Anatomy. Cell Biology and Maturation of the Serotonergic System:

Efrain C. Azmitia, Ph.D. and Patricia M. Whitaker-Azmitia, Ph.D.

The serotonin-producing neurons in the brainstem raphe nuclei have the largest and most complex efferent system in the human brain. It surpasses that described for the brainstem catecholamine-producing neurons or the descending cortical projecting system. The amino-acid transmitters are greater in absolute abundance throughout the brain, but they lack the strict anatomical boundaries characteristic of serotonin-producing neurons. In other words, the serotonergic neurons can exert a global influence, which is coordinated from a unified anatomical set of neurons. Glutamate, glycine, and GABA have neurons distributed throughout the brain, and these amino-acid transmitter neurons participate in local and projecting system that have no functional or anatomical cohesiveness. The special nature of the raphe brainstem neurons was recognized by the classical neuroanatomist Ramón y Cajal (90), who described these giant neurons in the brainstem midline but was unable to follow their extensive projections. Dahlstrom and Fuxe's work with histochemical fluorescence provided details concerning the anatomical architecture (45). It was immediately clear that these ancient neurons, located near the ventricular canals of the brain, innervated the entire neuraxis. Our work mapped five main ascending pathways and three descending pathways that provided redundant entry routes into terminal regions (14,20). The application of immunocytochemistry revealed the precise cellular details of the massive axonal branches flowing both up and down the brain (98,101).

Serotonin (5-hydroxytryptamine, 5-HT) was isolated by a chemist from the blood as a serum factor that increased smooth muscle tone (88). Professor DW Woolley, a chemist at The Rockefeller Institute, described the comparable actions of LSD and serotonin in the cortex of the cat. This finding so intrigued him that he wrote a prophetic book in 1962 entitled The Biochemical Basis of Psychosis; The Serotonin Hypothesis About Mental Disease (113). Brodie and Shore (37) proposed that serotonin and the catecholamines function as the autonomic nervous system of the brain by providing counteracting influences on affective states. Today, it is generally accepted that serotonin is important in a variety of psychiatric disorders (see clinical chapters).

The explosion of 5-HT pharmacology has provided clinicians with an array of drugs to manipulate the 5-HT system by interaction with individual 5-HT receptor-subtypes. The 5-HT1 receptors have a high affinity (nanomolar) for 5-HT, and several members of this family (A-F) have been cloned. This G protein-linked receptor has an inhibitory effect on adenylate cyclase activity and opens a K+ channel that produces hyperpolarization. The 5-HT2 receptors are low-affinity (micromolar) receptors, and several members of this group also have been cloned (A-D). Activation of this G protein-linked receptor increases Ca++ levels by stimulating phosphoinositol hydrolysis and depolarizes neurons by closing the K+ channel. The 5-HT3 receptor is a cation channel, and activation of this receptor results in neuronal depolarization. High numbers of this receptor are seen in limbic areas, where they may serve a role in anxiety and psychosis, and in the area postrema, where they play a role in chemically induced emesis. The 5-HT4-7 receptor families are G protein linked and positively coupled to adenylate cyclase. These receptors are found on neurons and glial cells and have varied distributions throughout the brain.

Drugs acting on particular receptors have proven useful for correcting chemical imbalances. Unfortunately, morphological deficits that produced the chemical imbalance often remain unchecked. In many instances, pharmacological treatment must be sustained indefinitely, with the added burden of increasing dosage due to the decreasing efficacy of the drug. Furthermore, treatment of the chemical imbalance may delay or prevent self-correction of the underlying morphological disorder. For example, if serotonin fibers in the rodent brain are partially removed, the animal's behavior is altered. It may become more aggressive or sexually receptive. Hormonal secretion and daily rhythms may be disrupted. Body temperature, eating and sleeping can become abnormal. However, given time (usually several weeks to months), the remaining 5-HT fibers can sense an increase in available growth factors and begin to re-colonize the vacant 5-HT tissue (11,12,21). The resetting of the trophic signal appears to be triggered when these receptors are unoccupied.

In addition to considering the role of serotonin, this neuronal system appears to have many interactions with the pituitary-adrenal axis. Adrenal steroids can influence the metabolism, pharmacology and growth of serotonergic neurons (9,17,18). Glucocorticoids and serotonin both function as maturational factors during early development (16). Therefore, both neural and endocrine factors are important in promoting recuperative sprouting.

