Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(βγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(βγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 μM, whereas slow mode targets neuronal NMDA receptors at around 1 μM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.

Key pointsr Here we show that glial gamma aminobutyric acid (GABA) is produced by monoamine oxidase B (MAOB), utilizing a polyamine, putrescine.r The concentration of GABA in Bergmann glial cells is estimated to be around 5-10 mM. r 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.r Glial-specific rescue of MAOB resulted in complete restoration of tonic GABA currents. r Our results identify MAOB as a synthesizing enzyme of glial GABA, which is released to mediate tonic inhibition in the cerebellum and striatum.Abstract GABA is the major inhibitory transmitter in the brain and is released not only from a subset of neurons but also from glia. Although neuronal GABA is well known to be synthesized by glutamic acid decarboxylase (GAD), the source of glial GABA is unknown. After estimating the concentration of GABA in Bergmann glia to be around 5-10 mM by immunogold electron microscopy, we demonstrate that GABA production in glia requires MAOB, a key enzyme in the putrescine degradation pathway. In cultured cerebellar glia, both Ca 2+ -induced and tonic GABA release are significantly reduced by both gene silencing of MAOB and the MAOB inhibitor selegiline. In the cerebellum and striatum of adult mice, general gene silencing, knock out of MAOB or selegiline treatment resulted in elimination of tonic GABA currents recorded from granule neurons and medium spiny neurons. Glial-specific rescue of MAOB resulted in complete rescue of tonic GABA currents. Our results identify MAOB as a key synthesizing enzyme of glial GABA, which is released via bestrophin 1 (Best1) channel to mediate tonic inhibition in the brain.

In mammalian brain, neurons and astrocytes are reported to express various chloride and anion channels, but the evidence for functional expression of Ca 2ϩ -activated anion channel (CAAC) and its molecular identity have been lacking. Here we report electrophysiological evidence for the CAAC expression and its molecular identity by mouse Bestrophin 1 (mBest1) in astrocytes of the mouse brain. Using Ca 2ϩ imaging and perforated-patch-clamp analysis, we demonstrate that astrocytes displayed an inward current at holding potential of Ϫ70 mV that was dependent on an increase in intracellular Ca 2ϩ after G ␣q -coupled receptor activation. This current was mediated mostly by anions and was sensitive to well known anion channel blockers such as niflumic acid, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and flufenamic acid. To find the molecular identity of the anion channel responsible for the CAAC current, we analyzed the expression of candidate genes and found that the mRNA for mouse mBest1 is predominantly expressed in acutely dissociated or cultured astrocytes. Whole-cell patch-clamp analysis using HEK293T cells heterologously expressing full-length mBest1 showed a Ca 2ϩ -dependent current mediated by mBest1, with a complete impairment of the current by a putative pore mutation, W93C. Furthermore, mBest1-mediated CAAC from cultured astrocytes was significantly reduced by expression of mBest1-specific short hairpin RNA (shRNA), suggesting that the CAAC is mediated by a channel encoded by mBest1. Finally, hippocampal CA1 astrocytes in hippocampal slice also showed mBest1-mediated CAAC because it was inhibited by mBest1-specific shRNA. Collectively, these data provide molecular evidence that the mBest1 channel is responsible for CAAC function in astrocytes.

OBJECTIVE To investigate the clinical outcome using ureteric stents to manage ureteric obstruction in advanced non‐urological malignancies. PATIENTS AND METHODS We retrospectively reviewed the use of ureteric stents (Endo‐sof, Cook Urological, Spencer, IN, USA) placed for malignant ureteric obstruction from June 2001 to September 2006. The clinical and radiological variables for predicting the failure of stent insertion, functional stent failure and death were analysed. RESULTS In all, 86 patients with a non‐urological malignant ureteric obstruction were treated by ureteric stenting; 13 (15%) had failure of retrograde stent insertion, and of the remaining 73, 12 (16%) had stent failures during the subsequent follow‐up. The risk of failure for stent insertion significantly increased with male gender (hazard ratio 6.45, P = 0.028) and the presence of bladder invasion (hazard ratio 27.04, P < 0.001). There was no independent predictor of stent failure in univariate analysis. Of the 86 patients, 54 (63%) died with a mean survival time of 8.6 months after an initial attempt to place a stent, and 41 (48%) died within 1 year. Multivariate analysis showed that low performance status, upper ureteric obstruction and no chemotherapy after stenting were independently associated with a poor prognosis (P = 0.03, 0.004 and 0.003, respectively). CONCLUSION The method of diversion for a malignant ureteric obstruction should be carefully discussed with male patients or if there is bladder invasion. Patients with a low performance status, upper ureteric obstruction and no scheduled chemotherapy after stenting had a poor survival time.

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.

Post-stroke neglect in the extrapersonal space can be easily and safely detected and measured using our three-dimensional immersive virtual street crossing program.

In patients with newly diagnosed T1 urothelial carcinoma of the bladder lymphovascular invasion in transurethral resection of bladder tumor specimens predicts disease progression and metastasis.

In this paper, we propose a system for training of stroke patients with unilateral neglect by using technology of virtual reality (VR). The proposed system is designed to compensate for unilateral neglect. This system contains the calibration of unilateral neglect and the training of this disease. The calibration procedure is implemented by aligning the virtual object at a subjective middle line. The training procedure is implemented by completing the missions that are used to keep the virtual avatar safe during crossing the street in a virtual environment. The results of this study show that the proposed system is effective to train unilateral neglect. The left to right ratio scores extracted from this system gradually decrease as the sessions of training are repeated. To validate the VR system parameters, the parameters are analyzed by correlation with those of traditional unilateral neglect assessment methods (such as the line bisection test and the cancellation test).