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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 |
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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 |