The following chapter outlines the anatomy of the 5-HT neurons that project to the forebrain and spinal cord. The synthesis and release of 5-HT are discussed. Many factors have been shown to influence the survival, metabolism and elongation of CNS serotonergic neurons (22,38,46,49,59,74,76,85,104). We first reviewed this area of trophic influences on serotonergic neurons nearly 10 years ago using primary neuronal tissue cultures as a method to study serotonergic maturation and survival. Neuropeptides, such as ACTH, leu-enkephalin, substance-P, and neuropeptide-Y, can influence the growth of serotonergic neurons in culture and during early development (22,46,104). Our laboratory and others have found evidence for trophic effects from a wide range of protein factors such as S100b (13,75), BDNF (49,76,85), and insulin-like factor (13,75). Transgenic animals that overexpress transforming growth factor-alpha (TGF alpha) show gender-specific changes in serotonin development (59). There is also evidence suggesting that the basic fibroblast growth factors FGF-2 (38) and FGF-5 (74) are trophic for serotonin neurons. In this chapter, we shall present evidence for the neurite-extension properties of S100b, a glial factor under the control of the 5-HT1A receptor (112), and also briefly present some of the evidence that BDNF functions as a survival factor (85). Finally, the trophic role of 5-HT on its target cells is presented. The purpose of this chapter is to emphasize the two roles of serotonin in the mammalian brain: neurotransmitter and neurotropic factor (8). It is important for the reader to appreciate that 5-HT plays both roles throughout the life of the brain. Moreover, both these roles should be kept in mind in order to understand serotonergic disorders and the actions of psychotropic drugs. The following sections, therefore, highlights not only the anatomy and cell biology, but also the development and plasticity of CNS serotonergic neurons and the cells they innervates.

The anatomy and development of the brainstem raphe nuclei that projection to the forebrain and spinal cord have been reviewed previously (14). The following is a brief summary of the main nuclear groupings, fiber pathways and terminal innervation patterns for the interested reader.

During early prenatal development according to Wallace and Lauder (105), two groups of serotonergic neurons are visible, a superior group at the boundary between the midbrain and pons, and a separate inferior group that stretches from the caudal pons to the cervical spinal cord.

The developing 5-HT superior neurons express 5-HT as they migrate and immediately begin to send fine processes. Within hours, 5-HT-IR fibers are seen crossing the midline (73,105). The superior group of 5-HT neurons has been described as having two collection of neurons, rostral and caudal. The rostral collection gives rise to the caudal linear nucleus and most of the dorsal raphe nucleus. The caudal collection descends from the ependymal zone in two streams of cells that meet in the midline to form the superior central nucleus (median raphe nucleus and the interfascicular portion of the dorsal raphe nucleus) [Fig. 1]. In the human, the dorsal raphe nucleus is the largest of the ascending nuclei, with 235,000 5-HT immunoreactive neurons (25). The 5-HT1A is the cell body autoreceptor, localized on raphe neurons and on target neurons, astrocytes and ependymal cells (23). The serotonergic neurons in the raphe nuclei normally have a slow, rhythmic firing rate when the animals are awake. The activity of most of these cells is linked to motor activity (brainstem and spinal cord) (63). Activation of the 5-HT1A autoreceptors results in a powerful inhibition of the firing rate. The 5-HT1D receptor has also been localized to serotonergic cell bodies and is believed to inhibit the local release of 5-HT (see Fig. 4).

The most rostral group is the CLN which starts at the level of the red nucleus . The 5-HT neurons are located between the rootlets of the oculomotor nuclei and extend dorsally from the anterior edge of the interpeduncular nucleus to blend with the rostral dorsal raphe nucleus. The neurons are often situated rostral to the median raphe nucleus (MRN) and are incorrectly considered as part of the MRN. The projections from these region extend to the thalamus and cortex.

The DRN is divided into medial, lateral (the wings), and caudal components. The medial component can be further divided into a mediodorsal (superior) and an interfascicular component (Fig. 2). The superior component is in the central gray, just below the cerebral aqueduct. The interfascicular component surrounds the MLF and is especially prominent between the fasciculi. These neurons blend with the caudal MRN. The lateral component (the wings) forms the larger division of the DRN and extends as far rostrally as the oculomotor nuclei. In the human, the lateral wings can be divided into a dorsal and ventral subdivision (101).

The MRN is a paramedian and median cluster of cells lying below and caudal to the superior cerebellar decussation (SCD) [Fig. 2)]. Scattered 5-HT cells of the MRN are seen ventrolateral to the MLF. These laterally situated cells lie in the nucleus pontis centralis oralis (87) and form a ring around the central tegmental tract, one of the most primitive ascending pathways carrying reticulothalamic axons. According to Olsewski and Baxter's human brain atlas (87), the MRN is but one part of the larger superior central nucleus (SCN), which includes the interfascicular aspect of the DRN. Although there is substantial anatomical, developmental and functional evidence to support including the DRN interfascicular neurons in the SCN, current usage prescribes keeping the original classification of DRN and MRN proposed by Dahlstrom and Fuxe (45).

This group (originally classified as B9) is located along the superior surface of the medial lemniscus, from the rostral border of the inferior olive to the level of the red nucleus. These cells are occasionally continuous with the cells of the MRN and form the ventral border of the ring of scattered cells that surrounds the central tegmental tract in the pontine reticular formation.

These collections of 5-HT neurons show a different developmental pattern from the superior group of serotonergic neurons. They express 5-HT phenotype after they have migrated into the tegmentum of the brainstem (105). These neurons show the characteristic localization along the midline, but in addition extend into several well known reticular nuclei.

This group (originally classified as B2) is a collection of large-medium multipolar neurons. They form a symmetrical paramedian cluster on either side of the midline. This dorsally situated nucleus extends from the caudal pons back into the cervical spinal cord. 5-HT neurons in the spinal cord lie ventral to the central canal and on the medial border of the ventral horn. The 5-HT neurons are commonly intermixed with the medial longitudinal fasciculus (MLF), the tectospinal tract (tst), and the dorsal aspect of the pyramidal decussation. The nucleus is more dense caudally in the medulla; at the level of cranial nerve VI, it is less densely packed than either the ventrally situated NRM or the NRPa.

