Jinhyun Kim

Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition.

A consortium of inhibitory neurons control the firing patterns of pyramidal cells, but their specific roles in the behaving animal are largely unknown. We performed simultaneous physiological recordings and optogenetic silencing of either perisomatic (parvalbumin (PV) expressing) or dendrite-targeting (somatostatin (SOM) expressing) interneurons in hippocampal area CA1 of head-fixed mice actively moving a treadmill belt rich with visual-tactile stimuli. Silencing of either PV or SOM interneurons increased the firing rates of pyramidal cells selectively in their place fields, with PV and SOM interneurons having their largest effect during the rising and decaying parts of the place field, respectively. SOM interneuron silencing powerfully increased burst firing without altering the theta phase of spikes. In contrast, PV interneuron silencing had no effect on burst firing, but instead shifted the spikes theta phase toward the trough of theta. These findings indicate that perisomatic and dendritic inhibition have distinct roles in controlling the rate, burst and timing of hippocampal pyramidal cells.

Regulation of Dendritic Excitability by Activity-Dependent Trafficking of the A-Type K+ Channel Subunit Kv4.2 in Hippocampal Neurons

Voltage-gated A-type K+ channel Kv4.2 subunits are highly expressed in the dendrites of hippocampal CA1 neurons. However, little is known about the subcellular distribution and trafficking of Kv4.2-containing channels. Here we provide evidence for activity-dependent trafficking of Kv4.2 in hippocampal spines and dendrites. Live imaging and electrophysiological recordings showed that Kv4.2 internalization is induced rapidly upon glutamate receptor stimulation. Kv4.2 internalization was clathrin mediated and required NMDA receptor activation and Ca2+ influx. In dissociated hippocampal neurons, mEPSC amplitude depended on functional Kv4.2 expression level and was enhanced by stimuli that induced Kv4.2 internalization. Long-term potentiation (LTP) induced by brief glycine application resulted in synaptic insertion of GluR1-containing AMPA receptors along with Kv4.2 internalization. We also found evidence of Kv4.2 internalization upon synaptically evoked LTP in CA1 neurons of hippocampal slice cultures. These results present an additional mechanism for synaptic integration and plasticity through the activity-dependent regulation of Kv4.2 channel surface expression.

Kv4 potassium channel subunits control action potential repolarization and frequency-dependent broadening in rat hippocampal CA1 pyramidal neurones

A‐type potassium channels regulate neuronal firing frequency and the back‐propagation of action potentials (APs) into dendrites of hippocampal CA1 pyramidal neurones. 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 the various neuronal functions performed by voltage‐gated K+ channels is lacking. Here we used a combination of molecular, electrophysiological and imaging techniques to show that one such gene, Kv4.2, controls AP half‐width, frequency‐dependent AP broadening and dendritic action potential propagation. Using a modified Sindbis virus, we expressed either the enhanced green fluorescence protein (EGFP)‐tagged Kv4.2 or an EGFP‐tagged dominant negative mutant of Kv4.2 (Kv4.2gW362F) in CA1 pyramidal neurones of organotypic slice cultures. Neurones expressing Kv4.2gW362F displayed broader action potentials with an increase in frequency‐dependent AP broadening during a train compared with control neurones. In addition, Ca2+ imaging of Kv4.2gW362F expressing dendrites revealed enhanced AP back‐propagation compared to control neurones. Conversely, neurones expressing an increased A‐type current through overexpression of Kv4.2 displayed narrower APs with less frequency dependent broadening and decreased dendritic propagation. These results point to Kv4.2 as the major contributor to the A‐current in hippocampal CA1 neurones and suggest a prominent role for Kv4.2 in regulating AP shape and dendritic signalling. 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.

mGRASP enables mapping mammalian synaptic connectivity with light microscopy.

The GFP reconstitution across synaptic partners (GRASP) technique, based on functional complementation between two nonfluorescent GFP fragments, can be used to detect the location of synapses quickly, accurately and with high spatial resolution. The method has been previously applied in the nematode and the fruit fly but requires substantial modification for use in the mammalian brain. We developed mammalian GRASP (mGRASP) by optimizing transmembrane split-GFP carriers for mammalian synapses. Using in silico protein design, we engineered chimeric synaptic mGRASP fragments that were efficiently delivered to synaptic locations and reconstituted GFP fluorescence in vivo. Furthermore, by integrating molecular and cellular approaches with a computational strategy for the three-dimensional reconstruction of neurons, we applied mGRASP to both long-range circuits and local microcircuits in the mouse hippocampus and thalamocortical regions, analyzing synaptic distribution in single neurons and in dendritic compartments.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection.

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Actin/alpha-actinin-dependent transport of AMPA receptors in dendritic spines: role of the PDZ-LIM protein RIL.

