Research report
Expression of serotonin transporter protein in developing rat brain

https://doi.org/10.1016/S0165-3806(99)00152-2Get rights and content

Abstract

Serotonin transporter (5-HTT), a transmembrane protein, has been shown in adult brain to be distributed not only on synaptic terminals but to a great extent on axons as well. Here we report the ontogeny of 5-HTT and its relationship with serotonin (5-HT) neurons using established 5-HTT and 5-HT antibodies. Both 5-HTT- and 5-HT-immunostaining (-im) appear in 5-HT neurons at embryonic day 12 (E12) in rostral raphe nuclei (RRN). Soon after appearing, 5-HTT-im is highly expressed on axons, similar to adult expression. However, in contrast to adult, 5-HTT-im also outlines the soma-dendrites. Rich 5-HTT-im appears along the entire length of projecting axons, extending to the growth tip. In the next 2 days, intensive 5-HTT-im axons from RRN travel a course preferentially in the floor plate and later, the medial forebrain bundle trajectory. A group of new 5-HT-im neurons and 5-HTT-im axons appear at E13 in caudal raphe nuclei. At E16–18, taking the exact trajectory course of 5-HT axons, 5-HTT-im axons reach ganglionic eminence, olfactory bulb, and cortex and disperse into many brain regions in E18-20. No 5-HTT-im cell bodies were seen in nigral, locus ceruleus, or hypothalamus. However, the transient expression of 5-HTT on non-serotonergic system was seen in cortical and striatal neuroepithelia at E12 and sensory thalamic pathways at P0–P10. Prominent 5-HTT-im fibers in thalamocortical bundles project from sensory thalamic nuclei through reticular nucleus, internal capsule bundle and form barrels in somatosensory cortices. No 5-HTT-im was seen in glia-like cells using currently available antibody. These observations indicate that 5-HTT is: (a) associated preferentially with 5-HT neurons in brainstem, (b) temporally co-expressed with 5-HT in 5-HT neurons, (c) expressed on axons prior to synaptical sites at target neurons, which strongly indicates a volumic (extrasynaptic) transmission, (d) expressed in non-5-HT neurons within a specific window, which may affect the development of the systems “borrowing” the 5-HT. The early appearance of 5-HTT may also endow functionality as well as vulnerabilities of 5-HT, sensory thalamic, and cortical neurons to 5-HTT targeting drugs during pregnancy and after birth.

Introduction

Serotonin (5-HT) is one of the earliest appearing transmitter systems during development. In rats, 5-HT neurons appear on embryonic day 12 (E12) 27, 35, 43, earlier than the genesis of most other transmitter neurons. 5-HT's early ontogenic appearance and distribution in the brain regions coincides with many neuronal differentiation and growth events. It is not surprising that 5-HT has been proposed as a signaling chemical for neuronal development [for review see Ref. [23]]. 5-HT has been shown to induce neurogenesis and neuronal differentiation 10, 22, 26, 44, affect cranial neural crest migration [32], induce synaptogenesis [44], and inhibit growth cone motility [17]. If 5-HT actively plays a role in these developmental events, an active regulation of 5-HT action most likely exists.

Serotonin transporter (5-HTT) is one of the most critical regulators, through removal of extracellular 5-HT, to fine-tune 5-HT signaling. The ontogeny of 5-HTT and its close relationship with 5-HT neurons is important for understanding the regulation of 5-HT and thus 5-HT activity during development.

On another spectrum, 5-HTT is a target of many 5-HT related drugs of abuse, from cocaine and amphetamine to methamphetamine. 5-HTT is also a major site of action of many antidepressants, including tricyclics and serotonin-specific re-uptake inhibitors (SSRIs). Growing concerns about prenatal exposure to drugs of abuse or even SSRIs during fetal development may be well grounded if 5-HTT appears early during brain development [38].

Currently the temporal appearance and the topographic distribution of the 5-HTT protein have not been reported. A functional binding site for 5-HTT in mouse [8]and a distribution of mRNA [15]in rats described in the developmental stage are available but with many incongruities. We previously reported that 5-HTT is not only distributed and functions near the synaptic terminal but also, for the most part, on the axonal membrane [47]. How 5-HTT is distributed during the growth of 5-HT neurons is among the questions that remain to be answered. The appearance and distribution of the 5-HTT protein in the developing brain is reported here, and the distribution of 5-HTT protein and mRNA are compared.

