Organotypic Hippocampal Brain Slices Research Articles

The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices.

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts - and knowing how to avoid them - takes on special urgency.We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a 'before-and-after' experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters.While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animals in vivo where the ECS typically becomes nearly depleted.Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues.Significance StatementChemical fixation of biological samples using PFA is a standard step routinely performed in neuroscience labs. However, it is known to alter various anatomical parameters ranging from protein distribution to cell morphology, potentially affecting our interpretation of anatomical data. With the increasing use of super-resolution microscopy, understanding the extent and nature of fixation artifacts is an urgent concern.Here, we use live STED microscopy to monitor in real time the impact of PFA on the microanatomy of organotypic hippocampal brain slices. Our results demonstrate that while PFA has little impact on the extracellular space and astrocytes, it compromises cell membranes and dendritic structures. Our study provides a strategy for a direct characterization of fixation artifacts at the nanoscale, facilitating the optimization of fixation protocols.

Extremely Low-Frequency Electromagnetic Stimulation (ELF-EMS) Improves Neurological Outcome and Reduces Microglial Reactivity in a Rodent Model of Global Transient Stroke.

Extremely low-frequency electromagnetic stimulation (ELF-EMS) was demonstrated to be significantly beneficial in rodent models of permanent stroke. The mechanism involved enhanced cerebrovascular perfusion and endothelial cell nitric oxide production. However, the possible effect on the neuroinflammatory response and its efficacy in reperfusion stroke models remains unclear. To evaluate ELF-EMS effectiveness and possible immunomodulatory response, we studied neurological outcome, behavior, neuronal survival, and glial reactivity in a rodent model of global transient stroke treated with 13.5 mT/60 Hz. Next, we studied microglial cells migration and, in organotypic hippocampal brain slices, we assessed neuronal survival and microglia reactivity. ELF-EMS improved the neurological score and behavior in the ischemia-reperfusion model. It also improved neuronal survival and decreased glia reactivity in the hippocampus, with microglia showing the first signs of treatment effect. In vitro ELF-EMS decreased (Lipopolysaccharide) LPS and ATP-induced microglia migration in both scratch and transwell assay. Additionally, in hippocampal brain slices, reduced microglial reactivity, improved neuronal survival, and modulation of inflammation-related markers was observed. Our study is the first to show that an EMF treatment has a direct impact on microglial migration. Furthermore, ELF-EMS has beneficial effects in an ischemia/reperfusion model, which indicates that this treatment has clinical potential as a new treatment against ischemic stroke.

Super-resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge.

The extracellular space (ECS) plays a central role in brain physiology, shaping the time course and spread of neurochemicals, ions, and nutrients that ensure proper brain homeostasis and neuronal communication. Astrocytes are the most abundant type of glia cell in the brain, whose processes densely infiltrate the brain's parenchyma. As astrocytes are highly sensitive to changes in osmotic pressure, they are capable of exerting a potent physiological influence on the ECS. However, little is known about the spatial distribution and temporal dynamics of the ECS that surrounds astrocytes, owing mostly to a lack of appropriate techniques to visualize the ECS in live brain tissue. Mitigating this technical limitation, we applied the recent SUper-resolution SHadow Imaging technique (SUSHI) to astrocyte-labeled organotypic hippocampal brain slices, which allowed us to concurrently image the complex morphology of astrocytes and the ECS with unprecedented spatial resolution in a live experimental setting. Focusing on ring-like astrocytic microstructures in the spongiform domain, we found them to enclose sizable pools of interstitial fluid and cellular structures like dendritic spines. Upon experimental osmotic challenge, these microstructures remodeled and swelled up at the expense of the pools, effectively increasing the physical interface between astrocytic and cellular structures. Our study reveals novel facets of the dynamic microanatomical relationships between astrocytes, neuropil, and the ECS in living brain tissue, which could be of functional relevance for neuron-glia communication in a variety of (patho)physiological settings, for example, LTP induction, epileptic seizures or acute ischemic stroke, where osmotic disturbances are known to occur.

Preparation and Culture of Organotypic Hippocampal Slices for the Analysis of Brain Metastasis and Primary Brain Tumor Growth.

