Organotypic Hippocampal Cultures Research Articles

Preconditioning effects of tumor necrosis factor-α and glutamate on calcium dynamics in rat organotypic hippocampal cultures

During cerebral ischemia, elevation of TNF-α and glutamate to pathophysiological levels in the hippocampus may induce dysregulation of normal synaptic processes, leading ultimately to cell death. Previous studies have shown that patients subjected to a mild transient ischemic attack within a critical time window prior to a more severe ischemic episode may show attenuation in the clinical severity of the stroke and result in a more positive functional outcome. In this study we have investigated the individual contribution of pre-exposure to TNF-α or glutamate in the development of ‘ischemic tolerance’ to a subsequent insult, using organotypic hippocampal cultures. At 6days in vitro (DIV), cultures were exposed to an acute concentration of glutamate (30μM) or TNF-α (5ng/ml) for 30min, followed by 24h recovery period. We then examined the effect of the pretreatments on calcium dynamics of the cells within the CA region. We found that pretreatment with TNF-α or glutamate caused in a significant reduction in subsequent glutamate-induced Ca2+ influx 24h later (control: 100.0±0.8%, n=7769 cells; TNF-α: 76.8±1.0%, n=5543 cells; glutamate: 75.3±1.4%, n=3859 cells; p<0.001). Antagonism of circulating TNF-α (using infliximab, 25μg/ml), and inhibition of the p38 MAP kinase pathway (using SB 203580, 10μM) completely reversed this effect. However glutamate preconditioning did not appear to be mediated by p38 MAP kinase signalling, or NMDAR activation as neither SB 203580 nor D-AP5 (100μM) altered this effect. Glutamate and TNF-α preconditioning resulted in small yet significant alterations in resting Ca2+ levels (control: 100.0±0.9%, n=2994 cells; TNF-α: 109.7±1.0%, n=2884 cells; glutamate; 93.3±0.8%, n=2899 cells; p<0.001), TNF-α's effect reversed by infliximab and SB 203580. Both TNF-α and glutamate also resulted in the reduction of the proportion (P) of responsive cells within the CA region of the hippocampus (control; P=0.459, 0.451≤x≥0.467, n=14,968 cells, TNF-α; P=0.40, 0.392≤x≥0.407, n=15,218; glutamate; P=0.388, 0.303≤x≥0.396, n=13,919 cells), and in the depression of the frequency of spontaneous Ca2+ events (vs. control: TNF-α: p>0.00001, D=0.0454; glutamate: p>0.0001, D=0.0534). Our results suggest that attenuation in resting Ca2+ activity and Ca2+ related responsiveness of cells within the CA region as a result of glutamate or TNF-α pre-exposure, may contribute to the development of ischemic tolerance.

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory. In addition, the well defined anatomy and connectivity of the hippocampus has made it a classical model system to study synaptic transmission and synaptic plasticity. As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods. Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection or biolistics. In addition, slices can be easily recovered for biochemical assays, or transferred to microscopes for imaging or electrophysiological experiments.

Induction of the Wnt Antagonist Dickkopf-1 Is Involved in Stress-Induced Hippocampal Damage

