Time course and manner of Purkinje neuron death following a single ethanol exposure on postnatal day 4 in the developing rat.
The present study was designed to evaluate the time course and manner of Purkinje cell death following a single ethanol dose delivered intragastrically on postnatal day (PN) 4 to rat pups. Analysis included immunolabeling of Purkinje cells with antibody specific for calbindin D28k and counting of Purkinje cells in each lobule of a mid-vermal slice. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis and immunodetection for cleaved (activated) caspase-3 enzyme was used to identify apoptosis, with calbindin D28k co-immunolabeling to identify apoptotic Purkinje cells. Finally, immunodetection for cytochrome c, again with co-labeling using calbindin D28k antibody, identified intracellular release of cytochrome c from the mitochondria into the cytoplasm of Purkinje cells. The data demonstrate that a single dose of ethanol results in a significant and extensive, lobular dependent loss of Purkinje cells within 24 h after administration. Extensive loss in the early developing lobules (I-III, VIII-X) and less to no loss in the later developing lobules (IV-VII) is consistent with prior literature reports on the ethanol-induced effects on Purkinje cells at this age. Clear and consistent evidence of apoptotic Purkinje cells was identified and the pattern was transient in nature. Finally, cytochrome c is released from the mitochondria of Purkinje cells in a time course consistent with the activation of the mitochondrial pathway of apoptosis. These data support the hypothesis that ethanol-induced loss of Purkinje cells involves apoptotic mechanisms. Furthermore, the initiation of apoptosis by ethanol is consistent with ethanol-induced interruptions of Purkinje cell neurotrophic support leading to activation of the mitochondrial pathway of apoptosis.
Light
K E
KE
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205, USA. lightkime@uams.edu
Belcher
S M
SM
Pierce
D R
DR
D020268:Alcohol-Induced Disorders, Nervous System / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D064092:Calbindin 1; D064026:Calbindins; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002525:Cerebellar Cortex / Q000187:drug effects* / Q000254:growth & development* / Q000473:pathology; D003574:Cytochrome c Group / Q000187:drug effects / Q000378:metabolism; D004334:Drug Administration Schedule; D000431:Ethanol / Q000633:toxicity*; D005260:Female; D063647:Fetal Alcohol Spectrum Disorders / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009410:Nerve Degeneration / Q000139:chemically induced* / Q000378:metabolism / Q000503:physiopathology; D011247:Pregnancy; D011297:Prenatal Exposure Delayed Effects; D011689:Purkinje Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D013997:Time Factors
C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D003574:Cytochrome c Group; D064030:S100 Calcium Binding Protein G; D000431:Ethanol; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases