Photobeams

For studies in 4-week old mice, the activity chamber consisted of a standard large cage crossed by infrared beams (Opto-Varimex, Columbus OH), as described previously (Sheleg et al., 2013 (link)). The number of horizontal motor movements was calculated by infrared beam breaks for a 30 minute session. For studies in adults, 4- month old mice were placed in sound-attenuated boxes equipped with photobeams (Med Associates, St. Albans, VT). Locomotor activity was quantified over 1–2 hours, with the first 30 min considered a habituation period, and calculated over 5 min blocks and summed across the session. For experiments with methylphenidate, mice were habituated to the open field by 4 daily 60 min sessions. On the fifth day, mice were gavaged with saline after a 30 min habituation period in the locomotor box, and locomotor activity monitored for an additional 60 min. On the sixth day, mice were gavaged with methylphenidate (1 mg/kg) after the 30 min habituation period and locomotor activity monitored for an additional 60 min. Locomotor activity was calculated as total distance traveled in cm.

On day 7 of chronic nicotine exposure, the rats were placed in the chambers and scored for baseline somatic withdrawal signs. After 30 minutes, the rats were given vehicle, AT-1001 (3 mg/kg, s.c.), or mecamylamine (1 mg/kg, s.c.), and scored for an additional 30 minutes (Figure 1D). The dose of AT-1001 used was the highest dose that blocked stable responding of nicotine self-administration [32 (link)], whereas the dose of mecamylamine used is known to produce robust precipitated withdrawal [47 (link)]. After drug treatment, behavior was digitally recorded and scored live by a blind observer for 10 seconds of every minute. Somatic nicotine withdrawal signs included teeth chatter/chewing, gasps/writhing, ptosis, “wet dog” shakes/tremors, and a miscellaneous category of less frequent symptoms such as hop, yawn, scratch, ejaculation, and dyspnea [based on Malin et al., 46 (link), 48 (link)]. Simultaneously, locomotor activity and center time were recorded in the open field chambers through 16 photobeams along the sides of each wall (MED Associates, Inc., St. Albans, VT).

mice were tested in a rat-sized open field (40 cm long × 40 cm wide × 37 cm high) using photobeams to detect and quantify movement (Med-Associates, Fairfax, VT). Three 90-minute sessions on consecutive days were averaged.