Brain metastasis is a major challenge for therapy and defines the end stage of tumor progression with a very limited patients' prognosis. Experimental setups that faithfully mimic these processes are necessary to understand the mechanism of brain metastasis and to develop new improved therapeutic strategies. Here, we describe an in vitro model, which closely resembles the in vivo situation. Organotypic hippocampal brain slice cultures (OHSCs) prepared from 3- to 8-day-old mice are well suited for neuro-oncology research including brain metastasis. The original morphology is preserved in OHSCs even after culture periods of several days to weeks. Tumor cells or cells of metastatic origin can be seeded onto OHSCs to evaluate micro-tumor formation, tumor cell invasion, or treatment response. We describe preparation and culture of OHSCs including the seeding of tumor cells. Finally, we show examples of how to treat the OHSCs for life-dead or immunohistochemical staining.

Noble gas neuroprotection: xenon and argon protect against hypoxic–ischaemic injury in rat hippocampus in vitro via distinct mechanisms

BackgroundNoble gases may provide novel treatments for neurological injuries such as ischaemic and traumatic brain injury. Few studies have evaluated the complete series of noble gases under identical conditions in the same model. MethodsWe used an in vitro model of hypoxia–ischaemia to evaluate the neuroprotective properties of the series of noble gases, helium, neon, argon, krypton, and xenon. Organotypic hippocampal brain slices from mice were subjected to oxygen-glucose deprivation, and injury was quantified using propidium iodide fluorescence. ResultsBoth xenon and argon were equally effective neuroprotectants, with 0.5 atm of xenon or argon reducing injury by 96% (P<0.0001), whereas helium, neon, and krypton were devoid of any protective effect. Neuroprotection by xenon, but not argon, was reversed by elevated glycine. ConclusionsXenon and argon are equally effective as neuroprotectants against hypoxia–ischaemia in vitro, with both gases preventing injury development. Although xenon's neuroprotective effect may be mediated by inhibition of the N-methyl-d-aspartate receptor at the glycine site, argon acts via a different mechanism. These findings may have important implications for their clinical use as neuroprotectants.

Fructose-1,6-Bisphosphate Protects Hippocampal Rat Slices from NMDA Excitotoxicity.

Effects of fructose 1,6-bisphosphate (F-1,6-P2) towards N-methyl-d-aspartate NMDA excitotoxicity were evaluated in rat organotypic hippocampal brain slice cultures (OHSC) challenged for 3 h with 30 μM NMDA, followed by incubations (24, 48, and 72 h) without (controls) and with F-1,6-P2 (0.5, 1 or 1.5 mM). At each time, cell necrosis was determined by measuring LDH in the medium. Energy metabolism was evaluated by measuring ATP, GTP, ADP, AMP, and ATP catabolites (nucleosides and oxypurines) in deproteinized OHSC extracts. Gene expressions of phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase were also measured. F-1,6-P2 dose-dependently decreased NMDA excitotoxicity, abolishing cell necrosis at the highest concentration tested (1.5 mM). Additionally, F-1,6-P2 attenuated cell energy imbalance caused by NMDA, ameliorating the mitochondrial phosphorylating capacity (increase in ATP/ADP ratio) Metabolism normalization occurred when using 1.5 mM F-1,6-P2. Remarkable increase in expressions of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase (up to 25 times over the values of controls) was also observed. Since this phenomenon was recorded even in OHSC treated with F-1,6-P2 with no prior challenge with NMDA, it is highly conceivable that F-1,6-P2 can enter into intact cerebral cells producing significant benefits on energy metabolism. These effects are possibly mediated by changes occurring at the gene level, thus opening new perspectives for F-1,6-P2 application as a useful adjuvant to rescue mitochondrial metabolism of cerebral cells under stressing conditions.

A 6-Step Approach to Gain Higher Quality Results From Organotypic Hippocampal Brain Slices in a Traumatic Brain Injury Model.