The extraraphe component of NRO was described in the human brain based on Nissl staining (87). These 5-HT neurons form a splinter group from NRO and lie just ventral to the fourth ventricle at the level of the VII cranial nucleus. These neurons were previously designated nucleus raphe ventricularis (14).

This group (originally designated as B1) is a group of medium sized multipolar 5-HT neurons in paramedian columns. The nucleus stretches from the cranial nerve XII to the anterior end of the inferior olive. The lateral aspects of the nucleus extend over the mediodorsal surface of the pyramidal tracts, while the main body of the nucleus lies between the pyramidal tracts. The cells appear to be contiguous with the NRM anteriorly and with the VLM laterally.

This collection of medium to large 5-HT neurons (originally classified as B3) extends from the rostral superior olive back to cranial nerve XII. This nucleus lies between NRPa and NRO, and at points the borders between these three nuclei are difficult to demarcate. The nucleus is invaded by both the trapezoid body and the dorsal border of the medial lemniscus. Occasionally, very large 5-HT neurons are seen more laterally in the boundary of the nucleus reticularis gigantocellularis.

A large number of medium-sized, multipolar 5-HT neurons (originally part of B1/B3) are seen in the ventral lateral medulla. The nucleus extends from the Inferior olive to cranial nerve XII. The neurons are closely associated with the pyramidal tract, trapezoid body, and medial lemniscus. This nucleus overlaps with two important reticular nuclei; rostrally it forms the medial component of the reticular lateral paragigantocellularis nucleus, while caudally it forms the medial part of the inferior reticular nucleus. At its most ventral position, the neurons lie against the pial surface and are closely intertwined with the large blood vessels entering the medulla.

This is a large collection of very small 5-HT neurons that lie ventral to the fourth ventricle and are associated with the parabrachial area. The neurons are considered immature and have a bipolar or simple oval shape.

The serotonergic fiber pathways are extremely complex to describe, since they include aspects of all the main pathways in the brain. There are five routes into the forebrain and three routes into the spinal cord (Fig. 3). These routes give rise to countless branches which follow other neuronal pathways, blood vessel ramifications, the ependymal lining of the ventricular system and even the pial surface. The molecules responsible for this expansion appear to be produced by both glial cells and neurons. Glial cells, especially immature ones, have laminin on their surfaces. This attachment factor has been shown to guide the growth of serotonergic fibers, even in the adult brain (116). S100b is an abundant soluble protein produced and secreted by glial cells (15,21). The secretion of S100b is decreased when 5-HT levels are reduced (19,55) and stimulated by a 5-HT1A agonist (112). S100b appears to function by stabilizing the cytoskeleton and permitting the formation of long neurites. Another protein that increases 5-HT neurons is BDNF. BDNF increases 5-HT synthesis, neuronal survival and sprouting (76,85). However, the increased sprouting may require S100b (85).

Detailed publications should be consulted to learn the precise local network of the 5-HT sprouts. In this review, the main pathways are listed for the descending and ascending projections, with some examples of local networks.

The innervation of the brainstem is very extensive. Motor and sensory nuclei are densely filled with 5-HT fibers and much is known about the local networks. The subnuclei of each nucleus have their own differential innervation patterns. Important functions for 5-HT neurons on brainstem systems regulating respiration, sleep, cardiovascular function, metabolism, temperature and cortical-reticular-activating effects are supported by selective innervation of the various subnuclei.

The serotonergic fibers projecting to the forebrain originate mainly in the superior group of raphe nuclei. At least five separate ascending pathways have been described in rats and primates (Fig. 3). In the rat, the largest pathway is the medial forebrain bundle, which carries fibers from the MRN and the DRN to a wide range of target areas in the forebrain. In primates, a significant number of these fibers (~25%) are heavily myelinated (14). Furthermore, in primates, the largest pathway appears to be the dorsal raphe cortical tract, which enters the cortex through the internal capsule network. 5-HT-containing fibers are seen associated with all fiber tracts in the brain, from the fornix to the pyramidal tracts. Furthermore, serotonergic fibers are seen along blood vessels, on the ependymal cells lining the ventricles, the pia mater, within the circumventricular organs (e.g., the area postrema, subcommissural organ, median eminence, and neurohypophysis) and the choroid plexus.

In the adult forebrain, a dense innervation is seen in the suprachiasmatic nucleus (rhythm center), the substantia nigra (source of dopamine neurons), the Papez circuit and related limbic centers, and around and in the ventricles. Serotonergic fibers in the cortex are abundant in limbic areas and the primary sensory and association areas (A13). The lowest levels are found in the motor regions of the frontal lobe. All these fibers have abundant branches, with thousands of varicosities and terminals filling the terminal areas. Virtually every cell in the brain is in close proximity to a 5-HT fiber and capable of responding to 5-HT by the process of volume diffusion. Seven families of 5-HT receptors have been identified in the forebrain and compliment the extensive distribution of fibers and 5-HT related functions.

