Organotypic Hippocampal Cultures Research Articles

Subregion-specific vulnerability to endoplasmic reticulum stress-induced neurotoxicity in rat hippocampal neurons

It is well known that in certain disease states, including ischemia and Alzheimer's disease, neurodegeneration occurs in the hippocampus and that vulnerability to neuronal death is area dependent. The present study investigated the mechanism of area-dependent vulnerability to neuronal death under endoplasmic reticulum stress conditions induced by tunicamycin (TM), using rat organotypic hippocampal cultures (OHC) and hippocampal slices. Analysis of propidium iodide uptake showed that TM-induced neuronal death in a concentration-dependent manner (20–80 μg/mL) and that the rank order of vulnerability among hippocampal subregions was dentate gyrus (DG) > CA1 > CA3. Results of immunohistochemistry using hippocampal slices also showed that procaspase-12-positive cells in area CA3 were significantly fewer than those in area CA1 and the DG. Moreover, procurement of neurons in areas CA1, CA3 and the DG by laser microdissection, followed by Western blot analysis, also revealed that the level of procaspase-12 in area CA3 was significantly lower than those in area CA1 and the DG. Pretreatment with z-ATAD-fmk, a cell-permeable caspase-12-selective inhibitor significantly attenuated the TM-induced increase of PI fluorescence in the CA1 and DG subregion but not in area CA3. These results suggest that TM elicits subregion-specific neuronal toxicity in OHC and that the vulnerability to TM-induced toxicity is at least partly dependent on the expression level of endogenous procaspase-12 in each area of the hippocampus.

Cadherin‐8 and N‐cadherin differentially regulate pre‐ and postsynaptic development of the hippocampal mossy fiber pathway

Cells sort into regions and groups in part by their selective surface expression of particular classic cadherins during development. In the nervous system, cadherin-based sorting can define axon tracts, restrict axonal and dendritic arbors to particular regions or layers, and may encode certain aspects of synapse specificity. The underlying model has been that afferents and their targets hold in common the expression of a particular cadherin, thereby providing a recognition code of homophilic cadherin binding. However, most neurons express multiple cadherins, and it is not clear whether multiple cadherins all act similarly in shaping neural circuitry. Here we asked how two such cadherins, cadherin-8 and N-cadherin, influence the guidance and differentiation of hippocampal mossy fibers. Using organotypic hippocampal cultures, we find that cadherin-8 regulates mossy fiber fasciculation and targeting, but has little effect on CA3 dendrites. In contrast, N-cadherin regulates mossy fiber fasciculation, but has little impact on axonal growth and targeting. However, N-cadherin is essential for CA3 dendrite arborization. Both cadherins are required for formation of proper numbers of presynaptic terminals. Mechanistically, such differential actions of these two cadherins could, in theory, reflect coupling to distinct intracellular binding partners. However, we find that both cadherins bind beta-catenin in dentate gyrus (DG). This suggests that cadherins may engage different intracellular signaling cascades downstream of beta-catenin, coopt different extracellular binding partners, or target distinct subcellular domains. Together our findings demonstrate that cadherin-8 and N-cadherin are critical for generating the mossy fiber pathway, but that each contributes differentially to afferent and target differentiation, thereby complementing one another in the assembly of a synaptic circuit.

Rab‐Mediated Endocytosis

Rab proteins are small GTPases involved in endocytosis and recycling of cell surface molecules. Recently they have been implicated in the etiopathogenesis of several neurodegenerative disorders including Alzheimer's and Lewy body disease. In experiments on organotypic hippocampal cultures, upregulation of Rab protein family member Rab5b after group I metabotropic glutamate receptor (mGluR) stimulation was associated with reduced neuronal vulnerability to excitotoxic injury. This mGluR-mediated neuroprotection was abolished by antisense-induced deficiency of Rab5b. Electrophysiological measurements of excitatory synaptic transmission in the Schaffer collateral-CA1 pathway revealed that mGluR activation that induces neuroprotection also induced long-term depression (LTD) of synaptic transmission. Similar to the neuroprotection, Rab5b deficiency abolished dihydroxyphenylglycine-induced LTD. Together, these findings support the idea that Rab proteins, and the Rab5b protein in particular, may provide a link between neurodegenerative disease, neuroprotection, and synaptic plasticity, as well as possibly being a useful target for pharmacological interventions.

