Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen-glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen-glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen-glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen-glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen-glucose deprivation induced necrotic cell death only. Addition of 10 microM of the N-methyl-D-aspartate glutamate receptor antagonist MK-801, and 20 microM of the non-N-methyl-D-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline to the culture medium confirmed that both N-methyl-D-aspartate and non-N-methyl-D-aspartate ionotropic glutamate receptors were involved in the oxygen-glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the DL-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of DL-threo-beta-benzyloxyaspartate in concentrations of 25 microM or more to otherwise untreated hippocampal slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen-glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10 microM) dose of DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation (but not during the next 48 h after oxygen-glucose deprivation) significantly reduced the oxygen-glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen-glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen-glucose deprivation-induced necrotic cell death in hippocampal slice cultures, which have proven to be a feasible tool in experimental studies on this topic.     

D2 autoreceptor inhibition reveals oxygen-glucose deprivation-induced release of dopamine in guinea-pig cochlea

Dopamine (DA), released from the lateral olivocochlear (LOC) efferent terminals, the efferent arm of the short-loop feedback in the cochlea, is considered as a protective factor in the inner ear since it inhibits auditory nerve dendrite firing in ischemia- or noise-induced excitotoxicity leading to sensorineural hearing loss (SNHL). In the present study we investigated the effect of oxygen-glucose deprivation (OGD), an in vitro ischemia model, on guinea-pig cochlear [(3)H]DA release in a microvolume superfusion system. We found that OGD alone failed to induce a detectable elevation of [(3)H]DA level, but in the presence of specific D(2) receptor antagonists, sulpiride and L-741,626, it evoked a significant increase in the extracellular concentration of [(3)H]DA. D(2) negative feedback receptors are involved not exclusively in the regulation of synthesis and vesicular release of DA, but also in the activation of its reuptake. Thus, D(2) receptor antagonism interferes with the powerful reuptake of DA from the extracellular space. To explore the underlying mechanism of this DA-releasing effect we applied nomifensine and found that the effect of OGD on cochlear DA release in the presence of D(2) antagonists could be inhibited by this selective DA uptake inhibitor. This finding indicates that the OGD-evoked DA release was mainly mediated through the reverse operation of the DA transporter. The two structurally different D(2) antagonists also augmented the electrical field stimulation-evoked release of DA proving the presence of D(2) autoreceptors on dopaminergic LOC terminals. Our results confirm the presence and role of D(2) DA autoreceptors in the regulation of DA release from LOC efferents, and suggest a protective local mechanism during ischemia which involves the direct transporter-mediated release of DA. Increasing the release of the protective transmitter DA locally in the inner ear may form the basis of future new therapeutic strategies in patients suffering from SNHL.     

Changes in secondary glutamate release underlie the developmental regulation of excitotoxic neuronal cell death

Vulnerability to excitotoxicity increases during development in vivo and in vitro. To determine whether the mere presence of mature N-methyl-D-aspartate (NMDA) receptors coincides with the emergence of excitotoxicity or whether post-receptor signaling processes may also contribute, we examined the temporal relationship of NMDA receptor expression, function and toxicity using cortical cell cultures. Surface expression of all NMDA receptor subunits increased with time in culture. This correlated with NMDA receptor function, assessed both biochemically and electrophysiologically, but not with the appearance of excitotoxicity. Specifically, cells at day in vitro (DIV) 10 were less susceptible to NMDA receptor-induced neurotoxicity than those cultured for 14 days, even though receptor expression/function was identical. In addition, cell-attached single channel recordings revealed that NMDA receptor conductance, open probability, and frequency of channel openings were not significantly different between the two days. Intriguingly, depolarization-induced release of glutamate from cultures grown for 10 days was significantly lower than that released from cultures grown for 14 days. Further, exogenous addition of glutamate receptor agonists immediately after removal of NMDA rendered cultures at DIV 10 susceptible to excitotoxicity, while toxicity was significantly reduced by addition of an NMDA receptor antagonist immediately after exposure to NMDA at DIV 14. These data are the first to demonstrate that the subsequent, secondary release of glutamate plays an equal, if not more important, role than NMDA receptor development per se, in mediating the enhanced vulnerability of neurons to excitotoxicity that occurs with age.     

