흰쥐 삼차신경절 신경세포에서 P2X3와 vesicular glutamate transporters의 발현양식 및 말초 염증에 따른 변화
Expression Patterns of P2X3 and Vesicular Glutamate Transporters in Rat Trigeminal Ganglion Neurons and Their Changes during Peripheral Inflammation
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일차감각신경세포에서 발현되는 P2X3의 활성화는 중추 및 말초 구심성신경섬유에서 glutamate 분비를 유도하며, 이는 급성 및 만성 통증에 관여한다. 그러나 P2X3 활성화와 관련된 glutamate 분비에 관여하는 vesicular glutamate transporters (VGLUTs)의 유형에 대해서는 거의 알려져 있지 않다. 본 연구에서는 정상 조건과 흰쥐의 vibrissa pad에 CFA를 주입하여 말초 염증 통증을 유발한 후, 삼차신경절 신경세포에서 P2X3, VGLUT1 및 VGLUT2의 발현을 면역형광염색법으로 조사하였다. 또한, 전자현미경을 사용하여 P2X3 면역양성 (+) 중추 신경종말의 초미세구조를 관찰하였다. P2X3+ 신경세포는 주로 작거나 중간크기이며, VGLUT1보다 VGLUT2를 함께 발현하였다. CFA 처리군에서는 대조군에 비해 P2X3+, VGLUT2+ 및 VGLUT2+/P2X3+ 신경세포의 수가 유의하게 증가하였다. 반대로, VGLUT1+ 및 VGLUT1+/P2X3+ 신경세포의 수는 CFA군과 대조군 사이에 유의한 차이를 보이지 않았다. 또한, 삼차신경꼬리핵에서 P2X3+ 축삭종말은 다양한 형태의 소포를 포함하는 축삭종말로부터 축삭 간 시냅스를 형성하였다. 이 결과는 1) 급성 및 염증성 통증 상태에서 P2X3 활성화와 관련된 glutamate 분비에는 VGLUT2가 관여하며, 2) 삼차신경꼬리핵에서 P2X3+ 일차구심성신경섬유는 다양한 연접이전 기전에 의해 정교하게 조절될 가능성이 있다는 것을 시사한다.
Abstract
Activation of purinergic receptor P2X3 expressed on the primary sensory neurons, induces the release of glutamate from their central and peripheral afferents, which is involved in both acute and chronic pain. However, little is known about the type of vesicular glutamate transporter (VGLUT) that is involved in the glutamate release associated with P2X3 activation. To address this issue, we investigated the expression of P2X3, VGLUT1, andVGLUT2 in rat trigeminal ganglion neurons by immunofluorescence under the normal condition and following inflammation induced by Complete Freund’s Adjuvant (CFA)-injection into the rat vibrissa pad. In addition, we examined the ultrastructure of P2X3-immunopositive (+) central terminals by electron microscopy. P2X3+ neurons were mostly small to medium-sized and frequently coexpressed VGLUT2, but rarely VGLUT1. In the CFA group, which exhibited thermal hyperalgesia, there was a significant increase in the number of P2X3+, VGLUT2+, andVGLUT2+/P2X3+ neurons compared with controls (P2X3+ neurons: 39.0% vs 28.4%, VGLUT2+ neurons:44.6% vs 34.9%, VGLUT2+/P2X3+ neurons: 33.8% vs 22.5%, p<0.05). Conversely, the numbers of VGLUT1+ and VGLUT1+/P2X3+ neurons did not significantly differ between the CFA group and controls (VGLUT1+ neurons: 28.8% vs 28.4%, VGLUT1+/P2X3+ neurons: 0.6% vs 0.5%). Additionally, P2X3+ axon terminals in the trigeminal caudal nucleus frequently received an axoaxonic synapse from an axon terminal containing pleomorphic vesicles, suggesting presynaptic modulation of P2X3-mediated sensory signals before synaptic transmission. These results indicate that 1) VGLUT2, rather than VGLUT1, is involved in glutamate release associated with P2X3 activation under acute and inflammatory pain conditions, and 2) P2X3+ primary afferents in the trigeminal caudal nucleus are finely regulated by various presynaptic mechanisms.
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