Hippocampal Slice Cultures Research Articles

Shank Proteins Differentially Regulate Synaptic Transmission.

Shank proteins, one of the principal scaffolds in the postsynaptic density (PSD) of the glutamatergic synapses, have been associated with autism spectrum disorders and neuropsychiatric diseases. However, it is not known whether different Shank family proteins have distinct functions in regulating synaptic transmission, and how they differ from other scaffold proteins in this aspect. Here, we investigate the role of Shanks in regulating glutamatergic synaptic transmission at rat hippocampal SC-CA1 synapses, using lentivirus-mediated knockdown and molecular replacement combined with dual whole-cell patch clamp in hippocampal slice culture. In line with previous findings regarding PSD-MAGUK scaffold manipulation, we found that loss of scaffold proteins via knockdown of Shank1 or Shank2, but not Shank3, led to a reduction of the number but not the unitary response of AMPAR-containing synapses. Only when both Shank1 and Shank2 were knocked down, were both the number and the unitary response of active synapses reduced. This reduction was accompanied by a decrease in NMDAR-mediated synaptic response, indicating more profound deficits in synaptic transmission. Molecular replacement with Shank2 and Shank3c rescued the synaptic transmission to the basal level, and the intact sterile α-motif (SAM) of Shank proteins is required for maintaining glutamatergic synaptic transmission. We also found that altered neural activity did not influence the effect of Shank1 or Shank2 knockdown on AMPAR synaptic transmission, in direct contrast to the activity dependence of the effect of PSD-95 knockdown, revealing differential interaction between activity-dependent signaling and scaffold protein families in regulating synaptic AMPAR function.

Higher Aminopeptidase Activity Determined by Electroosmotic Push–Pull Perfusion Contributes to Selective Vulnerability of the Hippocampal CA1 Region to Oxygen Glucose Deprivation

It has been known for over a century that the hippocampus, the center for learning and memory in the brain, is selectively vulnerable to ischemic damage, with the CA1 being more vulnerable than the CA3. It is also known that leucine enkephalin, or YGGFL, is neuroprotective. We hypothesized that the extracellular hydrolysis of YGGFL may be greater in the CA1 than the CA3, which would lead to the observed difference in susceptibility to ischemia. In rat organotypic hippocampal slice cultures, we estimated the Michaelis constant and the maximum velocity for membrane-bound aminopeptidase activity in the CA1 and CA3 regions. Using electroosmotic push-pull perfusion and offline capillary liquid chromatography, we inferred enzyme activity based on the production rate of GGFL, a natural and inactive product of the enzymatic hydrolysis of YGGFL. We found nearly 3-fold higher aminopeptidase activity in the CA1 than the CA3. The aminopeptidase inhibitor bestatin significantly reduced hydrolysis of YGGFL in both regions by increasing apparent Km. Based on propidium iodide cell death measurements 24 h after oxygen-glucose deprivation, we demonstrate that inhibition of aminopeptidase activity using bestatin selectively protected CA1 against delayed cell death due to oxygen-glucose deprivation and that this neuroprotection occurs through enkephalin-dependent pathways.

Impact of X-irradiation on microglia.

Irradiation is widely used to treat brain tumors, and also to create bone marrow (BM) chimeras. BM chimeras are widely used to dissect functions and origin of microglia and blood-derived mononuclear cells under homeostatic or pathological conditions. This is facilitated by the fact that microglia survive irradiation and are thus regarded radio-resistant. In this study, we tested whether microglia are indeed radio-resistant and looked for potential mechanisms that might explain this phenomenon. We analyzed the radio-resistance of microglia independently of their physiological brain environment compared to other mononuclear cells from spleen and brain after X-irradiation with 7 Gy or 30 Gy. Furthermore, we investigated long-term effects of X-irradiation on microglia using organotypic hippocampal slice cultures (OHSCs). We found a significant higher survival rate of isolated microglia 4 hr after X-irradiation with 30 Gy accompanied by a decreased proliferation rate. Investigations of apoptosis-related genes revealed no regulation of a specific antiapoptotic pathway but ataxia telangiectasia mutated (ATM), a DNA-repair-related gene, was significantly upregulated in isolated microglia 4 hr after 30 Gy. Irradiation of OHSCs with 7 and 30 Gy revealed a highly and significantly decreased cell number, morphological changes and an increase in migration velocity of microglia. Furthermore, cell loss, increased soma size and process length of microglia was also found in BM chimeras irradiated with 9.5 Gy 5weeks after irradiation. Here, we present new evidence implying that microglia are not a homogeneous population of radio-resistant cells and report on long-term alterations of microglia that survived irradiation.

