長期壓力導致過度疼痛之機制：環導水管灰質中內生性大麻酯所媒介之去極化壓抑抑制作用缺損

A mechanism of chronic stress-induced hyperalgesia: Impairment of endocannabinoid-mediated depolarization-induced suppression of inhibition in the periaqueductal gray

莊曜安(Yao-An Chuang)

指導教授 ： 邱麗珠

國立臺灣大學/醫學院/藥理學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201610148

摘要

內生性大麻酯 (endocannabinoids; eCBs)系統是由大麻酯1受體 (cannabinoid 1 receptors; CB1Rs)，大麻酯2受體 (cannabinoid 2 receptors; CB2Rs)以及其內生性之配體花生四烯乙醇胺 (anandamide; AEA)，花生四烯酸甘油酯 (2-arachidonoyl glycerol; 2-AG)所組成，而且，經由神經解剖的方式驗證了eCBs系統對於痛的刺激具有調節的功能。CB1Rs分布於許多腦區，包括：杏仁核 (amygdale)、海馬迴 (hippocampus)、小腦 (cerebellum)以及中腦環導水管灰質 (periaqueductal gray; PAG)。在cerebellum以及hippocampus中， eCBs已被證實在突觸間作為逆行信使，具有調節突觸間訊息傳導之功能。在大腦中，去極化壓抑抑制作用 (Depolarization-induced suppression of inhibition; DSI)是一種典型的以電生理學的方式去探討eCBs功能的例子。當突觸後神經元發生動作電位或去極化時，鈣離子流入，使胞內鈣離子濃度上升，誘發eCBs從突觸後神經元細胞膜的合成及釋放，隨後作用於位在突觸前神經元末梢的CB1Rs，抑制氨基丁酸 (aminobutyric acid; GABA)的釋放，進而降低對突觸後神經元的抑制性傳導訊息的輸入，此稱為DSI。直到現在，已在許多腦核區發現DSI的現象，包括：紋狀體 (striatum)、黑質 (substantia nigra)、amygdala以及下視丘 (hypothalamus)，但還未在PAG上探討。另一方面，許多研究指出，在長期接受壓力的情況下，會導致許多腦核區內eCBs訊息傳導的改變，包括：海馬迴 (hippocampus) 、前額葉 (prefrontal cortex)、striatum、丘腦 (thalamus)以及PAG，且伴隨著對於疼痛感受性的改變。因此，我們在這篇論文中透過電生理以及行為等研究法，探討在大鼠的掌管疼痛的中腦環導水管灰質腹外側區 (venrolateral periaqueductal gray; vlPAG)中是否存在由eCBs所調控的DSI現象，且在大鼠接受長期壓力之下，DSI如何被改變以及對於疼痛感受性的變化為何。
首先，我們發現在被記錄的vlPAG神經元上分別給予一個1、3、5秒的－70至0毫伏特的去極化後，發現IPSCs隨即被抑制，而且此抑制的程度大小與去極化的時間長短成正相關，代表DSI現象確實存在於vlPAG中。在mIPSCs的實驗中，我們發現去極化會減少mIPSCs的頻率但不改變振福，代表DSI的作用是經由突觸前的機制而來。接著，在大鼠腦薄片分別加入CB1R拮抗劑AM251、DAGL抑制劑THL、L型鈣離子通道阻斷劑nifedipine或PLC抑制劑U73122皆可顯著抑制DSI現象，代表在vlPAG中，DSI的作用是經由Ca2+–PCL–DAGL的途徑，進而合成2-AG而來。第二，在我們給予大鼠一個每天1小時且連續10天的束縛壓力(retrain stress)後，發現DSI顯著的減少。然而此現象即使再給予一個較密集的刺激，即給予連續3個去極化，也無法回復。此外，在大鼠腦薄片加入CB1R致效劑WIN 55,212-2後，發現在給予壓力的組別中，WIN 55,212-2抑制IPSCs的效果與控制組相比有顯著性的降低。這些結果指出在長期壓力下，vlPAG中的CB1Rs功能下降，進而導致DSI現象的損害。第三，在腳掌熱痛行為測試 (hot-plate test)的實驗中，我們發現在給予壓力的組別其縮腳潛伏期 (withdrawal latency)的時間與控制組相比有顯著性的降低。接著，以腹腔注射WIN 55,212-2，發現其止痛效果在給予壓力的組別也有顯著性的較差。這些結果指出vlPAG中的CB1Rs功能下降，進而導致DSI現象的損害可能為暴露在長期壓力下的大鼠產生過度疼痛且對於大麻酯類止痛劑效力降低的原因之一。
本篇論文的實驗結果指出經由Ca2+–PCL–DAGL的途徑，進而合成2-AG所媒介的DSI現象存在於大鼠的vlPAG中。而大鼠連續暴露在束縛壓力下，發現vlPAG中的CB1Rs功能下降，進而導致DSI現象的損害，以及過度疼痛且對於大麻酯類止痛劑效力降低的現象。這些發現與先前被提出的概念─長期壓力導致過度疼痛 (chronic stress-induced hyperalgesia)一致，且在vlPAG中的DSI現象可能在其中扮演重要的角色。

