Fig. 1.

Design of experiments within a test day. In the serial Pavlovian design, two CS+ sounds (first CS+1 and then CS+2) predicted a sucrose UCS. In the simple Pavlovian design, a single CS+ sound predicted sucrose pellet delivery as a UCS. On test days conducted after rats had learned the Pavlovian associations, microinjections of DAMGO, amphetamine, or vehicle were made in the hedonic hotspot of the NAc shell to activate NAc-VP circuits immediately before the test, and VP neural firing was recorded to all stimuli. For serial Pavlovian tests, serial CS+ cues were presented in extinction to prevent relearning about the UCS (20–45 min postinjection), and sucrose UCS was tested alone in a second block (45–60 min) to isolate hedonic signals. Reinforced Pavlovian approach trials with the single CS+ were presented in a third block (60–75 min). Consumption of M&Ms was then used to verify increases of behavioral food wanting (75–105 min).