Adrenergic transmission facilitates extinction of conditional fear in mice

Abstract

Extinction of classically conditioned fear, like its acquisition, is active learning, but little is known about its molecular mechanisms. We recently reported that temporal massing of conditional stimulus (CS) presentations improves extinction memory acquisition, and suggested that temporal spacing was less effective because individual CS exposures trigger two opposing processes: (1) fear extinction, which is favored by CS massing, and (2) fear incubation (increase), which is favored by spacing. We here report the effects of manipulating the adrenergic system during massed or spaced CS presentations in fear-conditioned mice. We administered yohimbine (5 mg/kg), an alpha(2)-receptor antagonist, or propranolol (10 mg/kg), a beta-receptor antagonist, systemically prior to CS presentation sessions and recorded both short- and long-term changes in conditional freezing. Yohimbine treatment facilitated extinction of both cue and context fear with massed protocols. When given before spaced CS presentations, propranolol led to a persistent incubation of cue fear, whereas yohimbine led to persistent extinction, compared with vehicle-treated animals, which showed no change in fear. These results suggest that norepinephrine positively modulates the formation of fear extinction memories in mice. They also provide clear evidence that spaced CS presentations trigger both fear-reducing (extinction) and fear-increasing (incubation) mechanisms.

Figure 1

Yohimbine facilitates long-term cue fear extinction with massed CS exposures. Separate experiments examined the effects of vehicle, yohimbine, and propranolol on extinction with 0 (A), 5 (B), 10 (C), 20 (D), or 30 (E) nonreinforced CS presentations (seven to eight mice/group). The data shown represent freezing during a single 2-min test CS given 1 d after the extinction sessions (drug free). The final summary panel (F) shows the results of the five experiments with fear normalized to freezing in groups of mice that were injected with vehicle and not presented with any extinction CSs on Day 2 (retention control mice, RC). (^*) p < 0.05 versus RC mice; (+) p < 0.05 versus vehicle-extinction mice.

Figure 2

Yohimbine facilitates long-term context fear extinction. Separate experiments examined the effects of vehicle, yohimbine, and propranolol on extinction with 0 (A), 20 (B), 40 (C), or 60 (D) min of context exposure (eight to 12 mice/group). The data shown represent freezing during a single 5-min context exposure given 1 d after the extinction sessions (drug free). The final summary panel (E) shows the results of the four experiments with fear normalized to freezing in groups of mice that were injected with vehicle and not exposed to the feared context on Day 2 (retention control mice, RC). (^*) p < 0.05 versus RC mice; (+) p < 0.05 versus vehicle-extinction mice.

Figure 3

Yohimbine facilitates extinction of cue and context fear acutely. CS-elicited freezing during the Day 2 extinction sessions from all of the experiments shown in Figures 1 and 2, 20 min after injection of vehicle, yohimbine, or propranolol (27-32 mice/group). Because mice were treated identically up to the fifth CS of the cue fear experiments (A) and the tenth minute of exposure in the context fear experiments (B), the data were combined for this analysis.

Figure 4

Yohimbine has no effect on cue fear extinction when injected immediately after massed CS presentations. Separate experiments examined the effect of injecting vehicle, yohimbine, or propranolol after 10 (A) or 20 (B) massed CS presentations (eight mice/group). The data shown represent freezing during a single 2-min CS given 1 d after the extinction sessions (drug free). Retention control (RC) mice received no CS presentations and were injected with vehicle on Day 2.

Figure 5

Yohimbine leads to extinction, whereas propranolol leads to incubation, of cue fear when given in conjunction with spaced CS presentations. Separate experiments examined the effect of vehicle, yohimbine, or propranolol on the effect of spaced CS presentations after weak (A,B; see Materials and Methods) or strong (C,D; see Materials and Methods) cue fear conditioning (eight mice/group). In each experiment, 1 d after acquisition, mice were injected with vehicle, yohimbine, or propranolol and presented with seven CSs (20 min ITI). (A,C) Freezing during the spaced CS sessions 20 min after injections. (B,D) Freezing 1 d later during the final 5-min CS tests (drug free). (^*) p < 0.05 versus RC mice; (+) p < 0.05 versus vehicle-exposure mice.

Figure 6

No effect of yohimbine or propranolol on gross locomotor activity. Separate groups of mice were injected with vehicle, yohimbine, or propranolol 20 min before placement in a novel chamber (eight mice/group). The total distance traveled was monitored by an automated system and expressed in arbitrary units.