Central role of lactic acidosis in cancer cell resistance to glucose deprivation-induced cell death

Solid tumours are dependent on glucose, but are generally glucose-deprived due to poor vascularization. Nevertheless, cancer cells can generally survive glucose deprivation better than their normal counterparts. Thus, to render cancer cells sensitive to glucose depletion may potentially provide an effective strategy for cancer intervention. We propose that lactic acidosis, a tumour microenvironment factor, may allow cancer cells to develop resistance to glucose deprivation-induced death, and that disruption of lactic acidosis may resume cancer cells' sensitivity to glucose depletion. Lactic acidosis, lactosis, or acidosis was generated by adding pure lactic acid, sodium lactate, or HCl to the culture medium. Cell death, cell cycle, autophagy, apoptosis, and gene expression profiling of the surviving cancer cells under glucose deprivation with lactic acidosis were determined. Under glucose deprivation without lactic acidosis, 90% of 4T1 cancer cells died within a single day; in a sharp contrast, under lactic acidosis, 90% of 4T1 cells died in a period of 10 days, with viable cells identified even 65 days after glucose was depleted. Upon glucose restoration, surviving cells resumed proliferation. Lactic acidosis also significantly extended survival of other cancer cells under glucose deprivation. G1/G0 arrest, autophagy induction, and apoptosis inhibition were tightly associated with lactic acidosis-mediated resistance to glucose deprivation. Lactosis alone had no effect on cell survival under glucose deprivation; acidosis alone can prolong cell survival time but is not as potent as lactic acidosis. Thus, the ability of cancer cells to resist glucose deprivation-induced cell death is conferred, at least in part, by lactic acidosis, and we envision that disrupting the lactic acidosis may resume the sensitivity of cancer cells to glucose deprivation.     

Altered glutamate and GABA release within thalamocortical circuitry in metabotropic glutamate receptor 4 knockout mice

The metabotropic glutamate receptor 4 is highly expressed presynaptically on thalamocortical neurons that are involved in the pathogenesis of generalized absence seizures. Mutant mice devoid of metabotropic glutamate receptor 4 are completely resistant to absence seizures induced by low doses of GABA type A receptor antagonists. The purpose of this study was to test the hypothesis that there is altered glutamate and GABA release within thalamocortical circuitry in mice devoid of metabotropic glutamate receptor 4. Extracellular GABA and glutamate release were determined in ventrobasal thalamus, the nucleus reticularis thalami and laminae I-III, and IV-VI of cerebral cortex (laminae I-III of cerebral cortex, and laminae IV-VI of cerebral cortex) using in vivo microdialysis techniques on awake, free moving mice. A significant increase of both basal and K(+)-evoked glutamate release was detected in the ventrobasal thalamus, the nucleus reticularis thalami and laminae IV-VI of cerebral cortex of mice devoid of metabotropic glutamate receptor 4 mice. There also was a significant increase in both basal and K(+)-evoked GABA release in the mice devoid of metabotropic glutamate receptor 4, but a significant decrease of GABA release in laminae IV-VI of cerebral cortex. However, there was no alteration of either GABA or glutamate release in laminae I-III of cerebral cortex, cortical laminae that are not involved in absence seizures. These data indicate that deletion of the metabotropic glutamate receptor 4 gene results in a selective perturbation of glutamate and GABA release within the thalamocortical circuitry involved in the pathogenesis of absence seizures.     