In the cortex, the 5-HT fibers are the first afferent system to arrive and the last to establish its innervation pattern (73). The 5-HT fibers stream across the superficial and deep layers of the primordial cortex to innervate all cortical layers diffusely. More extensive branching proceeds in the granular cell layers (layer IV of cortex) [7]. The close association between 5-HT fibers and granule neurons is seen in all cortical areas, even the hippocampus, where the granule neurons are confined to the dentate gyrus. These granule cells complete their final mitosis in the adult brain long after the pyramidal layer (2). Granule neurons receive direct thalamocortical connections, so 5-HT would be positioned to modulate the electrical entry into the cortex and influence cognitive functioning. The 5-HT2A receptor has a postsynaptic location on GABA interneurons in the pyriform cortex and large pyramidal neurons located in layer 5 (see Aghajanian chapter). It underlies many of the motor effects of 5-HT and is involved in the actions of the major hallucinogenic drugs. The 5-HT2A and 5-HT2C receptors are located on astroglial cells and regulate energy availability by stimulating the breakdown of glycogen (89).

The 5-HT projections from a single neuron or group of neurons can innervate several synaptically interconnected target regions (5, 62). For example, the MRN innervates the cingulate cortex, septal nuclei and the hippocampus, while the DRN innervates the substantia nigra, corpus striatum, amygdala and nucleus accumbens. Individual neurons in the DRN and MRN of rat may project to sensorimotor portions of both cerebral and cerebellar cortex, or to visual portions of both (106). The connections made by synaptically linked neurons could be matured by the collaterals of a single serotonergic neuron. When serotonergic neurons are active (e.g., during motor activity or arousal; see chapter by Jacobs, this volume), the connections would become stabilized, but when the 5-HT neurons are silent (e.g., during sleep), the target neurons would be exposed to a de-differentiating influence. As mentioned above, 5-HT fibers are the last extrinsic afferents to complete their innervation of the cortex and hippocampus (10). It was proposed that the serotonin innervation signals the completion of various neuronal circuits and is involved in verification and consolidation of interneuronal contacts (73). However, we propose that neuronal circuits are never complete (finished, stabilized) and advance the idea that neuronal circuits are normally capable of continual modification and correction to accommodate a changing environment.

There are at least three main entry routes into the spinal cord. The nucleus raphe obscrus innervates the ventral horn of the spinal cord using the posterior fasciculus of the spinal cord. The 5-HT fibers follow the MLF and tectospinal tract as these pathways sweep ventrally from their more dorsal position in the medulla. Many fine 5-HT varicose fibers are seen throughout the ventral horn. 5-HT-fibers densely innervate the primary alpha motoneurons, where the fibers encircle the entire motoneuron. The motoneurons contain both 5-HT1A and 5-HT2A receptors. Using an antipeptide antibody against the 5-HT1A receptor (24), we have seen receptor distribution on the dendrites, soma and most heavily on the proximal axonal segment (23,67).

The nucleus raphe magnus innervates the dorsal horn. A very dense plexus of fine varicose fibers extends through the substantia gelatinosa. A more sparse innervation exists in the other nuclei of the dorsal horn. This differential pattern extends into the medulla, where the nociceptive subnuclei of the trigeminal nuclei receive a very dense infiltration by 5-HT fibers (43).

The last major input into the spinal cord comes from the ventral lateral medullary 5-HT neurons. This fibers use the lateral fasciculus of the spinal cord to innervate the lateral horn. The 5-HT fibers use the medullary reticulo-spinal tract to reach the intermediate gray. The 5-HT fibers innervate both the sensory and motor nuclei of the autonomic system located at every spinal level.

Once the fibers have reached their targets, 5-HT must be released to exert both its electrical and neurotropic functions, but depolarization is not the only means of releasing 5-HT. It can also be released from vesicles and through the 5-HT transporter protein (Fig. 5). In the following section, the various local mechanisms influencing release are discussed. Many readers will be surprised to learn that 5-HT is released by the 5-HT transporter, working in reverse from its normally envisioned method. However, this form of release has been known for the dopamine system since the classic studies by Fisher using amphetamine to induce release (50 ). 5-HT can also be released by compounds such as methamphetamine, MDMA, MDA, and fenfluramine, and, to a much lesser degree, by cocaine and fluoxetine (29). Furthermore, this chapter will propose the interesting idea that vesicular and non-vesicular release are linked and in an intracellular balance (53). The consequence of this release coupling to neurotransmission after administration of 5-HT releasing compounds deserves further study, as it may have clinical relevance.

5-HT is synthesized from tryptophan, an essential amino acid that is preferentially taken up by serotonergic neurons. The levels of serotonin, and it fact its availability for release, are dependent on the levels of L-tryptophan. Tryptophan is also needed for the biosynthesis of a number of molecules that have important cellular and neural functions. These include melatonin, tryptamine, N-methyl-tryptamine, kynurenine, anthranilic acid, and quinolic acid. Furthermore, quinolic acid can combine with nicotinic acid to form the important cofactors NAD and NADP. Most of these metabolic pathways predominate over the pathway for serotonin.

