發育大鼠及Citalopram處理大鼠藍斑核與A7區域神經細胞的γ-氨基丁酸B型接受器所媒介之長期抑制

GABAB Receptor-Mediated Tonic Inhibition of Locus Coeruleus and A7 neurons in Developing and Citalopram-Treated Rat

王瀚潁(Han-Ying Wang)

指導教授 ： 閔明源

國立臺灣大學/生命科學院/動物學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.01371

摘要

γ-氨基丁酸(GABA)是中樞神經系統中最主要的抑制性神經傳導物質。許多研究雖然顯示GABA能顯著調控分泌正腎上腺素神經細胞(如藍斑核以及A7區域)的活性，然而此調控機轉目前還不清楚。在本論文中，我們指出在藍斑核以及A7區域內的神經細胞可以藉由突觸後GABA B型接受器(GABABR)來媒介長期性抑制作用，並進而調節自身的興奮性。當我們加入GABABR選擇性抑制劑將長期性抑制作用阻斷後，不僅能引發內流性電流，同時也加快了藍斑核神經細胞的自發性動作電位頻率(SFR)。這說明GABA確實能藉由GABABR來引發長期性抑制並調控其放電頻率。另外，為了提高藍斑核的細胞外GABA濃度，我們藉由藥物來抑制GABA傳輸蛋白(GAT 1, GAT 2/3)進而阻斷GABA的回收機制。在此狀況下，藍斑核正腎上腺素神經細胞的活性顯著地降低；此時當我們再加入GABABR選擇性抑制劑後發現，原本降低的活性不僅消失且伴隨著強烈反彈性上升現象，此說明了在藍斑核內的細胞外GABA濃度能調節GABABR所媒介的長期性抑制。我們接著利用發育過程以及長期Citalopram(一種血清素回收抑制劑)處理的大鼠來研究藍斑核正腎上腺素神經細胞的GABABR在功能性的表現上會否有所不同。在發育大鼠模式的實驗中，雖然藍斑核正腎上腺素神經細胞的SFR並沒有顯著差異，但當我們阻斷長期性抑制作用後，SFR會隨著發育增長而加快，這說明GABABR所媒介的長期性抑制除了能維持藍斑核神經細胞的興奮性在不同發育時期處於恆定狀態外，長期性抑制作用亦會隨著發育過程而表現出增量調節現象(upregulation)。在長期Citalopram處理大鼠的實驗中，我們發現雄性大鼠藍斑核神經細胞的興奮性會大幅上升，但雌性老鼠的藍斑核神經細胞興奮性則維持不變。我們也發現Citalopram處理的雄性大鼠，其藍斑核神經細胞的長期性抑制作用比控制組顯著性地降低。此結果暗示著在發育過程中，若長期注射血清素回收抑制劑會造成雄性大鼠藍斑核神經細胞的長期性抑制作用產生減量調節(downregulation)，進而造成藍斑核神經細胞興奮性不正常提高。
除了藍斑核外，我們的實驗也顯示另一群分泌正腎上腺素神經細胞(A7區域)也具有GABABR所媒介的長期性抑制作用。我們也估算了A7區域的細胞外GABA濃度大約為2.8 μM。在此GABA濃度下，我們發現自發性突觸後抑制電流(mIPSC)的振福及形狀與控制組相比並不會受到改變，這說明了A7神經細胞突觸中的GABA A型接受器(GABAAR)在我們所估算的細胞外GABA濃度下並不具有去敏感作用(desensitization)。另外，我們也發現GABABR能調節A7區域中的GABAergic抑制性輸入，但對於Glycinergic抑制性輸入則不具調節作用。
由此可知，GABABR所媒介的長期性抑制作用對於分泌正腎上腺素神經細胞的興奮性調節來說，扮演著一個非常重要的角色。

關鍵字

藍斑核 ； A7區域 ； 長期性抑制作用 ； GABA B型接受器 ； GABA A型接受器 ； 自發性動作電位頻率 ； GABA傳輸蛋白 ； 選擇性血清素回收抑制劑

並列摘要

GABA (γ- amino-butyric acid), the principal inhibitory neurotransmitter in the brain, has been reported to exert significant effect on excitability of norepinephrinergic (NEergic) locus coeruleus (LC) and A7 neurons; nevertheless, the underlying mechanisms remains unclear. Here we reported tonic inhibition of LC and A7 neurons mediated by postsynaptic GABAB receptors (GABABRs). Application of selective GABABR antagonists induced inward current and increased spontaneous firing rate (SFR) in LC neurons. These results show GABABR mediated a tonic inhibition in LC neurons and tuned their SFR. To further confirm this argument, we elevated ambient GABA by inhibiting GABA transporter 1 and 2/3 with bath application of (s)-SNAP5114 and NNC711, and found SFR of LC neurons was dramatically suppressed; subsequent application of CGP52466 not only reversed this effect, but also caused a rebound of SFR. In addition to ambient GABA, we also manipulated GABABR functionality in LC neurons by using development and chronic perinatal exposure of citalopram (CTM), a selective-serotonin-reuptake-inhibitor (SSRI), as models. We observed a developmental increase in GABABR functionality and tonic inhibition in LC neurons, which, interestingly, was not associated with developmental increase in SFR, unless tonic inhibition was removed by blocking GABABR with CGP 54626. These observations indicate that increasing tonic inhibition could keep SFR of LC neurons at constant level during development. In CTM treated male but not female rats, this developmental increase of GABABR functionality in LC neurons was retarded, corresponding to which was a reduction in tonic inhibition. This impairment of tonic inhibition in LC neurons during development might partially account for abnormal SFR found in CTM treated male rats. In conclusions, our results show GABABR-mediated tonic inhibition could effectively regulate SFR of LC neurons, which is important for normal development and might be a major player in pathophysiological processes in chronic preinatal CTM exposure condition.
We also showed GABABRs of the A7 area, another NEergic neuron group, were constitutively activated by an ambient GABA concentration of 2.8 μM. Since this ambient GABA concentration did not significantly affect the amplitude and the shape of GABAergic mIPSC, suggesting any desensitization of the GABAARs that were located at synaptic sites and mediated phasic transmission was minor in the estimation of ambient GABA concentration. Furthermore, we reported GABABR of the A7 area can regulate GABAergic instead of Glycinergic inhibitory inputs.
In summary, GABABRs-mediated tonic inhibition plays a significant role in regulation of NEergic neurons activity.

並列關鍵字

Locus Coeruleus ； A7 area ； tonic inhibition ； GABAB receptor ； GABAA receptor ； spontaneous firing rate ； GABA transporter ； SSRI

參考文獻

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