短期和長期安非他命改變小鼠嗅球多巴胺和迦瑪胺基丁酸系統的表現

Changes in Dopaminergic and GABAergic Expression of the Olfactory Bulb in Mouse after Acute or Chronic Amphetamine Treatment

鄭珮容(Pei-Jung Cheng)

指導教授 ： 尹相姝

國立臺灣大學/醫學院/解剖學暨生物細胞學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02695

摘要

嗅球內有許多含多巴胺 (dopamine)和GABA神經傳導物質的神經元，也許在精神疾病裡扮演重要的角色。安非他命注射的動物，可被用來作為研究藥物成癮或精神分裂症的動物模式。先前研究長期安非他命處理會改變大鼠內大腦皮質、紋狀體和海馬回的酪氨酸羥化酶 (tyrosine hydroxylase，TH)、麩胺酸脫羧酶 (glutamic acid decarboxylase67，GAD67)的表現。在中樞神經系統內，有一些抑制性GABAergic神經元會表現鈣結合蛋白 (calcium-binding proteins，CaBPs)，如parvalbumin (PV)和calretinin (CR)。另外，安非他命會改變許多腦區的cAMP反應結合蛋白磷酸化(pCREB)表現。然而較少研究探討，精神疾病或成癮對於嗅球內dopaminergic和GABAergic神經傳導物質的影響。所以本研究主要探討經一劑和長期安非他命處理後，小鼠嗅球TH、芳香族L-胺基酸脫羧酶(Aromatic L-amino acid decarboxylase，AADC)、GAD67、PV、CR和pCREB的變化。

本篇研究使用成年雄性小鼠，經腹腔內注射一劑或多劑(每天兩劑，共七劑)，5mg/kg安非他命或等容積的生理食鹽水(對照組)。最後一劑注射半小時或四小時後，小鼠接受灌流固定，以石蠟包埋嗅球，製成冠狀石蠟切片(7μm)，使用免疫化學染色法觀察上述蛋白質。免疫染色後再以影像分析系統定量，定量結果再以T test和two way ANOVA統計方法分析。

單染定量結果發現
(1)TH染色主要出現在嗅球小球層(glomerular layer，GL)的神經突起和細胞體。一劑藥物處理後沒有改變。長期藥物注射半小時後，小球層內TH細胞體的數目和神經突起染色程度，比對照組上升45%和160%。
(2)AADC染色主要出現在嗅球各層的細胞體和神經突起。一劑和長期藥物處理後，和對照組相似。
(3)GAD67染色出現在嗅球各層的神經末梢和細胞體。在一劑藥物注射半小時和四小時後，外叢狀層(External plexiform layer，EPL)內GAD67細胞體的數目比對照組增加60%和81%。在長期藥物注射半小時後，各層次內GAD67神經末稍染色程度，比對照組增加47%(GL)、34%(EPL)、40%(Mitral cell layer，ML)和29%(granule cell layer，GrL)。但在長期藥物注射四小時後，小球層和顆粒細胞層內GAD67細胞體的數目比對照組減少34%和44%；各層次內GAD67神經末稍染色程度，比對照組減少42%(GL)、51% (EPL)、55%(ML)和57%(GrL)。
(4)PV染色主要出現在外叢狀層的神經突起和細胞體。在一劑藥物注射半小時，外叢狀層內PV細胞體數目比對照組上升124%。一劑藥物注射四小時後，PV細胞體數和神經突起染色程度，皆比對照組上升155%和381%。
(5)CR染色出現在嗅球各層的神經突起和細胞體。在一劑藥物注射四小時後，各層內CR細胞體的數目和神經突起染色程度，皆比對照組上升75%(GL)、87% (EPL)、93%(ML)和116%(GrL)。
(6)pCREB免疫反應出現在嗅球各層神經元和神經膠細胞的細胞核中。在一劑藥物注射半小時後，顆粒細胞層內pCREB神經元的數目比對照組增加93%，在小球層內pCREB神經膠細胞的數目比對照組增加51%。但在長期藥物注射四小時後，顆粒細胞層內pCREB神經元的數目比對照組減少43%。

雙重染色的結果發現
(1)長期藥物注射半小時後，小球層內GAD67細胞體數目約有62%含有TH，對照組為53%，無顯著差異。TH細胞體數目約有72%含有GAD67，高於對照組59%。
(2)一劑安非他命和生理食鹽水注射半小時和四小時後，外叢狀層內100%PV細胞體數目含有GAD67。一劑藥物注射半小時和四小時後，GAD67細胞體數目約有56%含有PV，高於對照組31%。GAD67細胞體數目約有49%含有PV，與對照組39%無顯著差異。
(3)一劑藥物注射四小時後，小球層內大部分為單獨染上CR的細胞體。在外叢狀層內CR細胞體數目約有40%含有GAD67，高於對照組16%；GAD67細胞體數目約有68%含有CR，高於對照組36%。在僧帽細胞和顆粒細胞層內，有些為單獨染上CR的細胞，有些CR細胞體含有GAD67染色。
(4)長期藥物注射四小時後，有些細胞單獨染上核pCREB、有些細胞單獨染上GAD67，另外我們發現一些表現核pCREB的細胞膜外有GAD67神經末梢、有些表現核pCREB細胞的細胞質有GAD67染色的情形，藥物組可能比對照組少。