Activity‐dependent heteromerization of the hyperpolarization‐activated, cyclic‐nucleotide gated (HCN) channels: role of N‐linked glycosylation

Formation of heteromeric complexes of ion channels via co-assembly of different subunit isoforms provides an important mechanism for enhanced channel diversity. We have previously demonstrated co-association of the hyperpolarization activated cyclic-nucleotide gated (HCN1/HCN2) channel isoforms that was regulated by network (seizure) activity in developing hippocampus. However, the mechanisms that underlie this augmented expression of heteromeric complexes have remained unknown. Glycosylation of the HCN channels has been implicated in the stabilization and membrane expression of heteromeric HCN1/HCN2 constructs in heterologous systems. Therefore, we used in vivo and in vitro systems to test the hypothesis that activity modifies HCN1/HCN2 heteromerization in neurons by modulating the glycosylation state of the channel molecules. Seizure-like activity (SA) increased HCN1/HCN2 heteromerization in hippocampus in vivo as well as in hippocampal organotypic slice cultures. This activity increased the abundance of glycosylated HCN1 but not HCN2-channel molecules. In addition, glycosylated HCN1 channels were preferentially co-immunoprecipitated with the HCN2 isoforms. Provoking SA in vitro in the presence of the N-linked glycosylation blocker tunicamycin abrogated the activity-dependent increase of HCN1/HCN2 heteromerization. Thus, hippocampal HCN1 molecules have a significantly higher probability of being glycosylated after SA, and this might promote a stable heteromerization with HCN2.

ɛPKC phosphorylates the mitochondrial K+ATP channel during induction of ischemic preconditioning in the rat hippocampus

Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (epsilonPKC). A major goal in our laboratory is to define the cellular targets by which epsilonPKC confers protection. We tested the hypothesis that epsilonPKC targets the mitochondrial K(+)(ATP) channel (mtK(+)(ATP)) after IPC. Our results demonstrated a rapid translocation of epsilonPKC to rat hippocampal mitochondria after IPC. Because in other tissues epsilonPKC targets mtK(+)(ATP) channels, but its presence in brain mitochondria is controversial, we determined the presence of the K(+)(ATP) channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether mtK(+)(ATP) channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the K(+)(ATP) channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or epsilonPKC agonist-induced preconditioning. Blockade of mtK(+)(ATP) channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or epsilonPKC preconditioning. Both ischemic and epsilonPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK(+)(ATP) channel subunit Kir6.2. After IPC, selective inhibition of epsilonPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of epsilonPKC or its downstream effectors. Our results support the hypothesis that the brain mtK(+)(ATP) channel is an important target of IPC and the signal transduction pathways initiated by epsilonPKC.

Different effects of monocarboxylates on neuronal survival and β‐amyloid toxicity

Glucose is a principal metabolic fuel in the central nervous system, but, when glucose is unavailable, the brain can utilize alternative metabolic substrates such as monocarboxylates to sustain brain functions. This study examined whether the replacement of glucose with monocarboxylates (particularly pyruvate and lactate) had an equivalent effect of glucose on neuronal survival in rat hippocampal organotypic slice cultures, or ameliorate the neurotoxicity induced by amyloid beta-peptide (Abeta). The possible mechanism was also explored. We found that pyruvate and lactate alone increased cell death in the hippocampal slice cultures at 24 and 48 h. Supplementation of glucose-containing culture media and Abeta-treated culture media with pyruvate, but not lactate, attenuated cell death as strong as with trolox, known as a reactive oxygen species scavenger, and niacinamide, an NAD(+) precursor, and this protective effect was reversed by alpha-cyano-4-hydroxycinnamic acid. Pyruvate significantly increased the aconitase activity and the NAD(+) levels in the hippocampal slices in the presence of Abeta, but did not maintain the ATP levels. Our results indicate that pyruvate and lactate alone cannot replace glucose as an alternative energy source to preserve the neuronal viability in the hippocampal slice cultures. Pyruvate, in the presence of glucose, improves neuronal survival in the hippocampal slice cultures and also protects neurons against Abeta-induced cell death in which mitochondrial NAD(P) redox status may play a central role.

