安非他命對於中樞神經系統的作用，大都來自對於諸多神經傳導物質受體系統的影響，經由在貯存處游離釋放、阻斷回收、抑制代謝的方式，增加神經傳導物質的作用強度和時間，造成中樞神經的興奮、神經毒性和行為的變化。而目前已有許多研究證實安非他命對於norepinephrine 受體系統的作用，會導致中樞神經興奮；而對於dopamine 受體系統的作用則是產生快感和幻覺，並強化回饋成癮系統 (reward system) ；至於對於serotonin 的回收抑制則先是造成分泌增加，進而因回饋機制導致接受器的敏感度降低，造成serotonin急速消耗，這時候使用者會出現憂鬱、焦慮、注意力不集中、記憶力障礙和失眠問題，而長期使用則會導致腦功能受損和神經毒性。 CART的全名為Cocaine and Amphetamine-Regulated Transcript (古柯鹼和安非他命調控之轉錄因子)。目前研究顯示CART peptide與Amphetamine或古柯鹼等興奮性濫用藥物之作用有關，也有許多的文獻指出CART 55-102是調控著厭食反應最重要的區域。這結果顯示CART可能是一種內生性的神經傳遞物質，可能會影響許多與厭食反應有關的訊息傳遞，但其詳細機制及其相關的訊息傳遞路徑卻仍不清楚。雖然安非他命對中樞神經的影響已有許多相關的研究，但對於下視丘及其他腦區的影響及對於CART的影響則仍不清楚，因此本計畫擬利用大白鼠以「重複注射Amphetamine」的處理方式，每天一次，連續數天。在重複注射期間，每天記錄大白鼠的食物攝取量，完成由Amphetamine誘發之毒性及厭食的影響。並利用同樣的模式在重複注射的不同時間犧牲大白鼠，取腦內的下視丘及其他腦區，萃取其蛋白及mRNA，以western blotting及RT-PCR的分析方式，完成不同時間Amphetamine對下視丘及其他腦區中CART、SOD 、c-Jun及c-fos的基因變化及影響。我們利用不同濃度(2mg/kg及4 mg/kg)的Amphetamine每天注射一次，注射四天。於Amphetamine注射開始的第一天大白鼠便出現厭食的情況，且於第二到四天有慢慢恢復的情況。而此厭食情形亦具有濃度的相關效應。而以RT-PCR的分析方式，可發現在初期CART、SOD 、c-Jun及c-fos有明顯的上升，於第2-4天緩慢的下降。而我們設計CART的antisense oligodeoxynucleotide，以活體(In Vivo)的動物為材料，在Amphetamine注射前，將CART的antisense直接注入下視丘PVN部位，由攝食行為的改變及CART基因的表現，觀察改變CART的antisense對Amphetamine之影響。我們發現CART antisense的處理有回覆此厭食情形。因此我們發現，CART基因在由Amphetamine誘發之厭食反應中扮演一個很重要的角色。
Amphetamine (AMPH) and associated derivatives are the most commonly abused psychostimulant in the young people. AMPH is known for its suppressive effect on appetite. A daily treatment of AMPH for several days could induce a marked decrease of food intake on the first day of dosing and then the food intake returned gradually to the normal level (tolerant effect) on the followings. The mechanism for the anorectic action of AMPH was through the inhibitory action on hypothalamic neuropeptide Y, an orexigenic neurotransmitter in the brain. Amphetamine is an artificially synthesized psychostimulant. It could activate both central and autonomic nervous system. As we know, amphetamine acts on mesolimbic circuit or associated brain regions and influences on associated emotional circuit. Therefore, to investigate the modulation of amphetamine on emotional circuit is very important for current study. Cocaine and Amphetamine Regulated Transcript (CART), brilliantly identified by Douglass and co-workers is an anorectic peptide regulated by leptin. Fasting animals show a pronounced decrease in CART mRNA expression in the arcuate nucleus, an area of the brain known to be associated with feeding behavior. In animal models with disrupted leptin signalling, CART mRNA is almost absent in the arcuate nucleus. ICV administration of CART inhibits both normal and starvation-induced feeding and completely blocks the feeding response induced by NPY. Antisera directed against CART increases feeding in normal rats. Indications are that CART may be an endogenous inhibitor of food intake Therefore, to determine if CART was involved in the anorectic response, the intracerebroventricular infusions of antisense oligonucleotide (or missense control) were performed at 60 min before daily Amphetamine treatment in conscious rats, and results showed that CART knock down could block AMPH-induced anorexia. Furthermore, we found that c-fos or c-jun was also involved in the anorectic response of Amphetamine. It was suggested that c-fos/c-jun SOD-1/SOD-2 signaling might involve in the central regulation of AMPH-mediated feeding suppression via the modulation of CART gene expression.
