Selective excitotoxic pathology in the rat hippocampus

The pattern of cell loss and neuronal degeneration resulting from multiple microinjections of N -methyl-D-aspartate (NMDA), ibotenate (IBO), quisqualate (QUIS), and kainate (KA) into hippocampus was studied, together with the protection provided by the NMDA antagonist 3-(±)-2-carboxypiperazin-4-yl-propyl-l-phosphonate (CPP). Histological evaluation was carried out after 7 days of survival. NMDA and IBO resulted in an extensive loss of all cells in the hippocampus including dentate gyrus, hilar cells, and CA3-CA1 pyramidal cells, but there was an absence of damage to areas and structures outside hippocampus. After QUIS and KA injections the hippocampal damage was limited to hilar cells in the dentate gyrus, CA3 pyramidal cells, and partial loss of CA1 cells; there was extensive extrahippocampal damage including entorhinal cortex, amygdala, layers III, V, and VI of ventral neocortex, olfactory areas, and various thalamic nuclei. CPP provided almost complete protection from the effects of intrahippocampal injections of NMDA and IBO, but did not affect the hippocampal cell loss found after QUIS and KA (with the exception of minor protection of some CA1 cells). CPP protected most extrahippocampal sites from the damage resulting from QUIS and KA, indicating that such excitotoxic cell death is indirect and involves NMDA receptor activation by an endogenous agent. The use of multiple microinjections as opposed to single injections allows a clearer interpretation of selective excitotoxic vulnerability.     

Neuronal activity-dependent increase of net matrix metalloproteinase activity is associated with MMP-9 neurotoxicity after kainate

Matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs) are emerging as important modulators of brain physiopathology. Dramatic changes in the expression of MMPs and TIMPs occur during excitotoxic/neuroinflammatory processes. However, only the measurement of net protease activity is relevant physiologically, and the functional consequences of MMP/TIMP ratio modifications in the brain remain elusive. In order to assess MMP activity and effects in brain tissue, we combined in vivo and organotypic culture models of kainate (KA)-induced excitotoxicity to provoke selective neuronal death and neuroinflammation in the hippocampus. Using in situ zymography, we show that KA-induced excitotoxic seizures in rats increase net MMP activity in hippocampal neurons 8 h after seizures, before their death, and that this increase is neuronal activity-dependent. Three days after KA, proteolytic activity increases in blood vessels and reactive glial cells of vulnerable areas, in relation with neuroinflammation. At 7 and 15 days, proteolysis remains high in blood vessels whereas it is reduced in glia. In organotypic hippocampal cultures, which lack blood cell-mediated inflammation and extrinsic connections, a broad-spectrum inhibitor of MMPs (MMPI), but also a selective MMP-9 inhibitor, protect hippocampal neurons against KA-induced excitotoxicity. Moreover, recombinant MMP-9, but not MMP-2, induces selective pyramidal cell death in these cultures and KA-induced neuronal activity exacerbates the neuronal death promoting effects of MMP-9. These data strongly implicate MMPs, and MMP-9 in particular, in both excitotoxic neuronal damage and subsequent neuroinflammatory processes, and suggest that selective MMPIs could be therapeutically relevant in related neurological disorders.     

Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC(50) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 microM). In low GluR5 selective concentrations (0.3-3 microM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration-response curves with calculation of EC(50) values for glutamate receptor agonists are possible, as well as comparison of inhibition data for glutamate receptor antagonists. The observation that the slice cultures respond with more in vivo-like patterns of excitotoxicity than primary neuronal cultures, suggests that slice cultures are the best model of choice for a number of glutamate agonist and antagonist studies.     

