Inhibition of the Vesicular Glutamate Transporter (VGLUT) with Congo Red Analogs: New Binding Insights.

The vesicular glutamate transporter (VGLUT) facilitates the uptake of glutamate (Glu) into neuronal vesicles. VGLUT has not yet been fully characterized pharmacologically but a body of work established that certain azo-dyes bearing two Glu isosteres via a linker were potent inhibitors. However, the distance between the isostere groups that convey potent inhibition has not been delineated. This report describes the synthesis and pharmacologic assessment of Congo Red analogs that contain one or two glutamate isostere or mimic groups; the latter varied in the interatomic distance and spacer properties to probe strategic binding interactions within VGLUT. The more potent inhibitors had two glutamate isosteres symmetrically linked to a central aromatic group and showed IC50 values ~ 0.3-2.0 µM at VGLUT. These compounds contained phenyl, diphenyl ether (PhOPh) or 1,2-diphenylethane as the linker connecting 4-aminonaphthalene sulfonic acid groups. A homology model for VGLUT2 using D-galactonate transporter (DgoT) to dock and identify R88, H199 and F219 as key protein interactions with Trypan Blue, Congo Red and selected potent analogs prepared and tested in this report.

VGLUT substrates and inhibitors: A computational viewpoint.

The vesicular glutamate transporters (VGLUTs) bind and move glutamate (Glu) from the cytosol into the lumen of synaptic vesicles using a H+-electrochemical gradient (ΔpH and Δψ) generated by the vesicular H+-ATPase. VGLUTs show very low Glu binding and to date, no three-dimensional structure has been elucidated. Prior studies have attempted to identify the key residues involved in binding VGLUT substrates and inhibitors using homology models and docking experiments. Recently, the inward and outward oriented crystal structures of d-galactonate transporter (DgoT) emerged as possible structure templates for VGLUT. In this review, a new homology model for VGLUT2 based on DgoT has been developed and used to conduct docking experiments to identify and differentiate residues and binding orientations involved in ligand interactions. This review describes small molecule-ligand interactions including docking using a VGLUT2 homology model derived from DgoT.

LSP5-2157 a new inhibitor of vesicular glutamate transporters.

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

Glutamate transport system as a key constituent of glutamosome: Molecular pathology and pharmacological modulation in chronic pain.

Neural uptake of glutamate is executed by the structurally related members of the SLC1A family of solute transporters: GLAST/EAAT1, GLT-1/EAAT2, EAAC1/EAAT3, EAAT4, ASCT2. These plasma membrane proteins ensure supply of glutamate, aspartate and some neutral amino acids, including glutamine and cysteine, for synthetic, energetic and signaling purposes, whereas effective removal of glutamate from the synaptic cleft shapes excitatory neurotransmission and prevents glutamate toxicity. Glutamate transporters (GluTs) possess also receptor-like properties and can directly initiate signal transduction. GluTs are physically linked to other glutamate signaling-, transporting- and metabolizing molecules (e.g., glutamine transporters SNAT3 and ASCT2, glutamine synthetase, NMDA receptor, synaptic vesicles), as well as cellular machineries fueling the transmembrane transport of glutamate (e.g., ion gradient-generating Na/K-ATPase, glycolytic enzymes, mitochondrial membrane- and matrix proteins, glucose transporters). We designate this supramolecular functional assembly as 'glutamosome'. GluTs play important roles in the molecular pathology of chronic pain, due to the predominantly glutamatergic nature of nociceptive signaling in the spinal cord. Down-regulation of GluTs often precedes or occurs simultaneously with development of pain hypersensitivity. Pharmacological inhibition or gene knock-down of spinal GluTs can induce/aggravate pain, whereas enhancing expression of GluTs by viral gene transfer can mitigate chronic pain. Thus, functional up-regulation of GluTs is turning into a prospective pharmacotherapeutic approach for the management of chronic pain. A number of novel positive pharmacological regulators of GluTs, incl. pyridazine derivatives and ß-lactams, have recently been introduced. However, design and development of new analgesics based on this principle will require more precise knowledge of molecular mechanisms underlying physiological or aberrant functioning of the glutamate transport system in nociceptive circuits. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

C9orf72 Dipeptide Repeats Cause Selective Neurodegeneration and Cell-Autonomous Excitotoxicity in Drosophila Glutamatergic Neurons.

