Summary maps (A) show in sagittal section the motivated behaviors generated by microinjections in NAc medial shell of DNQX alone (left), DNQX plus SCH23390 (D1 antagonist, middle), or DNQX plus raclopride (D2 antagonist, right) in the Standard laboratory environment. Each subject (n=23) was designated as producing primarily appetitive (green symbols), fearful (red symbols) or mixed appetitive and fearful (yellow symbols) motivated behavior following DNQX microinjection. Purely appetitive eating behavior and food intake (criteria for including a site was a >200% increase in eating) was primarily stimulated in rostral shell by DNQX. Fearful distress calls, escape attempts and spontaneous emission of defensive treading-burying behavior directed toward specific targets (criteria for including a site was a >500% increase over vehicle levels) were primarily stimulated in caudal shell by DNQX. Intermediate sites in medial shell often met both appetitive and defensive criteria and were designated as generating ‘mixed valence’. Combining the D1 antagonist in the DNQX microinjection prevented the elicitation of all appetitive (rostral) and fearful (caudal) behaviors. By contrast, combining the D2 antagonist in the DNQX microinjection only prevented the elicitation of defensive behavior, but left appetitive behavior intact. Maximal Fos plumes (B) were analyzed for each drug microinjection condition. Fos labeled cells were individually counted within successive blocks (50 μm × 50 μm), along 8 radial arms emanating from the center of the site, with 10x magnification. Colors indicate levels of Fos expression of 3x (red), 2x (orange) and 1.5x (yellow) vehicle level Fos expression. Line graphs show that DNQX (red) produced elevated Fos expression starting at the center of the microinjection to zones ~0.3mm away. DNQX-induced increases in Fos expression were suppressed by adding D1 antagonist (SCH23390, blue) but were enhanced by adding D2 antagonist (raclopride, orange) to the DNQX microinjection.

Generation of increases in eating behavior (A), food intake (B), spontaneous defensive treading/burying behavior (C), incidence of distress vocalizations in response to human touch after the test (D), and of escape attempts in response to human touch (E). Effects are shown for microinjections of DNQX alone, DNQX plus SCH23390 (D1 antagonist), DNQX plus raclopride (D2 antagonist) and DNQX plus both D1 and D2 antagonists in rostral (n=9) and caudal (n=14) regions of medial NAc shell (relative to vehicle microinjections in the same rats). Data are presented as change from vehicle, errors bars indicate SEM, * p < 0.05, ** p < 0.01 change from vehicle, # p < 0.05, ## p < 0.01 change from DNQX, pairwise comparisons using Sidak corrections for multiple comparisons (eating, food intake and defensive treading) or McNemar’s test (distress vocalizations and escape attempts).

Fos plume maps (n=23) in sagittal plane of the generation in medial shell of eating (A), defensive treading (B), and fearful vocalizations and escape attempts (C) by DNQX (left), DNQX plus SCH23390 (D1 antagonist, middle), and DNQX plus raclopride (D2 antagonist, right). The D1 antagonist prevented DNQX from generating either eating or fear, while the D2 antagonist left DNQX-induced eating intact, but prevented DNQX-induced generation of fear. Histograms bars below the maps show behavior as percent of vehicle (eating, A; treading, B) or percent of subjects (vocalizations and escape attempts, C) for each behavior at rostrocaudal level as marked along the medial shell (error bars = SEM).

Summary maps (n=10) in sagittal plane show behavior produced by microinjections in the same rats of either DNQX alone or DNQX plus a D2 antagonist (raclopride), each tested both in the Home environment (A) and in the Stressful (C) environment. Each site was designated in a particular environment as producing primarily appetitive (green symbols), fearful (red symbols) or mixed valence (yellow symbols) motivated behavior following DNQX microinjection. Testing in the Home environment caudally expanded the area which DNQX generated purely appetitive behavior (criteria for including a site was a 200% increase in feeding behavior), and nearly eliminated the ability of DNQX to generate defensive treading behavior, distress calls or escape attempts at any site. Conversely, testing in the Stressful environment rostrally expanded the area of medial shell capable of generating intense fearful behaviors (criteria for including a site was a 500% increase in treading over vehicle levels), so that nearly every rostrocaudal location became able to generate fearful reactions after DNQX microinjections (relative to vehicle). The addition of the D2 antagonist to the microinjection blocked DNQX generation of defensive treading regardless of site. In contrast, the D2 antagonist never blocked the ability of DNQX to stimulate eating behavior and increase food intake. Histograms bars below the maps show mean behavior as percent of vehicle for each behavior at rostrocaudal level as marked along the medial shell, with the dominant behavior stimulated in each environment appearing along the top (Home environment: eating; Stressful environment: treading; error bars = SEM). Maps of fearful vocalizations indicate which rats emitted audible distress calls in response to the experimenter’s touch in the Home environment (B) and the Stressful environment (D) following DNQX alone or DNQX plus the D2 antagonist. Histograms bars above the maps show the percentage of subjects which vocalized at each rostrocaudal level.

Time course (A) of eating and defensive treading over the 1-hour trial (n=6): eating behavior peaks early in the trial, while defensive treading emerges towards the mid-point of the trial (average % of vehicle, error bars = SEM). During the period of greatest overlap (minutes 11 – 30, highlighted in A), most individual minutes (B) for a given rat consisted of purely appetitive or purely aversive, rather than mixed, and transitions between appetitive and defensive valence tend to be limited (<10 for most rats). Yet, even for the rat that demonstrated the greatest ambivalence (shown in C, ~31 transitions between appetitive versus defensive behaviors in the 1-hour test), the addition of the D2 antagonist (raclopride) completely eliminated fearful behavior, and left DNQX-stimulation of eating intact.

Close-up representation of synapses in NAc medial shell and position in larger circuits. D1 receptors are located postsynaptically on medium spiny neurons (red) that project via direct output path to ventral tegmentum (VTA), and via indirect output path to ventral pallidum (VP) and lateral hypothalamus (LH). D2 receptors are shown postsynaptically on medium spiny neurons that project via indirect output path to VP and LH (also D1–D2 co-expressing neurons). Dopamine receptors are also shown presynaptically in NAc on dopamine (black) and glutamate (green) neurons. Glutamatergic inputs (green) are shown from medial prefrontal cortex, orbitofrontal cortex, hippocampus, thalamus, and basolateral amygdala. Dopaminergic inputs (black) to NAc shell are shown from ventral tegmental area. GABAergic output pathways are shown to VP and LH (indirect path; D1, D2 and D1–D2 expressing neurons) and to VTA (direct path; D1).