Nicotine disrupts safety learning by enhancing fear associated with a safety cue via the dorsal hippocampus

Abstract

Learned safety, a learning process in which a cue becomes associated with the absence of threat, is disrupted in individuals with post-traumatic stress disorder (PTSD). A bi-directional relationship exists between smoking and PTSD and one potential explanation is that nicotine-associated changes in cognition facilitate PTSD emotional dysregulation by disrupting safety associations. Therefore, we investigated whether nicotine would disrupt learned safety by enhancing fear associated with a safety cue. In the present study, C57BL/6 mice were administered acute or chronic nicotine and trained over three days in a differential backward trace conditioning paradigm consisting of five trials of a forward conditioned stimulus (CS)+ (Light) co-terminating with a footshock unconditioned stimulus followed by a backward CS- (Tone) presented 20 s after cessation of the unconditioned stimulus. Summation testing found that acute nicotine disrupted learned safety, but chronic nicotine had no effect. Another group of animals administered acute nicotine showed fear when presented with the backward CS (Light) alone, indicating the formation of a maladaptive fear association with the backward CS. Finally, we investigated the brain regions involved by administering nicotine directly into the dorsal hippocampus, ventral hippocampus, and prelimbic cortex. Infusion of nicotine into the dorsal hippocampus disrupted safety learning.

Keywords: Learned safety; backward conditioning; hippocampus; nicotine; prelimbic cortex.

Figure 1

Design schematics for training and testing of backwards trace conditioned safety and fear. (a) Mice were trained over three days in context A with five trials. Exploded view shows temporal arrangement of cues during training, with a 30 s light co-terminating with 2 s footshock (dark gray with thunderbolt) followed by a 20 s trace interval and a 30 s tone (light gray). To test for learned safety, summation testing consisted of alternating presentations of light or light/tone. (b) To assess backwards trace conditioned fear mice where trained similarly, but on testing where only presented with the backward paired tone conditioned stimulus.

Figure 2

Cannula placement in hippocampus. Cannula tips location in relation to bregma in coronal sections (a) dorsal and (b) ventral hippocampus. Representative images of placements in dorsal (c) and ventral (d) hippocampus.

Figure 3

(a) Cannula placement in prelimbic cortex with (b) representative image. Cannula tips location in relation to bregma in coronal sections.

Figure 4

The effects of acute and chronic nicotine on backward trace conditioned safety. (a) Mice treated with acute nicotine, 0.09 mg/kg, did not freeze less during summation testing of the Light/Tone-Safety compound cue, but saline treated mice froze significantly less (data shown as % freezing in mice) during compound Light/Tone compared with Light alone (n = 9 per group). Error bars indicate SEM, * indicates within drug group difference between Light-Danger and Light/Tone-Safety, p < 0.05. (b) Mice in both chronic saline and chronic nicotine, 12.6 mg/kg/ per day, demonstrated reduced freezing during presentation of Light/Tone-Safety compared with Light alone (n = 14 per group). Mice treated with chronic nicotine showed greater generalized freezing during the Pre-CS period prior to stimulus presentation. Error bars indicate SEM, * indicates a main effect of testing condition Light-Danger versus Light/ Tone-Safety, p < 0.05. # indicates significant difference between chronic saline and nicotine during Pre-CS, p < 0.05. CS: conditioned stimulus

Figure 5

Backward trace fear conditioning control mice did not show any difference in freezing during the Pre-CS versus CS conditioning, indicating no fear association with the backward trace conditioned CS. In contrast, acute nicotine, 0.09 mg/kg, treatment resulted in greater freezing to the CS compared with Pre-CS, indicating a fear association with the backward trace conditioned CS (n = 7–8). Error bars indicate SEM, * indicates significant planned comparison paired samples t-test between Pre-CS and CS, p < 0.05. CS: conditioned stimulus

Figure 6

(a) Nicotine, in the dorsal hippocampus dose-dependently disrupts safety learning. Mice treated with 0.09 μg/side nicotine fail to show learned safety, with freezing levels similar between Light-Danger and Light/Tone-Safety. Mice treated with saline and nicotine, 0.18 μg/side, showed learned safety with decreased freezing during Light/Tone-Safety (n = 9–7 per group). Error bars indicate SEM, * indicates significant within group planned comparison paired samples t-test between Light-Danger and Light/Tone-Safety, p < 0.05. (b) Nicotine in the ventral hippocampus has no effect on backwards trace conditioned safety; all mice showed decreased freezing during Light/ Tone-Safety compared with Light-Danger (n = 9–7 per group). Error bars indicate SEM, * indicates significant within group difference between Light and Light/Tone, p < 0.05. (c) Nicotine in prelimbic cortex dose-dependently alters freezing to Light/Tone compound. Mice administered saline 0.09 μg/side and nicotine 0.18 μg/side all showed significantly less freezing during presentation of compound Light/Tone-Safety compared with Light-Danger. However, mice treated with 0.09 μg/side nicotine froze significantly more than saline treated mice during compound Light/Tone-Safety presentation (n = 8–9 per group). Error bars indicate SEM, * indicates planned comparison paired samples t-test p < 0.05, # indicates post-hoc Tukey’s HSD contrast p < 0.05. CS: conditioned stimulus; HSD: honest significant difference