Localization of vesicular glutamate transporters and neuronal nitric oxide synthase in rat nucleus tractus solitarii

Previously we reported that glutamate and neuronal nitric oxide synthase (nNOS) colocalize in neurons of the nucleus tractus solitarii (NTS). That finding provided anatomical support for the suggestion that nitric oxide and glutamate interact in cardiovascular regulation by the NTS. Here we test the hypothesis that nNOS colocalizes with vesicular glutamate transporters (VGluT1 and VGluT2) in the NTS. Immunoreactivity (IR) for VGluT better identifies glutamatergic terminals than does glutamate-IR, which may label metabolic as well as transmitter stores of the amino acid. We used fluorescent immunohistochemistry combined with confocal laser scanning microscopy to study IR for VGluT1, VGluT2 and nNOS in rat NTS. A high density of VGluT1-IR positive fibers was present in the gracilis and cuneatus nuclei while in the NTS we found a moderate density in the lateral and interstitial subnuclei and a low density in the dorsolateral, ventral and intermediate subnuclei. The medial, central, commissural and gelatinosus subnuclei contained few VGluT1-IR containing fibers. Thus, VGluT1 containing fibers are not prominent in portions of the NTS where cardiovascular afferent fibers terminate. In contrast, we found a high density of VGluT2-IR containing fibers in the gelatinosus subnucleus and subpostremal area and a moderate density in cardiovascular regions such as the dorsolateral and medial subnuclei as well as in the central and lateral subnuclei. We found a low density in the ventral, intermediate, interstitial and commissural subnuclei. VGluT1-IR and VGluT2-IR rarely colocalized in fibers within the NTS. VGluT1-IR did not colocalize with nNOS, but VGluT2-IR and nNOS-IR colocalized in fibers in all NTS subnuclei. When compared with the other NTS subnuclei, the dorsolateral, gelatinosus and subpostremal subnuclei had higher frequencies of colocalization of VGluT2-IR and nNOS-IR. VGluT2-IR positive fibers were also apposed to nNOS-IR positive fibers throughout the NTS. These data support our hypothesis and confirm that glutamatergic fibers in the NTS contain nNOS.     

Interleukin-1beta activates forebrain glial cells and increases nitric oxide production and cortical glutamate and GABA release in vivo: implications for Alzheimer's disease.

Interleukin-1beta (10 U) was injected into the nucleus basalis of adult male Wistar rats. The inflammation-induced changes in glial cell morphology and expression of inducible nitric oxide synthase in the injected area, the release of acetylcholine, GABA and glutamate from the ipsilateral cortex, the production of nitrite levels in the injected area and ipsilateral cortex, and changes in motor activity were investigated. Saline-injected rats were used as control. Interleukin-1beta induced an activation of both microglia and astrocytes which was already evident 24 h after injection. Seven days after injection, many reactive microglial cells and astrocytes were seen in the injected area and in other brain regions of the same hemisphere. Microglia reaction, but not astrocyte activation, disappeared 30 days post-injection. Seven days after interleukin-1beta injection, many cells immunopositive for inducible nitric oxide synthase were found surrounding the injection site. Inducible nitric oxide synthase-positive cells were identified, by double staining immunohistochemistry, in the reactive microglial cells and, by electron microscope examination, in the perineuronal subpopulation of resident activated microglia. Microdialysis investigations revealed a transient increase in reactive nitrogen intermediates (at seven days post-injection), a delayed (at 30 days post-injection) increase in GABA and glutamate release, and no changes in acetylcholine release in the ipsilateral cortex in interleukin-1beta, but not saline, injected rats. Inhibition of inducible nitric oxide synthase expression by N(G)-nitro-L-arginine methyl ester administration prevented the increase in nitrogen intermediates and GABA release, but not in glutamate release. Our findings suggest that an inflammatory reaction of the basal forebrain facilitates GABA release through the production of nitric oxide.     

