Nicotine infusions (0.02 mg/kg/infusion) were paired with a compound stimulus (light+tone), and was followed by a 20-s timeout period. Nicotine was delivered across a 5.9-s duration, at a total volume of 0.1 ml of nicotine was administered per infusion. Infusions were paired with cue lights, located above each lever, and a 2900-Hz tone was presented throughout the duration of the nicotine infusion. Active lever presses during the timeout period were recorded, but did not result in additional nicotine infusions. An inactive lever was extended at all times and responses were recorded; however, presses yielded no programmed consequences or rewards. Session duration was 2 h in length and a FR1 schedule of reinforcement was used. During the first two sessions of self-administration, nicotine infusions were capped at 25 to prevent aversive effects that can accompany high nicotine intake. All animals were required to complete a minimum of 10 sessions before moving into extinction with the following criteria: ≥10 nicotine infusions obtained and ≥2:1 active/inactive lever press ratio. Additionally, extinction can be defined in two ways: (1) either by the lack of delivery of a reinforcer that was previously delivered after a response (e.g., responses on the active lever no longer result in nicotine infusions), or (2) as the absence of a contingency between response and reinforcer (e.g., the nicotine infusion occurs regardless of an animals’ response; see Rescorla and Skucy, 1969; Lattal and Lattal, 2012). Given that the reinforcer here (the nicotine infusion) is not delivered following a time-out active lever press, the latter definition applies to these types of responses and are therefore distinct from active lever presses that directly result in the initiation of a nicotine infusion. Thus, these two types of lever presses were analyzed separately in the current study.

Following 10 non-consecutive criteria making sessions, animals were moved to the extinction phase which took place in the same boxes as self-administration. During the extinction phase, active lever presses no longer resulted in nicotine infusions or associated cues. A minimum of 14 2-h extinction sessions was required for each animal. Extinction criteria were set at <30 active lever presses on the last day of extinction. For reinstatement sessions, previously paired nicotine cues were presented on active lever pressing, however no nicotine infusions were delivered. Reinstatement sessions were 15 min in duration. Animals were then immediately killed for whole-cell electrophysiological recordings. Eight animals were removed from the current study because of not meeting self-administration criteria (N = 5) or lack of DREADD expression/improper cannula placement (N = 3).

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