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. 2020 Mar 5;13:13. doi: 10.1186/s13072-020-00332-0

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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