The identification of mechanisms that mediate stress-induced hippocampal damage may shed new light into the pathophysiology of depressive disorders and provide new targets for therapeutic intervention. We focused on the secreted glycoprotein Dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway, involved in neurodegeneration. Mice exposed to mild restraint stress showed increased hippocampal levels of Dkk-1 and reduced expression of β-catenin, an intracellular protein positively regulated by the canonical Wnt signalling pathway. In adrenalectomized mice, Dkk-1 was induced by corticosterone injection, but not by exposure to stress. Corticosterone also induced Dkk-1 in mouse organotypic hippocampal cultures and primary cultures of hippocampal neurons and, at least in the latter model, the action of corticosterone was reversed by the type-2 glucocorticoid receptor antagonist mifepristone. To examine whether induction of Dkk-1 was causally related to stress-induced hippocampal damage, we used doubleridge mice, which are characterized by a defective induction of Dkk-1. As compared to control mice, doubleridge mice showed a paradoxical increase in basal hippocampal Dkk-1 levels, but no Dkk-1 induction in response to stress. In contrast, stress reduced Dkk-1 levels in doubleridge mice. In control mice, chronic stress induced a reduction in hippocampal volume associated with neuronal loss and dendritic atrophy in the CA1 region, and a reduced neurogenesis in the dentate gyrus. Doubleridge mice were resistant to the detrimental effect of chronic stress and, instead, responded to stress with increases in dendritic arborisation and neurogenesis. Thus, the outcome of chronic stress was tightly related to changes in Dkk-1 expression in the hippocampus. These data indicate that induction of Dkk-1 is causally related to stress-induced hippocampal damage and provide the first evidence that Dkk-1 expression is regulated by corticosteroids in the central nervous system. Drugs that rescue the canonical Wnt pathway may attenuate hippocampal damage in major depression and other stress-related disorders.

A role for tumor necrosis factor-α in ischemia and ischemic preconditioning

During cerebral ischemia, elevation of TNF-α and glutamate to pathophysiological levels may induce dysregulation of normal synaptic processes, leading ultimately to cell death. Previous studies have shown that patients subjected to a mild transient ischemic attack within a critical time window prior to a more severe ischemic episode may show attenuation in the clinical severity of the stroke and result in a more positive functional outcome. Studies with organotypic hippocampal cultures and mixed primary hippocampal cultures have shown that prior incubation with low concentrations of glutamate and TNF-α increase the resistance of neurones to a subsequent insult from glutamate, AMPA and NMDA, while co-exposure of TNF-α and for example AMPA may have neuroprotective effects compared to cultures exposed to excitotoxic agents alone. In addition our work has shown that although glutamate and TNF-α pretreatment induces analogous levels of desensitisation of the intracellular calcium dynamics of neurons under resting conditions and in response to acute glutamate stimulation, their downstream signalling pathways involved in this response do not converge. Glutamate and TNF-α would appear to have opposing effects on resting Ca2+ levels which supports the proposal that they have distinct modes of preconditioning.

Mesenchymal Stem Cell-Conditioned Medium Triggers Neuroinflammation and Reactive Species Generation in Organotypic Cultures of Rat Hippocampus

Cell therapy using bone marrow-derived mesenchymal stem cells (MSCs) seems to be a new alternative for the treatment of neurodegenerative diseases. Despite several promising results with their use, possible side effects are still unknown. In a previous work, we have shown that MSC-conditioned medium is toxic to hippocampal slice cultures and aggravates cell death induced by oxygen and glucose deprivation. In this work, we investigated whether the inflammatory response and/or reactive species formation could be involved in that toxicity. Rat organotypic hippocampal cultures were exposed for 24 h to conditioned medium from MSCs isolated from rat bone marrow. A marked glial activation was observed after exposure of cultures to MSC-conditioned medium, as evidenced by glial fibrillary acid protein (GFAP) and isolectin B(4) increase. Tumor necrosis factor-α and interleukin-6 levels were increased in the culture medium, and 2,7-dihydrodichlorofluorescein diacetate oxidation (indicating reactive species generation) and inducible nitric oxide synthase (iNOS) immunocontent were also higher after exposure of cultures to MSC-conditioned medium. Antioxidants (ascorbic acid and TROLOX(®)), N(ω)-nitro-l-arginine methyl ester hydrochloride, and anti-inflammatory drugs (indomethacin and dexamethasone) reduced cell death in hippocampal organotypic cultures after their exposure to MSC-conditioned medium. The results obtained here suggest that MSC-secreted factors trigger reactive species generation and neuroinflammation in organotypic cultures of hippocampus, introducing a note of caution in the use of these cells for neurological application.

