Fig. 1

Procedural timeline for breeding and tissue collection. Beginning 30 days prior (PND 60) to crossing with drug-naïve sires (PND 90), C57BL/6J dams (zeroth generation, F0) underwent passive oral exposure to 0.2% saccharin (developmental vehicle exposure) or 0.2% saccharin containing 200 µg/mL nicotine (developmental nicotine exposure, DNE). Vehicle or nicotine treatment of F0 dams persisted through weaning of first-generation (F1) developmental vehicle-exposed (F1 Veh) or developmental nicotine-exposed (F1 NIC) offspring at PND 21. Thereafter, water was provided as the exclusive fluid source for all offspring. At PND 90, randomly selected female F1 NIC mice were crossed with drug-naïve sires to foster second-generation (F2) developmental nicotine-exposed (F2 NIC) offspring. To obtain tissue for subsequent immunoblot analyses, whole brains were extracted from PND 45 (adolescent) progeny belonging to each developmental exposure group (F1 Veh, F1 NIC, and F2 NIC), and bilateral frontal cortices, striata, and hippocampi were then collected by manual dissection. PND post-natal day, Veh 0.2% aqueous saccharin, NIC 200 µg/mL nicotine in 0.2% aqueous saccharin, F1 Veh first-generation developmental vehicle-exposed offspring, F1 NIC first-generation developmental nicotine-exposed offspring, F2 NIC second-generation developmental nicotine-exposed offspring