臺灣博碩士論文加值系統

帕金森氏症 (Parkinson’s disease)是神經退化性疾病。其主要病因為腦中黑質分泌多巴胺的神經元死亡影響基底核迴路運作以及動物運動功能失調。位於大腦深部的視丘下核 (subthalamic nucleus，簡稱 STN)，是大腦基底核迴路的重要結構。視丘下核神經元藉由分泌glutamate興奮性神經傳導物質，對基底核進行調控，並影響動物的運動功能。
目前對帕金森氏症研究，發現病人之視丘下核核區會出現叢集式放電表現增加的現象；另外，深部腦刺激術 (Deep Brain Stimulation, DBS)用高頻率刺激視丘下核，改變視丘下核電生理狀態，可有效改善帕金森氏症的症狀。然而，帕金森氏症症狀與視丘下核神經元叢及釋放電表現增加的因果關係至今尚未清楚。不僅如此，視丘下核神經元產生叢集式放電型態的機制以及深部腦刺激視丘下核如何改善帕金森氏症症狀可能的分子機制也未釐清。故解析視丘下核神經元各種放電特性之分子與細胞機制，應對了解大腦運動控制之迴路運作以及尋求相關運動疾病之治療方法相當關鍵。
為了解視丘下核神經元獨特電生理特性，由大鼠腦薄片分離出單一健康的視丘下核神經元，首先分別利用置換溶液、電生理特性以及藥理特性，區分出可能影響視丘下核神經元動作電位型式之鈣離子通道。我們的實驗結果顯示，視丘下核神經元有低電位活化型 (LVA或T-type)及高電位活化型(HVA或L-type)鈣離子通道。我們也發現對低電位活化型鈣離子通道具抑制作用之鎳離子、mibefradil和efonidipine可以抑制視丘下核神經元上低電位活化型鈣離子電流和視丘下核腦切片的自發性叢集式放電型態。另一方面，鎘離子和nifedipine這兩種高電位活化型鈣離子通道之選擇性抑制劑抑制視丘下核神經細胞上的高電位活化型鈣離子電流但不抑制視丘下核腦切片上之自發性叢集式放電產生。接下來將刺激電極放置視丘下核核區上給予胞外電流刺激，模擬活體腦深部電刺激治療帕金森氏症方式，並以全細胞記錄方式偵測視丘下核神經元膜電位變化。實驗發現，給予電流刺激視丘下核會改變其膜電位。此外，給予負電流刺激視丘下核可能藉由使視丘下核神經元之膜電位去極化使得低電位活化型鈣離子通道進入不活化態，而改變視丘下核神經元的膜電位與放電模式。另一方面，視丘下核神經元神經活動除了受到其本身膜特性影響也會接受來自其他核區之神經傳遞物質調節。我們刺激蒼白球，能在視丘下核記錄到抑制性的突觸後電流，且蒼白球與視丘下核所形成之突觸具有短期突觸衰減現象。總結，低電位活化型鈣離子通道在視丘下核叢集式放電扮演重要的角色，因此，調控視丘下核上之低電位活化型鈣離子電流，進而改變視丘下核的放電模式，可能可以提供未來治療帕金森氏症的新策略。

Parkinson’s disease (PD) is a neurodegenerative disorder associated with the loss of dopaminergic neurons in the substantia nigra (SN), which then results in basal ganglia dysfunction and motor deficits. The subthalamic nucleus (STN) is an important structure involved in the shaping of the basal ganglia activity. Increased burst firing activity in STN has been a hallmark in the pathophysiology of PD. Moreover, high-frequency deep brain stimulation (DBS) of the STN effectively improves the motor symptoms in many PD patients. However, the casual relation between subthalamic burst activity and motor disabilities of PD remains to be established. The cellular mechanisms underlying STN bursts and the DBS therapy are also unclear.
In this study, we first characterized Ca2+ channels in acutely dissociated STN neurons from the rat brain based on the electrophysiological and pharmacological properties of different Ca2+ channels. We demonstrated the existence of both low-voltage activated (LVA or T-type) Ca2+ channels and high-voltage activated (HVA) Ca2+ channels in dissociated STN neurons. We then investigated the possible contribution of T-type and HVA Ca2+ channels to the firing pattern of STN neurons in acute mouse brain slices. We found that T-type Ca2+ channel inhibitors (e.g. Ni2+ and miberfradil) but not HVA Ca2+ channel inhibitors (e.g. Cd2+ and nifedipine) suppress burst firing or even turn burst into spiking pattern of firing in STN slices. Subsequently, we performed whole-cell current-clamp recording of STN neurons to directly monitor the membrane potential changes with DBS-mimicking electrodes being placed onto STN. We found that extracellular injection of negative constant currents into STN by the DBS-mimicking electrodes depolarizes STN neurons and diminishes burst firing of STN, an effect ascribable to a decrease in the availability of T-type Ca2+ channels. Not only intrinsic neuronal membrane properties but also synaptic inputs that a neuron receives would, in theory, determine the discharge patterns in STN. STN neurons chiefly receive excitatory synaptic inputs from the cortex and inhibitory GABAergic inputs from the globus pallidus (GP). Our preliminary resluts showed that stimulation of GP would evoke GABAergic inhibitory postsynaptic currents (IPSC) in STN, and the GP-STN synapse exhibits short-term synaptic depression.
We conclude that T-type Ca2+ channel should essentially contribute to the burst pattern of firing in STN. Moreover, the effect of DBS therapy could rely on adequate membrane depolarization and thus T-type Ca2+ channel inactivation in STN neurons. Modulation of subthalamic firing pattern could therefore be a possible strategy for the treatment of Parkinson’s disease.

