EDITORIAL New Research Series Starting with this issue and extending over the next four months, Biological Psychiatry will publish thematicallygrouped articles on several dimensions of serotonergic neurotransmission. These articles derive from a meeting entitled, “A Decade of Serotonin Research,” held November 16 –18, 1997, sponsored by the Society of Biological Psychiatry with funds from an unrestricted educational grant generously provided by Eli Lilly and Company (Indianapolis, IN). This meeting gathered international leaders in this field of research. Biological Psychiatry is grateful to the attendees for preparing their presentations in written format to provide both an educational and critical overview for the readers. All manuscripts underwent peer review and have been rapidly prepared for publication. The first series of
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papers in this issue assess the role of serotonin in behavior, from gene “knock-out” studies in animals to implications for neuropsychiatric disorders. The Editors feel that critical and timely reviews like these are valuable to enhance high level, multidisciplinary communication among those with an active interest in the neurobiological basis of psychiatric disorders. We plan to provide additional such series in the coming years. Comments and recommendations from the readers are appreciated and can be sent via e-mail (
[email protected]) or FAX (203-764-4324). Robert B. Innis, MD, PhD Eric J. Nestler, MD, PhD Dennis S. Charney, MD PII S0006-3223(98)00140-1
SEROTONIN RESEARCH
Development of Serotonergic Neurons and Their Projections John L.R. Rubenstein Neurons producing serotonin are among the earliest to be born in the developing central nervous system. These cells are largely restricted to the hindbrain, where they form primarily in ventral regions. This review describes some of the mechanisms that regulate patterning and differentiation of the embryonic brain, which are implicated in neurogenesis of serotonergic neurons. It also covers the development of serotonergic axon pathways and the potential role of serotonin in regulating developmental processes. Biol Psychiatry 1998;44:145–150 © 1998 Society of Biological Psychiatry Key Words: Serotonin, hindbrain, development
From the Nina Ireland Laboratory of Developmental Neurobiology, Center for Neurobiology and Psychiatry, Department of Psychiatry, University of California, San Francisco, California. Address reprint requests to Dr. J.L.R. Rubinstein, Department of Psychiatry, University of California, San Francisco, 401 Parnassus Avenue, San Francisco, CA 94143-0984. Received January 29, 1998; accepted April 6, 1998.
© 1998 Society of Biological Psychiatry
Organization of the Embryonic Brain
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he neural plate, which is induced during gastrulation, is the embryonic precursor of the brain. Neurulation converts the neural plate into the neural tube, which is subdivided into a series of vesicles that are the primordia of the major brain regions: the forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon). As development proceeds, transverse constrictions further subdivide the brain into neuronal segments, or neuromeres (Vaage 1969). Neuromeres are present in the hindbrain, where they are called rhombomeres (reviewed in Lumsden and Krumlauf 1996) and also probably in the forebrain where they are known as prosomeres (Puelles et al 1987; Bulfone et al 1993; Puelles and Rubenstein 1993; Figdor and Stern 1993; Rubenstein et al 1994). The generation of the neuromeres is controlled by the anteroposterior (A/P) patterning system. A distinct patterning system (dorsoventral; D/V) induces the longitudinal organization of the central nervous system (for review see: Tanabe and Jessell 1996). D/V patterning generates 0006-3223/98/$19.00 PII S0006-3223(98)00133-4
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longitudinally aligned columns of cells that are parallel to the long axis of the central nervous system (Shimamura et al 1995). The four primary longitudinal columns (from dorsal to ventral) are the roof plate, alar plate, basal plate, and floor plate. The roof and floor plates contain nonneural cells that primarily regulate development of the nervous system. For example, the floor plate produces proteins that induce motor neuron development and chemotropic substances that direct the growth of axons. The basal plate contains motor neurons and other cell types such as the serotonergic and dopaminergic neurons. The alar plate contains the secondary sensory neurons. The neural crest comes from the dorsalmost region of the alar plate; these cells give rise to primary sensory neurons. This paper will briefly review where, when, and how serotonergic neurons, and their projections, are generated.
