(A) Schematic of single-unit recordings in the VTA. (B) Histological example showing an electrode placement in the VTA (white arrow). Red: immunostaining against tyrosine hydroxylase (TH), blue: DAPI staining. Scale bar: 0.5 mm. (C) Schematic coronal sections showing the location of the recording sites in the VTA. Numbers represent distance posterior to the bregma. (D) Schematic of the behavioral protocol. Hab: tone habituation, Fear Cond.: fear conditioning. (E) Schematic of CS and US presentations during tone habituation (Hab), fear conditioning (FC) and extinction (Ext). (F) Behavioral data. During tone habituation, mice (n = 11) showed low freezing levels in response to the CS. Twenty-four hours after fear conditioning, mice exhibited significant increase in freezing to the CS during E-Ext trials (first 10 CSs, paired t-test comparing E-Ext to Hab, t(10) = 8.30, p<0.0001). The CS evoked low freezing levels during L-Ext trials (last 10 CSs) indicating successful extinction learning (paired t-test: comparing L-Ext to Hab, t(10) = 1.32, p = 0.21; comparing L-Ext to E-Ext, t(10) = 11.9, p<0.0001). (***p<0.0001). (G) Raster plot (top) and peristimulus time histogram (1 s bins; bottom) of an example putative DA neuron (see inset; scale bar: 50 μV) responding to US omission during the extinction session. (H) The proportion of putative DA neurons that were significantly US omission (or CS offset for Hab) excited, inhibited or nonresponsive during the Hab, E-Ext and L-Ext trials. Proportion of US omission excited neurons significantly increased from Hab to E-Ext (Fisher’s exact test, p = 0.0028) and decreased back to Hab levels during L-Ext (Fisher’s exact test, p = 0.34). Note that there were no US omission inhibited neurons during E-Ext and L-Ext. (I) Peristimulus time histogram showing the z-scored population activity of all putative DA neurons that were significantly US omission excited during E-Ext (n = 10 of 40 putative DA neurons). (J) Peristimulus time histogram showing the z-scored population activity of all putative DA neurons during Hab (left), E-Ext (middle) and L-Ext (right). Note the significant increase in population activity at the time of the US omission during E-Ext (*p<0.05). Shaded regions represent mean ± s.e.m. across neurons.