Introduction:Organotypic Hippocampal Brain Slices (OHBS) provide an advantageous alternative to in vivo models to scrutinize Traumatic Brain Injury (TBI). We followed a well-established TBI protocol, but noticed that several factors may influence the results in such a setup. Here, we describe a structured approach to generate more comparable results and discuss why specific eligibility criteria should be applied.Methods:We defined necessary checkpoints and developed inclusion and exclusion criteria that take the observed variation in such a model into consideration. Objective measures include the identification and exclusion of pre-damaged slices and outliers. Six steps were outlined in this study.Results:A six-step approach to enhance comparability is proposed and summarized in a flowchart. We applied the suggested measures to data derived from our TBI-experiments examining the impact of three different interventions in 1459 OHBS. Our exemplary results show that through equal requirements set for all slices more precise findings are ensured.Conclusion:Results in a TBI experiment on OHBS should be analyzed critically as inhomogeneities may occur. In order to ensure more precise findings, a structured approach of comparing the results should be followed. Further research is recommended to confirm and further develop this framework.

Argon attenuates the emergence of secondary injury after traumatic brain injury within a 2-hour incubation period compared to desflurane: an in vitro study.

Despite years of research, treatment of traumatic brain injury (TBI) remains challenging. Considerable data exists that some volatile anesthetics might be neuroprotective. However, several studies have also revealed a rather neurotoxic profile of anesthetics. In this study, we investigated the effects of argon 50%, desflurane 6% and their combination in an in vitro TBI model with incubation times similar to narcotic time slots in a daily clinical routine. Organotypic hippocampal brain slices of 5- to 7-day-old mice were cultivated for 14 days before TBI was performed. Slices were eventually incubated for 2 hours in an atmosphere containing no anesthetic gas, argon 50% or desflurane 6% or both. Trauma intensity was evaluated via fluorescent imagery. Our results show that neither argon 50% nor desflurane 6% nor their combination could significantly reduce the trauma intensity in comparison to the standard atmosphere. However, in comparison to desflurane 6%, argon 50% displayed a rather neuroprotective profile within the first 2 hours after a focal mechanical trauma (P = 0.015). A 2-hour incubation in an atmosphere containing both gases, argon 50% and desflurane 6%, did not result in significant effects in comparison to the argon 50% group or the desflurane 6% group. Our findings demonstrate that within a 2-hour incubation time neither argon nor desflurane could affect propidium iodide-detectable cell death in an in vitro TBI model in comparison to the standard atmosphere, although cell death was less with argon 50% than with desflurane 6%. The results show that within this short time period processes concerning the development of secondary injury are already taking place and may be manipulated by argon.

Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds.

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo. Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance.

TRIO loss of function is associated with mild intellectual disability and affects dendritic branching and synapse function.

Recently, we marked TRIO for the first time as a candidate gene for intellectual disability (ID). Across diverse vertebrate species, TRIO is a well-conserved Rho GTPase regulator that is highly expressed in the developing brain. However, little is known about the specific events regulated by TRIO during brain development and its clinical impact in humans when mutated. Routine clinical diagnostic testing identified an intragenic de novo deletion of TRIO in a boy with ID. Targeted sequencing of this gene in over 2300 individuals with ID, identified three additional truncating mutations. All index cases had mild to borderline ID combined with behavioral problems consisting of autistic, hyperactive and/or aggressive behavior. Studies in dissociated rat hippocampal neurons demonstrated the enhancement of dendritic formation by suppressing endogenous TRIO, and similarly decreasing endogenous TRIO in organotypic hippocampal brain slices significantly increased synaptic strength by increasing functional synapses. Together, our findings provide new mechanistic insight into how genetic deficits in TRIO can lead to early neuronal network formation by directly affecting both neurite outgrowth and synapse development.

What Elements of the Inflammatory System Are Necessary for Epileptogenesis In Vitro?

Epileptogenesis in vivo can be altered by manipulation of molecules such as cytokines and complement that subserve intercellular signaling in both the inflammatory and central nervous systems. Because of the dual roles of these signaling molecules, it has been difficult to precisely define the role of systemic inflammation in epileptogenesis. Organotypic hippocampal brain slices can be maintained in culture independently of the systemic inflammatory system, and the rapid course of epileptogenesis in these cultures supports the idea that inflammation is not necessary for epilepsy. However, this preparation still retains key cellular inflammatory mediators. Here, we found that rodent hippocampal organotypic slice cultures depleted of T lymphocytes and microglia developed epileptic activity at essentially the same rate and to similar degrees of severity as matched control slice cultures. These data support the idea that although the inflammatory system, neurons, and glia share key intercellular signaling molecules, neither systemic nor CNS-specific cellular elements of the immune and inflammatory systems are necessary components of epileptogenesis.

