Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusions of D-cycloserine as assessed with fear-potentiated startle in rats

Abstract

NMDA receptor antagonists block conditioned fear extinction when injected systemically and also when infused directly into the amygdala. Here we evaluate the ability of D-cycloserine (DCS), a partial agonist at the strychnine-insensitive glycine-recognition site on the NMDA receptor complex, to facilitate conditioned fear extinction after systemic administration or intra-amygdala infusions. Rats received 10 pairings of a 3.7 sec light and a 0.4 mA footshock (fear conditioning). Fear-potentiated startle (increased startle in the presence vs the absence of the light) was subsequently measured before and after 30, 60, or 90 presentations of the light without shock (extinction training). Thirty non-reinforced light presentations produced modest extinction, and 60 or 90 presentations produced nearly complete extinction (experiment 1). DCS injections (3.25, 15, or 30 mg/kg) before 30 non-reinforced light exposures dose-dependently enhanced extinction (experiment 2) but did not influence fear-potentiated startle in rats that did not receive extinction training (experiment 3). These effects were blocked by HA-966, an antagonist at the glycine-recognition site (experiment 4). Neither DCS nor HA-966 altered fear-potentiated startle when injected before testing (experiment 5). The effect of systemic administration was mimicked by intra-amygdala DCS (10 microg/side) infusions (experiment 6). These results indicate that treatments that promote NMDA receptor activity after either systemic or intra-amygdala administration promote the extinction of conditioned fear.

Fig. 1.

Parametric evaluation of different amounts of extinction training. A, Timeline of the behavioral procedures for experiment 1. B. Percent fear-potentiated startle measured 24 hr before (pretest) and 24 hr after (post-test) extinction training or context exposure. The control group was tested 2 d after the pretest, with no intervening exposures. One session of non-reinforced cue exposure produced only modest levels of extinction. Two or three sessions more completely extinguished the fear response. *p < 0.05 versus context exposure group;⁺p < 0.05 versus control group.

Fig. 2.

Dose–response function for the effect of DCS on extinction. A, Timeline of the behavioral procedures for experiment 2. B, Percent fear-potentiated startle measured 24 hr before and 24 hr after a single session of extinction training in rats injected with saline or DCS (3.25, 15, or 30 mg/kg, i.p.) 30 min before non-reinforced cue exposure. DCS dose-dependently facilitated extinction learning. *p < 0.05 versus saline after extinction.

Fig. 3.

Effect of DCS in nonextinguished rats.A, Timeline of the behavioral procedures for experiment 3. B, Percent fear-potentiated startle measured 24 hr before and 24 hr after extinction training. Saline or DCS (15 mg/kg, i.p.) was administered 30 min before a single session of either extinction training (cue exposure) or context-alone exposure. Fear-potentiated startle was significantly lower in rats that received DCS plus extinction training compared with rats that received saline plus extinction training. Fear-potentiated startle was not appreciably affected by DCS in rats that did not receive extinction training. *p < 0.05 versus saline plus extinction training.

Fig. 4.

Effect of the strychnine-insensitive glycine-recognition site antagonist HA-966 on extinction and on the facilitation of extinction by DCS. A, Timeline of the behavioral procedures for experiment 4. B, Percent fear-potentiated startle measured 24 hr before (pre-extinction test) and 24 hr after (postextinction test) extinction training. Saline or HA-966 (6 mg/kg, i.p.) was administered 10 min before a second injection of saline or DCS, followed 30 min later by a single session of extinction training. HA-966 completely blocked the effects of DCS but did not, on its own, noticeably influence extinction at this dose. *p < 0.05 versus all other groups.

Fig. 5.

Effect of pretest DCS and HA-966 administration on fear-potentiated startle. A, Timeline of the behavioral procedures for experiment 5. B, Percent fear-potentiated startle measured 24 hr after fear conditioning in rats receiving pretest injections of saline, DCS (15 mg/kg), or HA-966 (6 mg/kg). Neither drug had any discernible effect on fear-potentiated startle.

Fig. 6.

Cannula tip placements transcribed onto atlas plates adapted from Paxinos and Watson (1997). The distance from bregma is indicated to the left; nuclei within the plane of section are identified to the right. BL, Basolateral amygdaloid nucleus; BLV, basolateral amygdaloid nucleus, ventral part; BM, basomedial amygdaloid nucleus; CeL, central amygdaloid nucleus, lateral division; CeM, central amygdaloid nucleus, medial division; ic, internal capsule;LA, lateral amygdaloid nucleus.

Fig. 7.

Effect of intra-amygdala DCS infusions.A, Timeline of the behavioral procedures for experiment 3. B, PBS or d-cycloserine (10 μg/side) was infused into the amygdala 15 min before extinction training. Other rats received DCS without extinction training. When tested 24 hr later, fear-potentiated startle was significantly lower in rats that received DCS plus extinction training compared with rats that received PBS plus extinction training. Fear-potentiated startle was not appreciably affected by DCS in rats that did not receive extinction training. For the group that received DCS without extinction training, the mean percent potentiation was calculated with and without data from a single outlier who had an atypically high percent potentiation score. *p < 0.05 versus all other groups.

Fig. 8.

Composite figure showing absolute startle values for all rats receiving drugs before extinction training. Black bars indicate baseline startle amplitude on noise-alone trials;white bars indicate startle amplitude on light–noise trials. The difference between these two (i.e., fear-potentiated startle) is indicated by the striped bars. In no case were significant differences found in baseline startle during the fear-potentiated startle test 24 hr after drug administration. Moreover the statistical results were similar when absolute difference scores (i.e., startle amplitude on light–noise trials minus startle amplitude on noise-alone trials) rather than percent potentiation scores were analyzed. *p < 0.05 (except Experiment 4,p = 0.087) versus leftmost bars.