Impaired fear extinction in serotonin transporter knockout rats is associated with increased 5-hydroxymethylcytosine in the amygdala

Abstract

Aims: One potential risk factor for posttraumatic stress disorder (PTSD) involves the low activity (short; s) allelic variant of the serotonin transporter-linked polymorphic region (5-HTTLPR), possibly due to reduced prefrontal control over the amygdala. Evidence shows that DNA methylation/demethylation is crucial for fear extinction in these brain areas and is associated with neuronal activation marker c-Fos expression. We hypothesized that impaired fear extinction in serotonin transporter knockout (5-HTT^-/- ) rats is related to changes in DNA (de) methylation and c-Fos expression in the prefrontal cortex (PFC) and/or amygdala.

Methods: 5-HTT^-/- and 5-HTT^+/+ rats were subjected to fear extinction. 2 hours after the extinction session, the overall levels of DNA methylation (5-mC), demethylation (5-hmC), and c-Fos in fear extinction and nonfear extinction rats were measured by immunohistochemistry.

Results: 5-HTT^-/- rats displayed decreased fear extinction. This was associated with reduced c-Fos activity in the infralimbic PFC. In the central nucleus of the amygdala, c-Fos immunoreactivity was increased in the fear extinction group compared to the no-fear extinction group, regardless of genotype. 5-hmC levels were unaltered in the PFC, but reduced in the amygdala of nonextinction 5-HTT^-/- rats compared to nonextinction wild-type rats, which caught up to wild-type levels during fear extinction. 5-mC levels were stable in central amygdala in both wild-type and 5-HTT^-/- extinction rats. Finally, c-Fos and 5-mC levels were correlated with the prelimbic PFC, but not amygdala.

Conclusions: In the amygdala, DNA demethylation, independent from c-Fos activation, may contribute to individual differences in risk for PTSD, as conferred by the 5-HTTLPR s-allele.

Keywords: 5-hydroxymethylcytosine (5-hmC); 5-methylcytosine (5-mC); DNA (de) methylation; epigenetics; fear extinction; serotonin transporter knockout.

Figure 1

Rats were exposed to 5 tone‐shock pairings. Data are expressed as mean ± SEM of percentage of freezing during tone (CS) presentation

Figure 2

Representative image of 5‐hydroxymethylcytosine (5‐hmC) immunoreactivity in rat brains. A, Prefrontal cortex (bregma 3.72 mm), the red box indicates the region for image analysis; B, manual delineation of the prelimbic prefrontal cortex (PrL) and infralimbic prefrontal cortex (IL). C, Amygdala region (bregma ‐1.92 mm), the red box indicates the region for image analysis; D, basolateral amygdala (BLA) and central nucleus of the amygdala (CA). Both 5‐methylcytosine (5‐mC) (not shown) and 5‐hmC showed a similar pattern of immunoreactivity. Previous colocalization studies have found that the densely stained 5‐mC 25 and 5‐hmC26 signals reflect nuclear staining. Abbreviations: a, the azygous pericallosal artery; CPu, caudate and putamen; fmi, forceps minor of the corpus callosum

Figure 3

Data represent mean percentage of freezing (± SEM) in 5‐HTT ^+/+ and 5‐HTT ^−/− rats during the 2 min prior to CS‐exposure (baseline, B), and during CS‐exposure. CSs were presented during 8 (1‐8) extinction trial blocks of 3 trials each. *P < 0.05 and **P < 0.01

Figure 4

c‐Fos‐ir in 5‐HTT ^+/+ and 5‐HTT ^−/− rats exposed to the fear conditioned stimulus in the absence of footshock in the test chamber (fear extinction group) or exposed to the test chamber only (nonfear extinction group). Data represent the mean relative percentage of matched nonextinction groups ± SEM at 2 h after the start of extinction session in the PrL (A), IL (B), CA (C) BLA (D). *Bonferroni‐corrected, P < 0.05

Figure 5

5‐hmC‐ir in 5‐HTT ^+/+ and 5‐HTT ^−/− rats exposed to the fear conditioned stimulus in the absence of footshock in the test chamber (fear extinction group) or rats exposed to the test chamber only (nonfear extinction group). Percentage ir (of nonextinction group set at 100%). Data represent the mean of 5‐hmC‐ir expressed as percentage of matched nonextinction groups ± SEM in the PrL (A), IL (B), CA (C) and BLA (D). *Bonferroni‐corrected, P < 0.05

Figure 6

5‐mC‐ir in 5‐HTT ^+/+ and 5‐HTT ^−/− rats exposed to the fear conditioned stimulus in the absence of footshock in the test chamber (fear extinction group) or exposed to the test chamber only (nonfear extinction group). Percentage ir (of nonextinction group set at 100%). Data represent the mean of 5‐mC‐ir expressed as percentage of matched nonextinction groups ± SEM in the PrL (A), IL (B), CA (C) BLA (D). *Bonferroni‐corrected, P < 0.05