The arginine-rich dipeptide repeats (DPRs) are highly toxic products from the C9orf72 repeat expansion mutations, which are the most common causes of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the effects of DPRs in the synaptic regulation and excitotoxicity remain elusive, and how they contribute to the development of FTD is primarily unknown. By expressing DPRs with different toxicity strength in various neuronal populations in a Drosophila model, we unexpectedly found that Glycine-Arginine/Proline-Arginine (GR/PR) with 36 repeats could lead to neurodegenerative phenotypes only when they were expressed in glutamatergic neurons, including motor neurons. We detected increased extracellular glutamate and intracellular calcium levels in GR/PR-expressing larval ventral nerve cord and/or adult brain, accompanied by significant increase of synaptic boutons and active zones in larval neuromuscular junctions. Inhibiting the vesicular glutamate transporter expression or blocking the NMDA receptor in presynaptic glutamatergic motor neurons could effectively rescue the motor deficits and shortened life span caused by poly GR/PR, thus indicating a cell-autonomous excitotoxicity mechanism. Therefore, our results have revealed a novel mode of synaptic regulation by arginine-rich C9 DPRs expressed at more physiologically relevant toxicity levels and provided a mechanism that could contribute to the development of C9-related ALS and FTD.SIGNIFICANCE STATEMENT C9orf72 dipeptide repeats (DPRs) are key toxic species causing ALS/FTD, but their roles in synaptic regulation and excitotoxicity are unclear. Using C9orf72 DPRs with various toxicity strength, we have found that the arginine-rich DPRs cause selective degeneration in Drosophila glutamatergic neurons and revealed an NMDA receptor-dependent cell-autonomous excitotoxicity mechanism. Therefore, this study has advanced our understanding of C9orf72 DPR functions in synaptic regulation and excitotoxicity and provided a new mechanism that could contribute to the development of C9-related ALS and FTD.

Cortical functional hyperconnectivity in a mouse model of depression and selective network effects of ketamine.

See Huang and Liston (doi:10.1093/awx166) for a scientific commentary on this article.Human depression is associated with glutamatergic dysfunction and alterations in resting state network activity. However, the indirect nature of human in vivo glutamate and activity assessments obscures mechanistic details. Using the chronic social defeat mouse model of depression, we determine how mesoscale glutamatergic networks are altered after chronic stress, and in response to the rapid acting antidepressant, ketamine. Transgenic mice (Ai85) expressing iGluSnFR (a recombinant protein sensor) permitted real-time in vivo selective characterization of extracellular glutamate and longitudinal imaging of mesoscale cortical glutamatergic functional circuits. Mice underwent chronic social defeat or a control condition, while spontaneous cortical activity was longitudinally sampled. After chronic social defeat, we observed network-wide glutamate functional hyperconnectivity in defeated animals, which was confirmed with voltage sensitive dye imaging in an independent cohort. Subanaesthetic ketamine has unique effects in defeated animals. Acutely, subanaesthetic ketamine induces large global cortical glutamate transients in defeated animals, and an elevated subanaesthetic dose resulted in sustained global increase in cortical glutamate. Local cortical inhibition of glutamate transporters in naïve mice given ketamine produced a similar extracellular glutamate phenotype, with both glutamate transients and a dose-dependent accumulation of glutamate. Twenty-four hours after ketamine, normalization of depressive-like behaviour in defeated animals was accompanied by reduced glutamate functional connectivity strength. Altered glutamate functional connectivity in this animal model confirms the central role of glutamate dynamics as well as network-wide changes after chronic stress and in response to ketamine.

Extrasynaptic glutamate release through cystine/glutamate antiporter contributes to ischemic damage.

During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and ischemic neuronal death.

Design, synthesis and biological evaluation of small-azo-dyes as potent Vesicular Glutamate Transporters inhibitors.