Functional tolerance to mechanical deformation developed from organotypic hippocampal slice cultures.

In this study, we measured changes in electrophysiological activity after mechanical deformation of living organotypic hippocampal brain slice cultures at tissue strains and strain rates relevant to traumatic brain injury (TBI). Electrophysiological activity was measured throughout the hippocampus with a 60-electrode microelectrode array. Electrophysiological parameters associated with unstimulated spontaneous activity (neural event firing rate, duration, and magnitude), stimulated evoked responses (the maximum response [Formula: see text], the stimulus current necessary for a half-maximal response [Formula: see text], and the electrophysiological parameter m that is representative of firing uniformity), and paired-pulse responses (paired-pulse ratio at varying interstimulus intervals) were quantified for each hippocampal region (CA1, CA3, and DG). We present functional tolerance criteria for the hippocampus in the form of mathematical relationships between the input tissue-level injury parameters (strain and strain rate) and altered neuronal network function. Most changes in electrophysiology were dependent on strain and strain rate in a complex fashion, independent of hippocampal anatomy, with the notable exception of [Formula: see text]. Until it becomes possible to directly measure brain tissue deformation in vivo, finite element (FE) models will be necessary to simulate and predict the in vivo consequences of TBI. One application of our study is to provide functional relationships that can be incorporated into these FE models to enhance their biofidelity of accident and collision reconstructions by predicting biological outcomes in addition to mechanical responses.

Neuroprotection against Traumatic Brain Injury by Xenon, but Not Argon, Is Mediated by Inhibition at the N-Methyl-d-Aspartate Receptor Glycine Site

Xenon, the inert anesthetic gas, is neuroprotective in models of brain injury. The authors investigate the neuroprotective mechanisms of the inert gases such as xenon, argon, krypton, neon, and helium in an in vitro model of traumatic brain injury. The authors use an in vitro model using mouse organotypic hippocampal brain slices, subjected to a focal mechanical trauma, with injury quantified by propidium iodide fluorescence. Patch clamp electrophysiology is used to investigate the effect of the inert gases on N-methyl-D-aspartate receptors and TREK-1 channels, two molecular targets likely to play a role in neuroprotection. Xenon (50%) and, to a lesser extent, argon (50%) are neuroprotective against traumatic injury when applied after injury (xenon 43±1% protection at 72 h after injury [N=104]; argon 30±6% protection [N=44]; mean±SEM). Helium, neon, and krypton are devoid of neuroprotective effect. Xenon (50%) prevents development of secondary injury up to 48 h after trauma. Argon (50%) attenuates secondary injury, but is less effective than xenon (xenon 50±5% reduction in secondary injury at 72 h after injury [N=104]; argon 34±8% reduction [N=44]; mean±SEM). Glycine reverses the neuroprotective effect of xenon, but not argon, consistent with competitive inhibition at the N-methyl-D-aspartate receptor glycine site mediating xenon neuroprotection against traumatic brain injury. Xenon inhibits N-methyl-D-aspartate receptors and activates TREK-1 channels, whereas argon, krypton, neon, and helium have no effect on these ion channels. Xenon neuroprotection against traumatic brain injury can be reversed by increasing the glycine concentration, consistent with inhibition at the N-methyl-D-aspartate receptor glycine site playing a significant role in xenon neuroprotection. Argon and xenon do not act via the same mechanism.

SAHA Enhances Synaptic Function and Plasticity In Vitro but Has Limited Brain Availability In Vivo and Does Not Impact Cognition

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer’s disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology.