Respiratory activity in brainstem of fetal mice lacking glutamate decarboxylase 65/67 and vesicular GABA transporter

The respiratory neural network in the mammalian medulla oblongata shows rhythmic activity before birth. GABA and glycine are considered to be involved in control of respiratory rhythm. Recently we have demonstrated respiratory failure in glutamic acid decarboxylase (GAD) 67-deficient mice [Tsunekawa N, Arata A, Obata K (2005) Development of spontaneous mouth/tongue movement and related neural activity, and their repression in mouse fetus lacking glutamate decarboxylase 67. Eur J Neurosci 21:173-178]. To further evaluate the involvement of GABA and glycine in fetal respiratory function, we studied neural activities in brainstem-spinal cord blocks prepared from GAD65-/-:67-/- and vesicular GABA transporter (VGAT)-/-mice on embryonic day 14 (E14)-E15 and E18. In these knockout mice, the synthesis of GABA and the vesicular release of GABA and glycine are completely absent, respectively. Spontaneous respiratory discharges were observed in the ventral roots at the cervical cord (C) 4 level from wild-type mice but not from the knockout mice on E18. Administration of substance P induced C4 discharges in GAD65-/-:67-/- preparations but not in VGAT-/- preparations. C4 discharges were observed in the knockout mice on E14-E15, although the frequency was lower than that in the wild-type. Neuronal activities in the respiratory network of the E18 brainstem were recorded using a "blind" patch-clamp technique. Expiratory and inspiratory neurons with their characteristic firing patterns were observed in the wild-type fetuses. Strychnine reversed inspiratory-phase hyperpolarization to large depolarization in expiratory neurons. On the other hand, neurons in the same area of the knockout mice fired spontaneously without any rhythm. Substance P induced hyperpolarizing potentials in medullary neurons of GAD65-/-:67-/- mice. Further administration of strychnine induced large depolarizing potentials. Rhythmic activities were not observed in VGAT-/- mice even in the presence of substance P and strychnine. These results indicate that the lack of GABA and glycine impairs the function of the respiratory network in mouse fetuses and the impairment progresses with fetal age.     

Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygen-glucose deprivation in preconditioned cortical cultures.

In vitro ischemic preconditioning induced by subjecting rat cortical cultures to nonlethal oxygen-glucose deprivation protects against a subsequent exposure to otherwise lethal oxygen-glucose deprivation. We provide evidence that attenuation of the postsynaptic N-methyl-D-aspartate (NMDA) receptor- and Ca(2+)-dependent neurotoxicity underlies oxygen-glucose deprivation tolerance. It is demonstrated that extended tolerance to otherwise lethal NMDA or oxygen-glucose deprivation can be induced by either of their sublethal forms of preconditioning. These four pathways are linked, since NMDA receptor blockade during preconditioning by oxygen-glucose deprivation eliminates tolerance. These results suggest that NMDA tolerance, induced by nonlethal activation of these receptors during oxygen-glucose deprivation preconditioning, underlies oxygen-glucose deprivation tolerance. Several neurotoxic downstream Ca(2+)-dependent signaling events specifically linked to NMDA receptor activation are attenuated during otherwise lethal oxygen-glucose deprivation in preconditioned cultures. Specifically, calpain activation, as well as degradation of microtubule-associated protein-2 and postsynaptic density-95, are attenuated 2 h following otherwise lethal NMDA treatment alone or oxygen-glucose deprivation in preconditioned cultures. Formation of microtubule-associated protein-2-labeled dendritic varicosities is also attenuated in preconditioned cultures within 1 h of lethal oxygen-glucose deprivation or NMDA application. Intracellular Ca(2+) levels, measured using the high- or low-affinity dyes Fluo-4 (K(d) approximately equal 345 nM) or Fluo-4FF (K(d) approximately equal 9.7 microM) respectively, are markedly attenuated during lethal oxygen-glucose deprivation in preconditioned cultures.Collectively, the results suggest the attenuation of the postsynaptic NMDA-mediated component of otherwise lethal oxygen-glucose deprivation through the suppression of Ca(2+)-dependent neurotoxic signaling, a mechanism that is initially induced by transient nonlethal activation of this receptor during ischemic preconditioning.     