Vesicular Glutamate Transporters (VGLUTs) allow the loading of presynapic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. VGLUTs have proved to be involved in several major neuropathologies and directly correlated to clinical dementia in Alzheimer and Parkinson's disease. Accordingly VGLUT represent a key biological target or biomarker for neuropathology treatment or diagnostic. Yet, despite the pivotal role of VGLUTs, their pharmacology appears quite limited. Known competitive inhibitors are restricted to some dyes as Trypan Blue (TB) and glutamate mimics. This lack of pharmacological tools has heavily hampered VGLUT investigations. Here we report a rapid access to small molecules that combine benefits of TB and dicarboxylic quinolines (DCQs). Their ability to block vesicular glutamate uptake was evaluated. Several compounds displayed low micromolar inhibitory potency when size related compounds are thirty to forty times less potent (i.e. DCQ). We then confirmed the VGLUT selectivity by measuring the effect of the series on vesicular monoamine transport and on metabotropic glutamate receptor activity. These inhibitors are synthesized in only two steps and count among the best pharmacological tools for VGLUTs studies.

Inhibition of vesicular glutamate transporters contributes to attenuate methamphetamine-induced conditioned place preference in rats.

Accumulating evidence suggests that glutamatergic system plays a crucial role in methamphetamine (METH) addiction. In the glutamatergic transmission, vesicular glutamate transporters (VGLUTs) are responsible for transporting glutamate into synaptic vesicles and affect the glutamate concentrations in the synaptic cleft. It is well documented that VGLUTs play an essential role in pathophysiology of several psychiatric and neurological diseases, however, whether VGLUTs also have a role in addiction caused by psychostimulant drugs is still unknown. The present study was underwent to investigate the effect of inhibition of VGLUTs on METH-induced induce conditioned place preference in rats. Rats were induced to conditioned place preference with METH (0.5, 1.0 and 2.0mg/kg) by intraperitoneal injection. Intracerebroventricular administration of 1.0 or 5.0µg Chicago sky blue 6B (CSB6B), a VGLUTs inhibitor, and 2.5h prior to METH was to observe its effect on METH-induced conditioned place preference in rats. The rats receiving METH showed stronger place preference at the dose of 1.0mg/kg than that of other doses. The intracerebroventricular administration of CSB6B (1.0, 5.0µg) 2.5h prior to the exposure to METH attenuated the acquisition of METH-induced conditioned place preference, while CSB6B itself had no effect on place preference. These results indicate that VGLUTs are involved in the effect of METH-induced conditioned place preference and may be a new target against METH addiction.

The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids.

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50∼70 µM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈20 µM.

A new VGLUT-specific potent inhibitor: pharmacophore of Brilliant Yellow.

The increased concentration of glutamate in synaptic vesicles, mediated by the vesicular glutamate transporter (VGLUT), is an initial vital step in glutamate synaptic transmission. Evidence indicates that aberrant overexpression of VGLUT is involved in certain pathophysiologies of the central nervous system. VGLUT is subject to inhibition by various types of agents. The most potent VGLUT-specific inhibitor currently known is Trypan Blue, which is highly charged, hence membrane-impermeable. We have sought a potent, VGLUT-specific agent amenable to easy modification to a membrane-permeable analog. We provide evidence that Brilliant Yellow exhibits potent, VGLUT-specific inhibition, with a Ki value of 12 nM. Based upon structure-activity relationship studies and molecular modeling, we have defined the potent inhibitory pharmacophore of Brilliant Yellow. This study provides new insight into development of a membrane-permeable agent to lead to specific blockade, with high potency, of accumulation of glutamate into synaptic vesicles in neurons.

The antinociceptive effects of intracerebroventricular administration of Chicago sky blue 6B, a vesicular glutamate transporter inhibitor.

Accumulating evidence suggests that vesicular glutamate transporters (VGLUTs), which control the storage and release of glutamate, may play a role in pain processing. Chicago sky blue 6B (CSB6B), which is structurally related to glutamate, is a competitive VGLUT inhibitor without affecting plasma membrane transporters. The present study was designed to investigate the antinociceptive effects of CSB6B in a number of pain models. The hot-plate test was used as an acute thermal pain test. Inflammatory pain was evaluated using acetic acid writhing, formalin, and complete Freund's adjuvant tests. Intracerebroventricular administration of CSB6B did not affect acute thermal pain responses in 50 or 55°C hot plate tests. However, CSB6B attenuated acetic acid-induced writhing in a dose-dependent and time-dependent manner. In addition, CSB6B reduced licking/biting behavior during the second phase, but not during the first phase, following an intraplantar injection of formalin. In the complete Freund's adjuvant test, a significant attenuation of thermal hyperalgesia was also observed in CSB6B-treated mice. At antinociceptive doses, CSB6B did not affect mice spontaneous locomotor activity. The present study shows that pharmacological inhibition of VGLUT activity was sufficient to attenuate experimental inflammatory pain and suggests that regulation of VGLUTs might be a novel therapeutic strategy for the treatment of pain.

