Junsung Woo

A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes

TWIK-1 is a member of the two-pore domain K(+) (K2P) channel family that plays an essential part in the regulation of resting membrane potential and cellular excitability. The physiological role of TWIK-1 has remained enigmatic because functional expression of TWIK-1 channels is elusive. Here we report that native TWIK-1 forms a functional channel at the plasma membrane of astrocytes. A search for TWIK-1-binding proteins led to the identification of TREK-1, another member of the K2P family. The TWIK-1/TREK-1 heterodimeric channel is formed via a disulphide bridge between residue C69 in TWIK-1 and C93 in TREK-1. Gene silencing demonstrates that surface expression of TWIK-1 and TREK-1 are interdependent. TWIK-1/TREK-1 heterodimers mediate astrocytic passive conductance and cannabinoid-induced glutamate release from astrocytes. Our study sheds new light on the diversity of K2P channels.

The amount of astrocytic GABA positively correlates with the degree of tonic inhibition in hippocampal CA1 and cerebellum

A tonic form of synaptic inhibition occurs in discrete regions of the central nervous system and has an important role in controlling neuronal excitability. Recently, we reported that GABA present in astrocyte is the major source of tonic inhibition in cerebellum and that GABA is released through Bestrophin-1 channel by direct permeation. In this study, we screened for the presence of astrocytic GABA in various brain regions such as hippocampus, thalamus, hypothalamus and cerebellum using immunohistochemistry. We found that astrocytic GABA was present in the regions that were reported to show tonic inhibition. Because the existence of tonic inhibition in hippocampal CA1 is somewhat controversial, we compared the amount of astrocytic GABA and tonic inhibition between the hippocampal CA1 pyramidal cell layer and the cerebellar granule cell layer. Unlike cerebellar glial cells, hippocampal astrocytes did not contain GABA. The tonic inhibition was also much lower in the pyramidal neurons of hippocampal CA1 compared to the granule cells of cerebellum. Nevertheless, most of the hippocampal astrocytes expressed Bestrophin-1 channel. These data indicate that the absence of astrocytic GABA results in a low level of tonic inhibition in hippocampal CA1 region.

Glial GABA, synthesized by monoamine oxidase B, mediates tonic inhibition

Here we show that glial gamma aminobutyric acid (GABA) is produced by monoamine oxidase B (MAOB), utilizing a polyamine, putrescine. The concentration of GABA in Bergmann glial cells is estimated to be around 5–10 mM. General gene silencing of MAOB resulted in elimination of tonic GABA currents recorded from granule cells in the cerebellum and medium spiny neurons (MSN) in the striatum. Glial‐specific rescue of MAOB resulted in complete restoration of tonic GABA currents. Our results identify MAOB as a synthesizing enzyme of glial GABA, which is released to mediate tonic inhibition in the cerebellum and striatum.

Channel-mediated astrocytic glutamate release via Bestrophin-1 targets synaptic NMDARs

BackgroundAstrocytes regulate neuronal excitability and synaptic activity by releasing gliotransmitters such as glutamate. Our recent study demonstrated that astrocytes release glutamate upon GPCR activation via Ca2+ activated anion channel, Bestrophin-1 (Best1). The target of Best1-mediated astrocytic glutamate has been shown to be the neuronal NMDA receptors (NMDAR). However, whether it targets synaptically or extra-synaptically localized NMDAR is not known.FindingsWe recorded spontaneous miniature excitatory postsynaptic currents (mEPSCs) from CA1 pyramidal cells to test whether Best1-mediated astrocytic glutamate targets synaptic NMDAR. An agonist of protease activated receptor 1 (PAR1) was used to induce astrocytic Ca2+ increase and glutamate release. Firstly, we found that activation of PAR1 and subsequent release of glutamate from astrocyte does not alone increase the frequency of mEPSCs. Secondly, we found that mEPSC rise time is variable depending on the different electrotonic distances from the somatic recording site to the synaptic region where each mEPSC occurs. Two subgroups of mEPSC from CA1 pyramidal neuron by rise time were selected and analyzed. One group is fast rising mEPSCs with a rise time of 1 ~ 5 ms, representing synaptic activities arising from proximal dendrites. The other group is slowly rising mEPSCs with a rise time of 5 ~ 10 ms, representing synaptic events arising from glutamate release at synapses located in the distal dendrites. We used cell-type specific Best1 gene silencing system by Cre-loxP cleavage to dissociate the effect of neuronal and astrocytic Best1. Astrocytic Best1-mediated glutamate release by PAR1 activation did not affect decay kinetics, frequency, and amplitude of fast rising mEPSC. In contrast, PAR1 activation resulted in an NMDA receptor component to be present on slowly rising mEPSC, but did not alter frequency or amplitude.ConclusionsOur results indicate that astrocytic glutamate via Best1 channel targets and activates synaptic NMDARs.

