Characterization of GABAergic Interneurons Within Locus Coeruleus Nucleus in Mice.

藍班核作為大腦中主要的正腎上腺素分泌之來源，先前的研究證實伽瑪氨基丁酸此醫神經傳導物質在調控藍斑核神經元活性上扮演重要角色。在(非)快速動眼睡眠期藍斑核區內伽瑪氨基丁酸的濃度顯著上升，且藍斑核區域伽瑪氨基丁酸的突觸前結構約占總數的三分之二。我們實驗室先前的研究成果指出，伽瑪氨基丁酸可透過B型受體以提供大鼠藍斑核細胞一個長期性的抑制性調控，且這個調控可能與細胞間伽瑪氨基丁酸濃度有關。本研究中我們以電顯觀察大鼠藍斑核的伽瑪氨基丁酸B型受體分布情形，發現他們主要分布於突觸外的位置，可推得細胞間伽瑪氨基丁酸濃度將透過突觸外之受體調控藍斑核細胞。先前的研究指出許多核區作為藍斑核伽瑪氨基丁酸來源，但近來的研究指出藍斑核區域內的中間神經元可能擔任調控藍斑核細胞的角色，而這些分泌伽瑪氨基丁酸中間神經元的電生理性質與突觸生理性質仍然未被報告。本研究以全細胞電生理技術紀錄藍斑核內分泌伽瑪氨基丁酸之中間神經元的基礎性質，另以過氧化酶染色技術重建此些中間神經元的型態構造，發現可以放電情況將這些中間神經元分成三群，且在型態重建中發現伸入該核區內的軸突結構上有被認為可能是突觸的結構。而在電生理雙紀錄的實驗中卻沒辦法發現藍斑核細胞與中間神經元的單突觸連結關係。The Locus coeruleus (LC) is a noradrenergic brainstem nucleus that is the major norepinephrine (NE) supply to the forebrain. It has been shown that GABA appears to be involved in the regulation of LC. Several studies have reported that extracellular GABA concentration is higher during REM/NREM sleep than wakefulness and that about two-third of boutons within LC contained GABA. Previous data of our lab showed that GABAB receptors could mediate tonic inhibition of LC neurons in rats, and this effect may link to ambient GABA concentration. Subcellular localization of GABAB receptors of LC nucleus in rats had been examined and found that most functional GABAB receptors are at extrasynaptic site, so ambient GABA could mediate tonic inhibition via extrasynaptic GABAB receptors here. However, the GABA source for tonic inhibition of LC neurons is still unclear. Many GABA afferents other brain nuclei have been reported previously, while recent studies found that local GABAergic interneuron may be a novel candidate of GABA supply to LC nucleus. However, the synaptic property and the role of LC GABAergic interneurons remain unknown. Thus we characterized these interneurons by using whole cell recording, following by staining of recorded interneurons for morphology reconstruction. Here we divided them into three group by their firing pattern and found some bouton-like structures on axons from the morphology reconstruction. But no functional pair has been found in the double patch experiments between LC neurons and GABAergic interneurons.目錄 口試委員審定書.............................................i 致謝.....................................................ii 中文摘要.................................................iii Abstract................................................iv 目錄.....................................................vi Introduction.............................................1 Locus Coeruleus….................................1 Spontaneous firing activity of LC neurons........2 Afferent to Locus Coeruleus……………………………………………………. 4 GABA : Major inhibitory neurotransmitter of LC...5 GABAergic interneurons within LC.................8 Objectives.......................................9 Material and Method.....................................10 Animals…........................................10 Preparation of brainstem slices.................10 Electrophysiology...............................11 Immunohistochemistry............................13 Immunohistochemistry after recordings...........14 Pre-embedding immunocytochemistry and electron microscopic techniques..................................14 Drugs and Chemicals.....................................16 Results.................................................17 Subcellular localization of GABAB receptors of LC nuclei in rat...........................................17 Distribution of GFP positive neurons in locus coeruleus area..........................................18 Both GABAA receptors and GABAB receptors mediate tonic inhibition of LC neurons in mice.....................................................19 Whole-cell recordings for GFP-positive neurons within LC.21 The firing characteristics of LC GABAergic interneurons..22 The membrane properties of LC GABAergic interneurons.....23 GABAergic interneurons project axons with bouton-like structure into LC........................................24 No functional pair could be found by double experiments between LC neuron and GABAergic interneuron................................25 Discussions..............................................27 EM observations of GABAB receptors...............28 Membrane properties of LC GABAergic interneurons.30 Morphology of LC GABAergic interneurons..........31 Future work......................................33 Figure legens............................................34 Reference................................................43 Figures..................................................48 Fig. 1.................................................. 48 Fig. 2...................................................50 Fig. 3...................................................51 Fig. 4...................................................52 Fig. 5...................................................53 Fig. 6...................................................54 Fig. 7...................................................55 Fig. 8...................................................56 Table 1..................................................5