Modulation of hippocampal synaptic transmission by the kynurenine pathway member xanthurenic acid and other VGLUT inhibitors.

Xanthurenic acid (XA), an endogenous kynurenine, is a known vesicular glutamate transport (VGLUT) inhibitor and has also been proposed as an mGlu2/3 receptor agonist. Changes in these systems have been implicated in the pathophysiology of schizophrenia and other psychiatric disorders; however, little is known of how XA affects synaptic transmission. We therefore investigated the effects of XA on synaptic transmission at two hippocampal glutamatergic pathways and evaluated the ability of XA to bind to mGlu2/3 receptors. Field excitatory postsynaptic potentials (fEPSPs) were recorded from either the dentate gyrus (DG) or CA1 region of mouse hippocampal slices in vitro. Addition of XA to the bathing medium (1-10 mM) resulted in a dose-related reduction of fEPSP amplitudes (up to 52% reduction) in both hippocampal regions. In the DG, the VGLUT inhibitors Congo Red and Rose Bengal, and the mGlu2/3 agonist LY354740, also reduced fEPSPs (up to 80% reduction). The mGlu2/3 antagonist LY341495 reversed the LY354740 effect, but not the XA effect. LY354740, but not XA, also reduced DG paired-pulse depression. XA had no effect on specific binding of 1 nM [(3)H]LY341495 to membranes with human mGlu2 receptors. We conclude that XA can modulate synaptic transmission via a mechanism that may involve VGLUT inhibition rather than activation of mGlu2/3 receptors. This could be important in the pathophysiology of nervous system disorders including schizophrenia and might represent a target for developing novel pharmacological therapies.

Chicago sky blue 6B, a vesicular glutamate transporters inhibitor, attenuates methamphetamine-induced hyperactivity and behavioral sensitization in mice.

Several lines of evidence demonstrate that glutamatergic system plays an important role in drug addiction. The present study was designed to investigate the effects of Chicago sky blue 6B (CSB6B), a vesicular glutamate transporters (VGLUTs) inhibitor, on methamphetamine (METH)-induced behaviors in mice. Mice were induced behavioral sensitization to METH by subcutaneous injection of 1mg/kg METH once daily for 7 days and then challenged with 1mg/kg METH in 14th day. Intracerebroventricular administration of CSB6B (7.5µg) 2.5h prior to METH was to observe its effects on METH -induced behavioral sensitization. Our results showed that the expressions of behavioral sensitization were significantly attenuated by intracerebroventricular administration of CSB6B 2.5h prior to METH either during the development period or before methamphetamine challenge in mice, while CSB6B itself had no effect on locomotor activity. Meanwhile, pretreatment of CSB6B also attenuated hyperactivity caused by a single injection of METH in mice. These results demonstrated that CSB6B, a VGLUTs inhibitor, attenuated acute METH-induced hyperactivity and chronic METH-induced behavioral sensitization, which indicated that VGLUTs were involved in the effect of chronic METH-induced behavioral sensitization and may be a new target against the addiction of METH.

Glutamate transporters control metabotropic glutamate receptors activation to prevent the genesis of paroxysmal burst in the developing hippocampus.