The synthesis of 5-HT, which is blocked by p-chlorophenylalanine (PCPA), a specific inhibitor of tryptophan hydroxylase (the rate limiting enzyme in the biosynthesis of serotonin), is increased by a Ca++-dependent stimulation in the presence of adrenal glucocorticoids (54). The newly synthesized 5-HT is stored in vesicular pools and can be released when the neuron fires. Extracellular 5-HT, the 5-HT which is released from a serotonergic neuron, is quickly and preferentially destroyed by monoamine oxidase (MAO) A (inhibited by clorgyline). The extracellular 5-HT must gain access to other cells in order to be destroyed by MAO-A. MAO-A is found in a wide variety of neurons (e.g., dopaminergic, pyramidal neurons) and non-neuronal cells (endothelial, astrocytes) [53,71]. 5-HT is taken up by both dopaminergic neurons and astrocytes (see chapter on transporter proteins).

Any 5-HT that escapes metabolism is quickly taken back into the cytoplasm of the 5-HT neuron (reuptake). This process is mediated by the transporter protein, which is driven by the Na+ gradient (32,50,53). The 5-HT in the cytoplasm can be stored, transported to vesicles, or degraded by monoamine oxidase-B (inhibited by deprenyl). The 5-HT is transported into the synaptic vesicles by a reserpine-sensitive transporter protein (96). It is degraded by MAO-B only when levels reach approximately 10-5 M, since this form of MAO has a lower affinity for 5-HT than MAO-A. The 5-HT remaining in the cytoplasm is available for an alternate form of release, that mediated by the serotonin transporter. This release can be initiated by a variety of drugs, including fenfluramine, p-chloroamphetamine and 3,4-dioxymethylene-methamphetamine (MDMA) [29]. Interestingly, these same drugs at low doses can inhibit the re-uptake of 5-HT. Cocaine and fluoxetine are mainly 5-HT reuptake blockers and can inhibit the release of 5-HT by fenfluramine, MDMA and PCA (29).

The 5-HT stored in cytoplasmic and vesicular pools is in a steady-state relationship (53,102). When 5-HT is released from one pool, it can be taken from the other pool (53). For example, neurons stimulated by MDMA release 5-HT through the 5-HT transporter. However, a component of the 5-HT released actually originates from the vesicular stores. Reserpine, an inhibitor of 5-HT uptake into vesicles, depletes the vesicular stores of 5-HT and also reduces the amount of 5-HT that exits the cell after MDMA exposure. Also, if MAO-B is inhibited by deprenyl, the depolarization-induced release of 5-HT is augmented. When depolarization and MDMA are combined, a greater-than-additive release is seen (53). Finally, stored 5-HT may actually be transported from a distant target area (e.g., hippocampus) back to the midbrain raphe cell bodies within 5-HT axons by retrograde flow (6).

The first studies showed that perinatal depletion of serotonin in rats with PCPA or 5,7-DHT delayed maturation and the postnatal period of neuronal proliferation in the forebrain (69), reduced the rate of increase in forebrain weight (60), and decreased the density of granule cell dendritic spines, conditions that worsened as the animals grew into adolescence (56). We have found permanent (up to 180 days) loss of dendrites, accompanied by profound changes in learning and memory, after PCPA injections during peak synaptogenesis (PND 10–20). Chubakov and his colleagues showed that serotonin plays a role in neurite outgrowth in the target region, increases electrical interconnections and promotes synaptogenesis in hippocampal cultures (41).

In adult animals, the loss of 5-HT results in a loss of protein and molecular markers associated with a mature neuronal phenotype. These losses are transient, and we have suggested that serotonin is needed to maintain neuronal maturation. In adult rats, PCPA, given for 8 days, produced up to a 50% decrease in the number of non-monoaminergic synapses in somatosensory cortex (40). It also produced a marked reduction in both MAP-2-IR and synaptophysin-IR in adult rat hippocampus (109). PCA, a 5-HT releaser that lesions serotonergic axons, resulted in the appearance of degenerating neurons seen throughout cortex (42) and produced substantial reduction of MAP-2-IR and synaptophysin-IR throughout the brain (19).

These studies indicated that 5-HT not only promoted neuronal maturation during development, but also maintained the maturational state in the adult. Growth factors are commonly accepted to have effects in the adult brain. In fact, many of the ideas of dementia associated with aging attribute the loss of neural function to a loss of trophic molecules or receptors. This implies, although it has not directly been stated before, that the growth factors must somehow be involved in maintaining the mature neuronal morphology. A simple analogy would be the effects of sunlight on plant growth. The light is needed to promote growth and to sustain it, and when light becomes limiting (for example, during winter), the plant regresses. It loses its morphology, the leaves fall and, in certain cases, the entire plant appears to have died. However, normally the root system is intact, and when conditions are favorable again (for example, during spring), the plant grows with greater vigor than before. Neurons, of course, are not plants, but they appear to grow in response to the presence of 5-HT. Growth in neurons consist of mitosis, migration and maturation. This last process is the one that is most obviously affected by serotonin, although there are many reports of the action of 5-HT on both migration and mitosis (see ref. 68). In term of maturation, the main processes are expression, elongation, elaboration and engagement. During early development, an immature neuron expresses the genes and proteins needed to produce, release and regulate neurotransmitter molecules. These small neuroblasts quickly begin to elongate their axons. In the case of 5-HT, this process begins in the first trimester, and the highly branched axons reach every area of the neuraxis. Dendritic elaboration occurs next, and the multipolar dendrites are studded with spines. The mature neurons, using receptors, form engagements with a variety of cells, both neuronal and non-neuronal. All these events are under the control of a host of genes and proteins (some of these will be discussed below). Interestingly, many of the events that govern growth occur independent of genetic control. Cajal, the great Spanish neuroanatomist, showed that axons could continue to grow and sprout for a time, even when the axon was severed from the cell body (91). Furthermore, many of the events involved in forming and stabilizing the cytoskeleton are regulated by phosphorylation mechanisms (see below). Thus, the process of maturation and de-maturation can be quickly and effectively regulated by non-genomic mechanism in the local environment. In fact, ribosomes for making proteins are abundantly distributed throughout the dendrites. The possible role of 5-HT in influencing the dynamic shifts of maturation of forebrain neurons is presented below, but the reader should understand that this new concept is not limited to the actions of 5-HT (as important as they are) but is applicable to all trophic and tropic molecules that have effects on neuronal maturation.

