長期給予嗎啡或安非他命對大白鼠或幼鼠腦部興奮性的表現與NMDA受體所產生的影響

The effect of chronic treatment of morphine or amphetamine on the expression of excitability and N-methyl-D-Aspartate (NMDA) receptor subunits in rat brain

莊玲菁(Ling-Jing Zhuang)

指導教授 ： 葉健全

臺北醫學大學/醫學院/醫學科學研究所/碩士(1998年)

摘要

第一部份 長期給予母鼠嗎啡對於其幼鼠腦部NMDA receptor 各亞型表現的影響 中文摘要 嗎啡成癮的母親所生的小孩常會有著長期神經、精神異常的現象，然而引發 的機制尚未明瞭。在過去本實驗室已經證實，長期投與嗎啡的母鼠所生的幼鼠腦中，N-me thyl-D-aspartate receptor (NMDA receptor,一種興奮性胺基酸的接受體之一)的密度在 出生後第十四天比起控制組有明顯的下降。由此顯示，長期嗎啡成癮的幼鼠腦部神經細胞 內的NMDA receptor會有減量調控(down-regulation)的現象。過去有實驗證實，NMDA rec eptor是由數個亞型所組合而成，包括NR1A, NR2A, NA2B, NR2C, NR2D。因此，長期給予 嗎啡可能與腦部NMDA receptor各亞型產生減量調控的變化有關。本研究於是利用西方墨 點法來定量幼鼠腦中NMDA receptor各亞型的變化，以求更進一步瞭解幼鼠腦中NMDA rece ptor表現的亞型專一性。我們採取遵循過去本實驗室的嗎啡給藥模式，並在幼鼠出生後的 第七天、十四天、二十八天、六十天將其予以斷頭犧牲，取腦分為大腦皮質、海馬迴等各 部位分別製備成非溶解性蛋白質溶液，利用NR1A, NR2A, NA2B之專一性抗體來量化各亞型 ，並以neuron-specific enolase (NSE)來偵測神經細胞量的多寡以作為內部標準控制。 本研究結果發現，在嗎啡組的大腦皮質中，NR1A的表現量在出生後第十四天大約少於對照 組40%，NR2A的表現量則是在出生後第七天大約比對照組增加2.8倍，而NR2B的表現量與對 照組間無明顯差異。在嗎啡組的海馬迴中，NR1A及NR2B的表現量與對照組間並無明顯差異 ，但是NR2A的表現量則是在出生後第十四天大約少於對照組34 %。此結果與本實驗室過去 利用受體結合試驗觀察NMDA receptor的變化結果相吻合；並且更進一步顯示長期曝露在 嗎啡下的幼鼠，其腦中NMDA receptor各亞型表現的減量調控，在大腦皮質中為NR1A，在 海馬迴中為NR2A。第二部份 長期給予安非他命對於kainic acid誘發大白鼠癲癇發作 行為變化及腦部NMDA receptor各亞型表現的影響中文摘要 安非他命，一種神經 興奮劑，是成癮與濫用的主要藥物之一，對於社會及公共衛生形成重要的衝擊。長期重覆 使用安非他命者，會產生非常類似妄想性精神分裂的精神病變，此種安非他命引起之精神 病經常在戒除藥物後即消失殆盡，然而有一部分安非他命成癮者，即使停藥了幾年後，當 再次投與低劑量的安非他命時，會出現劇烈反應，包括情緒不穩定、精神症或癲癇等的症 狀。引發對安非他命此種高度敏感化的機制目前仍未清楚，但大腦興奮性的改變可能是其 中一項因素。此外在過去本實驗室已經證實，重覆投與安非他命的大白鼠大腦皮質中，N- methyl-D-aspartate receptor (NMDA receptor,一種興奮性胺基酸的接受體之一) 的密 度在安非他命停藥後的第十四及二十八天，比起控制組有明顯的增高。如此顯示，重覆投 與安非他命的大白鼠腦部神經細胞的NMDA receptor會有增量調控(up-regulation)的現象 。所以我們推測，長期重覆對大白鼠給予安非他命可能會影響腦部NMDA receptor的表現 。因此本研究一方面利用kainic acid引發癲癇的行為變化，來探討重覆給與安非他命後 ，對於大腦興奮性是否有改變的現象。另一方面利用西方墨點法來定量大白鼠腦中的NMDA receptor各亞型的變化，以求更進一步瞭解大白鼠腦中NMDA receptor表現的亞型專一性 。我們採用Sprague-Dawley大白鼠每日給與一次腹腔注射安非他命(5mg/kg) 連續14天， 然後分別在安非他命停藥後的第一、十四、二十八天皮下注射一次劑量的kainic acid(12 mg/kg)，並且觀察3小時內kainic acid誘發抽搐的程度及其引發抽搐的潛伏時間。控制組 則每日腹腔注射生理食鹽水。隔日將大白鼠予以斷頭犧牲，取腦分為大腦皮質、海馬迴等 各部位分別製備成非溶解性蛋白質溶液，利用NR1A, NR2A, NR2B之專一性抗體來量化各亞 型，並以neuron-specific enolase (NSE)來偵測神經細胞量的多寡以作為內部標準控制 。 在本研究的大白鼠抽搐行為觀察中發現，在控制組由kainic acid引發產生wet-dog shakes的頻率，於停止生理食鹽水注射後的第一天及第二十八天，比起第十四天有減少的 現象。安非他命組則在停藥後的第一天及第二十八天，產生wet-dog shakes的頻率明顯的 高於同時期的控制組。安非他命組在停藥後的第十四天，引發產生wet-dog shakes的潛伏 時間比控制組慢了31.5 %。安非他命組在停藥後的第一天，由kainic acid引發嚴重抽搐 發作的隻數比例比起控制組有明顯的增加，然而在其餘停藥後的天數則沒有明顯變化。在 本研究西方墨點法的結果發現，在安非他命組的大腦皮質中，NR1A的表現量在停止給予安 非他命後的第一天，明顯的少於控制組約68 %。而NR2A, NR2B, NSE，以及在海馬迴中的N R1A, NR 2A, NR2B, NSE則無此現象發生。 由本研究以上的結果我們推測，長期給予安 非他命後會顯著增加對大腦的興奮性。並且KA 注射會選擇性的對安非他命組大腦皮質的N R 1A產生短暫性的減量調控。這種現象有可能是與慢性給與安非他命導致KA引發腦部興奮 性的增加有關。

