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PHYSIOLOGICAL REVIEWS Vol. 72, No. 1, January Printed 1992 in U.S.A. Structure and Function of the Brain Serotonin System BARRY L. JACOBS AND EFRAIN C. AZMITIA Program in Neuroscience, Department of Psychology, Princeton University, Princeton, New Jersey; and Center for Neural Science, Department of Biology, New York University, New York, New York I. INTRODUCTION In the course of reviewing the anatomy and physiology of central nervous system (CNS) serotonin [5=hydroxytryptamine (50HT)] in vertebrates, we focus on three major themes. First, although 5-HT has been implicated in a vast array of physiological and behavioral processes in vertebrates,’ it appears to be essential for ’ This may also apply to invertebrates, but their somatic diversity and the ganglionic organization of their nervous systems make it 0031-9333/92 $2.00 Copyright 0 1992 the American Physiological 165 166 166 166 16’7 171 172 175 177 179 180 180 180 182 183 185 187 188 188 188 189 190 194 194 194 198 209 209 210 210 211 211 212 212 213 214 215 Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 I. Introduction ......................................................................................... II. Historical Perspective ............................................................................... III. Anatomy ............................................................................................. A. Introduction ...................................................................................... B. Nuclei ............................................................................................ C. Afferents ......................................................................................... D. Pathways and terminals ......................................................................... E. Distribution of receptors ........................................................................ F. Ultrastructure ................................................................................... G. Summary ......................................................................................... IV. Development ......................................................................................... A. Introduction ...................................................................................... B. Nuclei ............................................................................................ C. Pathways and terminals ......................................................................... D. Tissue culture .................................................................................... E. Growth regulatory factors ....................................................................... F. Summary ......................................................................................... V. Adult Plasticity ...................................................................................... A. Introduction ...................................................................................... B. Regeneration ..................................................................................... C. Collateral sprouting .............................................................................. D. Transplantation .................................................................................. E. Summary ......................................................................................... VI. Cellular Neurophysiology ........................................................................... A. Unit activity in anesthetized animals or brain slices ........................................... B. Unit activity in behaving animals ............................................................... VII. Postsynaptic Actions ................................................................................ A. Introduction ...................................................................................... B. Inhibition/modulation ........................................................................... ........................................................................... C. Excitation/modulation D. Cotransmission .................................................................................. E. Latency and duration of action .................................................................. F. Summary ......................................................................................... VIII. Discussion and Speculation ......................................................................... A. Activity and actions of serotonergic neurons ................................................... B. Growth/plasticity and physiology ............................................................... C. Two serotonergic systems ....................................................................... none of them (92a, 236a, 507). This apparent contradiction is reconciled by the concept that the expansive system of serotonergic neurons exerts a tonic modulatory influence on its widespread targets. This is expressed primarily in association with the organism’s motor activity and is in phase with the sleep-wake-arousal cycle. difficult to encompass them within this generalization. Nonetheless, similar conclusions to those made here have been arrived at by a number of investigators working in this field. The reader is referred to articles by Kravitz (270), Kupfermann et al. (273), and Lent (287). Society 165 166 BARRY L. JACOBS AND II. HISTORICAL PERSPECTIVE The brief history in this section describes the seminal phases of research in this area, especially as they pertain to this review. The central or midline (raphe) location of the large neurons in the brain stem has attracted the attention of anatomists since the time of Ramon y Cajal (396), who described these cells as large multipolar neurons with uncertain projections. No one suspected that they contained the same chemical substance distributed throughout the body. For many years, investigators had known of a blood-borne chemical that produced vasoconstriction (a “serum” factor that affected blood vessel “tonus,” hence the name serotonin) and of a substance present in the gut that increased intestinal motility (enteramine). In the mid-twentieth century, 5-HT, the single compound producing both these effects, was isolated and synthesized, and its molecular structure was elucidated (204,399,400). Shortly thereafter, 5-HT was found to be present in the mammalian CNS in significant quantities and to be concentrated in varying amounts in different regions of the brain (18, 473). This led to the proposal of 5-HT as a CNS neurotransmitter. A great deal of interest soon focused on 5-HT because of its presumed involvement in the major psychoses. This hypothesis was based primarily on the finding that the actions of 5-HT in the periphery were antagonized by the powerful hallucinogenic (or psychotomimetic) drug d-lysergide (d-LSD) ( an extensive review of the relationship between the action of hallucinogenic drugs and 5-HT can be found in Refs. 232-234). Around the same time, Brodie and Shore (90) displayed extraordinary foresight in proposing that 5-HT and norepinephrine might act as opposing central neurochemical systems, somewhat akin to epinephrine and acetylcholine in the peripheral autonomic nervous system. As is common with many advances in neuroscience, the next significant step in 5-HT research was anatomic in nature. Employing the newly refined Falck-Hillarp histochemical fluorescence technique, Dahlstrom and C. AZMITIA Volume 72 Fuxe (117,118,173) described the localization of the cell bodies and axon terminals of rat brain neurons containing 5-HT. This description of serotonergic neurons answered Ramon y Cajal’s question about the extent of the projections of the raphe neurons. The information also provided a much needed “wiring diagram” for this neurochemical system. It is now accepted that this brain stem raphe system comprises the most expansive and complex anatomic/neurochemical system in the mammalian CNS (35). The next piece of information set the stage for much of the neurophysiological research on 5-HT over the ensuing 20 years. On the basis of the aforementioned histofluorescence results, that the cell bodies of 5-HT neurons were clustered along the midline in the brain stem, microelectrodes were lowered into this region of the rat brain. Aghajanian et al. (6) found that these neurons consistently displayed a distinctive slow and highly regular discharge pattern, which was established as a bioelectric “signature” for this neurochemical group of neurons. The last set of advances relevant to this review conterns the plastic nature of brain serotonergic neurons and involves three related research areas: development of specific chemical neurotoxins, regeneration and collateral sprouting in the CNS, and transplantation and culturing of fetal neurons. Neurotoxins to 5-HT enabled inputs from this system to be destroyed without producing mechanical injury to other neural and nonneural systems (57,58). This permitted researchers to study the process of regeneration of damaged serotonergic axons and to establish that new sprouts not only formed (70) but also reestablished functional contacts (363). By using intracerebral microinjections of neurotoxins into individual serotonergic pathways, homotypic collateral sprouting of undamaged serotonergic fibers was shown to restore the structure and function of lost serotonergic axons (41). The transplantation of fetal serotonergic neurons into the adult brain provided a method for replacing or augmenting the normal innervation and demonstrated that both fetal and adult neurons respond to the same trophic factors (46, 71). The ability of fetal serotonergic neurons to grow in tissue culture provided a controlled environment for quantifying the survival and growth of serotonergic neurons, making the search for endogenous trophic and toxic molecules feasible (49). Finally, the topic of CNS 5-HT receptor subtypes h as recently attracted a great deal of interest. Sections of this review describe their anatomic distribution and th e postsynaptic actions associated with them. However, the topics of their associated intracellular second messenger systems and ion channels and their molecular biology are beyond the scope of this paper. These latter two topics have been the subjects of recent reviews (169a 9 357a 9 507) . III. ANATOMY A Introduction ’ Visualization of neurotransmitters ch emical fluorescence, radioautography, in situ by histoand immunocy- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 Thus serotonergic neurons regulate the expression of neural circuits without being a necessary component of them. Second, although the firing rate of serotonergic neurons is unperturbed by a variety of physiological variables, the growth and terminal density of these neurons, both during development and after injury, are responsive to many of these same manipulations. We propose that the degree of serotonergic innervation of their target sites is, in fact, facilitated by this dissociation. Finally, there are evolutionary trends in the anatomy of this system: in lower vertebrates, serotonergic neurons have fine unmyelinated axons that are highly collateralized, whereas in higher vertebrates there is evidence for significant myelination of serotonergic axons and fewer collaterals. In this context, we also discuss the possibility of an evolutionary trend toward subsystems of serotonergic neurons with more precise anatomic projections subserving more specific physiological functions. EFRAIN January 1992 STRUCTURE AND FUNCTION B. Nuclei The 5-HT cell bodies in the brain are located in the brain stem on or near the midline and can be divided into superior and inferior groups. This division is based on the early developmental appearance of mesencephalic and myelencephalic groups (see sect. IvB). The original classification designated the 5-HT fluorescent clusters as Bl-B9 (117). The nomenclature used in this review has been agreed upon by a subcommittee of the Serotonin Club and does differ in some respects from published studies. We make an attempt in this section to clearly point out the changes. The superior group consists of four main nuclei: caudal linear nucleus (CLN: B8). median raphe nucleus BRAIN SEROTONIN SYSTEM 167 (MRN; B8 and B5, previously referred to as nucleus centralis superior) and its laterally displaced cells in nucleus pontis centralis oralis, the lateral B9 neurons lying just dorsal to the medial lemniscus (ML), and the dorsal raphe nucleus (DRN; B7 and B6). The inferior group consists of five main nuclei: nucleus raphe obscurus (NRO; BZ), nucleus raphe pallidus (NRPa; Bl and B4), nucleus raphe magnus (NRM; B3), the neurons in the ventrolateral medulla [lateral paragigantocellular nucleus (LPGN) and the intermediate reticular nuclei (IRN)] (Bl/B3), and the area postrema. The anatomy of these groups has been reviewed extensively in many species, including humans (465), Old and New World monkeys (45,150, ZZ), cats (54,236), and rats (305,374, 456). In this review, we survey the serotonergic neurons in rats, cats, and primates. The general location of these cell bodies within the mammalian brain can be seen in Figure 1, which displays their distribution in a sagittal view of the cat brain stem. In addition, Table 1 presents the approximate number of serotonergic neurons in each of the main clusters for both cats and rats. 1. Superior brain stem group I)CAUDAL LINEAR NUCLEUS(~8). The most rostra1 group of serotonergic neurons is in the CLN, which extends from the level of the red nucleus caudal to the anterior border of the superior cerebellar decussation (SCD). 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 the DRN. These neurons have similar efferents to those described for the DRN but not the MRN (229). Furthermore, there is a different dendritic morphology between the CLN and the MRN (222). Therefore these cells should be considered as distinct from the more caudal cells in the MRN and similar to the dorsal cells in the DRN (see sect. IvBI). II)MEDIANRAPHENUCLEUS(B~AND ~5). TheMRN is a paramedian and median cluster of cells lying below and caudal to the SCD (Fig. 2). The group has a rostrocaudal oblique orientation (34,305), and the paramedian columns are separated by as much as 400 pm in cats and monkeys. Rostrally, the cells from this group end around and within the caudal and lateral aspects of the interpeduncular nucleus. Caudally, the ventral border of this group is the trapezoid body, and the dorsal border fuses with the ventromedial (interfascicular) component of the DRN. In cats the MRN begins as a single cluster of cells at the level of the dorsal tegmental nucleus (236). The cluster lies in paramedian columns extending ventrally from the medial longitudinal fasciculus (MLF). The main cluster of neurons extends ventrolaterally from the SCD to an area just dorsal to the ML. The small paramedian cluster of 5-HT cells with a few cells lying directly on the midline formerly known as B5 (nucleus raphe pontis) can be classified as the caudal border of the MRN (B8). The cells are located ventral to the MLF and extend caudally to the rostra1 end of the abducens nucleus. At the caudal portion of this group, the cells are situated dorsal to the NRM (B3). Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 tochemistry changed our view of the nervous system. Before their introduction, brain anatomy could be summarized with black arrows indicating the connections between one group of cells and their targets along a particular pathway. With the advent of these new techniques, the dynamics and organization of specific neurochemical systems could be studied. Among the systems that have been elucidated, the serotonergic system is the most expansive. The serotonergic cell bodies are restricted to clusters of cells in the brain stem, but their fibers, using most of the known pathways, innervate nearly every area of the brain. In this section, we review the location of the cell bodies both of the superior (ascending) and inferior (descending) groups of serotonergic neurons. The afferents to these cells are described both anatomically and neurochemically. Associations between serotonergic neurons and nonneuronal cells are discussed. The efferents are reviewed to illustrate the size and characteristics of the axons, the wide number of pathways used, the general and precise patterns of innervation, and the evidence for both junctional and nonjunctional contacts. The anatomic distribution of the 5-HT receptor subtypes are reviewed. In addition there is evidence that the various serotonergic nuclei possess individual characteristics, such as the presence of cotransmitters, which may influence their anatomy and physiology. Before proceeding, it is important to obtain some general feeling for the numbers involved when we discuss 5-HT anatomy in the CNS. It may seem somewhat surprising that so much has been written about so little, i.e., the neurons in the entire mammalian CNS number in the billions, whereas serotonergic cells number in the thousands. Therefore they constitute -l/l,OOO,OOO of all CNS neurons. However, their influence on their target sites appears to go far beyond these numbers. In the rat brain it is estimated that there are -6 X lo6 serotonergic varicosities/mm3 cortical tissue. By extrapolation, this means that each serotonergic neuron projecting to the cortex may be responsible for 5 X lo5 serotonergic varicosities, that each of their cortical target and that serotonerneurons receives ~200 varicosities, gic terminals may account for as many as l/500 of all axon terminals in rat cortex (29). OF 168 BARRY L. JACOBS AND EFRAIN C. AZMITIA Volume 72 Scattered 5-HT cells of the MRN are seen ventrolateral to the MLF. These laterally situated cells, especially prominent in monkeys and cats, lie in the nucleus pontis centralis oralis (367) and form a ring around the central tegmental tract, one of the most primitive ascending pathways carrying reticulothalamic axons. III)DORSALRAPHENUCLEUS(B~AND ~6). TheDRN 1. Number (bilateral counts) and location of serotonergic neurons in cat and rat brains TABLE No. of Serotonergic Brain Region Linearis intermedius Raphe dorsalis Raphe medianus Raphe magnus Raphe pallidus Raphe obscurus Nonmidline* Total Cat 2,000 24,000 7,000 2,000 8,000 2,000 15,000 60,000 Neurons Rat (25) (70) (35) (15) (50) (35) 11,500 1,100 2,600 1,500 1,300 2,500 20,500 Numbers in parentheses are serotonergic neurons as a % total neuronal population. Linearis intermedius numbers for rat are included in raphe medianus and nonmidline. * At all brain stem levels from medulla oblongata to caudal mesencephalon, a band of scattered serotonergic cells forms a lateral extension from ventral raphe. The most lateral of these cells are located in relation to rubrospinal bundle (which serves as a good landmark at its location lateral to lateral reticular nucleus, facial nucleus, superior olive, etc.). From rostra1 medulla oblongata to mesencephalon there exists a lateral extension also from dorsal raphe, which consists of numerous serotonergic cells within periventricular gray and subjacent tegmentum. At pontine and most caudal mesencephalic levels, scattered serotonergic cells in lateral tegmentum connect lateral extensions from dorsal and ventral raphe. Thus, at pontine levels, distribution of serotonergic cells forms bilateral “rings” meeting in raphe nuclei in the midline. [From Wiklund et al. (511).] is divided into medial, lateral (the wings), and caudal components. The medial component can be further divided into a mediodorsal (superior) and a medioventral (interfascicular) component (Fig. 2). The superior component is in the central gray just below the cerebral aqueduct. It is much larger in its rostra1 aspect (level of the trochlear nuclei) and becomes smaller as it extends caudally. The cells extend rostrally to the caudal border of the oculomotor nuclei. The interfascicular component surrounds the MLF and is especially prominent between the fasciculi. It has been called the nucleus annularis, the interfascicular nucleus, or the pars dorsalis of the superior central nucleus (34,36). The neurons extend to merge with B6 caudally and lie just dorsal to MRN behind the SCD. There is developmental and morphological evidence for the caudal part of this group of 5-HT neurons being more closely related to MRN than the other components of DRN (see sect. IVES). The lateral component (the wings) forms the larger division of the DRN and extends as far rostrally as the oculomotor nuclei. This can be seen in rats and cats but is most prominent in primates. The wings are best developed at the level of the trochlear nucleus. This laterally situated group extends caudally and can be seen in the central gray of the pons just below the fourth ventricle. In humans, the lateral wings can be divided into a dorsal and ventral subdivision (465). IV) B9. This group is located along the superior surface of the ML (supralemniscal) from the rostra1 border of the inferior olive to the level of the red nucleus (Fig. 2). These cells are occasionally continuous with the paramedian cells of the MRN. The similar cytological and fluorescent characteristics of the cells in MRN and B9 were first noted in squirrel monkeys (227). In cats, these numerous cells are continuous with the MRN and form the ventral border of the ring of scattered cells that surrounds the central tegmental tract in the pontine Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 FIG. 1. Drawing of midsagittal view of cat brain stem with 5-hydroxytryptamine (5-HT)-immunoreactive cells indicated by black dots. Scale at bottom indicates anteroposterior levels in stereotaxic coordinates marked off in millimeters. AQ, cerebral aqueduct; CLN, caudal linear nucleus; DRN, dorsal raphe nucleus; MRN, median raphe nucleus; NRM, nucleus raphe magnus; NRO, nucleus raphe obscurus; NRPa, nucleus raphe pallidus; V4, 4th ventricle. [From Jacobs (234b).] January 1992 STRUCTURE AND FUNCTION OF BRAIN SEROTONIN SYSTEM 169 reticular formation. These findings confirm the similarity between these two nuclei in rats reported by DahlStrom and Fuxe (117). There is also evidence for a group of serotonergic neurons in the dorsomedial nucleus of the hypothalamus of rats. With the use of histochemical fluorescence after intraventricular infusion of 5-HT in rats pretreated with a monoamine oxidase inhibitor (MAOI), an additional group of 5-HT cells located in the dorsomedial nucleus of the hypothalamus was identified (175). This provided the first evidence that the hypothalamus contains an endogenous source of 5-HT. Biochemical support for the hypothalamus containing 5-HT-producing cells comes from the high concentrations of 5-HT and tryptophan hydroxylase present in the surgically isolated mediobasal hypothalamus (91). With the use of 5-rH]HT in vivo radioautography, a single cluster of labeled cells was found in the ventral part of the dorsomedial nucleus (60). A group of small round “immature” 5-HT-immunoreactive cells in the same area was observed only if the animal was pretreated with tryptophan and MAOI (165,166). Thus these neurons may have special characteristics unlike the other “traditional” groups and at this point should not be considered in the same category as the main nuclei. 2. Inferior brain stem group I) NUCLEUS RAPHE OBSCURUS (B2). The large COlleCtion of 5-HT neurons in monkeys, the NRO, lies in a symmetrical paramedian cluster that extends from the caudal border of the pons to the cervical spinal cord (Fig. 2). In the spinal cord, the cells are scattered in the central gray area just ventral to the central canal of the spinal cord and on the medial border of the ventral horn. The majority of these neurons are associated with the fibers of the MLF and the tectospinal tract as these tracts move from their superior position in the medulla to an inferior position in the spinal cord. The cells in the cervical spinal cord lying below the central canal were described in monkeys (276). In cats, at the level of the decussation of the pyramidal tract, 5-HT cells are seen off the midline along the border between the MLF and Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 FIG. 2. Adult rat brain coronal sections showing 5-HT-immunoreactive neurons. A: superior group: DRN, MRN, and B9 in low power (X15). B: DRN shown with its lateral (L), dorsomedial (DM), and ventromedial (VM) components in high power (~63). C: inferior groups: NRO, NRM, NRPa, and ventral lateral medulla (VLM) in low power (~15). D: inferior groups in high power (x63). 170 BARRY L. JACOBS AND C. AZMITIA Volume 7.2 At the rostra1 end of the inferior olive, the 5-HT cells move medially and become continuous with the NRPa and NRM. V) AREA POSTREMA. The 5-HT neurons in the area postrema of the brain are very small and densely packed with non-5-HT cells. They are apparent in both monkeys (45) and rats (305). In rats these cells project to the parabrachial area and appear to play a role in the neural circuitry involved in cardiovascular, respiratory, and ingestive control (277, 332). 3. Nonserotonergic neurons in raphe nuclei A substantial number of non-5-HT cells exist in many of the nuclei designated as serotonergic, especially the laterally placed neurons in B9 and the ventrolateral medulla. Even the MRN, the lateral wings of the DRN, and the medullary raphe nuclei have a sizeable number of non-5-HT neurons (Table 1). The surprising observation is that most studies find that nearly all the large cells with long projecting fibers in the raphe nuclei are serotonergic. For example, electrophysiological studies indicate that the vast majority of midline midbrain raphe cells can be identified as serotonergic (14). In anterograde tracing studies, the injection of 5,7-dihydroxytryptamine (5,7-DHT) into the medial forebrain bundle (MFB) eliminates nearly all the radioautographically labeled fibers after [3H]proline injections into the midline mesencephalic raphe nuclei (47). In retrograde transport studies, the connections to their forebrain targets from the midbrain raphe nuclei are nearly all serotonergic (34, 265, 266, 365, 453, 530). The observation that despite the heterogeneity of neurons in the ascending nuclei the majority of long projecting cells (except for BS-cortical projections) (365) are serotonergic is also seen with the descending raphespinal projections. For example, the NRM, NRPa, and NRO project largely to sensory, autonomic, and motor centers, respectively, in the spinal cord (21, 55, 84, 85, 311). The large percentage of BS-cortical projections may reflect the dispersed placement of the‘neurons, the great expansion of the cortical target area, or the methodology used to estimate retrogradely labeled 5-HT neurons. 4. Colocalixation of 5-hydroxytryptamine with neuropeptides At least three neuropeptides, substance P, thyrotropin-releasing hormone (TRH), and enkephalin, have been colocalized to the cell bodies of the medullary serotonergic neurons (217). Enkephalin, besides being localized to serotonergic neurons in the LPGN, is also found in certain neurons in the DRN (188). Single medullary neurons projecting to the spinal cord have been found to contain 5-HT, enkephalin, and TRH (243). It is generally believed that these neuropeptides function to modify Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 the pyramidal decussation just ventral to the central canal. Further rostrally at the exit of nerve VI, the nucleus is less densely packed than the NRPa and NRM. The neurons are medium sized, and at their most anterior extent they lie dorsal to the NRM. At the anterior aspect of NRO a smaller number of 5-HT cells have split dorsally from the main cluster and, in accordance to the nomenclature of Olszewski and Baxter (367), are designated as the extraraphe component of NRO. This group of 5-HT cells is seen in monkeys just ventral to the fourth ventricle at the level of the genu of nerve VII and extends caudally to join the NRO. This group was previously designated as the nucleus raphe ventricularis (45). The cells are small and located on the midline. The extraraphe component of NRO probably continues caudally to form the group of 5-HT neurons that surrounds the central canal at upper cervical levels of the spinal cord. II) NUCLEUS RAPHE PALLIDUS (~1). The ventral midline cluster of 5-HT neurons, the NRPa, extends from nerve XII to the rostra1 pole of the inferior olive (Fig. 2). The neurons lie in a compact group between the pyramidal tracts. The dorsal border is continuous rostrally with the less densely packed NRM. The majority of the 5-HT cells are found medial to the pyramidal tract. In cats, this is the largest nucleus in the inferior group, and the cells form paramedian columns. In rats the cells are fused on the midline and contain mediumsized multipolar cells. III) NUCLEUS RAPHE MAGNUS (~3). The midline group of large cells, the NRM, extends rostrocaudally from the emergence of the nerve XII roots to the rostra1 superior olive (Fig. 2). Its location overlaps the trapezoid body and the dorsal border of the ML. It is continuous at certain points with both the NRO and NRPa. In cats, at the level of the rostra1 half of the facial nucleus, the NRM is located on the midline between the ML. The cells of this nucleus remain largely on the midline, but scattered cells can be seen above the ML and superior olivary complex and within the trapezoid body. The group extends laterally into the nucleus reticularis gigantocellularis. 