Potassium Channels and Dendritic Function in Hippocampal

中枢神经系统构成了我们所有的经验、行动、情感、知识和记忆的基础。数十亿个神经元以数百赫兹的频率放电，大脑的复杂性令人惊叹。我们的方法是通过研究单个中枢神经元的工作原理来配对这项任务，单个中枢神经元是来自海马区CA1区的锥体神经元，这是大脑中对学习和记忆至关重要的区域，也是阿尔茨海默病-S病最早受到影响的区域之一。 在海马CA1区锥体神经元的树突中，非均匀密度的亚阈值，快速失活的钾通道调节信号的传播。这种不均匀的分布(在树突中的表达高于在胞体中的表达)意味着树突的电学性质与胞体的电学性质明显不同。传入的突触信号是由这些通道的活动形成的，一旦在轴突中启动，动作电位在传播回树突时逐渐降低幅度。结合膜片钳记录和分子生物学，分子神经生理学和生物物理学单位研究CA1树突表达的电压门控通道的电生理特性和分子性质，它们的表达是如何调节的，以及它们在学习和记忆的细胞模拟中所起的作用。 Kv4.2转基因小鼠的建立和鉴定。 霍夫曼 我们目前正在鉴定一只在电压门控钾通道亚单位Kv4.2中表达显性负孔突变的转基因小鼠，很可能与CA1树突中记录的瞬时电流的分子相同。该小鼠在四环素反式激活剂(TTA)反应启动子的控制下，表达突变的Kv4.2通道和GFP。表达受到一种新的TTA表达小鼠的空间控制，这些小鼠将TTA的活动限制在海马区的CA1和齿状回区域。多西环素的使用可以暂时控制其表达。本实验将利用这些小鼠的急性海马脑片来研究Kv4.2、S在调节AP向CA1树突的反向繁殖以及在突触整合和可塑性中的作用。 Kv4.2在CA1区锥体神经元树突的运输。 金姆 我们正试图在蛋白质和mRNA水平上表征控制Kv4.2的细胞分布(例如树突定位)和运输的机制。为了直观显示Kv4.2蛋白的分布，我们在细胞质C末端用增强型绿色荧光蛋白(EGFP)标记Kv4.2。EGFP标记的Kv4.2(Kv4.2g)在HEK 293细胞中表达时与野生型Kv4.2没有动力学差异，在培养的海马神经元中表达时模拟内源性Kv4.2的分布。我们发现神经元刺激导致Kv4.2G从突触位置到树突干的活性依赖的重新分配。这种再分布与长时程增强(LTP)有着共同的要求(NMDA受体激活和钙内流)，LTP是一种学习和记忆的细胞机制。我们正在确定Kv4.2内化的机制。活动诱导的Kv4.2重分布改变可能为神经元提供动态调节树突信号处理的手段。 现在人们相信树突有能力将信使核糖核酸局部翻译成蛋白质。信使RNA以高密度的RNA颗粒存在于海马树突内。我们通过RT-PCR从海马神经元树突RNA颗粒组分中检测到内源性Kv4.2 mRNA，表明Kv4.2可能在海马树突内局部翻译。为了可视化和跟踪Kv4.2mRNA，我们将报告基因mRNA(b-半乳糖苷酶和EGFP)与5？和/或3？Kv4.2基因的非翻译区。我们观察到3？而不是5英镑？UTR融合的报告基因产物以颗粒状点状分布于树突各处。目前，我们正在利用实时成像技术研究GFP标记的Kv4.2蛋白和3？UTR融合报告基因的活性依赖转运机制。 电压门控性钾通道在突触可塑性中的作用。 荣格 钾通道已被证明调节动作电位向CA1树突的反向传播。尽管动作电位反向传播的功能作用目前还不清楚，但最近有人提出，它们可能提供了解锁NMDA受体所需的去极化，从而诱导突触可塑性。我们目前正在研究钾通道突变对来自野生型和转基因小鼠的器官型切片培养中动作电位反向传播和LTP诱导的影响。 电压门控性钾通道在突触整合中的作用。 魏伟 我们正在使用钙离子成像作为一种指示剂来检测表达器官型切片的对照和突变Kv4.2之间的动作电位和突触反应的传播。由于我们对Kv4.2在树突整合中的作用感兴趣，我们实施了一种局部光解技术来选择性地激活倾斜的树突末端。利用这种技术，我们将确定Kv4.2通道微域表达模式(例如树突干、分支点和末端)是否对树突信号整合显示出不同的影响。 辅助蛋白在调节Kv4.2特性和表达水平中的作用。 雅兹达尼、金、霍夫曼 Kv4通道加速因子DPPX促进Kv4.2的表面表达，并重构异源表达系统中神经元电流的特性。与纽约大学的贝尔纳多·鲁迪？S博士合作，我们正在使用病毒介导的针对DPPX RNA的RNAi序列表达来观察对Kv4.2运输和突触信号的影响。

The central nervous system underlies all our experiences, actions, emotions, knowledge and memories. With billions of neurons firing at frequencies of hundreds of hertz, the complexity of the brain is stunning. Our approach is to pair down this task by studying the workings of a single central neuron, the pyramidal neuron from the CA1 region of the hippocampus, a region of the brain important for learning and memory and among the first affected in Alzheimer?s disease. In the dendrites of hippocampal CA1 pyramidal neurons, a nonuniform density of subthreshold, rapidly inactivating potassium channels regulates signal propagation. This nonuniform distribution (with higher expression in the dendrites than in the soma) means that the electrical properties of the dendrites are markedly different from those of the soma. Incoming synaptic signals are shaped by the activity of these channels, and action potentials, once initiated in the axon, progressively decrease in amplitude as they propagate back into the dendrites. Combining patch clamp recording with molecular biology, the Molecular Neurophysiology and Biophysics Unit investigates the electrophysiological properties and molecular nature of the voltage-gated channels expressed in CA1 dendrites, how their expression is regulated, and what their role is in a cellular analogue of learning and memory. Creation and characterization of Kv4.2 transgenic mice. Hoffman We are currently characterizing a transgenic mouse expressing a dominant negative pore mutation in the voltage-gated potassium channel subunit Kv4.2, likely the molecular identity of transient currents recorded in CA1 dendrites. This mouse expresses the mutant Kv4.2 channel along with GFP under control of a tetracycline transactivator (tTA) responsive promoter. Expression is spatially controlled by a new line of tTA expressing mice that limit tTA activity to the CA1 and dentate gyrus regions of the hippocampus. Expression can be controlled temporally by administration of