Involvement of three glutamate receptor ϵ subunits in the formation of N-methyl-d-aspartate receptors mediating excitotoxicity in primary cultures of mouse cerebellar granule cells

Didier, Michel; Xu, Min; Berman, Stephen; Saido, Takaomi C.; Bursztajn, Sherry

doi:10.1016/s0306-4522(96)00630-6

Cited by 12 publications

(14 citation statements)

References 59 publications

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“…NMDA receptor subunit expression is regulated developmentally and is associated with the development of susceptibility to glutamate excitotoxicity in cultured cerebral cortical (Mizuta et al, 1998; Cheng et al, 1999) and cerebellar neurons (Didier et al, 1997). We show that the essential NR1 subunit is expressed in our cultures, consistent with previous findings that the NR1 subunit appears early in culture before glutamate‐induced cell death is observed (Didier et al, 1997; Li et al, 1998; Mizuta et al, 1998). It is possible that a low level of NMDA receptor activation and/or ion influx is sufficient to alter dendrite growth, without inducing subsequent neuron death.…”

Section: Discussionmentioning

confidence: 99%

“…It is possible that a low level of NMDA receptor activation and/or ion influx is sufficient to alter dendrite growth, without inducing subsequent neuron death. The presence of NMDA receptor subtypes, composed of different subunit combinations, particularly NR2 subunits, which modulate NR1‐containing heteromeric receptor properties, which appear at different times, also may account for the absence of glutamate‐induced cell death in our system (Didier et al, 1997). Finally, the signaling cascades coupling activated NMDA receptors to downstream effectors in immature and mature neurons may differ in function or in the intracellular pathway that is involved.…”

Section: Discussionmentioning

confidence: 99%

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Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: Involvement of NMDA receptors

Monnerie

Shashidhara

Roux

2003

J of Neuroscience Research

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Glutamate is an important regulator of dendrite development; however, during cerebral ischemia, massive glutamate release can lead to neurodegeneration and death. An early consequence of glutamate excitotoxicity is dendrite injury, which often precedes cell death. We examined the effect of glutamate on dendrite growth from embryonic day 18 (E18) mouse cortical neurons grown for 3 days in vitro (DIV) and immunolabeled with anti-microtubule-associated protein (MAP)2 and anti-neurofilament (NF)-H, to identify dendrites and axons, respectively. Cortical neurons exposed to excess extracellular glutamate (100 microM) displayed reduced dendrite growth, which occurred in the absence of cell death. This effect was mimicked by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and blocked by the ionotropic glutamate receptor antagonist kynurenic acid and the NMDA receptor-specific antagonist MK-801. The non-NMDA receptor agonist AMPA, however, did not affect process growth. Neither NMDA nor AMPA influenced neuron survival. Immunolabeling and Western blot analysis of NMDA receptors using antibodies against the NR1 subunit, demonstrated that immature cortical neurons used in this study, express NMDA receptors. These results suggest that excess glutamate decreases dendrite growth through a mechanism resulting from NMDA receptor subclass activation. Furthermore, these data support the possibility that excess glutamate activation of NMDA receptors mediate both cell death in mature neurons and the inhibitory effect of excess glutamate on dendrite growth in immature neurons or in the absence of cell death.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: Involvement of NMDA receptors

Monnerie

Shashidhara

Roux

2003

J of Neuroscience Research

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“…Bessho et al reported that these treatments selectively induced NR2A subunit mRNA, and that the cells failed to acquire sensitivity to NMDA toxicity after depression of NR2A by antisense oligonucleotide treatment [16]. Furthermore, Didier et al recently reported that antisense oligonucleotide treatment of either m1-NR2A, m2-NR2B or m3-NR2C reduced protein expression, followed by attenuation of NMDA neurotoxicity to a similar extent [17]. Studies of cerebral cortical neurons reported that depression or destruction of the n1-NR1 subunit resulted in, respectively, reduced or no sensitivity to neurotoxicity [31,32],…”

Section: Discussionmentioning

confidence: 99%

“…However, molecular diversities of NMDA receptors in relation to glutamate neurotoxicity have been less thoroughly studied. Although some recent reports have referred to this issue by using cultures of cerebellar granule cells [16,17], little has been reported in the case of cerebral cortical neurons. The NMDA receptor subunits, especially the m-NR2 subunits, are expressed in a temporal-and spatial-specific manner [18][19][20][21].…”

