A Transient Placental Source of Serotonin for the Fetal Forebrain

A summary of the titular paper by Bonnin et al. published in Nature in 2011. Link

Introduction

• Serotonin and its Receptors Implicated in Psychiatric Disorders

Figure 1 -- Serotonin Target Molecules & Mutant Phenotypes in Mice; Adapted from Gaspar et al., 2003

Figure 1. Knockouts of 5-HT target molecules in mice can cause a variety of abnormal phenotypes, including anxiety, aggression, changes in drug response, and more. The links between specific knockouts and their effects are seen here. Adapted from Gaspar et al., 2003

Scientists are always looking for insight into the origins of various pathologies in order to effectively understand and treat them. This study by Bonnin et al. is particularly important because it investigates the origins of serotonin (5-hydroxytryptamine; 5-HT) in mammalian embryos. Serotonin is a neurotransmitter derived from the essential amino acid tryptophan, which is acquired through protein in the diet. It is involved in regulating mood, appetite, sleep, and social behavior. Serotonin and its various receptors are implicated in many human psychiatric disorders, including anxiety, drug addiction, autism, and schizophrenia (Gaspar et al., 2003). Figure 1 (on left) shows a more extensive list of phenotypes caused by knockouts of 5-HT target molecules in mice. Interestingly enough, changes in 5-HT-related neural connections (especially in the hippocampus) achieved at different periods of embryonic development can appear inoperative in that they don’t affect major developmental events, but later affect mental disorders that manifest in adulthood (Gaspar et al., 2003). The developmental periods in humans that correlate to the period that this study investigates primarily in mice are the 1st and early 2nd trimester of pregnancy, known to be particularly risky time periods for triggering mental illnesses.

• Exogenous Source for Serotonin During Early Embryonic Development

A known source of serotonin for developing mouse embryos is the Dorsal Raphe, located in the embryo’s brainstem. The transcription factor Pet1 is in control of this serotonin synthesis and disrupting Pet1 reduces 5-HT neurons to 20%  (Schaefer et al., 2009). However, the researchers tested levels of 5-HT in fetal Pet1 -/- mice compared to the wildtype, and although there was a significant difference at all ages for 5-HT levels in the hindbrain (see Fig. 2a), there was no difference in 5-HT levels in the forebrain between the two before 16.5 embryonic days (see Fig. 2b — the red box). This figure suggests that the Dorsal Raphe is indeed the major source of 5-HT in the hindbrain and in the forebrain at later stages, and Fig. 2 e,h,i,j display a reduction in 5-HT axon density in the Pet1 -/- forebrain. However, the lack of 5-HT depletion in the embryo forebrain (shown in the red box in Fig. 2b) suggests a 5-HT source besides the Dorsal Raphe at the early stage before 16.5 days, as forebrain serotonin production at this time is clearly not under the control of Pet1. The researchers were interested in finding out exactly what that exogenous source was.

 

Figure 2 -- Fetal serotonin levels in the forebrain and hindbrain. Adapted from Bonnin, et al. 2011
Figure 2.
a) Pet1 -/- mouse embryos show significantly different 5-HT concentrations compared to the wildtype in the hindbrain at all ages tested, suggesting that transcription initiated by Pet1 in the Dorsal Raphe is the main source of serotonin in the hindbrain.
b) Pet1 -/- mouse embryos show significantly different 5-HT concentrations compared to the wildtype in the forebrain after 16.5 embryonic days (see blue box), but not before this point.  Before embryonic day 16.5, no significant difference is seen (see red box). This suggests a source of forebrain serotonin that is independent of Pet1.
(e,h,i,j)  There is a reduction in 5-HT axon density in the Pet1-/- forebrain. Adapted from Bonnin et al., 2011.

 

Investigation and Experimentation

Investigating Potential Exogenous Sources of Serotonin

Three origins were considered as the main source of the 5-HT in the forebrain in early development: the embryonic gut, the maternal blood through the placenta, and the placenta. The embryonic gut was eliminated since its 5-HT biosynthetic enzyme, TPH1, is not expressed until embryonic day 15.5. Maternal blood as the source was ruled out through the use of SERT-/- dams. SERT is a serotonin transporter and would be necessary for transport of 5-HT from maternal blood through the placenta to the embryo; however, SERT knockout does not change the expression of 5-HT in the embryonic forebrain during the early stages.

