Novel Primate Model of Serotonin Transporter Genetic Polymorphisms Associated with Gene Expression, Anxiety and Sensitivity to Antidepressants

Genetic polymorphisms in the repeat upstream region of the serotonin transporter gene (SLC6A4) are associated with individual differences in stress reactivity, vulnerability to affective disorders, and response to pharmacotherapy. However, the molecular, neurodevelopmental and psychopharmacological mechanisms underlying the link between SLC6A4 polymorphisms and the emotionally vulnerable phenotype are not fully understood. Thus, using the marmoset monkey Callithrix jacchus we characterize here a new neurobiological model to help to address these questions. We first sequenced the marmoset SLC6A4 promoter and identified a double nucleotide polymorphism (−2053AC/CT) and two single-nucleotide polymorphisms (−2022C/T and −1592G/C) within the repeat upstream region. We showed their association with gene expression using in vivo quantitative PCR and with affective behavior using a primate test of anxiety (human intruder test). The low-expressing haplotype (AC/C/G) was linked with high anxiety while the high-expressing one (CT/T/C) was associated with an active coping strategy in response to threat. Pharmacological challenge with an acute dose of the selective serotonin reuptake inhibitor, citalopram, revealed a genotype-dependent behavioral response. While individuals homozygous for the high anxiety-related haplotype AC/C/G exhibited a dose-dependent, anxiogenic response, individuals homozygous for the low anxiety-related haplotype CT/T/C showed an opposing, dose-dependent anxiolytic effect. These findings provide a novel genetic and behavioral primate model to study the molecular, neurodevelopmental, and psychopharmacological mechanisms that underlie genetic variation-associated complex behaviors, with specific implications for the understanding of normal and abnormal serotonin actions and the development of personalized pharmacological treatments for psychiatric disorders.


Locomotion. Proportion of time spent in translational movements between locations (selfpropulsion involving all four limbs).
Head and body bobbing. Number of rapid and repetitive side-to-side movements of the upper body while sitting and staring at the object of interest (Barros, 2002).
Jumps. Number of jumps made to the front of the cage, towards the human intruder.
Number of vocalizations. Egg calls, associated with vigilant behavior (Bezerra and Souto, 2008). Tsik calls, uttered alone or in series, primarily mobbing calls (Cross and Rogers, 2006). Tsik-egg calls, usually one tsik followed by 1-3 eggs (Shiba et al, 2015). Tse calls, similar but distinguishable from tsik calls, emitted in less threatening situations. (Tse and Tseegg calls were highly correlated with one another so they were combined for purposes of analysis into Tse-like calls). The vocalizations were characterized using their sonograms (Fig.   S5D).
Vocalizations and head and body bobbing behavior were observed only in the presence of the human intruder.
For examples of the behaviors described above see Video S1 and Video S2. CT/C/G 3 (1,2) 0 0 Heterozygotes AC/C/G&CT/T/C 50 (28,22) 10 (4,6) 16 ( Table S1| Numbers of animals genotyped from our colony at the time (Population Genotypes column), numbers used in real time PCR assays (SLC6A4 Expression column), numbers in Human Intruder Test to measure anxiety phenotype (Anxiety phenotype column), and in the pharmacological study to measure anxiety in response to citalopram (Response to citalopram column). Numbers in () represent females and males, respectively.  Table S2| Primers SLC6A4-AF, SLC6A4-AR, SLC6A4-BF1, SLC6A4-BR2, SLC6A4-CF1, SLC6A4-CF2 and SLC6A4-CR were used to amplify three regions of the marmoset promoter (region A closer to Exon 1, B middle region and C further upstream exon 1). Primers IPCR-F1 and IPCR-R1 were used in inverse PCR to amplify the 5' end of the repeat region. Primers 5RACE5R, 5RACE6R and 5RACE7R were used as gene-specific, nested, reverse primers in the 5'RACE methods. Primers RPRF and RPRR were used for genotyping. Primers SeqF and SeqR were used to sequence the polymorphic region for genotyping. Primers SLC6A4-F and SLC6A4-R were used to amplify SLC6A4 RNA (gene of interest) and primers PBGD-F and PBGD-R to amplify PBGD RNA (reference gene) in real time PCR assays to assess SLC6A4 expression. CT/T/C 5 (2,3) CT/C/G 0 Heterozygotes AC/C/G&CT/T/C 12 (5,7) Total 26

Table S3|
Numbers of animals genotyped using genomic DNA from both hair follicles and brain tissue (brainstem). Numbers in () represent females and males, respectively.

Table S6|
Analysis of the effect of sex and age on the variables SLC6A4 relative gene expression, PC1 and PC2 in the human intruder test (HIT) and average distance in response to an acute dose of citalopram. First, sex has been analyzed as main factor, then as a second factor interacting with haplotype and finally added as a covariate. Age has been added as a covariate.   The 12 marmosets used in the pharmacological study with citalopram are a subgroup of this cohort.
Known genotype is indicated with a color code. Figure S2| Consensus sequence of the marmoset SLC6A4 promoter region. A 2.40 kb fragment from -2.3kb and the first exon was cloned, sequenced and aligned and a consensus sequence determined, accession number HG515029. The transcription start site that we determined experimentally is highlighted in black (+1A). Exon 1 is shaded yellow based on genebuild from the Ensembl database.