Nicotinamide reduces acute cortical neuronal death and edema in the traumatically injured brain

Previous studies have shown that administration of nicotinamide (Vitamin B(3)) in animal models of traumatic brain injury (TBI) and ischemia significantly reduced the size of infarction or injury and improved functional recovery. The present study evaluated the ability of nicotinamide to provide acute neuroprotection and edema reduction following TBI. Groups of rats were assigned to nicotinamide (500mg/kg) or saline (1.0ml/kg) treatment conditions and received contusion injuries or sham surgeries. Drug treatment was administered 15min following injury. Brains were harvested 24h later and either processed for histology or water content. Frozen sections were stained with the degenerating neuron stain (Fluoro-Jade B) (FJ) and cell counts were performed at the site of injury. Additional brains were processed for water content (a measure of injury-induced edema). Results of this study showed that administration of nicotinamide following TBI significantly reduced the number of FJ(+) neurons in the injured cortex compared to saline-treated animals. Examination of the water content of the brains also revealed that administration of nicotinamide significantly attenuated the amount of water compared to saline-treated animals in the injured cortex. These results indicate that nicotinamide administration significantly reduced neuronal death and attenuated cerebral edema following injury. The current findings suggest that nicotinamide significantly modulates acute pathophysiological processes following injury and that this may account for its beneficial effects on recovery of function following injury.     

Somatostatin presynaptically inhibits both GABA and glutamate release onto rat basal forebrain cholinergic neurons

A whole cell patch-clamp study was carried out in slices obtained from young rat brain to elucidate the roles of somatostatin in the modulation of synaptic transmission onto cholinergic neurons in the basal forebrain (BF), a region that contains cholinergic and GABAergic corticopetal neurons and somatostatin (SS)-containing local circuit neurons. Cholinergic neurons within the BF were identified by in vivo prelabeling with Cy3 IgG. Because in many cases SS is contained in GABAergic neurons in the CNS, we investigated whether exogenously applied SS can influence GABAergic transmission onto cholinergic neurons. Bath application of somatostatin (1 muM) reduced the amplitude of the evoked GABAergic inhibitory presynaptic currents (IPSCs) in cholinergic neurons. SS also reduced the frequency of miniature IPSCs (mIPSCs) without affecting their amplitude distribution. SS-induced effect on the mIPSC frequency was significantly larger in the solution containing 7.2 mM Ca(2+) than in the standard (2.4 mM Ca(2+)) external solution. Similar effects were observed in the case of non-NMDA glutamatergic excitatory postsynaptic currents (EPSCs). SS inhibited the amplitude of evoked EPSCs and reduced the frequency of miniature EPSCs dependent on the external Ca(2+) concentration with no effect on their amplitude distribution. Pharmacological analyses using SS-receptor subtype-specific drugs suggest that SS-induced action of the IPSCs is mediated mostly by the sst(2) subtype, whereas sst subtypes mediating SS-induced inhibition of EPSCs are mainly sst(1) or sst(4). These findings suggest that SS presynaptically inhibits both GABA and glutamate release onto BF cholinergic neurons in a Ca(2+)-dependent way, and that SS-induced effect on IPSCs and EPSCs are mediated by different sst subtypes.     

Reciprocal modulation of glutamate and GABA release may underlie the anticonvulsant effect of phenytoin

Although conventional wisdom suggests that the effectiveness of phenytoin as an anticonvulsant is due to blockade of Na+-channels this is unlikely to be it's sole mechanism of action. In the present paper we examined the effects of phenytoin on evoked and spontaneous transmission at excitatory (glutamate) and inhibitory (GABA) synapses, in the rat entorhinal cortex in vitro. Evoked excitatory postsynaptic potentials at glutamate synapses exhibited frequency-dependent enhancement, and phenytoin reduced this enhancement without altering responses evoked at low frequency. In whole-cell patch-clamp recordings the frequency of excitatory postsynaptic currents resulting from the spontaneous release of glutamate was reduced by phenytoin, with no change in amplitude, rise time or decay time. Similar effects were seen on miniature excitatory postsynaptic currents, recorded in the presence of tetrodotoxin. Evoked inhibitory postsynaptic potentials at GABA synapses displayed a frequency-dependent decrease in amplitude. Phenytoin caused a reduction in this decrement without affecting the responses evoked at low frequency. The frequency of spontaneous GABA-mediated inhibitory postsynaptic currents, recorded in whole-cell patch mode, was increased by phenytoin, and this was accompanied by the appearance of much larger amplitude events. The effect of phenytoin on the frequency of inhibitory postsynaptic currents persisted in the presence of tetrodotoxin, but the change in amplitude distribution largely disappeared. These results demonstrate for the first time that phenytoin can cause a simultaneous reduction in synaptic excitation and an increase in inhibition in cortical networks. The shift in balance in favour of inhibition could be a major factor in the anticonvulsant action of phenytoin.     