關鍵字

嗎啡 ； 腦部神經細胞 ； 大腦皮質 ； 海馬迴 ； 安非他命 ； NMDA受體 ； 醫學

並列摘要

Part 1 :abstract : Long-term neuropsychological abnormality has been documen ted in child born to morphine addicted mothers, but the mechanism of this phen omenon until now is not clear. Our previous ligand binding study has shown tha t combined prenatal and postnatal chronic treatment of morphine could alter th e ontogenic expression of the NMDA receptor. In that, the density of NMDA rece ptor of rats born to dam rats received morphine treatment were significantly l ess than the control rats on postnatal day 14 (PND 14) . It suggested that rat s born to chronic morphine addicted dam rats could induce the neuronal NMDA re ceptor downregulation. To further explore what kinds of NMDA receptor subuni ts are changed, we used western blotting to examine the expression of the NMDA receptor subunits, namely, (NR1A, NR2A, NR2B) proteins in the brain of rats b orn to morphine-treated dam rats. In cortex we found that the NR1A of morphine rats was decreased by 40 % on PND 14 as compare to that of control group. On the contrary, the NR2A of morphine rats on PND7 was increased 2.8 folds as com pared to that of control rats. But the NR2B of morphine rats was not different to that of control rats. In hippocampus, the NR1A and NR2B of morphine rats w ere not different to that of control rats. However, the NR2A of morphine rats was decreased by 34 % as compared to that of control rats. These results are i n consistent with our previous ligand binding assay, suggest that rats born to chronic morphine addicted dam rats induce cerebral NMDA receptor subunits dow nregulation, in the cortex that is NR1A and in the hippocampus that is NR2A. P art 2:abstract: Addition to the psychstimulant drugs, such as amphetamine (A MPH), is one of the important social and public health problems in Taiwan. Rep eated administration of amphetamine frequently develop a drug-induced paranoid psychosis, and the this phenomenon usually dissipates upon the cessation of d rug use. But there are some long-term sequel associated with amphetamine abuse , such as, re-exposure to a relatively low dose of amphetamine will often prec ipitate a psychotic episode in former amphetamine addicts who have been abstin ent for months to years. The mechanism of amphetamine produces hypersensitivit y is not clear, however, it may be associated with the change of cerebral exci tability. Previous investigations of the mechanism by AMPH induced addiction a nd long-term neurotoxicity is mainly focusing on dopaminergic neuron, however, recent evidenced have indicated that the N-methyl-D-aspartate receptor (NMDA receptor), one of the subtype receptor of glutamate receptors, which antagonis ts significantly attenuated AMPH-induced behavioral sensitization, glutamate r elease. To explore the amphetamine induced long-term neurotoxicity, we studi ed the effect of chronic treatment of amphetamine in kainic acid (KA) elicited seizure activity. Male Sprague-Dawley rats were received daily i.p. injection of 5 mg/kg amphetamine for 14 days. The animals were received a single dose o f kainic acid (12 mg/kg) s.c. injection 1, 14 and 28 days after the last injec tion of amphetamine. We observed the behavior change during the first 3h after . Control rats were received daily injection of normal-saline before injection of kainic acid. Furthermore, we used western-blotting to quantify the density of each NMDA receptor subunits, namely, the NR1A, NR2A, NR2B in the crude mem brane of cortex and hippocampus tissues prepared from rats received single dos e of KA after they had received 14 day injection of AMPH (AMPH group) or salin e (control) group. The results of kainic acid-induced seizure showed that i n control group rats, the wet-dog shake frequency was significant lower at 1 a nd 28 days than that of 14 days after the last injection of normal-saline. In amphetamine-treated group, the wet-dog shake frequency was significant higher than control group at 1 and 28 days after the last injection of amphetamine. T he latency of wet-dog shakes onset was later than control group about 31.5 % a t 14 days after the last injection of amphetamine. The percentage of rats had motor clonus of amphetamine group is significant higher than that of control g roup rats at 1 day after the last injection of amphetamine. The results of wes tern boltting showed a significant decrease in the density of NR1A in the cort ex of AMPH group one day after last injection of AMPH. However, no significant difference in the expression of this subunit in other time points or hippocam pus between control and AMPH group. Furthermore, there is no significant chang e in the expression of NR2A and NR2B of AMPH group. These results suggested that chronic administration of amphetamine lead to a transiently enhancing th e kainic acid-induced neuroexcitation, and demonstrated that chronic treatment of AMPH with single injection of KA may produce a transient down-regulation o f one NMDA receptor subunit. This receptor plasticity may be a consequence of chronic treatment of AMPH which result into an enhanced KA-induced neurotoxici ty.

並列關鍵字

NDMA ； Morphine ； Cortex ； Hippocampus ； Amphetamine (AMPH) ； NMDA receptor ； MEDICINE