臺灣博碩士論文加值系統

神經傳導物質乙醯膽鹼（acetylcholine, ACh）在痛覺的調控中扮演重要的角色。文獻指出，乙醯膽鹼參與鎮痛效果（analgesia），且在脊髓中，乙醯膽鹼及其他膽鹼性促效劑（cholinergic agonists）皆可與α2正腎上腺素受體（α2-adrenergic receptor）共同作用，以調控止痛效應。過去的行為實驗發現，蕈毒膽鹼性系統（muscarinic cholinergic system）可能藉由與正腎上腺素之下行性痛覺調控路徑（NAergic descending pain modulation pathway）交互作用，達到止痛的效果。形態及電生理的資料更顯示，包含了第五群膽鹼性神經元cholinergic neuron group CH5的腦核區─腳橋被蓋核區（pedunculopontine tegmental nucleus, PPTg），具有對正腎上腺素A7神經元（NAergic A7 neurons）之纖維投射。此位於腦幹的A7兒茶酚胺細胞群（catecholamine cell group）更能進一步投射其軸突至脊髓背角（dorsal horn），分泌正腎上腺素並進行痛覺訊息傳遞之調控。此外，電生理實驗也提出腳橋被蓋核區之麩胺酸性（glutamatergic）神經元具有對A7細胞群投射之可能性。在本實驗中，我們對幼鼠腦幹薄片的腳橋被蓋核區進行電刺激，並對其下游之A7細胞群進行全細胞紀錄，測量細胞膜電位被箝定在-70 mV時的刺激性突觸後電流（excitatory postsynaptic current, EPSC）。藉由提高胞外溶液之鈣離子濃度，非同步性神經傳導物質釋放（asynchronous neurotransmitter release）所佔之比例顯著提高，反之亦然。此結果顯示，胞外鈣離子環境之改變，可能成為神經細胞傾向於以同步性神經傳導物質釋放（synchronous neurotransmitter release）直接傳遞訊息、或以非同步性神經傳導物質釋放來調控突觸後刺激性（postsynaptic excitability）之重要變因。在電流箝制實驗中，由電刺激所引起的非同步動作電位可由慢性鈣離子螯合劑EGTA-AM抑制之，顯示非同步性神經傳導物質不僅可改變突觸後之活性，其累積也具直接傳遞神經訊息之功能。
　　藉由喜八辛（himbacine）在不同濃度下可抑制不同亞型之蕈毒膽鹼性受器的特性，腳橋被蓋核區對A7細胞群之投射被認為是藉由活化類M1亞型受器所調控。然而，在高鈣環境下對腳橋被蓋核區進行電刺激時加入蕈毒膽鹼性受器拮抗劑─阿托品（atropine），反而提升了非同步性神經傳導物質之釋放，顯示應有他種蕈毒膽鹼性亞型受器參與調控，我們認為此亞型受器藉由抑制腳橋被蓋核區之麩胺酸性投射，達到調節下游A7細胞群之功能。適當濃度的大豆異黃酮（Genistein）具有抑制酪胺酸激酶（tyrosine kinase）之效果。文獻指出G蛋白受器也可能透過酪胺酸激酶的Src family（Src family of tyrosine kinases, SFKs）調控下游的基因表現。然而，在本實驗中大豆異黃酮無法影響由膽鹼性促效劑─碳醯膽鹼素（carbachol, CCh）所引發的內流電流，顯示腳橋被蓋核區之蕈毒膽鹼性受器並非藉由SFKs對下游之A7細胞群做調控。

Acetylcholine (ACh) is one of principal neurotransmitters involved in pain modulation. By behavioral researches, it has been found that there might be some supraspinal interactions between muscarinic cholinergic system and noradrenergic (NAergic) pain descending pathway. Moreover, morphological and electrophysiological data indicates that the cholinergic neuron group CH5, which distributes most of its neurons in pedunculopontine tegmental nucleus (PPTg), projects its fibers to downstream NAergic neurons of A7 catecholamine cell group, which further project NAergic fibers to the dorsal horn of the spinal cord to modulate nociceptive signaling. By electrically stimulating PPTg, glutamatergic projection to NAergic A7 neurons can be recorded and further inhibited by DNQX. In this study, excitatory postsynaptic currents (EPSCs) on NAergic A7 neurons evoked by electrical stimulation at PPTg were recorded under whole-cell patch clamp with membrane voltage at -70 mV. By application of solution with high extracellular calcium concentration, the proportion of asynchronous neurotransmitter release, which is believed to allows for the modulation of postsynaptic excitability and the alteration of action potential firing patterns increased. This result suggests that the change of extracellular calcium concentration, which in turn influences the calcium influx and neurotransmitter release, may provide a way for neural activity regulation. Under current-clamp mode, delayed stimulation-evoked action potentials was blocked by EGTA-AM, indicates that asynchronous neurotransmitter release could not only modulates neural activity but communicate with downstream neurons. Previous studies showed that PPTg might regulate downstream NAergic A7 neurons through M1-like mAChRs. However, application of atropine, a non-selective mAChR antagonist, increased stimulation-evoked asynchronous neurotransmitter release, suggesting that there might be other types of mAChR between PPTg and NAergic A7 neurons, and these mAChRs might modulate downstream NAergic A7 neurons by blocking the glutamatergic neurons projected from PPTg. By applying genistein with the concentration to be a tyrosine kinase inhibitor, the inward current induced by carbachol, a cholinergic agonist did not changed, indicating that the mAChRs might not transmit signals by activating the Src family of tyrosine kinases (SFKs).

致謝 i
摘要 iii
Abstract v
Introduction 1
Noradrenergic system in central nervous system 1
The role of A7 catecholamine cell group in descending pain modulation pathway 3
The cholinergic system in the central nervous system 5
Cellular response to activation of muscarinic acetylcholine receptors 7
Role of cholinergic mechanism and pedunculopontine tegmental nucleus (PPTg) in pain modulation 7
Diverse projections of PPTg fibers to NAergic A7 neurons 10
Characteristics of synchronous and asynchronous neurotransmitter release 10
Aim of the study 11
Materials and Methods 12
Preparation of rat brain stem slices 12
Whole-cell patch clamp recording 12
Measurement of synchronous and asynchronous neurotransmitter release 14
Drug 15
Filling recorded neurons with biocytin and immunohistochemistry 15
Statistical analysis 16
Results 17
Identification and recording of NAergic neurons of the A7 cell group in sagittal rat brainstem slices 17
Extracellular Ca2+ concentration played a role in the modulation to NAergic A7 neurons by trains of stimulations at Pedunculopontine tegmental nucleus (PPTg) 18
Accumulative asynchronous neurotransmitter release of PPTg neurons could induce action potentials of NAergic A7 neurons. 19
Asynchronous neurotransmitter release from PPTg to NAergic A7 neurons could be blocked by mAChR inhibitor. 20
Tyrosine kinases were not involved in the activity of mAChR on NAergic A7 neurons 21
Discussion 23
The concentration of extracellular calcium affects the distribution of stimulation-evoked neurotransmitter release. 23
Accumulation of asynchronous neurotransmitter release is able to induce action potentials on downstream neurons. 24
PPTg projects the cholinergic fibers to its own glutamatergic fibers. 25
Tyrosine kinases are not involved in the activity of mAChRs on NAergic A7 neurons. 26
References 28
Figures 45

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