Reciprocal modulation of glutamate and GABA release may underlie the anticonvulsant effect of phenytoin

Although conventional wisdom suggests that the effectiveness of phenytoin as an anticonvulsant is due to blockade of Na⁺-channels this is unlikely to be it's sole mechanism of action. In the present paper we examined the effects of phenytoin on evoked and spontaneous transmission at excitatory (glutamate) and inhibitory (GABA) synapses, in the rat entorhinal cortex in vitro. Evoked excitatory postsynaptic potentials at glutamate synapses exhibited frequency-dependent enhancement, and phenytoin reduced this enhancement without altering responses evoked at low frequency. In whole-cell patch-clamp recordings the frequency of excitatory postsynaptic currents resulting from the spontaneous release of glutamate was reduced by phenytoin, with no change in amplitude, rise time or decay time. Similar effects were seen on miniature excitatory postsynaptic currents, recorded in the presence of tetrodotoxin. Evoked inhibitory postsynaptic potentials at GABA synapses displayed a frequency-dependent decrease in amplitude. Phenytoin caused a reduction in this decrement without affecting the responses evoked at low frequency. The frequency of spontaneous GABA-mediated inhibitory postsynaptic currents, recorded in whole-cell patch mode, was increased by phenytoin, and this was accompanied by the appearance of much larger amplitude events. The effect of phenytoin on the frequency of inhibitory postsynaptic currents persisted in the presence of tetrodotoxin, but the change in amplitude distribution largely disappeared.These results demonstrate for the first time that phenytoin can cause a simultaneous reduction in synaptic excitation and an increase in inhibition in cortical networks. The shift in balance in favour of inhibition could be a major factor in the anticonvulsant action of phenytoin.     

Zinc-induced cortical neuronal death with features of apoptosis and necrosis: mediation by free radicals

Some studies have provided evidence that delayed death of hippocampal CA1 neurons in transient global ischemia occurs by classical apoptosis. Recently, translocation of synaptic zinc has been shown to play a key role in ischemic CA1 neuronal death. With these two lines of evidence, we examined in mouse cortical cultures the possibility that zinc neurotoxicity, slowly triggered over a day, may occur by classical apoptosis. Exposure of cortical cultures to 30-35 microM zinc for 24 h resulted in slowly evolving death of neurons only, while exposure to zinc at higher concentrations ( > or = 40 microM) produced near-complete death of both neurons and glia. DNA agarose gel electrophoresis revealed internucleosomal DNA fragmentation, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method revealed DNA breaks in degenerating neurons after 24 h exposure to 30-35 microM zinc, suggesting that the death may occur by apoptosis. However, electron-microscopic examinations revealed ultrastructural changes clearly indicative of necrosis, such as marked swelling of intracellular organelles and disruption of cell membranes amid relatively intact nuclear membranes. Furthermore, the slowly triggered zinc neurotoxicity was not attenuated by cycloheximide, neurotrophins (brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5) or high potassium, all of which effectively reduced several forms of apoptosis in our cortical cultures. Interestingly, a vitamin E analogue trolox almost completely blocked slowly triggered zinc neurotoxicity, indicating that free radical injury is the main mechanism of zinc neurotoxicity. Consistently, exposure to zinc increased membrane lipid peroxidation assessed by the thiobarbituric acid reactive substance assay. Although zinc-induced neuronal death, slowly triggered over a day, is associated with DNA fragmentation, overall it exhibited features more typical of necrosis. This neuronal death is probably mediated by free radical injury. Further studies appear warranted to investigate the mechanistic link between toxic zinc influx and free radical generation and the possibility that selective neuronal death in transient global ischemia also occurs by zinc-triggered neuronal death exhibiting features of both apoptosis and necrosis.     

