In vitro evaluation of archaeosome vehicles for transdermal vaccine delivery.

Archaeosomes composed of archaeal total polar lipids (TPL) or semi-synthetic analog vesicles have been used as vaccine adjuvants and delivery systems in animal models for many years. Typically administered by intramuscular or subcutaneous injections, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses against entrapped antigens and provide protection in murine models of infectious disease and cancer. Herein, we evaluated various archaeosomes for transdermal delivery, since this route may help eliminate needle-stick injuries and needle re-use, and therefore increase patient compliance. Archaeosomes composed of TPL from different archaea (Halobacterium salinarum, Methanobrevibacter smithii, Haloferax volcanii) and various semi-synthetic glycolipid combinations were evaluated for their ability to diffuse across the skin barrier using an ex vivo pig skin model and the results were compared to conventional synthetic ester liposomes. Physicochemical characteristics were determined for selected formulations including vesicle size, size distribution, zeta potential, fluidity, antigen (ovalbumin) incorporation efficiency and release. Archaeosomes, in particular those composed of M. smithii TPL or the synthetic glycolipid sulfated S-lactosylarchaeol (SLA) mixed with uncharged glycolipid lactosyl archaeol (LA), appeared to be effective carriers for ovalbumin, achieving much better antigen distribution and vesicle accumulation in the skin epidermis than conventional liposomes. The enhanced skin permeation of archaeosomes may be attributed to their chemical structure and physicochemical properties such as particle size, surface charge, stability, and fluidity of their lipid bilayer.

Semaphorin3A elevates vascular permeability and contributes to cerebral ischemia-induced brain damage.

Semaphorin 3A (Sema3A) increased significantly in mouse brain following cerebral ischemia. However, the role of Sema3A in stroke brain remains unknown. Our aim was to determine wether Sema3A functions as a vascular permeability factor and contributes to ischemic brain damage. Recombinant Sema3A injected intradermally to mouse skin, or stereotactically into the cerebral cortex, caused dose- and time-dependent increases in vascular permeability, with a degree comparable to that caused by injection of a known vascular permeability factor vascular endothelial growth factor receptors (VEGF). Application of Sema3A to cultured endothelial cells caused disorganization of F-actin stress fibre bundles and increased endothelial monolayer permeability, confirming Sema3A as a permeability factor. Sema3A-mediated F-actin changes in endothelial cells were through binding to the neuropilin2/VEGFR1 receptor complex, which in turn directly activates Mical2, a F-actin modulator. Down-regulation of Mical2, using specific siRNA, alleviated Sema3A-induced F-actin disorganization, cellular morphology changes and endothelial permeability. Importantly, ablation of Sema3A expression, cerebrovascular permeability and brain damage were significantly reduced in response to transient middle cerebral artery occlusion (tMCAO) and in a mouse model of cerebral ischemia/haemorrhagic transformation. Together, these studies demonstrated that Sema3A is a key mediator of cerebrovascular permeability and contributes to brain damage caused by cerebral ischemia.

IGFBP-4 anti-angiogenic and anti-tumorigenic effects are associated with anti-cathepsin B activity.

Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our studies, CIBP-4 was shown to internalize and co-localize with lysosomal-like structures in both endothelial cells (ECs) and glioblastoma U87MG cells. CIBP-4 also inhibited both growth factor-induced EC tubulogenesis in Matrigel and the concomitant increases in intracellular cathepsin B (CatB) activity. In vitro assays confirmed CIBP-4 capacity to block recombinant CatB activity. Biodistribution analysis of intravenously injected CIBP-4-Cy5.5 in a glioblastoma tumor xenograft model indicated targeted accumulation of CIBP-4 in tumors. Most importantly, CIBP-4 reduced tumor growth in this animal model by 60%. Pleiotropic anti-angiogenic and anti-tumorigenic activities of CIBP-4 most likely underlie its observed therapeutic potential against glioblastoma.

Polymer peel-off mask for high-resolution surface derivatization, neuron placement and guidance.