Corticotropin Releasing Hormone (CRH) targets various neuroprotective mechanisms

The corticotropin releasing hormone (CRH) is a central mediator of the human stress response in the central nervous system by affecting the hypothalamic-pituitary adrenal axis. A chronic hyperdrive of the CRH system leads to anxiety-related disorders including depression. Therefore, a stable CRH level is necessary for an appropriate stress response. Furthermore, CRH also affects other central processes, such as learning and memory, synaptic plasticity and mediates neuroprotection. Indeed, CRH induces neuroprotective signalling in a brain region-specific manner by binding to its high-affinity cell surface receptor, the CRH receptor type 1, (Bayatti et al., 2003). Recent studies show that CRH exhibits neuroprotective properties in organotypic hippocampal slices, primary cultures and cell lines in response to certain toxins. Moreover, CRH can directly increase the expression of a potent neuroprotective factor, the brain-derived neurotrophic factor (BDNF) (Bayatti et al., 2005; Elliott-Hunt et al., 2002). Alternatively, neuroprotective effects of CRH may involve the direct modulation of pathways leading to cell death. We investigated activity of CRH in hippocampal slices and observed CRH receptor-mediated neuroprotective action, which may involve MAP kinase activation and BDNF upregulation. Interestingly, CRH unfolds a complex pattern of neuroprotective activities, new targets of CRH activity have been found and are currently under intensive investigation.

Inducible cAMP early repressor (ICER)‐evoked delayed neuronal death in the organotypic hippocampal culture

Programmed cell death involving gene regulation and de novo protein synthesis is a major component of both normal development and a number of disease conditions. Hence, knowledge of its mechanisms, especially transcription factors, that regulate expression of the genes involved in neurodegenerative disorders is of great importance. cAMP-responsive element-binding protein (CREB) has repeatedly been implicated in the neuronal survival. In the present study we showed that inducible cAMP early repressor (ICER), an endogenous CREB antagonist, is expressed during both excitotoxic and spontaneous neuronal cell death in organotypic hippocampal slice cultures in vitro. Furthermore, overexpression of ICER via an adenoviral vector evoked neuronal cell loss in such cultures. The time course of ICER-dependent cell death was hippocampal subdivision specific, with dentate gyrus neurons dying mostly 3-7 days after the adenovector infection, followed by CA3, where neuronal death peaked after 7 days, and then CA1, where most neuronal death occurred after 7-14 days. These results underscore the usefulness of the organotypic cultures for studies of neurodegeneration and point to neuronal loss having a multifaceted nature in a complex cellular environment.

Synaptic activity and triphenyltetrazolium chloride metabolism are correlated after oxygen–glucose deprivation in acute, but not cultured, hippocampal slices

The importance of the hippocampus to learning and memory has attracted significant attention to how the structure responds to damage. Although many studies have used either the acute hippocampal slice preparation or organotypic hippocampal slice cultures, little work has been done to determine if the choice of model is an important variable. The present study examined whether differences exist in how each model responds to a commonly studied ischemic-like paradigm, oxygen–glucose deprivation. Following the insult, synaptic activity was examined by recording orthodromically evoked CA1 subfield responses, while mitochondrial activity was assessed by spectrophotometric measurement of formazan produced by metabolism of 2,3,5-triphenyltetrazolium chloride. The insult significantly decreased both synaptic and mitochondrial activity within acutely prepared slices, but a disparity existed between these measures in cultured slices. While evoked activity was greatly reduced by an insult of moderate duration, a much longer period was required to cause a comparable decrease in formazan production. Quantitative immunoblotting revealed that one possible explanation for the discrepancy was an elevated expression of astrocytes, which display resistance to hypoxia–aglycemia. Our data indicate that acutely prepared and cultured slices respond differently to ischemic-like challenge; therefore, assays examining viability in these models must consider their innate differences.

The p21-activated Kinase 3 Implicated in Mental Retardation Regulates Spine Morphogenesis through a Cdc42-dependent Pathway

The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.

