The role of nicotinic acetylcholine receptors in the medial prefrontal cortex and hippocampus in trace fear conditioning

Abstract

Acute nicotine enhances multiple types of learning including trace fear conditioning but the underlying neural substrates of these effects are not well understood. Trace fear conditioning critically involves the medial prefrontal cortex and hippocampus, which both express nicotinic acetylcholine receptors (nAChRs). Therefore, nicotine could act in either or both areas to enhance trace fear conditioning. To identify the underlying neural areas and nAChR subtypes, we examined the effects of infusion of nicotine, or nicotinic antagonists dihydro-beta-erythroidine (DHβE: high-affinity nAChRs) or methyllycaconitine (MLA: low-affinity nAChRs) into the dorsal hippocampus, ventral hippocampus, and medial prefrontal cortex (mPFC) on trace and contextual fear conditioning. We found that the effects of nicotine on trace and contextual fear conditioning vary by brain region and nAChR subtype. The dorsal hippocampus was involved in the effects of nicotine on both trace and contextual fear conditioning but each task was sensitive to different doses of nicotine. Additionally, dorsal hippocampal infusion of the antagonist DHβE produced deficits in trace but not contextual fear conditioning. Nicotine infusion into the ventral hippocampus produced deficits in both trace and contextual fear conditioning. In the mPFC, nicotine enhanced trace but not contextual fear conditioning. Interestingly, infusion of the antagonists MLA or DHβE in the mPFC also enhanced trace fear conditioning. These findings suggest that nicotine acts on different substrates to enhance trace versus contextual fear conditioning, and that nicotine-induced desensitization of nAChRs in the mPFC may contribute to the effects of nicotine on trace fear conditioning.

Figure 1. Effects of nicotine infusion into the dorsal hippocampus on fear conditioning

A. Dorsal hippocampal nicotine infusion (0.09 μg/side contextual, 0.09 and 0.18 μg/side cued) at training and testing dose-dependently enhanced trace and contextual fear conditioning (n = 11–13). B. Infusion of 0.09 μg/side nicotine into the dorsal hippocampus enhanced 2-pairing trace fear conditioning, but not contextual conditioning, while 0.35 μg/side nicotine enhanced contextual but not trace conditioning (n = 8). C. Infusion of 0.35 μg/side nicotine into the dorsal hippocampus enhanced contextual but not 2-pairing delay cued fear conditioning (n= 8). D. Infusion of 0.09 μg/side nicotine into the dorsal hippocampus at training or at both training and testing enhanced both contextual and trace fear conditioning; however, infusion at testing had no effect (n = 8). Significant difference (p < 0.05) from the saline-infused control group denoted with (*); data are reported as mean ± standard error of the mean. Representations of cannula placements alongside data demonstrate that all infusions were directed into the dorsal hippocampus.

Figure 2. Effects of nicotinic antagonist infusion into the dorsal hippocampus on trace fear conditioning

A. Antagonism of high-affinity nAChRs (DHβE: 4.50 and 9.00 μg/side) in the dorsal hippocampus at training and testing produced dose-dependent deficits in trace fear conditioning (n = 7 – 8). B. Infusion of 9.00 μg/side DHβE into the dorsal hippocampus at training produced deficits in trace conditioning, while infusion prior to testing had no effect (n = 8). Significant difference (p < 0.05) from control group denoted with (*); data are reported as mean ± standard error of the mean. Representations of cannula placements to right of data demonstrate that all infusions were directed into the dorsal hippocampus.

Figure 3. Effects of nicotine infusion into the ventral hippocampus on fear conditioning

A. Infusion of nicotine into the ventral hippocampus at training and testing produced dose-dependent deficits in contextual (0.18 and 0.35 μg/side) and trace (0.35 μg/side) conditioning (n = 6 – 8). B. Infusion of nicotine into the ventral hippocampus produced deficits in contextual (0.09 and 0.35 μg/side) and trace (0.35 μg/side) conditioning in a 2-pairing trace fear conditioning paradigm (n = 8 – 9). C. Infusion of nicotine (0.09 or 0.35 μg/side) into the ventral hippocampus produced deficits in contextual fear conditioning, but had no effect on delay cued conditioning, using a 2-pairing 30 second CS training protocol (n = 8). D. Infusion of 0.35 μg/side nicotine into the ventral hippocampus at training or testing, or at both training and testing produced deficits in both trace and contextual fear conditioning (n = 8). Significant difference (p < 0.05) from the saline-infused control group denoted with (*); data are reported as mean ± standard error of the mean. Representations of cannula placements to right of data demonstrate that all infusions were directed into the ventral hippocampus.

Figure 4. Effects of nicotine infusion into the medial prefrontal cortex on fear conditioning

A. Infusion of nicotine (0.09 and 0.18 μg/side) into the medial prefrontal cortex at training and testing enhanced trace fear conditioning, but not contextual fear conditioning ( n = 13 – 14). B. Infusion of 0.09 μg/side nicotine into the medial prefrontal cortex at training and testing enhanced trace fear conditioning, but not contextual fear conditioning in 2-pairing trace fear conditioning (n = 7 – 8). C. Infusion of nicotine (0.09 or 0.35 μg/side) had no effect on contextual or delay cued fear conditioning (2 CS-US pairings, CS 30 second; n = 8). D. Infusion of 0.09 μg/side nicotine into the medial prefrontal cortex at training or at training and testing enhanced trace conditioning, but infusion at testing had no effect (n = 8). Significant difference (p < 0.05) from the saline-infused control group denoted with (*); data are reported as mean ± standard error of the mean. Representations of cannula placements to right of data demonstrate that all infusions were directed into the medial prefrontal cortex.

Figure 5. Effects of nicotinic antagonist infusion into the medial prefrontal cortex on trace fear conditioning

A. Infusion of DHβE into the medial prefrontal cortex at training and testing dose-dependently (9.00 and 18.00 μg/side) enhanced trace fear conditioning but not contextual conditioning (n = 8 – 9). B. Infusion of 9.00 μg/side DHβE into the medial prefrontal cortex at training enhanced trace conditioning, but infusion at testing produced deficits in both trace and contextual conditioning (n = 8). C. Infusion of MLA (13.50 and 27.00 μg/side) into the medial prefrontal cortex at training and testing enhanced trace fear conditioning but did not affect contextual conditioning (n = 9 – 10). D. Infusion of 13.50 μg/side MLA into the medial prefrontal cortex at training enhanced trace conditioning, but infusion at testing produced deficits in both trace and contextual conditioning (n = 8). Significant difference (p < 0.05) from the saline-infused control group denoted with (*); data are reported as mean ± standard error of the mean. Representations of cannula placements alongside data demonstrate that all infusions were directed into the medial prefrontal cortex.