decapitated and separated related brain areas after fully tested. The related brain areas included Hip and NAc. 2 Determination of protein levels in rat brain regions by Western-blot: Brain tissues were homogenized, then all the related proteins were detected. F-actin in cystoskeleton fraction was separated from G-actin in endochylema with different buffer, then detect the changes of expression of actin(G-actin and F-actin), actin binding protein(cofilin, drebrin A/E, cortactin, Glutamate Receptor 1, P-Glutamate Receptor 1 and β-actin) by western blot.Results: 1 The changes of CPP: Compared with the control group, the score of CPP in morphine dependence group was up-regulated by 400%(P<0.05). The score ofCPP in group of relapse was up-regulated by 1200%(P<0.01). The Latrunculin A group and FK-506 group were separately down-regulated by 1204%(P<0.001), 1260%(P<0.001). 2 The changes of protein levels in CPP models : 2.1 M/NS/M group compared with NS/NS group: in Hip, the ratio of F-actin/G-actin was down-regulated by 50%(P<0.05); the drebrin binding F-actin was down-regulated by 10%(P<0.05); the cortactin binding F-actin was down-regulated by 35%(P<0.05); the ratio of P-Glu R1/Glu R1 was up-regulated by 45%(P<0.05); WAVE-2 was up-regulated by 65%(P<0.05); Calcineurin A was up-regulated by 15%(P<0.05). In NAc, the ratio of P-Glu R1/Glu R1 was up-regulated by 20%(P<0.05). 2.2 M/Lat-A/M group compared with M/NS/M group: in Hip, the ratio of F-actin/G-actin was up-regulated by 47%(P<0.01); the cortactin binding F-actin was up-regulated by 35%(P<0.05); the ratio of P-Glu R1/Glu R1 was down-regulated by 65%(P<0.01). In NAc, the ratio of P-Glu R1/Glu R1 was down-regulated by 70%(P<0.01). 2.3 M/FK-506/M group compared with M/NS/M group: in Hip, in Hip, the ratio of F-actin/G-actin was up-regulated by 50%(P<0.05); the drebrin binding F-actin was up-regulated by 20%(P<0.05); the cortactin binding F-actin was up-regulated by 50%(P<0.05); the ratio of P-Glu R1/Glu R1 was down-regulated by 60%(P<0.05); Calcineurin A was down-regulated by 15%(P<0.05). In NAc, the ratio of P-Glu R1/Glu R1 was down-regulated by 50%(P<0.01).Conclusions: Actin remodeling is also observed in hippocampus of rats during reinstatement of CPP induced by morphine. The behavior of relapse can be inhibited by injecting Lantruculin A and FK506 in dorsal hippocampus. The mechanism may be related to actin remodeling in hippocampus and glutamatergic pathway from hippocampus to the nucleus accumbens.