Over the past 40 years, the field has embraced the concept that there are both fetal and maternal contributions of 5-HT during pregnancy. Serotonergic neurons appear early in the fetal hindbrain and could provide an endogenous source of 5-HT. Sources of 5-HT other than the fetal brain was assumed because of the observations that 5-HT receptors expression in the rostral forebrain, craniofacial and peripheral regions is evident several days before serotonergic axons even reach these regions (Buznikov et al., 2001
). Moreover, an exogenous source of 5-HT was consistent with the finding that in Pet-1
) mice, in which 70–80% of raphe neurons fail to develop 5-HT synthetic capacity, thalamic neurons were nonetheless normally targeted (Bonnin and Levitt, unpublished). Thus, all these data were consistent with the existence of an alternative, exogenous source of 5-HT at early stages of development. There are claims regarding multiple sources of the neurotransmitter during fetal development, including the fetal gut and maternal circulation (Lauder et al., 1981
, Yavarone et al., 1993
), prior to blood-brain barrier formation. But a careful examination of the literature indicated to us that the exact source of 5-HT in the early fetal brain was not completely clear. Some studies have suggested that maternal blood 5-HT could be transferred to the fetal circulation after crossing the placenta (Cote et al., 2003
, Cote et al., 2007
). Yet direct or indirect transfer was never demonstrated.
Given the lack of altered axonal guidance problems in the Pet-1−/−
mouse, and to take advantage of the deficiency of 5-HT levels in the adult brain (Hendricks et al., 2003
), we measured 5-HT concentrations throughout embryogenesis in the Pet-1−/−
and wild type littermate mice in order to gather direct evidence for source of 5-HT other than the fetal brain. We assayed different brain regions using high-pressure liquid chromatography (HPLC). The results uncovered a biphasic nature of the source of 5-HT specifically in the embryonic forebrain. In the hindbrain, the location of raphe neurons, we observed a consistent, massive reduction in 5-HT in the Pet-1−/−
mouse throughout fetal development. However, at early ages (E10.5 to E15.5) 5-HT levels are normal in the Pet-1−/−
mouse compared to wild type littermates. In contrast, from E16.5 on, 5-HT concentration is significantly decreased in the forebrain of Pet-1−/−
embryos compared to littermate controls, indicating that forebrain 5-HT becomes exclusively provided by DR serotonergic neurons; this corresponds to the timing of raphe axons reaching the forebrain in large numbers (Bonnin et al., 2011
). The differential accumulation of 5-HT in the forebrain, but not the hindbrain, in the Pet-1−/−
embryos early in development indicates that there is a striking transition from an early exogenous source to a later endogenous (DR serotonergic neurons) source. This switch during development allows the forebrain during early- and mid-embryogenesis to become dependent progressively on its own production of 5-HT ().
Figure 2 New model of placental contributions to the fetal brain and blood 5-HT. A, the source of 5-HT in the embryonic forebrain changes over time, from an early exogenous (placental) source to a later endogenous (dorsal raphe serotonergic neurons) source. This (more ...)
From E10.5–15.5, there is no synthetic capacity of the gut to produce 5-HT (Branchek and Gershon, 1989
). Myenteric plexus neurons express TPH2 and are able to uptake 5-HT at E12.5, and synthesize it at E18 (Rothman and Gershon, 1982
, Cote et al., 2007
). However, only enterochromaffin cells, which begin to express TPH1 at later stages (E15.5) (Cote et al., 2007
), are able to provide a significant source of 5-HT for the fetal blood (Chen et al., 2001
). We thus turned to the possibility that maternal 5-HT may be the source that is transferred to the fetal brain. We examined fetal brains of SERT knockout (SERT−/−
) dams in which maternal there is no detectable blood and platelets 5-HT (Chen et al., 2001
). Despite this status of the maternal blood, there was no impact on the concentration of 5- in the forebrain of SERT+/−
E12.5 embryos from SERT−/−
or wild type dams (Bonnin et al., 2011
). These data indicated that in contrast to prevailing dogma in the field, maternal blood 5-HT in the mouse is not the main source of fetal blood and forebrain 5-HT at early stages of development. Similar observations were made with another mouse mutant [the pallid mice – (Li et al., 2004
)], in which normal concentrations of fetal forebrain 5-HT were measured at E12.5, even though the maternal blood contains no 5-HT (Del Angelica and Bonnin, unpublished).
