CGRP antagonist infused into the bed nucleus of the stria terminalis impairs the acquisition and expression of context but not discretely cued fear

Abstract

Calcitonin gene-related peptide (CGRP) infusions into the bed nucleus of the stria terminalis (BNST) evoke increases in startle amplitude and increases in anxiety-like behavior in the plus maze. Conversely, intra-BNST infusions of the CGRP antagonist CGRP₈₋₃₇ block unconditioned startle increases produced by fox odor. Here we evaluate the contribution of CGRP signaling in the BNST to the development and expression of learned fear. Rats received five pairings of a 3.7-sec light and footshock and were tested for fear-potentiated startle one or more days later. Neither pre-training (Experiment 1) nor pre-test (Experiment 2) infusions of the CGRP antagonist CGRP₈₋₃₇ (800 ng/BNST) disrupted fear-potentiated startle to the 3.7-sec visual cue. However, in both experiments, CGRP₈₋₃₇ infusions disrupted baseline startle increases that occurred when rats were tested in the same context as that in which they previously received footshock (Experiment 3). Intra-BNST CGRP₈₋₃₇ infusions did not disrupt shock-evoked corticosterone release (Experiment 4). These data confirm previous findings implicating BNST CGRP receptors in fear and anxiety. They extend those results by showing an important contribution to learned fear and, specifically, to fear evoked by a shock-associated context rather than a discrete cue. This pattern is consistent with previous models of BNST function that have posited a preferential role in sustained anxiety as opposed to phasic fear responses. More generally, the results add to a growing body of evidence indicating behaviorally, possibly clinically, relevant modulation of BNST function by neuroactive peptides.

Figure 1.

Cannula tip placements of rats whose data are included in behavioral and corticosterone analyses. Circles indicate intra-BNST placements; squares indicate placements in Experiment 3 that did not meet inclusion criteria. The approximate distance posterior to bregma in millimeters is indicated to the lower right of each figure. Coronal sections are adopted from Paxinos and Watson (1998), with permission from Elsevier © 1998.

Figure 2.

Sequence and outline of behavioral procedures for Experiments 1 and 2.

Figure 3.

Startle amplitude across trials before and after fear conditioning. For all vehicle-infused rats from Experiments 1 and 2, mean startle amplitudes for the first 20 trials of the Pre-Conditioning Test are shown, together with mean startle amplitude from all 20 trials of the Post-Conditioning Context Test. The difference between startle amplitude from the pre- to post-conditioning test is also shown, together with 95% confidence intervals (i.e., overlap of the error bars with 0 on the x-axis indicates that the difference for that trial was not statistically significant). The increase in startle from the pre- to post-conditioning context test was most pronounced during the first 2 min of the test, but by the fifth startle response had become unreliable.

Figure 4.

Effect of intra-BNST CGRP_8–37 infusions on fear learning and expression. (A) When infused prior to training, the CGRP receptor antagonist significantly disrupted startle increases during the first 2 min of context exposure (i.e., the first four trials), though this difference was not maintained over the course of the entire session. (B) When infused prior to testing, the CGRP receptor antagonist significantly disrupted startle increases during the first 2 min of context exposure as well as the full test (note that the scaling of B is different to accommodate the greater range of scores). (*) P < 0.05 vs. vehicle-infused rats.

Figure 5.

Fear-potentiated startle as a function of similarity between shock and test contexts. Rats received light–shock pairings in one of two distinctive contexts (A or B) and were tested in B (i.e., the same context as for all other rats in this study). The baseline startle increase observed in rats trained and tested in the same context was largely eliminated when rats were trained and tested in different contexts, suggesting that they are primarily attributable to context fear. There was little, if any, effect on fear-potentiated startle to the 3.7-sec visual stimulus.

Figure 6.

Effect of intra-BNST CGRP_8–37 on stress-induced HPA activation. ANOVA indicated a significant effect of Stress (pre-shock to post-shock) but no significant Treatment effect or Treatment × Stress interaction. Thus, CGRP_8–37 did not disrupt the increase in plasma corticosterone that followed footshock.