Cathepsin D plays a crucial role in the trimethyltin-induced hippocampal neurodegeneration process

Trimethyltin chloride (TMT) is known to produce neuronal damage in the rat hippocampus, especially in the CA 1/CA 3 subfields, together with reactive astrogliosis. Previous studies indicate that in cultured rat hippocampal neurons the Ca 2+ cytosolic increase induced by TMT is correlated with apoptotic cell death, although some molecular aspects of the hippocampal neurodegeneration induced by this neurotoxicant still remain to be clarified. Cathepsin D (Cat D) is a lysosomal aspartic protease involved in some neurodegenerative processes and also seems to play an important role in the processes that regulate apoptosis. We investigated the specific activity and cellular expression of Cat D in the rat hippocampus in vivo and in cultured organotypic rat hippocampal slices. The role of Cat D in cell death processes and the mechanisms controlling Cat D were also investigated. Cat D activity was assayed in hippocampus homogenates of control and TMT-treated rats. In order to visualize the distribution of Cat D immunoreactivity in the hippocampus, double-label immunofluorescence for Cat D and Neu N, GFAP, OX42 was performed. In addition, in order to clarify the possible relationship between Cat D activity, neuronal calcium overload and neuronal death processes, organotypic hippocampal cultures were also treated with a Cat D inhibitor (Pepstatin A) or Calpain inhibitor (Calpeptin) or an intracellular Ca 2+ chelator (BAPTA-AM) in the presence of TMT. TMT treatment in rat hippocampus induced high levels of Cat D activity both in vivo and in vitro, in glial cells and in CA 3 neurons, where a marked TMT-induced neuronal loss also occurred. Cat D is actively involved in CA3 neuronal death and the protease increase is a calcium-Calpain dependent phenomenon.

Protective effects of indomethacin-loaded nanocapsules against oxygen-glucose deprivation in organotypic hippocampal slice cultures: Involvement of neuroinflammation

Targeted treatment of diseases of the central nervous system remains problematic due to the complex pathogenesis of these disorders and the difficulty in drug delivery. Here we investigate the neuroprotective effect of indomethacin-loaded nanocapsules (IndOH-NC) in an in vitro model of ischemia. For this purpose we used organotypic hippocampal cultures exposed to oxygen-glucose deprivation (OGD). When the cultures were exposed to 60 min of OGD, 54.5 ± 14.7% of the total area of the hippocampal slices was labeled with propidium iodide. On the other hand, when the cultures were treated with 50 or 100 μM of IndOH-NC the cell death was significantly reduced to 31 ± 7% ( P < 0.05) and 20 ± 4% ( P < 0.001), respectively. The treatment with IndOH-NC markedly inhibited the levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α levels even 48 h after OGD. Immunoblotting revealed that treatment with 100 μM of IndOH-NC was able to significantly reduce to the levels of control cultures the levels of ERK1/2 and JNK phosphorylation, as well as iNOS activation. Additionally, IndOH-NC prevented glial activation induced by OGD, as evidenced by a decrease of GFAP immunocontent and Isolectin B 4 reactivity. Our results clearly demonstrate that IndOH-NC might represent a promising pharmaceutical neuroprotective formulation for cerebral ischemia, most probably by inhibiting the inflammatory cascades.

In vitro interactions between bone marrow stromal cells and hippocampal slice cultures

Bone marrow stromal cells (BMSCs) are capable of differentiating into various cell types including brain cells. Several groups have also demonstrated trophic effects of MSC grafts in experimental ischemia models. However, the underlying molecular mechanisms of these effects are not fully understood. We developed an “ in vitro graft model” which consisted in a coculture of GFP-expressing BMSCs and hippocampal organotypic slice cultures. Total marrow cells (MCs) or BMSCs after one (BMSC 1P) or five passages (BMSC 5P) were transplanted on hippocampal slices. During the 10 days of our experiments, MCs and BMSC 1P migrated toward the tissue, but their total number remained constant. Conversely, the number of BMSC 5P decreased over the 10 days of the experiment, and no migration could be detected. Using immunohistochemistry, we observed that the hippocampal slices induced the expression of neural antigens in very few grafted cells, but MCs and BMSC 1P improved the conservation of the hippocampal slice culture. Similar experiments using BMSC 5P did not produce any significant change. We conclude that the number of passages greatly influence BMSCs survival rate, migration and neuroprotective capacities.

