Leptin 和 Neurotensin 在大鼠迷走神經運動神經核之作用

Effects of Leptin and Neurotensin in Dorsal Motor Nucleus of the Vagus in Rats

李紫鈴(Tzu-Ling Li)

指導教授 ： 黃玲玲博士

臺北醫學大學/醫學院/醫學科學研究所/碩士(2003年)

摘要

根據流行病學的調查，在許多國家包括台灣，近幾年之肥胖人口明顯成長，肥胖除了影響外觀外也會導致一連串的健康問題，如心血管疾病、高血壓、第2型糖尿病，因此，肥胖屬於全球性的健康問題。體重的控制是維持能量衡定的重要方式，下視丘為調節能量衡定之重要中樞，其能透過感測週邊的訊息，以瞭解目前身體之能量狀態，並藉由週邊與中樞之神經網路來調節食慾與能量消耗，進而維持體內能量衡定。證據顯示腦幹也是參與能量衡定之重要區域。 Leptin是由脂肪細胞以一定比例分泌而來，其能作用於下視丘進而抑制食慾;neurotensin是神經傳導物，從中樞給予neurotensin能抑制食慾，因此，leptin與neurotensin被認為參與能量衡定之月生月太。研究顯示下視丘與腦幹具有leptin與neurotensin receptor存在，其他研究顯示在腦幹中之迷走神經運動神經核(dorsal motor nucleus of vagus; DMNV)也是leptin與neurotensin作用之腦區。本研究是以腦幹組織切片為材料，以電生理之whole-cell patch-clamp之技術觀察leptin與neurotensin對迷走神經運動神經核之影響。 本研究結果顯示85個成功紀錄的神經元中，leptin的投予造成DMNV神經細胞去極化(19%)或引發內流電流，而且隨著leptin濃度增加(濃度範圍0.25-300nM)或leptin也能造成DMNV神經細胞過極化(15%)。河豚毒素存在下，leptin仍然可以引發DMNV 神經細胞產生去極化或內流電流反應，膜電位變化為8±1mV(n=9)，也能誘發過極化或外流電流，膜電位變化為11±3mV(n=10)，因此證明leptin能直接作用於DMNV 神經細胞造成膜電位變化而改變其興奮性。另外，河豚毒素存在下，投予neurotensin造成DMNV神經元去極化(52%)，平均膜電位變化為11?2 mV，證明 neurotensin能直接作用DMNV 神經細胞造成膜電位變化而改變其興奮性。 在電位嵌制下，觀察leptin或neurotensin誘發之I-V relationships，結果就leptin引起之去極化而言，leptin誘發之電流斜率為負值，得到反轉電位平均為-88±4 mV(n=12)；當神經細胞胞外鉀離子濃度增高時，此時leptin引發之內流電流變小，反轉電位為由-87 mV轉移至-60 mV (n=1)，因此可以確認leptin關閉鉀離子通道造成膜電位去極化反應。就leptin引起之過極化而言，leptin誘發之電流斜率為正值，得到反轉電位平均為-85±3mV(n=4)，由實驗條件推算出鉀離子通道平衡電位為-94mV，因此推測leptin開啟鉀離子通道造成膜電位過極化反應。就neurotensin引起之去極化而言，neurotensin誘發之電流斜率為負值，得到反轉電位平均為-92±3mV(n=9)，由實驗條件推算出鉀離子通道平衡電位為-94mV，因此推測關閉鉀離子通道造成膜電位去極化反應。 電生理紀錄前，從腹腔注射fluoro-gold，以反向運輸標定副交感節前神經細胞，電生理紀錄中，在電極溶液中加入lucifer yellow (0.2%)，以標定被記錄到神經細胞之位置，藉此方式確認被記錄到神經細胞之位置是否位於DMNV核區之副交感節前神經細胞。本研究顯示leptin或neurotensin作用而產生細胞膜去極化或過極化之DMNV神經細胞中，也包括副交感節前神經細胞。因此，本研究證實leptin或neurotensin可直接作用於腦幹之DMNV神經細胞，調節副交感神經活性。

關鍵字

肥胖 ； Leptin ； Neurotensin ； 迷走神經運動神經核 ； whole-cell patch-clamp recording

並列摘要

Obesity is one of the major public health problems in many countries in the world including Taiwan and is also a serious threat to health. Regulation of energy homeostasis is critical in body weight control. The hypothalamus, capable of detecting peripheral signals, and thus regulating energy intake (feeding) and expenditure by forming links between central and peripheral systems, appears to be the major integrating site of the adiposity signaling in the central nervous system (CNS). Evidences also suggest brain stem as an important structure involving in the regulation of energy balance. It has been shown that leptin is secreted in proportion to adiposity to serve as negative feedback signaling molecules on the regulation of body adiposity by acting in the hypothalamus. Neurotensin is a neurotransmitter known to be involved in energy homeostasis by reducing food intake. The receptors of leptin and neurotensin were found not only in the hypothalamus but also in the brain stem. Other anatomical evidences imply the dorsal motor nucleus of the vagus (DMNV) as a potential site of action for both leptin and neurotensin. By using whole-cell patch-clamp recording in brainstem slices, the effects of leptin and neurotensin were investigated in the present study. Leptin was found to either depolarize or hyperpolarize 19% and 15% of DMNV neurons, respectively, in a dose-dependent manner in the range of 0.5 nM to 300 nM. Neurotensin depolarize 52% of DMNV neurons. The effects of leptin and neurotensin persisted in the presence of tetrodotoxin, indicating direct effects of leptin and neurotensin on DMNV neurons. With the use of voltage-clamp recording techniques, the current-voltage (I-V) relationship of leptin- or neurotensin-induced currents were examined. Leptin-induced inward current exhibited a negative slope with a reversal potential of -87mV and the reversal potential was shift to -60mV in a high potassium Krebs solution. These results indicate that a decrease of potassium conductance may underlie leptin-induced inward current (or depolarization). Leptin-induced outward current exhibited a positive slope with a reversal potential of -85±3(n=4)mV implying that an increase of potassium conductance may be the major ionic mechanism of leptin-induced outward current (or hyperpolarization). Similar to the results of leptin-induced inward current a negative slope of the I-V curve and a reversal potential of -92±3mV(n=9) were obtained for neurotensin-induced inward current suggesting a decrease of potassium conductance as the underlying mechanism. By using retrograde labeling and intracellular staining techniques, the parasympathetic preganglionic neurons in the DMNV were identified. Our results show that leptin- and neurotensin-depolarized or hyperpolarized DMNV neurons include identified parasympathetic preganglionic neurons. The results suggest that DMNV is one of the target sites of leptin and neurotensin in the central nervous system and imply a role of leptin and neurotensin in the regulation of parasympathetic activity.

並列關鍵字

obesity ； Leptin ； Neurotensin ； dorsal motor nucleus of the vagus ； DMNV ； whole-cell patch-clamp recording

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