辣椒素在大鼠中腦環導水管灰質產生止痛作用之細胞機轉-mGluR, endocannabinoids和GABA系統所扮演的角色

Abstract

Transient receptor potential vanilloid type 1 (TRPV1) channel 在周邊和中樞神經系統的疼痛調節上皆扮演重要的角色。在動物疼痛模式的實驗中觀測到，將Capsaicin 打入大鼠中腦環導水管灰質背側區 (vlPAG)會產生止痛效果。本實驗利用細胞膜箝制記錄的電生理方法觀測Capsaicin對於環導水管灰質背側區的神經突觸可塑性的影響，以解釋Capsaicin在中樞產生止痛的機轉。本實驗結果發現，在中腦環導水管灰質背側區的腦薄片給予Capsaicin 1~10μM會抑制此區的evoked GABAergic inhibitory postsynaptic currents (IPSCs)。此抑制現象可以被SB 366791 (TRPV1的拮抗劑)防止 (前處理)或反轉 (後處理)，可推知此抑制現像是經由活化TRPV1 channels而來。當我們在70ms的間隔下，快速給予成對的兩個刺激，Capsaicin可以增加雙刺激的促進效果 (Paired-pulse facilitation)，這個現象暗示了其作用效果主要位於突觸前。由於在2002年有報導指出Capsaicin在環導水管灰質背側區引起的止痛反應可以被group Ⅰ metabotropic glutamate receptors (mGluRs) 所抑制。因此我們接著探討mGluRs在這裡和Capsaicin的交互作用。Capsaicin對於IPSCs的抑制現象可以被mGluR5 的拮抗劑 MPEP (2-methyl-6- (phenylethynyl) pyridine hydrochloride)反轉，而mGluR1拮抗劑CPCCOEt (7-(hydroxyimino) cyclopropa[b]chromen -1a-carboxylate ethyl ester)無法產生反轉。由此可知mGluR5是Capsaicin下游產生作用的主要調控者。在miniature EPSCs的實驗中，Capsaicin增加其頻率，但不影響其大小。此結果進一步說明了上述mGluR5的活化應是由Capsaicin釋放的glutamate引發的。在許多腦區，活化group I mGluRs 皆會抑制GABAergic transmission。這個現象目前已被廣泛接受是由Gqprotein-phospholipase C (PLC)-diacylglyceral (DAG)-DAG lipase的訊息傳遞路徑產生endocannabinoids。 Endocannabinoids接著逆行活化突觸前的Cannabinoid receptors subtype 1 (CB1)，而造成IPSCs的抑制現象。我們觀察到Capsaicin所抑制的IPSCs在AM251 (CB1拮抗劑)的作用下可以被反轉。上述的實驗結果顯示Capsaicin在中腦環導水管灰質背側區活化位於glutamatergic terminals的TRPV1 channels，促進glutamate釋放，進而活化mGluR5和其下游的Gqprotein- PLC- DAG-DAG lipase 傳導路徑，促進endocannabinoids的生成 ;再透過endocannabinoids逆行活化突觸前的CB1 cannabinoid receptors來抑制此區的GABAergic transmission。在evoked postsynaptic potentials (PSPs)的實驗中，Capsaicin在類似生理情況的條件下促進了中腦環導水管灰質背側區神經細胞的整體興奮性，亦即活化了這個下行性的疼痛抑制路徑 (descending pain inhibitory pathway)。本實驗結果提出一個嶄新的細胞內機轉來解釋Capsaicin在中樞神經系統所產生的止痛作用。

The transient receptor potential vanilloid type 1 (TRPV1) channel plays a role in pain modulation not only at the periphery, but also in the brain. It is a nonselective cation channel and can be activated by an irritant plant product, capsaicin, as well as the endovanilloid, anadamide (AEA). It was reported that capsaicin elicit antinociceptive response in vivo by activating the TRPV1 channels in the ventrolateral periaqueductal gray (vlPAG), a key midbrain region for initiating the descending pain inhibitory pathway. We, therefore, investigated how capsaicin, through activating TRPV1 channels, affects synaptic transmission in the vlPAG to produce its antinociception using visualized whole-cell patch clamp recording technique in brain slices containing the vlPAG. Capsaicin (1-10 μM) concentration-dependently depressed evoked GABAergic inhibitory postsynaptic currents (IPSCs) in vlPAG slices. This effect was competitively reversed and prevented by SB 366791, a selective TRPV1 antagonist. Capsaicin-induced depression of IPSCs was accompanied with an increase of the paired-pulse facilitation ratio, suggesting a presynaptic site of action. Capsaicin has been reported to facilitate neurotransmitter release via activating presynaptic TRPV1 channels and its central antinociceptive effect was blocked by the antagonists of group I metabotropic glutamate receptor (mGluR). We, therefore, examined if mGluRs were involved in the effect of capsaicin (3μM). Indeed, capsaicin-induced IPSC depression was reversed by 2-methyl-6- (phenylethynyl) pyridine hydrochloride (MPEP, 10 μM), a selective mGluR5 antagonist, but not by 7-(hydroxyimino) cyclopropa[b]chromen-1a- carboxylate ethyl ester (CPCCOEt, 10μM) a mGluR1 antagonist,. This suggests capsaicin has to facilitate glutamate release in order to activate mGluRs. This suggestion was supported by the finding that capsaicin dramatically increased the frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs). It has been demonstrated that activation of group I mGluRs, through the Gq-protein-phospholipase C (PLC)-diacylglyceral (DAG)-DAG lipase pathway, leads to synthesis of endocannabinoids which retrogradely inhibited GABAergic transmission in many brain regions, including the vlPAG. We, therefore, examine if endocannabinoids were involved in the action of capsaicin. We did find capsaicin-induced IPSC depression was reversed by AM251, a CB1 cannabinoid receptor antagonist. Furthermore, capsaicin produced an overall excitatory effect on the neuronal activity of vlPAG neurons when evoked postsynaptic potentials were recorded in the current clamp mode. This suggests that capsaicin can increase vlPAG neuronal excitability in physiological condition through this mechanism. It is concluded that capsaicin activates the TRPV1 channels at glutamatergic terminals to facilitate the release of glutamate which subsequently activates postsynaptic mGluR5. Activation of mGluR5 leads to synthesis of endocannabinoids, which retrogradely activates the CB1 receptors at nearby GABAergic terminals to inhibit GABAergic transmission in the vlPAG. The eventual effect gives rise to activation of descending pain inhibitory pathway, producing antinociception. This study reveals a novel cellular mechanism for the central antinociceptive action of capsaicin. To the best of our knowledge, this is the first study disclose the interplay between vanilloids and endocannabinoids, two pain-regulating players, in a pain-regulating brain locus.