Multiple mechanisms of dimethyl fumarate in amyloid β-induced neurotoxicity in human neuronal cells.

Alzheimer disease (AD) is characterized by a complex heterogeneity of pathological changes, and any therapeutic approach categorically requires a multi‐targeted way. It has been demonstrated that together with the hallmarks of the disease such as neurofibrillary tangles and senile plaques, oxidative and inflammatory stress covered an important role. Dimethyl fumarate (DMF) is an orally bioavailable methyl ester of fumaric acid and activator of Nrf2 with potential neuroprotective and immunomodulating activities. Therefore, the aim of the present work was to evaluate the potential beneficial effects of DMF, compared with its active metabolite monomethyl fumarate (MMF) (both at 30 μM) in an in vitro Alzheimer's model using SH‐SY5Y human neuroblastoma cell lines stimulated with amyloid‐beta (Aβ). Moreover, the effect of DMF, compared with MMF, was evaluate by an ex vivo model using organotypic hippocampal slice cultures stimulated with Aβ1‐42 (1 μg/ml), to better understand its action in a pathological setting. In both models, DMF pre‐treatment (30 μM) preserved cellular viability from Aβ stimulation, reducing tau hyper‐phosphorylation, much more efficiently then MMF (30 μM). Moreover, DMF was able to induce an activation of manganese superoxide dismutase (MnSOD) and heme‐oxygenase‐1 (HO‐1), decreasing the severity of oxidative stress. Our results showed important multi‐protective effects of DMF pre‐treatment from Aβ stimulation both in in vitro and ex vivo models, highlighting an Nrf2/NF‐κB‐dependent mechanism, which could provide a valuable support to the therapies for neurodegenerative diseases today.

Effects of neural stem cell media on hypoxic injury in rat hippocampal slice cultures

Neonatal hypoxic-ischemic brain injuries cause serious neurological sequelae, yet there is currently no effective treatment for them. We hypothesized that neurotrophic factors released into the medium by stem cells could supply hypoxia-damaged organotypic hippocampal slice cultures with regenerative abilities. We prepared organotypic slice cultures of the hippocampus of 7-day-old Sprague-Dawley rats based on the modified Stoppini method; slices were cultured for 14days in vitro using either Gahwiler’s medium (G-medium) or stem cell-conditioned medium (S-medium) as culture medium. At day 14 in vitro, hippocampal slice cultures were exposed to 95% N2 and 5% CO2 for 3h to induce hypoxic damage, the extent of which was then measured using propidium iodide fluorescence and immunohistochemistry images. We performed dot blotting to estimate neurotrophic/growth factor levels in the G- and S-media. Organotypic hippocampal slices cultured using S-medium after hypoxic injury were significantly less damaged than those cultured using G-medium. GLUT1, NGF, GDNF, VEGF, GCSF, and IGF2 levels were higher in S-medium than in G-medium, whereas FGF1, HIF, and MCP3 levels were not significantly different between media. In conclusion, we found that stem cell-conditioned medium had a neuroprotective effect against hypoxic injury, and that, of the various neurotrophic factors in S-medium, NGF, GDNF, and VEGF can contribute to neuroprotection.

