Study On Mechanism Of Over-activation In Direct-pathway In Rats With Levodopa-induced Dyskinesias

PART IChanges of dopamine and CAMP-regulated Phosphoprotein phosphorylation expression in a rat with lev odopa-induceddyskinesiasObjective To study the changes of opamine and CAMP-regulated Phosphoprotein (DARPP-32) phosphorylation and to explore the mechanism of over-activation in direct-pathway by dopamine D1 receptor mediated in rats with levodopa-induced dyskinesias (LID).Methods PD rats were established by 6-hydroxydopamine (6-OHDA) microinjection stereotaxically. They received twice daily L-3,4-dihydroxyphenylalanine methyl ester (L-dopa) celiac (10 mg/kg) injections for 28 days to get the LID rats. All rats were divided into control group (who received same course and dosage of L-dopa, n=8) , L-dopa treatment group (n=8), LID group. Behavior changes were observed. Protein and mRNA levels of total DARPP-32 and phospho-Thr-34 DARPP-32 level were measured by immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively.Results Pulsatile treatment with L-dopa induced contralateral forelimb, trunk and orofacial abnormal involuntary movement (AIM) in PD rats, similar to LID in PD patients. The levels of phospho-Thr-34 DARPP-32 increased significantly in LID group compared to control group and L-dopa treatment group (P<0.01,respetly), and there were no differences of mRNA and protein levels of total DARPP-32 among all groups (P>0.05).Conclusion Phospho-Thr-34 DARPP-32 level was increased in LID rats, which contribute to the over-activation in direct-pathway.PART IIEffect of antisense FosB and CREB on the expression ofprodynorphin Gene in rats with levodopa induced dyskinesiasObjective To explore the effects of antisense, sense FosB and response element-binding protein (CREB) respectively intra-striatum injection on expression of prodynorphin (PDyn) gene in striatal neurons of LID rats with PD.Methods LID rats were established by 6-OHDA microinjection stereotaxically and chronic intermittent L-dopa celiac injection. All rats were divided into control group (n=10), control + antisense FosB group (n=12), control + sense FosB group (n=13), control + antisense CREB group (n=11), control + sense CREB group (n=13), LID group (n=10), LID + antisense FosB group (n=9), LID + sense FosB group (n=7), LID + antisense CREB (n=8) group and LID + sense CREB group (n=8). Antisense FosB and CREB, sense FosB and CREB were injected into striatum of all rats respectively. Hybridization in situ was used to measure the changes in expression of PDyn mRNA in striatum and the behavior changes were observed.Results After administration of antisense FosB, scores of AIM was decreased in LID rats (12.5 ± 5.4 vs 40.1± 9.2, P <0.01). As compared with LID + sense FosB group, expression of PDyn mRNA on lesioned side of striatum was decreased in LID + antisense FosB group (0.2415 ± 0.0220 vs 0.4105 ± 0.0386, P <0.01). As compared with control group, expression of PDyn mRNA on lesioned side of striatum was decreased in control + antisense CREB group (0.1775 ± 0.0246 vs 0.2403 ± 0.0323 P <0.01). After administration of antisense CREB, LID rats showed no changes in scores of AIM (43.2 ± 11.8 vs 40.5 ±10.0, P >0.05). And compared with sense CREB treated LID group, antisense CREB treated LID group also showed no changes in expression of PDyn mRNA (0.3930 ±0.0332 vs 0.4087 ± 0.0440, P >0.05).Conclusion FosB protein, which replaced the CREB, might regulate the expression of PDyn mRNA and should be a critical role in pathogenesis of LID.PART IIIEffect of MK-801 on the Expressions of Prodynorphin Gene and DARPP-32 Phosphorylation in Rats with Levodopa InducedDyskinesiasObjective To explore the effects of MK-801 injection on the expression of PDyn mRNA and phospho-Thr-34 DARPP-32 in striatal neurons of LID rats.Methods PD rats received twice daily levodopa celiac (10 mg/kg) injections for 28 days to get the LID rats. On day 29, 8 rats in LID group were given an acute intraperitoneal . injection of MK-801 ( 0.1mg/kg ) 15 min before levodopa treatment(MK801 group, n=8). The normal rats received same course and dosage of levodopa as a control group (n=8). Hybridization in situ was used to measure the expression of PDyn mRNA in striatum. Protein and mRNA levels of total DARPP-32 and phospho-Thr-34 DARPP-32 level were measured by immunoblotting and RT-PCR, respectively.Results Scores of AIM was decreased in MK-801 treatment group markedly compared to LID group (P<0.01) . The levels of PDyn mRNA and phospho-Thr-34 DARPP-32 both decreased significantly in MK-801 group compared to LID group(P<0.05, respectly), and there were no differences of them between MK-801 group and control group. There were no differences of mRNA and protein levels of total DARPP-32 among three groups. Conclusion MK-801 could desease levels of PDyn mRNA and phospho-Thr-34 DARPP-32 in striatal neurons of LID rats. So MK-801 could reduce over-activation in direct-pathway by dopamine D₁ receptor mediated.