Microglia Actively Regulate the Number of Functional Synapses

Microglia are the immunocompetent cells of the central nervous system. In the physiological setting, their highly motile processes continually survey the local brain parenchyma and transiently contact synaptic elements. Although recent work has shown that the interaction of microglia with synapses contributes to synaptic remodeling during development, the role of microglia in synaptic physiology is just starting to get explored. To assess this question, we employed an electrophysiological approach using two methods to manipulate microglia in culture: organotypic hippocampal brain slices in which microglia were depleted using clodronate liposomes, and cultured hippocampal neurons to which microglia were added. We show here that the frequency of excitatory postsynaptic current increases in microglia-depleted brain slices, consistent with a higher synaptic density, and that this enhancement ensures from the loss of microglia since it is reversed when the microglia are replenished. Conversely, the addition of microglia to neuronal cultures decreases synaptic activity and reduces the density of synapses, spine numbers, surface expression of AMPA receptor (GluA1), and levels of synaptic adhesion molecules. Taken together, our findings demonstrate that non-activated microglia acutely modulate synaptic activity by regulating the number of functional synapses in the central nervous system.

Resolft Nanoscopy in Life Sciences: Unraveling Fine Details with Low Light Levels

Lens-based fluorescence microscopy, which has long been limited in resolution to > 200 nanometer by diffraction, is rapidly evolving into a nanoscale imaging technique. Here, we show that emergent RESOLFT fluorescence microscopy enables fast and continuous imaging of sensitive, nanosized features in living brain tissue. using low intensity illumination to switch photochromic fluorescent proteins reversibly between a fluorescent ON-state and a non-fluorescent OFF-state, we obtained more than a 3-fold increase in all three spatial dimensions over that of confocal microscopy. Dendritic spines located 10-50 μm deep inside living organotypic hippocampal brain slices were recorded for hours without signs of degradation. using a fast-switching fluorescent protein increased the imaging speed 50-fold over reported RESOLFT schemes, which in turn enabled us to record spontaneous and stimulated changes of dendritic actin filaments and spine morphology occurring on time scales from seconds to hours.

Nanoscopy of Living Brain Slices with Low Light Levels

Lens-based fluorescence microscopy, which has long been limited in resolution to about 200 nanometers by diffraction, is rapidly evolving into a nanoscale imaging technique. Here, we show that the superresolution fluorescence microscopy called RESOLFT enables comparatively fast and continuous imaging of sensitive, nanosized features in living brain tissue. Using low-intensity illumination to switch photochromic fluorescent proteins reversibly between a fluorescent and a nonfluorescent state, we increased the resolution more than 3-fold over that of confocal microscopy in all dimensions. Dendritic spines located 10-50 μm deep inside living organotypic hippocampal brain slices were recorded for hours without signs of degradation. Using a fast-switching protein increased the imaging speed 50-fold over reported RESOLFT schemes, which in turn enabled the recording of spontaneous and stimulated changes of dendritic actin filaments and spine morphology occurring on time scales from seconds to hours.

CGX-1007 prevents excitotoxic cell death via actions at multiple types of NMDA receptors

Glutamate induced excitotoxic injury through over-activation of N-methyl-D-aspartate receptors (NMDARs) plays a critical role in the development of many neurodegenerative diseases. The present study was undertaken to evaluate the role of CGX-1007 (Conantokin G) as a neuroprotective agent against NMDA-induced excitotoxicity. Conantokin G, a cone snail peptide isolated from Conus geographus is reported to selectively inhibit NR2B containing NMDARs with high specificity and is shown to have potent anticonvulsant and antinociceptive effects. CGX-1007 significantly reduced the excitotoxic cell death induced by NMDA in organotypic hippocampal brain slice cultures in a concentration-dependent manner. In contrast, ifenprodil, another NR2B specific antagonist failed to offer neuroprotection against NMDA-induced excitotoxicity. We further determined that the neuroprotection observed is likely due to the action of CGX-1007 at multiple NMDA receptor subtypes. In a series of electrophysiology experiments, CGX-1007 inhibited NMDA-gated currents in human embryonic kidney (HEK) 293 cells expressing NMDA receptors containing either NR1a/NR2B or NR1a/NR2A subunit combinations. CGX-1007 produced a weak inhibition at NR1a/NR2C receptors, whereas it had no effect on NR1a/NR2D receptors. Further, the inhibition of NMDA receptors by CGX-1007 was voltage-dependent with greater inhibition seen at hyperpolarized membrane potentials. The voltage-dependence of CGX-1007 activity was also observed in recordings of NMDA-gated currents evoked in native receptors expressed in cortical neurons in culture. Based on our results, we conclude that CGX-1007 is a potent neuroprotective agent that acts as an antagonist at both NR2A and NR2B containing receptors.

