Time course and manner of Purkinje neuron death following a single ethanol exposure on postnatal day 4 in the developing rat

• Published in: Neuroscience Vol. 114; no. 2; pp. 327 - 337
• Main Authors: Light, K.E, Belcher, S.M, Pierce, D.R
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.01.2002; Elsevier
• Subjects: Alcohol-Induced Disorders, Nervous System - metabolism; Alcohol-Induced Disorders, Nervous System - pathology; Alcohol-Induced Disorders, Nervous System - physiopathology; Alcoholism and acute alcohol poisoning; Animals; Animals, Newborn; apoptosis; Apoptosis - drug effects; Apoptosis - physiology; Biological and medical sciences; Calbindin 1; Calbindins; Caspase 3; Caspases - drug effects; Caspases - metabolism; Cerebellar Cortex - drug effects; Cerebellar Cortex - growth & development; Cerebellar Cortex - pathology; cerebellum; cytochrome c; Cytochrome c Group - drug effects; Cytochrome c Group - metabolism; Drug Administration Schedule; Ethanol - toxicity; Female; Fetal Alcohol Spectrum Disorders - metabolism; Fetal Alcohol Spectrum Disorders - pathology; Fetal Alcohol Spectrum Disorders - physiopathology; Immunohistochemistry; In Situ Nick-End Labeling; Male; Medical sciences; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondria - pathology; Nerve Degeneration - chemically induced; Nerve Degeneration - metabolism; Nerve Degeneration - physiopathology; neuronal development; Pregnancy; Prenatal Exposure Delayed Effects; Purkinje Cells - drug effects; Purkinje Cells - metabolism; Purkinje Cells - pathology; Rats; Rats, Sprague-Dawley; S100 Calcium Binding Protein G - metabolism; Time Factors; Toxicology; neuronal development; IE, intubated with ethanol; DAB, 3,3′-diaminobenzidine; TdT, terminal deoxynucleotidyl transferase; FITC, fluorescein isothiocyanate; apoptosis; TBS, Tris-buffered saline; IC, intubated with isocaloric control; MRC, mother-raised control; cerebellum; BDNF, brain-derived neurotrophic factor; PBS, phosphate-buffered saline; BEC, blood ethanol concentration; ANOVA, analysis of variance; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; cytochrome c; PN, postnatal day; caspase-3; Vertebrata; Mammalia; Ethanol; Rat; Cell death; Animal; Rodentia; Purkinje neuron; Postnatal development; Apoptosis
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/S0306-4522(02)00344-5

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.