The efficacy of excitatory transmission in the brain depends to a large extent on synaptic AMPA receptors, hence the importance of understanding the delivery and recycling of the receptors at the synaptic sites. Here we report a novel regulation of the AMPA receptor transport by a PDZ (postsynaptic density-95/Drosophila disc large tumor suppressor zona occludens 1) and LIM (Lin11/rat Isl-1/Mec3) domain-containing protein, RIL (reversion-induced LIM protein). We show that RIL binds to the AMPA glutamate receptor subunit GluR-A C-terminal peptide via its LIM domain and to α-actinin via its PDZ domain. RIL is enriched in the postsynaptic density fraction isolated from rat forebrain, strongly localizes to dendritic spines in cultured neurons, and coprecipitates, together with α-actinin, in a protein complex isolated by immunoprecipitation of AMPA receptors from forebrain synaptosomes. Functionally, in heterologous cells, RIL links AMPA receptors to the α-actinin/actin cytoskeleton, an effect that appears to apply selectively to the endosomal surface-internalized population of the receptors. In cultured neurons, an overexpression of recombinant RIL increases the accumulation of AMPA receptors in dendritic spines, both at the total level, as assessed by immunodetection of endogenous GluR-A-containing receptors, and at the synaptic surface, as assessed by recording of miniature EPSCs. Our results thus indicate that RIL directs the transport of GluR-A-containing AMPA receptors to and/or within dendritic spines, in anα-actinin/actin-dependent manner, and that such trafficking function promotes the synaptic accumulation of the receptors.

Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons.

Sindbis virus-based vectors have been successfully used for transient heterologous protein expression in neurons. Their main limitation arises from infection-associated cytotoxicity, attributed largely to a progressive shut down of host cell protein synthesis. Here we evaluated a modified Sindbis vector, based on a viral strain containing a point mutation in the second nonstructural protein, nsP2 P726S, described to delay inhibition of protein synthesis in BHK cells [Virology 228 (1997) 74], for heterologous expression in neurons in vitro and in vivo. First, we constructed an optimized helper vector, termed DH-BB(tRNA/TE12), for production of SINrep(nsP2S(726)) viral particles with low levels of helper RNA co-packaging and high neurospecificity of infection. Second, we determined that hippocampal primary neurons infected with SINrep(nsP2S(726)) virus expressing EGFP showed a delayed onset of viral induced cytotoxicity and higher levels of EGFP expression in comparison to cells infected with wild type SINrep5 EGFP-expressing virus. However, a strong decrease in protein synthesis still occurred by day 3 postinfection. The SINrep(nsP2S(726)) vector is thus well suited for rapid high level expression within this time window. As an experimental example, we demonstrate the applicability of this system for high-resolution two-photon imaging of dendritic spines in vivo.

Rapid, Bidirectional Remodeling of Synaptic NMDA Receptor Subunit Composition by A-type K+ Channel Activity in Hippocampal CA1 Pyramidal Neurons

The transient, A-type K+ current (IA) controls the excitability of CA1 pyramidal neuron dendrites by regulating the back-propagation of action potentials and by shaping synaptic input. Dendritic A-type K+ channels are targeted for modulation during long-term potentiation (LTP) and we have recently shown that activity-dependent internalization of the A-type channel subunit Kv4.2 enhances synaptic currents. However, the effect of changes in IA on the ability to induce subsequent synaptic plasticity (metaplasticity) has not been investigated. Here, we show that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced IA showed a decrease in relative synaptic NR2B/NR2A subunit composition and did not exhibit LTP. Conversely, reducing IA by expression of a Kv4.2 dominant-negative or through genomic knockout of Kv4.2 led to an increased fraction of synaptic NR2B/NR2A and enhanced LTP. Bidirectional synaptic remodeling was mimicked in experiments manipulating intracellular Ca2+ and dependent on spontaneous activation of NMDA receptors and CaMKII activity. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDAR activation to control synaptic NMDAR expression and plasticity.

Structured Synaptic Connectivity between Hippocampal Regions

The organization of synaptic connectivity within a neuronal circuit is a prime determinant of circuit function. We performed a comprehensive fine-scale circuit mapping of hippocampal regions (CA3-CA1) using the newly developed synapse labeling method, mGRASP. This mapping revealed spatially nonuniform and clustered synaptic connectivity patterns. Furthermore, synaptic clustering was enhanced between groups of neurons that shared a similar developmental/migration time window, suggesting a mechanism for establishing the spatial structure of synaptic connectivity. Such connectivity patterns are thought to effectively engage active dendritic processing and storage mechanisms, thereby potentially enhancing neuronal feature selectivity.

Potassium Channels: Newly Found Players in Synaptic Plasticity:

One of the major issues for modern neuroscience research concerns the molecular and cellular mechanisms that underlie the acquisition, storage, and recollection of memories by the brain. Regulation of the strength of individual synaptic inputs (synaptic plasticity) has, for decades, been the front-running candidate mechanism for cellular information storage, with some direct supporting evidence recently obtained. Research into the molecular mechanisms responsible for changing synaptic strength has, to date, primarily focused on trafficking and properties of the neurotransmitter receptors themselves (AMPARs and NMDARs). However, recent evidence indicates that, subsequent to receptor activation, synaptic inputs are subject to regulation by synaptically located K+ channels. It is therefore critical to understand the biophysical properties and subcellular localization (density and distribution) of these channels and how their properties are modulated. Here we will review recent findings showing that two different classes of K+ channels (A-type and small conductance, Ca2+ -activated K+ channels), beyond their traditional role in regulating action potential firing, contribute to the regulation of synaptic strength in the hippocampus. In addition, we discuss how modulation of these channels properties and expression might contribute to synaptic plasticity. NEUROSCIENTIST 14(3):276–286, 2008. DOI: 10.1177/1073858408315041