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長庚大學博碩士論文著作授權書 iii
致謝 iv
Abstract v
中文摘要 vii
目錄 x

實驗背景 1
1. 帕金森氏症 1
2. 大腦基底核迴路與帕金森氏症 3
3. 視丘下核與大腦基底核迴路 6
4. 視丘下核與帕金森氏症 7
5. 視丘下核神經元電生理特性 9
6. 電位依賴型鈣離子通道 12
7. 視丘下核與蒼白球連結 14
實驗方法 17
1. 視丘下核腦薄片的製備與分離單一視丘下核神經元 17
2. 單顆視丘下核神經元全細胞箝制記錄 18
3. 視丘下核腦薄片的製備與視丘下核神經元胞外記錄 19
4. 視丘下核腦薄片上的視丘下核神經元全細胞箝制記錄 21
5. 蒼白球與視丘下核的神經元連結測試 22
6. 數據分析 23
實驗結果 24
1. 以電生理及藥理學方式分析單顆分離之視丘下核神經元上鈣離子通道種類 24
2. 低電位活化型鈣離子通道與視丘下核神經元叢集式放電型態的關係 27
3. 全細胞紀錄方式研究低電位活化型鈣離子通道與視丘下核神經元叢集式放電型態的關係 29
4. 模擬深部腦刺激術(DBS)對視丘下核神經元放電模式影響 31
5. 蒼白球與視丘下核之突觸連結 33
討論 36
1. 低電位活化型鈣離子通道為視丘下核神經元叢集式放電形成的關鍵因子。 36
2. 低電位活化型鈣離子通道進入不活化態可能成為臨床上深部腦刺激治療帕金森氏症之機制 40
3. 蒼白球與視丘下核突觸對視丘下核放電型態之調節 41
實驗結果圖 45
(圖一)以電生理方式分離單一視丘下核神經元上低電位活化型鈣離子通道。 45
(圖二) 視丘下核神經元低電壓活化型鈣離子電流被抑制程度受鎳離子濃度影響。 46
(圖三) Mibefradil 對視丘下核神經元上低電壓活化型鈣離子電流有隨濃度抑制效果。 47
(圖四) Nifedipine、鎘離子與 efonidipine 對視丘下核神經元低、高電壓活化型鈣離子電流抑制情形。 48
(圖五) 腦薄片視丘下核神經元其自發性放電型態。 49
(圖六) 低電位活化型鈣離子通道抑制劑會抑制小鼠腦薄片之視丘下核神經元其棘波式與叢集式放電型態。 50
(圖七) 高電位活化鈣離子通道抑制劑:鎘離子、nifedipine 不抑制小鼠腦薄片之視丘下核神經元其棘波式與叢集式放電。 51
(圖八) efonidipine 對小鼠腦薄片之視丘下核神經元其棘波式與叢集式放電型態的影響。 52
(圖九) 低電位活化型鈣離子通道抑制劑 Ni2+會抑制小鼠腦薄片視丘下核神經元之叢集式放電型態。 53
(圖十)低電位活化型鈣離子通道抑制劑 Ni2+減少視丘下核神經元叢集式放電頻率及增加膜電位進入叢集式放電高原期斜率。 54
(圖十一)給予胞外負電流刺激視丘下核使其神經元膜電位去極化可能會減少膜上可活化的低電位活化型鈣離子通道，造成放電模式由叢集式放電變棘波式放電型態。55
(圖十二)蒼白球至視丘下核之抑制性 GABAergic 突觸。 56
(圖十三)蒼白球釋放抑制性神經傳遞物質活化突觸後視丘下核神經元之 GABA 受體(氯離子)通道。 57
(圖十四)蒼白球至視丘下核突觸有短時間突觸削減現象(short-term depression)。 58
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