Threat imminence dictates the role of the bed nucleus of the stria terminalis in contextual fear

Abstract

Recent work indicates that the bed nucleus of the stria terminalis (BNST) is critically involved in the regulation of conditioned fear responses to unpredictable threats. Here we examined whether the involvement of the BNST in contextual fear conditioning in male rats depends on the imminence of shock after placement in the conditioning chamber. Specifically, we hypothesized that the BNST supports contextual freezing after conditioning with delayed, but not imminent, footshock (relative to placement in the context). Rats were implanted with cannulae targeting the BNST and underwent a contextual fear conditioning procedure in which a single footshock unconditioned stimulus (US) was delivered either 1 min or 9 min after the rat was placed in the context; the rats received a total of four identical conditioning sessions over two days, all with equivalent exposure to the context. Contexts associated with either imminent or delayed US onsets produced distinct patterns of freezing and shock-induced activity but freezing in each case was context-dependent. Reversible inactivation of the BNST reduced the expression of contextual freezing in the context paired with delayed (9 min), but not imminent (1 min), footshock onset. Implications of these data are discussed in the light of recent conceptualizations of BNST function, as well as for anxiety behaviors.

Keywords: Anxiety; Bed nucleus of the stria terminalis; Context; Fear; Rat.

Figure 1.. Behavioral design for testing context discrimination following conditioning using imminent or delayed shock onset.

Rats were randomly assigned to undergo four separate sessions of contextual fear conditioning in which shock was delivered either 1 minute (1-MIN) or 9 minutes (9-MIN) after placement in the conditioning chamber. Each conditioning session was 10 min. After conditioning, rats received counterbalanced retrieval tests in the conditioning context (Ctx A) or a novel context (Ctxt B) for 10 min in the absence of shock.

Figure 2.. Context conditioning using imminent or delayed shock.

Mean percentage freezing (± s.e.m.) of 1-MIN (n = 16, top) and 9-MIN (n = 16, bottom) rats for each minute of the four conditioning sessions (I-IV). Shaded areas indicate the post-shock period for during each session.

Figure 3.. Context discrimination after imminent or delayed shock.

(A) Mean percentage freezing (± s.e.m.) of 1-MIN (n = 16) and 9-MIN (n = 16) rats during counterbalanced retrieval tests in a novel context (Context B) or the conditioning context (Context A). (B) Mean percentage freezing (± s.e.m.) of 1-MIN and 9-MIN rats across the entire test in each context. Shaded lines denote individual performance of each animal. (C) Mean freezing percentages (± s.e.m.) in Context B were subtracted from mean responding in Context A to generate a discrimination index for 1-MIN and 9-MIN rats. *p < 0.05.

Figure 4.. Shock-induced activity and conditioned freezing topography in animals trained with imminent or delayed shock.

(A) Left panel shows mean values of shock-induced activity (± s.e.m.) of 1-MIN (n = 16) and 9-MIN (n = 16) rats (freezing for these rats shown in Figure 2). Right panel shows the percentage change in magnitude of shock-induced activity (± s.e.m.) from shock 1 to shock 4. Note that the dotted line (at 100%) indicates the level at which there is no change in shock-induced activity. (B) Left panel depicts latency (in seconds; ± s.e.m.) of 1-MIN and 9-MIN rats to exhibit their first freezing bout in the conditioning context during testing (corresponding to Figure 3). Right panel shows mean duration of each bout (± s.e.m.) for the entire test. *p < 0.05.

Figure 5.. Behavioral design for contextual fear conditioning with imminent or delayed footshock.

BNST-cannulated animals were randomly assigned to undergo four separate sessions of contextual fear conditioning using 1-MIN (1-min placement-to-shock interval) or 9-MIN (9-min placement-to-shock interval) unsignaled shock. Each conditioning session was 10 min. After conditioning, 1-MIN and 9-MIN rats were infused with DRUG (MUS or NBQX) (collapsed into a single group in the figures) or vehicle (VEH) into the BNST prior to a 20-min shock-free retrieval session in the conditioning context.

Figure 6.. Bilateral cannula placements in the BNST.

Representative photomicrograph of a thionin-stained coronal section (40 μm) depicting bilateral cannula tracts and injector tips in the BNST (top image). Bottom image shows the positions of injector tips for each animal for each group included in the final analyses of the experiment (approximate borders of the BNST are outline by the dotted red line). 1-MIN-VEH (n = 13); 1-MIN-DRUG (n = 11); 9-MIN-VEH (n = 15); 9-MIN-DRUG (n = 9).

Figure 7.. Context conditioning using imminent or delayed shock onset.

Mean percentage freezing (± s.e.m.) of 1-MIN (top) and 9-MIN (bottom) rats at each minute of each conditioning session (I-IV). Shaded areas indicate minutes post-shock for both procedures at each session. Drug and vehicle assignments are shown for comparison but no BNST infusions occurred during this pre-test phase. 1-MIN-VEH (n = 13); 1-MIN-DRUG (n = 11); 9-MIN-VEH (n = 15); 9-MIN-DRUG (n = 9). *p < 0.05.

Figure 8.. BNST inactivation attenuates freezing in a context conditioned with delayed but not imminent shock onset.

Mean percentage freezing (± s.e.m.) of 1-MIN (top) and 9-MIN (bottom) rats in the conditioning context following intra-BNST microinfusions of VEH or DRUG. 1-MIN-VEH (n = 13); 1-MIN-DRUG (n = 11); 9-MIN-VEH (n = 15); 9-MIN-DRUG (n = 9). *p < 0.05.