伽瑪-氨基丁酸乙型接受器調控藍斑核強直性抑制實驗及在大鼠睡醒調控中所扮演的角色

GABAB Receptors Mediated Tonic Inhibition of Locus Coeruleus Neuron in Vivo and Its Role in Sleep-wakefulness Regulation in Rats

朱韻琳(Yun-Lin Chu)

指導教授 ： 閔明源

國立臺灣大學/生命科學院/生命科學系/碩士(2016年)

https://doi.org/10.6342/NTU201601328

摘要

藍斑核(Locus Coeruleus)大部分是由去甲腎上腺素神經元(noradrenergic neurons)所組成，是主要提供正腎上腺素(norepinephrine)至前腦(forebrain)的腦區，它調控了許多生理行為作用如焦慮、記憶、學習及清醒等等。之前我們實驗室的鼠腦片(brain slice)電生理的研究顯示，伽瑪-氨基丁酸乙型接受器(γ-Aminobutyric acid B receptors，GABAB receptors)是只要主要調控藍斑核的強直性抑制(tonic inhibition)的神經傳導物質接受器，並且可以藉由調控環境中的伽瑪-氨基丁酸可以調控藍斑核的神經放電速率(firing rate)。在本實驗中，我們測試了伽瑪-氨基丁酸乙型接受器是否也能夠在生物體的藍斑核中擁有調控其強直性抑制及神經放電速率的功能。
首先，在測試實驗中，我們先以伽瑪-氨基丁酸乙型接受器促效劑(agonist)經由外管直接注入至活動大鼠(Sprague-Dawley Rats)的藍斑核，在不同天的同一時間測試其睡眠以及清醒的時間比例差別，結果發現在藥物給予後的大鼠其睡眠時間有顯著性的增加(P=0.020526，N=4)。故我們推斷伽瑪-氨基丁酸對於藍斑核的抑制作用確實會影響生物體的睡醒比例。為了確定是伽瑪-氨基丁酸乙型接受器的作用造成此結果，我們將外管植入至藍斑核，在由異氟烷(isoflurane)氣體麻醉的誘導全身麻醉(general anesthesia)之下，將實驗老鼠四腳朝天以翻正反射(return of righting reflex)以及腦電波(Electroencephalography，EEG)來判定其由麻醉轉至清醒所需的時間。我們的結果顯示，在異氟烷停止給予後，經由內管對藍斑核的直接局部灌注伽瑪-氨基丁酸乙型接受器拮抗劑(antagonist)藥物CGP35348能夠降低結束麻醉至翻正反射的時間(845.89±98.73 seconds，N=9)，其數據顯著低於控制組(灌注人工腦脊髓液)(N=9)，以及沒有注射至藍斑核的組別(N=10)。
但是在局部灌注下，無法確定該行為上的改變是由於藍斑核或是其他鄰近藍斑核區域例如中腦三叉感覺核區(mesencephalic trigeminal nucleus neurons，Me5)等受到藥物影響而產生，故我們使用能夠同時記錄單一神經元神經放電速率以及注射藥物的電極，記錄在聚氨脂(urethane)或三氯乙醛(Chloral hydrate)麻醉下的老鼠其在緩慢注射伽瑪-氨基丁酸乙型接受器拮抗劑藥物CGP35348之前後藍斑核神經元神經放電速率的變化。此實驗數據顯示，藍斑核神經放電速率在藥物注射前300秒之神經放電速率為2.9158±0.9422Hz，在藥物注射後300秒則為9.8599±2.5460Hz，其神經放電速率在藥物注射後明顯高於注射前(P=0.01497，N=6)，而在其他型態染色上非位於藍斑核之神經元放電速率紀錄統計，其神經放電速率在藥物注射前以及注射後並無顯著差異(N=29)。且在藍斑核位置注射控制組磷酸根緩衝液生理食鹽水(phosphate buffer saline，PBS)之前後也並無顯著變化(N=6)。綜合上述實驗，我們的結果證實伽瑪-氨基丁酸乙型接受器對藍斑核的強直性抑制的確會對於生物體的睡醒調控有一定影響。

關鍵字

藍斑核 ； 伽瑪-氨基丁酸乙型接受器 ； 睡-醒調控

並列摘要

In mammalian brain, locus coeruleus (LC) provides most of norepinephrine (NE) for the whole brain. LC-NE system is involved in many brain behaviors, such as nociception, attention and wake-sleep cycle. We have previously shown a tonic inhibition of LC neurons mediated by GABAB receptor in acute brain slice, and that manipulating it by varying ambient GABA could effectively tune LC neuron firing rate. Furthermore, we predict that GABAB receptor-mediated tonic inhibition could play an important role in the wake-sleep cycle behavior. Here we tested whether GABAB receptor tonic inhibition of LC neurons also occurs in vivo.
Since ambient GABA in LC is reported to be higher in sleep than in wakefulness, we tested the role of GABAB receptor tonic inhibition of LC neurons in hypnosis induced by isoflurane. To investigate the hypothesis, righting reflex behavior test were made in SD (Sprague-Dawley) rats (male, 8-10 weeks) which implanted a cannula for drug injection and electrodes for EEG (electroencephalography) and EMG (electromyography) recording under isoflurane anesthesia. Righting reflex occurs during the position out of upright, was used to define emergence from general anesthesia. Local infusion of CGP35348 but not the vehicle into LC significantly reduced recovery latency from hypnosis. We found that the latency of righting reflex behavior decreased after GABAB receptor antagonist injection. A custom-made probe consisting of microwires and a fine cannula was implanted into LC to allow recording of LC single- unit and local drug application in rats. After application of CGP35348, a neutral GABAB receptor antagonist, robustly increases firing rate of LC single-unit about 3.382 times higher (n = 6 units; P=0.01497, paired-t test); In contrast, vehicle infusion did not have an effect. Above all, we suggest that not only the firing rate of LC neurons is inhibited by GABAB receptor in vivo, but also the activity of GABAB receptor on LC neurons play an important role in sleep-wakefulness regulation.

並列關鍵字

Locus coeruleus ； GABAB receptors ； sleep-wakefulness regulation

參考文獻

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