All experiments were carried out humanely and with regard for alleviation of suffering, with protocols approved by the Institutional Animal Care and Use Committee and in accordance with all federal and state guidelines. Timed-pregnant Sprague-Dawley rats (Charles River, Raleigh, NC) were housed in breeding cages, with a 12-hr light/dark cycle and free access to food and water. On the day of birth, all pups were randomized and redistributed to the dams with a litter size of 9–10 to maintain a standard nutritional status. Because of their poor water solubility, diazinon and parathion (both from Chem Service, West Chester, PA) were dissolved in dimethylsulfoxide (DMSO) to provide consistent absorption (Whitney et al. 1995 (link)) and were injected subcutaneously in a volume of 1 mL/kg once daily on postnatal days (PND)1–4; control animals received equivalent injections of DMSO vehicle, which does not itself produce developmental neurotoxicity (Song et al. 1998 (link); Whitney et al. 1995 (link)). Doses were chosen to lie below the threshold for signs of systemic toxicity as evidenced by impaired viability or reduced weight gain (Slotkin et al. 2006 (link)): 0.5, 1, and 2 mg/kg for diazinon, and 0.02, 0.05, and 0.1 mg/kg of parathion. The highest dose of each agent represents the maximum tolerated dose (Slotkin et al. 2006 (link)). On PND5, one male and one female pup were selected from each of at least six litters in each treatment group and were used for neurochemical evaluations. Animals were decapitated, the cerebellum was removed, and the brainstem and forebrain were separated by a cut made rostral to the thalamus. Tissues were weighed and flash frozen in liquid nitrogen and maintained at −45°C until analyzed. For a supplemental study determining the degree of cholinesterase inhibition immediately after treatment, additional animals were used to obtain samples 2 hr after the last injection of 2 mg/kg of diazinon or vehicle on PND4.
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