Behavioral phenotyping of a rat model of the BDNF Val66Met polymorphism reveals selective impairment of fear memory

Abstract

The common brain-derived neurotrophic factor (BDNF) Val66Met polymorphism is associated with reduced activity-dependent BDNF release and increased risk for anxiety disorders and PTSD. Here we behaviorally phenotyped a novel Val66Met rat model with an equivalent valine to methionine substitution in the rat Bdnf gene (Val68Met). In a three-day fear conditioning protocol of fear learning and extinction, adult rats with the Met/Met genotype demonstrated impaired fear memory compared to Val/Met rats and Val/Val controls, with no genotype differences in fear learning or extinction. This deficit in fear memory occurred irrespective of the sex of the animals and was not seen in adolescence (4 weeks of age). There were no changes in open-field locomotor activity or anxiety measured in the elevated plus maze (EPM) nor in other types of memory measured using the novel-object recognition test or Y-maze. BDNF exon VI expression in the dorsal hippocampus was higher and BDNF protein level in the ventral hippocampus was lower in female Val/Met rats than female Val/Val rats, with no other genotype differences, including in total BDNF, BDNF long, or BDNF IV mRNA. These data suggest a specific role for the BDNF Met/Met genotype in fear memory in rats. Further studies are required to investigate gene-environment interactions in this novel animal model.

Fig. 1. Fear conditioning in adult rats.

Fear learning in adult rats across three CS tone periods on Day 1 (A) and CS3 only (B) shows no difference between genotypes. While there was no difference between genotypes in extinction learning as shown by the decrease in freezing across four periods of 10 CS tones on Day 2 (C), fear memory was shown to be decreased in Met/Met rats during CS1–10 (D). There were also no genotype differences in extinction memory on Day 3 when looking at average freezing across four periods of 10 CS tones (E) or when comparing freezing for CS1–10 only (F). Data are presented as mean ± SEM. Individual data points are only shown for (B), (D), and (F) for clarity. There were no significant interactions with sex therefore all data are presented as sexes combined (males closed symbols, females open symbols). **p < 0.001 vs. Val/Val, *p < 0.05 vs. Val/Met.

Fig. 2. Fear conditioning in adolescent rats.

Fear learning across three CS tone periods on Day 1 (A), fear memory and extinction learning on Day 2 (B) and extinction memory on Day 3 (C). There were no significant differences between genotypes at any stage of testing in adolescent rats. Data presented as mean ± SEM. Individual data points for select periods can be found in supplementary Fig. 5. All data are presented as sexes combined.

Fig. 3. Anxiety-like behavior and learning and memory in adult rats.

Distance traveled (A) and time spent in the center of the open field (B), as well as total number of arm entries (C) and time spent in the open arms of the EPM (D) demonstrate no differences in baseline locomotor activity or anxiety-like behavior between genotypes. Time interacting with a novel compared to familiar object in the NORT (E) and time spent in novel compared to familiar arms of the Y-maze (F) showed that, while rats showed a significant preference for the novel object or arm, there were no differences between genotypes. Data presented as mean ± SEM with individual data points for all rats. There were no significant interactions between genotype and sex therefore all data are presented as sexes combined (males closed symbols, females open symbols). ***p < 0.001.

Fig. 4. BDNF gene expression in the dorsal hippocampus as measured by RT-qPCR.

We measured BDNF total mRNA (A), BDNF long 3′ UTR (B), BDNF exon IV (C) and BDNF exon VI (D, E) mRNA. Female Val/Met rats showed higher BDNF VI expression than Val/Val controls. Data are presented as % of controls (Male Val/Val rats) showing mean ± SEM of sexes combined with individual data points for all rats (males closed symbols, females open symbols). *p < 0.05 compared to Val/Val rats.

Fig. 5. BDNF levels in the ventral hippocampus as measured by ELISA.

Female Val/Met rats showed lower BDNF protein levels than Val/Val controls. Data are presented as mean ± SEM with individual data points for all rats. *p < 0.05 compared to Val/Val rats.