Markers for neuronal degeneration in organotypic slice cultures

This protocol describes ways of monitoring spontaneous or induced neuronal degeneration in organotypic brain slice cultures. Hippocampal cultures (4-week-old) are grown in normal serum-free control medium, or exposed to the neurotoxin trimethyltin (TMT) (0.5–100 μM) for 24 h or the excitotoxic glutamate agonist kainic acid (KA) (5–25 μM) for 48 h followed by 24 h or 48 h, respectively, in normal medium. Corticostriatal slice cultures (also 4-week-old) are exposed to KA (6–24 μM) for 48 h and normal medium for control. The resulting neurodegeneration is estimated by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux to the culture medium, (c) ordinary Nissl cell staining, (d) staining by the neurodegenerative marker Fluoro-Jade (FJ), (e) neuronal microtubule degeneration by immunohistochemical staining for microtubule-associated protein 2 (MAP2), and (f) Timm sulphide silver staining for heavy metal alterations. Both hippocampal and corticostriatal slice cultures show a dose- and time-dependent increase in PI uptake and LDH efflux after exposure to TMT and KA. The mean PI uptake and the LDH efflux into the medium correlate well for both types of cultures. Both TMT and KA exposed hippocampal cultures display in vivo patterns of differential neuronal vulnerability as evidenced by PI uptake, FJ staining and MAP2 immunostaining. Corticostriatal slice cultures exposed to a high dose of KA display extensive striatal and cortical degeneration in FJ staining as suggested by a high PI uptake. A change in Timm sulphide silver staining in deep central parts of some control cultures, corresponds to areas with loss of cells in cell staining, loss of MAP2 staining, PI uptake, and FJ staining. We conclude that organotypic brain slice cultures, in combination with appropriate markers in standardized protocols, represent feasible means for studies of excitotoxic and neurotoxic compounds.Themes: Disorders of the nervous systemTopics: Neurotoxicity     

Neuroprotection against NMDA excitotoxicity by group I metabotropic glutamate receptors is associated with reduction of NMDA stimulated currents

The neurotransmitter glutamate can have both excitotoxic and protective effects on neurons. The excitotoxic effects have been intensively studied, whereas the protective effects, including the involvement of metabotropic glutamate receptors (mGluRs), remain unclear. In the present study, we tested the protective effects of the group-I-mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) on organotypic hippocampal slice cultures exposed to excitotoxic concentrations of N-methyl-D-aspartate (NMDA). Effects of DHPG on electrophysiological responses induced by NMDA receptor activation were also recorded. Experiments were performed on organotypic hippocampal slice cultures derived from 7-day-old rats, with cellular uptake of propidium iodide as a marker for neuronal cell death. Slice cultures pretreated with DHPG (10 or 100 microM) for 2 h prior to exposure to 50 microM NMDA for 30 min displayed reduced propidium iodide uptake, compared to cultures exposed to NMDA only. The neuroprotective effect was confirmed by Hoechst 33342 staining, where the appearance of pycnotic nuclei after NMDA treatment was prevented by the DHPG pretreatment. Using caspase-3 activity to monitor the presence of apoptosis, failed to demonstrate this type of cell death in CA1 after NMDA application. The protective effect of DHPG was abolished by the mGluR1 selective antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385; 5 or 10 microM), whereas the mGluR5-selective antagonist 2-methyl-6-phenylethynylpyridine (MPEP; 1 microM) had no effect. Voltage-clamping of CA1 pyramidal cells in cultures treated with 10 microM DHPG for 2 h showed a significant depression of NMDA-induced inward currents compared to untreated controls. We conclude that neuroprotection induced by activation of group-I-mGluRs involve mGluR1 and is associated with decreased NMDA-stimulated currents.     

3-Nitropropionic acid toxicity in hippocampus: protection through N-methyl-D-aspartate receptor antagonism