One additional source of 5-HT had not been investigated – the placenta. We designed studies to examine the possibility that the precursor and essential amino acid tryptophan, originating from the pregnant dam, is converted to 5-HT in the placenta and delivered to the fetal circulation. Immunocytochemistry, RNAseq and qRT-PCR confirmed that 5-HT biosynthetic enzymes TPH1 and AADC are expressed in the syncytiotrophoblastic cell layer of the placenta at E10.5-E18.5 (Bonnin et al., 2011
). In vitro
placental 5-HT neosynthesis was confirmed biochemically at E10.5, E14.5 and E18.5. This synthetic capacity was also observed in human placental fetal villi at 11 weeks of gestation, suggesting that a placental source of 5-HT is important for human fetal development.
In order to prove neosynthesis and transport of placental 5-HT from maternal tryptophan precursor, we developed an ex vivo
technology for regulating live placental organ perfusion in the mouse (Bonnin et al., 2011
). Briefly, a mouse placenta is harvested and kept alive in an oxygenated and thermostated organ chamber. On the maternal side of the placenta, the uterine artery is cannulated and connected to a low-flow peristaltic pump; on the fetal side, the umbilical artery and veins are cannulated and connected to independent ultra-low flow peristaltic pumps. These independent perfusions allow recreating artificial maternal and fetal blood circulations in intact, live isolated placentas (Bonnin et al., 2011
). Effects of modifying the molecular composition of the artificial ‘maternal blood’ on 5-HT (or other molecules) release into the fetal blood through the umbilical vein are then quantified overtime. Within minutes after tryptophan injection through the maternal uterine artery, we observed large accumulation of newly synthesized 5-HT that was collected through the umbilical vein, demonstrating that the live placenta is able to convert tryptophan to 5-HT, and releases the neurotransmitter into the fetal circulation. In contrast, less than 1% of 5-HT injected on the maternal side could be transported to the fetal circulation. To establish placental synthetic capacity and transport of 5-HT to the fetus in vivo
, we blocked TPH1 enzymatic activity by microinjecting small volumes of the TPH inhibitor p-chlorophenyalanine (PCPA) directly into the labyrinth zone of E14.5 placentas in utero
(Bonnin et al., 2011
). This pharmacological manipulation significantly reduced 5-HT levels in the placenta and fetal forebrain but not in the fetal hindbrain. These in vivo
and ex vivo
experiments demonstrate that an exogenous source of 5-HT produced in the placenta is required to maintain normal levels of forebrain 5-HT during early stages of forebrain development (). Because the fetal blood brain barrier is not functional at early stages of brain development (Daneman et al., 2010
), we proposed that placental 5-HT could reach TCAs and other axons as they grow and influence their response to guidance cues (Bonnin et al., 2007
). This would be consistent with the diffusion of placenta-derived 5-HT within the fetal brain extracellular matrix, similar to bioactive molecules like growth factors and cytokines (see for instance (Li et al., 2009
)). The extracellular accumulation would be analogous in nature with the adult brain, in which 5-HT released through ‘volume transmission’ from axonal varicosities can have long-range effects (De-Miguel and Trueta, 2005
). Stability of the extracellular 5-HT accumulation might occur because of the requirement for uptake and degradation by cells that express MAOA (e.g. serotonergic DR and noradrenergic LC neurons); this is an unlikely route at early stages (<E15.5) of brain development, because serotonergic axons are just starting to reach the forebrain at that age, and they are few in number. Interestingly, SERT is expressed in TCAs (but MAOA is not) (Lebrand et al., 1996
), and therefore growing thalamic axons could provide an additional mechanism by which placental 5-HT is taken up and protected from degradation specifically in the forebrain. However, at these early stages of development (E10–E16), 5-HT immunoreactivity is not detectable above background in growing TCAs. The possibility that placental 5-HT affects TCA formation remains to be fully demonstrated and the impact of a placental-specific TPH1 gene deletion on fetal forebrain 5-HT levels and TCA pathway formation is currently under investigation.
Although a direct maternal source of 5-HT may also be provided to the embryo before and during placentation (e.g. before E10.5 in the mouse), and may affect very early events of embryonic development (Cote et al., 2007
), our data collectively provided the first evidence that a maternal precursor metabolized directly by the placenta influences fetal brain development. Because similar synthetic capability were observed in early human placenta, the extent to which there is a placental 5-HT influence on brain development during human pregnancy is key area for new investigations.