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 overwhelming. 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 and epilepsy. ? ? 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 techniques, 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 learning and memory. ? ? Kv4.2 control of firing patterns in hippocampal CA1 pyramidal neurons.? ? Although recent molecular cloning studies have found several families of voltage-gated K+ channel genes expressed in the mammalian brain, at present, information regarding the relationship between the protein products of these genes and their various neuronal functions is lacking. Our lab has used a combination of molecular, electrophysiological, imaging techniques to show that the voltage gated potassium channel subunit Kv4.2 controls AP half-width, frequency-dependent AP broadening and dendritic action potential propagation. As Ca2+ influx occurs primarily during AP repolarization, Kv4.2 activity can regulate cellular processes involving Ca2+-dependent second messenger cascades such as gene expression and synaptic plasticity. We are currently developing techniques which will enable us to directly record from dendrites where we have altered voltage-gated channel functional expression.? ? Kv4.2 trafficking in CA1 pyramidal neuron dendrites.? ? Using a modified Sinbis virus system to overexpress EGFP-labeled Kv4.2 (Kv4.2g) in cultured hippocampal neurons, we found that the EGFP fluorescence in dendritic spines of Kv4.2g expressing neurons appeared brighter than that from the adjacent dendritic shaft. The ratio of spine head to dendritic shaft fluorescence in Kv4.2g expressing neurons was approximately two-fold greater than in neurons expressing EGFP. Kv4.2 expression in spines was further shown using electronmicroscopy in collaboration with Ron Petralia here at the NIH.? ? We found stimulation (AMPA) to result in an activity-dependent redistribution of Kv4.2g away from spines to the dendritic shaft and a punctate accumulation of Kv4.2g within the soma. This AMPA-induced redistribution of Kv4.2g occurred within 15 min of stimulation and was reversible, indicating that the treatment was not excitotoxic (6 h washout). Co-expression with pre- and post-synaptic markers (synaptophysin and NR1) showed that Kv4.2 undergoes activity-induced redistribution without a gross change in synaptic architecture or number. We have confirmed these findings with live imaging of Kv4.2g removal from the spine in response to AMPA stimulation. The large number of synapses stimulated in these conditions enabled us to directly measure the effect of internalization as a decrease in the endogenous whole-cell transient K+ current from uninfected hippocampal neurons without a change in sustained or non-inactivating delayed rectifier-type voltage-gated K+ current amplitudes. Thus, activity-dependent Kv4.2 internalization occurs natively and is not an artifact of overexpression. ? ? Role of voltage-gated potassium channels in synaptic plasticity.? ? Using the Sindbis virus system to infect organotypic slice cultures with Kv4.2g and Kv4.2g(W362F), we have begun investigating the role of Kv4.2 in LTP using a depolarization pairing protocol. For the first 10 min after pairing, potentiation is similar in all three groups, achieving ~100% increase in EPSC size. After this period, however, Kv4.2 overexpressing neurons fail to maintain potentiation such that EPSC size is back to baseline after ~ 25 min. Conversely, expression of Kv4.2g(W362F) results in a potentiation, which reaches a greater level 40-50 min after initiation, compared to controls. These data indicate that Kv4.2 channels modulate the degree of LTP by influencing the induction of a late phase of potentiation or by controlling the mechanisms of LTP maintenance. ? ? Creation and characterization of Kv4.2 transgenic mice.? ? We are currently characterizing a transgenic mouse expressing a dominant negative pore mutation in the voltage-gated potassium channel subunit Kv4.2. 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. In collaboration with Dr. Anne Anderson, we are investigating seizure susceptibility in these mice. In addition, we are using these mice to investigate Kv4.2?s role in hippocampal dependent learning and memory in the Morris water maze. ? ? Role of auxiliary proteins in regulating Kv4.2 expression and function.? ? A-type K+ currents have unique kinetic and voltage-dependent properties that allow them to finely tune synaptic events, action potentials and neuronal firing. To achieve this diversity, different neuron types express specific complements of Kv4.2 auxiliary subunits. In hippocampal CA1 pyramidal neurons, DPPX is a prominently expressed subunit, which restores many properties of native CA1 A-type currents when co-expressed with Kv4.2 in heterologous systems.? ? To investigate the physiological role of DPPX in CA1 neurons we developed, in collaboration with Bernardo Rudy?s lab, short-interfering RNAs (siRNAs) to suppress the expression of all DPPX variants. To investigate whether DPPX alters the kinetics of A-type currents in a native system, we conducted voltage-clamp experiments in outside-out patches from CA1 pyramidal neurons in hippocampal organotypic slices infected with siDPPX using a modified Sindbis virus system. We found that siDPPX results in a delayed recovery from inactivation and rightward shifted the steady-state inactivation and activation curves for A-type currents. To determine the physiological effect of the A-type current kinetic modifications by siDPPX, we carried out current-clamp experiments in siDPPX expressing cells. Compared to negative control, siDPPX-infected neurons exhibited decreased input resistance, delayed time to AP onset, increased AP threshold, increased AP half-width and reduced fast AHP amplitudes. Thus siDPPX had contrasting effects, decreasing excitability subthreshold and increasing excitability suprathreshold. We are using computer modeling to determine whether or not these sub- and suprathreshold effects can be explained by the shift in inactivation and activation curves, respectively.
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