Origin, Ontogeny, and Regulation of Serotonin Neurogenesis Serotonin expressing cells in the adult central nervous system are found in the raphe nuclei that are largely restricted to the basal plate of the pons and medulla. The development of these cells has been studied in detail in the rat, whose gestational period is about 20 days. In this article, when describing the ontogeny of serotonin cells and their projections, I will refer to data obtained from studies of the rat (for reviews see: Molliver 1987; Wallace and Lauder 1992). There are two clusters of serotoninexpressing neurons in the ventral rhombencephalon (Figure 1C). The precise relationship of these clusters to the rhombomeres is uncertain. In general, these cells form in the basal plate; an exception are the serotonergic cells that contribute to the dorsal raphe nuclei. The rostral cluster is the first to be detectable using serotonin immunohistochemistry—these cells are apparent between embryonic days 12–15 (E12–15). Serotonin immunoreactivity appears in the caudal cluster around E14, although birthdating studies suggest that these cells are born around the same time as the rostral cells (E11–12). It is not known why the caudal cells have a delay in expressing serotonin relative to the rostral cells. The rostral cells are believed to give rise to the dorsal raphe (B7), median raphe (nucleus centralis superior, B8), and caudal dorsal linear raphe (B6) and the ventrolateral cell group (B9). The caudal cells contribute to the magnus, obscurrus, and pallidus raphe nuclei (B1–B3). Also note that cells with serotonergic properties are also detectable in the fetal hypothalamus (retrochiasmatic and suprachiasmatic areas) and adult dorsomedial hypothalamic nucleus. Finally, serotonergic cells are also found in the postnatal spinal cord. The mechanisms that regulate the induction and speci-
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fication of the serotonin neurons are beginning to be elucidated. As most of these cells are located in the ventral region of the neural tube, it is likely that their induction shares features with other ventral cell types such as motor neurons and dopamine neurons. There is evidence that molecules produced by the notochord can induce all three cell types (Yamada et al 1993; Tanabe and Jessell 1996; Hynes et al 1995). The notochord is a rodlike structure of mesendodermal origin that lies just under the floor plate (the ventral midline of the central nervous system) (Figure 2). The sonic hedgehog protein, which is produced in the notochord and floor plate, is now known to be necessary and sufficient for induction of ventral cell types (Tanabe and Jessell 1996; Chiang et al 1996), and therefore is likely to be essential for production of serotonin neurons. Because signals from the notochord and floor plate, such as sonic hedgehog, are present at all axial levels of the central nervous system (Figures 1A, 2C), it is unclear why serotonin neurons are present primarily in the hindbrain. Three mechanisms could contribute to this regional restriction. First, the ability of the neuroepithelium to respond to inductive signals may differ in different regions of the embryonic brain (e.g., only the hindbrain primordium is competent to produce serotonin neurons in response to sonic hedgehog and other inductive signals). Second, there may region-specific inductive signals produced by the notochord and neuroepithelium. Third, serotonin neurons may in fact be induced along the entire A/P axis of the central nervous system (CNS), but only those in the rhombencephalon survive. Available evidence does not definitively rule in, or rule out, any of these possibilities. Region-specific competence to induce regulatory genes and distinct cell types has been identified in several cases (e.g., see Shimamura et al 1997). For instance, sonic hedgehog appears to induce the expression of forebrain markers primarily in the primordia of the forebrain. In addition, fibroblast growth factor 8 (FGF8) induces forebrain markers only neuroectoderm isolated from the anterior CNS, whereas it induces midbrain/cerebellar markers in more posterior tissues. Genes that may regulate differential competence to FGF8 are beginning to be identified, and probably include transcription factors of the Otx class (Acampora et al 1997). Region-specific inductive substances also appear to have a role in patterning the brain. For instance, there is evidence that BMP7, produced by the prechordal mesendoderm, has a role in patterning the hypothalamus (Dale et al 1997). FGF8, which is implicated in patterning the forebrain, midbrain, and cerebellum, is expressed at local organizer tissues, such as the isthmus and anterior neural ridge (Crossley and Martin 1995; Shimamura et al 1997). A simple model for the regional restriction of serotonin
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the Otx genes in the midbrain and forebrain) or transcription factors that are permissive for serotonin neuron induction in the hindbrain. This model would explain why serotonin neurons form in the ventral hindbrain, but does not predict the actual genes that regulate specification and differentiation of serotonergic cells. There are now several candidates for genes that control this process. These include transcription factors that are expressed in the ventral central nervous system, such as genes in the Nkx family (Shimamura et al 1995; Qiu et al in press). These homeobox genes are expressed in bilateral longitudinal stripes in the basal plate of the embryonic and postnatal central nervous system (Figure 1B). Nkx2.1 is expressed in the forebrain (hypothalamus and basal telencephalon), Nkx6.1 is expressed in the caudal diencephalon, midbrain, hindbrain, and spinal cord, and Nkx2.2 is expressed as a thin stripe in all regions of the central nervous system. Mutation of Nkx2.1 disrupts differentiation of the hypothalamus (Kimura et al 1995).