The over-activation of glutamate receptors can lead to excitotoxic cell death and is believed to be involved in the progression of neurodegenerative events in the vulnerable hippocampus. Here, we used an in vitro slice model to study toxicity produced in the hippocampus by the mitochondrial toxin 3-nitropropionic acid (3-NP). The organotypic slice cultures exhibit native cellular organization as well as dense arborization of neuronal processes and synaptic contacts. The hippocampal slices were exposed to 3-NP for 2-20 days, causing calpain-mediated breakdown of the spectrin cytoskeleton, a loss of pre- and postsynaptic markers, and neuronal atrophy. The N-methyl-D-aspartate (NMDA) receptor antagonist memantine reduced both the cytoskeletal damage and synaptic decline in a dose-dependent manner. 3-NP-induced cytotoxicity, as determined by the release of lactate dehydrogenase, was also reduced by memantine with EC50 values from 1.7 to 2.3 microM. Propidium iodide fluorescence and phase contrast microscopy confirmed memantine neuroprotection against the chronic toxin exposure. In addition, the protected tissue exhibited normal neuronal morphology in the major hippocampal subfields. These results indicate that antagonists of NMDA-type glutamate receptors are protective during the toxic outcome associated with mitochondrial dysfunction. They also provide further evidence of memantine's therapeutic potential against neurodegenerative diseases.     

Potentiation of kainic acid epileptogenicity and sparing from neuronal damage by an NMDA receptor antagonist

The time course and severity of the excitotoxic syndrome induced in rats by s.c. injection of 10 mg/kg kainic acid (KA) was modified by pretreatment with MK801, a non-competitive inhibitor of the NMDA receptor, at doses of 0.1, 1 and 10 mg/kg. A dose-dependent increase in the severity of the KA-induced electrographic (EEG) manifestations of epilepsy was seen after MK801. This consisted of an earlier appearance and higher number of EEG seizures, longer time spent in seizures, and an earlier onset of status epilepticus. In contrast, behavioral seizures were increased only in the 0.1 mg/kg MK801 group, but abolished by higher doses. On the contrary, wet dog shakes were progressively reduced with increasing doses of MK801. Four of the 9 animals receiving KA-only group and 3 of the 10 animals in the 1 and 10 mg MK801 groups were sacrificed 5 days after KA. The brain of the KA-only rats presented diffuse gross and microscopic evidence of hemorrhagic necrosis and neuronal damage; the MK801 rats showed only minimal neuronal loss in the CA3 hippocampal sector. This study demonstrates that neuronal damage and epileptiform activity can be dissociated. Furthermore, it confirms the protective effect of MK801 against neuronal damage caused by multiple factors. Lastly, it emphasizes the need for EEG monitoring in order to accurately assess any epileptic/antiepileptic effect.     

The bisphosphonate clodronate depletes microglial cells in excitotoxically injured organotypic hippocampal slice cultures

The bisphosphonate clodronate, clinically used in the treatment of osteoporosis, is known to deplete cells of the monocytic lineage. Using an in vitro model of excitotoxic damage in organotypic hippocampal slice cultures (OHSC), we investigated whether clodronate can also prevent microglial activation that occurs in CNS pathologies. Lesioning of OHSC was performed by application of 50 microM N-methyl-D-aspartate (NMDA) for 4 h after 6 days in vitro (div). Treatment of lesioned OHSC with clodronate (1000, 100, or 10 microg/ml) resulted in an almost complete abrogation of the microglial reaction after 3 further div: Confocal laser scanning microscopy showed that the number of Griffonia simplicifolia isolectin B(4)-labeled (IB4+) microglial cells in the dentate gyrus (DG) was reduced to 4.25% compared with OHSC treated with NMDA alone. Continuous treatment with clodronate (100 or 10 microg/ml) of lesioned OHSC for 9 days resulted in a further reduction in the number of microglial cells (reduction to 2.72%). The number of degenerating, propidium iodide-labeled (PI(+)) neurons in lesioned OHSC that received clodronate treatment between 6 and 9 div was not significantly different from OHSC treated with NMDA alone. However, the number of PI(+) neurons in lesioned OHSC that received continuous clodronate treatment for 9 div was significantly higher when compared to NMDA-lesioned OHSC. In summary, clodronate is able to reduce microglial activation induced by excitotoxic neuronal injury. Our results demonstrate that clodronate is a useful tool in the investigation of neuron-glia interactions because it induces an efficient depletion of microglial cells that are activated after excitotoxic CNS injury.     

