Low resting potential and postnatal upregulation of NMDA receptors may cause Cajal-Retzius cell death.
Using in situ patch-clamp techniques in rat telencephalic slices, we have followed resting potential (RP) properties and the functional expression of NMDA receptors in neocortical Cajal-Retzius (CR) cells from embryonic day 18 to postnatal day 13, the time around which these cells normally disappear. We find that throughout their lives CR cells have a relatively depolarized RP (approximately -50 mV), which can be made more hyperpolarized (approximately -70 mV) by stimulation of the Na/K pump with intracellular ATP. The NMDA receptors of CR cells are subjected to intense postnatal upregulation, but their similar properties (EC50, Hill number, sensitivity to antagonists, conductance, and kinetics) throughout development suggest that their subunit composition remains relatively homogeneous. The low RP of CR cells is within a range that allows for the relief of NMDA channels from Mg2+ blockade. Our findings are consistent with the hypothesis that CR cells may degenerate and die subsequent to uncontrolled overload of intracellular Ca2+ via NMDA receptor activation by ambient glutamate. In support of this hypothesis we have obtained evidence showing the protection of CR cells via in vivo blockade of NMDA receptors with dizocilpine. (+info)
Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors.
Murine cortical cultures containing both neurons and glia (days in vitro 13-15) were exposed to periods of oxygen-glucose deprivation (5-30 min) too brief to induce neuronal death. Cultures "preconditioned" by sublethal oxygen-glucose deprivation exhibited 30-50% less neuronal death than controls when exposed to a 45-55 min period of oxygen-glucose deprivation 24 hr later. This preconditioning-induced neuroprotection was specific in that neuronal death induced by exposure to excitotoxins or to staurosporine was not attenuated. Neuroprotection was lost if the time between the preconditioning and severe insult were decreased to 7 hr or increased to 72 hr and was blocked if the NMDA antagonist 100 microM 3-((D)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid was applied during the preconditioning insult. This was true even if the duration of preconditioning was increased as far as possible (while still remaining sublethal). A similar preconditioning effect was also produced by sublethal exposure to high K+, glutamate, or NMDA but not to kainate or trans-1-aminocyclopentane-1, 3-dicarboxylic acid. (+info)
N-Methyl-D-aspartate antagonists and apoptotic cell death triggered by head trauma in developing rat brain.
Morbidity and mortality from head trauma is highest among children. No animal model mimicking traumatic brain injury in children has yet been established, and the mechanisms of neuronal degeneration after traumatic injury to the developing brain are not understood. In infant rats subjected to percussion head trauma, two types of brain damage could be characterized. The first type or primary damage evolved within 4 hr and occurred by an excitotoxic mechanism. The second type or secondary damage evolved within 6-24 hr and occurred by an apoptotic mechanism. Primary damage remained localized to the parietal cortex at the site of impact. Secondary damage affected distant sites such as the cingulate/retrosplenial cortex, subiculum, frontal cortex, thalamus and striatum. Secondary apoptotic damage was more severe than primary excitotoxic damage. Morphometric analysis demonstrated that the N-methyl-D-aspartate receptor antagonists 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonate and dizocilpine protected against primary excitotoxic damage but increased severity of secondary apoptotic damage. 2-Sulfo-alpha-phenyl-N-tert-butyl-nitrone, a free radical scavenger, did not affect primary excitotoxic damage but mitigated apoptotic damage. These observations demonstrate that apoptosis and not excitotoxicity determine neuropathologic outcome after traumatic injury to the developing brain. Whereas free radical scavengers may prove useful in therapy of head trauma in children, N-methyl-D-aspartate antagonists should be avoided because of their propensity to increase severity of apoptotic damage. (+info)
Necrosis and apoptosis after retinal ischemia: involvement of NMDA-mediated excitotoxicity and p53.