1V)VENTRALLATERAL MEDULLA (Bl/B3). Neurons in the ventral lateral medulla (VLM) are found in the reticular formation of the rostra1 and caudal ventrolatera1 medulla and extend laterally along the trapezoid body, ML, and pyramidal fibers (Fig. 2). Rostrally they are in the medial aspect of the LPGN and reach caudally into the ventral part of the IRN. This substantial group of cells extends from the emergence of the roots of nerve XII to the rostra1 part of the inferior olivary nucleus. The original classification proposed by Dahlstrom and Fuxe (117), in adult rats, assigned the cells to both NRPa and NRM. In cats, these cells were described as reaching the floor of the medulla (236,511). The 5-HT-immunoreactive cells in monkeys and cats reach the ventral pia and in certain cases are associated with the large blood vessels entering the medulla. The 5-HT cells are surrounded by a dense plexus of 5-HT fibers in this area. EFRAIN January 1992 STRUCTURE AND FUNCTION 5. Nonneuronal interactions Associations are also seen with nonneuronal cell types and serve to illustrate the possible role of 5-HT as a general trophic substance in addition to its role as a neurotransmitter. Neurons in the DRN and MRN are in apposition to oligodendrocytes (33,131), and their fibers in the MFB are usually in apposition to non-5-HT myelinated fibers (44). 5-Hydroxytryptamine cells from the DRN, area postrema, and NRO are associated with the ependymal cells of the cerebral aqueduct, fourth ventricle, and central canal. Rare ependymal cells in adult rats were immunoreactive to 5-HT antibody (E. C. Azmitia and P. J. Gannon, unpublished observations). This relationship between the DRN and the ventricular system is established very early in development when thick dendrites from 5-HT cells extend through the ependymal layer into the cerebrospinal fluid (CSF). Bundles of 5-HT dendrites and ventricular tanocytes have been reported (150). 5-Hydroxytryptamine cells are commonly associated with large blood vessels, with the cell body, or with processes in physical proximity to endothelial cells (33). This is most evident in the MRN and the LPGN. Tissue culture studies have also found evidence for a stimulatory role between astrocytes and fetal 5-HT neurons (491, 282a). During early development, 5-HT neurons are closely associated with astrocytes heavily stained by S-100 antibodies. Astrocytes also contain significant amounts of 5-HT, receptors. The possible implications of this 5-HT-astrocytic relationship are discussed in sections 1vE4 and vE. C. ARerents In this section the afferent connections of the raphe nuclei are described, and the neurotransmitter content of these afferent neurons is indicated. The first and largest afferent source is from the raphe nuclei themselves. BRAIN SEROTONIN SYSTEM 171 These connections actually begin in the embryo with the 5-HT commissural links between the paired bilateral neurons before they fuse along the midline (491). In adult animals, connections between DRN and MRN, B9, LPGN and NRM have been described in cats and rats by a variety of researchers using horseradish peroxidase (HRP) (13,250,257,343,417,456) and wheat germ agglutinin (WGA)-HRP (250). 5-Hydroxytryptamine autoreceptors have been demonstrated pharmacologically and physiologically (see sect. III&). The rest of the afferents to the raphe nuclei can be classified according to neurotransmitter type and/or anatomic location. Brain stem afferents to the DRN come from the superior vestibular nucleus via the MLF (250, 257) using acetylcholine as the neurotransmitter (327); the dorsal medulla in either the nucleus propositus hypoglossi (417), perihypoglossi (250), or nucleus of solitary tract (13) provides a source of epinephrine (218); locus coeruleus and subcoeruleus (13,229,250,458) bring norepinephrine (105,308,458); the substantia nigra and ventral tegmental area (13,250,417) provide dopaminergic afferents (252, 337); and finally the inputs from the periaqueductal gray (250,417) supply various neuropeptides (271). The inputs from the hypothalamus would be expected to complement the inputs derived from the brain stem. The most important of these comes from the medial preoptic area (13, 417) and lateral hypothalamus (13, 250, 417). Fewer labeled cells were reported in arcuate (417) and dorsomedial hypothalamus (250). These hypothalamic nuclei may be the source of neuropeptidergic (271) or histaminergic (371, 372, 451, 512) afferents to the raphe nuclei. Thus there is little doubt that the brain stem serotonergic neurons are in direct connection, by both their afferents and efferents, with the main brain stem and hypothalamic autonomic centers. Importantly, the serotonergic neurons also receive inputs concerned with the limbic forebrain. A major input from the lateral habenula has been described anatomically and electrophysiologically (13, 417) and has been shown to provide excitatory amino acid-containing fibers to both the MRN and DRN (250, 254). Some of these inputs probably end on the 5-HT neurons themselves, since these cells are sensitive to glutamate both neurophysiologically (see sect. VI) and metabolically (see sect. IV@. The physiological significance of this input appears to be complicated by ending on the GABAergic interneurons surrounding the MRN and DRN (284,359). The inputs to the GABA interneurons might provide an inhibitory feedforward mechanism on the 5-HT neurons excited by the habenular-glutamate connection. The pivotal position of the habenular nuclei in the limbic-midbrain system (355) places the serotonergic neurons in an ideal position to receive information concerning the activity of motor and limbic areas. Several other nuclei in the forebrain, which are part of the limbic system and contain cholinergic neurons, have also been reported to be sparsely labeled after HRP or WGA-HRP injections into the DRN (13,250). These include the prefrontal cortex, the diagonal band of Broca, Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 the action of the more classic type neurotransmitter on the postsynaptic cell (see sect. VIID). Another important action of neuropeptides is to regulate the growth of serotonergic neurons (49, 1%). Enkephalin has been shown to function as a negative growth regulatory factor on cultured fetal serotonergic neurons (124). Thus the neuropeptide that is colocalized may influence the innervation pattern of the parent neurons, which, in turn, determines the sites where 5-HT action will be strongest. This topic is discussed in greater detail in section V. There is evidence that nonneuropeptide neurotransmitters can be colocalized with 5-HT in certain neurons. For example, in the area postrema in rats, quantitative studies indicate that a large number of neurons contain both 5-HT and norepinephrine (332). In addition, studies of the DRN of rats suggest that certain neurons may contain both 5-HT and y-aminobutyric acid (GABA) (354). OF 172 BARRY L. JACOBS AND D. Pathways and Terminals Serotonergic neurons have extensive projections, both rostrally and caudally, from the superior and inferior groups, respectively (Fig. 3). C. AZMITIA Volume 72 FIG. 3. Summary diagram of primate serotonergic system. Main nuclei are shaded. Fiber pathways are shown as broken lines. AC, anterior commissure; Am, amygdaloid nuclei; CB, cingulum bundle; CC, corpus callosum; Cal, calcarine fissure; Cer, cerebellum; CLN, cauda1 linear nucleus; CQ, corpus quadrigimini; CSul, central sulcus; DG, dentate gyrus; DRCT, dorsal raphe cortical tract; F, fornix; FCtx, frontal cortex; H, hypothalamus; Hipp, hippocampus; IC, internal capsule; IO, inferior olivary nuclei; IP, interpeduncular nucleus; LC, locus coeruleus; MB, mammillary body; MFB, medial forebrain bundle; OB, olfactory bulb, P, pons; S, septum; SM, stria medullaris; SN, substantia nigra; T, thalamus; TCtx, temporal cortex; VAFP, ventro amygdalofugal pathway. [Modified from Azmitia and Gannon (45).] 1. Descending system The main nuclei of the inferior serotonergic clusters (NRO, NRM, NRPa, and ventrolateral medulla) account for the innervations seen in the spinal cord (Fig. 4). In studies in monkeys, two main descending projections were seen, a ventromedial pathway to the ventral horn and a lateral pathway to the central gray area of the spinal cord (45). The 5-HT projections to the intermediate lateral columns probably arise from the caudal extension of the extraraphe subnucleus of NRO that reaches the cervical spinal segments in layer X. Inputs to the intermediate columns appear to arrive also from the NRPa or LPGN (85). The fibers of NRO project caudally and ventrally to the motoneurons of the ventral horn via the descending MLF. These fibers begin dorsally at the level of the NRO and descend in the ventral columns of the spinal cord. The fibers reach this inferior position at the level of the decussation of the pyramidal tract along with the fibers comprising the MLF. This innervation of the motoneurons of the ventral horn is consistent with the other projections from NRO to the motoneurons of the brain stem nuclei of nerves X and XII (150). This ventral descending tract is called the raphe obscurus spinal tract (45). The 5-HT cells of the NRM heavily innervate the substantia gelatinosa of the spinal cord. Interestingly, Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 and the bed nucleus of the stria terminalis. These forebrain nuclei all contribute to the descending MFB (33) and would provide a direct but minor input from limbic centers. Immunocytochemical and receptor binding studies have been used to describe a number of peptidergic and hormonal afferents to the raphe nuclei. Almost this entire literature is based on studies carried out in the rat CNS (a few of the opioid analyses were conducted in monkeys). Enkephalin-like immunoreactivity has been reported in the raphe for both Old World monkeys (200) and for rats (259). The input to the DRN in monkeys was moderate, whereas that to the inferior raphe groups was described as sparse. ,&Endorphin-immunoreactive fibers are found in the DRN of rats, but the inferior raphe groups and the NRM were reported to be devoid of such fibers (77). With the use of rH]diprenorphine to label opioid receptors in the rat brain, the DRN and MRN were reported to have a low density of binding sites (28). Substance P immunoreactivity and receptor sites for substance P have been examined in studies of the rat brain, with surprisingly consistent results (309, 319,438). The DRN and MRN had moderate substance P inputs, whereas the inferior 5-HT nuclei (NRM, NRO, and NRPa) had only weak substance P inputs. Angiotensin II has been examined in the rat brain with immunohistochemistry (174) and autoradiography (331). The results of both types of analyses indicate little, if any, angiotensin II input to the raphe nuclei. In contrast, immunohistochemical analyses of neurotensin fibers in rats indicate moderate to strong inputs to several of the raphe nuclei (241, 474). Although the data are limited, there is some evidence for somatostatin inputs to the NRM (but not to any of the other 5-HT nuclei) and for ‘?-labeled somatostatin binding in the DRN of rats (151, 288). Autoradiographic analyses of cholecystokinin (CCK) indicate little [3H]CCK, binding in the raphe nuclei in rats (484) and some CCK-immunoreactive fibers in the DRN and NRM (477). Both immunohistological and neurochemical analyses indicate that in rats the DRN, but not the other raphe nuclei, receives a moderate neuropeptide Y input (104a). Substances related to the pituitary-adrenal axis have been studied. Autoradiographic analyses of receptors for corticotropin-releasing factor (CRF) (using ‘%ICRF) indicate a low level in the raphe nuclei in rats (133). Consistent with this, immunohistochemical analyses of CRF-immunoreactive fibers indicate that they are found in the various raphe nuclei of rats (459). All raphe groups in rats are reported to receive substantial adrenocorticotropic hormone (ACTH)-immunoreactive fibers (411). Two types of receptors for corticosterone have been studied in the rat brain (404). Those for glucocorticoids are present in moderate to high density in the raphe groups, whereas those for mineralocorticoids are absent in the raphe. EFRAIN January 1992 STRUCTURE AND FUNCTION the substantia gelatinosa might contain a pathway for descending serotonergic fibers to use for innervating lower spinal levels. This is based on studies of the descending noradrenergic (NA) fibers from LRN that suggest that once NA fibers innervate the substantia gelatinosa at the medullary-spinal cord interface, they descend for long distances within the nucleus itself (171). There is evidence that some of the 5-HT fibers in the lateral descending tract may be myelinated. Labeled myelinated fibers in the spinal cord in rats were seen after incubating the tissue with 5-[3H]HT and processing for electron microscopic radioautography (415). Labeled by 5-[3H]HT, myelinated fibers are also detected in the lower brain stem of monkeys (99). The frequency and distribution of these descending myelinated 5-HT fibers remain to be determined. Finally, the projection from the ventrolateral medulla probably innervates the intermediate lateral columns of the spinal cord (199). This projection would provide 5-HT fibers from the autonomic centers of the LPGN to the autonomic centers of the spinal cord. The fibers may use the lateral descending tract in the lateral funiculus or travel within the intermediate gray column itself. In the monkey spinal cord, a dense plexus of 5-HTimmunoreactive fibers is present in the substantia gelatinosa from the NRM, around the motoneurons in the ventral horn from the NRO, and in the intermediate sensory and motor zone of the autonomic neurons from the LPGN. Large injections of HRP into the lumbar spinal cord in Macaca fascicuZaris label cells in all three nuclei (85). A few cells with efferents to the spinal cord have been reported in the medial and lateral division of the DRN in squirrel monkeys and a baboon (320), although this has not been confirmed. Serotonergic endings from the NRM make direct contact with the neurons in the dorsal horn that give rise to the spinothalamic tract (228, 414) and are believed to be involved in modulating transmission of nociceptive information. BRAIN SEROTONIN SYSTEM 173 The distribution of 5-HT fibers in the brain stem has been described for rats (450), cats (511), and monkeys (150). Highest levels of immunoreactive terminal plexuses in rats were seen in areas of the interpeduncular nucleus, nucleus of the solitary tract, trigeminal motor nucleus, facial motor nucleus, substantia gelatinosa, and the ventral horn of the spinal cord. These studies have clearly shown that even within a particular nucleus there is a great deal of heterogeneity in fiber density. For example, the distribution of 5-HT-immunoreactive fibers in the trigeminal nuclear complex of the rat showed a dense innervation in the sensory nuclei, primarily associated with nociceptive activity, and sparse innervation in the sensory nuclei related to nonnociceptive afferent activity (114). Furthermore the marginal and gelatinosa layers of the spinal subnucleus caudalis had a dense number of fine fibers. The motor nucleus contained as many immunoreactive fibers as the subnucleus caudalis, but the fibers here were thicker and varicosities were more irregularly spaced than in the caudalis. 2. Ascending system Ascending 5-HT-containing fibers were first visualized with histochemical fluorescence methods (118,173), and using anterograde tracing methods, six ascending projections were described (33). Myelinated and unmyelinated 5-HT-containing axons in the MFB of rats and monkeys were visualized with immunocytochemistry (44). 5-Hydroxytryptamine immunoreactivity was seen predominantly in unmyelinated axons surrounded by unlabeled processes. More than half of these 5-HT-immunoreactive fibers were in apposition with unreactive myelinated axons in the hypothalamus of both rats and monkeys. In addition to the unmyelinated fibers, intensely labeled myelinated axons were seen in the MFB of both rats and monkeys. The percentage of 5-HT-immunoreactive myelinated axons was substantially greater in monkeys than in rats (25.4 vs. 0.7% of the total number of 5-HT-immunoreactive fibers, respectively). The serotonergic fibers projecting to the forebrain originate mainly in the superior group. Two main ascending bundles have been described in the primate brain (360,425,426). In human fetuses, 5-HT fibers were seen in the central gray near the ependyma of the fourth ventricle and the cerebral aqueduct and in a position between the medial raphe cells and the lateral cell system (360). In juvenile macaques, two main ascending bundles were described using histochemical fluorescence (426). A dorsal bundle was seen just ventral to the MLF, which originated at the level of locus coeruleus and received fibers from lateral wings of DRN and the ventromedial subnucleus of the DRN. These fibers appear to turn ventrally at the level of the red nucleus. A second bundle of ventrally flowing serotonergic fibers lateral to the MRN originated at the level of the trochlear nucleus and received fibers from the midline DRN and MRN. These Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 FIG. 4. Schematic drawing of sagittal section showing descending projections of inferior serotonergic neurons from medulla to spinal cord. Serotonergic soma are depicted as filled circles. Serotonergic cells are seen in VLM, NRM, and NRO with its dorsal subnucleus, extraraphe (NROer). 5-HT fibers are seen in tractus spinal thalamicus (tst) and spinal olivaris (tso) to intermediate gray from VLM, in medial longitudinal fasciculus (mlf) and tractus tectal spinalis (tts) to ventral horn from NRO, in substantia gelantinosa (SG) to dorsal horn from NRM, and in central gray from NROer. CC, corpus callosum. OF 174 BARRY L. JACOBS AND C. AZMITIA Volume 72 5-HT fibers extended to the external plexiform layer. The number of 5-HT-immunoreactive fibers was also numerous in the inner portions of the granular cell layer. In contrast to both rats and cats, the distribution of 5-HT-immunoreactive fibers in monkeys was scanty and partial in the glomerular layer. In this area, only a few of the glomeruli (no more than 5%) possessed 5-HTimmunoreactive axons. The heterogeneous distributio In of serotonergic fibers in the telencephalo n is briefly descr 1 bed for monkeys (45), si rice they show a more heterogeneous distribution than is seen in rats (144). Highest levels are seen in limbic area ,s (temporal lobe amygdaloid nucl .ei, and . hi ppocam PUS) and in sen sory areas (visual, cal carine; auditory, superior temporal gyrus; somatosensory, postcentral cortex; olfactory, amygdaloid nuclei and entorhinal cortex). The lowest levels are found in the motor regions in the fro ntal lobe (with the exception of the ret tus gyrus) . The distribut ion is qu ite varied even within the subregions of the hippocampal complex (dentate gyrus is greater than cornu Ammonis). As a general rule 5-HT levels are highest in those regions of cortex having more granule cells (layer IV). These cells are associated with sensory-receiving areas and association projections. Therefore the modulation of granule cell activity would affect the activity in all other cortical layers as well as corticocortical projections. Laminar distribution of 5-HT fibers has been described in the cerebral cortex. Although there are differences in the density of serotonergic innervation of different laminae of rat neocortex, this is seen most strikingly in monkey cortex. In monkeys, a clear lamination was observed with the highest densities in layer I and layer IV (45,80,342). The density in layer IV again reflects a preferential innervation of granule cells by 5-HT fibers. It is interesting that in rats, developing serotonergic fibers first project to the granular layer (IV), but as the brain matures the fibers become more localized to layer V (79). The most highly differential laminar specialization of all neocortical areas in monkeys is observed in primary visual cortex (also referred to as Vl and area 17) (268). 5-Hydroxytryptamine-immunoreactive fibers are present in all cortical layers of Vl in M fascicularis but form two especially prominent broad bands of fibers: one, extending from the deep half of layer III through IVC, reaches a peak in IVB, and the other begins in VA and goes through VI (Fig. 5). Between these two dense bands there is a band with a low density of serotonergic fibers in layer IVC (268). More recently, a quantitative analysis of the laminar density of innervation showed that layer IVC contained more varicosities per unit area than any other sublayer of primary visual cortex (126). In rats, there is evidence for two distinct populations of fibers in the cortex (264). They have been described as fine with many branches (D from dorsal raphe nucleus) and thick varicose fibers (M from median raphe nucleus) (267). The density of the fine fibers is heaviest in layers I and VA in adult rats (81). In cats, these fine fibers are widely spread throughout the corti9 Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 fibers appear to turn rostrally just dorsal to the nucleus interpeduncularis. In lK fascicularis, dense SHT-immunoreactive staining was present in the two main fiber tracts described above (45). In addition, fibers near the cerebral aqueduct in the hypothalamus were seen projecting lateroventrally from the DRN. Thus all six of the 5-HT fiber bundles observed in rats (47) can be observed in the primate brain stem. Furthermore, in monkeys, the main ascending bundle through the hypothalamus may not be the MFB, as it is in rats. Examination of the caudal hypothalamic area shows two main ascending bundles previously described in rats [the MFB and the dorsal raphe cortical tract (DRCT)]. However, in primates, the latter tract, which ascends to the cerebral cortex via the internal capsule, is larger than the MFB (44). The DRCT thus appears to have undergone a significant increase in absolute size when compared with the homologous pathway in rats. This probably represents an increase in the projection from the DRN to the cerebral cortex in primates. Several studies have used immunocytochemical methods to map the terminal distribution of 5-HT fibers (45, 304, 450). Highest levels of immunoreactive terminal plexuses were seen in hippocampus, suprachiasmatic nucleus of the hypothalamus, zona compacta of the substantia nigra, medial mammillary nucleus, lateral septum, periventricular nucleus of the thalamus, ventrolateral geniculate, and the medial nucleus of the amygdala. Lower, but clearly detectable, 5-HT-immunoreactive fibers were seen in virtually every area of the brain examined, except for major fiber tracts such as the corpus callosum and optic tract. Certain patterns in this vast innervation network can be seen. Innervation is greatest in those thalamic nuclei associated with limbic functions (e.g., nucleus reuniens and nucleus anterior dorsalis) and with sensory transmission, either nociceptive (e.g., posterior complex) or visual (e.g., dorsal lateral geniculate nucleus). In monkeys, 5-HT-immunoreactive axons were seen in both regions of the substantia nigra, although mainly in the pars reticulata (ZZO), whereas in rats the highest density was described in pars compacta (450). In the neostriatum of monkeys, 5-HT-immunoreactive fibers were particularly abundant in the ventromedial region of the caudate head bordering the nucleus accumbens where the plexus was heavier (378). The number of fibers in the caudate was reduced in more dorsal and lateral areas. The fibers were uniformly distributed in the putamen. Thus, despite the nearly ubiquitous distribution, selected areas receive much denser innervation. A comparative study in monkeys, cats, and rats of the olfactory bulb shows clear species differences. Serotonergic fibers were seen in all layers except the olfactory nerve layer in M. fuscata (460). 5-Hydroxytryptamine-immunoreactive fibers were observed to form a chain of large varicosities in the periglomerular region. The 5-HT distribution was predominantly in the internal plexiform layer. In some portions, a dense plexus of EFRAIN January STRUCTURE 1992 AND FUNCTION cal layers with increased branching in the upper layers (348,349). The large nonvaricose fibers (up to 2 pm wide) run in layer I and in the lower white matter of the cortex. They appear similar to the large fibers described in monkeys (342, 461). In cats, these large varicose fibers give off collaterals that form the basket terminals in the upper layers of the cortex (I-III) (348). The target cells for these basket terminals are heterogenous: both pyramidal and nonpyramidal (e.g., GABAergic) cells are included. A similar distribution of the fine and large varicose fibers is reported for marmosets (222a). The large varicose fibers are often seen surrounding certain cell bodies or proximal dendrites, possibly GABAergic or peptidergic. These fibers “may have a strong and specific influence on the cortical inhibitory circuitry, via relay through cortical inhibitory interneurons” (222a). 3. Nuclear organization of efferents The organization of the superior raphe projections indicates a rostrocaudal encephalotopy (229). The rostral group (DRN and the CLN) projects to basal ganglia-motor systems (e.g., corpus striatum and substantia nigra). The caudal group (MRN and the interfascicular aspect of the DRN) project to limbic structures (e.g., hippocampus and septum). The amygdala receives inputs from both groups, with the basolateral nucleus, which has projections to corpus striatum, belonging to the rostra1 group. The raphe nuclei with overlapping BRAIN SEROTONIN SYSTEM 175 terminal areas have a sizeable number of collateralized neurons to those terminal areas. This confirms and extends previous reports showing that serotonergic neurons are highly collaterized with innervations to more than one terminal area (19, 128, 266, 478). The projections to cortex show that the DRN projects heaviest to frontal cortex from the rostra1 and lateral wings of the nucleus (365). The lateral wings also project to the caudate putamen (452, 453). These projections would be compatible with this region being associated with the basal ganglia motor system. The MRN and B9 projected equally to parietal, occipital, and frontal cortex (365). This organizational pattern supports the idea that functionally related nuclei can be innervated by the same group of serotonergic neurons (33,229,334,489) or even the same individual neuron (498). The bilateral organization of the serotonergic neurons has been studied. There is general agreement that most of the serotonergic ascending projections from dorsal raphe are ipsilateral (235, 290). However, it should be noted that during development, a sizable number of serotonergic fibers cross at the midline (see sect. IvC). Thus contralateral or bilateral projecting neurons should be found. In a study of the dorsal hippocampal raphe afferents, -50% of the neurons were on or near the midline in both the DRN and MRN (34). Of the remainder not located on the midline, one-half of these cells were contralateral to the injected hippocampus in the MRN while only 17% were contralateral in the DRN. Furthermore, 10% of the labeled MRN neurons projected bilaterally to the hippocampus. When the projections to cortex were studied, a similar proportion of DRN and MRN ipsi- and contralateral cells to those to hippocampus were found (365). In the DRN, exclusive of the lateral wings, there is a predominately (3:l) ipsilatera1 projection with decreasing numbers of cells projecting to frontal, parietal, and occipital cortex. However, the MRN and B9 projections are laterally symmetric, with the MRN projecting throughout the neocortex and B9 projecting to selective cortical areas. E. Distribution of Receptors The availability of specific 5-HT ligands has made possible a study of the distribution of binding sites in the brain. The labeling is dependent on the particular ligand employed, and early studies used either [3H]LSD (330) or 5-[3H]HT (524). We briefly review both the anatomic and laminar distribution of the 5-HT,, 5-HT,, and the 5-HT, receptor families. Details of this classification can be found in recent reviews (169a, 369,385,507). 1. 5Hydroxytryptamine, The original classification of 5-HT, and 5-HT, receptors used 5-[3H]HT and [3H]spiroperidol as specific ligands (386). The radioautographic binding in rats showed a wide variation, with the highest levels in ven- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 FIG. 5. Distribution of 5-HT-immunoreactive fibers in primary visual cortex of cynomolgus monkey. A: bright-field photomicrograph of opercular surface of primary visual cortex Nissl stained to show laminar boundaries. B: dark-field nhotomicrogranh of same field in adjacent section immunostained for 5-HT visualization. Bar, 200 Frn. [From De Lima et al. (126).] OF 176 BARRY L. JACOBS AND C. AZMITIA Volume 72 hippocampus (225). In the cortex, layer II (322 fmol/mg protein) was higher than layers III and VI (40 and 87 fmol/mg protein, respectively). In the hippocampus, the pyramidal layer of CA1 (505 fmol/mg protein) was higher than CA3 (102 fmol/mg protein) with the DG having intermediate levels (280 fmol/mg protein). II) MINES,,. 