Section snippets

Materials and methods

Time pregnant Sprague–Dawley rats (Harlan, Indianapolis, IN) were used for the study. Animals were mated after dark, and the sperm check for indication of pregnancy was performed the next morning. The day of the positive sperm check was designated as gestation day 0 (E0). Animals were provided with normal rat chow and water ad lib. Fetuses of E12, 13, 14, 16, 18, and 20 were obtained from dams under deep ether anesthesia in the morning. At least three dams per age were used. Two embryos from

Results

No 5-HTT-im somas were seen in nigral, locus ceruleus, hypothalamus, or any forebrain structures in our prenatal samples, except for very light 5-HTT-im neurons in cortex in. Prominent 5-HTT-im cell bodies were encountered only in the raphe complex at the birthplace of 5-HT neurons. 5-HTT-im axons extended beyond raphe with very similar morphology of 5-HT-im axons. They are chronologically displayed hereafter. The feature expression is noted when they first appear in the age group. All sagittal

Discussion

The comparison of embryonic days varied depending on the definition of the sperm/plug confirmation day in the literature. Most of studies have adopted the mating period as beginning in late afternoon with sperm confirmation the next morning. The sperm/plug positive day is considered here as day 0. Comparisons with other literature were standardized for the convenience of the discussion. In addition, the embryonic day of rat is approximated one day behind mouse (i.e., rat E13 corresponds to

Acknowledgements

This study is supported by PHS RO3 MH 50602 and PHS P50AA07611-11 to F.C.Z.

References (49)

  • J.M. Lauder

    Neurotransmitters as growth regulatory signals: role of receptors and second messengers

    Trends Neurosci.

    (1993)
  • C. Lebrand et al.

    Transient uptake and storage of serotonin in developing thalamic neurons

    Neuron

    (1996)
  • K.P. Lesch et al.

    Genetically driven variation in serotonin uptake: is there a link to affective spectrum, neurodevelopmental, and neurodegenerative disorders?

    Biol. Psychiatry

    (1998)
  • H.G. Lidov et al.

    Immunohistochemical study of the development of serotonergic neurons in the rat CNS

    Brain Res. Bull.

    (1982)
  • H.G. Lidov et al.

    An immunohistochemical study of serotonin neuron development in the rat: ascending pathways and terminal fields

    Brain Res. Bull.

    (1982)
  • L.A. Mamounas et al.

    Evidence for dual serotonergic projections to neocortex: axons from the dorsal and median raphe nuclei are differentially vulnerable to the neurotoxin p-Chloroamphetamine (PCA)

    Exp. Neurol.

    (1988)
  • R. Mercado et al.

    A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding

    Brain Res. Dev., Brain Res.

    (1992)
  • J.H. Tao-Cheng et al.

    Differential polarization of serotonin transporters in axons vs. soma-dendrites: an immunogold electron microscopy study

    Neuroscience

    (1999)
  • M.V. Ugrumov et al.

    Development of the hypothalamic serotoninergic system during ontogenesis in rats. Immunocytochemical and radioautographic study

    Brain Res.

    (1986)
  • J.A. Wallace et al.

    Development of the serotonergic system in the rat embryo: an immunocytochemical study

    Brain Res. Bull.

    (1983)
  • F.C. Zhou et al.

    Serotonin transporters are located on the axons beyond the synaptic junctions: anatomical and functional evidence

    Brain Res.

    (1998)
  • F.C. Zhou et al.

    Serotonin transporter antibodies: production, characterization, and localization in the brain

    Mol. Brain Res.

    (1996)
  • M. Zoli et al.

    The emergence of the volume transmission concept

    Brain Res. Brain Res. Rev.

    (1998)
  • J. Altman et al.

    Development the of brainstem in the rat. V. Thymidine-radiographic study of the time of origin of neurons in the midbrain tegmentum

    J. Comp. Neurol.

    (1981)
  • Cited by (0)

    View full text