Astrocytes as GABA-ergic and GABA-ceptive Cells

GABA (gamma-aminobutyric acid) is considered to be the major inhibitory neurotransmitter that is synthesized in and released from GABA-ergic neurons in the brain. However, recent studies have shown that not only neurons but astrocytes contain a considerable amount of GABA, which can be released and activate the receptors responsive to GABA. In addition, astrocytes are themselves responsive to GABA by expressing GABA receptors. These exciting new findings raise more questions about the origin of GABA, whether it is synthesized or taken up, and about the role of astrocytic GABA and GABA receptors. In this review, we propose several potential pathways for astrocytes to accumulate GABA and discuss the evidence for functional expression of GABA receptors in astrocytes.

Imiquimod Enhances Excitability of Dorsal Root Ganglion Neurons by Inhibiting Background (K2P) and Voltage-Gated (Kv1.1 and Kv1.2) Potassium Channels

BackgroundImiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons.FindingsWhen IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K+ channels, Kv1.1 and Kv1.2 (voltage-gated K+ channels) and TREK1 and TRAAK (K2P channels). IQ effectively reduced the currents mediated by both K+ channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at Kv1.1 and Kv1.2.ConclusionsOur results demonstrate that IQ blocks the voltage-gated K+ channels to increase AP duration and K2P channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.

Channel-mediated astrocytic glutamate modulates hippocampal synaptic plasticity by activating postsynaptic NMDA receptors

BackgroundActivation of G protein coupled receptor (GPCR) in astrocytes leads to Ca2+-dependent glutamate release via Bestrophin 1 (Best1) channel. Whether receptor-mediated glutamate release from astrocytes can regulate synaptic plasticity remains to be fully understood.ResultsWe show here that Best1-mediated astrocytic glutamate activates the synaptic N-methyl-D-aspartate receptor (NMDAR) and modulates NMDAR-dependent synaptic plasticity. Our data show that activation of the protease-activated receptor 1 (PAR1) in hippocampal CA1 astrocytes elevates the glutamate concentration at Schaffer collateral-CA1 (SC-CA1) synapses, resulting in activation of GluN2A-containing NMDARs and NMDAR-dependent potentiation of synaptic responses. Furthermore, the threshold for inducing NMDAR-dependent long-term potentiation (LTP) is lowered when astrocytic glutamate release accompanied LTP induction, suggesting that astrocytic glutamate is significant in modulating synaptic plasticity.ConclusionsOur results provide direct evidence for the physiological importance of channel-mediated astrocytic glutamate in modulating neural circuit functions.

Networked neural spheroid by neuro-bundle mimicking nervous system created by topology effect

In most animals, the nervous system consists of the central nervous system (CNS) and the peripheral nervous system (PNS), the latter of which connects the CNS to all parts of the body. Damage and/or malfunction of the nervous system causes serious pathologies, including neurodegenerative disorders, spinal cord injury, and Alzheimer’s disease. Thus, not surprising, considerable research effort, both in vivo and in vitro, has been devoted to studying the nervous system and signal transmission through it. However, conventional in vitro cell culture systems do not enable control over diverse aspects of the neural microenvironment. Moreover, formation of certain nervous system growth patterns in vitro remains a challenge. In this study, we developed a deep hemispherical, microchannel-networked, concave array system and applied it to generate three-dimensional nerve-like neural bundles. The deep hemicylindrical channel network was easily fabricated by exploiting the meniscus induced by the surface tension of a liquid poly(dimethylsiloxane) (PDMS) prepolymer. Neurospheroids spontaneously aggregated in each deep concave microwell and were networked to neighboring spheroids through the deep hemicylindrical channel. Notably, two types of satellite spheroids also formed in deep hemispherical microchannels through self-aggregation and acted as an anchoring point to enhance formation of nerve-like networks with neighboring spheroids. During neural-network formation, neural progenitor cells successfully differentiated into glial and neuronal cells. These cells secreted laminin, forming an extracellular matrix around the host and satellite spheroids. Electrical stimuli were transmitted between networked neurospheroids in the resulting nerve-like neural bundle, as detected by imaging Ca2+ signals in responding cells.