We present a dry lift-off method using a chemically resistant spin-on plastic, polyimide, to pattern surfaces with high accuracy and resolution. Using well-known lithographic and reactive ion etching techniques, the spin-on polymer is patterned over a silicon dioxide surface. The plastic efficiently adheres to the silicon dioxide surface during the chemical modification and is readily lifted-off following the derivatization process, permitting highly reliable surface derivatization. The verticality of the reactive ion etch enables sub-micrometer features to be patterned, down to 0.8 µm. The technique is used to pattern neurons on silicon dioxide surfaces: efficient neuron placement over a 4 mm area is shown for patterns larger than 50 µm while process guidance is shown for 10 µm patterns.

Culturing and electrophysiology of cells on NRCC patch-clamp chips.

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons; recently, we have also reported progress towards interrogating mammalian neurons. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel .The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.

From understanding cellular function to novel drug discovery: the role of planar patch-clamp array chip technology.

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions - including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.

Recordings of cultured neurons and synaptic activity using patch-clamp chips.

Planar patch-clamp chip technology has been developed to enhance the assessment of novel compounds for therapeutic efficacy and safety. However, this technology has been limited to recording ion channels expressed in isolated suspended cells, making the study of ion channel function in synaptic transmission impractical. Recently, we developed single- and dual-recording site planar patch-clamp chips and demonstrated their capacity to record ion channel activity from neurons established in culture. Such capacity provides the opportunity to record from synaptically connected neurons cultured on-chip. In this study we reconstructed, on-chip, a simple synaptic circuit between cultured pre-synaptic visceral dorsal 4 neurons and post-synaptic left pedal dorsal 1 neurons isolated from the mollusk Lymnaea stagnalis. Here we report the first planar patch-clamp chip recordings of synaptic phenomena from these paired neurons and pharmacologically demonstrate the cholinergic nature of this synapse. We also report simultaneous dual-site recordings from paired neurons, and demonstrate dedicated cytoplasmic perfusion of individual neurons via on-chip subterranean microfluidics. This is the first application of planar patch-clamp technology to examine synaptic communication.

Cell to aperture interaction in patch-clamp chips visualized by fluorescence microscopy and focused-ion beam sections.

Patch-clamp is an important method to monitor the electrophysiological activity of cells and the role of pharmacological compounds on specific ion channel proteins. In recent years, planar patch-clamp chips have been developed as a higher throughput approach to the established glass-pipette method. However, proper conditions to optimize the high resistance cell-to-probe seals required to measure the small currents resulting from ion channel activity are still the subject of conjecture. Here, we report on the design of multiple-aperture (sieve) chips to rapidly facilitate assessment of cell-to-aperture interactions in statistically significant numbers. We propose a method to pre-screen the quality of seals based on a dye loading protocol through apertures in the chip and subsequent evaluation with fluorescence confocal microscopy. We also show the first scanning electron micrograph of a focused ion beam section of a cell in a patch-clamp chip aperture.

Synaptic neurotransmission depression in ventral tegmental dopamine neurons and cannabinoid-associated addictive learning.

Drug addiction is an association of compulsive drug use with long-term associative learning/memory. Multiple forms of learning/memory are primarily subserved by activity- or experience-dependent synaptic long-term potentiation (LTP) and long-term depression (LTD). Recent studies suggest LTP expression in locally activated glutamate synapses onto dopamine neurons (local Glu-DA synapses) of the midbrain ventral tegmental area (VTA) following a single or chronic exposure to many drugs of abuse, whereas a single exposure to cannabinoid did not significantly affect synaptic plasticity at these synapses. It is unknown whether chronic exposure of cannabis (marijuana or cannabinoids), the most commonly used illicit drug worldwide, induce LTP or LTD at these synapses. More importantly, whether such alterations in VTA synaptic plasticity causatively contribute to drug addictive behavior has not previously been addressed. Here we show in rats that chronic cannabinoid exposure activates VTA cannabinoid CB1 receptors to induce transient neurotransmission depression at VTA local Glu-DA synapses through activation of NMDA receptors and subsequent endocytosis of AMPA receptor GluR2 subunits. A GluR2-derived peptide blocks cannabinoid-induced VTA synaptic depression and conditioned place preference, i.e., learning to associate drug exposure with environmental cues. These data not only provide the first evidence, to our knowledge, that NMDA receptor-dependent synaptic depression at VTA dopamine circuitry requires GluR2 endocytosis, but also suggest an essential contribution of such synaptic depression to cannabinoid-associated addictive learning, in addition to pointing to novel pharmacological strategies for the treatment of cannabis addiction.

