A transient placental source of serotonin for the fetal forebrain

Abstract

Serotonin (5-hydroxytryptamine or 5-HT) is thought to regulate neurodevelopmental processes through maternal-fetal interactions that have long-term mental health implications. It is thought that beyond fetal 5-HT neurons there are significant maternal contributions to fetal 5-HT during pregnancy but this has not been tested empirically. To examine putative central and peripheral sources of embryonic brain 5-HT, we used Pet1(-/-) (also called Fev) mice in which most dorsal raphe neurons lack 5-HT. We detected previously unknown differences in accumulation of 5-HT between the forebrain and hindbrain during early and late fetal stages, through an exogenous source of 5-HT which is not of maternal origin. Using additional genetic strategies, a new technology for studying placental biology ex vivo and direct manipulation of placental neosynthesis, we investigated the nature of this exogenous source. We uncovered a placental 5-HT synthetic pathway from a maternal tryptophan precursor in both mice and humans. This study reveals a new, direct role for placental metabolic pathways in modulating fetal brain development and indicates that maternal-placental-fetal interactions could underlie the pronounced impact of 5-HT on long-lasting mental health outcomes.

Figure 1. Comparison of fetal 5-HT concentrations in the hindbrain and forebrain of Pet-1^−/− and wild type embryos from E10.5 to E17.5

a, 5-HT concentration in the Pet-1^−/− (KO) hindbrain is significantly lower than in wild type (WT) littermates at every age tested. b, In contrast 5-HT concentrations in the Pet-1^−/− forebrain are not significantly different from wild type littermates from E10.5 to E15.5 but become significantly lower at E16.5 and E17.5 (n>=6 embryos per genotype per age; *, p<0.005; one-way ANOVA; data are presented as means ± s.e.m.). c–h, Serotonergic axons (SERT+) and dorsal thalamic (DT) axons (NetG1a+) immunostained on sagittal sections at E14.5 in wild type (c) and Pet-1^−/− (f) embryos (regions shown correspond to the red box in the drawings). In wild type E14.5 embryos (d) SERT+ axons grow ventrally into the forebrain through the medial forebrain bundle (mfb, white arrowheads); SERT also labels DT axons at this age (open arrowhead). In comparison, very few SERT+ axons remain in the Pet-1^−/− (g). The pattern and density of SERT+ DT axons appear unaffected. Scale bars: 50 μm. The rostral-most extent of ingrowing serotonergic axons immunolabeled with 5-HT in the wild type (e) shows numerous 5-HT+ axons, some of which diverge toward the hypothalamus (Hyp). In contrast, only few 5-HT+ axons remain in the Pet-1^−/− forebrain (h). Scale bars: 20 μm. i, Densitometric analysis of 5-HT+ axons in the most rostral part of the mfb at E14.5 (region indicated in the right panel) confirms fewer axons in the Pet-1^−/−. j, AADC staining identifies DA neurons in the substantia nigra pars compacta (SNC) along with their and serotonergic axons coursing through the ventral forebrain at E14.5, and also AADC+ catecholaminergic neurons present in the hypothalamus (Hyp, black box). Scale bar: 100 μm. k-k″, AADC+ neurons in the hypothalamic region (red box in bottom right drawing; k′, white arrowheads) are 5-HTP-negative. Open arrowheads indicate fluorescence from blood vessels. Scale bar: 25 μm.

Figure 2. Placental synthesis of 5-HT in vitro and ex vivo

a, Placental extracts were incubated with the co-factor tetrahydrobiopterin (BH4; control) or BH4 and tryptophan (+Trp+BH4) and 5-HT neosynthesis measured after 30 min. b, Similar experiments conducted using human placenta tissue collected at 11 weeks of gestation (11 GW) show that human fetal villi synthesize 5-HTP and 5-HT (statistical significance versus control analyzed by Student’s t test; *, p<0.005; n=3; data are presented as means ± s.e.m.). c, Immunostaining for the monocarboxylate transporter MCT1, a marker of syncytiotrophoblastic cells (sc) in the labyrinth (la) region on the fetal side (fet) of an E14.5 mouse placenta. On the maternal side (mat) the decidua (dc) is devoid of staining. Asterisks indicate red blood cells of maternal origin. d–h, Higher magnifications of the region boxed in c; MCT1 is expressed on the apical side (arrows) of syncytiotrophoblasts facing the maternal blood space (mbs). The 5-HT synthetic enzymes TPH1 (f, g) and AADC (h) are expressed in overlapping patterns in the cytoplasm of syncytiotrophoblastic cells (arrows). i, Schematics of the ex-vivo dual perfusion system for the mouse placenta; UtA: uterine artery, UA umbilical artery, UV umbilical vein. g, 5-HT neosynthesis in ex vivo dually-perfused mouse placentas at E17.5. L-tryptophan (100 μM) was injected through the UtA. 5-HT, neo-synthesized from maternal tryptophan and released into the UV, is evident within 15 min of precursor injection (statistical significance of 5-HT levels variation across time was analyzed by one-way ANOVA; *, p<0.05; **, p<0.01; data from 3 independent experiments are presented as means ± s.e.m.).

Figure 3. HPLC measures of 5-HT concentrations in E14.5 hindbrain, forebrain and placenta of in utero PCPA-injected mice

a, Illustration of the in vivo injection procedure: PCPA or vehicle solution was injected into the labyrinth zone of the placenta. After 30 min, dams were euthanized and tissue collected and processed for HPLC. UtA: uterine artery; uc: umbilical cord; Ut: uterus. b, Compared to vehicle injection (untreated, n=6), placental PCPA injection (+PCPA, n=4), has no significant (n.s.) effect on 5-HT concentration in the hindbrain, whereas it significantly lowers 5-HT concentration in the forebrain and the placenta (statistical significance versus 5-HT levels in untreated tissue was analyzed by Student’s t test; *, p<0.05; data are presented as means ± s.e.m.). c, Model of the progressive switch of the source of 5-HT in the fetal forebrain, from an early exogenous (placental, blue line) to a later endogenous (5-HT axons, red line) source. The green boxes represent the amount of 5-HT measured in the Pet-1^−/− mice, which lack most of the endogenous neuronal source.