Ethanol withdrawal is required to produce persisting N-methyl-D-aspartate receptor-dependent hippocampal cytotoxicity during chronic intermittent ethanol exposure.
Chronic intermittent ethanol consumption is associated with neurodegeneration and cognitive deficits in preclinical laboratory animals and in the clinical population. While previous work suggests a role for neuroadaptations in the N-methyl-d-aspartate (NMDA) receptor in the development of ethanol dependence and manifestation of withdrawal, the relative roles of ethanol exposure and ethanol withdrawal in producing these effects have not been fully characterized. To examine underlying cytotoxic mechanisms associated with chronic intermittent ethanol (CIE) exposure, organotypic hippocampal slices were exposed to 1-3 cycles of ethanol (50 mM) in cell culture medium for 5 days, followed by 24 h of ethanol withdrawal, in which a portion of slices were exposed to competitive NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV; 40 μM). Cytotoxicity was assessed using immunohistochemical labeling of neuron-specific nuclear protein (NeuN; Fox-3), a marker of mature neurons, and thionine (2%) staining of Nissl bodies. Multiple cycles of CIE produced neurotoxicity, as reflected in persisting losses of neuron NeuN immunoreactivity and thionine staining in each of the primary cell layers of the hippocampal formation. Hippocampi aged in vitro were significantly more sensitive to the toxic effects of multiple cycles of CIE than were non-aged hippocampi. This effect was not demonstrated in slices exposed to continuous ethanol, in the absence of withdrawal, or to a single exposure/withdrawal regimen. Exposure to APV significantly attenuated the cytotoxicity observed in the primary cell layers of the hippocampus. The present findings suggest that ethanol withdrawal is required to produce NMDA receptor-dependent hippocampal cytotoxicity, particularly in the aging hippocampus in vitro.
University of Kentucky, Department of Psychology, 110 Kastle Hall, Lexington, KY 40506-0044, USA; University of Kentucky, Spinal Cord and Brain Injury Research Center, 741 South Limestone St., Lexington, KY 40536-0509, USA. Electronic address: arreyn3@uky.edu.
Anna R
AR
Reynolds
University of Kentucky, Department of Psychology, 110 Kastle Hall, Lexington, KY 40506-0044, USA; University of Kentucky, Spinal Cord and Brain Injury Research Center, 741 South Limestone St., Lexington, KY 40536-0509, USA.
Jennifer N
JN
Berry
University of Kentucky, Department of Psychology, 110 Kastle Hall, Lexington, KY 40506-0044, USA; University of Kentucky, Spinal Cord and Brain Injury Research Center, 741 South Limestone St., Lexington, KY 40536-0509, USA.
Lynda
L
Sharrett-Field
University of Kentucky, Department of Psychology, 110 Kastle Hall, Lexington, KY 40506-0044, USA; University of Kentucky, Spinal Cord and Brain Injury Research Center, 741 South Limestone St., Lexington, KY 40536-0509, USA.
Mark A
MA
Prendergast
D000818:Animals; D034961:Antigens, Nuclear / Q000187:drug effects / Q000378:metabolism; D002492:Central Nervous System Depressants / Q000008:administration & dosage / Q000009:adverse effects / Q000494:pharmacology*; D000431:Ethanol / Q000008:administration & dosage / Q000009:adverse effects / Q000494:pharmacology*; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism; D009562:Nissl Bodies / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013375:Substance Withdrawal Syndrome / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology
D034961:Antigens, Nuclear; D002492:Central Nervous System Depressants; D009419:Nerve Tissue Proteins; C546339:Rbfox3 protein, rat; D016194:Receptors, N-Methyl-D-Aspartate; D000431:Ethanol; C056639:2-amino-5-phosphopentanoic acid; D014633:Valine
APV; CIE; Hippocampus; NMDA receptor; NeuN; Thionine