Anticonvulsant activity, organotypic hippocampal neuroprotection assay and in-silico sodium channel blocking potential of 2-amino-6-nitrobenzothiazole derived semicarbazones

Epilepsy is one of the dreadful neurodegenerative disorder characterized by recurrent, unprovoked seizures. Currently available antiepileptic drugs are still associated with enormous side effects resulting in search of newer, more effective and safer agents. In view of this, we have investigated anticonvulsant activity of 2-amino-6-nitrobenzothiazole derived semicarbazones (7–32) in various in-vivo animal seizure models viz. maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) and 6Hz psychomotor seizure model. Neurotoxicity was estimated by rotarod test. The compounds were also assessed for their neuroprotective potential from excitotoxic insult using organotypic hippocampal slice culture neuroprotection assay. Several compounds exhibited excellent anticonvulsant activity in MES and scPTZ models compared to reference drugs, phenytoin and levetiracetam. The results of kainic acid (KA) – induced neuroprotection assay indicated that compounds 26 and 24 were found to be most potent with IC50 of 99.54±1.27 and 101.00±1.20μM respectively. Both the compounds attenuated KA-mediated cell death in organotypic hippocampal slice cultures. Some of the compounds were found to be good antidepressants, better than the reference drug citalopram, when analyzed in forced swim test. Since semicarbazones exhibited profile resembling phenytoin, an attempt was made to screen them against human neuronal sodium channel isoform (hNav1.2) by performing computational molecular docking using AutoDock 4.2. Compound 30, 1-(5-Chloro-2-oxoindolin-3-ylidene)-4-(6-nitrobenzothiazol-2-yl)semicarbazide emerged as lead candidate possessing excellent in-vivo MES activity and high binding affinity computationally, better than the reference drug phenytoin and also exhibited neuroprotection from excitotoxic insult in KA-induced neuroprotection assay (IC50=126.80±1.24μM). However, some of the active compounds were neurotoxic at their anticonvulsant doses. Further optimization studies are needed to reduce toxicity and develop them as novel therapeutic agents for epilepsy.

The Impact of Non-Lethal Single-Dose Radiation on Tumor Invasion and Cytoskeletal Properties.

Irradiation is the standard therapy for glioblastoma multiforme. Glioblastoma are highly resistant to radiotherapy and the underlying mechanisms remain unclear. To better understand the biological effects of irradiation on glioblastoma cells, we tested whether nonlethal irradiation influences the invasiveness, cell stiffness, and actin cytoskeleton properties. Two different glioblastoma cell lines were irradiated with 2 Gy and changes in mechanical and migratory properties and alterations in the actin structure were measured. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. Irradiation led to changes in motility and a less invasive phenotype in both investigated cell lines that were associated with an increase in a ”generalized stiffness” and changes in the actin structure. In this study we demonstrate that irradiation can induce changes in the actin cytoskeleton and motility, which probably results in reduced invasiveness of glioblastoma cell lines. Furthermore, “generalized stiffness” was shown to be a profound marker of the invasiveness of a tumor cell population in our model.

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Ethyl pyruvate does not require microglia for mediating neuroprotection after excitotoxic injury.

Ethyl pyruvate (EP) mediates protective effects after neuronal injury. Besides a direct conservation of damaged neurons, the modulation of indigenous glial cells has been suggested as one important mechanism for EP-related neuroprotection. However, the specific contribution of glial cells is still unknown. Organotypic hippocampal slice cultures (OHSC) were excitotoxically lesioned by 50μmol/L N-methyl-D-aspartate (NMDA, for 4hours) or left untreated. In an additional OHSC subset, microglia was depleted using the bisphosphonate clodronate (100μg/mL) before lesion. After removal of NMDA, EP containing culture medium (0.84μmol/L, 8.4μmol/L, 42μmol/L, 84μmol/L, 168μmol/L) was added and incubated for 72hours. OHSC were stained with propidium iodide to visualize degenerating neurons and isolectin IB4 -FITC to identify microglia. Effects of EP at concentrations of 0.84, 8.4, and 84μmol/L (0-48hours) were analyzed in the astrocytic scratch wound assay. EP significantly reduced neurodegeneration following induced excitotoxicity except for 168μmol/L. For 84μmol/L, a reduction in the microglia cells was observed. Microglia depletion did not affect neuronal survival after EP treatment. EP decelerated astrocytic wound closure at 48hours after injury. EP-mediated neuroprotection seems to be mediated by astrocytes and/or neurons.

Inhibitor of Endocannabinoid Deactivation Protects Against In Vitro and In Vivo Neurotoxic Effects of Paraoxon.

