臺灣博碩士論文加值系統

近期的研究顯示慢性疼痛可能和疼痛相關之大腦核區內的神經興奮性和突觸效力改變有關，即中樞敏感化。前腦島無顆粒皮質(RAIC)被發現在急性和慢性疼痛時都會被活化，且在我們之前的結果中發現肌肉痛小鼠的RAIC裡，神經活性提高的細胞大部分是錐狀細胞，極少數是抑制性中間神經元。因為RAIC中迴路的興奮性突觸訊號和抑制性突觸訊號不平衡會改變其疼痛訊息的輸出，所以此研究目的是比較肌肉痛小鼠和控制組的抑制性中間神經元到錐狀細胞的突觸效力是否不同。我先使用酸誘發肌肉痛模型，讓小鼠的兩隻後腳掌產生痛覺過敏的行為，再進一步用電生理結合光遺傳學的方式檢驗抑制性突觸的傳導。實驗結果顯示慢性疼痛小鼠的抑制性突觸後電流的paired-pulse ratio顯著地從控制組的0.66減少到0.37，且其突觸傳導的量子大小從控制組的12.88 pA增加到18.82 pA。這意味著在慢性疼痛的形成過程中，RAIC的抑制性中間神經元到第五層錐狀細胞的突觸功能產生變化。

It has been well demonstrated that the change of synaptic efficacy in neurocircuit of brain pain matrix is a cellular substrate for behavior hypersensitivity in animals with chronic pain. While most of previous studies focus on transmission at synapses between nociceptive inputs and principal neurons, the role of local GABAergic interneurons (IntNs) receives less attention. I address this issue by using the acid-induced muscle pain animal model (AIMP model) in mice and focusing on the rostral agranular insular cortex (RAIC). The RAIC is an important component of brain pain matrix as this cortical area is shown to tonically produce hyperalgesia signal and is a cortical area where nociceptive output originates. We propose that repeated acid saline injection may trigger a plastic change in synaptic efficacy of GABAergic IntNs onto pyramidal neurons (PNs) and cause an excitatory/inhibitory imbalance in neurocircuit in RAIC, which in turn alters cortical output of nociceptive signal in chronic pain. To test this possibility, dual-patch recording from a pair of IntN-PN in layer 5 was initially used to record unitary inhibitory postsynaptic current (IPSC) in previous experiments of our lab, and found that only 30% of all recorded IntN-PN pairs showed functional connectivity. To increase successful rate, here I employ optogenetic method to selectively active GABAergic IntNs. I injected a cre-dependent AAV that carries eYFP and channelrhodopsin2 sequences into RAIC in transgenic mice, in which the promoter of vesicular-GABA-transporter controls expression of cre recombinase. The animals were killed 2-3 weeks after AAV injection for brain slice preparation and whole-cell patch recording was made from PNs. Illuminating the slice with a single blue-light pulse (2 ms) evoked inhibitory postsynaptic current (IPSC) in PNs that was blocked by 20 uM bicuculline, a GABAA receptor antagonist. The paired-pulse ratio of the IPSC significantly reduced from 0.66 ± 0.10 (n = 13) in control mice to 0.37 ± 0.03 (n = 12) in muscle pain mice (P < 0.05; Mann-Whitney Test); the quantal size of the IPSC was significantly increased from 12.88 ± 1.22 pA (n = 13) in control mice to 18.82 ± 1.91 (n = 12) pA in muscle pain mice (P < 0.05; Mann-Whitney Test). These results show potential changes in synaptic function of GABAergic IntNs onto PNs in RAIC in chronic pain condition.

致謝 i
中文摘要 iii
Abstract iv
Content vi
List of Figures vii
Chapter 1 Introduction 1
1.1 Pain 1
1.2 Chronic muscle pain 2
1.3 Muscle pain pathway 3
1.4 Known mechanisms of chronic muscle pain by acid-induced muscle pain (AIMP) model 5
1.5 Rostral agranular insular cortex 8
1.6 Central sensitization and synaptic transmission 9
Chapter 2 Materials and Methods 12
2.1 Animals 12
2.2 Stereotaxic surgery for AAV injection 12
2.3 Acid-induced muscle pain model and behavioral test 13
2.4 Brain slice preparation 14
2.5 Whole-cell recording 15
2.6 Optogenetic stimulation 16
2.7 Drugs 16
2.8 Immunohistochemistry 17
2.9 Data analysis 17
2.10 Statistical analysis 18
Chapter 3 Results 19
3.1 Mechanical hyperalgesia of acid-induced muscle pain mice 19
3.2 The synaptic transmissions from GABAergic interneurons to pyramidal cells were investigated by paired whole-cell recording in muscle pain mice. 20
3.3 Monosynaptic IPSC of a pyramidal cell induced by photostimulation of GABAergic interneurons. 21
3.4 Increased quantal size of postsynaptic inhibitory current from GABAergic interneurons→ pyramidal cells in AIMP mice. 23
3.5 Increased release probability of GABAergic interneurons→ pyramidal cells in AIMP mice. 23
Chapter 4 Discussion 25
4.1 The appropriateness of using AIMP model to investigate widespread chronic muscle pain 25
4.2 The implied physiological explanations of synaptic plasticity in insular cortex in muscle pain mice 26
4.3 Diverse GABAergic neurons target to pyramidal cells 28
4.4 Significance and future works 29
Chapter 5 Reference 30

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