5-HYDROXYTRYPTAMINE&-HYDROXYTRYPTA- The 5-HT,, site has been defined in rat by 5[3H]HT binding in the presence of the 5-HT,, ligand 8OH-DPAT (382) or by the ligand [‘251]iodocyanopindolol ([1251]CYP) (142). Specific 5-HTlB binding was blocked by the P-adrenergic antagonist RU-21009 and RU-24969 (147). The 5-HT,, sites were particularly rich in the globus pallidus, dorsal subiculum, substantia nigra, and the olivary pretectal nucleus. 5-Hydroxytryptamine,, binding was performed on postmortem human tissue with ( -)-[1251]CYP in the presence of (-)-isoprenaline to suppress binding to ,&adrenergic receptors (224, 225), and specific sites were not detected. Other workers, using a similar technique, did report small nu mbers of these receptors in basal ganglia region s in hum an pos tmortem brains (68) . A complication in these studies is that 5-HT,, receptors may share many of the properties of the 5-HT,, receptors. III) 5-HYDROXYTRYPTAMINElc. The 5-HT,o site can be labeled by 5-[3H]HT, [3H]mesulergine, or ‘%I-LSD (216, 226, 381, 521). The labeling is almost exclusively confined to the choroid plexus. For example, in humans, 5-HT,c sites are highest in the choroid plexus (1,700 fmol/mg protein), and much less is found in the hippocampus, with the pyramidal layers of CA1 and CA3 having a higher density than the DG (170 vs. 80 fmol/mg protein). 2. 5-Hydroxytryptamine, Morphological evidence for a distinct low-affinity 5-HT receptor came from the differential anatomic distribution of 5-HT, receptors in the rat brain. This site has been studied using [3H]ketanserin, [3H]LSD, [3H]spiperone, and [3H]mesulergine (379), with ketanserin being the most selective (301). The 5-HT, sites are high to intermediate in the medial and posterior part of the medial mammillary nucleus, parafascicular nucleus of thalamus, inferior olivary nucleus, nucleus accumbens, body and tail of caudate, olfactory tuberc le, anterior olfactory nucleus, insulae Calleja, and in various areas of the cortex. In the cortex, high levels were seen in the entorhinal, pyriform, cingulate, frontal, and frontoparietal motor cortex with lower levels in the frontoparietal sensory, striate, and auditory cortex (380). The highest density reported was in lamina IV of the neocortex. The 5-HT, receptor distribution in rat neocortex was studied with M-methyl-2-1251-LSD (81). The anatomic distribution in cortex was frontal > parietal or occipital. A dense band of labeling was found in the upper part of lamina V and not lamina IV in the somatosensory cortex. In addition to the band seen in lamina Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 tral pallidum (3,600 fmol/mg protein) and the lowest levels in reticular and ventroposterior thalamic nuclei (180-190 fmol/mg protein) using 5-[“H]HT as a label. The density of 5-HT, receptors was high in the choroid plexus, lateral septal nuclei, dentate gyrus (DG), dorsal subiculum, olivary pretectal nucleus, substantia nigra, entorhinal cortex, Rexed layers I and IV of spinal cord, nucle us of the sol itary tract, gelatinosum area of nucleus of the spinal tract of V, amygdaloid nuclei (lateral and cortical highest), hypothalamus (ventromedial and dorsomedial highest), and DRN (67,380). In cortex, the density of receptors was entorhinal > anterior cingulate > frontal > somatosensory > motor > striate (382). This distribution is generally in good agreement with the known serotonergic fiber density in rat brain as described. A study of the distribution of 5-[3H]HT binding in rhesus monkey cortex showed higher levels in prefrontal than parietal or prestriate, with the somatosensory cortex higher than the motor cortex (306). The laminar distribution showed 185 fmol/mg tissue in layer II of the somatosensory cortex compared with 50 fmol/mg tissue in layers IIIB-V. In general, the 5-HT, receptors were located supragranularly (layers I and II). This general pattern was different in motor and visual cortex. In the motor cortex, the highest density was in layers I and II, and in the visual cortex, it was highest in layer IVC with high levels throughout layers III-VI. The distribution of the uptake sites as defined by [3H]paroxetine binding did not have strict correlation with the 5-HT, receptor distribution in cortex. The regional density of 5-[“H]HT binding varied markedly in the postmortem human brain: 1,151 fmol/ mg protein in frontal cortical layer I to 74 fmol/mg protein in ventral thalamus (66). Highest levels were seen in cortex (superior frontal > cingulate > medial frontal), subiculum, globus pallidus, claustrum, and midline nuclei of the thalamus (66). The laminar distribution in cortex showed that layers I and II were highest, with substantial labeling also in layer VI. I) 5-HYDROXYTRYPTAMINE,*. There is long-standing evidence for the existence of multiple high-affinity 5-HT receptors (502). The availability of specific ligands has made the study of their differential distribution in the brain possible. The 5-HT,, receptors are usually defined using 8-hydroxy-2(di-n-propylamino)tetralin (8OH-DPAT), either as the radiolabel or as the displacing (defining) ligand. With the use of this approach, the highest levels in rats of 5-HT,, receptors were seen in the midbrain raphe nuclei, DG, and lateral septum (382) and in the hippocampus and cortex (132, 193, 380,488). In the frontal cortex, the 5-HT,, receptors were reported to be highest in the deeper cortical layers (382). The highest binding in spinal cord was seen in layer I, which corresponds to substantia gelatinosum. The cellular binding of 8-OH-[3H]DPAT to midbrain raphe neurons has been shown to be on the soma and dendrites of 5-HT-immunoreactive neurons (16). In human postmortem brain, the binding of 8-OH[3H]DPAT was region specific in both frontal cortex and EFRAIN January 1992 STRUCTURE AND FUNCTION BRAIN SEROTONIN SYSTEM 177 (entorhinal > retrosplenic = frontal = cingulate = temporal > occipital = parietal). Recent studies with [3H]ICS 205-930 in the human brain show heavy labeling in the substantia gelatinosa of the spinal cord (219 fmol/mg protein), area postrema (729 fmol/mg protein), nucleus of solitary tract (255 fmol/mg protein), cranial nucleus of the vagus (493 fmol/mg protein), and the spinal trigeminal nucleus (178 fmol/mg protein) (490). Hippocampus and amygdala showed some labeling (61 and 72 fmol/mg protein, respectively), with the entorhinal cortex having much lower levels (18 fmol/mg protein). 4. Summary There is now evidence for at least six separate 5-HT receptors, each with a unique pharmacological, behavioral, and anatomic profile. The 5-HT,* is the cell body autoreceptor and is also responsible for some of the postsynaptic actions of 5-HT, especially in the hippocampus and cortex. The 5-HTlB/5-HTlD receptor (5HT,, in humans) is the axon terminal autoreceptor in rats and primates and is presumed to be involved in inhibiting the release of 5-HT from the axon terminals. The 5-HT,o receptor is most densely located in the choroid plexus, with much lower but detectable levels in the hippocampus and cortex. The 5-HT, receptor is postsynaptic and is associated with the fine serotonergic fibers in the middle layers of the cortex. It is responsible for many of the behavioral effects of 5-HT and has been shown to be involved in the action of the major hallucinogenic drugs. Finally, the 5-HT, receptor is found in limbic areas, where it may serve a role in anxiety and psychosis; in the substantia gelatinosa, where it may modulate sensory input; and in the area postrema, where it may inhibit emesis. The variety of 5-HT receptors may represent the distribution on the cell body versus the terminal or on the different population of cells (neurons and glial cells) that receive a 5-HT signal. Therefore, although the 5HT neurons show a rather simple pattern of firing (see sect. VIA) and can communicate through unmyelinated fibers onto nonspecialized postsynaptic sites, the receptors recognizing 5-HT are differentiated and highly specific. F. Ultrastructure 3. 5-Hydroxytryptamine, The most recently described CNS 5-HT receptor is 5-HT,, which is known to bind cocaine (86). This receptor is labeled with [3H]GR 65630A and has been localized to the brain with the highest levels in the entorhinal cortex (11.3 fmol/mg protein) and the lowest levels in the cerebellum (0 fmol/mg protein) (260). Other areas with dense 5-HT, binding are amygdala, hippocampus, nucleus accumbens/olfactory tuberculum, and cortex Ultrastructure of serotonergic neurons has been reviewed (33, 61). 5-Hydroxytryptamine neuronal perikarya in the DRN are medium-sized (15-25 pm mean diam) neurons with a relatively small but deeply indented nucleus. The cytoplasmic distribution of organelles depicts a metabolically active cell. There are numerous free ribosomes, a well-developed Golgi apparatus, numerous dense and multivesicular bodies, small clear vesicles, and occasional large dense-core vesicles. This descrintion cannot be generalized to all serotoner- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 VA, there was also heavy labeling in layer I just beneath the pia. Lower densities were seen in the deep half of the cortex. In the motor cortex, the middle band extended throughout layer V where the projecting pyramidal neurons are located. This region displayed more 5-HT, receptors than the somatosensory cortex. Interestingly, layer VA corresponded to the fine axons with pleomorphic varicosities that densely innervate this area from the DRN (267). The distribution of 5-HT, receptors as seen with [3H]ketanserin showed a moderate variation in rhesus monkeys (306). Highest levels (100 fmol/mg tissue) were observed in layers III and IV, and lowest levels (~25 fmol/mg tissue) were seen in layer I of prefrontal cortex. The density in the motor cortex was greater than in the somatosensory, similar to that seen in rats. Labeling in most cortical areas was heaviest in layers III and IV, with much lower levels in other laminae. In the visual cortex, the density of receptors was high in all laminae but was highest in lamina IVC. Interestingly, the receptors were higher in the supragranular layers (I-III) in the motor cortex than in other cortical areas. The localization of SHT, receptors in human postmortem tissue was studied with [3H]spiroperidol or E3H]ketanserin (66, 146, 226). The distribution determined by binding studies showed cortex and hippocampus > globus pallidus and caudate nucleus (146). The results with quantitative radioautography (range: 48 fmol/mg protein in lamina IV of cingulate cortex to 0 in ventral thalamus) showed the highest binding in cortex, amygdala, DG, anterior ventral nucleus of thalamus, and caudate/putamen (66). The cingulate cortex had more binding than the superior frontal cortex, and lamina IV was much higher than laminas I-III. In the spinal cord, highest levels (100 fmol/mg protein) were seen on motoneurons. A final study showed high levels in the cortex and hippocampus (226). Frontal cortex layers III and IV showed high levels (217-247 fmol/mg protein), whereas lower levels were seen in layer II. In the hippocampus, the pyramidal CA1 showed higher levels (190 fmol/mg protein) than were seen in CA3 or DG. Interestingly, the distribution of 5-[3H]HT uptake sites is highest in layer IV of primates, which agrees with laminar distribution of the 5-HT, receptor (44). The regional distribution of the uptake sites and the receptor binding does show many discrepancies in rank order, which may reflect the downregulation of the receptor by 5-HT release. OF 178 BARRY L. JACOBS AND C. AZMITIA Volume 72 443, 444). These percentages should be considered only rough approximations since synaptic contacts may be quite labile. In fact, the number of 5-HT junctional varicosities can be dramatically altered by changes in the local neuropil, as shown by studies performed with agranular rats where the number of specialized contacts was reduced (445). The factors that determine the frequency of junctional versus nonjunctional contacts are unknown. It has been suggested that fine fibers do not, but the thick fibers do, make specialized contacts (465). However, this view is not universally shared. This issue has received so much interest because the appearance of a specialized contact suggests relatively stable and strong associations between an afferent neuron and its target. Conversely, the lack of synaptic specialization implies a dynamic and possibly indiscriminate interaction with target neurons. Serotonergic neurons have long been described as a global modulatory system consistent with a nonjunctional framework. Furthermore, the regenerative capacity was initially said to be limited to these nonjunctional fibers (72). This view should be tempered; 5-HT specialized contacts do exist, but they are not rigid and permanent. Regenerative 5-HT fibers of the junctional types have been described (167,168). Some 5-HT fibers do lack specialized regions of contact and release 5-HT to nonsynaptically related neurons, glial cells, ependymal cells, endothelial cells, endocrine cells, and into the CSF (33,36,44,97,131,150). Thus the types of contacts made by these neurons throughout the brain reflect the varied nature of the action of serotonin. It seems clear from these ultrastructural data that, in most areas of the mammalian CNS, there are at least some sites where 5-HT is released and no evidence for synaptic specializations can be found. This type of arrangement, where neurotransmitter is released and then diffuses over distances as great as several hundred microns to affect target neurons, is well known in the mammalian peripheral autonomic nervous system. It implies the existence of a hormonelike form of neurotransmission, which is slow, global, and therefore carries only a primitive form of neural information. One of the more well-delineated examples of nonsynaptic release of 5-HT, with the accompanying physiological evidence, is in the spinal cord of a primitive vertebrate. In lampreys, serotonergic axonal varicosities are located in proximity to motoneurons and premotoneurons, but no synaptic specializations are observed. If 5-HT is applied during fictive locomotion in the spinal cord of the lamprey, it has profound effects on the motor pattern (for review see Ref. 492). Several additional points deserve discussion regarding the issue of nonsynaptic release. First, in a given species, the percent of 5-HT terminals associated with synaptic specializations apparently can vary from ~0 to lOO%, depending on the particular brain region. This may have important implications for the type of information processing in which 5-HT is involved in each of these brain areas in a given species. Second, it will be important to gather cross-species data on this Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 gic groups, since in the monkey LPGN they are mostly fusiform and somewhat larger than the serotonergic cells in DRN (98). The 5-HT neuronal dendrites have been shown to receive 5-HT terminals in monkeys (255) but not rats (131,383) or cats (101). However, many 5-HT processes lie in proximity to the 5-HT dendrites and provide a structural basis for homotypic interactions. Dendrodendritic appositions were frequently seen in all species with some evidence of specialization in cats and monkeys. However, with respect to the soma and dendrites, no unique elements were reported for separating 5-HT cells from the surrounding non-5-HT neurons. The sizes of the 5-HT-immunoreactive fibers in the MFB are 0.2-1.25 pm in diameter for unmyelinated axons and 1.0-2.1 pm for myelinated axons (44). The intraneuronal 5-HT-immunoreactive labeling was detected within small vesicles (30-35 nm) and the tubular profiles of smooth endoplasmic reticulum. These two sites comprise the membrane-enclosed compartments of 5-HT immunoreactivity. Evidence of 5-HT immunoreactivity was also found on the outer membranes of mitochondria (where monoamine oxidase is localized) and in discrete patches along the inner face of the axolemma. In the cat solitary tract, 5-HT immunoreactivity was reported in the large granule vesicles (80-150 nm) (317), and in the monkey it was associated with microtubules (377). There is a report that substance P and 5-HT can be contained within the same large granular vesicle (384). The localization of 5-HT to large granular vesicles is still controversial since not all studies have seen this localization. In certain brain nuclei (locus coeruleus, portions of the cerebellum, and the inferior olive), some of the 5-HT varicosities contain small vesicular organelles (15-25 nm) associated with tubular elements. These microcanaliculi (62,99,285) may represent a continuous intra-axonal network similar to the smooth endoplasmic reticulum. These may provide the pathway for the retrograde transport of 5-[3H]HT from the terminal to the cell body (25,43a). The projecting axons are either fine and varicose or large and smooth (113, 114, 118, 303, 450). The fibers were described as both unmyelinated and myelinated (44, 98, 99, 255, 415). There is now general agreement regarding whether serotonergic terminals make specialized synaptic contacts or release their transmitter by nonjunctional means. Both types of terminals have been clearly documented, suggesting both forms of release are present in the brain. The relative frequency of each type is region specific (33, 61). For example, in rats, there are very few postsynaptic specializations associated with serotonergic varicosities in layer I of the cerebral cortex (59,130), but in the deeper layers of somatosensory and visual cortex, the percent of junctional contacts can be as high as 50-90% (335,373). In the cat dorsal horn and in the rat hypothalamus, there are abundant specializations (167, 168, 414), but they are extremely rare in rat locus coeruleus (285). In the neostriatum, only 15% of all 5-HT varicosities made an asymmetrical synapse on dendritic shafts or spines (26, EFRAIN January 1992 STRUCTURE AND FUNCTION issue to see if there are any phylogenetic trends in this relationship (similarly, developmental data in a given species would be of interest). Finally, the dynamics of release at nonsynaptic sites remain to be delineated, such as the relative magnitude of these effects and their temporal characteristics. G. Summary BRAIN SEROTONIN SYSTEM 179 ation of the primate serotonergic neurons. However, and not surprisingly, this concept of an evolutionary stable system does not hold completely when all the comparative evidence is scrutinized. The nuclear organization in the primate brain does differ from that of rodents in several important aspects. First, in the rat brain, the majority of the serotonergic nuclear groups are indeed raphe nuclei; that is, the cells lie on the midline seam and extend laterally from there. In the primate, fewer cells lie directly on the midline, and most have at best a paramedian organization. “It appears the serotonin system undergoes a ‘lateralization’ as the phylogenetic scale is traversed towards primates” (441). Lateral cells in rats may in fact have fewer collaterals than the midline neurons (148) and would therefore have a more restricted terminal distribution. Second, the DRN is more highly organized in primates. The lateral wings have both a dorsal and ventral subdivision, and the ventromedial subnucleus includes the nucleus annularis rostrally and the interfascicular nucleus and the B6 group caudally. Third, the serotonergic cells from NRO extend far more caudally in the primate brain to reach the cervical levels of the spinal cord. The location of these cells in lamina X and the ventromedial fasciculus may indicate a more important relationship between serotonergic cells and spinal cord in the primate brain. Finally, the hypothalamic 5-HT-concentrating cells described by several groups in rats are not seen in the primate hypothalamus. Important differences also have been noted in the projection pathways. Descending fibers to the spinal cord in rats originate from NRM and NRPa but appear to originate only from NRM in primates (85). Ascending fibers to the forebrain in rats use mainly the MFB (33), but in monkeys, the DRCT may be quantitatively larger (44). Finally, fibers in the primate brain have been noted by several workers to be myelinated. Although a few myelinated 5-HT fibers have been noted in rodents (415), the occurrence in monkeys is 36 times greater than in rats (44). Differences between primates and subprimates in the termination pattern suggest that the primate has a more restricted target innervation. This can be seen in the distribution of 5-HT uptake sites in hippocampus where the CA fields are less innervated in monkeys compared with rats (45). In the cortex of primates, the laminar distribution is highest in layer IV (45, 80,342), whereas in rats it is in layer I and VA (81,130). Finally, in the olfactory bulb, the glomeruli are innervated in rats and cats but not monkeys (460). Whether this represents a true evolutionary trend, as has been suggested (36), or species differences in function remains unknown. To summarize, in higher mammals the cell bodies of the serotonergic system are more laterally situated (possibly associated with fewer collaterals), have a much greater proportion of their axons myelinated, and have a more restricted terminal innervation (type II). These properties are suited for rapid dissemination of information from the brain stem raphe to the related target Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 Serotonergic neurons do not comprise a homogenous collection of cells organized in specific nuclei. The various 5-HT-designated nuclei contain many nonserotonergic neurons. However, most studies have shown that the overwhelming majority of neurons within these nuclei with long projections are serotonergic. Furthermore, even the serotonergic neurons are not homogenous since many of the cells show that 5-HT is localized with particular neuropeptides, such as substance P, CCK, and TRH. Finally the axons of these neurons have been described as myelinated or unmyelinated, fine or coarse, and as making synaptic or nonsynaptic contacts. This morphological evidence supports the idea of two serotonergic systems (see sect. VIII). Serotonergic cell body distribution and their projection network appear to have remained remarkably stable across phylogeny. The main cellular nuclei located in the brain stem first described in rats are present in the monkey and the human brain. It has been concluded that “the pattern of organization of the three recognized aminergic systems of the brain have been preserved with remarkable consistency in the evolution of primates” (230). The stability of the cellular organization is consistent with the descriptions of the main descending and ascending projections. The four descending projection groups (NRO, NRPa, NRM, and ventrolateral medulla) in the primate brain are still believed to innervate the substantia gelatinosa of the dorsal horn, the motor neurons of the ventral horn, and the intermediate autonomic neurons of the spinal cord. The ascending nuclei (MRN, DRN, and B9) in the primate brain use multiple tracts to innervate most of the subcortical and cortical areas of the forebrain with a distribution pattern highly consistent with that reported in rats. Finally, the termination patterns seen are largely similar to those described in subprimates. The serotonergic fibers enter the various target structures through similar pathways and appear to innervate the same target neurons. For instance, the labeling patterns seen in the DG of monkeys are almost indistinguishable from those seen in rodents (45). Numerous types of morphologically distinct serotonergic terminals seen in the cerebellum and paratrigeminal nuclei were identified in both rats and monkeys (96, 99). The similarities between the primate and subprimate serotonergic systems are apparent in nuclear organization, major efferent pathways, target structures, and ultrastructural relationships. Thus it would appear that anatomic and physiological studies performed in subprimates would have a direct bearing on our appreci- OF 180 BARRY L. JACOBS AND IV. DEVELOPMENT A. Introduction The foundation of the organization of the adult CNS serotonereic svstem is embedded in the migration C. AZMITIA Volume 7.2 and outgrowth of serotonergic neurons. These fetal neurons are known to complete their final mitosis between days 12-14 of gestation in the rat brain (281). The early presence of 5-HT within these neurons was shown by histochemical fluorescence procedures (366a). High levels of 5-HT were detected within the cells before and during early neurite outgrowth. This observation raised the possibility that 5-HT might serve a trophic role during development before assuming its adult functions as a neurotransmitter. The 5-HT neurons are located in two separate clusters (Fig. 6) that show a different developmental pattern during early gestation. The superior group contains 5-HT stores before they migrate from the ependymal zone toward the DRN, MRN, and B9, but the neurons comprising the inferior group appear to produce 5-HT only after their migration is complete (491). Neurites of 5-HT cells have a robust outgrowth throughout the neuraxis. The pathways used correspond to many of the main interconnections established by nonserotonergic neurons (33, 304, 491). The ability of the serotonergic fibers to use preexisiting fiber pathways as guides was termed “epiphytic guidance” (33) and may provide a cellular basis for the many and varied connections formed by the 5-HT system. Interestingly, the formation of precise termination patterns often occurs long after the arrival of the 5-HT fibers. The terminal branching thus seems to depend on a different set of signals than those guiding the fibers to the terminal zone. The distinction between trophic and neurite-extension factors requires tissue culture methods. Serotonergic neurons show accelerated maturation in culture compared with other neural systems and respond to many of the same target cells that they encounter during normal development. A number of substances have been shown to function as serotonergic growth regulatory factors. In this section, these topics are discussed to illustrate the variety of steps required to construct a complex organization and how each step is dependent on both positional and chemical signals. B. Nuclei I. Superior group In the superior group, the first 5-HT-immunoreactive neurons are seen at 12 days gestation, 10 days before birth in rats (305,491). The cells are small (5-7 pm), and within 24 h they form long dendrites (up to 135 pm). These are typical isodendritic reticular formation developmental patterns. The cells are among the earliest mesencephalic neuroblasts to differentiate and the first to reach the mantle zone. This evidence of precocious neuronal maturation provides the framework for its later expansive network. At 14 days gestation, the MLF has appeared, and this tract is surrounded by 5-HT-immunoreactive neurons. The neurons have migrated ventrallv from the ven- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 areas. This description is at odds with the classic view of serotonergic fibers in the lower mammals of a diffuse, highly branched, unmyelinated midline group of neurons (type I). The possible functional implications for two types of serotonergic neurons, a slow expansive one (type I) and a fast precise one (type II), are discussed in section VIIIC. Most of the studies on the raphe afferents have been performed in rats. The most important innervation is probably the interraphe 5-HT connections. Numerous other afferents have been described using retrograde transport, immunocytochemistry, and receptor radioautography. These studies indicate that important nonserotonergic inputs come from the lateral habenula (glutaminergic) and the locus coeruleus (noradrenergic). Other neural afferents reported include various neurotransmitters (e.g., acetylcholine, GABA, dopamine, histamine, and epinephrine) and neuromodulators (enkephalin, endorphins, substance P, CCK, neurotensin, CRF, and ACTH). Nonneuronal cells also contact serotonergic neurons (e.g., oligodendroglial, ependymal, and endothelial cells). This convergence of neuronal and nonneuronal input provides the raphe cells with a broad sampling of information from many brain and endocrine systems that might have neuronotrophic as well as physiological relevance. An important topic covered in this section dealt with the anatomic distribution of 5-HT receptor subtypes. The raphe nuclei contained mainly the 5-HT,* receptor. Limbic areas such as the hippocampus, septum, amygdala, entorhinal, and pyriform cortices had high levels of the 5-HT,*, 5-HT,, and the 5-HT, receptors. The regional distribution of receptors can be further analyzed at the laminar level. For example, in the neocortex the 5-HT2 receptors are highest in primate layer IV and rat layer VA, whereas the 5-HT, receptor shows a more diffuse distribution. It has been suggested that particular 5-HT fiber types (e.g., fine from DRN) may be preferentially associated with particular 5-HT receptors (e.g., 5-HT,) (81). Whether the different receptor subtypes will correspond to separate morphologically identified serotonergic neurons is a new promising area of study. Finally, the restricted localization of serotonergic cell bodies exclusively in the brain stem implies a role in basic brain functions rather than involvement in higher order information processing. The relative paucity of cell bodies and their highly branched terminal domain are consistent with a more basic role in brain functioning and could imply that these neurons comprise a unifiying system for synchronization of functional centers from a restricted source. EFRAIN January 1992 STRUCTURE AND FUNCTION OF SEROTONIN 181 mesencephalic reticular formation and may form part of B9 (305). At 16 days gestation, the 5-HT cells are more prominent and less compact, forming four paired bilateral groups. These are the dorsal, medial, lateral, and ventrolateral (305). The dorsal group is mainly dorsal to MLF and forms the lateral wings of the DRN. The medial group is in the mantle zone and gives rise to the MRN. This group may contribute some neurons to B9 and nucleus pontis centralis oralis. The lateral group gives rise to the scattered neurons in the midbrain reticular formation and part of B9. Finally, the ventrolatera1 group develops into the main body of B9. Formation of a fused 5-HT-immunoreactive nucleus is apparent at 17 days gestation. This occurs because of a joining of the ependymal zone that gives rise to a thinning septum of nonneuronal cells (135 pm at 1’7 days gestation and 30 pm at 19 days gestation) (305). Neurons of the lateral wings of the DRN are seen in contact with the ependymal layer and have direct access to CSF. These cells probably contribute to the supraependymal layer of 5-HT-immunoreactive fibers observed in the third and lateral ventricles. The lateral and ventrolateral groups give rise to B9, which surrounds the newly formed medial lemniscal fibers. At 19 days gestation, the decussating fibers of the superior cerebellar peduncle separates the CLN rostrally, the MRN ventrally and caudally, and the DRN dorsally. Thus within 1 wk of the first appearance of 5-HT-containing neurons, all the major nuclei of the superior group can be observed in the fetal brain. 