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  Glia-neuron interaction by gliotransmitters 
   Role of gliotransmitters on synaptic transmission and plasticity 
   Mechanism of astrocytic release of gliotransmitters: glutamate, ATP, GABA, d-serine, taurine

  Anion channel function in glial cells and neurons:
   Glutamate release by permeation through anion channels.
   Tonic GABA release by anion channels.
   Bestrophin channel mediates AHP current in neuron.
   Volume regulation by anion channels.
   Developmental role of bestrophin channel in Drosophila

  Ca2+ signaling in glial cells through GPCR and Ca2+ dependent channels
   Pathological role of Ca2+ signaling and receptor/channels in Glioblastoma

  Interaction between GPCR and Ca2+ permeable channels.
   TRPV1 activation by DAG produced by GPCR.
   Role of histamine receptor and modulation of L-type Ca2+ channels in SCN neurons and circadian clock.
   GABAA receptor in modulation of L-type Ca2+ channels in SCN neurons and circadian clock.

  Development of molecular tools for the study of glial function.
   Selective knock down of gene expression in astrocytes using pSico-Cre system.
   Real time imaging of chloride concentration from brain slice using Two-photon microscope.
   Chloride channel blocker screening

  Glia-neuron interaction by gliotransmitters
   Role of gliotransmitters on synaptic transmission and plasticity
Astrocytes are known to release glutamate in Ca2+ dependent fashion. We have investigated the role of astrocytically released glutamate on synaptic transmission. We have utilized the PAR1 receptor agonist because PAR1 is a Gaq coupled receptor that induces Ca2+ release. Conveniently, PAR1 is exclusively expressed in astrocytes in the hippocampus, allowing us to selectively increase Ca2+ in astroyctes. Using this tool, we have found that astrocytically released glutamate binds to neuronal NMDA receptor preferentially over AMPA receptors due to the affinity, and potentiates NMDA receptor function, especially when there is a coincident activation by synaptic AMPA receptors (Lee et al, 2007). We demonstrated that astrocytically released glutamate enhances hippocampal synaptic plasticity by potentiating NMDA receptors. We further demonstrated that astrocytically released glutamate is mainly mediated by Ca2+ activated anion channel, encoded by bestrophin 1, and that blocking this channel eliminates the enhancement of synaptic plasticity.

   Mechanism of astrocytic release of gliotransmitters: glutamate, ATP, GABA, d-serine, taurine
Astrocytes are thought to release glutamate and other gliotransmitters in two ways: Ca2+ dependent and Ca2+ independent pathways. The Ca2+ dependent pathway can be divided into channel mediated and vesicular exocytotic mechanisms whereas Ca2+ independent pathway can be mostly channel and transporter mediated.
We have obtained compelling evidence that astrocytes release glutamate by channel mediated mechanism, by the recently characterized Ca2+ activated anion channel, Bestrophin-1. We have developed the “Sniffer Patch” technique to detect astrocytic glutamate at high temporal resolution (ms resolution) and high sensitivity (submicromolar concentration). Using this method we have detected various gliotransmitters including glutamate (Figure 2), ATP, d-serine and GABA and we are planning to detect taurine in near future.

  Anion channel function in glial cells and neurons:
   Brain distribution of mBest1.
Using in situ hybridization and immunohistochemistry we have determined the cellular distribution of mBest mRNA and protein in the brain. mBest1 is widely expressed in the CNS including astrocytes and neurons.

   Glutamate release by permeation through anion channels.
Using the sniffer patch technique we have directly demonstrated the release glutamate by permeation through mouse bestrophin 1 channel. We also developed the Lentivirus containing shRNA for mBest1 and demonstrated that mBest1 is responsible for majority of glutamate release from astrocytes.

   Tonic GABA release by anion channels.
In collaboration with Dr. George Augustine at Duke University, we have investigated the possibility of tonic GABA release mediated by anion channels. Using the Clomeleon transgenic mouse and patch clamping we have obtained evidence that tonic GABA release can be inhibited by anion channel blockers such as NPPB and niflumic acid. We are currently testing whether knock down of mBest1 by lentivirus containing mBest1-shRNA will inhibit the tonic GABA release.

   Bestrophin channel mediates AHP current in neuron.
Many neurons express Ca2+ activated potassium and chloride channels to modulate the action potential firing. The molecular identity of Ca2+ activated potassium channels are known but Ca2+ activated chloride channels are still unknown. We have tested whether bestrophin-1 channel is responsible for after-hyperpolarization due to Ca2+ activated chloride channels in thalamic relay neurons. First we characterized the Ca2+ activated chloride current by pharmacological tools and we are currently testing mBest1-shRNA-Lentivirus injection.

   Volume regulation by anion channels.
Astrocytes are known to dynamically control their cell volume by extruding and intruding various osmolytes and anions. The volume sensitive anion channels are thought to be involved in this process but the molecular identity of these anion channels remains undetermined. We have examined several candidate genes and molecules for volume regulated anion channels. During an intense synaptic transmission astrocytes dynamically control volume to remove and maintain the extracellular concentration of K+ ions. This phenomenon can be visualized by recording intrinsic optical signal. In collaboration with Dr. Whitsell of UNC Chapel Hill, we have investigated this dynamic process and determined the molecular mechanism of volume regulation during synaptic transmission. We have accumulated evidence that Ca2+ activated anion channel but not volume sensitive anion channel are mediating the volume recovery in astrocytes by extruding chloride and other osmolytes.

