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的理解不完全。TRP（V）离子通道在三叉神经感觉传导中起作用。TRPV 4是多模式激活的，例如通过机械、热和张力刺激，并且已经在三叉神经节感觉神经元及其外周投射中检测到trpv 4基因的产物。先前由PI产生的trpv 4-/-小鼠对于有害的机械和张力刺激具有升高的阈值。关键的问题仍然没有解决是否以及如何TRPV 4+三叉神经感觉神经元的刺激，引起伤害性行为。因此，本研究的目的是通过建立一种新的小鼠模型来研究frpv 4介导的三叉神经感觉神经元伤害性渗透和机械刺激转导的分子和细胞机制。待检验的假设是三叉神经感觉神经元中的trpv 4基因表达是否对伤害性刺激的神经感觉转导起关键作用。具体而言，（1）将通过膜片钳和Ca++成像研究来自trpv 4-/-小鼠的三叉神经元的原代培养物对张力和机械刺激的响应，所述张力和机械刺激由蛋白酶激活受体2（PAR-2）的激活（生理学相关的促痛觉通路）调节，以及它们对电压门控钠通道（Na）和辣椒素响应的“张力调节”;（2）与目的（1）相同，但对于来自感觉神经元特异性的、可诱导的trpv 4-/-小鼠的分离感觉神经元;（3）产生感觉神经元特异性的、可诱导的trpv 4-/-小鼠以测试这些小鼠对三叉神经外周的有害渗透刺激和辣椒素刺激的渗透调节（通过PAR-2的活化调节）的反应。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，该基因在小鼠的细菌T人工染色体转基因系中表达。对于目的（3），将对小鼠进行伤害反应行为测定和脑干三叉神经脊核对刺激的反应的c-FOS研究。因此，感觉转导和三叉神经伤害性刺激的传递对trpv 4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，将阐明trpv 4如何在辣椒素反应和INa-的“张力调节”中发挥作用。以及PAR-2对TRPV 4的调节。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伤害性刺激做出反应，以及trpv 4基因在这一过程中扮演的角色，有望为三叉神经介导的疼痛治疗开辟新的途径。

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.