Induction and Expression of Fear Sensitization Caused by Acute Traumatic Stress

Abstract

Fear promotes adaptive responses to threats. However, when the level of fear is not proportional to the level of threat, maladaptive fear-related behaviors characteristic of anxiety disorders result. Post-traumatic stress disorder develops in response to a traumatic event, and patients often show sensitized reactions to mild stressors associated with the trauma. Stress-enhanced fear learning (SEFL) is a rodent model of this sensitized responding, in which exposure to a 15-shock stressor nonassociatively enhances subsequent fear conditioning training with only a single trial. We examined the role of corticosterone (CORT) in SEFL. Administration of the CORT synthesis blocker metyrapone prior to the stressor, but not at time points after, attenuated SEFL. Moreover, CORT co-administered with metyrapone rescued SEFL. However, CORT alone without the stressor was not sufficient to produce SEFL. In these same animals, we then looked for correlates of SEFL in terms of changes in excitatory receptor expression. Western blot analysis of the basolateral amygdala (BLA) revealed an increase in the GluA1 AMPA receptor subunit that correlated with SEFL. Thus, CORT is permissive to trauma-induced changes in BLA function.

Figure 1

SEFL causes anxiety phenotype on the open field test, exaggerated startle to white noise, and a depressive-like phenotype in the forced swim test. (a) Experimental design. We developed a modified version of the open field test that has been validated for anxiety testing (Godsil and Fanselow, 2004; Godsil et al, 2005). The open field test consisted of three phases: (1) 4 min of dark, (2) 4 min of light and (3) 4 min of dark. Locomotion, defined as the number of crossovers, was quantified during the 12-min test. (b) Open field test. Pre-exposure to shock significantly decreased the number of crossovers during phase 1, the first four dark minutes of the open field, p<0.005. There was no effect of pre-exposure to shock on the number of crossovers during phase 2, minutes 5–8. Pre-exposure to shock decreased in the number of crossovers during phase 3, p<0.05. Therefore, previously shocked rats showed reduced exploratory activity than controls when placed in a dark open field. When bright lights turned on at one end, the rats retreated to the dark end and reduced activity similar to controls. However, when the lights went out, unlike controls, they remained in the dark corner and did not increase exploration. The open field test did not affect the context tests in either the stress or conditioning contexts (data not shown). (c) Experimental design for exaggerated startle test. Instead of 1-shock in conditioning context on Day 2, white noise (92 dB, 1 s) was given. (d) Freezing (+SEM) for baseline on Day 2, 5 min post-noise on Day 2, and context test on Day 3. Previously stressed rats showed a pronounced freezing response to a loud noise; this reaction was not seen in unstressed controls, **p<0.01 (mixed-factorial ANOVA). Upon return to the noise context the following day without noise, the stressed rats showed a small but statistically reliable increase in freezing relative to controls, *p<0.05. (e) Experimental design. Training (Day 4) and testing (Day 11) sessions for forced swim test were 5 min. Water temperature was 77 °F, and the apparatus dimensions are 74 cm × 36.5 cm. (f) Time spent immobilized (sec+SEM) on Day 4, and time spent immobilized (sec+SEM) on Day 11. *p<0.05 (one-way ANOVA).

Figure 2

Pre-stressor administration of metyrapone attenuates SEFL, and co-administration of metyrapone and CORT restores SEFL. (a) Experimental design for metyrapone administration. (b) Plasma CORT levels (ng/ml; mean+SEM) after the 15-shock trauma on Day 1. Main effect of stress, p<0.001; linear trend analysis for stressed animals, p<0.05 (two-way ANOVA, followed by planned contrasts). (c) The mean+SEM of freezing percentage in stress context test on Day 4. Drug × stress, p<0.0001 (two-way ANOVA), linear trend analysis for stressed animals, p<0.0001. (d) The mean+SEM of freezing percentage during the generalization test in the conditioning context on Day 5. No significant differences were found for any main effect or interaction (two-way ANOVA). (e) Freezing (mean+SEM) in conditioning context on Day 7. Drug × stress interaction, p<0.05, linear trend analysis for stressed animals, p<0.0005 (two-way ANOVA, followed by planned contrasts). (f) Experimental design for metyrapone and CORT co-administration. (g) Freezing (mean+SEM) in the stress context on Day 4. First digit in group designations=metyrapone dose (0 or 150 mg/kg); second=CORT dose (0 or 10 mg/kg). ***p<0.0001 (overall one-way ANOVA, followed by planned contrasts). (h) Freezing (mean+SEM) in conditioning context on Day 7. First digit in group designations=metyrapone dose (0 or 150 mg/kg); second=CORT dose (0 or 10 mg/kg). **p<0.01; ***p<0.0001 (overall one-way ANOVA, followed by planned contrasts).

Figure 3

Basolateral amygdala inactivation or intra-basolateral infusions of mifepristone reduces SEFL. (a) Experimental design for intra-amygdalar muscimol infusions. (b) Cannulae placement for amygdalar inactivation. (c) Freezing (mean+SEM) in the conditioning context on Day 3. **p<0.003 (one-way ANOVA, followed by planned contrasts). (d) Experimental design for mifepristone infusions. (e) Cannulae placement for mifepristone infusions. (f) Freezing (mean+SEM) in the conditioning context on Day 3. **p<0.001 (one-way ANOVA, followed by planned contrasts).

Figure 4

Increases in GluA1 after SEFL treatment are prevented by metyrapone. (a) Experimental design. (b, d, f) Representative western blot images of GluA1, GluA2, GluN1, and GAPDH from the BLA of stressed and unstressed rats receiving vehicle or 150 mg/kg metyrapone. (c) Mean GluA1: GAPDH OD ratios (+SEM). Main effect of drug, p<0.005, **p<0.01 (one-way ANOVA, followed by a priori planned comparisons). (e) Mean GluA2: GAPDH OD ratios (+SEM). (g) Mean GluN1: GAPDH OD ratios (+SEM).