Interleukin-1beta exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures

The effects of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1ra) on neurons and microglial cells were investigated in organotypic hippocampal slice cultures (OHSCs). OHSCs obtained from rats were excitotoxically lesioned after 6 days in vitro by application of N-methyl-D-aspartate (NMDA) and treated with IL-1beta (6 ng/mL) or IL-1ra (40, 100 or 500 ng/mL) for up to 10 days. OHSCs were then analysed by bright field microscopy after hematoxylin staining and confocal laser scanning microscopy after labeling of damaged neurons with propidium iodide (PI) and fluorescent staining of microglial cells. The specificity of PI labeling of damaged neurons was validated by triple staining with neuronal and glial markers and it was observed that PI accumulated in damaged neurons only but not in microglial cells or astrocytes. Treatment of unlesioned OHSCs with IL-1beta did not induce neuronal damage but caused an increase in the number of microglial cells. NMDA lesioning alone resulted in a massive increase in the number of microglial cells and degenerating neurons. Treatment of NMDA-lesioned OHSCs with IL-1beta exacerbated neuronal cell death and further enhanced microglial cell numbers. Treatment of NMDA-lesioned cultures with IL-1ra significantly attenuated NMDA-induced neuronal damage and reduced the number of microglial cells, whereas application of IL-1ra in unlesioned OHSCs did not induce significant changes in either cell population. Our findings indicate that: (i) IL-1beta directly affects the central nervous system and acts independently of infiltrating hematogenous cells; (ii) IL-1beta induces microglial activation but is not neurotoxic per se; (iii) IL-1beta enhances excitotoxic neuronal damage and microglial activation and (iv) IL-1ra, even when applied for only 4 h, reduces neuronal cell death and the number of microglial cells after excitotoxic damage.     

The calpain inhibitor MDL-28170 and the AMPA/KA receptor antagonist CNQX inhibit neurofilament degradation and enhance neuronal survival in kainic acid-treated hippocampal slice cultures

The cytoskeleton controls the architecture and survival of the central nervous system neurons by maintaining the stability of axons, dendrites and cellular architecture, and any disturbance in this genuine structure could compromise cell survival. The developmentally regulated intracellular intermediate filament protein neurofilament (NF), composed of the light (NF-L), medium (NF-M) and high (NF-H) molecular weight isoforms, is expressed abundantly in nerve cells but its significance in nerve cell survival in stress situations in the brain is unknown. We have used Western blotting, immunocytochemistry, and Fluoro-Jade B and thionine stainings to clarify the effect of kainic acid (KA) treatment on NF protein stability, and its importance for neuronal survival in hippocampal slice cultures. The contribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA glutamate receptor subtypes, calpain proteases and L-type Ca2+-channels to these processes were also assessed. Our results indicated that KA-induced degradation of NF was a fast process, similarly affecting all three NF proteins. It was effectively inhibited by the AMPA/KA receptor antagonist CNQX and the calpain inhibitor MDL-28170, whereas the Ca2+-channel blocker nifedipine and the NMDA receptor antagonist MK-801 had no significant effect. Moreover, KA-induced neuronal damage was effectively decreased in cultures treated with CNQX and MDL-28170. Our results suggest that the stability of NF proteins is an important factor contributing to neuronal survival after excitotoxic injury, and that both AMPA/KA receptor antagonists and calpain inhibitors might serve as neuroprotectants against this type of insult in the immature hippocampus.     