doxycycline. Experiments in acute hippocampal slices from these mice will be used to investigate Kv4.2?s role in regulating AP backpropagation into CA1 dendrites and in synaptic integration and plasticity. Kv4.2 trafficking in CA1 pyramidal neuron dendrites. Kim We are attempting to characterize the mechanisms that govern the cellular distribution (e.g. dendritic localization) and trafficking of Kv4.2, both at the protein and mRNA level. To visualize Kv4.2 protein distribution, we tagged Kv4.2 with the enhanced green fluorescent protein (EGFP) at the cytoplasmic C-terminus. EGFP-tagged Kv4.2 (Kv4.2g) showed no kinetic differences from wild type Kv4.2 when expressed in HEK 293 cells and it mimics endogenous Kv4.2 distribution when expressed in cultured hippocampal neurons. We have found neuronal stimulation to result in an activity-dependent redistribution of Kv4.2g away from synaptic sites to the dendritic shaft. This redistribution shares common requirements (NMDA receptor activation and calcium influx) with long-term potentiation (LTP), a cellular mechanism for learning and memory. We are determining the mechanisms by which Kv4.2 is internalized. Activity-induced change in Kv4.2 redistribution could provide neurons with the means dynamically regulate dendritic signal processing. It is now believed that dendrites have capability to locally translate mRNA into proteins. Messenger RNA exists in hippocampal dendrites as highly dense RNA granules. We detected endogenous Kv4.2 mRNA from dendritic RNA granule fractions of hippocampal neurons by RT-PCR, suggesting that Kv4.2 may be locally translated in hippocampal dendrites. To visualize and track Kv4.2 mRNA, we fused report gene mRNA (b-galactosidase and EGFP) with the 5? and/or 3? untranslated region (UTR) of Kv4.2 mRNA. We observed that 3? but not 5? UTR-fused reporter gene products were detected throughout dendrites in the form of granule-like puncta. We are currently investigating the mechanisms of activity-ddependent trafficking of both the GFP-tagged Kv4.2 protein and 3?UTR-fused reporter mRNA using live imaging. Role of voltage-gated potassium channels in synaptic plasticity. Jung Potassium channels have been shown to regulate the back-propagation of action potentials into CA1 dendrites. Although the functional role of back-propagation of action potentials is unclear at this time, it has recently been suggested that they may provide the depolarization necessary to unblock NMDA receptors allowing for the induction of synaptic plasticity. We are currently investigating the effect of potassium channel mutations on back-propagation of action potentials and in the induction of LTP in organotypic slice cultures from wild type and transgenic mice. Role of voltage-gated potassium channels in synaptic integration. Wei We are using Ca2+ imaging as an indicator to examine the propagation of action potentials and synaptic responses between control and mutant Kv4.2 expressing organotypic slices. Since we are interested in the role of Kv4.2 on the dendritic integration, we have implemented a localized photolysis technique to selectively activate oblique dendrite terminals. Using such a technique we will determine if Kv4.2 channel microdomain expression patterns (e.g. dendritic trunk, branch points, and terminals) show different effects on dendritic signal integration. Role of auxiliary proteins in regulating Kv4.2 properties and expression levels. Yazdani, Kim, Hoffman Kv4 channel accelerating factor DPPX facilitates Kv4.2 surface expression and reconstitutes the properties of the neuronal currents in heterologous expression systems. In collaboration with Dr. Bernardo Rudy?s lab from NYU we are using virus-mediated expression of RNAi sequences against DPPX RNA to look at the effect on Kv4.2 trafficking and synaptic signaling.