Figure 1. Schema showing a lateral view of a generic midgestation mammalian or avian embryo at the early neural tube stage (;E10 mouse). (A) Expression of sonic hedgehog in the floor plate of the spinal cord and hindbrain and in the basal plate of the midbrain and forebrain. (B) Expression of three Nkx homeobox genes (Nkx2.1, 2.2, and 6.1). (C) Approximate location of serotonergic nuclei. Abbreviations: di, diencephalon; HY, hypothalamus; is, isthmus; mes, mesencephalon; MGE, medial ganglionic eminence; OS, optic stalk; r, rhombomeres; sc, spinal cord; sp, secondary prosencephalon; Tel, telencephalon; ZL, zona limitans, the boundary that separates the dorsal and ventral thalamic nuclear complexes (prosomeres 2 and 3, respectively).
neurons would include the combination of local inductive substances and regional competence to respond to these signals. Figure 2 shows such a model. For instance, ventral specification signals (e.g., sonic hedgehog) from the notochord and floor plate induce basal plate type neurons, including the progenitors for serotonin neurons. Dorsal signaling centers that produce bone morphogenetic proteins (BMPs) may repress serotonin neurogenesis in the alar plate. Furthermore, additional signals that may regulate serotonin neurogenesis are produced by other patterning centers, such as the isthmic region at the midbrain/ hindbrain transition. For instance, FGF8 and WNT1 are produced in the embryonic isthmus region. Finally, region-specific competence to form serotonin neurons is controlled through the regional expression of transcription factors that either repress the serotonin phenotype (such as
Figure 2. Organization and patterning of the neural plate. (A) Schematic view of the upper surface of a rodent neural plate around the 1 somite stage of development (;E8.0 mouse). The major brain regions are indicated: pr, prosencephalon; me, mesencephalon; rh, rhombencephalon. The nonneural ectoderm (ec) that is lateral to, and continuous with, the neural ectoderm is also shown. (B) A transverse section plane through the neural plate is indicated in A, and is shown in B. The midline of the neural plate is above the notochord. The paraxial mesoderm that gives rise to somitic tissues is also shown. (C) Schema showing the relationship of the neural plate to the axial mesendodermal organizers (prechordal plate and notochord), which induce ventral structures in the neural plate (bp, basal plate) via the sonic hedgehog protein (black arrows). Location of the alar plate (ap), telencephalon (tel), and eyes (e) are also indicated. (D) Schema showing the location of local organizers within the neural plate that produce FGF8 (gray areas that have white arrows); one is in the anterior neural ridge and the other is in the isthmus. These organizers appear to regulate patterning of the forebrain, midbrain, and hindbrain.
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Analysis of the Nkx2.2 and Nkx6.1 mutants is in progress (Sussel, Sander, German, and Rubenstein, in progress). Preliminary evidence suggests that Nkx2.2 mutants lack many of the serotonergic neurons. Additional studies of the Nkx2.2 mutants are needed to establish whether these cells are never specified or whether their differentiation is disrupted. It is clear that we are at the very beginning of understanding the molecular basis for serotonin neuron development, as there will undoubtedly be many transcription factors, and other molecules that are required for the formation of serotonergic neurons. For instance, there is evidence that neurotrophic proteins can regulate serotonergic neuronal development. Brain-derived neurotrophic factor (BDNF) appears to promote the survival of serotonergic neurons (Eaton and Whittemore 1996), whereas ciliary neurotrophic factor (CNTF) promotes raphe cells to become cholinergic neurons and reduces the number of serotonergic neurons (Rudge et al 1996). In addition, other levels of regulation must act to control the synthesis and synaptic release of serotonin as well as control how serotonergic axons find their appropriate targets.