Astrocytic activation and an inhibition of MAP kinases are required for proteinase‐activated receptor‐2‐mediated protection from neurotoxicity

Proteinase-activated receptor-2 (PAR-2) expression levels are altered in several CNS disorders with these changes being proposed to either exacerbate or diminish the disease state depending on the cell type in which this occurs. Here we present data investigating the consequence of PAR-2 activation on kainate (KA)-induced neurotoxicity in organotypic hippocampal slices cultures (OHSC). Exposure of OHSC to the PAR-2 activators trypsin or Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL) induced no neurotoxicity when applied alone but was neuroprotective against KA-induced neurotoxicity. SLIGRL-mediated neuroprotection involved astrocytic activation as the neuroprotective effect was abolished following OHSC pre-treatment with fluoroacetate. Moreover, co-application of either reparixin or LY341495, antagonists of the CXCR2 chemokine receptor and metabotropic glutamate receptors respectively, inhibited the SLIGRL-mediated neuroprotection. SLIGRL application inhibited both p38 MAPK and ERK activity in OHSC, but not the JNK 1/2 signalling pathway. Accordingly, the co-application of the p38 MAPK and ERK inhibitors SB203580 and UO126 reduced KA-induced cell death, mimicking PAR-2-mediated neuroprotection. These data indicate that PAR-2 activation is neuroprotective and involves astrocytic activation, gliotransmitter release, and the subsequent inhibition of MAPK signalling cascades, providing further evidence for PAR-2 as an interesting therapeutic target in certain CNS disorders.

38 miR-449 induces apoptosis while triggering a stress and DNA damage response

Synaptic vesicle docking, priming, and fusion at active zones are orchestrated by a complex molecular machinery. We employed hippocampal organotypic slice cultures from mice lacking key presynaptic proteins, cryofixation, and three-dimensional electron tomography to study the mechanism of synaptic vesicle docking in the same experimental setting, with high precision, and in a near-native state. We dissected previously indistinguishable, sequential steps in synaptic vesicle active zone recruitment (tethering) and membrane attachment (docking) and found that vesicle docking requires Munc13/CAPS family priming proteins and all three neuronal SNAREs, but not Synaptotagmin-1 or Complexins. Our data indicate that membrane-attached vesicles comprise the readily releasable pool of fusion-competent vesicles and that synaptic vesicle docking, priming, and trans-SNARE complex assembly are the respective morphological, functional, and molecular manifestations of the same process, which operates downstream of vesicle tethering by active zone components.

Hydroxylase inhibition reduces synaptic transmission and protects against a glutamate-induced ischemia in the CA1 region of the rat hippocampus

The discovery of hydroxylases as oxygen sensors and key regulators of hypoxia-induced gene expression has made them a novel target for manipulating the transcriptional response to hypoxia for therapeutic benefit. In this study we have investigated the effect of prolyl hydroxylase inhibition on synaptic activity in hippocampal slices and compared this to the changes occurring following exposure to hypoxia. Furthermore, we investigated a potentially protective role for hydroxylase inhibition against a glutamate-induced ischemic insult in the CA1 region of organotypic hippocampal cultures. Application of the hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), depressed synaptic transmission. Both hypoxia and DMOG induced a reversible reduction in synaptic transmission, enhanced paired pulse facilitation (P<0.05) and inhibited N-methyl d-aspartate receptor (NMDAR) activity (P<0.01). However the effects of DMOG were adenosine A1 receptor independent. Our results also suggest a potential therapeutic application for prolyl 4-hydroxylase (PHD) inhibitors in cerebral ischemia, since DMOG protected the CA1 region in organotypic hippocampal slices against a glutamate-induced ischemic insult.

