?班核 (Locus ceoruleus; 簡稱LC) 位於橋腦背側面，主要含有分?正腎上腺素的?經元 (簡稱 LC ?經元)。LC ?經元對大腦有非常廣泛的正腎上腺素纖維投射，因此在大腦功能?態的調節上扮演極重要的角色，包括睡眠-清醒?態與痛覺傳遞的調節。過去的研究顯示，LC ?經元具有節?性自發放電的?為，並且其頻?與型式特徵具有日週期性的變化。愈?愈多的研究證據顯示，LC ?經元放電?為的日週期性變化，其節?訊號的?源是?自下視丘的視上核 (Suprachiasmatic nucleus；簡稱SCN) ，經由下視丘背中側核(dorsomedial nucleus of the hypothalamus；簡稱DMH) 的?經投射到LC，而DMH ?經投射的主要?經傳遞物質即為食慾素(orexin)。?過，現今的?經細胞學知?對於LC ?經元如何節?性的自發放電？?麼樣的機轉可以造成LC ?經元自發節?放電的頻?與型式改變？又如何具有日週期性？ 還有 SCN-DMH 投射與食慾素在這些變化過程中扮演?麼角色？以上等等問題的?解?相當缺乏。?經細胞是否具有節?性自發放電的能?，其細胞膜上是否表現低活化閾值與能持續活化的膜電位依賴型鈉?子電?(INaP)的?子通道蛋白是主要的關鍵，並且INaP 通道也?與控制這些中樞?經元的放電頻?與型式。據此，本計畫的假?為：1. LC ?經元表現有INaP 通道，故具有節?性自發放電的?為；2. LC ?經元節?性自發放電的頻?與型式的日週期性變化是因為LC ?經元的INaP 通道表現?也具有日週期性變化；3. INaP 通道表現?的日週期性變化是受到SCN-DMH 投射與食慾素的調節。針對以上?點，我們設計以下實驗： 1. 檢視 LC ?經元的電位依賴型鈉?子電?的動?學特徵；並著重於是否有INaP 特徵的存在。 2. 檢視 LC ?經元表現哪些Nav 蛋白的次單元。 3. 檢視何種 Nav 蛋白的次單元能媒介INaP 。此部份實驗將針對在LC ?經元中表現有Nav 的次單元分別加以剃除後，檢視何種Nav 次單元的剃除能阻斷LC ?經元的節?性自發放電?為。 4. 檢視實驗動物的 LC ?經元在夜間與日間，其節?性自發放電?為與細胞膜上媒介INaP 的Nav 通道蛋白表現?是否有差?。 5. 檢視在光剝奪或 SCN 破壞後，LC ?經元在夜間與日間的節?性自發放電?為與細胞膜上媒介 INaP 的Nav 通道蛋白表現?差?是否仍存在。 6. 檢視食慾素蛋白纖維剃除對 LC ?經元節?性自發放電?為與細胞膜上媒介INaP 的Nav 通道蛋白表現?的影響。我們相信這些問題的解答，?但能??解?班核對大腦清醒與睡眠等功能?態週期的調節，也可使我們??解中樞?經系統如何透過Nav 通道的角色?調整?經細胞的放電?為，進而調整生?的功能。 The locus coeruleus (LC) consists of tightly packed noradrenergic neurons located in the dorsol pontine tegmentum. LC neurons give rise to extensive axonal projections throughout many regions of the central nervous system and play an important role in regulation of multiple physiological processes, including sleep-waking cycle and pain control. Noradrenergic neurons in the LC appear to fire action potentials spontaneously and exhibit a circadian rhythm in impulse activity, which peaks with the active period. Previous studies have shown that the noradrenergic LC neurons participate in the circadian regulation of the sleep-waking rhythm through a suprachiasmatic nucleus (SCN) - dorsomedial nucleus of the hypothalamus (DMH)-LC circuit. DMH is one of the few brain areas containing neurons that express the neuropeptide orexin. At the present time, the ionic mechanisms that drive spontaneous firing of LC neurons at any time of day is still not fully understood, and the physiological and molecular evidences for the action of SCN-DMH projection on LC neurons have not been addressed. One candidate for inward current driving spontaneous firing of LC neurons is a low threshold activated and persistently activated voltage-dependent sodium current (INaP). Therefore, the hypotheses of this project are: (1) INaP drives pacemaking in noradrenergic LC neurons, (2) LC neurons exhibiting a circadian rhythm in impulse activity is dependent on the level of INaP expression, (3) the level of INaP expression is regulated by the SCN-DMH projection and orexin. The aims of this project are: (1) to characterize the voltage-dependent sodium currents on LC neurons, and determine whether INaP is expressed in the LC neurons, (2) to examine which Nav channel subunits can mediate pacemaking currents, (3) to determine which Nav channel subunits can mediate INaP, and test the effect of knockdown of certain Nav channel subunit on the LC neurons, (4) to determine whether there is any difference on rhythmically spontaneous firing and the level of INaP related Nav channel proteins expression between day and night time, (5) to examine whether there is any difference on rhythmically spontaneous firing and the level of INaP related Nav channel proteins expression after light deprivation or the lesion of SCN, (6) to test the effect of orexinergic fibers depletion on rhythmically spontaneous firing and the level of INaP related Nav channel proteins expression. At the completion of these studies, we anticipate decoding the role of Nav channels on noradrenergic LC neurons which provide a circadian regulation of the sleep-waking rhythm. The results will be high significance because it will improve our understanding of how the nervous system mediates the neuronal firing by Nav channels, and regulate the physiological function.
