内源性大麻素系统介导的脊髓前馈抑制回路LTD在神经病理性痛觉超敏发生机制中的作用

Melzak and Wall (Science, 1965) proposed a well-known hypothesis, the gate theory of pain, which is highly influential for 50 years. However, the theory remains a conjecture because of insufficient evidence to substantiate the neural circuitry underlying pain signaling and modulation in the spinal dorsal horn. The applicant have reported the functional organization of nociceptive component in the gate theory related neuronal circuits (Lu et al. J Neurosci. 2003,2005). Our recent study (Lu et al. J Clin Invest. 2013) found that the spinal dorsal horn contains a feed-forward inhibitory circuit that can gate incoming low-threshold Abeta input to nociceptive pathway. This study identified a novel “gate” with definite morphological and functional details in the spinal dorsal horn, in which activation of low-threshold innocuous Abeta fibers in neuropathic pain condition “opens” the gate to elicit mechanical allodynia. Based on our previous findings and preliminary data, we propose herein a new hypothesis on how the “allodynia gate” works: The peripheral nerve injury elevates the content of endocannabinoids (eCBs) in spinal dorsal horn, which activats CB1 receptors on the spinal astrocyte and/or inhibitory neurons. The activated astrocyte releases glutamate and activates NMDA receptors on inhibitory neurons. NMDA receptor activation induces the endocytosis of postsynaptic glutamate AMPA receptors of inhibitory neurons. These molecular cascades ventually mediate the development of the eCB dependent LTD (eCB-LTD) in the feed-forward inhibitory circuit, and then, open the “allodynia gate” contributing to the development of neuropathic pain. The present proposal will utilize paired patch-clamp whole-cell recordings, morphological, molecular biological and behaviour techniques to study the mechanisms of eCB-LTD and its potential role in the development of neuropathic allodynia induced by peripheral nerve injury in SD rat and conditional CB1 KO mice.

疼痛闸门控制学说已发表50周年，其推测的脊髓闸门回路组成及功能尚未完全阐明。申请人在发表闸门控制回路中痛觉回路构成（J Neurosci. 2003,2005）之后，新近研究发现，脊髓后角前馈抑制回路可发挥“痛觉超敏闸门”作用，神经损伤可开启闸门，使触觉信息传递到痛觉通路产生痛觉超敏（J Clin Invest. 2013），丰富和发展了闸门控制学说。本课题提出“痛觉超敏闸门”开启机制假说：内源性大麻素（eCBs）激活星形胶质细胞或/和抑制性神经元的CB1受体，通过突触外NMDA受体活化诱发AMPA受体内吞，产生抑制回路特异性LTD，参与痛觉超敏形成。本课题拟用双膜片钳技术、形态学、分子生物学和行为学技术，以大鼠和细胞特异性CB1基因敲除小鼠为研究平台，深入探讨eCBs参与“痛觉超敏闸门”抑制性回路LTD机制，及其在痛觉超敏中的作用，为阐明神经病理性痛觉超敏的神经学机制提供结构与功能基础。

本课题联合应用膜片钳电生理学、形态学、分子生物学，化学遗传学，液相色谱-质谱联用（HPLC-MS/MS），在体脊髓基因调控技术，疼痛行为学等技术方法，以本课题组构建的15个品系的细胞特异性基因编辑小鼠为研究平台，系统阐明了内源性大麻素在脊髓“痛觉超敏闸门”的开启机制中的关键作用。脊髓后角星形胶质细胞和甘氨酸（Gly）神经元上的CB1受体的激活是“闸门”开启的始动因素，Gly神经元和PKCγ+神经元之间形成的前馈抑制回路的功能降低，造成PKC+神经元“脱抑制”，Aβ纤维介导的非伤害信息经由伤害性通路上传，最终产生痛觉超敏。 . 我们首先应用两种不同的方法验证了外周神经损伤后脊髓背角内Gly神经元，PKC+ 神经元和星形胶质细胞周围内源性大麻素显著升高，证明了痛觉超敏闸门开启的物质基础。我们发现构成“痛觉超敏闸门”的前馈抑制回路关键组成部分的PKCγ+ 神经元接受Aβ纤维介导的先兴奋后抑制的双向调控，而GlyT2+ 神经元主要接受Aβ纤维介导的兴奋性调控。然后发现脊髓背角内升高的内源性大麻素通过作用于GlyT2+ 神经元上的CB1R抑制GlyT2+-PKC++ 前馈抑制回路的甘氨酸释放，进而提高PKCγ+ 神经元的兴奋性，使得Aβ纤维介导的非伤害信息经由PKCγ+ 神经元传递到痛觉通路，最终导致痛觉超敏的发生。这个前馈抑制回路功能降低的机制是发生了大麻素介导的抑制性突触短时程和长时程抑制（eCBs-STD/LTD）。应用药理学和基因敲除技术证实闸门回路的eCBs-STD/LTD是发生神经病理性痛觉超敏的神经学基础。此部分实验验证了本课题假说1。. 假说2涉及了星形胶质细胞细胞上的CB1R的作用。星形胶质细胞细胞上的CB1R激活诱发星形胶质细胞释放谷氨酸，引起GlyT2+ 神经元上的GluR2内吞。 GluR2内吞是诱发GlyT2+-PKCγ+ 神经元前馈抑制回路突触可塑性发生长时程改变的另一个途径。.本研究为阐明神经病理性慢性疼痛的脊髓机制提供了结构与功能基础，特异性干预星形胶质细胞和脊髓前馈抑制回路上的CB1受体可能成为治疗神经病理性痛觉超敏的特异性药物靶点。. .