P-192: Tramiprosate decreases amyloid-β induced ERK1/2 activity in primary rat neurons by a GABA-independent pathway

The mitogen-activated protein kinase (MAPK) family members, including extracellular signal regulated kinase (ERK1/2), are involved in neuronal cell death mediated by amyloid-β (Aβ). Aβ has been shown to induce activation of ERK1/2 in primary neurons (Rapoport. J Neurochem 2000, 74:125). Treatment of organotypic hippocampal cultures (OHC) with soluble oligomeric Aβ has been reported to result in ERK1/2 activation and contribute to cell death (Chong. J Biol Chem 2006:281). Tramiprosate (3-amino-1-propanesulfonic acid), presently in phase III trials for Alzheimer's disease, is neuroprotective against Aβ42-mediated toxicity in rat primary neurons and OHC. This study assessed the role of MAPK in this neuroprotective activity. Rat primary cortical/hippocampal neurons were treated with soluble Aβ42, and ERK1/2 phosphorylation and caspase 3/7 activity were assessed. OHC were treated with soluble Aβ42 ± U0126 (MEK1 inhibitor). Cellular mortality was assessed by propidium iodide uptake. Treatment of rat primary neurons with Aβ42 induced ERK1/2 phosphorylation, while coincubation with tramiprosate significantly reduced this effect. Tramiprosate preferentially inhibits ERK1/2 activation induced by an Aβ42 preparation that is predominantly in a β-sheet conformation rather than random coil. U0126 completely blocked Aβ42-induced ERK1/2 phosphorylation while muscimol, a potent GABAA receptor agonist, was inactive. Together these results demonstrate the GABAA-independent activity of tramiprosate on ERK1/2. Treatment of OHC with Aβ42 induced ERK1/2 phosphorylation in all hippocampal regions while U0126 was completely protective against Aβ42-induced cell death. The effect of tramiprosate on Aβ42-mediated ERK1/2 activation in OHC is under investigation. Tramiprosate is neuroprotective in rat primary neurons at least in part via inhibition of Aβ42-induced activation of ERK1/2 by a GABAA-independent mechanism. Interestingly, tramiprosate binds to the GABAA receptor with high affinity and inhibits Aβ42-induced caspase-3/7 activation in rat primary neurons through a GABAA-dependent mechanism (See Galarneau et al., poster presentation at this meeting). Because muscimol was inactive against Aβ42-induced ERK1/2 phosphorylation, and U0126 did not protect against Aβ42-induced caspase 3/7 activation, these results suggest that the neuroprotective activity of tramiprosate involves at least two potentially independent and complementary mechanisms, one GABAergic and the other non-GABAergic.

Modeling Neural Injury in Organotypic Cultures by Application of Inertia-Driven Shear Strain

In vitro models of traumatic brain injury (TBI) are indispensable to explore the effects of mechanotrauma on neurological injury cascades and injury thresholds. This study characterizes a novel in vitro model of neural shear injury, which for the first time subjects organotypic cultures to inertia-driven shear strain. In this model, organotypic cultures preserved a high level of biological heterogeneity and spatial cytoarchitecture, while inertia-driven shear strain represented a tissue-level insult typical for closed head TBI in vivo. For neural injury simulation, organotypic hippocampal cultures derived from rats were inserted in an inertial loading module, which was subjected to impacts at five graded impact velocities ranging from 2 to 10 m/sec. The mechanical insult was quantified by measuring the transient shear deformation of the culture surface during impact with a high-speed camera. The resultant cell death was quantified with propidium iodide (PI) staining 24 hours following shear injury. Increasing impact velocities of 2, 4.6, 6.6, 8.1, and 10.4 m/sec caused graded peak shear elongation of 2.0 +/- 0.9%, 5.4 +/- 3.8%, 15.1 +/- 14.6%, 25.4 +/- 14.7%, and 56.3 +/- 51.3%, respectively. Cell death in response to impact velocities of 6.6 m/sec or less was not significantly higher than baseline cell death in sham cultures (4.4 +/- 1.5%). Higher impact velocities of 8.1 and 10.4 m/sec resulted in a significant increase in cell death to 19.9 +/- 12.9% and 36.7 +/- 14.2%, respectively (p < 0.001). The neural shear injury model delivered a gradable, defined mechanotrauma and thereby provides a novel tool for investigation of biological injury cascades in organotypic cultures.