Competitive Inhibition at the Glycine Site of the N -Methyl-d-Aspartate Receptor Mediates Xenon Neuroprotection against Hypoxia–Ischemia

The general anesthetic gas xenon is neuroprotective and is undergoing clinical trials as a treatment for ischemic brain injury. A small number of molecular targets for xenon have been identified, the N-methyl-D-aspartate (NMDA) receptor, the two-pore-domain potassium channel TREK-1, and the adenosine triphosphate-sensitive potassium channel (KATP). However, which of these targets are relevant to acute xenon neuroprotection is not known. Xenon inhibits NMDA receptors by competing with glycine at the glycine-binding site. We test the hypothesis that inhibition of the NMDA receptor at the glycine site underlies xenon neuroprotection against hypoxia-ischemia. We use an in vitro model of hypoxia-ischemia to investigate the mechanism of xenon neuroprotection. Organotypic hippocampal brain slices from mice are subjected to oxygen-glucose deprivation, and injury is quantified by propidium iodide fluorescence. We show that 50% atm xenon is neuroprotective against hypoxia-ischemia when applied immediately after injury or after a delay of 3 h after injury. To validate our method, we show that neuroprotection by gavestinel is abolished when glycine is added, confirming that NMDA receptor glycine site antagonism underlies gavestinel neuroprotection. We then show that adding glycine abolishes the neuroprotective effect of xenon, consistent with competitive inhibition at the NMDA receptor glycine site mediating xenon neuroprotection. We show that xenon neuroprotection against hypoxia- ischemia can be reversed by increasing the glycine concentration. This is consistent with competitive inhibition by xenon at the NMDA receptor glycine site, playing a significant role in xenon neuroprotection. This finding may have important implications for xenon's clinical use as an anesthetic and neuroprotectant.

Formic Acid, a Novel Metabolite of Chronic Ethanol Abuse, Causes Neurotoxicity, Which Is Prevented by Folic Acid

Methanol is endogenously formed in the brain and is present as a congener in most alcoholic beverages. Because ethanol is preferentially metabolized over methanol (MeOH) by alcohol dehydrogenase, it is not surprising that MeOH accumulates in the alcohol-abusing population. This suggests that the alcohol-drinking population will have higher levels of MeOH's neurotoxic metabolite, formic acid (FA). FA elimination is mediated by folic acid. Neurotoxicity is a common result of chronic alcoholism. This study shows for the first time that FA, found in chronic alcoholics, is neurotoxic and this toxicity can be mitigated by folic acid administration. To determine if FA levels are higher in the alcohol-drinking population and to assess its neurotoxicity in organotypic hippocampal rat brain slice cultures. Serum and CSF FA was measured in samples from both ethanol abusing and control patients, who presented to a hospital emergency department. FA's neurotoxicity and its reversibility by folic acid were assessed using organotypic rat brain hippocampal slice cultures using clinically relevant concentrations. Serum FA levels in the alcoholics (mean +/- SE: 0.416 +/- 0.093 mmol/l, n = 23) were significantly higher than in controls (mean +/- SE: 0.154 +/- 0.009 mmol/l, n = 82) (p < 0.0002). FA was not detected in the controls' CSF (n = 20), whereas it was >0.15 mmol/l in CSF of 3 of the 4 alcoholic cases. Low doses of FA from 1 to 5 mmol/l added for 24, 48 or 72 hours to the rat brain slice cultures caused neuronal death as measured by propidium iodide staining. When folic acid (1 micromol/l) was added with the FA, neuronal death was prevented. Formic acid may be a significant factor in the neurotoxicity of ethanol abuse. This neurotoxicity can be mitigated by folic acid administration at a clinically relevant dose.