Stimulation of N-methyl-d-aspartate receptors induces apoptosis in rat brain

To evaluate the contribution of apoptotic mechanisms to excitotoxin-induced neurodegeneration as well as to characterize the glutamate receptor subtypes involved, biochemical and morphological effects of intrastriatally administered NMDA receptor agonist N-methyl-d-aspartate (NMDA) or quinolinic acid (QA) were studied. Receptor autoradiography showed that NMDA (75–300 nmol) caused a loss of 18–68% of striatal D₁ dopamine (DA) and 10–43% of NMDA receptors 7 days after drug administration. Treatment with QA (60–240 nmol) also led to a loss of 60–73% of D₁ DA and 37–44% of NMDA receptors in the ipsilateral striatum. Agarose gel electrophoresis revealed that both NMDA and QA induced internucleosomal DNA fragmentation in the striatum 12 to 48 h after drug administration. NMDA- and QA-induced internucleosomal DNA fragmentation was attenuated by the protein synthesis inhibitor cycloheximide in a dose-dependent manner. Terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-digoxigenin nick end labeling (TUNEL)-positive nuclei were found in the ipsilateral striatum in response to NMDA or QA treatment. In addition, many fragmented nuclei were observed in the NMDA or QA-treated striatum and propidium iodide staining showed profound nuclear condensation in the NMDA or QA-treated striatum. NMDA- and QA-induced internucleosomal DNA fragmentation and TUNEL-positive nuclei as well as nuclear condensation were abolished by the NMDA receptor antagonist MK-801, but not by the AMPA/KA receptor antagonist NBQX. MK-801, but not NBQX, also prevented NMDA or QA-induced striatal cell death. These results suggest that apoptotic mechanisms are involved in excitotoxin-induced striatal cell death. The initiation of an apoptotic cascade by NMDA or QA appears to be mediated by stimulation of NMDA but not AMPA/KA receptors.     

IL-18 deficiency aggravates kainic acid-induced hippocampal neurodegeneration in C57BL/6 mice due to an overcompensation by IL-12

The role of interleukin-18 (IL-18) in excitotoxic neurodegeneration is largely unknown. To address this issue, we used kainic acid (KA)-induced hippocampal neurodegeneration in IL-18 knockout (KO) mice. One day after KA administration, clinical symptoms and histopathological changes did not differ between IL-18 KO mice and wild-type mice. However, 7 days after KA application the hippocampal neurodegeneration was markedly severe in IL-18 KO mice as demonstrated by increased locomotion and prominent histopathological changes including neuronal cell loss, microglia activation and astrogliosis. Surprisingly, when wild-type mice received recombinant mouse IL-18 (rmIL-18) in advance, after KA treatment both the clinical and pathological signs were dose-dependently aggravated compared to mice without rmIL-18 pre-treatment. To clarify the mechanism behind this, we assessed the expression of the IL-18 associated cytokines IL-12, IL-1beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) in the hippocampus by immunohistochemistry and flow cytometry. IL-12 and IFN-gamma expression was strongly increased in IL-18 KO mice when compared to wild-type mice 7 days after KA treatment in agreement with increased microglia activation. These results suggest that the role of IL-18 in excitotoxic injury in IL-18 deficient mice may be overcompensated by increased IL-12 secretion.     

Involvement of cyclin-dependent kinase-5 in the kainic acid-mediated degeneration of glutamatergic synapses in the rat hippocampus

Increased levels of glutamate causing excitotoxic damage accompany neurological disorders such as ischemia/stroke, epilepsy and some neurodegenerative diseases. Cyclin‐dependent kinase‐5 (Cdk5) is important for synaptic plasticity and is deregulated in neurodegenerative diseases. However, the mechanisms by which kainic acid (KA)‐induced excitotoxic damage involves Cdk5 in neuronal injury are not fully understood. In this work, we have thus studied involvement of Cdk5 in the KA‐mediated degeneration of glutamatergic synapses in the rat hippocampus. KA induced degeneration of mossy fiber synapses and decreased glutamate receptor (GluR)6/7 and post‐synaptic density protein 95 (PSD95) levels in rat hippocampus in vivo after intraventricular injection of KA. KA also increased the cleavage of Cdk5 regulatory protein p35, and Cdk5 phosphorylation in the hippocampus at 12 h after treatment. Studies with hippocampal neurons in vitro showed a rapid decline in GluR6/7 and PSD95 levels after KA treatment with the breakdown of p35 protein and phosphorylation of Cdk5. These changes depended on an increase in calcium as shown by the chelators 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N ′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM) and glycol‐bis (2‐aminoethylether)‐N,N,N ′,N ′‐tetra‐acetic acid. Inhibition of Cdk5 using roscovitine or employing dominant‐negative Cdk5 and Cdk5 silencing RNA constructs counteracted the decreases in GluR6/7 and PSD95 levels induced by KA in hippocampal neurons. The dominant‐negative Cdk5 was also able to decrease neuronal degeneration induced by KA in cultured neurons. The results show that Cdk5 is essentially involved in the KA‐mediated alterations in synaptic proteins and in cell degeneration in hippocampal neurons after an excitotoxic injury. Inhibition of pathways activated by Cdk5 may be beneficial for treatment of synaptic degeneration and excitotoxicity observed in various brain diseases.     