In the next few paragraphs, we will focus on S100b, because it appears to be critical in stabilizing the cytoskeleton and because its release is regulated by the 5-HT1A receptor. However, it is important to note that S100b does not appear to be a survival factor (13,75). Brain-derived neuronal growth factor does promote survival of 5-HT neurons in the brain. The serotonergic neurons contain the mRNA for trk-B receptors, the signal transducing receptors for BDNF (79). When BDNF (10–100 ng/ml) is applied to primary brainstem raphe cultures, the number and size of serotonergic neurons are dramatically increased (85). Interestingly, BDNF also promotes sprouting of serotonergic neurons, both in culture and in vivo (76). In culture, application of antibodies raised against S100b blocks the increased neurite elongation (85). Thus, the actions of BDNF on serotonergic sprouting could be mediated via release of S100b from glial cells, either directly via trk-B receptors, or indirectly via release of 5-HT from serotonergic neurons. A model of the potential interactions between S100b and BDNF has been proposed of ref. 85).

Many of the trophic effects of serotonin on target tissues are produced by the 5-HT1A receptor. The 5-HT1A receptor plays a role in migration of cranial neural crest cells (81), is associated with synaptic plasticity in the visual cortex of dark-reared rats (82), increases choline acetyltransferase activity and neurite outgrowth and branching in cultured septal cholinergic neurons (93) and stimulates growth of synaptophysin-IR varicosities in hippocampal cell cultures (84). The effects of PCPA depletion in the neonate are reversed by the 5-HT1A agonist buspirone (57). Neonatal rats treated with a 5-HT-depleting drug or the 5-HT-1A antagonist NAN-190 show a loss of spines on hippocampal pyramidal neurons (114).

There is evidence for the involvement of other 5-HT receptors. The 5-HT1B receptor may be involved in postnatal regulation of pattern formation in somatosensory cortex (30,70,92). Several researchers have also suggested a trophic role for the 5-HT2A receptor, including actions on synaptogenesis (85).

5-HT receptors are distributed on many cells types in the brain and body (23, 63,110). Astroglial cells possess a number of different neurotransmitter receptors, including 5-HT1A, 5-HT2A, 5-HT2C and 5-HT7 receptors. When the 5-HT1A receptor is stimulated, the astroglial cell responds by releasing S-100b and attaining a mature morphology, with a shift from a flattened morphology to a process-bearing morphology (112). In this treatment, there is an effective feedback inhibition, since, as the glial cell matures, it binds to the 5-HT-1 receptors (108). The 5-HT2A receptor on astroglial cells upregulates glycogenolysis (89). Thus, 5-HT, by stimulating both the 5-HT-1A receptor and the 5-HT2A/C receptor regulates the release of both S100b and glucose, two factors necessary for neuronal sprouting. The pharmacology of these glial receptors has been discussed (107). The expression of these glial receptors can be downregulated by 5-HT1A receptor agonists and cyclic-AMP or upregulated by dexamethasone and PCPA (72,84,108,111).

Much of serotonin's influence on target tissues on the mature brain appears to relate to actions of the 5-HT1A receptor. In adult rats, the decreases in MAP-2-IR and synaptophysin-IR seen after PCA injections are rapidly restored towards control levels after three days of injections with a 5-HT-1A agonist (19). Removal of glucocorticoids, by adrenalectomy, results in loss of the adult phenotype of granule neurons in the dentate gyrus (72,97) and a re-expression of the transitory peak in the 5-HT-1A receptor (39,77). The morphological changes induced by the loss of glucocorticoids can be rapidly (24–72 hr) reversed both by steroid replacement or by a 5-HT-1A agonist (60). There is also evidence for a trophic role for 5-HT2A/C receptors in the adult spinal cord (94).

The loss of synapses, terminals and dendrites in adults can be considered a retraction of the mature state rather than evidence of neuropathological degeneration. This is consistent with the rapid reversibility of the morphology when 5-HT is replaced or the 5-HT1A receptor is activated (16). Serotonin maintains the adult morphology by stabilization of the cytoskeleton of the cell, either by decreasing the levels of cAMP or by releasing the glial factor S-100b.