The anticholinesterase paraoxon (Pxn) is related to military nerve agents that increase acetylcholine levels, trigger seizures, and cause excitotoxic damage in the brain. In rat hippocampal slice cultures, high-dose Pxn was applied resulting in a presynaptic vulnerability evidenced by a 64% reduction in synapsin IIb (syn IIb) levels, whereas the postsynaptic protein GluR1 was unchanged. Other signs of Pxn-induced cytotoxicity include the oxidative stress-related production of stable 4-hydroxynonenal (4-HNE)-protein adducts. Next, the Pxn toxicity was tested for protective effects by the fatty acid amide hydrolase (FAAH) inhibitor AM5206, a compound linked to enhanced repair signaling through the endocannabinoid pathway. The Pxn-mediated declines in syn IIb and synaptophysin were prevented by AM5206 in the slice cultures. To test if the protective results in the slice model translate to an in vivo model, AM5206 was injected i.p. into rats, followed immediately by subcutaneous Pxn administration. The toxin caused a pathogenic cascade initiated by seizure events, leading to presynaptic marker decline and oxidative changes in the hippocampus and frontal cortex. AM5206 exhibited protective effects including the reduction of seizure severity by 86%, and improving balance and coordination measured 24h post-insult. As observed in hippocampal slices, the FAAH inhibitor also prevented the Pxn-induced loss of syn IIb in vivo. In addition, the AM5206 compound reduced the 4-HNE modifications of proteins and the β1 integrin activation events both in vitro and in vivo. These results indicate that Pxn exposure produces oxidative and synaptic toxicity that leads to the behavioral deficits manifested by the neurotoxin. In contrast, the presence of FAAH inhibitor AM5206 offsets the pathogenic cascade elicited by the Pxn anticholinesterase.

Aβ42-mediated proteasome inhibition and associated tau pathology in hippocampus are governed by a lysosomal response involving cathepsin B: Evidence for protective crosstalk between protein clearance pathways.

Impaired protein clearance likely increases the risk of protein accumulation disorders including Alzheimer’s disease (AD). Protein degradation through the proteasome pathway decreases with age and in AD brains, and the Aβ42 peptide has been shown to impair proteasome function in cultured cells and in a cell-free model. Here, Aβ42 was studied in brain tissue to measure changes in protein clearance pathways and related secondary pathology. Oligomerized Aβ42 (0.5–1.5 μM) reduced proteasome activity by 62% in hippocampal slice cultures over a 4-6-day period, corresponding with increased tau phosphorylation and reduced synaptophysin levels. Interestingly, the decrease in proteasome activity was associated with a delayed inverse effect, >2-fold increase, regarding lysosomal cathepsin B (CatB) activity. The CatB enhancement did not correspond with the Aβ42-mediated phospho-tau alterations since the latter occurred prior to the CatB response. Hippocampal slices treated with the proteasome inhibitor lactacystin also exhibited an inverse effect on CatB activity with respect to diminished proteasome function. Lactacystin caused earlier CatB enhancement than Aβ42, and no correspondence was evident between up-regulated CatB levels and the delayed synaptic pathology indicated by the loss of pre- and postsynaptic markers. Contrasting the inverse effects on the proteasomal and lysosomal pathways by Aβ42 and lactacystin, such were not found when CatB activity was up-regulated two-fold with Z-Phe-Ala-diazomethylketone (PADK). Instead of an inverse decline, proteasome function was increased marginally in PADK-treated hippocampal slices. Unexpectedly, the proteasomal augmentation was significantly pronounced in Aβ42-compromised slices, while absent in lactacystin-treated tissue, resulting in >2-fold improvement for nearly complete recovery of proteasome function by the CatB-enhancing compound. The PADK treatment also reduced Aβ42-mediated tau phosphorylation and synaptic marker declines, corresponding with the positive modulation of both proteasome activity and the lysosomal CatB enzyme. These findings indicate that proteasomal stress contributes to AD-type pathogenesis and that governing such pathology occurs through crosstalk between the two protein clearance pathways.

Impact of neonatal hypoxia-ischaemia on oligodendrocyte survival, maturation and myelinating potential.