Glutamate-Mediated Excitotoxic Death of Cultured Striatal Neurons Is Mediated by Non-NMDA Receptors

Considerable interest has focused on the role of glutamate-mediated excitotoxicity in neurodegenerative disorders of the basal ganglia. The in vitro data on the receptor mechanisms involved in this process, however, have been inconclusive. Some studies have indicated that excitotoxins acting at NMDA receptors kill striatal neurons and others have indicated that NMDA receptor-mediated excitotoxic death of striatal neurons is minimal in the absence of cortex. In the present study, we used a pharmacological approach to carefully reexamine this issue in 2-week-old cultures of striatal neurons dissociated from E17 rat embryos. The sensitivity of these neurons to glutamate agonists and antagonists was determined by monitoring cell loss in identified regions of the growth dishes. We found that glutamate killed striatal neurons with an EC₅₀ of 100 μM. This loss was not mediated by NMDA receptors, since it was not reduced by the NMDA receptor antagonist APV (0.1-1.0 mM). Consistent with this result, up to 50 mM NMDA receptor-specific excitotoxin quinolinic acid (QA) did not affect neuronal survival. Depolarizing the QA-exposed neurons with 35 mM potassium chloride to enhance NMDA receptor activation by QA also did not produce neuron loss. The metabotropic glutamate receptor antagonist AP3 (500 μM) also had no effect on the striatal neuron loss produced by 100 μM glutamate. In contrast, the non-NMDA antagonist GYKI 52466 (100 μM) did block the excitotoxic effect of glutamate (100 μM). Specific AMPA and KA receptor agonists and the non-NMDA antagonist GYKI 52466 revealed that the non-NMDA receptor-mediated excitotoxic effect of glutamate was mediated by KA receptors. These results suggest that cultured striatal neurons are directly vulnerable to non-NMDA glutamate agonists, but not to NMDA and metabotropic glutamate agonists. Thus, non-NMDA receptors may play a greater role in the excitotoxic death of striatal neurons in disease and experimental animal models than previously realized.     

Kainate excitotoxicity in organotypic hippocampal slice cultures: evidence for multiple apoptotic pathways

The mechanisms underlying kainate (KA) neurotoxicity are still not well understood. We previously reported that KA-mediated neuronal damage in organotypic cultures of hippocampal slices was associated with p53 induction. Recently, both bax and caspase-3 have been demonstrated to be key components of the p53-dependent neuronal death pathway. Caspase activation has also been causally related to the release of mitochondrial cytochrome c (Cyto C) in the cytoplasm as a result of the collapse of the mitochondrial membrane potential (Deltapsi(M)) and the opening of mitochondrial permeability transition pores (mPTP). In the present study, we observed a rapid induction of bax in hippocampal slice cultures after KA treatment. In addition, the levels of Cyto C and caspase-3 were increased in the cytosol while the level of the caspase-9 precursor was decreased. There was also a complete reduction of Rhodamine 123 fluorescence after KA treatment, an indication of Deltapsi(M) dissipation. Furthermore, inhibition of mPTP opening by cyclosporin A partially prevented Cyto C release, caspase activation and neuronal death. These data suggest the involvement of bax, several caspases, as well as Cyto C release in KA-elicited neuronal death. Finally, inhibition of caspase-3 activity by z-VAD-fmk only partially protected neurons from KA toxicity, implying that multiple mechanisms may be involved in KA excitotoxicity.     

