Nicotine modulates contextual fear extinction through changes in ventral hippocampal GABAergic function

Abstract

Numerous studies have attributed the psychopathology of anxiety and stress disorders to maladaptive behavioral responses such as an inability to extinguish fear. Therefore, understanding neural substrates of fear extinction is imperative for developing more effective therapies for anxiety and stress disorders. Although several studies indicated a role for cholinergic transmission and nicotinic acetylcholine receptors (nAChRs) in anxiety and stress disorder symptomatology, very little is known about the specific contribution of nAChRs in the fear extinction process. In the present study, we first examined the involvement of several brain regions essential for fear extinction (i.e., dorsal and ventral hippocampus, dHPC and vHPC; infralimbic, IL, and prelimbic, PL of the medial prefrontal cortex, mPFC; basolateral nucleus of the amygdala, BLA) in the impairing effects of a nAChR agonist, nicotine, on contextual fear extinction in mice. Our results showed that systemic administration of nicotine during contextual fear extinction increased c-fos expression in the vHPC and BLA while not affecting dHPC, IL or PL. In line with these results, local nicotine infusions into the vHPC, but not dHPC, resulted in impaired contextual fear extinction. Interestingly, we found that local nicotine infusions into the PL also resulted in impairment of contextual fear extinction. Second, we measured the protein levels of the GABA synthesizing enzymes GAD65 and GAD67 in the dHPC and vHPC during contextual fear extinction. Our results showed that in the group that received acute nicotine, both GAD65 and GAD67 protein levels were downregulated in the vHPC, but not in dHPC. This effect was negatively correlated with the level of freezing response during fear extinction suggesting that the downregulated GAD65/67 levels were associated with disrupted fear extinction. Finally, using c-fos/GAD65/67 double immunofluorescence, we showed that nicotine mainly increased c-fos expression in non-GABAergic ventral hippocampal cells, indicating that acute nicotine increases vHPC excitability. Overall, our results suggest that acute nicotine's impairing effects on fear extinction are associated with ventral hippocampal disinhibition. Therefore, these results further our understanding of the interaction between nicotine addiction and anxiety and stress disorders by describing novel neural mechanisms mediating fear extinction.

Keywords: Fear extinction; GABA; Hippocampus; Nicotinic receptors; PTSD.

Figure 1.. Acute nicotine administration during contextual fear extinction increases vHPC and BLA c-fos expression.

A. Normalized %freezing scores across initial testing and 3 extinctions sessions (n=6 per group). B. Acute nicotine increased the number of c-fos IR cells within the vHPC and vCA1, VCA3, and vDG but did not affect c-fos expression in the dHPC and its subregions. Right panel shows representative c-fos immunohistochemistry images from dHPC and vHPC of the Saline-Extinction and Nicotine-Extinction groups. C. BLA c-fos expression was increased in the group that received acute nicotine during contextual fear extinction. Right panel shows representative c-fos immunohistochemistry images from BLA of the Saline-Extinction and Nicotine-Extinction groups. D. Acute nicotine did not affect IL or PL c-fos expression, but extinction training increased c-fos expression in these brain regions. Right panel shows representative c-fos immunohistochemistry images from IL and PL of the Saline-Extinction and Nicotine-Extinction groups. E. Correlation plots between dHPC, vHPC, PL, IL, and BLA c-fos IR cell numbers and normalized %freezing scores for each subject. Error bars show standard error of the mean. *p<0.05; **p<0.01; ***p<0.001 compared to Saline-Extinction controls.

Figure 2.. Local nicotine infusion into the vHPC and PL result in contextual fear extinction deficits.

A. Local nicotine infusion into the dHPC did not affect normalized %freezing scores across 4 extinctions sessions (n=7 per group). B. vHPC local nicotine infusions disrupted contextual fear extinction (n=7 per group). C. IL nicotine infusions had no effect on contextual fear extinction (n=4–6 per group). D. Local nicotine infusions into the PL also resulted in impaired extinction of contextual fear. (n=5 per group). Right panels show placement of guide cannulas. Error bars show standard error of the mean. *p<0.05; **p<0.01; ***p<0.001 compared to Saline controls.

Figure 3.. Acute nicotine decreased protein levels of vHPC GAD65 and GAD67 following the 3^rd extinction session.

A. GAD65, B. GAD67, and C. ChAT levels in the dHPC and vHPC were not altered by acute nicotine administration after the 1^st contextual fear extinction session (n=7–8 per group). Acute nicotine decreased vHPC levels of D. GAD65 and E. GAD67, but not F. ChAT (n=6–8 per group). dHPC levels of these proteins were not altered. Lower panels show representative immunoblots for each target protein and brain region in the Nicotine and Saline treated groups. G. Correlation plots between vHPC GAD67, GAD65, ChAT optical density values and normalized %freezing scores after the 3^rd extinction session for each subject. Error bars show standard error of the mean. *p<0.05 compared to Saline controls.

Figure 4.. vHPC c-fos expression in non-GAD cells was enhanced by acute nicotine administration during contextual fear extinction.

A. Behavioral results showing that acute nicotine injections increased normalized %freezing response to the context and impaired contextual fear extinction. B. Representative image showing c-fos-GAD65/67 double immunofluorescent labeled cells in the hippocampus. Arrows show cells double labeled for GAD65/67 and c-fos, while arrowheads show cells labeled for only GAD65/67. C. Number of dHPC c-fos and c-fos+GAD65/67 labeled cells was not altered by acute nicotine during contextual fear extinction (n=6–7 per group). D. Acute nicotine increased non-GAD65/67 c-fos expression in the vHPC without altering c-fos expression in the GAD65/67 cells. E. dHPC c-fos+GAD/c-fos ratios were not affected by acute nicotine. F. Acute nicotine decreased c-fos+GAD/c-fos ratios in the vHPC during contextual fear extinction. Error bars show standard error of the mean. *p<0.05; **p<0.01; ***p<0.001 compared to Saline controls.