Altered expression of Bcl 2, Bad and Bax mRNA occurs in the rat cerebellum within hours after ethanol exposure on postnatal day 4 but not on postnatal day 9

Abstract

Previous studies have demonstrated that ethanol exposure during the vulnerable postnatal (PN) day 4–6 period results in a dose-dependent loss of Purkinje neurons in rats by apoptosis. Although the mechanism of ethanol action and the reasons for Purkinje cell vulnerability are unknown, we hypothesize that during the PN4–6 vulnerable period Purkinje cells are dependent on active trophic factor suppression of apoptosis. Furthermore, ethanol acts to prevent the reception of this trophic signaling resulting in the execution of the apoptotic pathway that includes specific alterations of proteins in the Bcl2 gene family. Ethanol exposure that occurs after this vulnerable period (i.e. PN9) would not be expected to demonstrate alterations in these apoptotic proteins since the Purkinje cells no longer demonstrate vulnerability to ethanol. The current study was undertaken to identify the alterations in mRNA expression for members of the Bcl2-family within the initial hours following ethanol administration on PN4 or PN9. Semi-quantitative reverse transcriptase with polymerase chain reaction (PCR) techniques were used to determine the expression levels of pro-apoptotic factors Bad and Bax, and anti-apoptotic Bcl 2 mRNA. Ethanol was administered at four different doses (1.5, 3.0, 4.5, and 6.0 g/kg) on PN4 and analyses of whole cerebellar mRNA was conducted at 1, 4, 6, and 8 h after treatment. Doses greater than 1.5 g/kg produced significant decreases in Bcl 2 and significant increases in Bad and Bax mRNA during the 8-h period after treatment. In stark contrast, when ethanol was administered at 3.0 or 6.0 g/kg to PN9 pups, no significant alterations of these apoptotic factors were identified at either 1 or 4 h after treatment. These results are in agreement with and provide further support for our hypothesis that ethanol interrupts the active suppression of apoptosis that is a crucial feature of Purkinje cell vulnerability during this time period.