Separate populations of receptor cells and presynaptic cells in mouse taste buds
Separate populations of receptor cells and presynaptic cells in mouse taste buds
复制标题
DOI：
10.1523/jneurosci.0515-06.2006
复制
发表时间：
2006-04-12
影响因子：
5.3
通讯作者：
Chaudhari, N
中科院分区：
文献类型：
--
作者：
DeFazio, RA;Dvoryanchikov, G;Chaudhari, N
味蕾由 50-100 个细胞组成，其中只有一小部分表达味觉受体和细胞内信号蛋白的基因。我们将功能性钙成像与单细胞分子分析相结合，证明小鼠味蕾中存在两种不同的细胞类型。钙成像显示，分离的味觉细胞对促味剂或 KCl 去极化的响应是细胞质 Ca2+ 短暂升高，但不会同时响应两者。使用单细胞逆转录 (RT)-PCR，我们发现单个味觉细胞表达磷脂酶 C beta 2 (PLC beta 2)（一种重要的味觉转导效应子）或突触体相关蛋白 25 (SNAP25)（钙触发的递质胞吐作用的关键成分）。钙成像揭示的两个功能类别映射到单细胞 RT-PCR 确定的两个基因表达类别。具体而言，对促味剂做出反应的细胞表达 PLC beta 2，而对 KCl 去极化做出反应的细胞表达 SNAP25。我们通过两种方法证明了这一点：首先，通过连续钙成像和单细胞 RT-PCR；其次，通过对转基因小鼠切片中的味蕾进行钙成像，其中表达 PLC beta 2 的味觉细胞被绿色荧光蛋白标记。为了评估 SNAP25 表达细胞的重要性，我们使用单细胞的 RNA 扩增，然后进行 RT-PCR。我们发现SNAP25阳性细胞还表达典型的突触前蛋白，包括电压门控钙通道（α1A）、神经细胞粘附分子、突触蛋白-II以及神经递质合成酶谷氨酸脱羧酶和芳香氨基酸脱羧酶。通过扩增 RNA 分析或免疫细胞化学，未在 PLC beta 2 细胞中检测到突触标记。这些数据证明存在至少两种分子上不同的味觉细胞功能类别：受体细胞和突触形成细胞。
Taste buds are aggregates of 50-100 cells, only a fraction of which express genes for taste receptors and intracellular signaling proteins. We combined functional calcium imaging with single-cell molecular profiling to demonstrate the existence of two distinct cell types in mouse taste buds. Calcium imaging revealed that isolated taste cells responded with a transient elevation of cytoplasmic Ca2+ to either tastants or depolarization with KCl, but never both. Using single-cell reverse transcription (RT)-PCR, we show that individual taste cells express either phospholipase C beta 2 (PLC beta 2) ( an essential taste transduction effector) or synaptosomal-associated protein 25 (SNAP25) ( a key component of calcium-triggered transmitter exocytosis). The two functional classes revealed by calcium imaging mapped onto the two gene expression classes determined by single-cell RT-PCR. Specifically, cells responding to tastants expressed PLC beta 2, whereas cells responding to KCl depolarization expressed SNAP25. We demonstrate this by two methods: first, through sequential calcium imaging and single-cell RT-PCR; second, by performing calcium imaging on taste buds in slices from transgenic mice in which PLC beta 2-expressing taste cells are labeled with green fluorescent protein. To evaluate the significance of the SNAP25-expressing cells, we used RNA amplification from single cells, followed by RT-PCR. We show that SNAP25-positive cells also express typical presynaptic proteins, including a voltage-gated calcium channel (alpha 1A), neural cell adhesion molecule, synapsin-II, and the neurotransmitter-synthesizing enzymes glutamic acid decarboxylase and aromatic amino acid decarboxylase. No synaptic markers were detected in PLC beta 2 cells by either amplified RNA profiling or by immunocytochemistry. These data demonstrate the existence of at least two molecularly distinct functional classes of taste cells: receptor cells and synapse-forming cells.