Metabotropic glutamate receptors (mGluR) can control neuronal excitability by modulating several ionic channels. In hippocampal pyramidal cells, groups I/II mGluR are located extrasynaptically, suggesting that their endogenous activation is dependent on the glutamate clearance rate and therefore on excitatory amino-acid transporters (EAAT) efficiency. Deficiency of glutamate uptake can generate seizures in rodents and has been suggested as a mechanism of seizure generation in some human epileptic syndromes. However, the cellular mechanisms linking EAAT dysfunction and pathological cortical activities remain elusive. Here, we investigate the possible role of mGluR on paroxysmal burst of multiple unit activities (MUA) generated in the CA1 region of developing hippocampal slices using an EAAT inhibitor, TBOA. These bursts are generated by a synaptic release of glutamate and involve extrasynaptic NMDA receptors (NMDAR) activated by transmitter spillover. Here, we show that postsynaptic mGluR (groups I/II) are tonically activated by the rise in ambient glutamate concentration after EAAT inhibition and strongly contribute to paroxysmal burst genesis. The inhibition of mGluR with broad spectrum antagonists or addition of a glutamate scavenger strongly reduced the occurrence of paroxysmal burst and the frequency/number of MUA during the burst. Moreover, this endogenous activation of groups I/II mGluR leads to (i) the reduction of the slow afterhyperpolarization current (I(sAHP)), increasing the firing pattern of pyramidal cells, and (ii) the potentiation of extrasynaptic NMDAR-mediated responses, enabling glutamate spillover to generate a suprathreshold depolarization for several seconds. Our data show that an insufficient buffering of extracellular glutamate enables a cross talk between groups I/II mGluR and NMDAR, which, combined with a decrease of I(sAHP), leads to the hyperexcitability of the hippocampal network, facilitating the genesis of epileptical-like activity in response to glutamate release. These findings highlight the importance of the control exerted by EAAT on mGluR.

Marked synergism between mutant SOD1 and glutamate transport inhibition in the induction of motor neuronal degeneration in spinal cord slice cultures.

Loss of astrocytic glutamate transport capacity in ALS spinal cord supports an excitotoxic contribution to motor neuron (MN) damage in the disease, and dominant gain of function mutations in Cu/Zn superoxide dismutase (SOD1) cause certain familial forms of ALS. We have used organotypic slice cultures from wild type and G93A SOD1 mutant rat spinal cords to examine interactions between excitotoxicity and the presence of mutant SOD1 in the induction of MN degeneration. Slice cultures were prepared from 1 week old pups, and after an additional week in vitro, some were exposed to either a low level (30 µM) of the glutamate uptake inhibitor, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) for 3 weeks, or a higher level (50 µM) for 48 h, followed by histochemical labeling to assess MN injury. In wild type animals these exposures caused relatively little MN degeneration. Similarly, little MN degeneration was seen in slices from SOD1 mutant animals that were not exposed to PDC. However, addition of PDC to SOD1 mutant slices resulted in substantial MN injury, which was markedly attenuated by a Ca2+ permeable AMPA-type (Ca-AMPA) glutamate channel blocker, or by a nitric oxide synthase antagonist. These observations illustrate the utility of the organotypic culture model for the investigation of intracellular interactions underlying MN degeneration in ALS, and support the hypothesis that activation of Ca-AMPA channels on MNs provides a metabolic burden that synergizes with deleterious effects of mutant SOD1 in the induction of MN injury.

Mechanisms of GABA release from human astrocytes.

We have previously demonstrated that human astrocytes are GABAergic cells. Throughout the adult human brain, they express the GABA synthesizing enzyme GAD 67, the GABA metabolizing enzyme GABA-T, and the GABA(A) and GABA(B) receptors. GABA modulates the actions of microglia, indicating an important role for astrocytes beyond that of influencing neurotransmitter function. Here we report on the mechanisms by which astrocytes release GABA. Astrocytes were found to express the mRNA and protein for multiple GABA transporters, and multiple receptors for glutamate, GABA, and glycine. In culture, untreated human astrocytes maintained an intracellular GABA level of 2.32 mM. They exported GABA into the culture medium so that an intracellular-extracellular gradient of 3.64 fold was reached. Inhibitors of the GABA transporters GAT1, GAT2, and GAT3, significantly reduced this export in a Ca(2+)-independent fashion. Intracellular GABA levels were enhanced by treatment with the GABA-T inhibitors gabaculine or vigabatrin. Treatment with glutamate increased GABA release in a concentration-dependent fashion. This was partially inhibited by blockers of N-methyl-D-aspartate and kainate receptors. Conversely, glycine and D-serine, co-agonists of NMDA receptors, enhanced the GABA release. GABA release was accompanied by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and was reduced by adding the Ca(2+) chelator, BAPTA-AM to the medium. These data indicate that astrocytes continuously synthesize GABA and that there are multiple mechanisms which can mediate its release. Each of these may play a role in the physiological functioning of astrocytes.

