Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear

Abstract

Dihydropyridine (DHP) L-type Ca(2+) channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. Ca(V)1.2 and Ca(V)1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive Ca(V)1.2 LTCCs (Ca(V)1.2DHP(-/-) mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in Ca(V)1.2DHP(-/-) mice, indicating that it is mediated by Ca(V)1.2, but not by Ca(V)1.3 LTCCs. Supporting this conclusion, Ca(V)1.3-deficient mice (Ca(V)1.3(-/-)) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral Ca(V)1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in Ca(V)1.2DHP(-/-) mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the Ca(V)1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly Ca(V)1.3) is not sufficient to accelerate extinction of conditioned fear in mice.

Figure 1.

Ca_V1.2 LTCCs modulate contextual fear memory extinction. (A) Nifedipine (40 mg/kg) blocked extinction of contextual fear in WT mice (n = 6–7). (B) Nifedipine and BayK (4 mg/kg) had no effect on extinction in Ca_V1.2DHP^−/− mice (n = 7). Data are presented as mean ± SEM. Statistical analysis was performed using 2-way ANOVA, *P < 0.05, **P < 0.01.

Figure 2.

Ca_V1.2 LTCCs modulate cued fear memory extinction. (A) Nifedipine (40 mg/kg) blocked extinction of cued fear in WT mice (n = 6–7). (B) Nifedipine and BayK (4 mg/kg) had no effect on extinction in Ca_V1.2DHP^−/− mice (n = 8–9). Note that no, or only very low levels of freezing were observed in all groups of cued-conditioned mice upon exposure to context alone (trial 0). Data are presented as mean ± SEM. Statistical analysis was performed using 2-way ANOVA. WT mice treated with vehicle showed significantly enhanced freezing behavior compared with nifedipine-treated animals from the sixth presentation onward (6th CS, P < 0.05; 7th–15th CS, P < 0.001). Asterisks indicating significant differences were omitted for clarity.

Figure 3.

Nifedipine influences spontaneous locomotor activity in the conditioning chamber. (A) Total distance traveled in an open field during a 40-min session. WT mice were injected 50 min before the session with vehicle (n = 7) or 40 mg/kg of nifedipine (n = 7). (B) Total distance traveled in the conditioning chamber during a 40-min session. WT mice were injected 50 min before the session with vehicle (n = 10) or 40 mg/kg of nifedipine (n = 9). Data are presented as mean ± SEM. Statistical analysis was performed using a Mann Whitney U-test. **P < 0.01, WT nifedipine vs. vehicle. (C) Distance traveled in the conditioning chamber broken down to 5-min time bins. WT mice were injected 50 min before the session with vehicle (n = 9) or 40 mg/kg of nifedipine (n = 10). Data are presented as mean ± SEM. Statistical analysis was performed using 2-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, WT nifedipine vs. vehicle.

Figure 4.

Ca_V1.3 LTCCs are not involved in fear memory extinction. Ca_V1.3^−/− (n = 12) as the WT animals (n = 11) showed a similar, normal extinction of contextual fear memory. Data are presented as mean ± SEM. No significant differences were found (2-way ANOVA).

Figure 5.

Inhibition of fear memory extinction is not centrally mediated. (A) Experimental design. (B,C) Percentage of freezing during contextual fear acquisition and extinction sessions. WT mice were injected i.c.v. 10 min before the extinction session with vehicle (n = 7) or 50 μg/kg of nifedipine (n = 7). (D,E) Percentage of freezing during contextual fear acquisition and extinction sessions. WT mice were injected i.c.v. 10 min before the extinction session with vehicle (n = 10) or 200 μg/kg of nifedipine (n = 9). (F,G) % of freezing during contextual fear acquisition and extinction sessions. WT mice were injected i.c.v. 10 min before the extinction session with vehicle (n = 10) or 1 mg/kg of nifedipine (n = 8). (H) Percent of freezing during a long-term extinction session. Vehicle- and nifedipine- (1 mg/kg) treated groups when tested 24 h later in drug-free conditions. Data are presented as mean ± SEM. Statistical analysis was performed using a 2-way ANOVA. *P < 0.05, WT nifedipine vs. vehicle.