2, Inferior tricular surface to take up their positions as bilateral groups near the midline. However, there are a number of axons and dendrites forming a 5-HT-immunoreactive commissural system connecting these bilateral groups at this early period. Thus 5-HT intercommunication is one of the first and most basic forms of neuronal interactions (491). Major advances are seen by 15 days gestation. The 5-HT-immunoreactive cells are clustered into two groups, dorsolateral andventrolateral(491). In the bilateral group dorsolateral to the MLF, the neurons are seen in the rostra1 aspect near the mesencephalic flexure and do not extend very far caudally. These cells are characterized as having thick dendrites still connected to the ventricular zone and most probably give rise to the lateral wings of the DRN. In the bilateral group located on the ventrolateral aspects of the floor plate just beneath the MLF, the cells form a continuous longitudinal column that extends to the pontine flexure. The importance of this observation is that later (17 days gestation) the ventrolateral group splits to form the MRN and the interfascicular portion of the DRN. This provides developmental support for considering these two groups of 5-HT neurons as part of a larger functional grouping, as previously suggested (45). More laterally displaced cells become dispersed and give rise to the neurons in the SYSTEM group The inferior group of serotonergic cells forms -2 days after the superior group (305, 491). They are seen for the first time as a ventral group at the end of 14 days gestation. The cells are believed to migrate away from the ependymal zone and to synthesize 5-HT once they have reached their ventral placement. The caudal group at 15 days gestation is completely separate from the superior group with no 5-HT-immunoreactive cells or fibers between them. The NRM is the first nucleus to form in the inferior group, and in whole mount preparations NRM shows more 5-HT immunoreactivity than NRO and NRP (15b). It begins in the rostra1 myelencephalon while soon afterward (16 days gestation) NRO appears in the middle to caudal myelencephalon at a slightly more dorsal position. These nuclei have a fair number of non-5-HT-immunoreactive cells mixed with them. At 1’7 days gestation, the three main caudal nuclei are visible: the NRM, NRO, and NRPa. The cells of NRM have begun to form as a midline group, and processes are seen extending across the midline. The neurons are part of the pontine reticular formation, with some cells extending fairly laterally. The major change at 19 days gestation is the appearance of a well-developed group of 5-HT-immunoreactive neurons in the ventrolateral medulla near the inferior olive in the ventromedial aspect Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 FIG. 6. Sagittal section of brain stem of 15-day-gestation rat fetus. Note appearance of superior (1) and inferior (2) cell group. IF, isthmic fovea. Bar, 100 pm. [Modified from Lidov and Molliver (305).] BRAIN 182 BARRY L. JACOBS AND C. AZMITIA Volume ?Z? medullaris to the lateral habenula (17 days gestation), the external capsule to the claustrum and the internal capsule to the globus pallidus (17 days gestation) and the lateral neocortex (18 days gestation), and the ansa peduncularis and the ventroamygdalofugal pathway to the pyriform cortex and amygdala. The ability of 5-HT-immunoreactive fibers to utilize so many different tracts suggests that these fibers are using other fibers as mechanical guides to innervate a target area. This process has been termed epiphytic guidance (33). The molecule responsible for this form of guidance is believed to be a general type of pathway substrate guidance cue that would direct both 5-HT and non-5-HT fibers (256,304). A likely candidate for such a substrate factor is laminin, which acts as an effective adhesive for growing neurites (141, 318, 410). Laminin immunoreactivity is observed in the prenatal brain colocalized with 5-HT-immunoreactive fibers (F. C. Zhou, personal communication). The precocious development of the 5-HT neurons strategically places them in the midline of the floor of the brain stem where they can C. Pathways and Terminals contact a large number of laminin-rich pathway primordia. These metabolically active neurons quickly utilize many tracts to reach the entire neuraxis. I. Subcortical It is interesting to note that the 5-HT-immunoreactive fibers very early extend into the ventricular system Almost as soon as the 5-HT-immunoreactive neuto grow as supraependymal axons. Although the lateral rons are seen (12 days gestation), fine processes can be wings of the DRN have thick processes attached to the followed rostrally for a distance (50-75 pm), and by 13 ventricular zone as early as 14 days gestation, axonallike projections were not verified on the ependymal surdays gestation, 5-HT-immunoreactive fibers are seen crossing the midline (491). These fibers are nonvaricose face until 17 days gestation. This innervation continues straight axons that form tight fascicles as they pass until the entire ventricular formation is covered by 5HT-immunoreactive fibers at 6 postnatal days. These over the mesencephalic flexure. These fascicles extend toward the mammillary complex, possibly following the fibers have direct access to CSF and can be seen in cerpreviously formed mammillotegmental tract [lZ days tain areas to pass back into brain tissue (43a, 491). The fibers are most likely not transmitting or receiving elecgestation (304)]. This latter tract is the mesencephalic component of the MFB (33). trical information from the ventricle, so the information they process may be trophic in nature. The samThe first connections are those seen decussating the midline and linking the paired bilateral groups of 5-HT pling of CSF by the fibers and growth back into neural tissue would constitute a nonelectrical means of conveyneuronal groups at E-13 days gestation (491). Fibers ing or receiving information on the metabolic state of enter the ependymal surface by 17 days gestation. There is a slow innervation of the various nuclei as they form the organism. The arrival of serotonergic fibers near a terminal so that by postnatal day I, the cochlear nucleus, the spinal trigeminal, and the inferior olivary nucleus are area does not necessarily mean that they immediately densely innervated (304). The first fibers are seen in the will proceed to form a terminal plexus there. For examcerebellum at 21 days gestation, and the innervation of ple, 5-HT-immunoreactive fibers pass just caudal to the the internal granular layer (postnatal day 1) and the optic chiasm at 16 days gestation (491), yet these fibers Purkinje cells and molecular layer (10 postnatal days) do not innervate the suprachiasmatic nucleus until 10 postnatal days (304). Interestingly, the optic innervaare reached postnatally. The signals for terminal sprouting that occasionally are delayed after the 5-HT tion of the suprachiasmatic nucleus arrives at 4 postnatal days (149, 448). The neuronal packing in the suprafibers reach a terminal area are not known, but clearly they represents a timing inherent to the target cells and chiasmatic nucleus declines as the target cells mature not to the innervating 5-HT afferents themselves. and grow dendrites. Then at 10 postnatal days the serotonergic fibers innervate the suprachiasmatic nucleus The 5-HT fibers quickly begin to colonize the forebrain by extending along newly formed pathways (305, itself. This is also true of the mammillary complex, 366a, 491). These include the fasciculus retroflexus to which is not innervated until -3 postnatal days. The the lateral habenula (15 days gestation), supraoptic de- substantia nigra does not begin to receive 5-HT terminal fibers until after this time. An interesting contrast cussation into the supraoptic region (16 days gestation), the same fibers traverse caudally with the optic tract to is seen in the striatum, which is innervated prenatally by dopaminergic fibers (190,435) but does not receive a the lateral geniculate body (18 days gestation), the stria Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 of the LPGN. These neurons are a lateral migration from the neurons comprising the NRM. Medially, the neurons of the more rostrally positioned NRM appear to border the neurons of NRPa at the rostra1 inferior olivary nucleus. In summary, the various raphe nuclei are phenotypically similar, but during development they show differential migratory patterns. The inferior groups are parceled into separate nuclei at the same time as the superior group, although they do not contain 5-HT until 2 days after the superior group. These differences in staining and migratory pattern between the two major groups may underlie the other differences seen in the adult brain (e.g., eolocalization with peptides). The superior groups give rise to a ventrolateral cluster that forms the MRN and the interfascicular portion of DRN and the lateral wings of DRN. Finally, it should be emphasized that the fetal neurons begin laterally and move toward the midline as they mature. EFRAIN January 1992 STRUCTURE AND FUNCTION substantial 5-HT innervation until 14 postnatal days (491). This represents an interesting difference between serotonergic and dopaminergic fetal neurons. 2. Hippocampus 3. Neocortex The neocortical innervation deserves special note. 5-Hydroxytryptamine fibers reach the frontal cortex by 17 days gestation via a rostra1 route through the septum. They form a bilaminar distribution above and just below the developing cortical plate (79, 304). Occasionally, some fibers traverse the cortical plate. However, not until 3 postnatal days is the entire cortex reached. The adult laminar pattern is not established until at least 2 wk postnatally. This should be contrasted with the NA system that innervates frontal cortex at 16 days gestation and extends over the entire cortex by 20 days gestation (424, 435). The locus coeruleus-cortical connection reaches the adult pattern by 7 postnatal days (303,336). This differential sequence between 5-HT and BRAIN SEROTONIN SYSTEM 183 NA immature neurons suggests the neurons might be responding to different signals in the target and may show different growth rates because of a presynaptic signal. Cortical SHT-immunoreactive axons show a patchy distribution in the somatosensory cortex of neonatal mice (172) where a transient dense patch of SHTimmunoreactive fibers and [3H]citalopram (binds to the reuptake site) labeling was seen in layers IV and VI (119). This increased patchy labeling was seen from 3 to 17 postnatal days and disappeared by 2 1 postnatal days. These patches closely overlap the prim .ary sensory area of cortex . Th e den se patches seen in somatosensory cortex were not seen in visual (area 17) or auditory cortex (area 41) where continuous bands of dense axons were seen i n layers IV and VI. In contrast, motor areas as well as parietal and visual association cortex had a sparse 5-HT-immunoreactive axonal distribution. In the somatosensory vibrissae barrel fields, the patches of 5-HTimmunoreactive axons in layers IV and VI are in vertical register. After 5,7-DHT neurotoxin treatment, the patches of serotonergic axons and the density of [3H]citalopram binding is greatly diminished. Recen .tlY 9 it has been observed that removal of the serotonergic patches in the barrel fields actually suppresses the formation of the barrel fields themselves (80). Thus 5-HT innervatio In in the cortex appears to serve a trophic influence on the deve lopment of specific sensory receptive areas. This supports the general hypothesis that the CNS serotonergic system exerts powerful effects by virtue of its innervation density, a chemical determinant of the m orphological organization that underlies the physi01WY of the brain This distinctive temporal innervation pattern serves to underscore th e various steps necessary for development of a 5-HT terminal connection. First, the 5HT neurons migrate from the ventricular surface to assume a paramedian position. This most likely is in response to the presence of tran sient gli al radial fibers (486). The neu.rons then begin to form neurites in response to a soluble trophic signal (see sect. IV&) and to be guided to a variety of target areas by laminin tracts. The final innervation density requires the production of a local trophic signal that depends on the maturation of target cells (neurons and glia). 0 Tissue Culture 1. I~~~oduCtio~ Fetal neurons can survive and mature outside their normal brain environment. The first cultured serotonergic CNS neurons were explants of midbrain raphe taken from newborn rats (202). These postnatal serotonergic neurons survived for up to 16 days in culture. The neurons synthesized and metabolized 5-HT. Detectable levels of 5-[3H]hydroxyindole acetic acid (5[3H]HIAA), the major 5-HT metabolite, were measured Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 The hippocampus illustrates an example of a structure innervated by separate 5-HT pathways that arrive at different times during development (304, 491). The 5-HT-immunoreactive fibers that first reach the hippocampus enter from the dorsal supracallosal route. They pass through the diagonal band, septum, and in the supracollosal stria in the developing cingulum bundle beof the corpus callosum . These fibers fore the formation enter th e retrosplenial primordial cortex, pass in to the subiculum, and finally into the cornu Ammonis of the hippocampus by 18 days gestation. The 5-HT-immunoreactive fibers lie above (stratum oriens) and below (straturn radiatum) the pyramidal neurons. The fornix-fimbria route appears to be utilized 1 day later by fibers to the d.orsal hippocampus. Finally, the third and final entry into the hippocampal formation via the ventral amygdalofugal pathways (47) is by laterally coursing 5-HT-immunoreactive fibers that also project to the pyriform cortex and the ventrobasal amygdaloid nuclei. It should be noted that the two dorsal pathways originate in the MRN and interfascicular part of DRN, whereas the ventral pathway originates in the lateral wings of the DRN (530). The 5-HT-immunoreactive fibers at 3 postnatal days are seen in two dense bands in the CA field, a superficial band. in the stratum oriens (over basal dendrites) and a deeper band between the stratum radiuturn and stratum laconosum. This pattern slowly matures into the adult pattern where a dense innervation is seen over the stratum lacunosum molecular by 10 postnatal days. The innervation of the DG begins at 1 postnatal day with a few fibers in the hilus, and by 2 wk postnatal it has not yet reached adult patterns. The 5HT innervation of the hippocampus is still increasing 3 mo postnatally in rats (40). OF 184 BARRY L. JACOBS AND 2. Accelerated maturation Serotonergic neurons develop more rapidly in culture than neurons in most other neurotransmitter systems. Process outgrowth occurs within hours in culture (48), and Ca2+-dependent uptake and release are functional as early as 2-3 days in culture (48,403a). Noradrenergic neurons from fetuses of 18 days gestation show an immature phenotype and do not show substantial uptake for at least 3 wk, while specific uptake into dopaminergic neurons occurs after 5 days in culture (391, 392). Aggregate cultures of dopaminergic neurons after 4 days in culture (300) and explant cultures of locus coeruleus neurons after 8-14 days in culture (138, 423) contain fluorescent cells with fine fibers. Dissociated mesencephalic cultures do not have fluorescent cell bodies or axonal varicosities for up to 3 wk in culture (403a). These studies illustrate the variation in morphogenesis of identified transmitter systems in culture (391, 392). In general, serotonergic neurons appear more precocious than catecholaminergic neurons. This differential temporal pattern is consistent with developmental findings in the rodent fetal brain (see sect. IvB). However, the results in culture should be viewed cautiously since procedures vary dramatically among laboratories. C. AZMITIA Volume 72 3. Target cell interaction Serotonergic cell growth in culture can be stimulated by a variety of normal target and nontarget cells (48). Hippocampal cells, a normal target for the superior groups, induce the greatest increase of 5-[3H]HT uptake capacity by mesencephalic raphe cells. Immunocytochemical staining shows that coculturing with hippocampus did not significantly change the survival of 5HT-immunoreactive cells between 48 and 96 h. Thus the increase in accumulation represents a maturation of the uptake and storage of the existing cells. A similar result was achieved in cocultures of dopaminergic neurons with striatal cells (35). The stimulation of 5-HT neurons by hippocampal neurons occurs under conditions where glial cell proliferation is suppressed (48). Thus the target cell stimulation is probably due to neuronal-neuronal interactions or to interactions with postmitotic glial cells. Other target areas, normally innervated by mesencephalic raphe cells (cortex, caudate, and olfactory bulb), can stimulate the development of the amount of 5-rH]HT accumulated at high plating densities. In addition, serotonergic neurons from the superior group are stimulated by spinal cord cells that they do not normally innervate. The degree of stimulation observed is not related to innervation reached in the adult brain since hippocampal 5-HT levels and fiber density are relatively low (304, 416,450). However, the stimulation by target tissue may reflect 5-HT receptor density since hippocampal 5-HT receptors are among the highest in the CNS (67, 357). Pharmacological studies are consistent with this latter explanation (44.2, 503, 505, 506). Several neurotransmitter systems show a tight coupling between the afferent neurons and their normal target cells. Dopaminergic neurons respond to their normal target cells (corpus striatum) but not to nontarget cells (parietal cortex) by an increased capacity for uptake and storage for [3H]dopamine (135, 391, 392). This stimulation has been replicated by another laboratory (269). Further evidence for target specificity for dopaminergic neurons is shown by the stimulation produced by the striatal membrane preparations but not by preparations from the hippocampus, cortex, mesencephalon, or cerebellum, all areas area normally devoid of a dopaminergic innervation (391). These results indicate that dopaminergic neurons respond only to their normal target cells in the striatum. Similarly, rat sympathetic substance P-containing neurons are stimulated by their normal target cells (pineal and salivary gland) but not by nontarget cells (heart or intestine) (258). These patterns are consistent with a point-to-point mechanism in brain development. In summary, cultured serotonergic neurons respond to a wide variety of target neurons within a wide developmental window. The combination of diverse target cells and extended time frames could explain the expansive distribution of serotonergic fibers in the vertebrate brain. In other words, the serotonergic fibers have a long period during development in which to lo- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 in the medium after addition of radioactive tryptophan. In a dissociated embryonic (14-15 days gestation) rat brain stem raphe, 5-HT neurons were visualized by immunocytochemistry and by specific uptake of 5-[3H]HT (522, 282b, 390a). Within 24 h of plating, 3.3% of the surviving neurons were immunoreactive to the 5-HT antibody (522). The percent of surviving neurons immunoreactive for 5-HT increased over time, reaching up to 36% of the surviving neurons by 28 days in culture. This observation emphasizes the hardy nature of serotonergic neurons when compared with other brain stem neurons, including dopaminergic and noradrenergic. These cells in culture synthesized, stored, and released 5[3H]HT after exposure to [3H]tryptophan. It is interesting to note that a large number of GABAergic neurons were observed in this raphe culture system (up to 40% of the neurons in 14-day-old cultures). However, these neurons were smaller in somal size and had much shorter processes than the serotonergic neurons. Furthermore, GABA was not colocalized with 5-HT in the larger neurons. Dissociated mesencephalic raphe cells from fetal rat (14-18 days gestation) were grown in microcultures (39,48). The maturation of serotonergic cells was studied qualitatively using immunocytochemistry with a 5HT antibody and quantitatively by measuring the retention of radioactivity after incubation in the presence of a low concentration of 5-PH]HT. The 5-HT-immunoreactive neurons showed specific staining in the perikarya, nuclei, dendrites, axons, and growth cones. These neurons formed varicose fibers and growth cones within hours and survived for up to 3 wk in culture. Each serotonergic neuron concentrated ,- 1 fmol5-rH]HT after 20 min of incubation. EFRAIN January 1992 STRUCTURE AND FUNCTION cate and interact with one of their many target neurons. The characteristics of the target cell stimulation described for 5-HT neurons are uncommon. These findings hold out the possibility that a widespread maturational factor exists for CNS serotonergic neurons. E. Growth Regulatory Factors 1. Ions and small molecules Calcium has a myriad of biologic effects, such as inhibition of phosphorylation, uncoupling of mitochondria electron transport, activation of phospholipase and proteases, cell adhesion, and process outgrowth (186, 518, 523). In cultures of mouse cortical neurons or molluscan ganglion neurons, low Ca” results in decreased process outgrowth (323,518). Conversely, high Ca2+levels intracellularly result in neurite retraction and eventual neuronal death (104, 106). Serotonergic CNS neurons grown in low-Ca2+ medium have fewer 5-HT-immunoreactive cells with smaller somas and fewer processes after 3 days in culture (49). Conversely, high levels of Ca2+ result in neuronal death. If a specific Ltype Ca2+ channel antagonist (nimodipine, lo-’ M) is added to the cultures at the time of plating, then a substantial potentiation in serotonergic survival and process outgrowth is seen over normal conditions (38). This indicates that regulating the L-type Ca2+ channel can produce supranormal growth. In vivo studies support this concept of the L-type Ca2+ channel stimulating serotonergic growth. Treatment of pregnant rats with nimodipine results in accelerated outgrowth of serotonergic fibers to forebrain structures in the offspring (362). Neurons in culture are assumed to favor supranorma1 glucose concentrations since most of their energy is derived from the oxidation of glucose (83). Cerebellar neurons have been shown to survive better in 0.3% (19 mM) glucose than in 0.1% (6 mM) glucose (388). In studies of myelination of central and peripheral neurons, a glucose concentration of 0.6% (37.5 mM) was found to be best (350). Likewise, cerebellar explants require 40 mM BRAIN SEROTONIN SYSTEM 185 glucose for optimal development (286). Several reports on serum-free conditions also advise using glucose concentrations between 25 and 33 mM for optimal neuronal growth (15, 313, 356). Thus it would be expected, given their robust outgrowth and high metabolic activity, that serotonergic neurons would require a similar high-glucose condition. In fact, serotonergic neurons can survive for up to 2 days, but with reduced outgrowth, when glucose is absent from the medium at the time of plating (39). As the concentration of glucose is increased to 0.15% (8.3 mM), the growth rate for the cultured serotonergic neurons shows a sharp increase. However, higher levels of glucose (O&0.5%; 12.5-31 mM) reduce the development of the serotonergic neurons by as much as 75%. The decreased outgrowth in high glucose is probably due to increased acidosis from the enhanced production of lactate (see Ref. 361). Interestingly, the glucose level where serotonergic neurons grow best is similar to the Michaelis constant (K,) of glucose for uptake into neurons and glial cells (0.18 and 0.16%, respectively). Free hydroxyl radicals are believed to be a major cause of neuronal death (203,206). It has been proposed that the production of H,O, by monoamine oxidase can contribute to the generation of free radicals inside neurons (107). Various neurotoxins, such as l-methyl-4 phenyl-l&3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA), appear to act by producing an excess of free hydroxy radicals (352,463), and it has been suggested that neurotransmitters themselves can be converted to potent oxidizing neurotoxins under certain conditions (195,434). In these studies, the addition of free radical scavengers (e.g., pyruvate, vitamin E) or antioxidant enzymes (e.g., supraoxide dismutase, catalase, or glutathione peroxidase) prevents the buildup of oxidants and corresponding cell death (see also Ref. 321). Serotonergic neurons in culture are suppressed by increased levels of H202 in the medium and are enhanced by addition of catalase and glutathione peroxidase (Azmitia, unpublished observations). These results indicate that serotonergic neurons are adversely affected by conditions that generate hydroxy radicals or are stimulated by conditions where free radical buildup is prevented. Increased levels of free hydroxy radicals in the aged brain (as evidence by the buildup of lipofuscin) (440) may offer an explanation for the decrease in longterm survival of transplanted fetal neurons in aged rats (37). 2. Neurotransmitters Serotonergic neuronal maturation appears to be under the influence of its own neurotransmitter during development. Mesencephalic raphe cells from rats of 14 days gestation cocultured with hippocampal cells from 18 days gestation were grown for up to 4 days in the presence of various agents known to alter serotonergic function in the mature brain (505). Pargyline (a nonspecific MAOI) alone and with 5-HT (10-8-10-6 M) inhibited growth of serotonergic neurons as assessedby uptake of Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 Discussion of development would be incomplete without attention to the factors that regulate the growth response of serotonergic neurons. In this section, we focus on a wide range of factors that have significant effects on the survival, attachment, biochemical maturation, neurite extension, and death of fetal serotonergic neurons in dissociated tissue culture. Our selection of factors is broadened to include ions, intermediate metabolites, antioxidants, neuropeptides, gangliosides, neurotransmitters, and proteins. A detailed treatment of this topic has been published (49) so the effects of these factors on cultured serotonergic neurons are only briefly covered. Furthermore, the results of prior depletion of 5-HT on general brain maturation have been extensively reviewed (see Refs. 279, 280, 282) and are not discussed here. OF 186 BARRY L. JACOBS AND C. AZMITIA Volume 72 important functions in regulating the growth of serotonergic neurons. It might be proposed that the vigorous and precocious innervation of the lateral habenula may be related to the glutamate stored in these neurons and the presence of appropriate excitatory amino acid receptors on the growing serotonergic fibers. The role of neuropeptides as neuronal growth-regulating factors has been reviewed (49, 125). Neuropeptides comprise the largest group of putative neurotransmitters (neuroregulators) in the brain and serve as growth factors that are multifunctional (446). Their first appearance in brain during early development is variable and depends on the peptide and region. For instance, vasoactive intestinal peptide (VIP) and bombesin are not measurable in the brain before birth, whereas substance P and somatostatin occur in all regions examined by 14 days gestation. Somatostatin and VIP show large increases in mesencephalon and forebrain, bombesin shows a similar but less dramatic pattern, whereas substance P displayed its largest developmental increases in diencephalon and mesencephalon. Thus brain chemical systems begin to develop early and increase in complexity as the brain slowly matures. In dissociated cultures of mesencephalic neurons, low doses of ACTH (l-10 rig/ml) can stimulate serotonergic development (43). The ACTH family of peptides [ACTH-(4-IO), ACTH-(l-23), ACTH-(l-35), and Organon 27661 are all effective in culture. However, if hippocampal fetal neurons (target cells) are present, exogenous ACTH is no longer stimulatory on serotonergic maturation. This overriding of the trophic peptide effect by target neurons supports the idea that positive neuropeptide growth factors are general signals and may have no direct action on neuronal maintenance after the neurons reach their target. Enkephalin was found to be inhibitory to these same 5-HT neurons at low concentrations (~1 rig/ml) (124). Interestingly, the addition of fetal hippocampal or spinal cord neurons to the raphe cultures enhanced the inhibitory properties of this opioid. This result, with a negative neuropeptide growth factor, indicates that it may have the ability to counteract the trophic effect of the target and halt or repel the growing fiber. In summary, these studies raise the intriguing possibility that neuropeptides may function as positive and negative growth regulatory factors in the brain. Their presence during early development is consistent with this idea. Therefore changes in peptide levels in the brain may contribute to the termination pattern of the developing 5-HT system. 