Nucleus accumbens GABAergic inhibition generates intense eating and fear that resists environmental retuning and needs no local dopamine

Abstract

Intense fearful behavior and/or intense appetitive eating behavior can be generated by localized amino acid inhibitions along a rostrocaudal anatomical gradient within medial shell of nucleus accumbens of the rat. This can be produced by microinjections in medial shell of either the γ-aminobutyric acid (GABA)A agonist muscimol (mimicking intrinsic GABAergic inputs) or the AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione), disrupting corticolimbic glutamate inputs). At rostral sites in medial shell, each drug robustly stimulates appetitive eating and food intake, whereas at more caudal sites the same drugs instead produce increasingly fearful behaviors such as escape, distress vocalizations and defensive treading (an antipredator behavior rodents emit to snakes and scorpions). Previously we showed that intense motivated behaviors generated by glutamate blockade require local endogenous dopamine and can be modulated in valence by environmental ambience. Here we investigated whether GABAergic generation of intense appetitive and fearful motivations similarly depends on local dopamine signals, and whether the valence of motivations generated by GABAergic inhibition can also be retuned by changes in environmental ambience. We report that the answer to both questions is 'no'. Eating and fear generated by GABAergic inhibition of accumbens shell does not need endogenous dopamine. Also, the appetitive/fearful valence generated by GABAergic muscimol microinjections resists environmental retuning and is determined almost purely by rostrocaudal anatomical placement. These results suggest that nucleus accumbens GABAergic release of fear and eating are relatively independent of modulatory dopamine signals, and more anatomically pre-determined in valence balance than release of the same intense behaviors by glutamate disruptions.

Figure 1. Summary graphs of the effect of local dopamine blockade on muscimol-generated eating and fearful behaviors

Time spent eating (A), amount of food intake (B), time spent defensive treading/burying (C), as well as incidence of distress vocalizations in response to human touch (D), elicited by vehicle (grey), dopamine antagonist combination of raclopride and SCH23390 (black), muscimol (red) and mixture of muscimol and dopamine antagonist (yellow) in rostral (n=9) and caudal (n=7) regions of medial NAc shell. Errors bars indicate SEM, * p < 0.05, ** p < 0.01 change from vehicle, # p < 0.05, ## p < 0.01 change from dopamine antagonists alone, pairwise comparisons using Sidak corrections for multiple comparisons (eating, food intake and defensive treading) or McNemar’s test (distress vocalizations). Muscimol alone and mixture were never significantly different. Summary maps below show the localization of microinjections that produced primarily appetitive (green symbols), defensive (red symbols) or mixed appetitive and defensive (yellow symbols) in rostral (white) versus caudal (grey) shell.

Figure 2. Effects of dopamine antagonism on muscimol-induced eating and defensive fearful behaviors

Fos plume maps (n=16) of the generation in medial shell of eating (A), defensive treading (B), and fearful calls, escape attempts and bite attempts (C) by muscimol (left) and a mixture of muscimol plus dopamine antagonists (right). Local dopamine blockade failed to prevent muscimol generated eating or fearful behaviors, despite its previously reported ability to prevent DNQX-induced eating and fear, and its generally suppressive effects. Histograms bars below the maps show behavior as percent of vehicle (eating, A; treading, B) or percent of subjects (calls, escape attempts and bite attempts, C) for each behavior at rostrocaudal level as marked along the medial shell (error bars = SEM).

Figure 3. Summary maps of behavior and percent flipped by changes in ambience

Summary maps (A) show behavior produced by muscimol (top) or DNQX (bottom) in either the Home (left), Standard (middle) or Stressful (right) environments. Each subject (n=59) was designated as producing primarily appetitive (green symbols), defensive (red symbols) or mixed appetitive and defensive (yellow symbols) motivated behavior following DNQX microinjection. In a Standard environment, 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 and muscimol. Fearful distress calls, escape attempts and spontaneous emission of defensive treading-burying (criteria for including a site was a >500% increase in treading over vehicle levels, or emission of a defensive reaction to the experimenter) were primarily stimulated in caudal shell by DNQX. Mixed sites met criteria for both motivations. Testing in the Stressful environment shifted fearful behavior produced by DNQX into more rostral regions, whereas testing in the Home environment virtually eliminated all defensive behavior produced by DNQX. Muscimol produced a similar rostrocaudal gradient of eating and fear regardless of environment.