Development of Serotonergic Projections The rostral raphe nuclei produce axonal projections that ascend to the midbrain and forebrain, whereas the caudal raphe nuclei produce axons that descend to the spinal cord (Molliver 1987; Aitken and Tork 1988; Wallace and Lauder 1992). The rostral projections become visible soon after serotonin immunoreactivity is present in the brain stem. These unbranched fibers grow in the marginal zone as a fascicle within the medial forebrain bundle, and by E15 they reach the diencephalon. Here the fibers spread out; some follow preexisting axon pathways such as the fasciculus retroflexus. There is evidence that within the medial forebrain bundle, the medial fibers project to the frontal pole of the telencephalon, whereas the lateral fibers project to the hypothalamus. These latter fibers reach the rostral end of the brain around E17, where some cross the midline in the supraoptic commissure. Also on E17, the serotonin fibers enter the telencephalon. The majority of these fibers pass through the diagonal bands of Broca, the septal areas, and then into the cerebral cortex, while a minority of fibers enter the telencephalon through the ganglionic eminences. Within the cerebral cortex, the fibers segregate into two groups; one superficial (within the marginal zone) and the other deep to the cortical plate. The hippocampal cortex receives fibers that follow a dorsomedial course through the cingulate cortex. Finally, there is a distinct set of late arriving serotonin-containing fibers that preferentially innervate the sensory cortices— these will be discussed below. Descending serotonin fibers enter the spinal cord as early as E14. These axons
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innervate the intermediolateral column (preganglionic sympathetic neurons) and somatic motor neurons, where they begin to form synapses by E17. Later, fibers innervate the dorsal horn neurons. As noted above, there is a set of serotonin immunoreactive fibers in the sensory neocortex that appear just after birth (D’Amato et al 1987). These fibers have recently received a great deal of attention, because it appears that they are not raphe projections. Rather, they are thalamocortical axons that contain serotonin (Lebrand et al 1996). Because thalamic neurons do not make serotonin, it was a mystery how the thalamic afferents could be immunoreactive for serotonin. Available evidence now suggests that these fibers absorb serotonin from the neocortex and perhaps the reticular nucleus using the serotonin transporter (Lebrand et al 1996). Serotonin has been implicated in the development of synaptogenesis. For instance, mice that have a mutation in the monoamine oxidase A gene, have excess levels of serotonin in their brain. These mice also lack the formation of normal synaptic architecture within the somatosensory cortex, resulting in the lack of so-called vibrissae-related barrel fields (Cases et al 1996). On the other hand, a depletion of serotonin also affects various aspects of barrel field development, although the barrels do form (Osterheld-Haas and Hornung 1996; Bennett-Clarke et al 1994a,b, 1995). The fact that the thalamocortical fibers incorporate serotonin further implicates this neurotransmitter in regulating the development of thalamocortical circuitry.
Serotonin as a Regulator of Development As noted above, there is evidence implicating serotonin in regulating the generation of the neocortical circuitry within the somatosensory cortex. Serotonin also appears to affect other developmental processes such as differentiation and cell migration (reviewed in Lauder 1995). For instance, decreased levels of serotonin delay the onset of differentiation of neurons that are found along the pathway where serotonin fibers grow (Lauder et al 1985). Several studies have demonstrated that serotonin receptors are expressed early enough to mediate these effects (Roth et al 1991; Hellendall et al 1993; Tecott et al 1995). In addition, nonneural brain tissues also express serotonin receptors. For instance, the choroid plexus has high levels of the serotonin 2C receptor (Hellendall et al 1993). It has been postulated that serotonin may regulate the production of neurotrophic factors from the choroid plexus (discussed by Lauder 1995). In addition, ependymal cells and radial glia express serotonin 1A receptors (Lauder 1995). Glial cells also express the S-100 beta protein, which acts as a trophic substance for serotonin neurons. It has been suggested that
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serotonin neurons stimulate adjacent glial cells to produce S-100 beta, and thereby provide trophic support (reviewed by Lauder 1995). Perhaps the clearest evidence that serotonin can regulate developmental processes comes from studies of craniofacial development. Ectomesenchymal cells express serotonin transporter and serotonin receptors (Lauder 1993; Shuey et al 1993). It is believed that they are exposed to serotonin derived from the maternal circulation. Drugs that impede serotonin transport or binding to receptors cause craniofacial malformations (see Moiseiwitsch and Lauder 1995). Serotonin can also regulate the migration of the cranial neural crest cells that produce most of the mesenchyme of the craniofacial primordia (Moiseiwitsch and Lauder 1995). It is possible that serotonin may have related effects within the central nervous system.
Perspective Evidence is accumulating that implicates serotonin in regulating developmental processes, including differentiation, cell migration, and synaptogenesis. Thus, this simple molecule appears to regulate both the development and function of the brain. An important goal for future studies will be to understand in detail how disrupting development of the serotonin system affects assembly and function of the brain. This is particularly germane, as there is evidence that the serotonin system is abnormal in a number of neurodevelopmental disorders that cause autism and mental retardation (Lauder 1995). This work was presented at the Neuroscience Discussion Forum “A Decade of Serotonin Research” held at Amelia Island, Florida in November 1997. The conference was sponsored by the Society of Biological Psychiatry through an unrestricted educational grant provided by Eli Lilly and Company.
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