Transcriptomics of Traumatic Brain Injury: Gene Expression and Molecular Pathways of Different Grades of Insult in a Rat Organotypic Hippocampal Culture Model

Traumatic brain injury (TBI) is the one of the most common forms of head trauma, and it remains a leading cause of death and disability. It is known that the initial mechanical axonal injury triggers a complex cascade of neuroinflammatory and metabolic events, the understanding of which is essential for clinical, translational, and pharmacological research. These can occur even in mild TBI, and are associated with several post-concussion manifestations, including transiently heightened vulnerability to a second insult. Recent studies have challenged the tenet that ischemia is the ultimate modality of tissue damage following TBI, as metabolic dysfunction can develop in the presence of normal perfusion and before intracranial hypertension. In order to elucidate the cellular and molecular changes occurring in TBI as a direct result of neuronal injury and in the absence of ischemic damage, we performed a microarray analysis of expressed genes and molecular interaction pathways for different levels of severity of trauma using an in-vitro model. A stretch injury, equivalent to human diffuse axonal injury, was delivered to rat organotypic hippocampal slice cultures, and mRNA levels following a 10% (mild) and 50% (severe) stretch were compared with controls at 24 h. More genes were differentially expressed following 10% stretch than 50% stretch, indicating the early activation of complex cellular mechanisms. The data revealed remarkable differential gene expression following mTBI, even in the absence of cell damage. Pathway analysis revealed that molecular interactions in both levels of injury were similar, with IL-1beta playing a central role. Additional pathways of neurodegeneration involving RhoA (ras homolog gene family, member A) were found in 50% stretch.

Differential expression of TRPM2 and TRPV4 channels and their potential role in oxidative stress-induced cell death in organotypic hippocampal culture

TRPM2 and TPPV4 channels, two members of TRP channel family, are known to be widely expressed in the brain but their exact expression pattern and function are not well understood. Due to their high Ca 2+ permeability and gating by reactive oxygen species (TRPM2), or cell swelling, low pH and high temperature (TRPV4), they are likely to be involved in cell damage associated with various brain pathologies. The aim of this study was to investigate the expression of these channels and their potential role in oxidative stress-induced cell damage in organotypic hippocampal slice cultures, a model that retains the complex interaction between neurons and astrocytes. Channel expression was confirmed with RT-PCR and western blotting, while immunocytochemistry demonstrated TRPM2 in CA1–CA3 pyramidal neurons and TRPV4 in astrocytes. Oxidative stress induced by exogenous application of H 2O 2 (600 μM) caused preferential damage of pyramidal neurons, while oxidative stress evoked with mercaptosuccinate (MCS; 400 μM) or buthionine sulfoximine (BSO; 4 μM) mainly damaged astrocytes, as identified by propidium iodide fluorescence. Antioxidants (Trolox 500 μM; MitoE 2 μM) reduced both neuronal and astrocytic cell death. Blockers of TRPV4 channels (Gd 3+ 500 μM; Ruthenium red 1 μM) increased the viability of astrocytes following MCS or BSO treatments, consistent with the expression pattern of these channels. Blockers of TRPM2 channels clotrimazole (20 μM), N-( p-amylcinnomoyl)anthranilic acid (ACA, 25 μM) or flufenamic acid (FFA, 200 μM) failed to protect pyramidal neurons from damage caused by exogenous H 2O 2, and increased damage of these neurons caused by MCS and BSO. The differential expression of stress-sensitive TRPM2 and TRPV4 channels in hippocampal neurons and astrocytes that show distinct differences in vulnerability to different forms of oxidative stress suggests the specific involvement of these channels in oxidative stress-induced cell damage. However, the exact relationship between TRPM2 channel activation and cell death still remains to be determined due to the lack of protective effects of TRPM2 channel blockers.

Argon neuroprotection

Certain noble gases, though inert, exhibit remarkable biological properties. Notably, xenon and argon provide neuroprotection in animal models of central nervous system injury. In the previous issue of Critical Care, Loetscher and colleagues provided further evidence that argon may have therapeutic properties for neuronal toxicity by demonstrating protection against both traumatic and oxygen-glucose deprivation injury of organotypic hippocampal cultures in vitro. Their data are of interest as argon is more abundant, and therefore cheaper, than xenon (the latter of which is currently in clinical trials for perinatal hypoxic-ischemic brain injury; TOBYXe; NCT00934700). We eagerly await in vivo data to complement the promising in vitro data hailing argon neuroprotection.