關鍵字

內生性大麻酯 ； 中腦環導水管灰質 ； 慢性壓力

並列摘要

The endocannabinoid (eCB) system consists of cannabinoid 1 receptors (CB1Rs) and CB2 receptors (CB2Rs), and their endogenous ligands, such as N-arachidonylethanolamide (anandamide) and 2-arachidonoyl glycerol (2-AG), and is present throughout the central nervous system, including the ventrolateral periaqueductal gray (vlPAG), an midbrain region for initiating desceding pain inhibition. eCBs are synthesized on demand and, especially 2-AG, are believed to function as retrograde inhibitory messengers at glutamatergic and GABAergic synapses in the brain. Depolarization-induced suppression of inhibition (DSI) is a classical and original electrophysiological example of eCB function in the brain. It is initiated postsynaptically by an elevation of intracellular Ca2+ concentration following action potential firing or depolarization, and is expressed presynaptically as a suppression of the GABAergic transmission through activating the CB1R on presynaptic GABAergic terminals. DSI has been reported in various brain regions such as the hippocampus, cerebellum, striatum, substantia nigra, amygdala, and hypothalamus, but not yet in the PAG. On the other hand, it has been reported that repeated exposures to stress modulates pain responses and alters eCB signalings in several brain regions, including the PAG. We, therefore, investigated whether eCB-mediated DSI exists in the vlPAG and is involved in pain regulation, and how chronic stress affects DSI in the vlPAG and the nociceptive response in the rat hot-plate test.
Visualized whole cell patch-clamp recordings were conducted in vlPAG slices of rats (P14-P18). Inhibitory postysynaptic currents (IPSCs) and excitatory PSCs (EPSCs) evoked at 0.05 Hz (but evoked at 0.33 Hz in DSI and DSE experiments) were recorded at -70 mV. IPSCs and EPSCs were recorded in the presence of 2 mM kynurenic acid and 10 M bicuculline, respectively. Miniature IPSCs (mIPSCs) were recroded in the presence of 1 M tetrodotoxin and kynurenic acid. The hot-plate test was used to measure the nociceptive threshold and the antinociceptive response of rats.
First, we found that when the membrane potential of the recorded vlPAG neuron was depolarized from －70 to 0 mV for 1, 3, 5 s, IPSCs were suppressed in a manner depending on the duration of depolarization, suggesting DSI indeed can be induced in vlPAG slices. The postsynaptic depolarizing pulse (-70 to 0 mV, 5 s) that reliably induced DSI significantly reduced the frequency, but not amplitude, of mIPSCs, suggesting that DSI in vlPAG slices is mediated through a pre-synaptic mechanism. When the vlPAG slice was in the presence of AM251, a CB1R antagonist, tetrahydrolipstatin (THL) that inhibits 2-AG synthesizing enzyme, diacylglycerolipase (DAGL), nifedipine, an inhibitor of L-type voltage-dependent calcium channels (L-VDCC), or U73122, a PLC inhibitor, DSI was significantly abolished. These results suggest that DSI in the vlPAG is an eCB-mediated retrograde inhibition of IPSCs. This eCB is 2-AG that was synthesized via a Ca2+–phospholipase C–DAGL enzymatic cascade, which is activated postsynaptically following depolarization-induced L-VDCC activation.
When the rat was exposed to a restraint stress for 1 hour every day for 10 consecutive days, the DSI in the vlPAG was significantly decreased, even a more intensive stimulation (with 3 depolarizing pulses) was given. In the stress group, WIN 55,212-2, a CB1R agonist, produced a smaller IPSC depression in vlPAG slices than in the naïve group. These results suggest that chronic stress impairs DSI in the rat vlPAG, and this may result from a hypofunction of CB1Rs in the vlPAG. In the stress group of rats, the hot-plate withdrawal latency was significantly lower than the naïve group. Besides, the antinociception effect of WIN 55,212-2 (i.p., 2 mg/kg) in the stress group was also significantly smaller than naïve rats, suggesting that the hypofunction of CB1R leads to the impairment of DSI in the vlPAG of rats after chronic stress may contribute to hyperalgesia and smaller sensitivity to the antinociception of cannabinoid, WIN 55,212-2.
In summary, we found that DSI exists in the vlPAG of rats and is mediated by 2-AG generated postsyantpically through a Ca2+–PCL–DAGL cascade following depolarization-induced L-VDCC activation. This DSI in the vlPAG may serve as an endogenous analgesic protector to maintain the function of descending analgesic pathway. Chronic stress can lead to an impairment of DSI and the decreased CB1R response in the vlPAG as well as a lower nociceptive threshold and a smaller antinocicpeitve effect of WIN 55,212-2. Thus, DSI impairment in the vlPAG, possibly due to down regulation of CB1Rs, may contribute to chronic stress-induced hyperalgesia.

並列關鍵字

endocannabinoid ； ventrolateral periaqueductal gray ； chronic stress ； depolarization-induced suppression of inhibition

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