S-100b is an important factor in regulating the development of cortical neurons and astrocytes (15,21). Therefore, any factor which influences the release of S-100b from the astrocytes where it is produced also may regulate the maturation of neurons and their supporting cells by stabilizing the cytoskeleton. Most of the cortical release of this factor appears to be through stimulation of 5-HT1A receptors. Perinatal injections of PCPA, PCA, 5,7-DHT or cocaine all reduce the levels of S100b (1,19,55). Thus, 5-HT, by acting on glial receptors linked to S100 release, can maintain the mature cytoskeleton. The clinical implications of accelerated activation of this system are discussed below.

Stress can have profound effects on the 5-HT system. A relationship between stress, steroids and 5-HT synthesis and turnover was first reported nearly 30 years ago (9,18). Recently, using a specific antipeptide antibody against a unique region of the tryptophan hydroxylase protein, an increase in the amount of protein in adrenalectomized animals was confirmed by both Western immunoblots and immunocytochemistry of the raphe nuclei after exposure to dexamethasone (17). Stress has significant effects on brain 5-HT metabolism which are blocked by adrenalectomy (ADX) and restored by corticosterone or dexamethasone replacement (31,47). In addition, the growth of 5-HT fibers during development and after damage may require the presence of adrenal steroids (99,115).

The increase in synthesis and turnover of 5-HT is associated with changes in the 5-HT1A receptor. Short-term adrenalectomy increases the binding of 8-OH-DPAT to hippocampal membranes (78). In situ hybridization studies indicated an increased expression of 5-HT1A receptor mRNA in the cornu Ammonis of the hippocampus. This appeared to be mediated by the type-1 mineralocorticoid receptor (MR) [39]. In contrast, long-term adrenalectomy resulted in decreased expression of 5-HT1A mRNA in the dentate gyrus (72). This decrease was reversed by dexamethasone, an agonist at the type-2 glucocorticoid receptor (GR). It is possible that these changes reflect a neuronal vs. a glial localization of the 5-HT1A receptor, with the glial 5-HT1A receptor regulated by GR and the neuronal receptor by MR (84). This would be consistent with the block of the 5-HT1A receptor mediated change in a Ca++-independent K+ channel on neurons by mineralocorticoids (65). The multiple interactions between 5-HT and stress makes this area of neuroscience a challenge for devising effective, long-term therapy.

In humans, 5-HT1A receptors are highest early in gestation (26) and decrease with age (44,80). The potential for plasticity shows a corresponding decrease as the brain matures. Developmental disorders, such as Down's syndrome, are associated with changes in the levels of 5-HT1A receptors. Given the recent interest in schizophrenia as a developmental disorder, it may be pertinent, that 5-HT1A receptors actually increase dramatically in the schizophrenic brain (66).

Serotonin manipulations produce transient changes in the brain morphology of experimental animals. There are a number of disorders in which similar changes may be occurring in humans. Serotonin uptake inhibitors are effective in the treatment of anorexia nervosa (3). In MRI studies of anorexic children, a dramatic loss of brain volume occurs, which normalizes when the children are well (58,95). This observation indicates that pharmacologically induced changes in human brain volume are possible, especially since recovery can be enhanced with 5-HT1A agonists.

Prenatal exposure to alcohol or cocaine disrupts the normal developmental maturation of serotonergic fibers and thus may contribute to some of the damage seen in crack babies or fetal alcohol syndrome. In both of these models, 5-HT1A agonists reverse the loss of 5-HT fibers and the retarded brain maturation seen in neonatal rats (1,100). Alcohol-preferring rats show marked disruption of serotonergic forebrain innervation (117). In human alcoholics, there is a significant loss of brain volume which is inversely correlated with the disease state (64). These examples of chemically-induced loss of brain volume and its return to the normal range indicates that severe morphological shrinkage is not terminal.

Patients with posttraumatic stress disorder (PTSD) with alcohol dependency have been reported to respond well to sertraline, an SSRI (35). Patients with PTSD show a loss of brain volume (36) and respond well to serotonergic drugs (35). Other types of depression result from a loss of brain 5-HT levels. PCPA and a tryptophan-free drink induce a rapid onset of clinical depression in patients with previous depressive episodes which responded to treatment with 5-HT drugs (48). This indicates that loss of serotonin itself, can produce clinical evidence of depression. In addition, suicide is often considered as an extreme form of depression. Selected brain regions from suicide patients show reduced 5-HT fibers and increased 5-HT1A receptor binding (5). These studies indicate a correlation between loss of 5-HT fibers and depression. In the case of PTSD, a corresponding (transient) loss of brain volume has been suggested.

Based on the trophic role of 5-HT presented in this volume, it is tempting to speculate that the loss of 5-HT may be more than a correlation. If there is a corresponding loss of neuronal morphology in human forebrain associated with PTSD, it would not be unreasonable (if loss of 5-HT was persistent in certain brain regions) to assume that severe depression might also be associated with loss of neuronal morphology in these same regions. This hypothesis would explain many of the persistent clinical symptoms of depression, including withdrawal, lethargy, loss of rhythms, vegetative state, inability to feel pleasure and reward, loss of pleasant memories, cognitive deficits and loss of appetite. It would also provide an explanation for the "lag-time" evident in most cases of clinical recovery which are based on monoamine treatment.

