Neurotransmission in tastebuds

项目摘要 III型“突触前”味觉细胞对酸和一些咸刺激有动作电位和释放反应 神经递质与传入神经纤维的常规突触。有关发射器的身份， 但目前还不清楚。我们以前已经证明，所有的味觉刺激（包括酸和咸）都需要ATP 信号传导至神经P2 X受体以传递至传入纤维。尽管如此，从III型中释放的ATP 没有检测到细胞。相反，III型细胞释放5-羟色胺（5-HT），激活5-HT 3 受体的传入神经纤维，但既没有敲除或药理学抑制5-HT 3完全 消除了对酸或盐的反应，这表明其他神经递质也参与其中。中的一个问题 研究III型细胞的传播是用来激活这些细胞的传统刺激--酸， 盐和KCl --都对味蕾中的其他细胞类型有非特异性的影响。为了避免这个问题， 我们采用光遗传学策略，允许用光直接刺激III型细胞，而不是 化学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， pkd 2l 1作为Cre驱动程序。当这些Pkd 2l 1-Cre小鼠与“floxed”通道视紫红质（ChR 2）小鼠杂交时， ChR 2在PKD 2L 1免疫反应性味觉细胞中忠实表达，在其他味觉细胞中无脱靶表达。 味觉细胞类型将470 nm的光闪烁到鼓索中的舌反射反应上， 舌咽神经是健壮的和可重复的，类似于对酸和咸刺激的反应。在 我们将利用这些Pkd 2l 1-ChR 2小鼠和其他基因靶向小鼠来研究 这些细胞使用神经递质与神经系统和感知系统进行交流。 通过选择性刺激PKD 2l 1 III型细胞诱发的质量。目的1将研究ATP的作用， 其它神经递质（即，5-HT，GABA和NE）激活膝状体神经节神经元， 神经支配III型细胞。孤立的膝状体神经节细胞的钙成像将识别 激活这些神经元，鼓索和舌咽神经对光的反应在Pkd 2l 1- ChR 2小鼠将使我们能够测试其同源受体在体内的作用。目标2将解决感知 使用行为范例，通过刺激Pkd 2l 1-ChR 2细胞引起的质量和cFos激活将是 用于解决表达PKD 2L 1的III型细胞的中心代表。在目标3中，我们将测试 假设PKD 2L 1-III型细胞参与芽内（味蕾内）回路，导致 调节其他味觉品质，特别是由II型细胞转导的那些。这些研究的结果 将提供有关转导，传入神经传递和胚内的重要新信息， III型味觉细胞中的信号传导。

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.