Figure 3 -- Tryptophan increases levels of placental serotonin in mice and humans.  Adapted from Bonin, et al. 2011

Figure 3.
a) Synthesis of serotonin occurs in the mouse placenta when the placenta is treated with tryptophan. With this treatment, serotonin synthesis also occurs in the hindbrain, but this synthesis is delayed compared to placental synthesis.
b) Ex vivo experimentation in human placental tissue from 11 weeks of gestation also show placental synthesis of serotonin with treatment of tryptophan.  The control in both studies consisted of treatment with tetrahydrobiopterin (BH4). Adapted from Bonnin et al., 2011

As previously stated, tryptophan is an amino acid precursor of serotonin and the researchers considered that it may be transferred from the mother to the placenta for serotonin synthesis in the placenta. Tryptophan injection into pregnant dams does, in fact, increase 5-HT concentration in the fetal brain and a set of proteins sufficient for 5-HT synthesis, TPH1 and amino acid decarboxylase (AADC), are present in the placenta by embryonic day 10.5. The researchers were able to demonstrate that the mouse placenta could synthesize 5-HT when incubated with tryptophan by 10.5 embryonic days (Fig. 3a). Furthermore, human placentas were also able to synthesize 5-HT (Fig. 3b). This was accomplished by the 11th week of gestation.

Confirmation

• Blocking Serotonin Synthesis in the Placenta

Figure 4 -- PCPA inhibits serotonin levels in the placenta and the forebrain. Adapted from Bonnin et al. 2011.

Figure 4
(a) This shows the injection of PCPA into the placenta in vivo.
b) Injection of PCPA (meant to block placental TPH1 activity) into the placenta significantly decreased placental and forebrain levels of serotonin, but did not affect hindbrain serotonin levels. Adapted from Bonnin et al., 2011.

To confirm that the placenta was the exogenous source of 5-HT in the embryonic forebrain during days 10.5-16.5, the researchers blocked placental TPH1 activity using the TPH inhibitor p-chlorophenylalanine (PCPA). Recall that TPH is necessary for serotonin synthesis. By limiting exposure time, the PCPA was kept from inhibiting TPH2 activity in the Dorsal Raphe, meaning that any changes in serotonin levels must be due to changes in serotonin synthesis in the placenta, not the Dorsal Raphe. Both placental and fetal forebrain 5-HT levels were significantly decreased in the PCPA-treated embryos, suggesting that, as expected, the placenta is responsible for the maintenance of 5-HT in the forebrain during early development (see Fig. 4b). Levels of 5-HT in the hindbrain were not affected, supporting the idea that the main source of serotonin in the hindbrain is the Dorsal Raphe.

• Serotonin vs. Tryptophan from Maternal Blood

The researchers also managed to perform live transfers of tryptophan and serotonin to pregnant mice. Serotonin quickly (<15 minutes) accumulates in the fetal umbilical vein after an injection of tryptophan to the maternal uterine artery. When the researchers tried injecting serotonin directly into the maternal uterine artery and observing the transfer of this serotonin to the fetus over a 30 minute period, very little serotonin was transferred to the fetal umbilical vein , as compared to the serotonin that accumulates in this vein after an injection of tryptophan. This gives additional support to the idea of placental synthesis of serotonin using maternal tryptophan and discredits the theory of maternal blood as an exogenous source for serotonin.

Conclusions

• Summary of Main Findings

The main conclusion of the paper is that serotonin is produced in the placenta in both mice and humans. The placenta is the main source of serotonin in the fetal forebrain up until embyronic day 16.5 in mice. This corresponds to the first and early 2nd human trimesters and is a critical period for mental health. Serotonin plays a crucial role in mental health, and understanding its origins at all stages of development, but particularly during the first and second trimesters, is extremely important.