Hypoxic‐ischaemic episodes experienced at the perinatal period commonly lead to a development of neurological disabilities and cognitive impairments in neonates or later in childhood. Clinical symptoms often are associated with the observed alterations in white matter in the brains of diseased children, suggesting contribution of triggered oligodendrocyte/myelin pathology to the resulting disorders. To date, the processes initiated by perinatal asphyxia remain unclear, hampering the ability to develop preventions. To address the issue, the effects of temporal hypoxia‐ischaemia on survival, proliferation and the myelinating potential of oligodendrocytes were evaluated ex vivo using cultures of hippocampal organotypic slices and in vivo in rat model of perinatal asphyxia. The potential engagement of gelatinases in oligodendrocyte maturation was assessed as well. The results pointed to a significant decrease in the number of oligodendrocyte progenitor cells (OPCs), which is compensated for to a certain extent by the increased rate of OPC proliferation. Oligodendrocyte maturation seemed however to be significantly altered. An ultrastructural examination of selected brain regions performed several weeks after the insult showed however that the process of developing central nervous system myelination proceeds efficiently resulting in enwrapping the majority of axons in compact myelin. The increased angiogenesis in response to neonatal hypoxic‐ischaemic insult was also noticed. In conclusion, the study shows that hypoxic‐ischaemic episodes experienced during the most active period of nervous system development might be efficiently compensated for by the oligodendroglial cell response triggered by the insult. The main obstacle seems to be the inflammatory process modulating the local microenvironment.

INVESTIGATING PRESYNAPTIC DISRUPTION IN ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES FROM HU-APP TRANSGENIC MICE

Loss of presynaptic proteins has been reported as an early and clinically-relevant alteration in the brains of patients with Alzheimer's disease. Understanding the mechanisms by which synapses are disrupted requires a model system that represents all major CNS cell types, is amenable to repeated analysis, and can be readily manipulated. Long-term organotypic hippocampal slice cultures (OHSCs) from neonatal amyloid mice offer an excellent compromise between in vivo and primary culture studies, largely retaining the cellular composition and neuronal architecture of the in vivo hippocampus, but with the in vitro advantages of accessibility to live imaging, sampling and intervention. OHSCs are made using tissue from P6-P9 TgCRND8 mice and maintained on membrane inserts for up to 2 months. Pathology development was closely monitored using Aβ ELISA, western blotting for synaptic proteins, and immunofluorescence for Aβ, neurons and glia. The mechanisms behind presynaptic disruption were probed via pharmacological manipulation of Aβ production and genetic knockout of endogenous mouse tau. Aβ1–40 and Aβ1–42 rise progressively in transgenic slice culture medium and stabilise when regular feeding balances continued production. In contrast, intra-axonal Aβ continues to accumulate in close correlation with a specific decline in presynaptic proteins. There are no fibrillar plaques under these conditions indicating that plaques are not needed for synaptic disruption. Beta secretase inhibitor treatment completely abolished the accumulation of Aβ1–42 in the medium but surprisingly this did not rescue synaptophysin levels, even when continually present from before synaptophysin declines. This raises the question of whether BACE1-independent APP products underlie some synaptic defects and provides an effective experimental system to test this. Interestingly, genetic deletion of tau does rescue the loss of synaptophysin, opening lines of investigation for potential therapeutics. Organotypic brain slices from TgCRND8 mice represent an important new system for understanding mechanisms of presynaptic disruption induced by the presence of mutant huAPP. The pathology observed in these cultures will allow for rapid assessment of disease modifying compounds in a system amenable to manipulation and observation. Future work will involve examination of the mechanisms resulting in synaptophysin depletion, particularly in relation to the involvement of tau and alternative APP processing products.

