ATF6 and caspase 12 expression in Purkinje neurons in acute slices from adult, ethanol-fed rats

The purpose of this study was to determine, whether previously reported ethanol-induced alterations to the smooth endoplasmic reticulum (SER), predispose Purkinje neurons (PN) to thapsigargin-induced endoplasmic reticulum (ER) stress. Thapsigargin blocks the sarco/endoplasmic Ca(2+) ATPase pump (SERCA 2), depleting the SER of calcium. Forty-one, eight month old Fischer 344 male rats were treated with either the AIN (American Institute of Nutrition) liquid control or ethanol diets for 10 (n=14), 20 (n=10), or 40(n=17) weeks. At the end of treatment, acute cerebellar slices were prepared by standard means. Cerebellar slices were treated with thapsigargin or as controls for three hours in oxygenated (95% CO2, 5% O2) ACSF (artificial cerebrospinal fluid). Slices were then fixed in 4% paraformaldehyde and sectioned on a freezing microtome. Free floating sections were stained with antibodies against activating transcription factor 6 (ATF6) or activated caspase 12 and calbindin. Results showed a significant increase in the activated caspase+PN dendrites in the EF rats along with a significant interaction due to enhanced expression of activated caspase 12 at 20 weeks. The density of ATF6 labeling was not different between the EF and PF groups and was confined to the PN soma. The finding of activated caspase and ATF6 expression in PN within both the EF and PF groups supports the finding of thapsigargin-induced ER stress. The finding of increased activated caspase 12 in the dendrites supports an increased tendency to ER stress and other dendritic deficits in the ethanol rats.