Supplementary Figure Legends Van Der Doelen Et Al., 2014

Van der Doelen et al.: Early life adversity and serotonin transporter gene variation interact at the level of the adrenal gland to affect the adult hypothalamo-pituitary-adrenal axis.

Supplementary figure legends Van der Doelen et al., 2014.

Figure S1. Body weight (± SEM) development from weaning, postnatal day (PND) 22, until adulthood (PND79). Independently, early life stress (ELS) and serotonin transporter (5-HTT) genotype affected body weight. The maternally separated (MS180, n = 50) animals developed a significantly lower body weight compared to control animals (MS0, n = 45) from PND30 onwards (A). Furthermore, while serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-, n = 24) rats had significantly lower body weight than 5-HTT heterozygous knockout (5-HTT+/-, n = 42) and wild-type (5-HTT+/+, n = 29) rats at every time point (B). *p < 0.05.

Figure S2. Glucocorticoid receptor (GR) mRNA levels in the paraventricular nucleus of the hypothalamus of serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-), heterozygous knockout (5-HTT+/-) and wild-type (5-HTT+/+) rats (n = 5-9) exposed to daily 3 h separations (MS180) or a control treatment (MS0). Factorial ANOVA revealed a main effect of 5-HTT genotype (G: p 0.05) and post-hoc testing showed that 5-HTT-/- rats have significantly lower GR mRNA levels than 5-HTT+/+ rats (p 0.05). Data were normalized to the average of the MS0-5-HTT+/+ group.

Figure S3. Corticotropin-releasing factor (CRF), mineralocorticoid receptor (MR) and FK506-binding protein 51 (FKBP5) mRNA levels in the paraventricular nucleus of the hypothalamus of serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-), heterozygous knockout (5-HTT+/-) and wild-type (5-HTT+/+) rats (n = 5-9) exposed to 3 h daily separations (MS180) or a control treatment (MS0). Data were normalized to the average of the MS0-5-HTT+/+ group.

Figure S4. Pro-opiomelanocortin (POMC), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA levels in the pituitary glands of serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-), heterozygous knockout (5-HTT+/-) and wild-type (5-HTT+/+) rats (n = 5-9) exposed to 3 h daily separations (MS180) or a control treatment (MS0). Data were normalized to the average of the MS0-5-HTT+/+ group.

Figure S5. Corticotropin-releasing factor receptor 1 and FK506-binding protein 51 (FKBP5) mRNA levels in the pituitary glands of serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-), heterozygous knockout (5-HTT+/-) and wild-type (5-HTT+/+) rats (n = 5-9) exposed to daily 3 h separations (MS180) or a control treatment (MS0). Factorial ANOVA revealed a main effect of early life treatment (E: p 0.05), with 5-HTT+/+ and 5-HTT-/- rats showing decreased FKBP5 mRNA levels after exposure to early life stress. Data were normalized to the average of the MS0-5-HTT+/+ group.

Figure S6. Steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) mRNA levels in the adrenal glands of serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-), heterozygous knockout (5-HTT+/-) and wild-type (5-HTT+/+) rats (n = 7) exposed to 3 h daily separations (MS180) or a control treatment (MS0). The mRNA levels of both StAR and 3β-HSD1 were found to be significantly affected by the interaction of 5-HTT genotype and ELS (p < 0.05, p < 0.001 respectively). Data were normalized to the average of the MS0-5-HTT+/+ group. *: p < 0.05, **: p < 0.01.

Figure S7. Adrenal weight expressed as percentage of body weight of serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-), heterozygous knockout (5-HTT+/-) and wild-type (5-HTT+/+) rats (n = 5-9) exposed to 3 h daily separations (MS180) or a control treatment (MS0).

Figure S8. Plasma corticosterone (CORT) levels of serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-), heterozygous knockout (5-HTT+/-) and wild-type (5-HTT+/+) rats (n = 7) which were reared in standard animal facility conditions. The plasma CORT levels were significantly affected by 5-HTT genotype (F2,20 = 5.029, p < 0.05), with 5-HTT-/- rats showing higher levels than 5-HTT+/- (p = 0.053) and 5-HTT+/+ rats (p < 0.05).

Figure S9. Plasma corticosterone (CORT) response to 10-12 M adrenocorticotropic hormone, of adrenal tissue derived from serotonin transporter (5-HTT) homozygous knockout (5-HTT-/-), heterozygous knockout (5-HTT+/-) and wild-type (5-HTT+/+) rats (n = 6) which were reared in standard animal facility conditions. The total amount of secreted CORT, i.e. the area under the curve is shown, which was not found to be significantly altered by 5-HTT genotype (F2,17 = 2,368, p > 0.05). However, as it was hypothesized that adrenal tissue derived from AFR 5-HTT-/- rats would show a significantly higher CORT response than adrenal tissue derived from AFR 5-HTT+/+ rats we also performed a one-sided t-test, confirming the a priori hypothesis (p < 0.05).