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip.

We present a polymer microchip capable of monitoring neuronal activity with a fidelity never before obtained on a planar patch-clamp device. Cardio-respiratory neurons Left Pedal Dorsal 1 (LPeD1) from mollusc Lymnaea were cultured on the microchip's polyimide surface for 2 to 4 hours. Cultured neurons formed high resistance seals (gigaseals) between the cell membrane and the surface surrounding apertures etched in the polyimide. Gigaseal formation was observed without applying external force, such as suction, on neurons. The formation of gigaseals, as well as the low access resistance and shunt capacitance values of the polymer microchip resulted in high-fidelity recordings. On-chip culture of neurons permitted, for the first time on a polymeric patch-clamp device, the recording of high fidelity physiological action potentials. Microfabrication of the hybrid poly(dimethylsiloxane)-polyimide (PDMS-PI) microchip is discussed, including a two-layer PDMS processing technique resulting in minimized shrinking variations.

A novel silicon patch-clamp chip permits high-fidelity recording of ion channel activity from functionally defined neurons.

We report on a simple and high-yield manufacturing process for silicon planar patch-clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high-quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high-impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high-fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole-cell current recordings obtained from a voltage-clamp stimulation protocol, and in current-clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch-clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high-information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity.

Cell placement and guidance on substrates for neurochip interfaces.

Interface devices such as integrated planar patch-clamp chips are being developed to study the electrophysiological activity of neuronal networks grown in vitro. The utility of such devices will be dependent upon the ability to align neurons with interface features on the chip by controlling neuronal placement and by guiding cell connectivity. In this paper, we present a strategy to accomplish this goal. Patterned chemical modification of SiN surfaces with poly-d-lysine transferred from PDMS stamps was used to promote adhesion and guidance of cryo-preserved primary rat cortical neurons. We demonstrate that these neurons can be positioned and grown over microhole features which will ultimately serve as patch-clamp interfaces on the chip.

In vitro studies of antifreeze glycoprotein (AFGP) and a C-linked AFGP analogue.

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found in deep sea Teleost fish. These compounds have the ability to depress the freezing point of the organism such that it can survive the subzero temperatures encountered in its environment. This physical property is very attractive for the cryopreservation of cells, tissues, and organs. Recently, our laboratory has designed and synthesized a functional carbon-linked (C-linked) AFGP analogue (1) that demonstrates tremendous promise as a novel cryoprotectant. Herein we describe the in vitro effects and interactions of C-linked AFGP analogue 1 and native AFGP 8. Our studies reveal that AFGP 8 is cytotoxic to human embryonic liver and human embryonic kidney cells at concentrations higher than 2 and 0.63 mg/mL, respectively, whereas lower concentrations are not toxic. The mechanism of this cytotoxicity is consistent with apoptosis because caspase-3/7 levels are significantly elevated in cell cultures treated with AFGP 8. In contrast, C-linked AFGP analogue 1 displayed no in vitro cytotoxicity even at high concentrations, and notably, caspase-3/7 activities were suppressed well below background levels in cell lines treated with 1. Although the results from these studies limit the human applications of native AFGP, they illustrate the benefits of developing functional C-linked AFGP analogues for various medical, commercial and industrial applications.

Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.