3. Gangliosides Gangliosides are glycosphyngolipids, which are a major component of brain membranes. Ganglioside administration after brain damage has been shown to promote axonal sprouting during nerve regeneration and to enhance recovery of function. Following partial lesions of the hippocampus, both aeetylcholinesterase and 5-HT uptake are reduced (194). Daily injections of the GM, Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 5-[“H]HT. High-affinity 5-HT receptors in fetal brain stem tissue and in fetal forebrain tissue may regulate direction and extent of growth. The presence of these receptors in the fetal brain was confirmed using a direct binding assay (505). 5-Methoxytryptamine (5-MT), a 5HT agonist with selectivity for 5-HT autoreceptors (322), inhibited growth at nanomolar concentrations. 5Methoxytryptamine was observed to produce stunted processes, increase autoinnervation, and lead to the death of 5-HT-immunoreactive neurons. These inhibitory effects on the growth of cultured 5-HT neurons were reversed at micromolar concentrations of 5-MT, where an actual stimulation was found. In vivo confirmation of these results used pregnant rats treated with 5-MT. The pups showed suppressed outgrowth of 5-HT fibers toward the hippocampus (2 wk to 1 mo) after treatment with low doses of 5-MT (1 mg/kg SC) (436). The precise mechanism for these 5-HT receptormediated stimulatory or inhibitory effects has received considerable attention. It is known that glial cells play an important role since they produce laminin and a host of trophic factors. High-affinity serotonergic receptors exist on brain astroglia (504). The expression of these receptors varies inversely with the maturational state of the astrocyte. Furthermore, stimulation of astroglial cells by specific 5-HT,, agonists produces a conditioned medium that can stimulate the maturation of cultured serotonergic neurons (506). The substance produced by the astrocytes responsible for this effect is believed to be the S-100 protein (see sect. 1vE4). Autoregulation of fetal serotonergic neuronal development provides a unique way for the CNS 5-HT system to innervate the brain. This mechanism implies that a serotonergic neuron will grow until it reaches a brain region where it produces excess 5-HT because of the conditions encountered in the target area (e.g., cofactor availability). The 5-HT could leak out to interact with terminal autoreceptors and stop its own growth. Because serotonergic fibers contain terminal autoreceptors (191), such a mechanism is feasible as a feedback brake on neurite extension. The excitatory amino acid glutamate can produce a Ca2+-related cell death on neurons containing an Nmethyl-D-aspartate (NMDA) receptor (104, 263). The serotonergic raphe neurons have NMDA receptors in association with the excitatory input derived from the lateral habenula (see sect. IIIC). Addition of 0.5 mM glutamate to serotonergic cultures raised with low glucose produces evidence of neuropathology (39). The addition of MK-801, a specific NMDA antagonist, prevents the decrease seen in the development of cultured serotonergic neurons. Interestingly, nimodipine, the specific Ltype Ca2+ channel antagonist, also attenuated the effects of glutamate on serotonergic maturation. These results indicate that excess Ca2+ entry, via a variety of channels, is additive in producing neuronal toxicity. In fact, addition of phencyclidine (PCP) to normal cultures can enhance serotonergic maturation in an analogous fashion as observed with nimodipine. Thus voltage-sensitive and chemically mediated Ca2+ channels may serve EFRAIN January 19% STRUCTURE AND FUNCTION OF BRAIN SEROTONIN SYSTEM 187 HIAA. A similar lack of NGF on fetal mouse brain stem catecholaminergic neurons in culture has been reported (139). The results with the 5-HT,,-induced release of astrocytic S-100 have interesting implications. For one, it has been reported that the 5-HT receptors on astrocytes are reduced when the cells are matured by exposure to adenosine 3’,5’-cyclic monophosphate (504). Thus serotonergic neurons may determine their own innervation density by stimulating the release of trophic molecules when the appropriate receptors are available. When the astrocytes become mature, the availability of receptors for trophic stimulation is greatly reduced. Interestingly, damage to the 5-HT fibers in the adult brain may upregulate the 5-HT receptors (164, 357) and permit new fibers to stimulate trophic factor release. This might account for the various reports of hyperinnervation after damage to adult 5-HT fibers (46, 168, 169, 528, 529, 533). 4. Proteins F. Summary Partial reduction of brain 5-HT fibers by chemical toxins will eventually result in the collateral sprouting of undamaged 5-HT fibers (41,531). The signal responsible for this growth of 5-HT fibers is believed to be a protein molecule (45b, 49). When a soluble fraction is prepared from 5,7-DHT-lesioned hippocampi, a stimulation of fetal serotonergic neurons is observed in tissue culture. The identity of this protein remains unknown. However, recent findings suggest that the molecule may be S-100 (P. M. Whitaker-Azmitia and E. C. Azmitia, unpublished observations). S-100 is found in brain astrocytes, and the P-subunit has been shown to have neurotrophic activity in chick embryo cerebral cortical neurons (261). S-100b is very active in stimulating serotonergic uptake capacity in a microculture system. Levels as low as 10 rig/ml of the ,&subunit can increase the uptake capacity for 5-[“H]HT by fetal serotonergic neurons by as much as 100%. In astroglial cultures, the addition of a specific 5-HT,* receptor agonist, ipsapirone, results in the release of a stimulatory molecule into the medium (506). This 5-HT,* neuronotrophic factor can increase neurite outgrowth in cultured serotonergic neurons. In addition, all of this trophic activity is blocked by treatment with a specific S-100 antibody. The results with S-100 stand in marked contrast to the lack of serotonergic trophic activity seen with nerve growth factor (NGF), a well-recognized peripheral nervous system (PNS) growth factor (73). Electrolytic lesions of the serotonergic input to the hippocampus resulted in a marked decrease in 5-HT and 5-HIAA. The administration of NGF (10 pg/rat icv twice a week for 2 wk) increased cortical choline acetyltransferase activity but had no effect on the lesion-induced decrease in hippocampal 5-HT and 5-HIAA (312). Conversely, injections of GM gangliosides (30 mg/kg ip for 2 wk) significantly reduced the hippocampal loss of 5-HT and 5- Serotonergic neurons differentiate very early into two main clusters of cells, a superior and an inferior group (305,491), and begin immediately to produce neurites that extend anteriorly from the superior group and caudally from the inferior group (305). These fibers use other nonserotonergic pathways, blood vessels, and the ependymal cells as guides for innervating their target areas. This ability to utilize a variety of substrates for the growing processes has been termed epiphytic guidance (33). Serotonergic fibers reach their target very early in development. They reach the hypothalamus by 16 days gestation and the cortex by 17 days gestation (304). However, the final termination density is not reached in these structures until the second week of postnatal development. The reason for this delayed maturation may involve production of some local growth regulatory substance (49). It has been demonstrated by tissue culture studies that serotonergic neurons respond to a variety of substances produced by neurons, glia, or endocrine tissue. The timing of the availability of these factors may act as the signal for the completion of innervation. There is evidence that this might occur following enhanced terminal activity (see Ref. 304). The studies indicating that 5-HT can function as a trophic factor indicate that a shift in the timing of serotonergic ingrowth may have important consequences on the maturation of the target neurons. The delayed onset of barrel fields in the cortex produced by 5,7-DHT injections supports this idea (80). A number of different factors have been described that can function as negative or positive serotonergic growth factors. The factors can often be stimulatory at one and inhibitory at another concentration or condition. Calcium, for instance, can produce accelerated growth at 0.5 mM but actually suppress growth at 2.0 mM. The same is true for glucose, 5-MT, gangliosides, Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 ganglioside resulted in enhancement of recovery in both neuronal systems at 6 and 21 days postlesion. In another paradigm, electrolytic lesions in the midbrain caused a significant decrease in hippocampal 5-HT and its major metabolite 5-HIAA (312). Daily systemic injections of the GM, ganglioside significantly reduced the injury-induced decrease in hippocampal 5-HT and 5-HIAA at 6-14 days. Similar results have been seen with the dopaminergic neurons (464). Low doses of GM,, gangliosides (pg/ml) were stimulatory on both serotonergic and dopaminergic neurons in low-density cultures (E. C. Azmitia and E. Yavin, unpublished observations). However, in high-density cultures, a higher dose of gangliosides (pg/ ml) was inhibitory. The effects of GD1 gangliosides, which have two sialic acid residues, are much more potent than GM1 on serotonergic neurons. These results imply that gangliosides can be beneficial when a neuron has been stressed but may be inhibitory when a neuron is growing under optimal conditions. 188 BARRY L. JACOBS AND and neuropeptides. It appears that the conditions of growth are finely tuned so that imbalances on either side of the ideal condition can suppress the maturational rate. The manipulation of these factors during development to produce a change in the final innervation density and behavior holds promise for correcting developmental disorders (436). V. ADULT PLASTICITY A. Introduction B. Regeneration The ability of injured neurons to send their axons back into the same area they vacated following axotomy C. AZMITIA Volume 72 is termed regeneration. This process had been well characterized in the PNS by a variety of researchers, but in the CNS many scientists still adhere to the words of Ramon y Cajal (397): “once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. In adult centers, the nerve paths are something fixed, ended, and immutable. Everything may die, nothing may be regenerated.” Early studies of 5-HT regeneration in spinal cord and brain stem have been previously reviewed (33, 72). A case of serotonergic regeneration is discussed: the morphological and functional recovery after 5,7-DHT lesions in the hypothalamus, where the regrowing fibers not only reestablish their normal synaptic contacts but can also hyperinnervate the target. Unilateral microinjection of 5,7-DHT into the MFB of adult female rats results in the destruction of the serotonergic fibers traveling in this pathway. Within 1 wk, the hypothalamus is largely devoid of 5-HT-immunoreactive fibers (166), and levels of 5-HT and 5-HIAA are greatly reduced (e.g., 90% decrease in the dorsomedial nucleus) (169). Behaviorally, progesterone-primed ovariectomized rats show a facilitated lordosis response to injected estrogen after bilateral 5,7-DHT injection (314). Between 14 and 19 days after the neurotoxin injection, 5-HT-immunoreactive axons in the caudal hypothalamus have smooth bulbous tips that begin to emit fine wispy processes. The 5-HT-immunoreactive fibers extend back into the previously denervated hypothalamic nuclei by 1 mo, with the levels of 5-HT increasing to 50% of control. The facilitated lordosis begins to be attenuated at this time. The results at 8 wk after lesion show that 5-HT-immunoreactive fibers have hyperinnervated the hypothalamus and 5-HT and 5-HIAA have returned to control levels in many areas. The facilitated lordosis response to estrogen is once again normal after 9 wk. This series of studies indicates that damaged serotonergic fibers can survive an injury and with time send back their axons to reinnervate the denervated region. The hyperinnervation observed with immunocytochemistry is confirmed by light- and electron-microscopic radioautography (167,168). At the ultrastructural level, 50 days after 5,7-DHT neurotoxin injection, there was no significant difference in the types of appositions or synaptic frequency between 5-[3H]HT-labeled axons and the postsynaptic neurons compared with sham injected. Thus the regenerating serotonergic axons grow back to some of the same nuclei (dorsomedial hypothalamus and zona incerta), reestablish synaptic and neurotransmitter function, and restore the behavior to normal levels. The ability to achieve this complicated sequence of events in an adult brain emphasizes a target-directed regrowth signal for serotonergic fibers should exist. Successful regeneration in the adult CNS, because it must occur in an environment deprived of signals available to developing fibers, requires months. The time required for the fibers to reach their target is much longer than that needed by the growing fetal neurons, which can innervate the hypothalamus within a few Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 The adult nervous system loses many of the properties associated with the developing brain. There is a more complex and varied collection of neuronal and glial cells in the adult. Many of the radial glial cells clearly visible during early development are gone. Substrate molecules like laminin seen throughout the immature brain are no longer present (307). Myelinated pathways not seen prenatally are abundant in every region of the adult brain. The number of synaptic contacts observed in the mature neuropil illustrates how extensively interconnected the adult brain has become. Thus it is not surprising that the dynamic plastic nature of adult neurons has been difficult to study. Serotonergic neurons are exceptional in their regenerative capacity in the adult brain. Damage to fine 5-HT fibers produces only temporary decreases in the levels of 5-HT in target areas. Given sufficient time (several months) the damaged neurons will reestablish both a structural and a functional presence. Adult target cells can provide many of the crucial molecules to promote sprouting, although key guidance cues (e.g., laminin) may be absent. This may explain why regenerating fibers require more time to innervate an area than do the fetal cells during development. Undamaged serotonergic fibers in the brain can also expand when neighboring 5-HT fibers are removed. This process of homotypic collateral sprouting was established for the first time with 5-HT innervation of the hippocampus by utilizing a specific neurotoxin for 5-HT fibers (41). The characteristics of this form of adult plasticity are discussed in section vC. Neuronal transplantation enables fetal neurons to be inserted into the mature environment of the adult brain. This interaction of neurons of different ages provides unique insights into the process of adult plasticity. This procedure underscores the tremendous capacity of the adult brain for reorganization, both in structure and function. Locally placed fetal serotonergic neurons can substitute for the loss of normal afferent input arriving from the brain stem raphe nuclei. Furthermore, the response of fetal neurons to the adult neuropil may suggest which factors are needed to facilitate regrowth of damaged adult neurons. EFRAIN January 1992 STRUCTURE AND FUNCTION days. The temporal discrepancy might be due to the lack of a proper substrate in the adult for the growing axonal sprouts. For example, the substrate molecule laminin is not detected in the adult neuropil (205, 307). However, injections of laminin can accelerate this process (182). The fibers perform properly when they arrive at the target site as indicated by the ability to restore morphological, biochemical, and functional connections. The problem is getting to the target, and laminin may be an important chemical substrate to direct the growing sprouts. OF BRAIN SEROTONIN A 5,7-DHT C Symmetry Asymmetry Supracallosal 5,7-DHT C (14 days) LTCB (42 days) +STFI (14 days) FIG. 7. Schematic representation of 5-HT innervation of hippocampus by supracallosal fibers traveling in cingulum bundle (CB) and by infracallosal fibers traveling in fimbria (FI). These fibers originate from different neurons in median raphe (MR) nucleus and ascend to forebrain in medial forebrain bundle (MFB). A: degenerating 5-HT axons (dashed line) after unilateral short-term cingulum bundle (STCB) microinjection of 5,7-dihydroxytryptamine (5,VDHT) and resultant anatomic asymmetry produced. B: collateral sprouting of infracallosal5-HT axons into dorsal hippocampus, which restores symmetry [after long-term cingulum bundle (LTCB) injection of 5,7DHT]. C: degenerating 5-HT axons (dashed line) produced by midline microinjection of 5,7-DHT into fimbria (STFI) of LTCB rats and resultant asymmetry produced. Midline microinjection into fimbria of normal rats (not shown) produced no such asymmetry. [From Azmitia et al. (41).] are densely distributed in the infragranular layer of DG, in the stratum lacunosum-molecular of the cornu Ammonis, and in the area fasciola cinerea. The density of the 5-HT-immunoreactive fibers in the dorsal hippocampus is greatly decreased but maintains a similar laminar pattern 3 days after lesioning by 5,7-DHT into the CB. An apparently normal density and distribution pattern of 5-HT-immunoreactive fibers is seen by 42 days postlesion. The fasciola cinerea in the hippocampus is among the first regions reinnervated by 5-HT-immunoreactive fibers with very dense and large varicosities. Restitution of 5-HT-immunoreactive fibers in the dorsal hippocampus after the 5,7-DHT lesion in the CB is accompanied by a marked increase in the number and intensity of 5-HT-immunoreactive fibers in the FF. No evidence of a regeneration of 5-HT-immunoreactive fibers is seen distal to the injection site. These observations provide direct evidence for serotonergic collateral sprouting (SCS) in the CNS induced by removal of 5-HT fibers. Collateral sprouting of cholinergic and noradrenergic fibers after partial deafferentation of the hippocampus by lesions has also been demonstrated (137, 176, 184). In general, these studies show a time course of sprouting similar to the more specific chemical lesions. The phenomenon of homotypic collateral sprouting is slower than seen by reactive synaptogenesis (RS) where the sprouting begins within days and is essentially complete by 2 wk (see Ref. 110). Several other differences between SCS and RS have been noted (50). One is that the chemical specificity of the lesioned fiber is critical Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 Homotypic collateral sprouting is the ability of an undamaged neuron to expand its terminal innervation density in response to removal of fibers containing the same neurotransmitter. This plastic response was first shown in the CNS of adult rats by 5,7-DHT-selective destruction of the serotonergic hippocampal afferents traveling in the cingulum bundle (CB) and by monitoring the induced expansion of the undamaged serotonergic fibers traveling in the fornix-fimbria (FF) (Fig. 7). Serotonergic fibers were traced by anterograde transport after injections of [3H]proline in the midbrain raphe nuclei by the radioautographic technique. After a Z-wk delay, the labeled fibers in the FF began to grow into the space left by the lesioned CB fibers, and within 42 days the innervation appeared normal. Evidence for a hyperinnervation after 90 days was noted. These unilateral CB-lesioned animals also showed an asymmetrical turning response to a 5-HT agonist that returned to normal after 42 days. To demonstrate that the anatomic and functional recoveries were due to sprouting fibers from the FF pathway, the midline injection of 5,7-DHT into the FF at 4.2 days after a previous unilateral CB lesion reinstated both the functional and anatomic deficits. Further evidence in support of this phenomenon involved HRP-retrograde transport studies (531). With this procedure a significant increase was seen in HRPlabeled neurons in the MRN at 21 and 42 days compared with 3 days after CB lesion, with the number of cells at 4.2 days actually being supranormal (121% of sham injected). Neurons in the interfascicular subnucleus of the DRN that normally travel in the CB to innervate the dorsal hippocampus were not labeled at any time after the injection of 5,7-DHT in the CB. In addition, there was no increase in cell labeling for 2 mo after combined CB-FF 5,7-DHT lesions. These results indicate that the CB midbrain raphe fibers do not regenerate into the dorsal hippocampus, but FF fibers can increase their territory by collateral sprouting in response to homotypic denervation. The availability of sensitive antibodies against 5HT enables these neurons to be observed directly without the use of transported material (533). The 5-HT fibers are distributed in a laminar pattern in the hippocampus (Fig. 8). These fibers have large varicosities and 8 Asymmetry STCB C Collateral Sprouting 189 SYSTEM 190 BARRY L. JACOBS AND for SCS but not for RS. In RS the proximity of the fiber to the site of lesion is critical, whereas in SCS the collateral sprouts can travel long distances (up to a centimeter). Further evidence that SCS is a separate process from RS is their differential response to adrenal steafferents after a lesion of roids. In RS of hippocampal C. AZMITIA Volume 7.2 the entorhinal cortex, the removal of circulating adrenal steroids enhances the appearance of new sprouts (421), whereas in SCS the adrenal steroids are necessary for sprouting (532). The opposite actions of growth modulatory substances are an important principle for understanding recovery of function. Furthermore, the innervation density of a particular system should be viewed as being in a dynamic equilibrium, and increasing the amount of target area (partial deafferentation) or of the afferent input (see next section) can change the level of innervation. Established contacts are fairly stable to changes in growth regulatory factors (e.g., glucocorticoid). These factors are effective when the system is in flux. Thus the maintenance of neuronal connections is resistant to daily changes in the environment, but when these connections are severed a new balance can be easily created. A final word should be mentioned concerning the signal for SCS. When 5-HT fibers are damaged, there is a period of time (2-3 wk) before new collateral sprouts are formed (50). This appears to be due to the formation of a soluble trophic factor by the target cells. The hippocampus can be removed at various times after 5,7-DHT lesions and a soluble extract can be studied in culture. No significant enhancement of trophic activity is seen until 2 wk postlesion (45b). This is in marked contrast to the production of NGF, which increases within a few days and peaks in the hippocampus at 2 wk postlesion (116). Furthermore, when 5-HT fibers begin to grow there is evidence that they can overshoot their normal levels and hyperinnervate their target neurons. D. Transplantation Integration of fetal cells with adult neurons is probably the most extreme form of brain plasticity. Therefore this topic is reviewed in some detail because it illustrates several important anatomic and physiological aspects of both the immature and mature serotonergic system. I. Introduction The first transplantation of serotonergic neurons in the brain was made into a cavity in the retrosplenial cortex of rats (71). Serotonergic neurons from fetal embryonic pontine raphe region (15 days gestation) grew along the lesioned temporoammonic perforant path to innervate the partly denervated hippocampus. At l-3 mo, 5-HT fibers were seen to conform to the normal distribution of terminals of the perforant path. The 5,7DHT-lesioned and nontreated hosts were reported to support the same outgrowth pattern. Transplantation of serotonergic neurons into the spinal cord was first performed by Nygren et al. (364). The fetal neurons (17-19 days gestation; crown-rump length 19-26 mm) were inserted into the lumbar spinal cord with a glass pipette. With the use of fluorescence Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 FIG. 8. Schematic drawings of 5-HT fiber distribution in coronal sections of dorsal hippocampi of normal (A), 3 days after 5,7-DHT lesion lesion in cingulum bundle (CB) (B), and 42 days after 5,7-DHT in CB (C). A: dorsal hippocampus receives 5-HT innervation via supracallosal pathway [medial CB-indesium griseum (IG)] and infracallosal pathway [fornix-fimbria (FF)]. These afferent 5-HT fibers mainly terminate in stratum lacunosum-moleculare of cornu Ammonis (CA), infragranular layer of dentate gyrus (DG), and area fasciola cinerea (FC). Less dense 5-HT fibers are distributed in stratum oriens and stratum radiatum of CA areas. Density of 5-HT fibers in CA areas appears in sequence of CAl, CA4, and CA3. B: 3 days after 5,7-DHT lesion in CB, degenerated fibers are observed in CB, IG, and CA areas and in DG. Density of 5-HT-immunoreactive fibers are significantly decreased in CB, IG, infragranular layer of dentate, stratum lacunosum-moleculare of CA, and FC. 5-HT-immunoreactive fibers in FF appear normal. C: 42 days after lesion, 5-HT-immunoreactive fibers are restored to normal density and pattern except for a greater than normal fiber density in FC. On lesion side, 5-HT fibers regenerate from degenerated stumps but do not extend far from lesion site. Reactive growth of compensatory 5-HT fibers is observed in FF, which results in abnormally high density of 5-HT-immunoreactive fibers in ipsilateral FF. ALV, alveus; CC, corpus callosum; Fi, fimbria; G, granular cell layer; LM, stratum lacunosum-moleculare; LV, lateral ventricle; M, stratum moleculare; 0, stratum oriens; P, stratum pyramidalis; PI, stratum polymorphic; Pm, pyramidal cell layer; R, stratum radiaturn. [From Zhou and Azmitia (533).] EFRAIN January STRUCTURE 19% AND FUNCTION histochemistry, the fate of the donor neurons was studied. Only 25% of the transplants were successful. Serotonergic neurons survived for up to 3 mo, and fluorescent fibers could be traced 10 mm cranially or caudally from the cell bodies. The reason for the low survival was attributed to the graft being extruded from the spinal cord because of pressure and bleeding. 2. Descriptive studies OF BRAIN SEROTONIN RAPHE SYSTEM & LC TRANSPLANTS IN 5,7-DHT HIPPOCAMPUS and hyperinnervation N DHT DYT DkjT R-TP LC-TP Ml N DHT DYT DYT R-TP LC-TP FIG. 9. 