Opioids inside Organelles

生物膜对外源性阿片类药物--两种植物来源的分子--具有渗透性 比如吗啡，以及成百上千个合成分子。现在一种基因编码的 荧光生物传感器技术使我们能够测量中性细胞器中的阿片类物质，如 内质网(ER)。我们称这些分子为基于强度的阿片类药物敏感 荧光记者，iOpioidSnFR，家族(图1)。正在进行的实验，在项目之前 开始，将把iOpioidSnFR家族扩展到主要类别的微阿片激动剂。 AIM 1进一步扩展了iOpioidSnFR，用于酸性细胞器中的测量，如 内小体和突触小泡。Aim 1a利用目前的循环置换绿 荧光蛋白(CpGFP)部分。目的1b开发新型循环排列的HaloTag，它 对pH不敏感。目标1c，扩大现有措施，以衡量阿片类药物的进入 进入细胞器，以及它们离开细胞器。量化既涉及动态，也涉及稳态-- 州政府的测量。 目的2验证阿片类药物在突触小泡后产生某些作用的假设 通过酸捕获积聚类阿片。突触小泡随后将阿片类药物释放到 突触前刺激。这一机制将扩展外源性药物的病理药理作用。 阿片类药物从许多类型的突触前神经元释放--即使是那些没有 释放内源性阿片肽。Aim 2a进一步进化了iOpioidSnFR对 所需的纳摩尔水平。Aim 2b确定测试突触前神经元最敏感的方法 放手。 Aim 3验证了这一假设，即大脑表达µ-阿片受体的区域在程度和程度上存在差异 细胞器阿片类药物的时间。AIM 3a产生腺相关病毒载体，编码“FLOXED” IOpioidSnFR。它们将在囊泡GABA转运体(VGAT)的控制下表达 Cre重组酶在合适的小鼠系中的表达。目的3b测量腹侧被盖脑片的含量 黑质网状部(SNR)、中脑导水管周围灰质(PAG)和腹侧 梅毒(VP)。结果将有助于正在进行的努力了解细胞和分子 对µ-阿片配体的耐受性基础。

Biological membranes are permeable to exogenous opioid drugs--both plant-derived molecules such as morphine, and synthetic molecules of which hundreds exist. Now a genetically encoded fluorescent biosensor technique allows us to measure opioids within neutral organelles such as the endoplasmic reticulum (ER). We term these molecules the intensity-based opioid-sensitive fluorescent reporter, iOpioidSnFR, family (Figure 1). Ongoing experiments, before the project begins, will extend the iOpioidSnFR family to the major classes of µ-opioid agonists. Aim 1 Further extends the iOpioidSnFRs for measurements within acidic organelles such as endosomes and synaptic vesicles. Aim 1a utilizes the present circularly permutated green fluorescent protein (cpGFP) moiety. Aim 1b develops novel circularly permuted HaloTags, which are pH-insensitive. Aim 1c, Extends the existing measurements to measure the entry of opioids into organelles, and their exit from organelles. Quantification involves both dynamics and steady- state measurements. Aim 2 tests the hypothesis that some effects of opioid drugs result after synaptic vesicles accumulate opioids via acid trapping. The synaptic vesicles would then release the opioids upon presynaptic stimulation. This mechanism would extend the patho-pharmacology of exogenous opioids to their release from many types of presynaptic neurons—even those neurons that do not release endogenous opioid peptides. Aim 2a evolves iOpioidSnFR sensitivity further, to the required nanomolar levels. Aim 2b Identifies the most sensitive method for testing presynaptic release. Aim 3 tests the hypothesis that brain regions expressing µ-opioid receptors vary in the extent and timing of organellar opioids. Aim 3a generates adeno-associated viral vectors that encode “floxed” iOpioidSnFRs. These will be expressed under the control of vesicular GABA transporter (vGAT) cre recombinase in suitable mouse lines. Aim 3b measures in brain slices from ventral tegmentum area (VTA) / substantia nigro pars reticulata (SnR), periaqueductal gray (PAG), and ventral pallidum (VP).The results will aid in the ongoing efforts to understand the cellular and molecular basis of tolerance to µ-opioid ligands.