Factors that enhance ethanol inhibition of N-methyl-D-aspartate receptors in cerebellar granule cells.
The objective of this study was to identify factors that influence ethanol (EtOH) inhibition of the N-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.
Popp
R L
RL
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee, USA.
Lickteig
R L
RL
Lovinger
D M
DM
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000502:physiology*; D004357:Drug Synergism; D000431:Ethanol / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D005998:Glycine / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000502:physiology; D011994:Recombinant Proteins / Q000187:drug effects; D013997:Time Factors; D014162:Transfection
D018691:Excitatory Amino Acid Antagonists; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D000431:Ethanol; D016202:N-Methylaspartate; C010739:ifenprodil; D005998:Glycine