Perinatal asphyxia induces neurogenesis in hippocampus: an organotypic culture study

There is clinical and experimental evidence indicating that neurocircuitries of the hippocampus are vulnerable to hypoxia/ischemia occurring at birth, inducing, upon re-oxygenation/re-circulation, delayed neuronal death, but also compensatory mechanisms, including neurogenesis. In the present report, perinatal asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath at 37 degrees C for 20 min. Some pups were delivered immediately after the hysterectomy to be used as non-asphyxiated caesarean-delivered controls. The pups were sacrificed after seven days for preparing organotypic hippocampal cultures. The cultures were grown on a coverslip in a medium-containing culture tube inserted in a hole of a roller device standing on the internal area of a cell incubator at 35 degrees C, 10% CO2. At days in vitro (DIV) 25-27, cultures were fixed for assaying cell proliferation and neuronal phenotype with antibodies against 5-bromo-2'deoxyuridine (BrdU) and microtubule associated protein-2 (MAP-2), respectively. Confocal microscopy revealed that there was a 2-fold increase of BrdU-positive, but a 40% decrease of MAP-2-positive cells/mm3 in cultures from asphyxia-exposed, compared to that from control animals. Approximately 30% of BrdU-positive cells were also positive for MAP-2 (approximately 4800 cells), mainly seen in the dentate gyrus of the hippocampus, demonstrating a 3-fold increase of postnatal neurogenesis, when the total amount of double-labelled cells seen in cultures from asphyxia-exposed animals is compared to that from control animals.

Neuroprotective activity of selective mGlu1 and mGlu5 antagonists in vitro and in vivo

The neuroprotective potential of allosteric mGlu5 and mGlu1 antagonists such as 6-methyl-2-(phenylethynyl)-pyridin (MPEP)/[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) and (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM), was tested in vitro in organotypic hippocampal cultures and in the middle cerebral artery occlusion model of stroke in vivo. Both classes of agent have high selectivity toward mGlu sub-types and are active in animal models of various diseases indicating satisfactory CNS penetration. In organotypic hippocampal cultures MPEP showed high neuroprotective potency against sub-chronic (12 days) insult produced by 3-NP with an IC 50 of c.a. 70 nM. In contrast, although the mGlu1 antagonist EMQMCM was also protective, it seems to be weaker yielding an IC 50 of c.a. 1 μM. Similarly, in the transient (90 min) middle cerebral artery occlusion model of ischaemia in rats, MTEP seems to be more effective than EMQMCM. MTEP, at 2.5 mg/kg and at 5 mg/kg provided 50 and 70% neuroprotection if injected 2 h after the onset of ischaemia. At a dose of 5 mg/kg, significant (50%) neuroprotection was also seen if the treatment was delayed by 4 h. EMQMCM was not protective at 5 mg/kg (given 2 h after occlusion) but at 10 mg/kg 50% of neuroprotection was observed. The present data support stronger neuroprotective potential of mGlu5 than mGlu1 antagonists.

The growth compromised HSV-2 mutant ΔRR prevents kainic acid-induced apoptosis and loss of function in organotypic hippocampal cultures

We have previously shown that the HSV-2 anti-apoptotic protein ICP10PK is delivered by the replication incompetent virus mutant ΔRR and prevents kainic acid (KA)-induced epileptiform seizures and neuronal cell loss in the mouse and rat models of temporal lobe epilepsy. The present studies used ΔRR and the ICP10PK deleted virus mutant ΔPK to examine the mechanism of neuroprotection. ΔRR-infected neuronal cells expressed a chimeric protein in which ICP10PK is fused in frame to LacZ (p175) while retaining ICP10PK kinase activity. ΔPK-infected neuronal cells expressed a mutant ICP10 protein that is deleted in the PK domain and is kinase negative (p95). p175 and p95 were expressed in CA3 (86 ± 3%) and CA1 (69 ± 7%) cells from ΔRR or ΔPK-infected organotypic hippocampal cultures (OHC) and 80–85% of the ICP10 positive cells co-stained with antibody to β III Tubulin (neuronal marker). ΔRR, but not ΔPK, inhibited KA-induced cell death and caspase-3 activation in CA3 neurons, an inhibition seen whether ΔRR was delivered 2 days before or 2 days after KA administration (95% neuroprotection). Neuroprotection was associated with ERK and Akt activation and was abrogated by simultaneous treatment with the MEK (U0126) and PI3-K (LY294002) inhibitors. ΔRR-mediated neuroprotection was associated with increased expression of the anti-apoptotic protein Bag-1 and decreased expression of the pro-apoptotic protein Bad. The surviving neurons retained normal synaptic function potentially related to increased expression of the transcription factor CREB. The data indicate that ΔRR is a promising platform for neuroprotection from excitotoxic injury.

