Year of Publication
Doctor of Philosophy (PhD)
Arts and Sciences
Dr. Mark Prendergast
Chronic ethanol (EtOH) exposure produces neuroadaptations within the NMDA receptor system and alterations in HPA axis functioning that contribute to neurodegeneration during ethanol withdrawal (EWD). Chronic EtOH exposure and EWD, as well as corticosteroids, also promote increased synthesis and release of polyamines, which allosterically potentiate NMDA receptor open-channel time at the NR2B subunit. The current studies investigated effects of 10 day EtOH and corticosterone (CORT) co-exposure on toxicity during EWD in rat organotypic hippocampal slice cultures, and alterations in function and/or density of the NR2B subunit of the NMDA receptor that may mediate CORT-potentiation of toxicity during EWD. We hypothesized that toxicity during withdrawal following EtOH and CORT co-exposure would be greatest in the CA1 region due to increased NMDA NR2B receptor abundance and/or function. Cultures were exposed to CORT (0.01–1 μM) during 10 day EtOH exposure (50 mM) and 1 day EWD. Additional EtOH-naïve cultures were exposed to CORT for 11 days. Propidium iodide (PI) was used to measure toxicity in the CA1, CA3, and DG hippocampal regions. In EtOH-naïve cultures, 11 day exposure to CORT (0.01 – 1 μM) produced modest toxicity and in all regions. Exposure to CORT during EtOH exposure/EWD potentiated CORT-toxicity at all concentrations in the CA1 region. Ifenprodil, an NR2B polyamine site antagonist, significantly reduced toxicity from EtOH and CORT (0.1 μM) co-exposure during withdrawal. Immunohistochemistry and Western blot analyses were conducted for measurement of NR2B immunoreactivity in organotypic cultures, and autoradiography studies were conducted for measurement of polyamine-sensitive NR2B subunits with [3H]ifenprodil. Consistent increases in NR2B subunit protein were not detected with use of any methodology. Additional studies exposed cultures to a membrane impermeable form of CORT (BSA-conjugated CORT; 0.1 μM) with or without EtOH exposure and withdrawal. BSA-CORT exposure did not produce toxicity in any hippocampal region, suggesting that CORT toxicity was not mediated by membrane bound substrates. These data suggest that CORT and EtOH co-exposure result in increased function of polyamine-sensitive NR2B subunits, but this toxicity does not appear dependent on the number of hippocampal NMDA NR2B subunits.
Butler, Tracy Renee, "EFFECTS OF CORTICOSTERONE AND ETHANOL CO-EXPOSURE ON HIPPOCAMPAL TOXICITY: POTENTIAL ROLE FOR THE NMDA NR2B SUBUNIT" (2011). University of Kentucky Doctoral Dissertations. 146.