Mechanistic distinctions between excitotoxic and acidotic hippocampal damage in an in vitro model of ischemia

Excitotoxicity is believed to underlie the selective loss of vulnerable neurons after transient ischemia, while lactic acidosis seems to be the principal feature and probable cause of tissue infarcts. Primary hippocampal cultures containing both neurons and astrocytes derived from fetal rats were used to examine the relative contributions of and interactions between excitotoxic and acidotic cell injury. Hypoxia-induced damage was energy dependent and involved the N-methyl-D-aspartate (NMDA) receptor. Glucose above 1 mM could completely protect against hypoxia-induced injury in a pH range of 7.4-6.5, while the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (500 microM) during the posthypoxic period provided only partial protection in the absence of glucose. Astrocyte cultures were undamaged by ischemic-like treatment in this pH range, suggesting that hypoxia-induced cell death in mixed cultures was restricted to neurons. Lowering the extracellular pH to 7.0 and 6.5 caused no neuronal damage in normoxic controls, but in each case provided significant protection against hypoxic neuronal injury. In contrast, a second type of neurotoxicity was observed after a 6-h exposure to pH 6.0, while exposure to pH 5.5 was required to kill astrocytes. This acidotic damage appeared to be energy independent and did not involve the NMDA receptor. These results suggest that excitotoxic neuron death has an energetic component and that acidosis may produce both protective and damaging effects in the hippocampus during ischemic insults.     

Activated microglial cells migrate towards sites of excitotoxic neuronal injury inside organotypic hippocampal slice cultures

The aim of this study was to analyse microglial reactions to excitotoxic N ‐methyl‐ d ‐aspartic acid (NMDA)‐induced degeneration of rat dentate and hippocampal neurons in vitro . We used a migration model combining the techniques of microglial single cell culture and organotypic hippocampal slice culture (OHSC). Site‐specific oxidative damage in OHSCs was induced by pretreatment with 50 μ m NMDA. Neuronal injury determined by propidium iodide (PI) uptake included the hippocampal cell layers of the dentate gyrus (DG) and the cornu ammonis (CA). Fluorescence‐prelabelled microglial cells with ameboid morphology were transferred onto the OHSC and migrated predominantly to the prelesioned cell layers of DG and CA when compared with unlesioned areas of the OHSC. In NMDA pretreated slices, microglial cells clustered around degenerating granule cells in the DG and pyramidal cells in the CA. This effect was significantly inhibited in unlesioned slice cultures and in NMDA‐exposed cultures that were pretreated with the NMDA‐antagonist MK‐801. Our observations suggest that microglia – attracted by the presence of stimuli provided by NMDA‐induced neuronal death – migrate specifically towards these lesioned neurons.     

Mitochondrial complex I inhibition produces selective damage to hippocampal subfield CA1 in organotypic slice cultures

The effects of mitochondrial respiratory chain inhibitors and the excitotoxinN-methyl-D-aspartate (NMDA) on cell death in hippocampal subfields CA1 and CA3 were examined in hippocampal organotypic slice cultures. Slice cultures, 2–3 week old, were exposed for 1 h to either the Complex 1 inhibitors, rotenone or 1-methyl-4-phenylpyridium (MPP⁺), the Complex II inhibitor 3-nitropropionic acid (3-NP), or the excitotoxin NMDA. Cell death was examined 24 and 48 h following treatment, by measuring propidium iodide (PI) fluorescence. Treatment with 1 μM rotenone caused greater cell death in hippocampal subfield CA1 than CA3. Exposure of hippocampal slice cultures to 10 μM rotenone, to MPP⁺ or to NMDA resulted in damage to both CA1 and CA3 subfields. 3-NP produced little damage in either subfield. The data suggest that mitochondrial complex I inhibition can produce selective cell damage in hippocampus and in this regard is similar to that observed following hypoxia/ischemia.