Chronic nicotine induces the changes of locomotor activities in rats by increasing the mRNA levels of dopamine D1 receptor in striatum / 中国组织工程研究

ABSTRACT

BACKGROUND: Nicotine, which is a known central nervous system stimulant, appears to be the neuroprotective factor of Parkinson disease(PD). It has been reported that PD patients' symptoms such as trembling,rigor, hypokinesia are ameliorated during smoking, but its mechanism still keeps unclear.OBJECTIVE: To observe the effects of nicotine on gene expression levels of dopamine D1 and D2 receptors (D1R,D2R)in striatum of rats and analyze the possible mechanism of behavioral changes of rats induced by nicotine.DESIGN:Randomized and controlled experiment.SETTING:Institute of Neurology, Xiangya Hospital, Central South University.MATERIALSTwenty-four SD rats aged at 10 weeks were chosen,weighing 180-200 g. Nicotine (Sigma),revert AidTM M-Mulv reverse transcriptase (MBI Fermentas,USA), polymerase chain reaction (RCR,Beckman),densitometric scanning imaging system (Stratagene Eagle Eye Ⅱ ,USA).METHODS:This experiment was carried out in the Laboratory of Institute of Neurology, Xiangya Hospital,Central South University from July 2001 to July 2002. These rats were divided into two groups control group (n=12)and nicotine group(n=12). The level of D1 and D2 receptors on striatum of rats was estimated at the timepoint of thirty-minute after chronic nicotine administration (4 mg/kg per day s.c.), and the behavioral activities were also recorded at the same timepoint for thirty minutes. The functional behavioral activities recorded included rearing up repeatedly, moving about, provoking, climbing, grooming, yawning, rotating, smelling and vomiting. At the fourteenth day, all rats were killed after thirty minutes of nicotine injection,the brains were dissected out and the region of striatum was separated immediately. Total RNA was extracted from striatum by RNeasy Total RNA Kit. PCR amplification was performed at special condition. For semi-quantitative analysis, 10 μ L of PCR products for each was examined by electrophoresis on 12 g/L agarose gel containing 0.5 mg/L ethidium bromide,and absorbance (A value) was quantitated by using densitometric scanning imaging system, thuse D1R,D2R mRNA expression were determined. Differences between means were analyzed with two-tailed student's t test.MAIN OUTCOME MEASURFS Changes of locomotor activities and the gene mRNA expression levels of D1 R and D2R in the regions of striatum in rats.RESULTS: Totally 24 SD rats were involved in the final results.① Locomotor activities of rats become more active after 3-day nicotine administration and reach the top during 7-14 days.②The A value of total RNA ratio of A260/A280 ＞1.8, and the total RNA had no degradation with 12 g/L agarose gels electrophoresis. ③As expected, PCR amplification product lengths of D1R, D2R,βA were 350 bp, 399 bp, 218 bp respectively. A significant increase of 23% of D1R mRNA expression in the region of striatum detected in the nicotine group compared with that of control group (98.63±1.13 and 65.29±1.45 seperately,P ＜ 0.01), no difference was detected on the level of D2R mRNA expression in the same regions above (76.73±1.45 and 78.21±1.69 respectively ,P ＞ 0.05 ).CONCLUSION: Nicotine may induce changes of locomotor activities of rats by up-regulating D1R mRNA expression in striatum.