We can speculate that loss of 5-HT leads to neuronal de-differentiation (de-maturation) and then depression. This would be consist with the animal literature, where it was noted that loss of 5-HT produced decreases in synapses and a variety of neuronal marker proteins, such as MAP-2 and synaptophysin (19). In animal studies, the loss is transient and depends upon the decreased availability of S100b. In humans, the loss of cortical gray matter seen in PTSD may reflect the instability of the mature neuronal phenotype. As explained above, when the cytoskeleton is disrupted, neuronal dendritic processes collapse and the neuron assumes a smaller and less-elaborate morphology. Full recovery would require morphological restoration of retracted neural connections.

Recent work has shown that activation of the 5-HT-S100b link can result in accelerated maturation and premature aging. This appears to be particularly relevant to the development seen in S100b transgenic mice, where synaptic markers in the hippocampus show a greatly accelerated maturation but also an early decline (Whitaker-Azmitia, in preparation). Since Down's syndrome is believed to result from trisomy of chromosome 18, where the S100b gene is localized (28), accelerated development may be responsible for many of the deficits seen in these children (34). It is noteworthy that all Down's patients eventually develop Alzheimer's disease if they survive into mid-life (52). Thus, premature or overstimulation of S100b release can have long-term deleterious effects.

The links between 5-HT and Down's syndrome are strong. Twenty-five years ago, scientists recognized that Down's patients have attenuated 5-HT systems. In 1965, Tu and Zellweger (103) reported low blood levels of 5-HT in Down's children, and Bazelon (27) showed that 5-HTP could reverse hypotonia in these infants. Researchers described these children as showing accelerated aging (34), and the trophic 5-HT1A receptor does show a very early prenatal peak in Down's fetuses (27). Thus, it appears that the premature activation of the 5-HT system results in accelerated aging and lower 5-HT levels at birth. Although there is long-term improvement with 5-HT drugs or special high-tryptophan diets (51), the progression of the disease is unaltered. Interestingly, cholinergic enzymes such as choline acetyltransferase and acetylcholinesterase are not abnormal in young children with Down's syndrome (28).

In summary, we have provided evidence that loss of morphology may be central to the etiology of many clinical disorders associated with an altered 5-HT system. The challenge is to recognize and utilize the trophic and tropic properties of serotonin and other brain neurotransmitters and factors to encourage regrowth of lost neuronal connections without producing a condition of hyperinnervation.

The serotonergic system is both phylogenetically and ontogenetically ancient. Two main groups of neuronal cell bodies produce serotonin: the superior and inferior raphe nuclei. The superior group innervates the midbrain and forebrain, while the inferior group innervates the cerebellum, pons, medulla and spinal cord. The serotonergic neurons in the superior group are in the caudal linear nucleus, the dorsal raphe nucleus, median raphe nucleus, and the supralemniscal nucleus. The inferior group consists of the nucleus raphe obscrus, nucleus raphe magnus, nucleus raphe pallidus and the reticular nuclei in the ventral lateral medulla.

The superior raphe nuclei use five pathways to innervate the forebrain: the two main ones are the medial forebrain bundle and the dorsal raphe cortical tract. The inferior raphe nuclei innervate the entire spinal cord via the medial longitudinal fasciculus, tectospinal tract, and reticulospinal tract. Many branches form from these main pathways. The smaller 5-HT branches grow along other neuronal pathways, blood vessels, ependymal cells lining the ventricles and along the pial surface surrounding the brain and spinal cord. There is evidence for laminin and S-100 being chiefly responsible for the extensive network created by the 5-HT neurons.

The 5-HT network permits 5-HT to be released throughout the brain and spinal cord. 5-HT release is dependent upon the firing of the brainstem raphe cell bodies and on local synthesis of serotonin, which can escape through the 5-HT transporter protein. The firing is regulated mainly by the 5-HT1A receptor, while release is regulated by 5-HT1B/D receptor. Evidence suggests that raphe neuronal firing is influenced by descending corticospinal pathways associated with locomotor activity, while local synthesis is influenced by such factors as substrate availability (tryptophan, oxygen, biopterin), steroids, energy and phosphorylation.

In the last few years, it has become increasingly evident that communication between neurons and their supporting glial cells is crucial for normal functioning of the mammalian brain. The functions thus regulated include not only the normal homeostatic mechanisms for providing energy and removing wastes, but also more vital functions—the development and aging of the entire brain. One result of signaling between serotonergic neurons and astrocytes may be the release of S-100b. This soluble protein is a glial mitogen and a potent neurite extension factor for serotonergic neurons, cortical neurons and spinal motoneurons (15,21). 5-HT can then regulate its own growth (autotrophic) and induce the maturation of a variety of target cells through this glial protein.

Stress-, drug-, and steroid-induced 5-HT imbalances can alter the architecture of the brain and result in mental disorders that can be treated by encouraging selective serotonergic sprouting. The strong 5-HT link between the structure and function is an important insight to neuropsychopharmacologists interested in the permanent redesigning of the brain in order to correct mental disorders.

published 2000