β-Amyloid peptide toxicity in organotypic hippocampal slice culture involves Akt/PKB, GSK-3β, and PTEN

In the present study we investigated the toxicity induced by exposing organotypic slice culture to β-amyloid peptide 25–35 (25 μM) for 1, 3, 6, 12, 24 and 48 h. To elucidate a mechanism involved in its toxicity, we studied the PI3-K cell signaling pathway, particularly Akt/PKB, GSK-3β, and PTEN proteins. Cell death was quantified by propidium iodide uptake and proteins were analyzed by immunoblotting. Our results showed a significant cell death after 48 h of β-amyloid 25–35 peptide exposition. The exposition of cultures to β-amyloid peptide resulted in an increase in the phosphorylation state of Akt and GSK-3β proteins after 6 h, followed by a decrease of the phosphorylation state of these proteins after 12 h of exposition. However, after 24 h of peptide treatment, the phosphorylation of GSK-3β presented a new increase while the phosphorylation of Akt remained down. The immunocontent of the PTEN protein, an indirect Akt phosphatase, increased after 24 and 48 h of β-amyloid exposition. These results suggest an involvement of Akt dephosphorylation/inactivation in the toxicity induced by the β-amyloid 25–35 peptide in organotypic slice hippocampal culture, probably induced by increasing PTEN immunocontent. Taken together, our results provide more information about the molecular mechanisms involved on β-amyloid peptide toxicity.

Potential Value of Changes in Cell Markers in Organotypic Hippocampal Cultures Associated With Chronic EtOH Exposure and Withdrawal: Comparison with NMDA‐Induced Changes

Previous studies have shown that withdrawal from ethanol (EtOH) exposure induces neuronal damage, as indicated by propidium iodide (PI) uptake, in organotypic hippocampal slice cultures. This is prevented by MK801, suggesting that damage is "excitotoxic," resulting from activation of N-methyl-d-aspartate (NMDA) receptors by endogenous glutamate. To avoid reliance on a single indicator, and to enable assessment of recovery from the EtOH withdrawal (EWD) insult, we assessed changes in cell markers for neurons and glia, as well as cell division, following either EWD or NMDA challenge (as a positive control). Organotypic cultures from postnatal day (PND) 8 rats were cultured for 5 days before exposure to EtOH (mean concentration approximately 65 mM) for 10 days before EWD. Cultures of the same "days in vitro" age (DIV16) were exposed to NMDA (200 microM) for 1 hour. Neuronal injury was visualized using PI and indices of neurons, glia, or cell division were measured at intervals up to 10 days following the neurotoxic insults. Each time point and measurement used separate slice cultures, and these were treated as separate experiments with paired controls. Regional neuronal content was assessed by neuronal nuclear protein (NeuN) and calbindin D28k (Calb), glial content by glial fibrillary acidic protein (GFAP), and cell division by bromodeoxyuridine (BrdU) incorporation, all measured immunohistochemically. Chronic exposure to EtOH was associated with a dramatic reduction in BrdU incorporation in all regions of cultures. Propidium iodide fluorescence in the CA1 region was elevated significantly after EWD and more so after NMDA challenge. Reduced immunoreactivity (IR) of NeuN and Calb suggested that loss of neurons resulted from the EWD insult. Bromodeoxyuridine incorporation was initially depressed even further by EWD, but had returned to control levels after 3 days. In contrast, following NMDA insult, BrdU incorporation was significantly and persistently elevated above control levels after 3 days. Glial fibrillary acidic protein was reduced immediately after both EWD and NMDA challenge. Several days after EWD, expression of neuronal and glial markers, although variable, had generally returned to control levels. In contrast, NeuN IR remained significantly reduced after NMDA challenge. In general, the use of additional markers supports data obtained with PI uptake alone and suggests that neurons (and glia) are lost from the culture following EWD or NMDA challenge. These cell markers recover several days after EWD, but it is unclear whether functional recovery accompanies these changes. If the dramatic effect of EtOH exposure and EWD on BrdU incorporation reflects reduced neuro- and gliogenesis, it is likely that this adversely affects long-term recovery from EWD. Finally, some markers showed significant and consistent changes after EWD, whereas others did not. This information may facilitate the use of this model in evaluation of potential medications that protect against and/or promote recovery from neurotoxicity.

