Genetic background modulates phenotypes of serotonin transporter Ala56 knock-in mice

Abstract

Background: Previously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes.

Methods: To explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity.

Results: One consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior.

Conclusions: Our findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be especially sensitive to changes in SERT function. Finally, these results provide a platform for the identification of genes that may modulate the risk of ASD in humans.

Figure 1

Activity and anxiety-like behavior in male B6 SERT Ala56 knock-in mice. A) Time (mean, standard error of the mean) spent ambulating during the first 5 minutes in the open field for wildtype littermate controls (G/G, n = 20), heterozygous (A/G, n = 16), and homozygous (A/A, n = 20) SERT Ala56 mice; B) Time spent in the center (>1 cm away from the side) of the open field divided by total time in the open field; C) Time spent in the open zone divided by total time in the elevated zero maze; D) Time spent in the light chamber divided by total time in the light–dark test. *P <0.05.

Figure 2

Social behaviors and pup ultrasonic vocalizations in male B6 SERT Ala56 knock-in mice. A) Time (mean, standard error of the mean) spent in the social stimulus chamber (novel mouse), center chamber, or inanimate stimulus chamber (novel object) in the three-chamber sociability test for wildtype littermate controls (G/G, n = 20), heterozygous (A/G, n = 16), and homozygous (A/A, n = 20) SERT Ala56 mice; B) Time spent within 1 cm of the social stimulus or inanimate stimulus in the three-chamber sociability test; C) Total number of wins (frontward exit) for pairings of wildtype littermate controls (G/G) and homozygous SERT Ala56/Ala56 (A/A) mice in the tube test confrontation; D) Total number of wins for heterozygous (A/G) versus homozygous SERT Ala56 (A/A) mice when paired in the tube test; E) Total number of wins for wildtype (G/G) versus heterozygous SERT Ala56 (A/G) mice when paired in the tube test; F) Latency to attack a wildtype 129S4 intruder mouse introduced into the cage of a resident subject mouse who had been singly housed for 7 days (n = 16 per genotype); G) Pup vocalizations upon separation from the dam for 5 min at postnatal day 7 for wildtype littermate controls (G/G, n = 36), heterozygous (A/G, n = 65), and homozygous SERT Ala56 (A/A, n = 33) mouse pups. *P <0.05.

Figure 3

Home cage, exploratory, and sensory behavior in male B6 SERT Ala56 knock-in mice. A) Time (mean, standard error of the mean) spent performing individual non-sleep behaviors over 24 hours in the home cage in wildtype littermate controls (G/G, n = 12) and homozygous (A/A, n = 12) SERT Ala56 mice; B) Number of bouts of hanging behavior in 24 hours in the home cage; C) Ratio of spontaneous alternations, defined as entering a different arm than on the previous two entries, divided by total entries in the Y maze (n = 20 per genotype); D) Force threshold at which the paw is moved upon bending of the von Frey filaments (n = 20 per genotype).

Figure 4

Assays of the serotonin system in B6 SERT Ala56 knock-in mice. A) Whole blood serotonin levels (mean, standard error of the mean) in adult male wildtype littermate control (G/G, n = 6) and homozygous SERT Ala56 (A/A, n = 6) mice; B) Whole blood serotonin levels in male pups at postnatal day 7 (n = 12 per genotype); C) Synaptosome uptake of 20 nM ^3H5-HT during a 5-minute assay at 37°C, (n = 6 per genotype, with 3 replicates for each subject); D) Synaptosome uptake of 100 nM ^3H5-HT during a 5-minute assay at 37°C (n = 6 per genotype, with 3 replicates for each subject); E) Head twitch response to injection of the 5-HT₂ agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) during a 15-minute observation period (n = 12 per genotype); F) Decrease in rectal temperature from baseline in response to injection of the mixed 5-HT_1A/7 agonist 8-OH-DPAT (n = 10 per genotype).