Abstract
The objective of this study was to identify factors that influence ethanol (EtOH) inhibition of theN-methyl-d-aspartate receptor (NMDAR) in primary cultured cerebellar granule cells. Several factors contributing to the inhibitory effects of EtOH on NMDAR function were assessed using both whole-cell and perforated patch-clamp recordings. The NMDAR subunit composition was examined by Western blot analysis using NR2 subunit-specific antibodies and pharmacological manipulation with the NR2B-specific antagonist infenprodil. Western blot analysis indicated that NMDAR subunit composition changed from a combination of NR2A and NR2B containing NMDARs to primarily NR2A with increasing days in vitro (DIV). Although the NR2B subunit was detectable until 21 DIV, there was a significant decrease in ifenprodil sensitivity after 7 DIV. EtOH sensitivity did not change with an increasing DIV. A high concentration of glycine reversed EtOH inhibition of steady-state, but not peak, NMDA-induced current during whole-cell recordings. Significant glycine reversal of effects of a low concentration of EtOH on peak current was observed under perforated patch-clamp conditions. A 30-s EtOH pretreatment significantly enhanced EtOH inhibition of NMDA-induced peak current. Collectively, these results indicate that EtOH sensitivity of the NMDAR in primary cultured cerebellar granule cells is not related to subunit composition nor ifenprodil sensitivity, involves a kinetic interaction with glycine, and can be enhanced by a slowly developing transduction mechanism that occurs within tens of seconds.
Footnotes
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Send reprint requests to: R. Lisa Popp, Ph.D., Department of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt School of Medicine, Nashville, TN. E-mail:lisa.popp{at}mcmail.vanderbilt.edu
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↵1 This research was supported by Grants AA08986 and AA05458 from the National Institute of Alcohol Abuse and Alcoholism.
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↵2 Current address: Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, OR 97201.
- Abbreviations:
- CGC
- cerebellar granule cell
- DIV
- days in vitro
- EtOH
- ethanol
- NMDAR
- N-methyl-d-aspartate receptor
- NMDA
- N-methyl-d-aspartate
- Pk
- peak
- SS
- steady state
- WC
- whole cell
- PP
- perforated patch
- Received August 19, 1998.
- Accepted January 26, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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