Neurotransmission in tastebuds

项目总结 III型“突触前”味觉细胞通过动作电位对酸味和盐味刺激作出反应并释放 常规突触与传入神经纤维的神经递质。所涉及的发射机的身份， 然而，目前仍不清楚。我们以前已经证明，所有的味觉刺激(包括酸和咸)都需要三磷酸腺苷 向神经P2X受体发出信号，以传递到传入纤维。尽管如此，从III型释放的ATP 尚未检测到细胞。相反，III型细胞确实会释放5-羟色胺(5-HT)，从而激活5-HT3 传入神经纤维上的受体，但不能完全敲除5-HT3，也不能完全抑制5-HT3 消除对酸或盐的反应，表明其他神经递质参与其中。有一个问题是 研究III型细胞的传播是用来激活这些细胞的常规刺激--酸， 盐和KCl--对味蕾中的其他细胞类型都有非特异性的影响。为了绕过这个问题， 我们采用一种光遗传策略，允许用光直接刺激III型细胞，而不是 化学制品。我们开发了一种在III型味觉细胞中选择性表达Cre重组酶的小鼠，依赖于 PKD2L1作为CRE驱动程序。当这些PKD2L1-Cre小鼠与通道视紫红质(ChR2)小鼠杂交时 ChR2在对PKD2L1免疫反应阳性的味觉细胞中得到忠实表达，在其他味觉细胞中无非靶向表达 味觉细胞类型。用470 nm的光照射舌头可引起鼓索和 舌咽神经强健且可重复，类似于对酸和咸刺激的反应。在……里面 在这项建议中，我们将利用这些PKD2L1-ChR2小鼠加上其他基因靶向的小鼠来研究 这些细胞用来与神经系统沟通的神经递质，以及知觉 选择性刺激PKD2L1III型细胞的特性。目标1将调查ATP的作用和 其他神经递质(即5-羟色胺、GABA和NE)在激活膝状神经节神经元时选择性地 支配III型细胞。对分离的膝状神经节细胞进行钙成像将识别 激活这些神经元，鼓索和舌咽神经对光的反应在PKD2L1- ChR2小鼠将允许我们在体内测试它们的同源受体的作用。目标2将解决知觉问题 使用行为范式刺激PKD2L1-ChR2细胞所引起的质量，以及CFOS的激活 用于解决表达PKD2L1的III型细胞的中心表达。在目标3中，我们将测试 假设PKD2L1-III型细胞参与宝石内(味蕾内)环路，导致 其他口味的调节，特别是那些由类型II细胞转导的味道。这些研究的结果 将提供有关转导、传入神经传递和宝石内的重要新信息。 III型味觉细胞中的信号。

PROJECT SUMMARY Type III “presynaptic” taste cells respond to sour and some salty stimuli with action potentials and release neurotransmitter at conventional synapses with afferent nerve fibers. The identity of the transmitters involved, however, is still unclear. We have shown previously that all taste stimuli (including sour and salty) require ATP signaling to neural P2X receptors for transmission to afferent fibers. Nonetheless, ATP release from Type III cells has not been detected. Conversely, Type III cells do release serotonin (5-HT) which activates 5-HT3 receptors on afferent nerve fibers but neither knockout nor pharmacological inhibition of 5-HT3 completely eliminates responses to acids or salts, suggesting other neurotransmitters are involved. One problem in studying transmission from Type III cells is that the conventional stimuli used to activate these cells -- acids, salts, and KCl -- all have non-specific effects on other cell types in the taste bud. To circumvent this problem, we employ an optogenetic strategy permitting direct stimulation of Type III cells with light rather than chemicals. We developed a mouse that expresses Cre recombinase selectively in type III taste cells relying on Pkd2l1 as a Cre driver. When these Pkd2l1-Cre mice are crossed with “floxed” channelrhodopsin (ChR2) mice ChR2 is faithfully expressed in taste cells immunoreactive for PKD2L1 with no off-target expression in other taste cell types. Flashing 470 nm light onto the tongue elicits responses in the chorda tympani and glossopharyngeal nerves that are robust and repeatable, resembling responses to sour and salty stimuli. In this proposal we will utilize these Pkd2l1-ChR2 mice plus other gene-targeted mice to investigate the neurotransmitters that are used by these cells to communicate with the nervous system, and the perceptual qualities evoked by selective stimulation of PKD2l1 Type III cells. Aim 1 will investigate the role of ATP and other neurotransmitters (i.e., 5-HT, GABA, and NE) in activating geniculate ganglion neurons that selectively innervate Type III cells. Calcium imaging of isolated geniculate ganglion cells will identify transmitters that activate these neurons, and chorda tympani and glossopharyngeal nerve responses to light in the Pkd2l1- ChR2 mice will allow us to test the role of their cognate receptors in vivo. Aim 2 will address the perceptual quality elicited by stimulation of Pkd2l1-ChR2 cells, using behavioral paradigms, and cFos activation will be used to address the central representation of PKD2L1-expressing Type III cells. In Aim 3 we will test the hypothesis that PKD2L1-Type III cells participate in an intragemmal (intra taste bud) circuit, resulting in modulation of other taste qualities, particularly those transduced by Type II cells. Results from these studies will provide important new information about transduction, afferent neurotransmission, and intragemmal signaling in Type III taste cells.