Insulin/PI3K signaling protects dentate neurons from oxygen–glucose deprivation in organotypic slice cultures

It is known that ischemia/reperfusion induces neurodegeneration in the hippocampus in a subregion-dependent manner. This study investigated the mechanism of selective resistance/vulnerability to oxygen-glucose deprivation (OGD) using mouse organotypic hippocampal cultures. Analysis of propidium iodide uptake showed that OGD-induced duration- and subregion-dependent neuronal injury. When compared with the CA1-3 subregions, dentate neuronal survival was more sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling under basal conditions. Dentate neuronal sensitivity to PI3K/Akt signaling activation was inversely related to its vulnerability to OGD-induced injury; insulin/insulin-like growth factor 1 pre-treatment conferred neuroprotection to dentate neurons via activation of PI3K/Akt signaling. In contrast, CA1 and CA3 neurons were less sensitive to disruptions of endogenous PI3K/Akt signaling and protective effects of insulin/insulin-like growth factor 1, but more vulnerable to OGD. OGD-induced injury in CA1 was reduced by inhibition of NMDA receptor or mitogen-activated protein kinase signaling, and was prevented by blocking NMDA receptor in the presence of insulin. The CA2 subregion was distinctive in its response to glutamate, OGD, and insulin, compared with other CA subregions. CA2 neurons were sensitive to the protective effects of insulin against OGD-induced injury, but more resistant to glutamate. Distinctive distribution of insulin receptor beta and basal phospho-Akt was detected in our slice cultures. Our results suggest a role for insulin signaling in subregional resistance/vulnerability to cerebral ischemia.

NADPH oxidase mediates β-amyloid peptide-induced activation of ERK in hippocampal organotypic cultures

BackgroundPrevious studies have shown that beta amyloid (Aβ) peptide triggers the activation of several signal transduction cascades in the hippocampus, including the extracellular signal-regulated kinase (ERK) cascade. In this study we sought to characterize the cellular localization of phosphorylated, active ERK in organotypic hippocampal cultures after acute exposure to either Aβ (1-42) or nicotine.ResultsWe observed that Aβ and nicotine increased the levels of active ERK in distinct cellular localizations. We also examined whether phospho-ERK was regulated by redox signaling mechanisms and found that increases in active ERK induced by Aβ and nicotine were blocked by inhibitors of NADPH oxidase.ConclusionOur findings indicate that NADPH oxidase-dependent redox signaling is required for Aβ-induced activation of ERK, and suggest a similar mechanism may occur during early stages of Alzheimer's disease.

Bilateral Interaction Between Cord Blood–Derived Human Neural Stem Cells and Organotypic Rat Hippocampal Culture

The umbilical cord blood-derived neural stem/progenitor cells (HUCB-NSCs) potentially represent a rich source of transplantable material for treatment of a wide range of neurological diseases. Although, recently reported effects of their implementation in animal models of brain pathology are still controversial. As a simplified alternative to in vivo transplantation in this work we have applied a long-term organotypic rat hippocampal slice culture (OHC) as a recipient tissue to study bilateral graft/host cells interactions ex vivo. This type of culture can be considered as a kind of reductionistic model of brain transplantation where direct influence of systemic immunological responses to transplanted human cells would be excluded. The transplantation material derived from a HUCB-NSC line developed and characterized in our laboratory and delivered to the slices either as a single-cell suspension or after formation of typical neurospheres in serum-free medium in vitro (N-HUCBs). Experiments were focused on space-temporal context of cell transplantation in relation to their ability to ingrown, migrate, and differentiate within the slice cytoarchitecture. We gain evidences that these responses are strictly dependent on the engraftment site and that cell movement reflects typical routes used for migratory neuroblasts in vivo. The cells implanted at the second week of slice cultivation ingrown readily and deeply into host cytoarchitecture then matured to the level never observed in our transplantation animal models in vivo. Importantly, transplanted neurospheres, in addition to yield exogenous migratory cells to the host tissue can locally inhibit astrocytosis and promote outgrow of DCX-reactive neuroblasts in the surrounding OHC tissue.

