Identification of the Kenyon Cell Neurotransmitter

 描述(申请人提供)：与哺乳动物的海马体相似，果蝇蘑菇体(MBS)对学习和记忆至关重要。令人惊讶的是，大脑皮层固有神经元--凯尼恩细胞(KCs)储存和释放的神经递质(S)尚不清楚。我们最近发现了一种新的囊泡神经递质转运蛋白portabella(PRT)，它在KCs中表达。我们建议使用PRT作为一种工具来识别储存在KCs中的神经递质。我们将使用两种互补的方法。在目标1中，我们将使用表达PRT的细胞进行体外转运分析。在目标1A中，我们将测试有无线电标记的考生的交通工具。AIM 1B描述了一种使用不需要放射性标记化合物的技术的后备计划。由于1B的风险比1A大，因此只有在目标1A和目标2都失败的情况下，才会采用1B。在目标2中，我们将使用电化学检测和质谱仪来检测分泌囊泡的管腔含量。在目标2a中，我们将对分泌囊泡进行生化分离。我们假设Prt在wt果蝇体内储存的小分子将在来自prt突变体的囊泡中减少。作为后备计划(目标2B)，我们将检测培养的KCs分泌囊泡的胞外释放内容。我们假设，由wt KCs释放到介质中的小分子不会被Prt突变神经元释放。在目标2(或1B)中确定的候选分子将像在目标1A中一样，在运输分析中直接进行测试。这些实验的结果将决定PRT底物。由于PrT是唯一在KCs中表达的囊泡性神经递质，这些实验将确定KCs中储存的神经递质(S)。

 DESCRIPTION (provided by applicant): Similar to the mammalian hippocampus, Drosophila mushroom bodies (MBs) are critical for learning and memory. Surprisingly, the neurotransmitter(s) stored and released by the intrinsic neurons of the MBs, the Kenyon cells (KCs) are not known. We recently identified a novel vesicular neurotransmitter transporter, portabella (prt) that is expressed in KCs. We propose to use prt as a tool to identity the neurotransmitter stored in KCs. We will use two complementary methods. In Aim 1, we will perform in vitro transport assays using cells that express prt. In Aim 1A, we will test transport o radiolabeled candidates. Aim 1B describes a backup plan using a technique that does not require radiolabeled compounds. Since 1B is more risky than 1A it will only be employed if both Aim 1A and Aim 2 fail. In Aim 2 we will assay the lumenal contents of secretory vesicles using electrochemical detection and mass spectrometry. In Aim 2A we will biochemically fractionate secretory vesicles. We hypothesize that the small molecule stored by prt in wt flies will be reduced in vesicles derived from the prt mutant. As a backup plan (Aim 2B), we will assay the exocytotically released contents of secretory vesicles from cultured KCs. We hypothesize that a small molecule released into the media by wt KCs will not be released by prt mutant neurons. Candidate molecules identified in Aim 2 (or 1B) will be directly tested in transport assays as in Aim 1A. The results of these experiments will determine the prt substrate. Since prt is the only vesicular neurotransmitter that is expressed in KCs, these experiments will identify the neurotransmitter(s) that is stored in KCs.