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confidence: 99%

Developmental expression of NMDA receptor subunits and the emergence of glutamate neurotoxicity in primary cultures of murine cerebral cortical neurons

Mizuta

Katayama

Watanabe

et al. 1998

Cellular and Molecular Life Sciences (CMLS)

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Using primary cultures of murine cerebral cortices, we investigated the developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits in relation to the appearance of NMDA receptor-mediated glutamate neurotoxicity. The cultures were not affected by glutamate exposure on culture days 7-9, but became sensitive to glutamate neurotoxicity on day 11. The expression of NMDA receptor subunit messenger RNAs (mRNAs) was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The epsilon 3-NR2C and epsilon 4-NR2D transcripts could not be detected in the culture. The epsilon 2-NR2B and zeta 1-NR1 subunit mRNAs, on the other hand, could be detected clearly and continuously from the culture initiation, and the epsilon 1-NR2A subunit mRNA became clearly detectable on culture day 4. The expression of these three subunits' proteins in the glutamate-insensitive stage (culture day 8) and the sensitive stage (day 11) were studied by means of Western blotting. The epsilon 2-NR2B and zeta 1-NR1 subunit proteins were clearly expressed on culture days 8 and 11, but the epsilon 1-NR2A subunit protein could hardly be detected on either day 8 or day 11. These results suggest that the glutamate neurotoxicity in the primary culture was mediated mainly by epsilon 2/zeta 1 NMDA receptors. The time lag between the protein expression of the epsilon 2-NR2B and zeta 1-NR1 subunits and the emergence of glutamate neurotoxicity may be necessary for the maturation of functional NMDA receptor systems, including heteromeric receptor formation, increase in receptor density and maturation of the postreceptor signal transduction system.

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“…Nevertheless, studies that provide direct physical evidence for ODN͞mRNA hybridization in vivo are rare. Decrement in targeted mRNA levels (12)(13)(14) or down-regulation of targeted protein (15)(16)(17)(18) is widely used to infer duplex formation but RNA fragments consistent with ODN-guided cleavage have been described only in Xenopus oocytes (19) and hematopoietic cells (10). Whether such fragments truly represent the result of hybridization in the living cell is uncertain.…”

mentioning

confidence: 99%

Real time detection of DNA⋅RNA hybridization in living cells

Sokol

Zhang²,

Lu³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

355

234

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Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized ''molecular beacon'' (MB) reporter oligodeoxynucleotides with matched f luorescent donor and acceptor chromophores on their 5 and 3 ends. In the absence of a complementary nucleic acid strand, the MB remains in a stem-loop conformation where f luorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing f luorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. Solution hybridization studies revealed that in the presence of a complementary strand targeted MB could yield up to a 60-fold increase in f luorescence intensity in comparison to control MB. By using a f luorescence microscope fitted with UV f luoride lenses, the detection limit of preformed MB͞target sequence duplexes microinjected into cells was found to be >1 ؋ 10 ؊1 ag of MB, or ϳ10 molecules of mRNA. On the basis of this exquisite sensitivity, real-time detection of MB͞target mRNA hybridization in living cells was attempted by microinjecting MB targeted to the vav protooncogene, or control MB, into K562 human leukemia cells. Within 15 min, confocal microscopy revealed f luorescence in cells injected with targeted, but not control, MB. These studies suggest that realtime visualization and localization of oligonucleotide͞mRNA interactions is now possible. MB could find utility in studying RNA processing, trafficking, and folding in living cells. We hypothesize that MB may also prove useful for finding targetable mRNA sequence under physiologic conditions.

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Involvement of three glutamate receptor ϵ subunits in the formation of N-methyl-d-aspartate receptors mediating excitotoxicity in primary cultures of mouse cerebellar granule cells

Cited by 12 publications

References 59 publications

Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: Involvement of NMDA receptors

Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: Involvement of NMDA receptors

Developmental expression of NMDA receptor subunits and the emergence of glutamate neurotoxicity in primary cultures of murine cerebral cortical neurons

Real time detection of DNA⋅RNA hybridization in living cells

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