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.

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U.S. National Inst/Child Hlth/Human Dev
United States
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Kim, Jinhyun; Jung, Sung-Cherl; Clemens, Ann M et al. (2007) Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons. Neuron 54:933-47
Kwon, Oh-Bin; Longart, Marines; Vullhorst, Detlef et al. (2005) Neuregulin-1 reverses long-term potentiation at CA1 hippocampal synapses. J Neurosci 25:9378-83
Kim, Jinhyun; Wei, Dong-Sheng; Hoffman, Dax A (2005) Kv4 potassium channel subunits control action potential repolarization and frequency-dependent broadening in rat hippocampal CA1 pyramidal neurones. J Physiol 569:41-57
Krestel, Heinz E; Mihaljevic, Andre L A; Hoffman, Dax A et al. (2004) Neuronal co-expression of EGFP and beta-galactosidase in mice causes neuropathology and premature death. Neurobiol Dis 17:310-8
Johnston, Daniel; Christie, Brian R; Frick, Andreas et al. (2003) Active dendrites, potassium channels and synaptic plasticity. Philos Trans R Soc Lond B Biol Sci 358:667-74