(−)-Nicotine tartrate (MP Biomedicals, Solon, OH, USA) was dissolved in sterile 0.9% sodium chloride solution and then infused subcutaneously via osmotic minipumps (Alzet model 2002; DURECT Corporation, Cupertino, CA, USA) at a dose of 18 mg kg−1 day−1 for 15 days. Chronic treatment with nicotine at this dose yields a plasma level of approximately 0.3 μM in mice (reported as nicotine free base molecular weight), a concentration comparable to that observed in human smokers consuming an average of 17 cigarettes a day (plasma levels between 0.06 and 0.31 μM) (64). Furthermore, in vivo nicotine delivery in mice has shown this low-moderate dose to be the most effective in maintaining face validity with clinical withdrawal symptomology. Our past studies have used chronic nicotine doses ranging from 6 to 36 mg kg−1 day−1 (1115), with the dose of 18 mg kg−1 day−1 being the lowest tested concentration able to elicit all the major hallmarks of nicotine withdrawal observed clinically (510). Control animals were implanted with osmotic minipumps containing only sterile 0.9% sodium chloride solution. Before the start of surgery, mice were anesthetized with isoflurane/oxygen mixture (1–3%), and minipumps were inserted using aseptic techniques. Surgical wounds were closed with 7-mm stainless steel wound clips (Reflex, Cellpoint Scientific, Gaithersburg, MD, USA), after which mice were left to recover on the recovery pad before they were returned to their individual cages. After 14 days of chronic administration of either saline or nicotine via osmotic minipumps, mice in the withdrawal groups (withdrawal from either nicotine or saline) were subjected to spontaneous withdrawal by the removal of their osmotic minipumps using a similar aseptic surgical approach as above. Animals in the chronic nicotine or chronic saline treatment groups underwent sham surgery, where an incision was made and restapled, without removal of the pump. No molecular or behavioral differences were observed between the saline and saline withdrawal groups; therefore, these groups were combined during analysis.

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