Use of the hydantoin isostere to produce inhibitors showing selectivity toward the vesicular glutamate transporter versus the obligate exchange transporter system x(c)(-).

Evidence was acquired prior to suggest that the vesicular glutamate transporter (VGLUT) but not other glutamate transporters were inhibited by structures containing a weakly basic α-amino group. To test this hypothesis, a series of analogs using a hydantoin (pK(a)∼9.1) isostere were synthesized and analyzed as inhibitors of VGLUT and the obligate cystine-glutamate transporter (system x(c)(-)). Of the hydantoin analogs tested, a thiophene-5-carboxaldehyde analog 2l and a bis-hydantoin 4b were relatively strong inhibitors of VGLUT reducing uptake to less than 6% of control at 5mM but few inhibited system x(c)(-) greater than 50% of control. The benzene-2,4-disulfonic acid analog 2b and p-diaminobenzene analog 2e were also good hydantoin-based inhibitors of VGLUT reducing uptake by 11% and 23% of control, respectively, but neither analog was effective as a system x(c)(-) inhibitor. In sum, a hydantoin isostere adds the requisite chemical properties needed to produce selective inhibitors of VGLUT.

Distinct pharmacological and functional properties of NMDA receptors in mouse cortical astrocytes.

BACKGROUND AND PURPOSE: Astrocytes of the mouse neocortex express functional NMDA receptors, which are not blocked by Mg(2+) ions. However, the pharmacological profile of glial NMDA receptors and their subunit composition is far from complete. EXPERIMENTAL APPROACH: We tested the sensitivity of NMDA receptor-mediated currents to the novel GluN2C/D subunit-selective antagonist UBP141 in mouse cortical astrocytes and neurons. We also examined the effect of memantine, an antagonist that has substantially different affinities for GluN2A/B and GluN2C/d-containing receptors in physiological concentrations of extracellular Mg(2+). KEY RESULTS: UBP141 had a strong inhibitory action on NMDA receptor-mediated transmembrane currents in the cortical layer II/III astrocytes with an IC(50) of 2.29 µM and a modest inhibitory action on NMDA-responses in the pyramidal neurons with IC(50) of 19.8 µM. Astroglial and neuronal NMDA receptors exhibited different sensitivities to memantine with IC(50) values of 2.19 and 10.8 µM, respectively. Consistent with pharmacological differences between astroglial and neuronal NMDA receptors, NMDA receptors in astrocytes showed lower Ca(2+) permeability than neuronal receptors with P(Ca) /P(Na) ratio of 3.4. CONCLUSIONS AND IMPLICATIONS: The biophysical and pharmacological properties of the astrocytic NMDA receptors strongly suggest that they have a tri-heteromeric structure composed of GluN1, GluN2C/D and GluN3 subunits. The substantial difference between astroglial and neuronal NMDA receptors in their sensitivity to UBP141 and memantine may enable selective modulation of astrocytic signalling that could be very helpful for elucidating the mechanisms of neuron-glia communications. Our results may also provide the basis for the development of novel therapeutic agents specifically targeting glial signalling.

Rose Bengal analogs and vesicular glutamate transporters (VGLUTs).

Vesicular glutamate transporters (VGLUTs) allow the loading of presynaptic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. Rose Bengal (RB) is the most potent known VGLUT inhibitor (Ki 25 nM); therefore we designed, synthesized and tested in brain preparations, a series of analogs based on this scaffold. We showed that among the two tautomers of RB, the carboxylic and not the lactonic form is active against VGLUTs and generated a pharmacophore model to determine the minimal structure requirements. We also tested RB specificity in other neurotransmitter uptake systems. RB proved to potently inhibit VMAT (Ki 64 nM) but weakly VACHT (Ki>9.7 microM) and may be a useful tool in glutamate/acetylcholine co-transmission studies.