5-HT and norepinephrine (NE) levels in control hippocampus (N), hippocampus with 5,7-DHT lesions (DHT), and after raphe (R-TP) or locus coeruleus transplantation (LC-TP) in hippocampus with lesion. When hippocampus (Hipp) was deprived of 5-HT innervation by 5,‘7-DHT lesion, growth of transplanted fetal 5-HTraphe neurons is stimulated, but growth of transplanted fetal NE-LC neurons was not facilitated (see drawing). A: 5-HT levels. R-TP had greater increase in level of 5-HT when placed in hippocampus with lesion than when placed in control hippocampus. LC-TP did not increase 5-HT level in hippocampus with 5,7-DHT lesion. B: NE levels. There was no difference in NE levels between control hippocampus and hippocampus with 5,7-DHT lesion nor LC-TP or R-TP in hippocampus with 5,7-DHT lesion. NE levels were not higher in LC-TP in 5,7-DHT-lesioned hippocampus compared with LC-TP in control hippocampus. [From Zhou et al. (528).] fetal serotonergic neurons. Fetal raphe neurons (16-18 days gestation) were transp lanted into the entorhinal cortex of 6-day-old neonatal rat recipients that had received a fimbria and entorhinal cortex ablation 3 days earlier (219). One week after transplant, some 5-HT-immunoreactive fibers were seen in the host cortex but none in the hippocampus. Between 14-21 days, some 5HT-immunoreactive fibers were seen in CAZ-CA3 and some in infragranular DG. After 3 wk, a hyperinnervation was noted in the infra- and supragranular layers of the DG, especially in the hilus area. The general lamination throughout the hippocampus was not clear, but this was attributed to the extensive denervation by the fornix and entorhinal lesions. The authors noted that their implants decreased in size between 30 and 60 days. The 5-HT fetal transplants (E-14 days gestation; crown-rump length 8-12 mm) were placed into adult hippocampus and examined with immunocytochemistry after 5,7-DHT lesions. The authors observed that the number of surviving fetal neurons was stable for -7 wk and then decreased at 5 mo to 37% of the number of cells seen at 3 wk. Nevertheless. at the longest nostonerative Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 The first microinjections of fetal serotonergic neurons were made into the hippocampus of adult and aged animals (46). Isogeneic mice (16 days gestation) were used, and the transplanted fetal raphe area was examined with an antibody raised against 5-HT. The 5-HTimmunoreactive fibers hyperinnervated both cornu Ammonis (stratum lacunosum-molecular) and the DG (both the polymorphic and the molecular layers). This ability to supercede the normal levels of innervation is unique. If fetal hippocampus was transplanted, then the adult host serotonergic fibers grew into the donor target tissue. These studies demonstrated that the innervation density of the brain by serotonergic neurons can exceed normal levels when fetal neurons are supplied. Furthermore, fetal hippocampus can induce an expansive response from the adult neurons. It can be concluded that neuronotrophic interactions are not governed solely by the ages of the cells. This is especially noteworthy in the aged brain where the endogenous serotonergic fibers are degenerating (40,47). Unlike most neuronal systems, hyperinnervation of target areas by fetal serotonergic neurons is a well-confirmed event by anatomic and biochemical measures of 5-[3H]HT uptake and by 5-HT and 5-HIAA levels. If transplants were made into unlesioned brains, then high-affinity uptake of 5-[3H]HT increased by 53% over normal levels, 5-HT levels increased by 50%) and 5-HTimmunoreactive cells and fiber density of 5-HT-immunoreactive fibers were supranormal after 1 mo (32). A prior 5,7-DHT lesion reduced both uptake of 5-[3H]HT and 5-HT levels to 36 and 30% of normal values, respectively (Fig. 9). If transplants were made into these animals, then a dramatic increase of high-affinity uptake (345% over control and 946% over 5,7-DHT-primed hippocampus) and 5-HT levels (225% over control and 850% over 5,7-DHT-primed hippocampus) was found. Similar results were seen 2 wk after intracisternal injection of 5,7-DHT (472). Levels were 205 and 234% above normal levels and 3,383 and 1,504% above the 5,7DHT-treated levels for hippocampal5-HT and 5-HIAA, respectively. There was also evidence for a change in rotational behavior after ipsilateral transplantation, which was significantly correlated with the asymmetry in 5-HT levels. These results demonstrate that transplanted fetal neurons can replace adult hippocampal5HT in a motor response. Correlation between hippocamI pal 5-HT levels and locomotor activity has been previously demonstrated in normal and lesioned rats (515). Other workers have noted a hvnerinnervation bv 192 BARRY L. JACOBS AND 3. Role of target sites With the use of a transplantation procedure with cell suspensions, fetal serotonergic neurons (14 days gestation) from the forebrain projecting raphe nuclei were transplanted into the spinal cord (389,390), which normally is innervated by the descending medullary raphe nuclei. The spinal cord was previously transected at the lower thoracic level, and after laminectomy a lmm thick segment was excised. After 1 wk, the raphe cell suspension was injected into the distal cord. Within 10 days, 5-HT-immunoreactive fibers were seen 15 mm from the transplant site, and by 60 days, fibers were seen 20 mm from the site. The fibers appeared to travel near the central canal and heavily innervated the nucleus intermediolateralis. Recent work suggests that 5HT transplants in damaged spinal cord preparations in primates may result in the return of some of the lost function (A. Privat, unpublished observations). It is interesting to note that the fetal serotonergic cells can innervate and restore functions lost by transection of the spinal cord, even if those 5-HT cells would normally have projected to the forebrain. Similar evidence for neuronotrophic interactions between the rostra1 raphe nuclei and the spinal cord was provided by coculturing methods (48). The developmental maturation of the superior group of serotonergic neurons was stimulated by the presence of spinal cord neurons when grown together for 3 days in culture. An extensive series of papers dealt with 5-HT-, subneurons transstance P-, and TRH-immunoreactive planted from medullary or mesencephalic (rostra1 brain C. AZMITIA Volume 7.2 stem) raphe nuclei into the spinal cord, hippocampus, or corpus striatum (160-163). Adult rats were pretreated with 5,7-DHT, and fetal raphe cells (13-14 days gestation) injected into target sites. In the spinal cord, mesencephalic cells grew out ~15 mm after 2-12 mo and did not innervate motoneurons. Some small substance P cells with intratransplant fibers were seen. No TRH fibers were seen. In the hippocampus, there was a substantial outgrowth of 5-HT-immunoreactive fibers but no detectable substance P or TRH. Finally, in the striaturn, the mesencephalic 5-HT-immunoreactive neurons grew better than in the spinal cord. Colocalization of 5-HT/substance P was found in this area for the first time. Very few fibers for substance P were seen in the graft, and no TRH fibers were seen. The situation with medullary transplants was different. In the spinal cord, the 5-HT-immunoreactive neurons innervated motoneurons. Colocalizations of 5-HT with substance P and TRH were seen. The 5-HT-immunoreactive fibers extended up to 15 mm. Some outgrowth of substance P and TRH fibers was seen. In hippocampus, the medullary 5-HTimmunoreactive neurons behaved like the mesencephalic raphe group where no substance P or TRH fibers were seen outside the graft. Finally, in the striatum, 5-HT, substance P, and TRH fibers were restricted to the graft. This series of studies emphasizes the importance of considering not only the source and age of the donor fetal neurons but also trying to match the fetal cell with its proper target area to achieve the most satisfactory result. Neuronotrophic interactions can be very specific but not always logical. For instance, medullary 5-HT neurons normally innervate spinal cord, but if transplanted into hippocampus, they grow out, whereas if transplanted into striatum, they will not grow out. The role of neuropeptides as neuronal growth regulating factors is discussed in section IVE. 4. Host trophic factor Partial removal of 5-HT fibers from the adult hippocampus by injections of 5,7-DHT can induce homotypie collateral sprouting of the undamaged 5-HT fiber (41, 50, 533). These results indicate the presence of an endogenous trophic factor for 5-HT fibers that can induce adult 5-HT sprouting. Can fetal 5-HT neurons be similarly affected? This question of a lesion-induced neuronotrophic response was studied in greater detail. Transplants of raphe neurons into an intact adult hippocampus showed increases in high-affinity uptake of 5-[“H]HT, 5-HT levels, and 5-HT-immunoreactive fiber density above normal but not as high as that seen if the transplants were made into brains previously injected with 5,7-DHT (32, 528). Although the extent of fiber outgrowth of the 5HT-immunoreactive neurons was increased, the number of surviving 5-HT-immunoreactive cells was not changed significantly. The neuronotrophic effect of the 5,7-DHT lesion was attributed to a soluble factor since Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 times (7 wk and 5 mo), some of the animals exhibited extensive hyperinnervation patterns throughout the dorsal part of Ammon’s horn and the DG. In ultrastructural studies of raphe transplants, 15% of 5-HT-immunoreactive fibers in the hippocampus formed asymmetrical contacts in normal rats. This percentage was not changed after fetal raphe transplants, even though the number of fibers was greatly increased (22). These results concerning hyperinnervation of denervated adult tissue by transplanted fetal 5HT neurons illustrate the robust trophic and dynamic interactions of serotonergic neurons and their targets. The ability of sprouts to reach the proper target sites (and even make appropriate synaptic contacts) in the absence of the developmental guidance cues is remarkable. What trophic factors are these neurons seeking? There are many trophic factors that stimulate 5-HT neurons. It is interesting that the trophic signals must be present in great supply despite the age of the animal (see sect. W4). How can a fetal neuron substitute for the adult neuron equipped with its normal complement of afferents obtained from the brain stem raphe? The ability of transplanted fetal 5-HT neurons to restore normal behavior lost by deafferentation of the adult 5-HT neuron emphasizes the basic information conveyed by these neurons. EFRAIN January 1992 STRUCTURE AND FUNCTION 5. Electrophysiological maturation The development of mature electrophysiological properties by transplanted serotonergic cells was studied in a hippocampal slice preparation (430, 431). The serotonergic neurons from 15- to 19-day-gestation rat embryos showed few of the adult characteristics. After transplantation, 5-HT neurons develop all the normal characteristics associated with the raphe neurons that BRAIN SEROTONIN SYSTEM 193 develop in situ. In this work, it was also demonstrated that electrical stimulation of the transplant caused a marked hyperpolarization of hippocampal pyramidal neurons. This established the existence of a connection between host and donor tissue and verified that the electrophysiological characteristics are similar to those seen for the normal raphe-hippocampal serotonergic projection. Again, evidence is obtained that an ectopitally situated fetal serotonergic neuron can substitute functionally for adult brain stem raphe neurons. Electrophysiological studies have also been carried out on a cotransplant of fetal raphe and spinal tissue into the anterior chamber of the eye (210). Dorsal raphe neurons from the midbrain formed cellular plexuses around large spinal cord neurons. Firing rates of both 5-HT and spinal cord neurons were obtained. In the spinal cord, glutamate triggered cell firing and 5-HT (3 x 10e4 M) accelerated the rate. Metergoline, a 5-HT receptor antagonist, blocked the response to exogenous 5-HT. In certain cotransplants, 5-HT terminals hyperinnervated the spinal cord and slowed spontaneous cell firing. If the dorsal raphe grafts were stimulated, the spinal cord neurons’ firing rates were increased over the elevated glutamate-induced rate. Thus a mini-brain circuit can be established in the eye, and the physiology of 5-HT on motoneurons can be studied. 6. Functional recovery In the most complete study of functional recovery by transplanted fetal serotonergic neurons, it was first shown that neurotoxin-induced destruction of serotonergic terminals in the medial hypothalamus facilitated lordosis, decreased 5-HT-immunoreactive fiber density, and reduced 5-HT and 5-HIAA levels (314). Transplantation of serotonergic neurons then restored 5-HT and 5-HIAA levels 50 days later and completely restored the change in lordosis to control levels or below. The 5-HTimmunoreactive fibers were also seen at normal or hyperinnervated levels in the medial hypothalamus. These studies demonstrate that not only motor behavior but complex hormonal-dependent sex-linked behaviors require the presence of 5-HT, whether derived from normal midbrain raphe neurons or from transplanted fetal neurons. Several other complex behaviors can function when fetal 5-HT neurons are substituted for those of the adult. Transplantation of fetal serotonergic neurons into the fourth ventricle may correct sleep disturbances (181, 189, 329). The 5,7-DHT lesions in neonates significantly decrease paradoxical sleep in adult rats. Three months after intracisternal 5,7-DHT, 16-day-gestation raphe tissue was transplanted into the fourth ventricle. Paradoxical sleep in these animals was normal. A few 5-HT-immunoreactive cells and fibers were seen near the transplant site. In another study, 5,7-DHT microinjection into the FF resulted in a facilitation of complex maze learning by reducing the mean number of errors and trials to criterion in rats (17). The transplantation Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 an extract from a lesioned-primed hippocampus could stimulate the development of transplanted 5-HT neurons when transplanted to an intact hippocampus (528). Hyperinnervation of the transplanted neurons did not appear to displace the normal afferent inputs to the hippocampus from the midbrain raphe neurons (529). Thus fetal and adult afferents can coexist in an adult target area and establish a much higher degree of innervation. It is important to note that the enhanced development of fetal serotonergic neurons after lesions with 5,7-DHT did not enhance the development of fetal locus coeruleus neurons (528). In fact, the noradrenergic neurons grew more poorly into the 5-HT-denervated than into the normal hippocampus (Fig. 9). This provides evidence that the damaged adult brain can produce specific neuronotrophic or neuronotoxic factors that differentiate between 5-HT- and norepinephrine-producing neurons. One of the problems with fetal axonal growth into adult tissue may be the lack of a suitable substrate for the axons to attach to when growing. To test whether laminin has a similar role in the adult brain, sequentially injected laminin and transplanted fetal neurons were made into various brain regions to determine if the fetal neurons would preferentially grow along a laminin injection tract. Serotonergic neurons were transplanted to the motor cortex, neostriatum, or hippocampus of adult animals (534). The tract used for microinjection of cell suspension was then immediately filled with suspension medium or laminin. In other animals, laminin or control solution was injected in a separate needle tract displaced 0.3-l mm from the transplant injection tract. Straight and thick 5-HT-immunoreactive fibers (stained with anti-5-HT antiserum) were predominant within the laminin-treated tracts or were directed toward the laminin-treated parallel tracts when it was positioned ~0.5 mm from the transplant site. The density of 5-HT-immunoreactive fibers in the injection tracts in all three brain areas was much higher for laminin than that seen with control medium. Thin axonal fibers of fetal 5-HT neurons were observed surrounding the laminin-treated tracts but not in vehicle-treated tracts. Finally, laminin injection to the hippocampus, motor cortex, or neostriatum of the adult brain did not stimulate sprouting of undamaged adult 5-HT fibers. These results suggest that purified laminin can facilitate and guide process outgrowth of 5-HT neurons during an early developmental stage but does not induce sprouting on these same fiber types in the adult brain. OF 194 BARRY L. JACOBS AND VI. C. AZMITIA CELLULAR Volume 72 NEUROPHYSIOLOGY A. Unit Activity in Anesthetized Animals or Brain Slices I. Introduction Neurophysiological experiments carried out in anesthetized animals or in tissue slices maintained in vitro are of limited value in elucidating questions regarding organismic physiology or behavior. Nonetheless, the simplicity and/or stability afforded by these preparations have proven invaluable for examining the synaptic physiology and membrane properties of neurons. Several recent papers have been devoted exclusively to this aspect of serotonergic neurons (5,10, g&115,429, 516). E. Summary 2. Intrinsic activity Serotonergic neurons, because they have extensive projections and can regulate a wide range of biological functions, are an excellent neuronal system to use for the study of adult plasticity. Undamaged serotonergic neurons will respond to the loss of neighboring 5-HT fibers by collateral sprouting (41). This demonstrates that the adult serotonergic system has not reached its growth potential but is fact limited by the environment of the target structure. What does determine the innervation density? This particular question has intrigued investigators for many years, and transplantation studies have provided some clues. Serotonergic neurons are a good source of cells for transplantation work. The cells can be easily dissected, accurately measured by biochemical methods, sensitively stained by immunocytochemistry, specifically destroyed by neurotoxins, and reliably transplanted into cerebral tissue. A major conclusion from this work is that the age of the host or donor tissue does not provide a complete barrier to communication between serotonergic neurons and billions of its target neurons. For example, they can quickly contact the hippocampal pyramidal cells and spinal cord motoneurons and establish electrical and chemical connections. This colonization can regulate complex functions such as sexual behavior and cognitive processing. These transplantation studies indicate that the innervation density of 5-HT in the adult brain can be experimentally manipulated by increasing either the number of 5-HT neurons or by enlarging the target area. The lack of a suitable substrate for fiber attachment in the adult brain can hinder the rate of fetal neurite growth (534). Recent evidence indicates that astrocytes that produce a number of serotonergic trophic molecules, such as S-100, undergo aging and may lose the receptors necessary for proper trophic responses (504,506). Interestingly, the 5,7-DHT lesion of the adult hippocampus appears to reactivate the glial cells to once again produce S-loop in response to a 5-HT signal (45a). In the late 196Os,Aghajanian et al. (6,7) descri.bed a distinctive type of neuronal activity recorded in the area of the DRN or MRN of rats anesthetized with chloral hydrate. They consistently recorded cells with a slow (1-Z spikes/s) and highly regular discharge pattern (Fig. lo), whose activity was completely suppressed by systemic administration of LSD. On the basis of these initial results, they hypothesized that these slow and regular firing cells were serotonergic, since this type of activity was found only in brain areas known to contain serotonergic cells and since only this type of neuron showed a consistent depressant response to low dosesof LSD (an effect predicted on the basis of previous neurochemical studies examining the effects of LSD on brain 5-HT). The assumption that these were serotonergic neurons has now been supported by a large body of experimental evidence (for review see Ref. 238). The characteristic slow and regular discharge of serotonergic neurons led to the hypothesis that their activity might be endogenous, driven by an intrinsic pacemaker mechanism. In the first attempt to explore this issue, rats were prepared with a complete transection of the neuraxis, immediately rostra1 to the DRN, thus isolating it from the influence of the entire forebrain (347). The activity of DRN neurons in these brain stem-transected animals was virtually indistinguishable from that found in intact animals. Although their discharge rate was ~30% higher than that of a corresponding group of neurons in intact animals (possibly representing removal of a tonic forebrain inhibitory influence), these cells still displayed the slow rhythmic discharge pattern that characterizes serotonergic neurons. In an attempt to explore this issue more directly, the activity of serotonergic neurons in the rat DRN was examined in vitro (346). By cutting a 400-pm thick section through the rat brain stem, the serotonergic neurons in this tissue slice were isolated from neurons in the remaining forebrain and brain stem. In vitro, most DRN neurons displayed discharge patterns indistin- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 of these animals with fetal serotonergic neurons reversed this facilitation (395). Finally, it has recently been shown that cognitive functions associated with the cortex and hippocampus are dependent on both the serotonergic and cholinergic systems (358, 483). Rats injected with 5,7-DHT or administered atropine (a cholinergic antagonist) show only slight dysfunction in spatial memory, but when both treatments are given there is a severe loss of spatial memory (432). If these 5,7-DHT-lesioned animals receive a serotonergic fetal neuronal transplant into the dorsal hippocampus, then the atropine injection is no longer effective at disrupting memory. Thus in cortically based cognitive behaviors, fetal serotonergic neurons provide the input for normal responses. The only requirement is a trophic one: the growth of the fetal 5-HT neurons into the proper target zone. Once the 5-HT fibers are in place, the brain appears to function normally once again. EFRAIN January 1992 STRUCTURE AND FUNCTION OF BRAIN SEROTONIN SYSTEM 195 pacemaker cells studied in both vertebrate and invertebrate preparations (310). 3. Development from typical rat. Each guishable from those observed in vivo. They uniformly fired with a regular pattern; some fired at a rate substantially higher than that observed in vivo, but over 60% of the cells had a mean discharge rate between 0.8 and 2.5 spikes/s (Fig. 11). The most direct and compelling evidence for the intrinsic activity of brain serotonergic neurons came in a study employing intracellular recordings in vivo. This analysis revealed that these cells display a large afterhyperpolarization (AHP) followed by a gradual depolarization, without evidence of excitatory postsynaptic potentials (EPSPs), eventuating in the succeeding spike (lla). This same pattern was also found in subsequent intracellular analyses of serotonergic unit activity recorded in DRN tissue slices (115, 481). These neurons also had very high membrane input resistances and very long time constants (both -3-4 times that of neighboring neurons). Further studies in tissue slices led to the following proposed sequence of membrane events generating this slow rhythmic activity (5, 92, 429). During an action potential, calcium enters serotonergic neurons through a high-threshold calcium channel. This is followed by a large (15-20 mV) AHP generated by a calcium-activated potassium conductance. This AHP results in a long relative refractory period, thus preventing discharges in bursts and insuring slow rates of firing. As the AHP decreases (due to sequestration and/ or extrusion of calcium), it deinactivates a low-threshold calcium current and an early transient outward potassium current (IA). The currents generated by the activation of these two voltage-dependent channels are opposed, with IA tending to slow the rate of depolarization and the low-threshold calcium conductance increasing it. Under normal conditions, the calcium conductance is stronger, thus leading to a shallow ramp which ultimately reaches threshold, depolarization, fires, and, as the calcium enters the cell, reinitiates the sequence of events. The slope of this ramp is what determines the rate of discharge of serotonergic neurons. This sequence of events is similar to that seen for other FIG. 11. Spontaneous single-unit activity of rat DRN neurons recorded in vitro. Single oscilloscope sweep traces of 3 different units are shown at left (bar, 1 s). Interspike interval histograms corresponding to same 3 units are shown at right (bars, 1 s and 25 spikes). [From Mosko and Jacobs (346).] Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 FIG. 10. Oscilloscope trace of single-unit recording serotonergic neuron in DRN of chloral hydrate-anesthetized trace is 10 s long. [From Jacobs et al. (238a).] Developmental analyses of the single-unit activity of DRN serotonergic neurons indicate that they take on adult characteristics very early postnatally or even prenatally. Embryonic rat brain serotonergic neurons have been studied in slices (431). No activity was observed at embryonic days 14-16, but the characteristic activity of brain serotonergic neurons was seen on embryonic days 17-18 (i.e., 3-4 days prenatal). The slow and regular discharge that characterizes adult DRN serotonergic neurons was also seen in anesthetized 3-day-old rat pups (278,177). Additionally, these cells displayed a suppression of activity in response to a 5-HT autoreceptor agonist drug, a response characteristic of DRN serotonergic neurons in adults. Finally, as early as the first postnatal week in kittens, DRN cells displayed the slow and regular activity and pharmacological response characteristic of adults. These cells also showed the adultlike pattern of activity across the sleep-wake cycle (2). Thus this group of neurons is among the first in the CNS to take on adultlike activity both spontaneously as well as dur- 196 BARRY L. JACOBS AND ing a physiologically driven change. This is consistent with the aforementioned precocial morphological development of neurons in the superior group of serotonergic cells. 4. Feedback regulation C. AZMITIA Volume 72 gic neurons in the same, as well as other, raphe nuclei, it was proposed that these cells are under local, rather than long-loop, feedback control (343). Third, Aghajanian has further refined and elaborated this latter concept by providing physiological evidence that serotonergic neurons are under the influence of axon collateral inhibition (493) and anatomic evidence that neighboring serotonergic neurons make dendrodendritic and dendrosomatic connections (4; see sect. IIIF). Apparently, this feedback exerts a tonic influence since administration of 5-HT autoreceptor antagonist drugs increases the firing rate, and regularizes the discharge pattern, of serotonergic neurons in behaving animals (159; Fig. 12). Interestingly, when this same type of experiment was conducted in anesthetized animals, no increase in discharge rate was seen (74, 315). Additionally, as would be predicted on theoretical grounds, the feedback becomes increasingly manifested as the basic neuronal activity increases. Thus under conditions where serotonergic neurons are known to be relatively inactive, for example, during sleep, autoreceptor blockade has little, if any, effect on neuronal activity. However, under conditions where serotonergic neurons are activated and 5-HT release would be expected to exert feedback inhibition, for example, during arousal, autoreceptor blockade is manifested as a significantly larger increase in the activity of serotonergic neurons (159). These results provide evidence for the physiological significance of this feedback inhibition. Finally, there is indirect evidence that the density of cell body autoreceptors on individual neurons may be a factor in determining both the level of spontaneous activity of serotonergic neurons as well as the degree to which they respond to 5-HT agonist drugs (212,237). Thus neurons with few autoreceptors would be faster firing, due to less feedback inhibition, and less responsive to 5-HT agonist drugs, due to fewer sites for drug-receptor interactions. Neighboring serotonergic neurons in the DRN (two cells were recorded simultaneously with the same electrode) display complex patterns of interactions (496). Baseline 8-OH-DPAT (5pg/kg,i.v.) Spiperone (1 mg/kg,i.v.) 8-OH-DPAT (5yg/kg,i.v.) FIG. 12. Polygraph traces displaying single-unit activity of serotonergic neuron in DRN of cat, following systemic administration of 5-HT,, agonist drug [Ghydroxy-2-(di-n-propylamino)tetralin @-OH-DPAT)] and a 5-HT,, antagonist drug (spiperone). Note rapid and potent effect of agonist. Note also that antagonist not only blocks effect of agonist (bottom trace), but it significantly increases baseline level of neuronal activity (middle trace). III I I + ~ 4 ,,,, ,,,,, ,,,,. .. .,,. .. . ... .. . .. . .... .... .... .. . .. . . .. . .. ... ... . . .... . 