Figure 4. Maps of changes in motivational valence between the Home and Stressful environment

Summary maps show sites where DNQX (A) or muscimol (B) generated either intense eating behavior (top; green; > 200% of vehicle) in the Home environment but not in the Stress environment, or intense defensive behavior (bottom; red; > 500% of vehicle level treading or defensive reaction to the experimenter) in the Stressful environment but not in the Home environment. Sites mapped in white produced the same valence of behavior in both the Home and Stressful environment. Criteria for designating a site as appetitive was a >200% increase in eating, criteria for designating a site as defensive was a >500% increase in treading over vehicle levels, or emission of a defensive reaction to the experimenter. The percentage of rats that “flipped” between mainly defensive/mixed in the Stressful environment and purely appetitive in the Home environment was significantly greater in rats that received DNQX (C) than rats that received muscimol (D), and the number of rats who “flipped” when given muscimol was not significant.

Figure 5. Effect of changing environmental ambience on defensive treading produced by DNQX or muscimol

Fos plume maps (n=59) of the generation of defensive treading by muscimol (top) or DNQX (bottom) in the Home (left), Standard (middle), or Stressful (right) environments. Testing in the Stressful environment had no effect on muscimol-generated treading, but the Home environment nearly eliminated muscimol-generated treading. This may be due to the removal of all visual cues that the animals usually tread toward. DNQX treading was produced at more rostral locations in a Stressful environment, but was nearly eliminated by testing in a Home environment. Histogram bars show treading as percent of vehicle at each rostrocaudal level as marked along the medial shell (error bars = SEM).

Figure 6. Effect of changing environmental ambience on defensive reactions to the experimenter produced by DNQX or muscimol

Fos plume maps (n=59) of the generation of defensive reactions to the experimenter by muscimol (top) or DNQX (bottom) in the Home (left), Standard (middle), or Stressful (right) environments. Changing environment between the familiar Home or the aversive Stressful environments had no effect on defensive reactions produced by muscimol, which were robustly generated regardless of environment ambience. In the Stressful environment, defensive reactions to the experimenter produced by DNQX treading were generated at more rostral locations in shell, but were nearly eliminated by testing in a Home environment. Histogram bars show percent of rats emitting distress calls (yellow), distress calls and escape attempts (red) and distress calls, escape attempts and bite attempts (red) at each rostrocaudal level as marked along the medial shell.

Figure 7. Effect of changing environmental ambience on eating produced by DNQX or muscimol

Fos plume maps (n=59) of the generation of eating by muscimol (top) or DNQX (bottom) in the Home (left), Standard (middle), or Stressful (right) environments. Changing environmental ambience had inconsistent effects on eating produced by DNQX or muscimol. Histogram bars show treading as percent of vehicle at each rostrocaudal level as marked along the medial shell (error bars = SEM).

Figure 8. Fos plume analysis

Fos expression was assessed following microinjections of vehicle, muscimol, dopamine antagonists alone, or mixture of muscimol and dopamine antagonists. 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. For vehicle microinjections colors indicate levels of Fos expression of 3x (red), and 2x (orange) levels of Fos expression found in normal (uninjected) tissue. For drug microinjections, colors indicate levels of Fos expression of 1.5x (yellow), .75x (light blue), and .50x (darker blue) vehicle level Fos expression. Line graphs show that muscimol (red) reduced Fos expression approximately ~.35 mm away from the microinjection center, and that dopamine antagonists (black) and mixture (yellow) reduced Fos expression from .15 mm to .40 mm away from the microinjection center.