Columnar distribution of activity dependent gabaergic depolarization in sensorimotor cortical neurons.

BackgroundGABA, the major inhibitory neurotransmitter in CNS, has been demonstrated to paradoxically produce excitation even in mature brain. However activity-dependent form of GABA excitation in cortical neurons has not been observed. Here we report that after an intense electrical stimulation adult cortical neurons displayed a transient GABA excitation that lasted for about 30s.ResultsWhole-cell patch recordings were performed to evaluate the effects of briefly applied GABA on pyramidal neurons in adult rodent sensorimotor cortical slice before and after 1 s, 20 Hz suprathreshold electrical stimulation of the junction between layer 6 and the underlying white matter (L6/WM stimulation). Immediately after L6/WM stimulation, GABA puffs produced neuronal depolarization in the center of the column-shaped region. However, both prior to or 30s after stimulation GABA puffs produced hyperpolarization of neurons. 2-photon imaging in neurons infected with adenovirus carrying a chloride sensor Clomeleon revealed that GABA induced depolarization is due to an increase in [Cl-]i after stimulation. To reveal the spatial extent of excitatory action of GABA, isoguvacine, a GABAA receptors agonist, was applied right after stimulation while monitoring the intracellular Ca2+ concentration in pyramidal neurons. Isoguvacine induced an increase in [Ca2+]i in pyramidal neurons especially in the center of the column but not in the peripheral regions of the column. The global pattern of the Ca2+ signal showed a column-shaped distribution along the stimulation site.ConclusionThese results demonstrate that the well-known inhibitory transmitter GABA rapidly switches from hyperpolarization to depolarization upon synaptic activity in adult somatosensory cortical neurons.

α-pinene, a major constituent of pine tree oils, enhances non-rapid eye movement sleep in mice through GABAA-benzodiazepine receptors

α-Pinene is a major monoterpene of the pine tree essential oils. It has been reported that α-pinene shows anxiolytic and hypnotic effects upon inhaled administration. However, hypnotic effect by oral supplementation and the molecular mechanism of α-pinene have not been determined yet. By combining in vivo sleep behavior, ex vivo electrophysiological recording from brain slices, and in silico molecular modeling, we demonstrate that (−)-α-pinene shows sleep enhancing property through a direct binding to GABAA-benzodiazepine (BZD) receptors by acting as a partial modulator at the BZD binding site. The effect of (−)-α-pinene on sleep-wake profiles was evaluated by recording electroencephalogram and electromyogram. The molecular mechanism of (−)-α-pinene was investigated by electrophysiology and molecular docking study. (−)-α-pinene significantly increased the duration of non-rapid eye movement sleep (NREMS) and reduced the sleep latency by oral administration without affecting duration of rapid eye movement sleep and delta activity. (−)-α-pinene potentiated the GABAA receptor-mediated synaptic response by increasing the decay time constant of sIPSCs in hippocampal CA1 pyramidal neurons. These effects of (−)-α-pinene on sleep and inhibitory synaptic response were mimicked by zolpidem, acting as a modulator for GABAA-BZD receptors, and fully antagonized by flumazenil, an antagonist for GABAA-BZD receptor. (−)-α-pinene was found to bind to aromatic residues of α1- and -γ2 subunits of GABAA-BZD receptors in the molecular model. We conclude that (−)-α-pinene enhances the quantity of NREMS without affecting the intensity of NREMS by prolonging GABAergic synaptic transmission, acting as a partial modulator of GABAA-BZD receptors and directly binding to the BZD binding site of GABAA receptor.