Role of trpv4 in trigeminally mediated nociception

描述(申请人提供)：三叉神经介导的疼痛障碍在分子水平上还没有完全被理解。Trp(V)离子通道在三叉神经感觉传导中起重要作用。TRPV4可被机械刺激、热刺激和紧张性刺激等多种方式激活，在三叉神经节感觉神经元及其周围投射中检测到TRPV4基因的产物。先前由PI产生的TRPV4-/-小鼠对有害的机械刺激和紧张性刺激具有更高的阈值。关键问题仍未解决，TRPV4+三叉神经感觉神经元是否以及如何转导这种刺激，从而引发伤害性行为。因此，本研究的目的是通过建立一种新的小鼠模型，研究Frpv4介导的伤害性渗透和机械刺激在三叉神经感觉神经元中转导的分子和细胞机制。有待检验的假设是，TRPV4基因在三叉神经感觉神经元中的表达是否对伤害性刺激反应的神经感觉转导起关键作用。具体地说，(1)TRPV4-/-小鼠三叉神经细胞的原代培养将通过膜片钳和钙成像来研究其对张力和机械刺激的响应，受生理上相关的前痛觉通路--蛋白酶激活受体2(PAR-2)的激活及其电压门控钠通道(LNA)和辣椒素反应的“张力调节”的调节；(2)与AIM(1)相同，但从感觉神经元特异性的、可诱导的TRPV4-/-小鼠分离的感觉神经元；(3)将建立感觉神经元特异性、可诱导的TRPV4-/-小鼠，以测试这些小鼠对伤害性渗透刺激和三叉神经外周辣椒素刺激的渗透调节的反应，这些刺激受PAR-2的激活调节。对于AIMS(1)-(2)，原代培养的神经元将被一个遗传编码的荧光报告基因标记，该基因在细菌人工染色体转基因小鼠系中表达。对于Aim(3)，小鼠将接受对刺激反应的脑干三叉神经脊束核的积极行为分析和c-fos研究。因此，三叉神经伤害性刺激的感觉转导和传递对TRPV4的依赖性将在细胞水平上被解构，从而阐明三叉神经感觉神经元的相对贡献。此外，还将阐明TRPV4如何在辣椒素反应和INA-的“调谐”中发挥作用。以及PAR-2对TRPV4的调控作用。因此，我们的新小鼠模型将有助于我们更好地了解三叉神经节神经元对伤害性刺激的反应以及TRPV4基因在这一过程中所起的作用，有望为三叉神经痛的治疗开辟新的途径。

DESCRIPTION (provided by applicant): Trigeminally-mediated pain disorders are incompletely understood at the molecular level. TRP(V) ion channels have been implicated to function in trigeminal sensory transduction. TRPV4 is multimodally activated, e.g. by mechanical, thermal and tonicity stimuli, and products of the trpv4 gene have been detected in trigeminal ganglion sensory neurons and their peripheral projections. trpv4-/-mice, previously generated by the PI, have an elevated threshold for noxious mechanical and tonicity stimuli. The critical question remains unresolved whether and how TRPV4+ trigeminal sensory neurons transduce such stimuli that elicit nociceptive behavior. Thus, the objective of this proposal is to investigate molecular and cellular mechanisms of frpv4-mediated transduction of noxious osmotic and mechanical stimuli in trigeminal sensory neurons by development of a novel mouse model. The hypothesis to be tested is whether trpv4 gene expression in trigeminal sensory neurons is critical for neurosensory transduction in response to noxious stimuli. Specifically, (1) primary cultures of trigeminal neurons from trpv4-/- mice will be investigated by patch-clamp and Ca++ imaging in response to tonicity and mechanical stimuli, modulated by activation of proteinase-activated receptor 2 (PAR-2), a physiologically relevant pro-algesic pathway, and for their "tonicity-tuning" of voltage-gated sodium channels (lNa) and capsaicin-response; (2) same as in Aim (1), but for dissociated sensory neurons from sensory-neuron-specific, inducible trpv4-/- mice; (3) sensdry-neuronspecific, inducible trpv4-/- mice will be generated to test the response of these mice to noxious osmotic stimuli and osmotic modulation of capsaicin stimuli of trigeminal peripherals, modulated by activation of PAR-2. For Aims (1)-(2), primary cultured neurons will be labeled by a genetically-encoded fluorescent reporter gene, expressed in a bacterialTartificial chromosome transgenic line of mice. For Aim (3), mice will be subjected to nocifensive behavioral assays and c-FOS studies of the brain stem trigeminal spinal nucleus in response to stimulation. Thus, the dependence of sensory transduction and transmission of noxious trigeminal stimuli on trpv4 will be deconstructed at the cellular level, clarifying the relative contribution of the trigeminal sensory neuron. Moreover, light will be shed on how trpv4 functions in "tonicity-tuning" of the capsaicin response and of INa-. and on modulation of TRPV4 by PAR-2. Thus, our new mouse model will help us better understand how trigeminal ganglion neurons respond to noxious stimuli and which role the trpv4 gene plays in this process, hopefully opening up new avenues for treatment of trigeminally-mediated pain.