L-type voltage-gated calcium channels are required for extinction, but not for acquisition or expression, of conditional fear in mice

Abstract

It has been shown recently that extinction of conditional fear does not depend acutely on NMDA-type glutamate receptors, although other evidence has led to the hypothesis that L-type voltage-gated calcium channels (LVGCCs) play a role in conditional fear. We therefore tested the role of LVGCCs in the acquisition, expression, and extinction of conditional fear of cue and context in mice. Using systemic injections of two LVGCC inhibitors, nifedipine and nimodipine, which both effectively cross the blood-brain barrier, we show that LVGCCs are essential for the extinction, but not for the acquisition or expression, of conditional fear in mice.

Fig. 1.

Nifedipine does not affect cue fear acquisition.A, Experimental design (7–8 mice per group).Open circles, Vehicle; filled circles, nifedipine. B, Freezing during the five cues that preceded each shock for mice injected with nifedipine (40 mg/kg dose shown, 20 min pretreatment) or vehicle. C, Freezing 24 hr later during a 2 min, drug-free CS test. D, Freezing during the five cues that preceded each shock for mice injected with nifedipine (40 mg/kg dose shown, 50 min pretreatment) or vehicle. Also included is freezing during the 2 min stimulus-free period after the last CS–US pairing (Ctxt). E, Freezing 24 hr later during a 2 min acclimation period (Pre-CS), followed by a 2 min, drug-free CS test. F, Freezing during a 2 min, drug-free CS test, 24 hr after injections (50 min pretreatment) and a single cue–shock pairing. *p< 0.05 versus vehicle.

Fig. 2.

Nimodipine does not affect cue fear acquisition, although recall appears to be state dependent. A, Experimental design (8 mice per group). B, Freezing during the five cues that preceded each shock for mice injected with nimodipine (15 mg/kg, 20 min pretreatment) or vehicle.C, Freezing 24 hr later during a 2 min, drug-free CS test. D, Freezing during a second state-dependence test (3 hr after test 1) after reinjections of drug or vehicle (20 min pretreatment). E–G, Repeat of the above experiment (A–C) using a weaker training protocol (2 cue–shock pairings). *p < 0.05 versus vehicle.

Fig. 3.

Recall of fear is partially state dependent with nimodipine. A, Experimental design (8 mice per group; between-subjects design). B, Freezing during the two cues that preceded each shock for mice injected with nimodipine (16 mg/kg, 20 min pretreatment; n = 16) or vehicle (n = 16). Freezing 24 hr later during a 2 min CS test, 20 min after drug or vehicle injections. One-half of the mice from each of the treatment groups of the previous day received nimodipine (16 mg/kg) or vehicle. *p < 0.05 versus 0–0 group; +p < 0.05 versus 16–0 group.

Fig. 4.

LVGCC inhibitors do not prevent acquisition of context fear. A, Experimental design (7–8 mice per group). B, Freezing shown in 2 min blocks during the entire 12 min session for mice injected with nifedipine (40 mg/kg, 50 min pretreatment) or vehicle. Unsignaled foot shocks occurred at the 2nd, 4th, 6th, 8th, and 10th minutes. C, Freezing 24 hr later during a 5 min drug-free context exposure. D,E, Identical experiment conducted with nimodipine (16 mg/kg, 20 min pretreatment).

Fig. 5.

Nifedipine blocks extinction, but not expression, of cued fear. A, Experimental design. Extinction sessions began 24 hr after cue fear acquisition (5 cue–shock pairings). B, Freezing during the first 15 CS presentations, after injections of nifedipine (40 mg/kg dose shown, 20 min pretreatment) or vehicle. C, Freezing 24 hr later during a 2 min, drug-free CS test (n values = 8 for extinction groups). Retention control mice (RC;n = 16) were injected with vehicle and placed in the extinction chamber on day 2 but were not exposed to any CS.D, E, Identical experiment with a longer pretreatment (50 min) of a single nifedipine dose (40 mg/kg) or vehicle (n values = 8). Freezing was also scored during a 2 min acclimation period (Pre-CS). *p < 0.05 versus retention control; +p < 0.05 versus vehicle–extinction.

Fig. 6.

Nimodipine blocks extinction, but not expression, of cued fear. A, Experimental design (12 mice per group). Extinction sessions began 24 hr after cue fear acquisition (5 cue–shock pairings). B, Freezing during the first 15 CS presentations, after injections of nimodipine (15 mg/kg, 20 min pretreatment) or vehicle. C, Freezing 24 hr later during a 2 min, drug-free CS test. Retention control mice (RC) were injected with vehicle and placed in the extinction chamber on day 2 but were not exposed to any CS. D, E, Identical experiment with several doses of nimodipine. Only the 16 mg/kg dose is shown for acute extinction. *p < 0.05 versus retention control; +p < 0.05 versus vehicle–extinction.

Fig. 7.

Extinction with LVGCC blockers is not state dependent. A, Experimental design (7–8 mice per group). The 3 d experiment was identical to previous experiments (Figs. 5, 6), with the exception that drugs and vehicle were injected 20 min before both day 2 extinction and the day 3 test.B, Freezing during a 2 min CS test, 24 hr after extinction. *p < 0.05 versus retention control (RC); +p < 0.05 versus vehicle–extinction.

Fig. 8.

LVGCC inhibitors block extinction, but not expression, of context fear. A, Experimental design (16 mice per group). Extinction was conducted 24 hr after context fear acquisition (5 unsignaled foot shocks, 2 min ITI). B, Freezing in 5 min blocks for the first 30 min of context exposure (120 total). Mice were injected with nifedipine (40 mg/kg, 50 min pretreatment), nimodipine (16 mg/kg, 20 min pretreatment), or vehicle. Retention control mice were injected with vehicle and placed in dissimilar chambers for 120 min. C, Freezing 24 hr later during a 5 min drug-free context fear test (data were lost for 1 mouse in the nimodipine group attributable to a camera failure).

Fig. 9.

Effects of nifedipine and nimodipine on spontaneous locomotor activity in an open field. A, Experimental design (6 mice per group). B,Left, Total distance traveled during a 60 min session in a novel chamber (arbitrary units). Mice were injected 20 min before the session with nifedipine (40 mg/kg), nimodipine (16 mg/kg), or vehicle.Right, An identical test of the same mice 24 hr later in the drug-free state. p < 0.05 versus vehicle.