A regulatory role for actin in dendritic spine proliferation

Dendritic spines are small protrusions that receive 90% of excitatory cortical synapses and are critically important to neural function. Each dendritic spine is supported by a dynamic actin cytoskeleton that responds to internal and external cues to allow spine development, elongation, retraction and movement. Multiple proteins have roles in spinogenesis, but until now, a regulatory role for actin itself has not been established. Here, we show that, in the acute slice preparation, actin expression increases during a period of rapid spinogenesis. Furthermore, actin overexpression in organotypic hippocampal cultures leads to a significant increase in spine density on CA1 pyramidal cells. Specifically, the number of filopodia (long, thin protrusions without heads) increases by 38% on secondary apical dendrites and 88% on basal dendrites and the number of elongated spines with heads increases by 162% on secondary apical dendrites and 113% on basal dendrites. Synapsin-I immunostaining demonstrated that the majority of filopodia and elongated spines are apposed by axon terminals. Additionally, we show that overexpressed actin enters both new and established spines within 24 h. These data demonstrate that neurons undertaking spinogenesis upregulate actin expression, that actin overexpression per se increases spine density, and that both new and established spines incorporate exogenous actin.

Protective effect of resveratrol against oxygen–glucose deprivation in organotypic hippocampal slice cultures: Involvement of PI3-K pathway

Here we investigated the neuroprotective effect of resveratrol in an in vitro model of ischemia. We used organotypic hippocampal cultures exposed to oxygen-glucose deprivation (OGD). In OGD-vehicle exposed cultures, about 46% of the hippocampus was labeled with PI, indicating a robust percentage of cell death. When cultures were treated with resveratrol 10, 25 and 50 microM, the cell death was reduced to 22, 20 and 13% respectively. To elucidate a possible mechanism by which resveratrol exerts its neuroprotective effect, we investigated the phosphoinositide3-kinase (PI3-k) pathway using LY294002 (5 microM) and mitogen-activated protein kinase (MAPK) using PD98059 (20 microM). The resveratrol (50 microM) neuroprotection was prevented by LY294002 but was not by PD98059. Immunoblotting revealed that resveratrol 50 microM induced the phosphorylation/activation of Akt and extracellular signal-regulated kinase-1 and -2 (ERK1/2) and the phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK-3beta). Our results suggest that PI3-k/Akt pathway are involved in the neuroprotective effect of resveratrol.

Long-term soluble Aβ 1–40 activates CaM kinase II in organotypic hippocampal cultures

Recent findings suggested a role for soluble amyloid-β (Aβ) peptides in Alzheimer's disease associated cognitive decline. We investigated the action of soluble, monomeric Aβ 1–40 on CaM kinase II, a kinase involved in neuroplasticity and cognition. We treated organotypic hippocampal cultures short-term (up to 4 h) and long-term (5 days) with Aβ 1–40 (1 nM–5 μM). Aβ did not induce cell damage, apoptosis or synaptic loss. Short-term treatment down-regulated enzymatic activity of the kinase, by reducing its Thr 286 phosphorylation. In contrast, long-term treatment (1 nM–5 μM) markedly and significantly up-regulated enzymatic activity, with peak stimulation at 10 nM (three-fold). Up-regulation of activity was associated with increased expression of the α-isoform of CaM kinase II, increased phosphorylation at Thr 286 (activator residue) and decreased phosphorylation at Thr 305–306 (inhibitory residues). We investigated the effect of glutamate on CaM kinase II following exposure to 1 or 10 nM Aβ 1–40. As previously reported, glutamate increased CaM kinase II activity. However, the glutamate effect was not altered by pretreatment of slices with Aβ. Short- and long-term Aβ treatment showed opposite effects on CaM kinase II, suggesting that long-term changes are an adaptation to the kinase early down-regulation. The marked effect of Aβ 1–40 on the kinase suggests that semi-physiological and slowly raising peptide concentrations may have a significant impact on synaptic plasticity in the absence of synaptic loss or neuronal cell death.