Role of trpv4 in trigeminally mediated nociception

描述（由申请人提供）：三叉神经介导的疼痛疾病在分子水平上尚不完全了解。TRP(V)离子通道与三叉神经感觉传导有关。TRPV4是多模态激活的，如机械、热和强直刺激，TRPV4基因的产物在三叉神经节感觉神经元及其外周投射中被检测到。先前由PI产生的trpv4-/-小鼠对有害机械和强直刺激的阈值升高。TRPV4+三叉感觉神经元是否以及如何传导这种刺激引发伤害性行为的关键问题仍未解决。因此，本研究的目的是通过建立一种新的小鼠模型来研究frpv4介导的有害渗透和机械刺激在三叉感觉神经元中的传导的分子和细胞机制。要验证的假设是三叉神经感觉神经元中的trpv4基因表达是否对有害刺激反应的神经感觉传导至关重要。具体而言，(1)将通过膜片钳和Ca++成像研究trpv4-/-小鼠三叉神经原代培养对强直性和机械刺激的反应，由蛋白酶激活受体2 （PAR-2）的激活调节，这是一种生理相关的促痛觉途径，以及它们的电压门控钠通道（lNa）和辣椒素反应的“强直性调节”；(2)与Aim(1)相同，但用于从感觉神经元特异性，可诱导的trpv4-/-小鼠中分离的感觉神经元；(3)产生感觉神经元特异性的，可诱导的trpv4-/-小鼠，以测试这些小鼠对有害渗透刺激的反应和三叉神经外周辣椒素刺激的渗透调节，由PAR-2的激活调节。对于目的(1)-(2)，原代培养的神经元将被遗传编码的荧光报告基因标记，在细菌人工染色体转基因小鼠系中表达。对于Aim(3)，将对小鼠进行有害行为分析和脑干三叉脊髓核对刺激反应的c-FOS研究。因此，将在细胞水平上解构trpv4对三叉神经有害刺激的感觉传导和传递的依赖，阐明三叉神经感觉神经元的相对贡献。此外，还将揭示trpv4在辣椒素反应和INa-的“张力调节”中如何起作用。以及PAR-2对TRPV4的调制。因此，我们的新小鼠模型将帮助我们更好地了解三叉神经节神经元如何对有害刺激作出反应，以及trpv4基因在这一过程中发挥的作用，有望为治疗三叉神经节介导的疼痛开辟新的途径。

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.