Contrasting patterns of Bim induction and neuroprotection in Bim-deficient mice between hippocampus and neocortex after status epilepticus

Prolonged seizures (status epilepticus) are associated with brain region-specific regulation of apoptosis-associated signaling pathways. Bcl-2 homology domain 3-only (BH3) members of the Bcl-2 gene family are of interest as possible initiators of mitochondrial dysfunction and release of apoptogenic molecules after seizures. Previously, we showed that expression of the BH3-only protein, Bcl-2 interacting mediator of cell death (Bim), increased in the rat hippocampus but not in the neocortex after focal-onset status epilepticus. In this study, we examined Bim expression in mice and compared seizure damage between wild-type and Bim-deficient animals. Status epilepticus induced by intra-amygdala kainic acid (KA) caused extensive neuronal death within the ipsilateral hippocampal CA3 region. Hippocampal activation of factors associated with transcriptional and posttranslational activation of Bim, such as CHOP and c-Jun NH(2)-terminal kinases, was significant within 1 h. Upregulation of bim mRNA was evident after 2 h and Bim protein increased between 4 and 24 h. Hippocampal CA3 neurodegeneration was reduced in Bim-deficient mice compared with wild-type animals after seizures in vivo, and short interfering RNA molecules targeting bim reduced cell death after KA treatment of hippocampal organotypic cultures. In contrast, neocortical Bim expression declined after status epilepticus, and neocortex damage in Bim-deficient mice was comparable with that in wild-type animals. These results show region-specific differential contributions of Bim to seizure-induced neuronal death.

Neuroprotection by a novel NMDAR functional glycine site partial agonist, GLYX-13

GLYX-13 (threonine-proline-proline-threonine-amide) is an amidated di-pyrrolidine that acts as a functional partial agonist at the glycine site on N-methyl-D-aspartate glutamate receptors (NMDARs). GLYX-13 can both increase NMDAR conductance at NR2B-containing receptors, and reduce conductance of non-NR2B-containing receptors. Here, we report that GLYX-13 potently reduces delayed (24 h) death of CA1 pyramidal neurons produced by bilateral carotid occlusion in Mongolian gerbils, when administered up to 5 h post-ischemia. GLYX-13 also reduced delayed (24 h) neuronal death of CA1, CA3, and dentate gyrus principal neurons elicited by oxygen/glucose deprivation in in-vitro hippocampal organotypic slice cultures, when applied up to 2 h post-oxygen/glucose deprivation. The glycine site full agonist D-serine completely occluded neuroprotection, indicating that GLYX-13 acts by modulating activation of this site.

Optogenetic control of epileptiform activity

The optogenetic approach to gain control over neuronal excitability both in vitro and in vivo has emerged as a fascinating scientific tool to explore neuronal networks, but it also opens possibilities for developing novel treatment strategies for neurologic conditions. We have explored whether such an optogenetic approach using the light-driven halorhodopsin chloride pump from Natronomonas pharaonis (NpHR), modified for mammalian CNS expression to hyperpolarize central neurons, may inhibit excessive hyperexcitability and epileptiform activity. We show that a lentiviral vector containing the NpHR gene under the calcium/calmodulin-dependent protein kinase IIalpha promoter transduces principal cells of the hippocampus and cortex and hyperpolarizes these cells, preventing generation of action potentials and epileptiform activity during optical stimulation. This study proves a principle, that selective hyperpolarization of principal cortical neurons by NpHR is sufficient to curtail paroxysmal activity in transduced neurons and can inhibit stimulation train-induced bursting in hippocampal organotypic slice cultures, which represents a model tissue of pharmacoresistant epilepsy. This study demonstrates that the optogenetic approach may prove useful for controlling epileptiform activity and opens a future perspective to develop it into a strategy to treat epilepsy.