PURPOSE: Accumulated evidence has shown that apoptosis and necrosis contribute to neuronal death after ischemia. The present study was performed to study the temporal and spatial patterns of neuronal necrosis and apoptosis after ischemia in retina and to outline mechanisms underlying necrosis and apoptosis. METHODS: Retinal ischemia was induced by increasing intraocular pressure to a range of 160 mm Hg to 180 mm Hg for 90 minutes in adult rats. The patterns of neuronal cell death were determined using light and electron microscopy and were visualized by TdT-dUTP nick-end labeling (TUNEL). The mRNA expression profile of p53 was examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. Immunohistochemistry was performed using anti-p53, anti-microtubule associated protein-2, and anti-glial fibrillary acidic protein antibodies. RESULTS: Within 4 hours after ischemia, neurons in the inner nuclear cell layer (INL) and ganglion cell layer (GCL) underwent marked necrosis, made apparent by swelling of the cell body and mitochondria, early fenestration of the plasma membrane, and irregularly scattered condensation of nuclear chromatin. After 3 days, the INL and GCL neurons showed further degeneration through apoptosis marked by cell body shrinkage, aggregation, and condensation of nuclear chromatin. Apoptotic neurons were also observed sparsely in the outer nuclear cell layer. Intravitreal injections of MK-801 prevented early neuronal degeneration after ischemia. Of note, mRNA and protein levels of p53, the tumor suppressor gene known to induce apoptosis, were increased in the retinal areas undergoing apoptosis 1 to 3 days after ischemic injury. CONCLUSIONS: Ischemia produces the N-methyl-D-aspartate-mediated necrosis and slowly evolving apoptosis of neurons in the retina. The latter may depend on the expression of the p53 proapoptosis gene. (+info)
Nitric oxide mediates cerebral ischemic tolerance in a neonatal rat model of hypoxic preconditioning.
Neuroprotection against cerebral ischemia can be realized if the brain is preconditioned by previous exposure to a brief period of sublethal ischemia. The present study was undertaken to test the hypothesis that nitric oxide (NO) produced from the neuronal isoform of NO synthase (NOS) serves as a necessary signal for establishing an ischemia-tolerant state in brain. A newborn rat model of hypoxic preconditioning was used, wherein exposure to sublethal hypoxia (8% oxygen) for 3 hours renders postnatal day (PND) 6 animals completely resistant to a cerebral hypoxic-ischemic insult imposed 24 hours later. Postnatal day 6 animals were treated 0.5 hour before preconditioning hypoxia with the nonselective NOS inhibitor L-nitroarginine (2 mg/kg intraperitoneally). This treatment, which resulted in a 67 to 81% inhibition of calcium-dependent constitutive NOS activity 0.5 to 3.5 hours after its administration, completely blocked preconditioning-induced protection. However, administration of the neuronal NOS inhibitor 7-nitroindazole (40 mg/kg intraperitoneally) before preconditioning hypoxia, which decreased constitutive brain NOS activity by 58 to 81%, was without effect on preconditioning-induced cerebroprotection, as was pretreatment with the inducible NOS inhibitor aminoguanidine (400 mg/kg intraperitoneally). The protective effects of preconditioning were also not blocked by treating animals with competitive [3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate; 5 mg/kg intraperitoneally] or noncompetitive (MK-801; 1 mg/kg intraperitoneally) N-methyl-D-aspartate receptor antagonists prior to preconditioning hypoxia. These findings indicate that NO production and activity are critical to the induction of ischemic tolerance in this model. However, the results argue against the involvement of the neuronal NOS isoform, activated secondary to a hypoxia-induced stimulation of N-methyl-D-aspartate receptors, and against the involvement of the inducible NOS isoform, but rather suggest that NO produced by the endothelial NOS isoform is required to mediate this profound protective effect. (+info)
Changes in the diffusion of water and intracellular metabolites after excitotoxic injury and global ischemia in neonatal rat brain.
