DETECTION OF CYCLIC NUCLEOTIDES IN INTACT NEURONS

完整神经元中环状核苷酸的检测

• 批准号: 2268653
• 负责人: RICHARD H KRAMER
• 金额: $ 9.82万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2268653
• 关键词: Aplysia; action potentials; alternatives to animals in research; behavioral habituation /sensitization; biological signal transduction; conditioning; cyclic AMP; cyclic GMP; fluorescent dye /probe; gene expression; hormone regulation /control mechanism; membrane channels; neural transmission; neurons; neurotransmitters; synapses; voltage /patch clamp

Our long-term goal is to understand intracellular signalling by cyclic nucleotides (cyclic AMP and cyclic GMP) in neurons. Cyclic nucleotides are crucial regulators of cellular processes including metabolism, cell growth and synaptic transmission. Cyclic AMP plays a major role in triggering changes in synaptic structure and function that occur during short and long term learning. Disruption of cyclic nucleotide signalling is likely to contribute to many clinical disorders, and cyclic nucleotide signalling cascades are the targets of many drugs. While much is known about the biochemistry of cyclic nucleotide signalling cascades, we know little about the dynamics and spatial distribution of these signal in neurons. This proposal is focused on a newly-discovered class of ion channels in olfactory receptor cells that are gated directly by cyclic nucleotides. These channels are crucial for converting a change in cyclic nucleotide concentration into an electrical signal during olfactory signal transduction. In addition to studying the channels per se, we shall use the channels as novel tools for detecting cyclic nucleotides in other cell types. Membrane patches containing the channels will be obtained using the patch clamp technique, and these patches will inserted into molluscan (Aplysia and Helix) neurons, where the activity of the detector channels will reflect the local intracellular concentrations of cyclic AMP and cyclic GMP. We will measure the kinetics of elevation and decay of cyclic nucleotides during neurotransmitter responses that modulate electrical activity and synaptic transmission. In addition we will examine changes in cyclic nucleotides when transmitter is applied paired with action potentials in the neuron. Such pairing is thought to alter cyclic nucleotide metabolism, resulting in long-term synaptic plasticity which contributes to associative conditioning. The ability to monitor cyclic nucleotides with olfactory patches, combined with the use of pharmacological agents, will enable us to determine which steps in the cyclic nucleotide cascade are rate-limiting for the onset and decay of transmitter responses, which steps persist during long-lasting responses, and which steps are altered during pairing. In addition a cloned gene encoding the olfactory cyclic nucleotide-gated channel will be expressed in these neurons, essentially directing the neurons to synthesize their own directly-gated cyclic nucleotide channel detectors. Activation of the expressed channels will be assessed with patch clamp measurements, and alternatively using Ca2+ influx through the channels in cells loaded with Ca2+, indicator dyes. Expression of the cloned channel by the neurons will allow the investigation of the spatial distribution and intracellular spread of cyclic nucleotide signals in response to neurotransmitters.

我们的长期目标是通过环状了解细胞内信号传导 神经元中的核苷酸（环状AMP和环状GMP）。 环状核苷酸是 细胞过程的关键调节剂，包括代谢，细胞生长 和突触传输。 循环AMP在触发中起主要作用 短和长时间发生的突触结构和功能的变化 学期学习。 循环核苷酸信号的破坏可能是 有助于许多临床疾病和环状核苷酸信号传导 级联是许多药物的靶标。 虽然对 环状核苷酸信号传导级联的生物化学，我们对 这些信号在神经元中的动力学和空间分布。 该提议的重点是新发现的一个离子渠道类 嗅觉受体细胞直接通过循环核苷酸门控。 这些通道对于转换环状核苷酸的变化至关重要 嗅觉信号期间集中到电信号 转导。 除了研究渠道本身，我们还将使用 通道作为用于检测其他细胞中环核苷酸的新工具 类型。 使用包含通道的膜贴片将使用 补丁夹技术，这些贴片将插入软体动物 （Aplysia和Helix）神经元，其中检测器通道的活性 将反映局部循环AMP的局部细胞内浓度和 循环GMP。 我们将测量循环的高程和衰变的动力学 神经递质反应期间调节电递质的核苷酸 活动和突触传播。 此外，我们将检查更改 在循环核苷酸与作用配对时，循环核苷酸 神经元中的电势。 这种配对被认为改变了循环 核苷酸代谢，导致长期突触可塑性 有助于关联条件。 监测环状的能力 具有嗅觉斑块的核苷酸，结合使用 药理学剂将使我们能够确定 环状核苷酸级联对于发作和衰减的速率限制 发射器响应（在长期响应中）持续存在的发射机响应， 以及在配对过程中更改了哪些步骤。 另外一个克隆基因 编码嗅觉循环核苷酸门控通道将在 这些神经元基本上指示神经元合成自己的 直接门控环核苷酸通道检测器。 激活 表达的通道将通过贴片夹测量评估，并将 或者，通过加载的单元中的通道使​​用Ca2+涌入 Ca2+，指示染料。 神经元克隆通道的表达将 允许研究空间分布和细胞内 响应神经递质的循环核苷酸信号传播。