Neuroprotection of brain-permeable iron chelator VK-28 against intracerebral hemorrhage in mice

Iron overload plays a key role in the secondary brain damage that develops after intracerebral hemorrhage (ICH). The significant increase in iron deposition is associated with the generation of reactive oxygen species (ROS), which leads to oxidative brain damage. In this study, we examined the protective effects of VK-28, a brain-permeable iron chelator, against hemoglobin toxicity in an ex vivo organotypic hippocampal slice culture (OHSC) model and in middle-aged mice subjected to an in vivo, collagenase-induced ICH model. We found that the effects of VK-28 were similar to those of deferoxamine (DFX), a well-studied iron chelator. Both decreased cell death and ROS production in OHSCs and in vivo, decreased iron-deposition and microglial activation around hematoma in vivo, and improved neurologic function. Moreover, compared with DFX, VK-28 polarized microglia to an M2-like phenotype, reduced brain water content, deceased white matter injury, improved neurobehavioral performance, and reduced overall death rate after ICH. The protection of VK-28 was confirmed in a blood-injection ICH model and in aged-male and young female mice. Our findings indicate that VK-28 is protective against iron toxicity after ICH and that, at the dosage tested, it has better efficacy and less toxicity than DFX does.

Differentiation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons in Mouse Hippocampal Slice Cultures.

Potential clinical applications of neurons derived from human induced pluripotent stem cells (hiPSC-neurons) for drug screening and transplantation therapies have received considerable attention. However, it remains unclear whether and how transplanted hiPSC-neurons are incorporated into pre-existing neural circuits. Here we developed a co-culture system of hiPSC-neurons and mouse hippocampal slices to examine the differentiation of hiPSC-neurons in pre-existing neural circuits. hiPSC-neurons transplanted in mouse hippocampal slices expressed the hippocampal neuron-specific markers HuB and Prox1 after 7 days of culture, while those markers were scarcely expressed in hiPSC-neurons cultured on glass dishes. Furthermore, hiPSC-neurons transplanted in the dentate gyrus (DG) of slice cultures grew to exhibit dentate granule cell-like morphologies, including besom-shaped dendrites. Similarly, hiPSC-neurons transplanted in the CA1 region of slice cultures grew to exhibit CA1 pyramidal cell-like morphologies, including primary apical and multiple basal dendrites with synaptic spines. Additionally, these cells projected axons toward the entorhinal cortex (EC) as observed in vivo. These data suggest that hiPSC-neurons were anatomically integrated into pre-existing neural circuits in a region-specific manner. Thus, the co-culture system will be useful for the study of efficient strategies to differentiate transplanted hiPSC-neurons.

Inhibition of neuronal ferroptosis protects hemorrhagic brain.

Intracerebral hemorrhage (ICH) causes high mortality and morbidity, but our knowledge of post-ICH neuronal death and related mechanisms is limited. In this study, we first demonstrated that ferroptosis, a newly identified form of cell death, occurs in the collagenase-induced ICH model in mice. We found that administration of ferrostatin-1, a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin in organotypic hippocampal slice cultures (OHSCs). Mice treated with ferrostatin-1 after ICH exhibited marked brain protection and improved neurologic function. Additionally, we found that ferrostatin-1 reduced lipid reactive oxygen species production and attenuated the increased expression level of PTGS2 and its gene product cyclooxygenase-2 ex vivo and in vivo. Moreover, ferrostatin-1 in combination with other inhibitors that target different forms of cell death prevented hemoglobin-induced cell death in OHSCs and human induced pluripotent stem cell-derived neurons better than any inhibitor alone. These results indicate that ferroptosis contributes to neuronal death after ICH, that administration of ferrostatin-1 protects hemorrhagic brain, and that cyclooxygenase-2 could be a biomarker of ferroptosis. The insights gained from this study will advance our knowledge of the post-ICH cell death cascade and be essential for future preclinical studies.

Formation and Maintenance of Functional Spines in the Absence of Presynaptic Glutamate Release

SummaryDendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release.

