Cocaine exposure in vitro induces apoptosis in fetal locus coeruleus neurons through TNF-α-mediated induction of Bax and phosphorylated c-Jun NH2-tenninal kinase

Cocaine exposure results in aberrant outgrowth and survival for locus coeruleus (LC), a noradrenergic population of neurons that putatively regulates attentional function; however, the underlying mechanisms for these events are not known. We previously showed that cocaine exposure in vitro activates pro-apoptotic Bax, caspase-9, and caspase-3 in LC neurons dissected from embryonic day 14 rats, implicating that apoptosis may be orchestrated via signal transduction events. In the current study in vitro, we examined upstream events to determine the role of the pro-inflammatory cytokine, tumor necrosis factor alpha TNF-alpha, on LC signal transduction, because cocaine exposure to LC neurons triggered TNF-alpha expression at 30 min as measured by ELISA. Exposure of LC neurons to recombinant-TNF-alpha resulted in decreased metabolic activity, an indicator of reduced neuron viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], and increased apoptosis (terminal deoxynucleoticlyl transferase- mediated DNA nick end labeling assay). Pro-apoptotic caspase-3 was induced by cocaine starting at 30 min. Recornbinant-TNF-alpha induced caspase-3 activity earlier than cocaine (15 and 20 min). The caspase-3 levels were significantly reduced when cocaine and TNF-alpha were combined with neutralizing-TNF-alpha (nTNF-alpha), respectively. Further, cocaine alone elevated phospho-p38-mitogen-activated protein kinases that persisted when combined with nTNF-a. However, both cocaine and TNF-alpha independently increased phospho-c-Jun NH2-terminal kinase and Bax levels at concurrent time periods (30 min and 1 h), and this elevation was attenuated in the presence of nTNF-a. These simultaneous molecular events triggered by cocaine and TNF-a implicate a potential apoptotic signal transduction pathway via induction of phospho-c-Jun NH2-terminal kinase and Bax that may lead to caspase-3 activation and apoptosis in cocaine-exposed fetal LC neurons.