Prolonged exposure to organophosphate (OP) pesticides may produce cognitive deficits reflective of hippocampal injury in both humans and rodents. Recent work has indicated that microtubule trafficking is also adversely affected by exposure to the OP pesticide chlorpyrifos, suggesting a novel mode of OP-induced neurotoxicity. The present studies examined effects of prolonged exposure to chlorpyrifos-oxon (CPO) on acetylcholinesterase (AChE) activity, immunoreactivity (IR) of microtubule-associated proteins, neuronal injury, and tubulin polymerization using in vitro organotypic slice cultures of rat hippocampus and bovine tubulin. Cultures were exposed to CPO (0.1-10 μM) in cell culture medium for 1-7 days, a regimen producing progressive reductions in AChE activity of 15-60%. Cytotoxicity (somatic uptake of the non-vital marker propidium iodide, as well as, IR of α-tubulin and microtubule-associated protein-2 (a/b) were assessed 1, 3, and 7 days after the start of CPO exposure. As early as 24-hr after the start of exposure, CPO-induced deficits in MAP-2 IR were evident and progressive in each region of slice cultures at concentrations as low as 0.1 μM. CPO exposure did not alter α-tubulin IR at any time point. Concentration-dependent injury in the CA1 pyramidal cell layer and to a lesser extent, CA3 and dentate cells, was evident 3 days after the start of CPO exposure (≥ 0.1 μM) and was greatest after 7 days. Tubulin polymerization assays indicated that CPO (≥ 0.1 μM) markedly inhibited the polymerization of purified tubulin and map-rich tubulin, though effects on MAP-rich tubulin were more pronounced. These data suggest that exposure to CPO produces a progressive decrease in neuronal viability that may be associated with impaired microtubule synthesis and/or function.The broad spectrum organophosphorus (OP) insecticide chlorpyrifos (O,O-diethyl O-3,5,6trichloro-2-pyridinyl phosphorothioate; CPF) is an inhibitor of cholinesterases, including acetylcholinesterase (AChE), and has until recently been widely used in residential settings in the United States. This compound remains one of the most widely used pesticides in agricultural settings. Emerging evidence, obtained largely through the use of rodents, suggests that acute or prolonged exposure to CPF and/or its metabolic product(s) may overtly injure the central nervous system or produce marked changes in neuronal function that persist after exposure has ceased, particularly during the early postnatal period (Olivier et al. 2001;Slotkin et al. 2001;Zheng et al. 2000). However, it must be noted that not all studies find evidence of persisting CNS abnormalities following the cessation of CPF exposure (Padilla et al. 2005).* Corresponding Author: Department of Psychology, University of Kentucky, B363 BBSRB, 741 S. Limestone, University of Kentucky, Lexington, KY 40536-0509, Telephone: (859) 257-6120, Email: prender@uky.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our custome...