Opioids inside Organelles

生物膜对外源性阿片类药物是可渗透的--两者都是植物来源的分子 如吗啡和合成分子，其中存在数百种。现在一个基因编码的 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药物， 内质网（ER）。我们称这些分子为基于强度的阿片类药物敏感分子 荧光报告基因iOpioidSnFR家族（图1）。正在进行的实验，在项目之前 将iOpioidSnFR家族扩展到主要类别的μ-阿片类激动剂。 目的1进一步扩展iOpioidSnFR用于酸性细胞器内的测量，例如 内体和突触囊泡。目标1a利用目前循环排列的绿色 荧光蛋白（cpGFP）部分。Aim 1b开发了新颖的循环排列HaloTags， pH不敏感。目标1c，扩展现有测量方法，以测量类阿片的入境量 进入细胞器，以及它们从细胞器中的出口。量化包括动态和稳态- 国家测量。 目的2验证阿片类药物的某些作用是在突触囊泡形成后产生的假说 通过酸捕获积累阿片类药物。突触囊泡会释放阿片类物质， 突触前刺激这一机制将扩大外源性的病理药理学 阿片类药物从许多类型的突触前神经元释放-即使是那些不 释放内源性阿片肽。目标2a进一步发展iOpioidSnFR敏感性， 需要纳摩尔的水平。Aim 2b是检测突触前神经元的最敏感方法 release. 目标3检验了一个假设，即表达μ-阿片受体的大脑区域在程度上是不同的， 细胞器阿片类药物的时机。Aim 3a产生编码“floxed”的腺相关病毒载体 iOpioidSnFR。这些将在囊泡GABA转运蛋白（vGAT）的控制下表达。 CRE重组酶在合适的小鼠品系中的表达。腹侧被盖脑切片中的Aim 3b测量 区域（VTA）/黑质网状部（SnR）、导水管周围灰质（PAG）和腹侧 结果将有助于正在进行的努力，了解细胞和分子的苍白球（VP）。 对μ-阿片配体耐受的基础。

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.