Perisomatic glutamatergic axon terminals: a novel feature of cortical synaptology revealed by vesicular glutamate transporter 1 immunostaining

The cellular localization of the vesicular glutamate transporter 1, VGLUT1, was studied in the rat cerebral cortex with immunocytochemical techniques. VGLUT1 immunoreactivity (ir) was localized to punctate structures dispersed in the neuropil of all cortical layers as well as around the profile of somata and proximal dendritic segments of virtually all pyramidal neurons. Using a correlative light and electron microscopic method, we found that VGLUT1 ir is expressed in axon terminals forming synapses exclusively with dendritic shafts and spines. Perisomatic VGLUT1-positive terminals never formed synapses with the pyramidal cell bodies to which they were in apposition, but formed asymmetric synapses with adjacent neuropilar dendritic elements. The high probability of a close spatial relationship between glutamatergic and GABAergic terminals in perisomatic regions suggests that spilled-out glutamate may act on inhibitory axon terminals innervating the soma of cortical pyramidal neurons.     

Effects of anticonvulsants on veratridine- and KCl-evoked glutamate release from rat cortical synaptosomes

The rat pheochromocytoma PC12 cells have been employed as a useful model to study neuronal differentiation. To gain insight into the molecular events involved in extension of neurites, the differential expression of phospholipase D (PLD) isozymes were examined in cyclic AMP- or nerve growth factor (NGF)-treated PC12 cells. When the cells were incubated with dibutyryl cyclic AMP (dbcAMP), almost all the cells displayed prominent neurite outgrowth at 24 h. The neurites developed in NGF-treated cells at 24 h were shorter than those in the cells treated with dbcAMP. However, most of the NGF-treated cells extended long neurites at day 5. The rPLD1b mRNA increased within 6 h following dbcAMP treatment and maintained a high level up to 24 h. In contrast, the levels of rPLD1a and rPLD2 mRNAs were rather consistent throughout the time course examined. However, when the cells were treated with NGF, rPLD1a and rPLD1b mRNAs, but not rPLD2 mRNA, increased within 2 days and remained elevated up to 5 days. These results suggest the possible implication of PLD1 in PC12 cell differentiation.     

Post-insult activity is a major cause of delayed neuronal death in organotypic hippocampal slices exposed to glutamate

We investigated the pathophysiological mechanisms of glutamate-induced delayed neuronal damage in rat hippocampal slice cultures [Stoppini et al. (1991) J. Neurosci. Methods 37, 173-182], with propidium iodide as a marker of cell death. Exposure of the cultures to growth medium containing 10 mM glutamate for 30 min resulted in a slowly developing degeneration of hippocampal principal cells, starting from the medial end of the CA1 region and reaching the dentate gyrus by 48 h. By 24 h, most pyramidal cells in CA1 were damaged. An acute phase of degeneration preceded the delayed damage at 2-6 h, affecting cells in a spatially diffuse manner. When tetrodotoxin (0.5 microM) was present during the glutamate insult, a marked protection (mean 57%, P<0.001) of the CA1 damage was observed. Rather strikingly, when tetrodotoxin was applied immediately following or even with a delay of 30 min after the insult, a similar amount of protection was achieved. In field recordings carried out after the insult, the glutamate-treated slices exhibited spontaneously occurring negative shifts with a duration of 1-10 s and an amplitude of up to 400 microV in the CA3 region, whereas the control slices were always quiescent. Taken together, the results suggest that post-insult neuronal network activity, rather than the direct action of exogenous glutamate, is a major cause of delayed CA1 pyramidal cell death in the organotypic slices. These observations may have implications in the design of neuroprotective strategies for the treatment of brain traumas which are accompanied by delayed and/or distal neuronal damage.