Although organotypic hippocampal slice cultures (OHSCs) are used to study function within the hippocampus, the effect of maintenance in vitro upon protein expression is not fully understood. Therefore, we examined developmental changes in cultures prepared from P8 rats and maintained on porous membranes between medium and atmosphere. Between 7 and 28 days following explantation, altered hippocampal morphology could not be detected despite a significant decrease in both MAP-2c and a mid-range tau isoform by 21 DIV. During the same period, lower GFAP expression was observed, and GFAP labeling suggested a migration of astrocytes to the slice-atmosphere interface. In contrast, levels of the synaptic proteins synaptophysin and PSD-95 were significantly increased, but GAP-43 was not. The preservation of myelinated axons and synapses, along with glial and endothelial cells, was confirmed by ultrastructural analysis. Furthermore, intranuclear inclusion bodies, which are associated with normal aging in vivo, were detected in the CA1 pyramidal layer in cultures older than 14 DIV. When OHSCs were maintained for approximately 3, 4, and 10 weeks, a rise and then fall in the expression of synaptophysin and, especially, PSD-95 were found, and the biphasic trend paralleled by significant changes in Schaffer collateral-evoked excitatory post-synaptic potentials from CA1 neurons. Our data not only describe changes in cytoskeletal, synaptic, and nuclear proteins related to the maintenance of interface OHSCs, but also emphasize the potential of the model for the study of age-related phenomena within the hippocampus.

Cobra cardiotoxin-induced cell death in fetal rat cardiomyocytes and cortical neurons: different pathway but similar cell surface target.

Cobra cardiotoxins (CTXs) are basic polypeptides with diverse pharmacological functions that are cytotoxic to many different cell types through both necrotic and apoptotic cell death pathways. In this comparative study of the action of CTX A3 from the Taiwan cobra (Naja atra) on fetal rat cardiomyocytes and cortical neurons, it was shown that CTX A3 induced different patterns of elevation of intracellular Ca2+ concentration ([Ca2+]i), CTX internalization, caspase-3 activity and viability. Application of an anti-sulfatide monoclonal antibody, O4 specific for 3-sulfo-galactose lipid, but not in the control experiments using anti-GM3 monoclonal antibody, reduces CTX-induced [Ca2+]i elevation, CTX internalization and toxicity. Therefore, CTX may target similar sulfo-containing cell surface receptors in both fetal rat cardiomyocytes and cortical neurons, but induce cell death through different pathways specific to each cell type.

An alternative Ca2+-dependent mechanism of neuroprotection by the metalloporphyrin class of superoxide dismutase mimetics.

This study challenges the conventional view that metalloporphyrins protect cultured cortical neurons in models of cerebral ischemia by acting as intracellular catalytic antioxidants [superoxide dismutase (SOD) mimetics]. High SOD-active Mn(III)porphyrins meso-substituted with N,N'-dimethylimidazolium or N-alkylpyridinium groups did not protect neurons against oxygen-glucose deprivation (OGD), although lower SOD-active and -inactive para isomers protected against N-methyl-D-aspartate (NMDA) exposure. Mn(III)meso-tetrakis(4-benzoic acid)porphyrin (Mn(III)TBAP), as well as SOD-inactive metalloTBAPs and other phenyl ring- or beta-substituted metalloporphyrins that contained redox-insensitive metals, protected cultures against OGD and NMDA neurotoxicity. Crucially, neuroprotective metalloporphyrins suppressed OGD- or NMDA-induced rises in intracellular Ca2+ concentration in the same general rank order as observed for neuroprotection. Results from paraquat toxicity, intracellular fluorescence quenching, electrophysiology, mitochondrial Ca2+, and spontaneous synaptic activity experiments suggest a model in which metalloporphyrins, acting at the plasma membrane, protect neurons against OGD by suppressing postsynaptic NMDA receptor-mediated Ca2+ rises, thereby indirectly preventing accumulation of neurotoxic mitochondrial Ca2+ levels. Though neuroprotective in a manner not originally intended, SOD-inactive metalloporphyrins may represent promising therapeutic agents in diseases such as cerebral ischemia, in which Ca2+ toxicity is implicated. Conventional syntheses aimed at improving the catalytic antioxidant capability and/or intracellular access of metalloporphyrins may not yield improved efficacy in some disease models.