The reduction of the apparent diffusion coefficient (ADC) of brain tissue water in acute cerebral ischemia, as measured by diffusion-weighted magnetic resonance imaging, is generally associated with the development of cytotoxic edema. However, the underlying mechanism is still unknown. Our aim was to elucidate diffusion changes in the intracellular environment in cytotoxic edematous tissue. The ADC of intracellular metabolites was measured by use of diffusion-weighted 1H-magnetic resonance spectroscopy after (1) unilateral N-methyl-D-aspartate (NMDA) injection and (2) cardiac arrest-induced global ischemia in neonatal rat brain. The distinct water ADC drop early after global ischemia was accompanied by a significant reduction of the ADC of all measured metabolites (P < 0.01, n = 8). In the first hours after excitotoxic injury, the ADC of water and the metabolites taurine and N-acetylaspartate dropped significantly (P < 0.05, n = 8). At 24 and 72 hours after NMDA injection brain metabolite levels were diminished and metabolite ADC approached contralateral values. Administration of the NMDA-antagonist MK-801 1.5 hours after NMDA injection completely normalized the water ADC but not the metabolite ADC after 1 to 2 hours (n = 8). No damage was detected 72 hours later and, water and metabolite ADC had normal values (n = 8). The contribution of brain temperature changes (calculated from the chemical shift between the water and N-acetylaspartate signals) and tissue deoxygenation to ischemia-induced intracellular ADC changes was minor. These data lend support to previous suggestions that the ischemia-induced brain water ADC drop may partly be caused by reduced diffusional displacement of intracellular water, possibly involving early alterations in intracellular tortuosity, cytoplasmic streaming, or intracellular molecular interactions. (+info)
Evidence for a novel glutamate-mediated signaling pathway in keratinocytes.
Phenotypic alterations in keratinocyte behavior are essential for maintaining epidermal integrity during growth and wound repair and rely on co-ordinated cell signaling events. Numerous growth factors and cytokines have been shown to be instrumental in guiding such changes in keratinocyte activity; here we provide evidence which proposes a novel epidermal signaling pathway mediated by the excitatory amino acid glutamate. Glutamate is the major excitatory neurotransmitter at synaptic junctions within the central nervous system; however, we have identified expression in vivo of several regulatory molecules associated with glutamate signaling in keratinocytes. In resting rat skin epidermis, different classes of glutamate receptors, transporters, and a recently described clustering protein were shown to display distinct distribution patterns, supportive of a multifunctional cellular communication pathway. Immunoreactive N-methyl-D-aspartate-type, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type, and metabotropic-type glutamate receptors were colocalized with the specific glutamate transporter EAAC1 in basal layer keratinocytes, and GLT-1, a related transporter, was expressed suprabasally. In full-thickness rat skin wounds, marked modifications in the distribution of N-methyl-D-aspartate receptors and EAAC1 were observed during re-epithelialization, and alterations in N-methyl-D-aspartate receptor expression accompanied embryonic epidermal development, implicating glutamate signaling in these important biologic events. Furthermore, we provide evidence that these receptors are functional in vitro. These data provide strong evidence to support a role for glutamate in the control of epidermal renewal, and therefore suggest potentially novel therapeutic targets for the treatment of skin disease and enhancement of wound healing. (+info)
Effects of (+)-HA-966, CGS-19755, phencyclidine, and dizocilpine on repeated acquisition of response chains in pigeons: systemic manipulation of central glycine sites.
The effects of i.m. injections of (+)-HA-966, a glycine-site antagonist at the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor, its enantiomer (-)-HA-966, the competitive glutamate antagonist CGS-19755, the uncompetitive glutamate antagonists phencyclidine and dizocilpine, and the micro opioid agonist morphine were evaluated in a repeated acquisition task in pigeons. All of the drugs produced dose-dependent decreases in rates of responding. The NMDA receptor and channel blockers and (+)-HA-966 appeared to have a greater effect on acquisition than did morphine at doses that did not fully suppress responding. The rate suppression and learning impairment produced by a large dose of (+)-HA-966 (100 mg/kg) were completely prevented by coadministration of the glycine-site agonist D-serine (560 mg/kg) but not by its enantiomer, L-serine (1000 mg/kg). D-Serine, however, produced incomplete antagonism of the effects of dizocilpine and phencyclidine and failed to alter those of CGS-19755. These findings provide evidence that reducing the activity of the NMDA subtype of the glutamate receptor through pharmacological action at any of three sites produces similar decrements in acquisition, and those produced through antagonism of the glycine site are differentially sensitive to the glycine-site agonist D-serine. (+info)