Unit on Molecular Neurophysiology and Biophysics

hippocampal interneurons and their role in controlling

excitability

Dax Hoffman, PhD, Head, Unit on Molecular Neurophysiology and Biophysics

Sung-Cherl Jung, PhD, Postdoctoral Fellow

Jinny Kim, PhD, Postdoctoral Fellow

Dongsheng Wei, PhD, Postdoctoral Fellow

Arrash Yazdani, BS, Biologist

With billions of neurons firing at frequencies of hundreds of hertz, the complexity of the brain is so stunning that even a rudimentary comprehension seems unattainable. Our approach is to pare down the task of comprehension 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 regulate signal propagation. This nonuniform distribution (with higher expression in the dendrites than in the soma) means that the electrical properties of the dendrites differ markedly 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, we investigate the electrophysiological properties and molecular nature of the voltage-gated channels expressed in CA1 dendrites, the regulation of their expression, and their role in synaptic integration and plasticity.

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, the likely molecular substrate of transient currents recorded in CA1 dendrites. The mouse expresses the mutant Kv4.2 channel along with GFP under the control of a tetracycline transactivator–responsive promoter. Expression is spatially controlled by a new line of tetracycline transactivator–expressing mice that limit tetracycline transactivator (tTA) activity to the CA1 and dentate gyrus regions of the hippocampus. Expression can be controlled temporally by administering doxycycline. We will undertake experiments in acute hippocampal slices from the mice to investigate Kv4.2’s role in regulating AP back-propagation 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, at both the protein and mRNA levels. 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, when expressed in cultured hippocampal neurons, mimics endogenous Kv4.2 distribution. We have found that neuronal stimulation results in an activity-dependent redistribution of Kv4.2g away from synaptic sites to the dendritic shaft. The redistribution of Kv4.2g is not blocked by TTX and appears to be NMDA receptor–dependent. An activity-induced change in Kv4.2 redistribution could provide neurons with the means for dynamically regulating dendritic signal processing.

It is now believed that dendrites have the ability to translate mRNA into proteins locally. 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 reporter gene mRNA (beta-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. Using live imaging, we are currently investigating the mechanisms of activity-dependent trafficking of both the GFP-tagged Kv4.2 protein and 3´ UTR–fused reporter mRNA.

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, thus allowing for the induction of synaptic plasticity. We are currently investigating the effect of potassium channel mutations on back-propagation of action potentials and on the induction of LTP in organotypic slice cultures from wild-type and transgenic mice.

Hoffman DA, Sprengel R, Sakmann B. Molecular dissection of hippocampal theta-burst pairing potentiation. Proc Natl Acad Sci USA 2002;99:7740-7745.

Johnston D, Christie BR, Frick A, Gray R, Hoffman DA, Schexnayder LK, Watanabe S, Yuan LL. Active dendrites, potassium channels and synaptic plasticity. Philos Trans R Soc Lond B Biol Sci 2003;358:667-674.

Watanabe S, Hoffman DA, Migliore M, Johnston D. Dendritic K⁺ channels contribute to spike-timing dependent long-term potentiation in hippocampal pyramidal neurons. Proc Natl Acad Sci USA 2002;99:8366-8371.

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. Given that we are interested in the role of Kv4.2 in dendritic integration, we will implement a localized photolysis technique to activate oblique dendrite terminals selectively. Using such a technique, we hope to determine whether 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, Hoffman

Kv4 channel accelerating factor (KAF) facilitates Kv4.2 surface expression and reconstitutes the properties of the neuronal currents in heterologous expression systems. We are attempting to localize Kv4.2’s site of interaction with KAF. Co-expression of Kv4.2 and KAF in HEK 293 cells results in an increase in cell surface expression and accelerated channel inactivation. A comparison of current levels and properties of serial Kv4.2 deletion mutations suggests an N-terminal binding site for KAF on Kv4.2.

For further information, contact hoffmand@mail.nih.gov