Kappa opioid receptor signaling in the basolateral amygdala regulates conditioned fear and anxiety in rats

Abstract

Background: The kappa opioid receptor (KOR) system contributes to the prodepressive and aversive consequences of stress and is implicated in the facilitation of conditioned fear and anxiety in rodents. Here, we sought to identify neural circuits that mediate KOR system effects on fear and anxiety in rats.

Methods: We assessed whether fear conditioning induces plasticity in KOR or dynorphin (the endogenous KOR ligand) messenger RNA (mRNA) expression in the basolateral (BLA) and central (CeA) nuclei of the amygdala, hippocampus, or striatum. We then assessed whether microinfusions of the KOR antagonist JDTic (0-10 μg/side) into the BLA or CeA affect the expression of conditioned fear or anxiety. Finally, we examined whether fear extinction induces plasticity in KOR mRNA expression that relates to the quality of fear extinction.

Results: Fear conditioning upregulated KOR mRNA in the BLA by 65% and downregulated it in the striatum by 22%, without affecting KOR levels in the CeA or hippocampus, or dynorphin levels in any region. KOR antagonism in either the BLA or CeA decreased conditioned fear in the fear-potentiated startle paradigm, whereas KOR antagonism in the BLA, but not the CeA, produced anxiolytic-like effects in the elevated plus maze. Effective fear extinction was associated with a 67% reduction in KOR mRNA in the BLA.

Conclusions: These findings suggest that fear conditioning and extinction dynamically regulate KOR expression in the BLA and provide evidence that the BLA and CeA are important neural substrates mediating the anxiolytic-like effects of KOR antagonists in models of fear and anxiety.

Figure 1. Control data for quantitative PCR analysis of gene expression

(A and B) Micrographs of coronal rat brain sections showing prodynorphin immunoreactivity (methods in the Supplement) and the approximate location and size of bilateral tissue punches (1mm³) taken from (A) the hippocampus (solid-line) and (B) the central nucleus of the amygdala (CeA, solid-line), basolateral nucleus of the amygdala (BLA, dashed-line), and striatum (STR, dotted-line). (C) Rats that received Light+Shock training (n=12) showed increased fear-potentiated startle (FPS) compared to those that received Light Alone training (n=6) (mean ± S.E.M.; Student’s t test). (D) Levels of glutamic acid decarboxylase 65 and 67 kDa (GAD65, GAD67) and prodynorphin (PDyn) were higher in tissue punches taken from the CeA compared to the BLA (n=20; mean ± S.E.M.; Mann-Whitney tests). *P<0.05 vs. Light Alone (C), **P<0.01 vs. BLA tissue punches (D).

Figure 2. Fear conditioning increases the relative quantity of kappa opioid receptor (KOR) mRNA in the basolateral nucleus of the amygdala (BLA)

(A) Fear conditioning increased KOR mRNA and decreased glutamic acid decarboxylase 65 kDa (GAD65) mRNA in the BLA, without affecting prodynorphin (PDyn) or glutamic acid decarboxylase 67 kDa (GAD67) mRNA expression (n=10/group; mean ± S.E.M.; Mann-Whitney tests). Fear conditioning did not affect mRNA quantities in the central nucleus of the amygdala (CeA) (B) or in the hippocampus (HIP) (C), but decreased KOR mRNA in the striatum (STR), without affecting PDyn mRNA (D). *P<0.05, **P<0.01 vs. Light Alone control.

Figure 3. Kappa opioid receptor (KOR) antagonism in the basolateral (BLA) and central (CeA) nuclei of the amygdala affects the time course of fear-potentiated startle (FPS)

Microinfusions of the KOR antagonist JDTic or vehicle were administered 24 hr before FPS testing. The effects of JDTic in the BLA (A), CeA (B), and striatum (STR) (C) on FPS were most apparent within the first 20 min (blocks 1–2) of the 30-min test session (n=5–12/group; mean ± S.E.M.). FPS was significantly reduced at the 30-min time point (block 3) in rats that received microinfusions of vehicle into the BLA (A) (Student’s t tests, # denotes significant difference from block 2, P<0.05). Significant reductions in FPS were not detected in vehicle treated rats in the CeA (B) and STR (C) groups, which may be due to increased levels of fear in these groups. The magnitude of FPS in block 1 tended to be higher in vehicle treated rats with cannulas in CeA (B) than in the BLA (A), and the extinction profile for vehicle treated rats with cannulas in the STR (C) suggests an inverted U-shaped function relating FPS to fear that is characteristic of high fear levels (58).

Figure 4. Kappa opioid receptor (KOR) antagonism in the basolateral (BLA) and central (CeA) nuclei of the amygdala decreases fear-potentiated startle (FPS)

Microinfusions of the KOR antagonist JDTic or vehicle were administered 24 hr prior to FPS testing. JDTic in the BLA (A) or CeA (B) decreased FPS (mean ± S.E.M.; Dunnett’s tests), without affecting baseline startle (not shown). (C) JDTic in the striatum (STR) did not affect FPS (Student’s t test). (D) Summary of cannula tip placements in the BLA (black circles; n=10–12/group), CeA (gray triangles; n=5–8/group), or STR (gray squares; n=6–7/group). *P<0.05 vs. vehicle. Images in D published in The Rat Brain in Stereotaxic Coordinates, 3rd ed. (93) and reprinted with permission, Copyright Elsevier (1996).

Figure 5. Kappa opioid receptor (KOR) antagonism in the basolateral (BLA) and central (CeA) nuclei of the amygdala increases open arm exploration in the elevated plus maze (EPM)

Microinfusions of the KOR antagonist JDTic or vehicle were administered 24 hr prior to EPM testing. (A) JDTic in the BLA increased the percentage of time rats spent in the open arms and the number of open arm entries (mean ± S.E.M.; Student’s t tests). JDTic in the CeA (B) or striatum (STR) (C) did not affect the percentage of time rats spent in the open arms or the number of open arm entries. None of the drug treatments affected closed arm entries or maze crosses (not shown). (D) Summary of cannula tip placements in the BLA (black circles; n=7–9/group), CeA (gray triangles; n=10/group), or STR (gray squares; n=7–9/group). *P<0.05, **P<0.01 vs. vehicle. Images in D published in The Rat Brain in Stereotaxic Coordinates, 3rd ed. (93) and reprinted with permission, Copyright Elsevier (1996).

Figure 6. Effective fear extinction decreases the relative quantity of kappa opioid receptor (KOR) mRNA in the basolateral nucleus of the amygdala (BLA)

Rats were subjected to fear conditioning (10 light-shock pairings) and tested 24 hr later for fear-potentiated startle (FPS). One day later, rats were given extinction training (60 presentations of the light alone) and the next day they were retested for FPS. Rats with the lowest (“good” extinction; n=4) or highest (“poor” extinction; n=4) levels of FPS were killed immediately after testing by decapitation and gene expression in the BLA was analyzed using qPCR. (A) Rats that had good extinction learning had significantly less FPS during the retest than rats with poor extinction learning (mean ± S.E.M.). (B) Rats that had good extinction learning had 67% less KOR mRNA in the BLA than those that had poor extinction learning. *P<0.05.