一劑藥物處理後，造成PV細胞體、神經突起和CR細胞體的增加，可能因為安非他命使鈣離子流入細胞內增加，使細胞興奮，所以造成PV和CR表現增加以保護細胞。已知PV神經元會和嗅球內興奮性叢狀(Tufted cells)和僧帽神經元互相形成突觸，所以在外叢狀層內，GABAergic PV和CR的細胞體數目增加，可能是增加製造GABA去抑制這些興奮性神經元。

長期藥物處理半小時後，可能因為安非他命使神經末梢內多巴胺和GABA枯竭，補償反應導致TH和GAD67表現增加。長期藥物處理四小時後，神經末梢內多巴胺和GABA量可能足夠，所以TH表現回復，GAD67回復甚至向下調節，以致GAD67表現下降。在一劑藥物處理半小時後，pCREB神經元上升可能因為多巴胺結合到突觸後D1受體，活化Adenylate cyclase使cAMP增加，導致下游CREB磷酸化增加。但在長期藥物處理四小時後，pCREB神經元減少可能因為多巴胺結合到突觸後D2受體，導致下游CREB磷酸化減少。我們推測一劑和長期藥物處理下，多巴胺可能結合在不同的多巴胺受體，或是PKA、CaM和MAPK路徑的活化或抑制，進而影響CREB磷酸化。

我們的研究結果指出一劑和長期安非他命處理下，會改變小鼠嗅球內神經傳導物質系統和神經元活性，可能導致嗅覺功能改變，並可能代表精神疾病早期致病機轉的一種情形。

關鍵字

安非它命 ； 嗅球 ； 多巴胺 ； 迦瑪胺基丁酸

並列摘要

The olfactory bulb (OB) contained dopaminergic and GABAergic neurons and may play important roles in affective disorders. Amphetamine (Amph) treated animals have been used as models to study pathogenesis of addiction and schizophrenia. Previous work using animal model has demonstrated that chronic administration of Amph alters the expression of tyrosine hydroxylase (TH)、glutamic acid decarboxylase (GAD) and phosphorylated CREB (pCREB )in neocortex, striatum, hippocampus of the rat. In addition, calcium-binding proteins (CaBPs) were useful markers of some populations of GABAergic neurons in the central nervous system. Thus, this study investigated the role of olfactory bulb plays in the mechanisms for the action of acute and chronic Amph treatment, by examining the expression of TH, AADC, GAD67, CaBPs and pCREB.

In this study, male adult mice were received single or multiple (2 doses/day, 7 doses in total) intraperitoneal injections of saline or Amph, 5mg/kg. At 0.5 or 4 h after the last injection, the mice were perfused with Bouin’s fixative, followed by preparation of paraffin OB sections and immunohistochemistry.

Our results revealed that：
(1)At 0.5 h after chronic Amph treatments, the number of TH-positive somata and the ratio of cell processes were increased in the glomerular layer (GL).
(2)At 0.5 h and 4h after acute and chronic Amph treatment, AADC-immunoreactivity (IR) was unchanged in all layer of OB.
(3) At 0.5 h and 4 h after acute Amph treatment, the number of GAD67-positive somata was increased in the external plexiform layer (EPL). At 0.5h after chronic Amph treatments, the ratio of GAD67-positive terminals was increased in all layers. At 4 h after chronic Amph treatments, the number of GAD67-positive somata was decreased in the GL and granule cell layer (GrL), and the ratio of GAD67-positive terminals was decreased in all layers.
(4) At 0.5 and 4 h after acute Amph treatment, the number of PV-positive somata and the ratio of cell processes were significantly increased in the EPL.
(5) At 4 h after acute Amph treatment, the number of CR-positive somata was significantly increased in all layers.
(6) At 0.5 after acute Amph treatment, the number of pCREB-positive glia was increased in the GL, and the number of pCREB-positive neurons was increased in the GrL. At 4 h after chronic Amph treatments, the number of pCREB-positive neurons was decreased in the GrL.

The results of double-staining：
(1) At 0.5 h after chronic saline and Amph treatments, about 59% or 72% TH- positive somata were GAD67-IR in the GL. About 53% or 62% GAD67-somata were TH-IR in the GL.
(2) After acute saline and Amph treatment, all the PV-positive somata contained GAD67-IR in the EPL. At 0.5 h after saline and Amph treatment, about 31% or 56% GAD67-somata contained PV-IR in the EPL. At 4 h after saline and Amph treatment, about 39% or 49% GAD67-somata contained PV-IR in the EPL.
(3) At 4 h after acute saline and Amph treatments, about 16% or 40% CR-positive somata were GAD67-IR in the EPL. About 36% or 68% GAD67-somata were CR-IR in the EPL. Some of CR-somata were GAD67-IR in the ML and GrL.
(4) In the GrL, at 4 h after chronic Amph treatments, some pCREB-expression cells were surrounded by GAD67-terminals. Some pCREB-expression cells showed GAD67 in their cytoplasm.

Our results suggested that up-regulation of TH and GAD67 at 0.5 h after chronic Amph treatments could imply compensation to long-term depletion of GABA and dopamine by Amph. The acute Amph induced increase in PV and CR expression may indicate inhibitory regulation of the OB to the stimulation of the psychostimulant. Changes in the expression of pCREB may be induced by activation or inhibition of protein kinase A, CaM or MAPK pathway.

The data supported that amphetamine induces changes in neurotransmitter systems and neuronal activity in the OB, leading to olfactory functional changes, and may implicate early mechanisms of pathogenesis of psychosis.

並列關鍵字

Dopamine ； GABA ； Olfactory Bulb ； Amphetamine

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