   Developmental role of bestrophin channel in Drosophila
In order to study the function of bestrophin channel, we have resorted to the Drosophila system where the wealth of molecular and genetic tools provides us opportunity to address the developmental role of bestrophin channels. We have been examining the role of dBest1 and dBest2 in the synapse formation and synaptic transmission at the neuromuscular junction of Drosophila larva. We found that dBest1 and dBest2 are expressed in neurons and muscles cells and control the number and morphology of presynaptic boutons as well as the expression of postsynaptic glutmate receptors.

  Ca2+ signaling in glial cells through GPCR and Ca2+ dependent channels
   Pathological role of Ca2+ signaling and receptor/channels in Glioblastoma
Glioblastoma is the most deadly brain cancer known with only less than a year of survival time after diagnosis. The glioblastoma originates from the dormant astrocytomas which are basically cancerous astrocytes. The most prominent feature of these glioblastoma cells is that they are very invasive and migratory. The complete resection is impossible due to these properties. We found that blocking the Ca2+ release pathway can greatly inhibit the invasiveness and migratory behavior of these cells. We have by serendipity found caffeine which is well know activated Ca2+ release channel Ryanodine receptor potently inhibited invasion and migration of glioblastoma cells. We have demonstrated that this caffeine effect is through an unexpected blocking effect of caffeine on another Ca2+ release channel IP3R subtype 3. We discovered that glioblastoma cells show elevevated level of molecules in Ca2+ signaling pathway, including 2 fold increase in IP3R3 expression. We further demonstrate that caffeine treatment reduces tumor mass and extends the survival time of nude mouse injected with glioblastoma cells. From this study we found a novel therapeutic target for glioblastoma and a novel use of caffeine for the treatment of this deadly disease.

  Interaction between GPCR and Ca2+ permeable channels.
   TRPV1 activation by DAG produced by GPCR.
In collaboration with Dr. Seog Bae Oh at Seoul National University, we have investigated the possible activation mechanism of the highly Ca2+ permeable TRPV1 channels. We found that similar to some TRPC channels, TRPV1 can also be activated by DAG that is produced by activation of Gaq coupled receptors such as group I mGluR and muscarinic receptors (Woo et al, 2008).

   Role of histamine receptor and modulation of L-type Ca2+ channels in SCN neurons and circadian clock.
H1 histamine receptors are involved in maintaining wakefulness. In suprachiasmatic nucleus(SCN), histamine receptors are suggested to be involved in phase shift of circadian clock. We have found that activation of H1, which is Gaq coupled receptor causes a robust Ca2+ increase in these cells surprisingly not by Cca2+ release but by Ca2+ influx through L-type Ca2+ channels. We investigated the possible mechanism of H1 receptor activation leading to L-type Ca2+ channel activation. We found that SCN neurons mainly express Cav1.3 but not much of Ca1.2. H1 linked to Cav1.3 could be the possible mechanism of H1 mediated phase shift in circadian clock.

   GABAA receptor in modulation of L-type Ca2+ channels in SCN neurons and circadian clock.
In collaboration with Dr. Yang In Kim at Korea University, we have investigated the possible interaction of GABA and L-type Ca2+ channels in SCN. We found that during the night the chloride concentration in certain population of neurons in SCN is increased by the activity of NKCC1 and this causes GABA to be excitatory (Choi et al., 2008). When GABAA receptor is activated during this time, SCN neurons show robust increase in Ca2+ due to an influx Ca2+ through L-type Ca2+ channels. These results suggest that in adult animal chloride concentration can be dynamically regulated and through interaction with L-type Ca2+ channels GABA can provide source of Ca2+ required for circadian rhythm phase shift.

  Development of molecular tools for the study of glial function.
   Selective knock down of gene expression in astrocytes using pSico-Cre system.
Recent advances in small interference RNA technique allowed us to consider a strategy to cell-type specifically knock down gene expression. We have utilized the pSico system which has a shRNA expression under the U6 promoter with loxP sites to conditionally remove the U6-shRNA cassette. We are developing the one construct containing the pSico with the NLS-Cre under the cell-type specific promoter such as GFAP for astrocyte and Synapsin for neuron.

   Real time imaging of chloride concentration from brain slice using Two-photon microscope.
Imaging astrocytes in living brain slice or in vivo requires state of the art imaging facility. We recently acquired TriMScope which can image at high speed (200 Hz) for long time. We have used this system to image chloride concentration from a single astrocyte in living brain slice for the first time. This technique will be very useful in our future study of anion channel function.

   Chloride channel blocker screening
Ca2+ activated Cl- channels (CaCCs) participate in many important physiological processes. However, the lack of effective and selective blockers has hindered the study of these channels, mostly due to the lack of good assay system. We have developed a reliable drug screening method for better blockers of CaCCs, using the endogeneous CaCCs in Xenopus laevis oocytes and two-electrode voltage-clamp (TEVC) technique. Using this technique, we have synthesized and tested various compounds. As a result, newly synthesized N-(4-trifluoromethylphenyl)anthranilic acid with trifluoromethyl group (–CF3) at para position on the benzene ring showed the lowest IC50.
Our results provide an optimal drug screening strategy suitable for high throughput screening, and propose N-(4-trifluoromethylphenyl)anthranilic acid as an improved CaCC blocker. (Oh et al, 2008)

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