• Model of Fetal Serotonin Sources

Using the data from this paper and information from previous labs’ studies, the researchers proposed the following model for maternal and placental contributions to fetal 5-HT, as shown in Figure 5.

Tryptophan from maternal blood is transferred to the placenta, where it is both retained and sent to the fetal hindbrain. The tryptophan in the placenta is converted to 5-HT via TPH1 and AADC. During early development, this 5-HT is sent to the forebrain and to the hindbrain. In addition, the tryptophan sent directly to the hindbrain is converted to 5-HT via TPH2 and AADC. This 5-HT is maintained in the hindbrain throughout the developmental period in question, and 5-HT is sent to the forebrain from the Dorsal Raphe during later development (16.5 embryonic days in mice).

Trace amounts of 5-HT are also taken up by the placenta from maternal blood, but they are found to be degraded by placental MAOA and this placental 5-HT does not constitute a significant source of 5-HT in the embryo.

Model of maternal and placental contributions to the fetal brain and blood stream 5-HT.

Figure 5.
Tryptophan from maternal blood (left) is transferred to the placenta (middle), where it is both retained and sent to the fetal hindbrain (lower right, grey dashed arrow).  The tryptophan in the placenta is converted to 5-HT via TPH1 and AADC. During early development, this 5-HT is sent to the forebrain and the hindbrain (red pathway).  The tryptophan sent directly to the hindbrain is converted to 5-HT via TPH2 and AADC. This 5-HT is maintained in the hindbrain throughout the developmental period in question, and 5-HT is sent to the forebrain from the Dorsal Raphe during later development (black arrow from hindbrain to forebrain).  Trace amounts of 5-HT are also taken up by the placenta from maternal blood, but they are found to be degraded by placental MAOA and this placental 5-HT does not constitute a significant source of 5-HT in the embryo. From Bonnin et al., 2011.

• Evaluation of Paper

The researchers were very thorough in their investigation of the exogenous source of serotonin for the fetal forebrain. Their experimental methods were clear and they made good use of controls. In addition, the researchers did an exemplary job of explaining why maternal blood and the embryonic gut were not found to be exogenous sources of fetal serotonin in the forebrain. The embryonic gut was ruled out because TPH1 in this region does not begin until embryonic day 15.5 and TPH is necessary for serotonin synthesis. The possibility of maternal blood as a source for fetal serotonin was evaluated in two ways. First, it was determined that SERT -/- knockout dams, which have almost no serotonin, produced fetuses with no significant drops in forebrain serotonin. Second, it was determined that the injection of serotonin directly into the maternal uterine artery transferred very little serotonin to the fetal umbilical vein over a 30 minute period, as compared to an injection of tryptophan.

In general, the researchers’ overall model of serotonin contributors followed their research results, but the researchers did not show that placental serotonin goes into the hindbrain, in addition to the forebrain, as is laid out in their model. In order to evaluate the truth of this aspect of their model, an experiment could be set up using an inhibitor of TPH2 in the Dorsal Raphe (perhaps PCPA injected directly into the Dorsal Raphe). After a brief period of time, levels of serotonin in the hindbrain could be evaluated, both in a control and in an experimental condition in which tryptophan was provided to the placenta (as in Figure 3). It is also important to remember that although the researchers were able to show that synthesis of serotonin occurs in human placentas, as it does in mice, some results may not be transferable.

Resources

Bonnin, A., Goeden, N., Chen, K., Wilson, M.L., King, J., Shih, J.C., Blakely, R.D., Deneris, E.S., Levitt, P., 2011. A transient placental source of serotonin for the fetal forebrain. Nature 472, 347-350.

Gaspar, P., Cases, O., Maroteaux, L., 2003. The developmental role of serotonin: news from mouse molecular genetics. Nature Reviews Neuroscience 4, 1002-1012.

Schaefer, T.L., Vorhees, C.V., Williams, M.T., 2009. Mouse plasmacytoma-expressed transcript 1 knock out induced 5-HT disruption results in a lack of cognitive deficits and an anxiety phenotype complicated by hypoactivity and defensiveness. Neuroscience 164, 1431-1443.

There are also hyperlinks to Wikipedia.

 

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