Abstract
Alterations in N-methyl-d-aspartate receptor (NMDAR) protein levels or subcellular localization in brain after chronic ethanol exposure may contribute to withdrawal-associated seizures and neurotoxicity. We have investigated synaptic localization of NMDARs in cultured hippocampal pyramidal neurons after prolonged (7 days) exposure to, and acute withdrawal from, 80 mM ethanol using fluorescence immunocytochemistry techniques. After chronic ethanol exposure, there was a significant increase in the clustering of NR1 and NR2B subunits and their colocalization with the synaptic proteins synaptophysin and postsynaptic density protein 95, respectively. There was also increased expression of NR1 variants containing the C2′ cassette after chronic ethanol exposure. The ethanol-induced synaptic clustering and colocalization were rapidly reversed within 4 h after ethanol withdrawal. Surface labeling of NR2B subunits suggested that this rapid reversal involved lateral receptor movement to extrasynaptic sites rather than internalization of receptors. Receptor removal from the synapse during ethanol withdrawal was associated with changes in the phosphorylation state of NR2B Ser1480, controlled by the protein kinase CK2. The redistribution of NMDAR to synapses produced by long-term ethanol exposure, as well as the rapid removal during withdrawal, may not only affect neuronal withdrawal hyperexcitability but also may sensitize the system to subsequent synaptic plasticity.
Footnotes
This work was supported in part by the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grants AA14101, AA007464]; and the Banbury Fund.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.158741.
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ABBREVIATIONS:
- NMDAR
- N-methyl-d-aspartate receptor
- LTP
- long-term potentiation
- PSD
- postsynaptic density
- Ur+FdUr
- uridine + fluorodeoxyuridine
- AP5
- 2-amino-5-phosphonopentanoate
- H89
- N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline
- PKA
- protein kinase A
- TBB
- 4,5,6,7-tetrabromobenzotriazole
- PBS
- phosphate-buffered saline
- AMPAR
- α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
- BSA
- bovine serum albumin
- syp
- synaptophysin
- PSD-95
- postsynaptic density protein 95
- D
- dendrite
- P
- puncta
- C
- colocalization
- pSer1480
- phospho-Ser1480
- ANOVA
- analysis of variance
- KT-5720
- (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester.
- Received July 9, 2009.
- Accepted December 11, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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