Neurogenesis and neuronal communication on micropatterned neurochips.

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.

Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling.

Transient exposure of rat cortical cultures to nonlethal oxygen-glucose deprivation (OGD preconditioning) induces tolerance to otherwise lethal oxygen-glucose deprivation (OGD) or N-methyl-D-aspartate 24 h later. This study evaluates the role of cytosolic and mitochondrial Ca2+-dependent cellular signaling. Mechanistic findings are placed in context with other models of ischemic preconditioning or known neurotoxic pathways within cortical neurons. Tolerance to otherwise lethal OGD is suppressed by performing OGD preconditioning in the presence of the broad-scope catalytic antioxidants Mn(III)tetra(4-carboxyphenyl)porphyrin (MnTBAP) or Zn(II)tetra(4-carboxyphenyl)porphyrin [Zn(II)TBAP], but not by a less active analog, Mn(III)tetra(4-sulfonatophenyl)porphyrin, or a potent superoxide scavenger, Mn(III)tetra(N-ethyl-2-pyridyl)porphyrin chloride. Inhibitors of adenosine A1 receptors, nitric oxide synthase, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase fail to suppress OGD preconditioning despite possible links with reactive oxygen species in other models of ischemic preconditioning. Preconditioning is suppressed by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which has been ascribed elsewhere to inhibition of superoxide transport to the cytosol through mitochondrial anion channels. However, although it induces mitochondrial Ca2+ uptake, neuronal preconditioning is largely insensitive to mitochondrial uncoupling with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone or 2,4-dinitrophenol. Un-couplers will prevent production of mitochondrial reactive oxygen species, implying nonmitochondrial targets by MnTBAP, Zn(II)TBAP, and DIDS. Emphasizing the importance of an increase in cytosolic Ca2+ during preconditioning, a Ca2+/calmodulin-dependent protein kinase II inhibitor, KN-62, suppresses development of subsequent tolerance. Summarizing, only those cellular transduction pathways that have the potential to be neurotoxic may be activated by preconditioning in cortical neurons. Finally, a marked decrease in extracellular glutamate is observed during otherwise lethal OGD in preconditioned cultures, suggesting that this end effector may represent a point of convergence across different preconditioning models.

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10.

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal-glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24-48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 microg/ml of coenzyme Q(10) (functions both as a ROS scavenger and co-factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q(10) in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.

Protection of cortical neurons against oxygen-glucose deprivation and N-methyl-D-aspartate by DIDS and SITS.

The Cl(-) channel blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) dose-dependently protected against oxygen-glucose deprivation in cultured rat cortical neurons. DIDS or SITS attenuated oxygen-glucose deprivation-induced increases in extracellular glutamate concentrations and intracellular Ca(2+). DIDS or SITS provided moderate protection against N-methyl-D-aspartate (NMDA) toxicity and decreased NMDA receptor-mediated increases in intracellular Ca(2+). Whole-cell NMDA receptor currents were attenuated 39+/-2% and 21+/-3% by 1 mM DIDS and SITS, respectively. Other Cl(-) channel blockers as equipotent as DIDS and SITS did not decrease oxygen-glucose deprivation- or NMDA-mediated neuronal Ca(2+) influx or toxicity. Neurotoxicity by exogenous glutamate was not prevented by SITS and was exacerbated by DIDS. Reductions in oxygen-glucose deprivation-induced increases in intracellular Ca(2+) levels underlie neuroprotection by DIDS and SITS. This was a reflection of lower extracellular [glutamate], direct inhibition of Ca(2+) influx through postsynaptic NMDA receptors, and possibly through other protective properties associated with DIDS and SITS.