2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660084摘要：精神症的病因，被認為是中樞神經細胞之間的突觸連結異常。的確，多巴胺，GABA，glutamate 等傳導系統功能的異常，已被提出在此疾病的病因扮演重要角色。但詳細病理機制則未知。本實驗室先前以安非他命處理動物，作為研究安非他命精神症，即妄想性精神分裂症，與成癮的模式，發現其紋狀體多巴胺含量改變；再者，其GABAB受體活性，和GAD67 蛋白質與鈣結合蛋白parvalbumin(PV)的表現，在運動與體感覺皮質，紋狀體等區均異常；PV 可代表GABAergic 細胞的活性。我們也觀察另一種動物模式，即NRG1 基因變異小鼠(NRG1+/-)，因為NRG1 基因與神經細胞發育相關，又是精神分裂症的易感基因之一。初步結果顯示，此動物在行為上也出現過度興奮的情形；另外，GABA 及多巴胺含量，NRG1 的受體ErbB4 和PV 蛋白質的表現在NRG1+/-的特定腦區似乎均異於野生型。我們提出的假說是：NRG1 基因活性與維持腦神經細胞之間的正常GABA 與多巴胺傳導功能有關。本計劃將使用經安非他命重複注射小鼠與NRG1 基因變異(+/-)小鼠，探討精神症病因機制，主要觀察GABA 系統與NRG1 基因的角色，並兩者的關係。我們將使用行為測試，生化，形態等方法，研究這些小鼠腦部GABA系統的表現及NRG1 活性與行為的關連性。觀察腦區將主屬額葉，包括扣帶，運動與體感覺皮質，紋狀體等區，因這些區與運動，感覺，記憶，情緒等功能密切相關。我們將使用HPLC 測量這些腦區的GABA，glutamate，多巴胺等含量；亦將使用免疫細胞化學染色方法，進行單一及雙重染色，探討以下蛋白質在皮質各層次和紋狀體的表現：GAD67，PV,GABA 受體，NRG1,ErbB4。也將觀察GABAB受體或ErbB4 活化後，相關胞內訊息分子Akt,ERK1/2,CREB, cAMP 等的活化程度。亦將使用免疫電子鏡術觀察GABA系統與含NRG1 或ErbB4 的細胞形成突觸情形，並與多巴胺末稍的突觸關係。再者，我們的初步資料發現NRG1+/-與安非他命處理成年小鼠的體感覺皮質的以doublecortin 標定的新生神經細胞和GABAergic 細胞的數量改變。的確，先前其他研究發現，精神分裂症病人的齒狀回新生細胞數量降低；又已知側腦室新生細胞會移至大腦新皮質，分化成GABAergic 細胞。但NRG1 活性是否參與此新生，遷移，分化過程的調節則不清楚。故本計劃也將主要觀察成鼠神經細胞新生的調節與NRG1 的相關性，將以bromodeoxyuridine(BrdU)注射動物，觀察側腦室與齒狀回新生細胞，並追蹤細胞遷移分化情形，包含形成GABAergic 細胞的比率，將使用不同標誌抗體觀察細胞發育成熟各個時期。我們將整合形態及生化結果，以期了解GABA系統與NRG1 基因活性和細胞新生的關連，及在精神症病因中的重要性。其中有關GABAB受體傳導與神經新生方面，具有藥物療效的潛力，可做為發展未來臨床治療的基礎。<br> Abstract: The psychiatric disorders are thought to be caused by abnormality in synapticconnectivity in the brain. Indeed, malfunction of dopamine, GABA, and glutamatesystems have been hypothesized to contribute to the pathogenesis of the disorders.However, detailed mechanisms await clarification. We have used amphetamineadministeredanimals as a study model for amphetamine psychosis (paranoidschizophrenia) and addiction; compared with the control, in the drug-treated animal,there were changes in the dopamine content, the activity of GABAB receptor (R), andthe expression levels of GAD67 and the calcium binding protein, parvalbumin (PV) inthe motor and somatosensory cortices, and striatum. We also observed anothermodel, the neuregulin 1 (NRG1) heterozygous mutant (+/-) mice for psychiatricdisorder, as NRG1 activity is critically involved in neural development and is one of theimportant susceptibility genes of schizophrenia. Our preliminary data revealedbehavioral hyperactivity, and altered dopamine and GABA content, and the proteinexpression of PV and ErbB4, the receptor of NRG1, in the specific brain regions of themutant. Thus we propose that the activity of NRG1 is associated with maintaining thenormal transmission of GABA and dopamine systems of brain neurons.This proposal plans to use the amphetamine-treated and NRG1+/- mutant mousemodels to explore causes of psychosis. Our main observations include the roles ofGABA system and NRG1 activity, and relations between the two parameters. We willemploy behavioral tasks, and biochemical and morphological methods to correlatebehavior with the activity of GABA and NRG1. The frontal lobe regions will beexamined, including the cingulate, motor and somatosensory cortices and striatum, asthese areas are closely associated with motor and sensory functions, memory andemotions. The GABA, dopamine and glutamate content will be measured in theseregions by using HPLC method. Single and double immunostaining will be used toanalyze the following protein expression and interrelations: GAD67, PV, GABAreceptors, NRG1, and ErbB4. Levels of activated intracellular signaling molecules, Akt,Erk1/2, CREB and cAMP will also be investigated via activation of GABABR or ErbB4.Synaptic connections will be monitored among the GABA system elements andNRG1/ErbB4 of the cells, or dopaminergic terminals by double immuno-electronmicroscopy.Furthermore, our preliminary data have revealed that the number of neuroblasts,labeled with doublecortin, was increased in the somatosensory cortex 1 of the adultNRG1 mutant and amphetamine-treated mice and the number of GABAergic neuronschanged as well. Previous studies indeed showed decreased number of neuroblastsin the dentate gyrus of postmortem brains of schizophrenic patients. Moreover, newneurons generated from the subventricular zone (SVZ) have been found to migrate tothe neocortex to become GABAergic neruons, in addition to the olfactory bulb.However, the regulatory role of NRG1 activity in this neocorical migration anddifferentiation remains unknown. Thus we will also explore the relations amongregulation of adult neurogenesis, GABAergic neuron maturation and NRG1.Bromodeoxyuridine will be applied to the animals to label newly born cells in the SVZand dentate gyrus, and various markers will be used to trace cells of distinctdevelopmental stages in the migrating pathways, including the maturation rate of theneocortical GABAergic cells. We will integrate results from the biochemical andmorphological approaches. Through understanding changes in the expression ofGABA system and its relations with NRG1 activity and neurogenesis in the animalmodels, the significance of these parameters will be better understood in thepathogenesis of psychosis. In particular, the GABABR transmission and postnatalneurogenesis have potentials for future clinical therapeutic development.