Effects of Brevetoxin and NMDA on Dendritic Arborization in Murine Organotypic Hippocampal Slice Culture

BackgroundDuring neuronal development dendritic arbors become complex as function of both intrinsic genetic factors and neuronal activity. The N‐methyl‐D‐aspartate receptor (NMDAR) is essential for activity‐dependent neurite outgrowth, dendritic arborization and spine formation. Dysfunction in NMDARs is associated with diverse neuropsychiatric disorders such as depression and autism spectrum disorder. High and low NMDAR activity, respectively, leads to excitotoxity and failure of neuronal development. Voltage‐gated sodium channel (VGNaC) activation using gating modifiers such as brevetoxin (PbTx‐2) is one pathway to influence NMDAR function. PbTx‐2 augments NMDAR function in dissociated cerebrocortical neurons. Here we assessed the effect of PbTx‐2 and NMDA on dendritic arbor complexity in organotypic hippocampal slice cultures (OHSC). The activity of the NMDAR is also linked to its phosphorylation status. Phosphorylation of tyrosine residues on the NMDAR increases the open probability and mean open time of the NMDAR leading to increased Ca2+ levels. Calcineurin (CaN) regulates NMDAR function by dephosphorylating the tyrosine residues. In cerebrocortical neurons, FK‐506, an inhibitor of calcineurin, eliminated the reduction in neurite outgrowth observed at high concentrations of PbTx‐2. Here, we determined the influence of FK‐506 alone on dendritic arborization in OHSC.Hypothesis1) VGNaC activation or direct activation of NMDARs will promote dendritic arborization in OHSC. 2) FK‐506 alone will not affect dendritic arbor complexity in OHSC.MethodsThe Thy‐1 YFP transgenic mice were used. Hippocampal slices were prepared from postnatal day 5 mouse pups. Slices were treated 18 hours after plating with control, (10–1000 nM) PbTx‐2, (0.01 – 100 μM) NMDA or (0.001–10 μM) FK‐506 for a period of 4 days. A Leica SP8 microscope was used to generate Z‐stack confocal images and Imaris‐XT software was used create 3D‐reconstructed images of the dendritic arbor to run a sholl analysis.ConclusionThe results demonstrated that both 100 nM PbTx‐2 and 1 μM NMDA treatment produced significant increases in dendritic arbor complexity. The OHSC appeared more sensitive to NMDA than dissociated cerebrocortical neurons inasmuch as 100 μM NMDA was extremely toxic to OHSC neurons. The use of the OHSC confirmed that VGNaC activation augments dendritic arbor complexity and this effect generalizes to hippocampal pyramidal neurons. Our results also indicate that (0.001–10 μM) FK‐506 alone had no effect on dendritic arborization in OHSC. Overall these studies suggest that VGNaC activators may represent a novel pharmacological strategy to regulate NMDAR function and promote neuronal structural plasticity.Support or Funding InformationNIH‐RO1NS053398

Phosphodiesterase-4 inhibition restored hippocampal long term potentiation after primary blast

Due to recent military conflicts and terrorist attacks, blast-induced traumatic brain injury (bTBI) presents a health concern for military and civilian personnel alike. Although secondary blast (penetrating injury) and tertiary blast (inertia-driven brain deformation) are known to be injurious, the effects of primary blast caused by the supersonic shock wave interacting with the skull and brain remain debated. Our group previously reported that in vitro primary blast exposure reduced long-term potentiation (LTP), the electrophysiological correlate of learning and memory, in rat organotypic hippocampal slice cultures (OHSCs) and that primary blast affects key proteins governing LTP. Recent studies have investigated phosphodiesterase-4 (PDE4) inhibition as a therapeutic strategy for reducing LTP deficits following inertia-driven TBI. We investigated the therapeutic potential of PDE4 inhibitors, specifically roflumilast, to ameliorate primary blast-induced deficits in LTP. We found that roflumilast at concentrations of 1nM or greater prevented deficits in neuronal plasticity measured 24h post-injury. We also observed a therapeutic window of at least 6h, but <23h. Additionally, we investigated molecular mechanisms that could elucidate this therapeutic effect. Roflumilast treatment (1nM delivered 6h post-injury) significantly increased total AMPA glutamate receptor 1 (GluR1) subunit expression, phosphorylation of the GluR1 subunit at the serine-831 site, and phosphorylation of stargazin at the serine-239/240 site upon LTP induction, measured 24h following injury. Roflumilast treatment significantly increased PSD-95 regardless of LTP induction. These findings indicate that further investigation into the translation of PDE4 inhibition as a therapy following bTBI is warranted.