Cocaine exposure in vitro induces apoptosis in fetal locus coeruleus neurons by altering the Bax/Bcl-2 ratio and through caspase-3 apoptotic signaling.
Cocaine inhibits survival and growth of rat locus coeruleus (LC) neurons, which may mediate alterations in attention, following in utero exposure to cocaine. These effects are most severe in early gestation during peak neuritogenesis. Prenatal cocaine exposure may specifically decrease LC survival through an apoptotic pathway involving caspases. Dissociated fetal LC neurons or substantia nigra (SN) neurons (control) were exposed in vitro to a pharmacologically active dose of cocaine hydrochloride (500 ng/ml) and assayed for apoptosis using terminal deoxynucleotidyl transferase mediated DNA nick end labeling and Hoechst methodologies. Cocaine exposure decreased survival and induced apoptosis in LC neurons, with no changes in survival of SN neurons. Activation of apoptotic signal transduction proteins was determined using enzyme assays and immunoblotting at 30 min, 1 h, 4 h and 24 h. In LC neurons, Bax levels were induced at 30 min and 1 h, following cocaine treatment, and Bcl-2 levels remained unchanged at all time points, altering the Bax/Bcl-2 ratio. The ratio was reversed for SN neurons (elevated Bcl-2 levels and transient reduction of Bax levels). Further, cocaine exposure significantly increased caspase-9 and caspase-3 activities at all time points, without changes in caspase-8 activity in LC neurons. In addition, cleavage of caspase-3 target proteins, alpha-fodrin and poly (ADP-ribose) polymerase (PARP) were observed following cocaine treatment. In contrast, SN neurons showed either significant reductions, or no significant changes, in caspase-3, -8 or -9 activities or caspase-3 target proteins, alpha-fodrin and PARP. Thus, cocaine exposure in vitro may preferentially induce apoptosis in fetal LC neurons putatively regulated by Bax, via activation of caspases and their downstream target proteins.
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Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536-0298, USA. sdey0@uky.edu
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D002452:Cell Count; D003042:Cocaine / Q000494:pharmacology*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D066298:In Vitro Techniques; D008125:Locus Coeruleus / Q000166:cytology*; D008954:Models, Biological; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D011247:Pregnancy; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
D018765:Dopamine Uptake Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D053148:Caspase 3; D003042:Cocaine