Cannabinoid Signaling in the dPAG: Specific Analgesic and Autonomic Functions

描述(由申请人提供)： 神经性疼痛在退伍军人中很常见，严重阻碍了他们恢复功能的努力。已经确定了许多起作用的机制，但还没有带来任何新的治疗方法。初步观察表明，大麻类药物可能为新的治疗方法带来希望。内源性大麻素(ECs)是大麻素受体(CB1R)的内源性激动剂，越来越多的人认识到内源性大麻素(ECs)通过下行抑制感觉通路(抗伤害性感觉)参与疼痛的中枢调节，尽管其作用部位尚未确定。有证据表明，中脑导水管周围灰质背侧(DPAG)的EC系统可能参与了抗伤害性感觉。来自我们实验室的有希望的初步数据显示，在神经损伤后没有出现神经病理性疼痛的一组大鼠中，DPAG中的大麻素信号上调。因此，我们建议探索DPAG作为一个潜在的疼痛控制位点，并检验DPAG中EC系统的激活驱动下行止痛信号从而抑制神经病理性疼痛的总体假设。由于DPAG神经元的激活和EC调节可增加交感神经活动和血压，因此DPAG具有协调自主神经控制和镇痛机制的潜力。我们之前通过证明初始交感神经张力升高的大鼠在接受脊髓神经结扎(SNL)时不会出现痛敏反应，建立了神经病理性疼痛的易感性和自主神经激活之间的联系。在慢性疼痛的背景下，交感神经兴奋通常不是EC诱导的止痛所期望的副作用。因此，我们还将探索DPAG中的ECs选择性地调节自主神经激活而不是镇痛的具体机制。该计划被组织成三个具体目标。在特定的目标1中，我们将描述DPAG回路的解剖和药理学特征，该回路专用于抗伤害感受和交感兴奋。这些探索将在新的细节水平上提供对DPAG回路的关键见解，并确定背角感觉神经元(DHN)激活和交感神经流出的变化是否显示出差异控制的模式。抗伤害性感觉将被定义为DHN神经元活动的抑制，而交感兴奋将被记录为交感神经活动的增加。药物将被微量注射到DPAG中，以兴奋神经元并激活或减弱大麻系统功能。了解DPAG将抗伤害感受与交感兴奋相结合的机制将提供机会，将理想的大麻类抗伤害感受与不希望看到的心血管效应分开。在具体目标2中，我们将评估伤害诱导的DPAG大麻素系统各组成部分表达的变化。在SNL后表现出一系列痛觉过敏的大鼠身上，将描述DPAG对疼痛的特定分子后果。DPAG中大麻素系统成分的基因和蛋白表达水平将与个体大鼠痛觉过敏的发展程度相关。这些实验将检验这样的假设，即DPAG中上调的EC信号可以防止神经损伤后痛觉过敏的发展，这表明DPAG大麻素具有治疗潜力。最后，在具体目标3中，我们将确定DPAG大麻素系统在神经病理性疼痛发展中的作用。我们的初步发现支持DPAG大麻素信号在神经病理性疼痛的产生中的一个以前未被认识到的重要作用。在神经病理性疼痛模型SNL中，微量注射CB1R拮抗剂或通过RNA干扰下调CB1R可使DPAG中的大麻素系统功能急剧减弱，其影响与痛觉过敏程度有关。这些实验将验证这样一个假设，即DPAG中CB1R功能的丧失有助于神经病理性疼痛的发展。

DESCRIPTION (provided by applicant): Neuropathic pain is common among Veterans, substantially impeding their attempts to rehabilitate function. Numerous contributing mechanisms have been identified, but have not led to any new therapies. Initial observations show that cannabinoids may hold promise for new therapeutic approaches. There is growing recognition of the participation of endocannabinoids (ECs), which are endogenous agonists of cannabinoid receptors (CB1R), in the central regulation of pain by descending inhibition of sensory pathways (antinociception), although their site of action has not been determined. There is evidence that the EC system in the dorsal periaqueductal gray (dPAG), a key pain regulatory center, may contribute to antinociception. Promising preliminary data from our lab shows an upregulation of cannabinoid signaling in the dPAG in a subset of rats that did not develop neuropathic pain after nerve injury. Therefore, we propose to explore the dPAG as a potential locus for pain control, and test the overall hypothesis that activation of the EC system in the dPAG drives descending analgesic signaling that suppresses neuropathic pain. The dPAG has the potential to coordinate analgesic mechanisms with autonomic control since activation and EC-modulation of dPAG neurons increases sympathetic nerve activity and blood pressure. We have previously established a link between susceptibility to development of neuropathic pain and autonomic activation by showing that rats with elevated initial sympathetic tone do not develop hyperalgesia when subjected to spinal nerve ligation (SNL). Sympathoexcitation is typically not a desired side effect of EC-induced analgesia in the setting of chronic pain. We will therefore also explore specific mechanisms whereby ECs in the dPAG could selectively regulate autonomic activation versus analgesia. The program is organized into three Specific Aims. In Specific Aim 1, we will characterize anatomic and pharmacologic features of dPAG circuitry specific to antinociception and sympathoexcitation. These explorations will provide critical insights into dPAG circuitry at a novel level of detail and establish whether changes in dorsal horn sensory neuron (DHN) activation and sympathetic outflow show patterns of differential control. Antinociception will be defined by inhibition of DHN neuron activity while sympathoexcitation will be recorded as elevated sympathetic nerve activity. Agents will be microinjected into the dPAG that excite neurons and activate or attenuate cannabinoid system function. An understanding of the dPAG mechanisms that integrate antinociception with sympathoexcitation will provide opportunities to dissociate the desirable cannabinoid antinociception from unwanted cardiovascular effects. In Specific Aim 2, we will evaluate injury-induced changes in the expression of components of the dPAG cannabinoid system. Specific molecular consequences underlying dPAG contributions to pain will be characterized in rats showing a range of hyperalgesia after SNL. Gene and protein expression levels of components of the cannabinoid system in the dPAG will be correlated with the degree to which hyperalgesia develops in individual rats. These experiments will test the hypothesis that upregulated EC signaling in the dPAG prevents the development of hyperalgesia following nerve injury, suggesting a therapeutic potential of dPAG cannabinoids. Finally, in Specific Aim 3, we will identify the role of the dPAG cannabinoid system in the development of neuropathic pain. Our preliminary findings support an important and previously unrecognized role of dPAG cannabinoid signaling in the generation of neuropathic pain. Cannabinoid system function in the dPAG will be attenuated acutely by microinjection of a CB1R antagonist or chronically by RNA interference to downregulate CB1R in rats undergoing SNL, a model of neuropathic pain, and the effects correlated with levels of hyperalgesia. These experiments will test the hypothesis that loss of CB1R function in the dPAG contributes to the development of neuropathic pain.