Ethanol withdrawal is required to produce persisting N-methyl-d-aspartate receptor-dependent hippocampal cytotoxicity during chronic intermittent ethanol exposure

Anna R. Reynolds, Jennifer N. Berry, Lynda Sharrett-Field, Mark A. Prendergast

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


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 (50mM) in cell culture medium for 5 days, followed by 24h of ethanol withdrawal, in which a portion of slices were exposed to competitive NMDA receptor antagonist (2. R)-amino-5-phosphonovaleric acid (APV; 40μM). 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 invitro 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 invitro.

Original languageEnglish
Pages (from-to)219-227
Number of pages9
Issue number3
StatePublished - May 1 2015

Bibliographical note

Funding Information:
This research was supported by Grant AA013388 from the National Institute on Alcohol and Alcoholism (NIAAA) awarded to MAP and National Institute on Drug Abuse (NIDA) DA035200 . This funding agency had no role in study design, data collection, or analysis or preparation and submission of the manuscript. The authors declare no conflicts of interest relevant to this research.

Publisher Copyright:
© 2015 Elsevier Inc.


  • APV
  • CIE
  • Hippocampus
  • NMDA receptor
  • NeuN
  • Thionine

ASJC Scopus subject areas

  • Health(social science)
  • Biochemistry
  • Toxicology
  • Neurology
  • Behavioral Neuroscience


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