10 set Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 A major variable that controls the activity of brain serotonergic neurons is negative neuronal feedback. Thus as the level of brain 5-HT increases, for example, following administration of its precursors, L-tryptophan or L-5hydroxytryptophan, the activity of brain serotonergic neurons correspondingly decreases (3,467). It is presumed that this neuronal decrease is a homeostatic response that acts to compensate for increases in synaptic levels of 5-HT. The activity of serotonergic neurons is also strongly inhibited by 5-HT autoreceptor agonist drugs such as 8-OH-DPAT (Fig. 12). During the past several years, a good deal of evidence has been accumulated to indicate that the nature of this feedback is a short loop, or local, rather than a long loop, involving the response of postsynaptic target neurons. Increased levels of synaptic 5-HT appear to inhibit serotonergic neurons by a direct action at somatodendritic 5-HT receptors (the 5-HT,* autoreceptors). The following evidence indicates that this is a result of 5-HT being released from axons or dendrites of neighboring serotonergic neurons or via axon collateral feedback. First, studies employing iontophoresis show that serotonergic neurons are directly responsive to the application of 5-HT. For example, the ejection of 5-HT onto serotonergic neurons in the DRN strongly depresses their rate of firing (8). Second, drugs that release the endogenous stores of 5-HT, or block its reuptake, depress serotonergic unit activity in the DRN independent of any feedback from the forebrain, since the effectiveness of these compounds was undiminished in animals whose neuraxes were transected immediately rostra1 to the nucleus (347). When these neurophysiological findings were coupled with anatomic evidence that serotonergic neurons make connections with serotoner- EFRAIN January 19% STRUCTURE AND FUNCTION 5. Aflerent control Physiological analysis of the control of serotonergic neurons by afferent inputs follows from anatomic studies. There is evidence for afferents from GABAergic interneurons in the area of the DRN (64,192), from catecholaminergic neurons (52,105,173,252), from neurons utilizing excitatory amino acids as their neurotransmitter (250), and from histaminergic neurons (462). A large number of other afferent inputs to raphe nuclei have been demonstrated through the use of retrograde tracing techniques and were discussed in section IIIC. These additional inputs are not discussed here because their lack of neurochemical identity precludes their manipulation by pharmacological means. Furthermore, in many cases, neurochemically identified afferents have simply not been studied electrophysiologically. The studies on afferent control have focused primarily on the issue of whether the input increases or decreases the activity of serotonergic neurons and the strength and/or temporal characteristics of the effects. In some cases, the ionic bases of these effects have begun to be investigated; however, this is not described in detail here because it is not of primary importance to our central themes (see Refs. 10, 516). Stimulation of the pontine reticular formation has been found to produce a short-latency suppression of serotonergic unit activity in the DRN (497). The effects were blocked by systemic administration of the GABA antagonist picrotoxin but not by the glycine antagonist strychnine. There was some specificity to this action, since stimulation of the neighboring locus coeruleus did not produce these suppressive effects (20,497). The lateral habenula constitutes the densest input to the DRN, thus providing a pathway for extrapyramidal and limbit systems to exert control over the brain stem serotonergic system. This led to the examination of the effects of electrical stimulation of this region in anesthetized rats (455, 495). Once again a strong suppressive effect (latency of 15 ms and duration of 50-400 ms) on serotonergic neurons was seen. This effect was also blocked by picrotoxin but not by strychnine (495). On the basis of these data, the authors hypothesized that the habenu- BRAIN SEROTONIN SYSTEM 197 loraphe pathway was GABAergic in nature. Other evidence, however, indicates that the GABAergic cells involved in this response are not long-axoned neurons located in the habenula but are small interneurons found in the area of the DRN (64, 192). Habenula lesions increase choline acetyltransferase activity in the DRN, thus suggesting that the habenuloraphe pathway must be cholinergic (180). Intracellular recording of DRN neurons in anesthetized rats indicates that stimulation of the habenula produces inhibitory postsynaptic potentials mediated by an increased chloride conductance (375). Somewhat complicating the issue, these latter results also indicate that these effects are both mono- and polysynaptically mediated. It has been proposed, on the basis of anatomic and neurochemical studies, that the lateral habenula also exerts a direct excitatory influence on the DRN, which is mediated by an excitatory amino acid (250, 254). These results regarding GABA and excitatory amino acids are consistent with recent intracellular analyses of rat DRN neurons studied in vitro. Electrical stimulation just below the cerebral aqueduct evoked a short-latency (0.5-5.0 ms) and longduration (20-200 ms) GABA-mediated inhibition and excitatory amino acid-mediated excitation (370). The issue of neurochemical identity of afferent inputs to serotonergic neurons in the DRN has also been addressed by means of studies employing microiontophoresis. Application of either GABA or glycine suppressed the activity of serotonergic neurons, and these effects were blocked by their respective antagonists, picrotoxin and strychnine (179). More recently, iontophoretie application of histamine has been found to exert a depressant effect on serotonergic unit activity in the DRN (275). Iontophoretic application of norepinephrine had no effect on serotonergic unit activity (178, 457); however, the application of noradrenergic antagonists strongly suppressed the activity of these neurons, an effect that could be reversed by the iontophoretic application of norepinephrine (51). These data imply that noradrenergic neurons provide a tonic excitatory input to serotonergic neurons in the DRN. [Voltage-clamp studies indicate that this is mediated by a suppression of two potassium currents, a resting conductance and a voltage-dependent transient conductance (5, lo).] Because systemic or iontophoretic application of a variety of cyadrenoceptor antagonist drugs (also reserpine and low doses of clonidine) can completely suppress the activity of serotonergic neurons in the DRN of chloral hydrateanesthetized rats, it was proposed that the activity of serotonergic neurons is dependent on a continued adrenergic input (51,53,178,457). Similar experiments have also been conducted in freely moving cats (214). When the change in behavioral state produced by systemic administration of adrenergic drugs was taken into account, however, little or no net change in activity of serotonergic neurons was seen. On the basis of these latter results, it appears that an excitatory adrenergic input to the DRN influences the activity of serotonergic neurons under physiological conditions, but their con- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 They exhibit synchronization of activity, inhibition, or mutual inhibition of each other. These data are consistent with the aforementioned concept that serotonergic neurons influence the activity of other serotonergic neurons. The fact that neighboring neurons rarely fire simultaneously or on a one-to-one basis (496) indicates that they are not electrotonically coupled (there is also no ultrastructural evidence to support this type of interaction). The release of 5-HT is also believed to be modulated by local feedback inhibition operating at the axon terminal (for review see Refs. 100,449). However, there is no evidence to indicate that this action at an axon terminal autoreceptor (5-HT& (145,224) is not related to neuronal activity or release in a linear manner. OF 198 BARRY L. JACOBS AND 6. Environmental/physiological manipulations Serotonergic unit activity in anesthetized animals (primarily rats) has been studied in conjunction with the manipulation of several different physiological systems (e.g., body temperature and blood pressure), several pituitary-adrenal hormones (e.g., ACTH and corticosterone), and with stimuli from several sensory modalities (e.g., light flash and electrical shocks to peripheral nerves). In general, the studies have revealed that these manipulations produce either no effect on serotonergic unit activity or the results of several studies on the same issue have been contradictory. Experiments utilizing a variety of physiological and pharmacological approaches have implicated brain 5-HT in the regulation of body temperature (351). Therefore it is not surprising that a number of studies have explored the relationship between elevation of body temperature and raphe unit activity. The first experiment to do so reported that the single-unit activity of midbrain raphe neurons in chloral hydrate-anesthetized rats was significantly increased in response to elevated body temperature induced by radiant heat (500). However, a subsequent study, utilizing the identical procedure, failed to find any evidence for such a relationship, despite elevations of body temperature as great as 4OC (468). This latter result is consistent with another study that failed to find an increase in DRN unit activity desnite a 12°C increase in bode temperature (88). Addi- C. AZMITIA Volume 72 tional studies on this issue have been inconsistent. Heat-sensitive neurons were found in the MRN; however, no attempt was made to identify these neurons as serotonergic (239). Indeed, in all likelihood, they were nonserotonergic cells since they fired at high rates (~6 spikes/s) and were not regular in their discharge pattern. Another study in urethan-anesthetized rats reported that slow and regular firing neurons in the DRN, MRN, NRM, and nucleus raphe pontis were unaffected by changes in body temperature (134). However, some cells, especially in the NRM were found to be responsive to increases or decreases in skin temperature independent of changes in core temperature. Finally, slow and regular firing midbrain raphe neurons in chloral hydrate-anesthetized cats were reported to increase their activity in response to locally induced elevation of brain temperature (112). Although not examined extensively, two studies have reported a lack of relationship between DRN unit activity and variations in blood pressure in anesthetized rats. Intravenous administration of l-norepinephrine had no effect on unit activity, despite an increase in blood pressure (154). The other study reported that no relationship existed between spontaneous discharge rate of DRN neurons and basal fluctuations in blood pressure (88). A large literature exists relating 5-HT to pituitaryadrenal function. Two studies have investigated the effect of pituitary-adrenal hormones on the unit activity of serotonergic neurons in the mesencephalon of chloral hydrate-anesthetized rats. Intravenous administration of hydrocortisone was reported to produce a decrease in the activity of these neurons (153). In an attempt to follow up these results, a similar study reported no effect on serotonergic unit activity of various intravenous doses of corticosterone, hydrocortisone, and ACTH (345). No ready explanation is available to explain these differences. Finally, the activity of midbrain raphe neurons in anesthetized rats has been examined in response to sensory afferent input. Repetitive presentation of light flashes had no effect on neuronal activity (344), whereas direct stimulation of a peripheral nerve did elicit activation of DRN serotonergic neurons (14). 7. Summary Most salient is the fact that serotonergic neurons have endogenous biological mechanisms for generating their slow and highly rhythmic activity. This characteristic pattern of neuronal activity is seen not only in the adult but is manifested early in development. This basic activity is then available for modification by the variety of afferent inputs received by serotonergic neurons. B. Unit Activity in Behaving Animals I. Introduction Because of the general lack of response of serotonergic neurons in anesthetized animals to a varietv of Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 tinued activity does not seem to be dependent on this tonic input. To summarize, 5-HT, GABA, and histamine exert depressive effects on serotonergic neurons. Evidence also indicates that glycine may be an inhibitory neurotransmitter on these neurons. Norepinephrine is the transmitter best known to exert an excitatory influence on serotonergic neurons; however, a glutaminergic input has been described, and this is also likely to exert an excitatory effect. The noradrenergic input seems to be a tonic one operating at a near maximal level, since the iontophoretic application of norepinephrine on spontaneously active cells has little further effect. These data are of obvious relevance to pharmacological studies, since it can reasonably be assumed that drugs known to affect serotonergic, GABAergic, histaminergic, glutaminergic, and noradrenergic neurotransmission will also engage or disengage the activity of brain serotonergic neurons. These results set the stage for a set of related questions, for example, when is the powerful influence of the habenula exerted, under what conditions is the GABAergic inhibitory influence exerted or lifted, and when is the excitatory noradrenergic input engaged or disengaged? As described in section VI& we know that serotonergic neurons in behaving animals can be inhibited (e.g., during sleep) or excited (e.g., in response to phasic sensory input), but studies are just beginning to explore the CNS sites from which these influences originate and the neurochemical systems that mediate them. EFRAIN January 1992 STRUCTURE AND FUNCTION 2. Neurochemical identi$cation Because single-unit studies of serotonergic neurons in behaving animals examine extracellularly recorded action potentials, the identification of neurons as “serotonergic” is necessarily indirect. The criteria used in this identification are derived largely from studies carried out in anesthetized rats. As discussed in section VIA& a population of neurons found within the midbrain raphe nuclei consistently displayed the following characteristics: discharging in a slow (-1-Z spikes/s) and highly regular manner, having biphasic action potentials of long duration (>2 ms), and having activity that is selectively and completely suppressed by low doses of serotonergic agonist drugs (6,10,127). Neurons displaying similar characteristics have been observed in all the brain areas where serotonergic neurons are localized and are not found in regions devoid of serotonergic neurons. These initial results were strengthened by subsequent studies providing direct evidence for the serotonergic identity of these neurons. With the use of a double-labeling technique, experiments were c.arried out in which the slow and regular firing DRN cells were injected intracellularly with fluorescent dye, then localized, and subsequently examined for the presence of 5HT by histochemical fluorescence (11). All cells that displayed this characteristic activity were found to contain 5-HT. Additional indirect evidence in support of the serotonergic identity of these neurons has been reviewed in detail (238). Studies in behaving animals capitalize on this foregoing body of evidence. Serotonergic neurons are initially identified on-line by their slow and regular activity and their characteristic action potential waveform BRAIN 9 SEROTONIN 199 SYSTEM C 200 > =I 0 v) 1TE B 200 0.5 400 600 set INTER-SPIKE INTERVAL (met) FIG. 13. A: oscilloscope trace of extracellular action potential recorded from serotonergic NRM neuron recorded in behaving cat (positive is down). B: oscilloscope trace of slow and regular discharge of serotonergic NRM neuron. C: interspike interval histogram generated from activity of same serotonergic NRM neuron during quiet waking. Histogram was constructed from 1,024 intervals using bin width of 10 ms. [From Fornal et al. (155).] and duration (Fig. 13). Further confirmation of the serotonergic identity of these neurons derives from evidence that neuronal activity is completely suppressed by systemic administration of 5-HT somatodendritic autoreceptor agonists, such as 5-methoxy-N,N-dimethyltryptamine or GOH-DPAT. After completion of an experiment, the localization of presumed serotonergic neurons is examined histologically to determine whether they were in areas known to contain dense concentrations of serotonergic neurons. 3. Basic characteristics I) SLEEP-WAKE-AROUSAL CYCLE. The first study to examine the activity of serotonergic neurons in behaving animals was carried out in the DRN of cats (328). During quiet waking behavior these cells displayed the same slow (ml-3 spikes/s) and highly regular activity found in earlier studies of the DRN in anesthetized rats or in tissue slices (328, 469). This stable pattern of slow and highly regular activity during quiet waking progressively changes across the sleep-wake-arousal cycle (469). As the animals become drowsy and then enter slow-wave sleep, neuronal activity displays a corresponding decrease in rate and loss of regularity. This culminates during rapid-eyemovement (REM) sleep, where a complete cessation of DRN neuronal activity is seen (Fig. 14). Because it is well known that a primary feature of REM sleep is centrally induced atonia, this decreased neuronal activity might, in some way, be related to this loss of muscle tone. This issue has been examined from several perspectives, and the results provide general support for the existence of a relationship between tonic level of motor activity and DRN serotonergic unit activity in Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 manipulations, and/or the equivocal nature of these findings, investigators began studying the activity of serotonergic neurons in unanesthetized freely moving animals. To date, such studies have been confined exclusively to one species, domestic cats. In cats, the vast majority of serotonergic neurons are found in four main clusters that correspond, roughly, to the midline DRN, MRN, NRM, and NRO/NRPa (236,511). Single-unit activity has been examined, at least to some extent, in all four of these groups. In addition, however, ~25% of brain serotonergic neurons are located at some distance from these midline groups (236, 511). These “pararaphe” serotonergic neurons have not been studied in terms of their single-unit activity. The preponderance of these single-unit studies have examined the activity of serotonergic neurons in the DRN. Therefore most of our discussion focuses on this group. However, some attention has also been devoted to the NRM because of its involvement in nociception and analgesia. Finally, because the MRN and NRO/ NRPa groups have been the subject of very few experiments, these data are discussed primarily for the purpose of comparison and contrast with the DRN and NRM. OF Volume X? EOG EMG UNIT 111 ‘1I iI’ I -11 REM SLEEP r I I SLEEP I dim EEG 11 5 SEC FIG. 14. Polygraph traces displaying single-unit activity of DRN serotonergic neuron recorded across sleep-wake-arousal cycle in cat. Note how slow and regular activity is maintained during active and quiet waking but then falls off during slow-wave sleep. Cell became totally silent during rapid-eye-movement (REM) sleep. EEG, electroencephalogram; EOG, electrooculogram; EMG, electromyogram. behaving cats. Destruction of a discrete area of the dorsomedial pontine reticular formation in cats produces REM sleep without atonia (209, 248). An animal with such a lesion has epochs that, by all criteria, appear to be REM sleep, except that antigravity muscle tonus is present and the animals are therefore capable of moving and even engaging in complex motor acts, including locomotion. When the activity of DRN serotonergic neurons was examined during REM sleep in these animals, it was found to be increased well above that of normal animals in REM sleep (471). In fact, the level of neuronal activity was directly related to the degree of restored motor activity during REM sleep. During other phases of the sleep-wake-arousal cycle in these animals, the activity of DRN neurons was similar to that observed in normal animals. A second series of experiments examined this issue in a somewhat reciprocal manner. When cholinomimetic agents such as carbachol are injected directly into the dorsomedial pontine reticular formation, they can produce atonia in an otherwise awake cat. Dorsal raphe nucleus serotonergic unit activity during this centrally triggered atonia was found to be completely suppressed (454). Carbachol injections into nearby areas, which failed to induce atonia, had no effect on DRN unit activity. Several additional experiments in this series led to the conclusion that the critical variable was centrally induced atonia rather than muscle paralysis per se. Systemic administration of dantrolene, a peripherally act- ing muscle relaxant, or succinylcholine, an antagonist at the neuromuscular junction, produced no change in DRN unit activity despite a profound loss of muscle tonus (the animals given succinylcholine had to be artificially respirated). However, systemic administration of mephenesin, a centrally acting muscle relaxant, once again produced a large decrease in DRN unit activity. Several points should be made regarding these data. First, it is clear that this relationship is not one where a decrease in serotonergic neuronal activity causes atonia. When serotonergic neurotransmission is blocked by any of a variety of pharmacological means (inhibition of 5-HT synthesis, blockade of 5-HT receptors, or central application of a serotonergic neurotoxin), muscle paralysis or atonia does not ensue. Similarly, the decreased neuronal activity cannot be attributed to diminished proprioceptive input since succinylcholine produces muscle flaccidity but no change in DRN unit activity. Thus it appears that some CNS group(s) of neurons that produces atonia also causes these serotonergic neurons to decrease their activity. Second, if a functional relation does exist between central motor systems and serotonergic unit activity, it is not a simple linear relation, since small changes in muscle tone are frequently unaccompanied by changes in serotonergic unit activity (316). Thus the relationship appears to be at a more gross level, perhaps one reflective of the general behavioral state of the organism. Although serotonergic neurons in the other raphe groups Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 SLOW-WAVE January 19% STRUCTURE AND FUNCTION OF BRAIN SEROTONIN UNIT FIG. 15. Polygraph record showing activity of mesencephalic 5-HT neuron during transition from REM sleep to wakefulness. Note resumption of neuronal activity before arousal from REM sleep [as indicated by return of neck electromyogram (EMG) activity] and high level of this activity. Pontogeniculate occipital waves can be seen in latera1 geniculate (LGN) trace. EEG, electroencephalogram; EOG, electrooculogram. EEG 201 SYSTEM lrn , ,I .‘- I’ . ’ ‘IW 10 set have not been studied under these conditions, the fact that they are all REM-off leads to the prediction that they would also be atonia-off. A final feature of DRN neuronal activity during sleep is seen in a resumption of the activity of these neurons signaling the end of a REM sleep epoch. Over 50% of DRN cells examined displayed a dramatic increase in neuronal activity as much as 10 s before the end of a REM period (Fig. 15), as defined by behavioral and/or polygraphic criteria (the mean lead time was 3.2 s). Most interesting was the fact that the level of this resumed activity was approximately twice that observed during a quiet waking state and was approximately equivalent to that seen during phasic arousal. This high level of neuronal activity in anticipation of awakening may serve to prepare the various CNS target systems of the organism for responding to external stimuli. Reciprocal to the decrease in activity that DRN cells display during sleep, an increase in activity is seen as the animal becomes behaviorally activated (469). Increases above the level of neuronal activity during quiet waking are seen during an alert or active waking state (-20%) and during an aroused state (-40%). In response to an arousing stimulus, such as a click that elicits an orienting response, DRN neuronal activity displays a phasic activation (l-2 s in duration) to a level approximately twice that of the quiet waking level. As the ability of a stimulus to elicit an orienting response wanes with repeated presentations, so does the concomitant phasic activation of DRN cells. In summary, a dramatic, almost linear, positive relationship exists between the level of tonic motor activity/behavioral activation of the organism that is seen spontaneously, across the sleep-wake-arousal cycle, and the discharge rate of DRN serotonergic neurons. These results have been confirmed by several laboratories (93, 316,437). In section VI, B5 and B6, we review how these neurons respond to more extreme changes in behavioral state, such as those induced by painful stimuli or a varlety of stressors and during specific complex behaviors. When the same type of analysis was extended to serotonergic neurons in the MRN, NRM, and NRO/ NRPa of behaving cats, a generally similar pattern of state-related neuronal activity was observed (93, 155, 211,401, 418; Fig. 16). However, there were several important differences, especially between the superior group of serotonergic neurons (DRN and MRN) and the inferior group of serotonergic neurons (NRM and NRO/ NRPa). For example, medullary serotonergic neurons generally display a higher (30-60%) spontaneous firing rate during comparable behavioral states. In addition, mesencephalic serotonergic neuronal activity exhibits a strong inverse relationship to the occurrence of sleep spindles and pontogeniculate-occipital cortex (PGO) waves, two events associated with slow-wave sleep and REM sleep, respectively.2 In contrast, the activity of medullary serotonergic neurons is unrelated to these events. The activity of NRO/NRPa neurons, as well as a subset of MRN neurons, is not as strongly related to behavioral state as DRN, NRM, and the majority of MRN cells. Thus they did not display as great a decline in activity across the sleep-wake cycle. Their activity, although significantly reduced during REM sleep, was not completely suppressed. They were generally unresponsive to arousing stimuli, whereas DRN, NRM, and the majority of MRN cells were often strongly activated by such stimuli. A subgroup of DRN neurons whose activity often deviates from the highly regular discharge pattern that 2 This is consistent with neuropharmacological evidence that PGO waves are held under inhibitory serotonergic control (413). In addition, these data are consistent with the concept that decreased serotonergic (and noradrenergic) tone, in conjunction with increased brain stem cholinergic activity, influence thalamocortical mechanisms responsible for important physiological and behavioral aspects of sleep (454a). Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 EOG 202 BARRY 6 L. JACOBS AND EFRAIN C. AZMITIA DRN 3 6 4 i::iii~i~~-~~~iiiiii~. QW DRO SWS, SWS2 SWS3 PREREM REM AW QW DRO SWS, SW!39 SWS2 SWS3 PREREM REM PREREM REM NRPa QW DRO generally characterizes serotonergic neurons has been described. Accordingly, these cells have been described as “nonclocklike” (353,437). As with other serotonergic neurons, the activity of these cells changes dramatically across the sleep-wake cycle. They become silent during REM sleep; however, they often display little change in activity in going from quiet waking to slow-wave sleep. Additionally, as with other serotonergic neurons, their activity is strongly suppressed by the systemic administration of 5-HT,* agonist drugs. They do not appear to be localized to any particular portions of the DRN but instead are scattered throughout the nucleus (437). As described in section VIB~, many neurons in this subgroup have a specific relationship to particular behaviors in cats (406). Finally, an attempt to understand the neurochemical mechanisms l mediating the suppression of DRN neuronal activity during sleep has recently been initiated. With the use of single-unit recordings in conjunction with multibarrel microiontophoresis in the conscious but head-restrained cat, preliminary evidence suggests that a GABA-mediated inhibition may be responsible (289a). When the GABA antagonist bicuculline was applied onto DRN serotonergic neurons during sleep, in some cases their activity was restored to waking levels. However, when bicuculline was applied during waking, there was no change in neuronal activity, suggesting that this GABAergic influence was selectively activated during sleep. II)CIRCADIANVARIATION. A basic feature of many biological systems is variation in a circadian manner. Because brain 5-HT metabolism is strongly influenced by the light-dark cycle (for review see Ref. 470) (true circadian rhythmicity has not been examined directly), SWS, SWS2 SWS3 PREREM FIG.16.Meandischargeratesof serotonergic neurons in 2 mesencephalic [dorsalis (DRN) and medianus (MRN)] and 2 medullary [magnus (NRM) and pallidus (NRPa)] raphe nuclei across sleep-waking continuum in cat. Each bar represents mean * SE; n = 18-47 cells/nucleus. AW, active waking; QW, quiet waking; DRO, drowsy; SWS,, SWS,, SWS,, beginning, middle, and end of slow-wave sleep epoch, respectively; PRE-REM, minute before REM onset. [From Fornal and Jacobs (156).] REM it is of interest to ask whether the activity of serotonergic neurons varies as a function of environmental lighting. If behavioral state is held constant, i.e., when the activity of a given neuron is studied repetitively during REM sleep or quiet waking at various times across a 12:12-h light-dark cycle, the activity of DRN serotonergic neurons is invariant (470). Thus the activity of these cells is not influenced by the light-dark cycle nor, by inference, the circadian cycle. How then can we account for the numerous reports that 5-HT metabolism does vary as a function of environmental lighting? There are two obvious, non-mutually exclusive, possibilities. First, because neurotransmitter metabolism occurs predominantly at the axon terminal, local conditions at these sites -may modulate metabo llism somewhat independently of constant neuronal activ ity at the cell body. Such -modulatory influences are known to exist (1,W Second, the variation in 5-HT metabol ism across the light-dark cycle may simply be secondary to the wellknown distribution of sleep and waking across this cycle. For example, rats are known to spend most of their time in the dark, awake and active. During this time brain serotonergic neuron s would also be active and metabolism would be increased. In contrast, rats in the light spend most of their time sleeping. During this period the activity of serotonergic neurons would be decreased, as would metabolism. In all likelihood, a combination of these two factors accounts for the observed variation in brain 5-HT metabolism across the lightdark cycle. III)SENSORY STIMULATION. As discussed in section VIkt6, ‘in anesthetized animals serotonergic neurons show no response to ph asic environmental stim .uli, such as light flashes. In contrast, in conscious animals, repeti1. Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 SWS, SWS3 - NRM liw6 c35 DRO 72 MRN 5 it 3; z Volume January 199.2 STRUCTURE AND FUNCTION OF SEROTONIN SYSTEM 203 Finally, confirming the speculation that the response of DRN neurons in conscious animals might be blunted by anesthesia, it has been shown experimentally that chloral hydrate anesthesia completely blocks the response of these neurons to either clicks or flashes (213). An important aspect of this study was the fact that the responses of the same neuron were examined under identical conditions while the animal was initially unanesthetized and then anesthetized. 4. Physiological manipulations I) INTRODUCTION. The next two sections review the data on the response of serotonergic neurons to a variety of physiological manipulations and behavioral/environmental stressors. Many of these physiological manipulations can also be considered to be stressors, as indicated by their ability to produce increased plasma catecholamine levels and increased tonic heart rate. There are two reasons for carrying out these studies. First, it enables us to explore, at the single cell level, issues in which 5-HT previously has been implicated, e.g., thermoregulation or nociception. Second, the possibility of noting a change in serotonergic neuronal activity would be enhanced by exposing the animal to strong stimuli from a variety of domains. Thus the neuronal responses to these strong stimuli might provide clues to other conditions that could influence serotonergic activity in a more subtle manner. II) THERMOREGULATORY CHALLENGES. Historically, brain 5-HT has been most closely tied to the central mechanisms underlying thermoregulation (351). The primary focus of this research has been on the anterior hypothalamus/preoptic area, which receives its serotonergic input from neurons lying within the mesencephalic groups of serotonergic neurons (47,82,476). Hypothalamic 5-HT has been implicated in both heat loss responses (those occurring in reaction to a heated environment, e.g., panting and vasodilation) and heat gain responses (such as those occurring during the febrile response of disease states, e.g., shivering and vasoconstriction). A comparison of these two situations is of interest because they both involve increased body temperature but are produced as opposite thermoregulatory mechanisms are engaged. The previous research in this area, employing primarily pharmacological approaches, does not provide a clear-cut consistent picture (for review see Refs. 156, 157). This may be attributable, in part, to the nonphysiological nature of such experiments. Until recently, single-cell studies of serotonergic neurons and thermoregulation were carried out primarily in anesthetized animals, and the results were contradictory or, at best, equivocal. Experiments conducted in behaving animals have examined the response of DRN neurons to both increased ambient temperature and pyrogen-induced fever (157). For environmental heating, the temperature in the experimental chamber was maintained at 43 t Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 tive presentation (once every 2 s) of clicks or flashes produces an excitation of DRN neurons that is followed by a period of inhibition (215,402). The excitation has a latency of ~40-50 ms and a duration of ~60-80 ms. The inhibitory period that follows the excitation has a mean duration of -250 ms and closely corresponds to the interspike interval that normally follows the discharge of these highly regular firing neurons. For this reason, it has been proposed that this does not constitute a true excitation of these neurons but simply a resetting of their discharge pattern. Thus these phasic sensory inputs may cause the entire group of DRN serotonergic neurons to fire and then pause in unison. A similar type of response to sensory stimuli was observed in most MRN serotonergic neurons (401). Interestingly, a subgroup of MRN serotonergic neurons displayed the opposite response, i.e., an inhibition followed by a modest excitation. In contrast to DRN and MRN cells, the activity of caudal medullary NRO/NRPa cells was either unaffected or only weakly responsive to either clicks or flashes (211). Finally, the response of rostra1 medullary NRM cells was intermediate between the strong excitation of DRN and MRN cells and the weak excitatory response of NRO/NRPa cells (155). Thus a gross caudalto-rostra1 brain stem gradient of increasing excitation of serotonergic neurons by sensory stimuli appears to exist. This may be related to the predominantly ascending projections of the rostra1 groups and their resulting influence on forebrain target sites, which may be more closely involved in processing exteroceptive sensory inputs. The fact that the response of DRN serotonergic neurons to sensory stimuli does not habituate, even over the course of hundreds of repetitions, may be important (215, 402). It is well known that many nonserotonergic mesencephalic neurons also respond to simple sensory stimuli, but this response invariably displays habituation (65, 422, 433). Thus the maintained responsiveness of DRN serotonergic neurons to repeated sensory stimuli is rare among brain stem neurons. The possible functional significance of the maintained responsiveness of these neurons, in the face of a declining response of virtually all other brain stem neurons, is discussed in section VIIIA. Recently, an attempt to understand the neurochemical mechanisms mediating the sensory-induced excitation of DRN serotonergic neurons has been initiated. With the use of single-unit recordings in conjunction with multibarrel microiontophoresis in the conscious but head-restrained cat, preliminary evidence suggests that an excitatory amino acid mediated effect may be responsible (E. S. Levine and B. L. Jacobs, unpublished observations). When the excitatory amino acid antagonist kynurenine was applied onto DRN neurons during exposure to a series of clicks, the typically observed excitation was significantly diminished. However, this same treatment had no effect on the spontaneous activity of these cells, suggesting that this excitatory amino acid influence was at least somewhat selective to this sensorv-induced activation. BRAIN BARRY L. JACOBS AND EFRAIN : : 0’ 8’ : i : : 8’ 8’ ,’ a 0 ! 6 3 ’ 0 ! FIG. 17. Example of heating trial showing effects of acute heat stress on brain temperature, respiratory rate, and activity of single serotonergic DRN neuron in awake cat. Firing rate of this neuron was net significantly different from baseline at any time during heating trial, although respiratory rate was increased 2- to 12-fold, and brain temperature was elevated by as much as 13°C. [From Fornal et al. Wm.1 Volume 72 Body/brain temperature began to increase within 30 min, reached peak at I-2 h, and returned to predrug level by 6 h. The peak elevation of body temperature that was attained was typically 1.5~25°C. Once again, no change in DRN single-unit activity was observed during any phase of the pyrogen-induced febrile response when compared with the level of activity measured during a comparable control behavioral state. These data indicate that at the single-unit level of analysis, DRN serotonergic neurons are not involved in the mechanisms for either heat gain or heat loss. Additionally, they do not appear to be heat sensitive, i.e., responding to elevations in skin, body, or brain temperature. Overall, these results do not support a specific role for these neurons in thermoregulation. However, they may be involved in the well-known state-dependent changes in thermoregulation, since it is clear that serotonergic neuronal activity does vary dramatically across the sleep-wake-arousal cycle. Furthermore, these data do not rule out the possibility that the release of 5-HT in the hypothalamus may be controlled locally by circulating neurohormones, neurotransmitters, or by changes in brain temperature. Exploration of the possibility that, under certain conditions, there may be a dissociation between single-unit activity and axon terminal release of 5-HT is described in section VIB7. III) CARDIOVASCULAR CHALLENGES. Both the ascending serotonergic system, in its projections to cortex, limbic system, and hypothalamus, and the descending serotonergic system, in its projections to autonomic regulatory regions of the brain stem and spinal cord, have been implicated in cardiovascular control (24,272,517). As with any complex system, such as the cardiovascular, attempts to relate a central neurotransmitter to its control or modulation are difficult to do with any degree of precision or certainty. This is attributable both to the inherent nonspecificity in some experimental approaches, such as electrical stimulation of the brain or systemic drug treatment, and to the multiple sites of possible interaction between serotonergic neurons and the cardiovascular system. As discussed, previous studies examining DRN unit activity and blood pressure in anesthetized rats failed to find any relationship (88,154). l°C until each cat displayed continuous panting (respiratory rate --150/min). The activity of DRN neurons remained unaffected during the interval when ambient temperature was increased from 25 to 43°C (Fig. 17). During this initial phase of heating, no appreciable behavioral or physiological responses were seen. However, following prolonged heat exposure, cats displayed intense panting, relaxation of posture, and a progressive rise in body/brain temperature (range: 0.520°C). Once again, however, no change in DRN single-unit activity was observed in comparison to the level of activity measured during a comparable behavioral state in the absence of heat. In studies of DRN neuronal activity during the febrile response, the synthetic pyrogen muramyl dipeptide was administered svstemicallv (50 ue/ke iv) (157). The first study to examine the relationship between the cardiovascular system and DRN and NRM unit activity in behaving animals found that, under resting conditions, the discharge of these neurons was unrelated to the cardiac cycle (338). This finding becomes more meaningful when one considers that a neighboring group of neurochemically identified neurons, the noradrenergic cells of the locus coeruleus, did display a cardiac-related periodicity (338). These data indicate that these serotonergic neurons do not receive direct afferent input from the cardiovascular system, at least under resting conditions. The response of DRN neurons and a small group of NRM neurons has also been studied in relation to changes in blood pressure induced by peripherally acting drugs. A iueular catheter was used to administer Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 38.5 C. AZMITIA January 1992 STRUCTURE AND FUNCTION OF BRAIN SEROTONIN 205 V) DISCUSSION. These data indicate that, at least at the single-unit level of analysis, brain serotonergic neurons are not changed from their basal level of activity by manipulations of several important physiological regulatory systems. Several things militate against simply dismissing these data as trivial or uninteresting. First, this lack of neuronal change from that seen during a comparable baseline behavioral state occurs in the face of significant physiological responses of the organism to defend against these challenges (339,341). Second, these studies manipulated a number of different physiological regulatory systems. Finally, in a parallel series of studies, a neighboring group of neurochemically identifiable neurons, the noradrenergic cells of the locus coeruleus, were found to be highly responsive to the same thermoregulatory (339), cardiovascular (340), and glucoregulatory (341) challenges. The implications of the differential responsiveness of these two widely projecting brain stem neurochemical systems is discussed in section VIIIA. 5. Environmental stressors I) INTRODUCTION. Paralleling the experiments just described, the studies in this section explore, at the single-cell level, behavioral and environmental issues in which 5-HT has been historically implicated, e.g., aggression and nociception. These studies also utilized strong stimuli from several domains in the hope that this would optimize the possibility of finding a change. II) STRESSORS. This series of studies examined the responses of DRN serotonergic neurons to three environmental challenges from differing domains: noise, restraint, and exposure to a natural enemy. Exposure of various species, including humans, to loud noise for a sustained period of time is known to be stressful. When cats are exposed to loud white noise (100 dB) for 15 min, the stimulus elicits strong sympathetic activation, as indicated by a 30-50% increase in tonic heart rate and a lOO-150% increase in plasma norepinephrine. It also evokes a stereotyped behavioral response of crouching, with ears flattened (I, 514). Despite this strong organismic activation, the activity of a group of DRN neurons was not significantly different from that observed during an undisturbed active waking baseline (514). Because serotonergic neurons are unaffected by a stressor that impinges on the organism via its telereceptors (i.e., white noise), it was of interest to examine the effects of a proximal stressor, such as restraint. When cats are restrained for 15 min, a strong sympathetic activation is once again seen. Struggling and vocalizations during the restraint provide additional behavioral evidence for the stressful nature of the stimulus (1, 514). Despite this behavioral and physiological activation, the activity of a group of DRN neurons was not significantly different from that observed during an undisturbed active waking baseline (166). Confronting an animal with one of its natural ene- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 phenylephrine, to induce increases in blood pressure; sodium nitroprusside, to phasically decrease blood pressure; and hydralazine, to produce prolonged hypotension. The drugs were administered while the animals were in a quiet waking state. Over a range of ZO- to 70-mmHg increases and IO- to 50-mmHg decreases in mean arterial pressure, there was no change in the discharge rate of DRN or NRM serotonergic neurons (158, 158a). This is despite the fact that these blood pressure changes were of sufficient magnitude to produce significant reflexive changes in heart rate and plasma catecholamines. These data are another indication that serotonergic neurons are unresponsive to afferent input deriving from the cardiovascular system. This lack of change in activity of serotonergic neurons to manipulations of the cardiovascular system is also consistent with the results of a recent study of medullary serotonergic neurons in anesthetized cats (326). As discussed with regard to thermoregulation, these data do not preclude the possibility that changes in serotonergic neuronal activity across the sleep-wake cycle may be involved in the well-established state-dependent changes in the cardiovascular system (368,525). This conceptualization of the functional role of the serotonergic system is more fully developed in section VIII. It should also be noted that the serotonergic neurons in the ventrolateral medulla, an area known to be important in cardiovascular regulation, have not been studied under these conditions. IV)GLUCOREGULATORYCHALLENGE. Glucoserepresents the main source of energy for the brain. Therefore it is of interest to manipulate blood glucose levels in both directions and to examine the effects on DRN single-unit activity in behaving animals. This issue is of interest for two reasons. The central serotonergic system is thought to be a broad neuromodulatory system, and therefore its neuronal activity might be responsive to this fundamental biochemical variable. In addition, brain 5-HT has been implicated specifically in the control of food intake. The activity of serotonergic DRN neurons in behaving cats was not significantly altered by bolus injections of glucose (500 mg/kg iv) that elevated blood glucose levels three- to fivefold (158). Likewise, the activity of these neurons was not significantly affected by the administration of a dose of insulin (2-4 IU/kg iv) that lowered blood glucose by 250% or after the rapid reversal of this hypoglycemia by subsequent glucose administration. These results indicate that the activity of serotonergic DRN neurons is uninfluenced by alterations in blood glucose, nor is it sensitive to elevations of endogenous circulating insulin levels or to the administration of exogenous insulin. Furthermore, changes in the activity of these neurons do not appear to be a component of glucoregulatory mechanisms evoked by either hyper- or hypoglycemia. This is despite strong pituitary and adrenal activation by insulin-induced hypoglycemia and is therefore unexpected since brain 5-HT has often been implicated in the neuroendocrine response to stress. SYSTEM 206 BARRY L. JACOBS AND C. AZMITIA Volume 72 stimulation (Fig. 18, top). In contrast, some neighboring nonserotonergic neurons were dramatically activated when the stimulus reached a noxious level but showed a minimal response at nonnoxious levels (Fig. 18, bottom). The response of these latter neurons to noxious stimuli was blocked by a systemic injection of morphine (Fig. 18, bottom right). The response of NRM serotonergic neurons has also been studied during the application of a more prolonged painful stimulus. A subcutaneous injection of dilute Formalin solution produces a series of stereotyped behavioral responses in cats: initially it elicits licking, biting, and shaking of the injected paw; with time this abates and the paw is simply held aloft; then it is placed on the floor but no weight is applied; finally, after -30 min, use of the paw returns to normal. Throughout this 30-min episode the activity of NRM serotonergic neurons was no different from that seen during a comparison undisturbed active waking state (30). Because a critical link in opioid-induced analgesia may be the release of 5-HT in the spinal cord, the response of NRM neurons to systemic administration of morphine has been examined. Across a dose range of 0.5-4.0 mg/kg ip, the response of NRM neurons in behaving cats was unchanged from that observed during a control waking condition, despite clear-cut behavioral evidence of drug-induced analgesia (30). These results are consistent with those seen in similar studies of NRM neurons conducted in anesthetized rats (103). These results do not exclude the possibility that opioid drugs may exert an action directly on serotonergic nerve terminals in the spinal cord (69). However, it is also worth noting that several studies have questioned the involvement of descending serotonergic neurons in opioid-induced analgesia (242, 393, 394). To summarize, morphine-induced analgesia does not appear to be dependent on the activation of serotonergic NRM neurons and, at the single-unit level, serotonergic NRM neurons do not appear to be specifically involved in pain modulation, since they respond to noxious and nonnoxious stimuli to approximately the same degree. This suggests that they are not an essential component of the analgesic system. However, the activation of these neurons in response to arousing stimuli could be part of a more general sensory modulatory system operating during periods of increased motor activity and behavioral arousal. This is consistent with one of the central themes of this review, i.e., that serotonergic neurons exert modulatory control over a large variety of sensory, motor, and endocrine functions. Another possibility is that painful stimuli can directly increase the release of 5-HT at the nerve terminal, thus bypassing an action on the cell body, leaving single-unit activity unchanged. This issue is examined below in experiments described in section VIB7. 6. Behavior One major domain remains to be discussed with respect to its relationshiD to serotonergic neuronal activ- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 mies represents a powerful stimulus of particular biologic relevance. In this experiment, cats were exposed to a dog while physiological measures were taken and DRN single-unit activity was recorded. When a dog was brought into view of the cat for 5 min, it evoked the typical stereotyped feline defense reaction of arched back, facing broadside, piloerection, and often growling and hissing. Sympathetic activation, as indicated by an increase in tonic heart rate and an increase in plasma norepinephrine level, was once again seen. Despite this behavioral and physiological activation, the discharge rate of a group of DRN serotonergic neurons was unchanged from that observed during an undisturbed, active waking baseline condition (514). These negative results with DRN neurons are somewhat surprising in light of the literature implicating 5-HT in aggression and/or defense reactions in a variety of mammalian species. Additionally, as with physiological challenges, when the activity of locus coeruleus noradrenergic neurons was examined in response to these same behavioral/environmental stressors, large increases in activity were consistently observed (1, 289). III) NOCICEPTION/ANALGESIA. Much attention has been devoted to the role of central 5-HT in nociception and analgesia, especially because of the important social and clinical relevance of this subject. The bulk of the data in support of an important role for 5-HT in analgesia derive from anatomic, pharmacological, and brain stimulation studies (reviewed in Refs. 30,156,283). Very few studies have addressed this question at the singlecell level, and only one has done so in behaving animals. Most of the experimental attention regarding the role of 5-HT in nociception and analgesia has been focused on the medullary serotonergic neurons of the NRM. It has been proposed that painful stimuli activate brain stem opioid-containing neurons, which in turn activate spinal cord-projecting NRM serotonergic neurons, thus suppressing pain (56). If opioid- and nociception-induced analgesia are dependent on activation of these neurons, then both painful stimuli and opioid drugs should increase their activity. As the series of studies described in this section shows, however, the experimental data do not provide support for this hypothesis. If radiant heat is applied to an animal’s tail, the animal will move its tail to escape this stimulus as it becomes painful. When the activity of cat NRM serotonergic neurons was studied during such a procedure, it was found that there was no consistent increase in discharge during the application of the heat (30). NRM neurons were equally activated by arousing but nonpainful stimuli, such as tapping on the experimental chamber or entering the experimental room. In an attempt to provide a quantification of the response of these neurons to painful stimuli, the inferior alveolar (dental) nerve was electrically stimulated while the neuronal response was analyzed (30). The data indicated that NRM serotonergic neurons show no greater response to high-intensity noxious stimulation of the alveolar nerve than to low-intensitv nonnoxious EFRAIN January 1992 STRUCTURE AND FUNCTION OF BRAIN SEROTONERGIC 30 z z \ SYSTEM 207 NEURON 1 NOCIRESPONSIVE NEURON II 100 ( NON-NOXIOUS UA 400 1 ity. Do serotonergic neurons display changes in activity in association with any specific behaviors? Recently, a group of DRN and MRN 5-HT neurons have been found to undergo dramatic alterations in neuronal activity in relation to two types of behavior, oral-buccal movements and orienting responses. A substantial number (as many as 3050%) of serotonergic neurons in the DRN and MRN of cats are tonically activated (Z- to &fold) during repetitive motor activities involving the oral-buccal region: chewing/biting (Fig. 19, top and middle), lapping, and grooming the body surface with the tongue (406). During these periods of activation the discharge pattern of these neurons frequently becomes extremely regular (i.e., clocklike). Interestingly, these neurons do not appear to be activated during any other types of behavior; in fact, their activity typically decreases during many active behaviors (Fig. 19, bottom), such as locomotion and orientation. Individual neurons within this subpopulation may display characteristics not shared by the others within this group. Some may increase their activity in anticipation of initiating the behavior, for example, in response to seeing or smelling food. Others may increase their activity in phase with the licking or biting responses (most, however, appear to be tonically activated during these behaviors). Finally, many of these neurons can also be activated by somatosensory stimuli, especially those applied to the head and neck area (C. A. Fornal and B. L. Jacobs, unpublished observations). Many (perhaps all) of the aforementioned serotonergic neurons that increase their activity during oral- ( NON-NO lJA XIOUS ) 800 ( NOXIOUS A uA f 800 1 POS - 200 msec UA T MORP HINE buccal movements display the opposite response during orientation to an external stimulus (Fornal and Jacobs, unpublished observations). Phasic auditory or visual stimuli often elicit the typical feline orienting response, which is to foveate the source of the stimulus by moving the head and eyes toward it. This is usually accompanied by a suppression of all other overt movement. During this brief orientation, and for several seconds following it, the activity of some DRN and MRN neurons is suppressed, often completely. This may be related to the previously known fact that 5-HT neuronal activity is suppressed during PGO waves during deep slow-wave sleep and REM sleep (156). Morrison and colleagues (341b) have shown that PGO waves during sleep are a sign of internal alerting. Of more importance to the present discussion, they also demonstrated that PG 0 waves could be evoked by phasic stimuli during the waking state, where they were frequently associated with startle or orienting responses (external alerting). Completing this picture, the suppression of 5-HT unit activity during orientation is frequently accompanied by PGO waves. 7. In vivo release Up to this point, our discussion of functional analyses of serotonergic neurons has focused on studies of single-unit activity. A basic assumption inherent to the usefulness of single-unit activity is that such activity is tightly coupled to the axon terminal release of neuro- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 18. Computer-generated peristimulus time histograms of simultaneously recorded responses of serotonergic neuron of NRM (top) and neurochemitally unidentified nociresponsive neuron (bottom) to repetitive stimulation (constant current, monophasic pulses of 1.4ms duration once every 2 s) of alveolar nerve. Serotonergic neuron and nociresponsive neuron were recorded on adjacent leads of same microelectrode bundle in behaving cat. Current intensity of 100 PA was threshold for eliciting jaw-opening reflex, and 400 PA elicited a strong jaw-opening reflex without any behavioral signs of pain. Current intensity of 800 PA elicited signs of pain. Current intensity of 800 PA was repeated 30 min after administration of morphine (2 mg/kg ip), a dose that eliminated behavioral signs of pain. Occurrence of stimulation is marked by arrow. Each histogram was constructed from 64 trials using bin width of 10 ms. See text for discussion. [From Auerbach et al. (30).] FIG. SEROTONIN 208 BARRY QUIET L. JACOBS AND EFRAIN C. AZMITIA Volume 72 WAKING The most serious is the tissue damage that results from having fluid pushed into the brain with a high flow rate ti II, I I I I ,Ir\ and under positive pressure and then withdrawn under negative pressure. EM More recently, two other approaches to this problem have been developed, voltammetry and intracereEOO G ---+ bra1 microdialysis. In vivo voltammetry is based on the principle that electrooxidizable substances, such as 5HT, can be oxidized by applying a specific voltage between an electrode in the brain area of interest and FEEDING some neutral point (262, 447). The resulting current is assumed to be proportional to the concentration of the substances oxidizing at the electrode surface. At present, the major impediment to studying 5-HT with this approach is that both 5-HT and its major metabolite, SHIAA, are oxidized at the same potential. However, a number of laboratories are currently attempting to develop a treated electrode that would not oxidize the metabolite, thus leaving 5-HT to be measured. A recent ACTIVE WAKING study reports some success in this endeavor (111). In vivo brain dialysis can be likened to a push-pull IIIB,l\llii\l\Illlli\lllI\\\ !I II lllrn II !lIl!mmmI!!l III I \I III!\ Imu II e II lnrrirmrrrr! cannula approach conducted inside of a dialysis membrane (for review see Refs. 262,475,501). Thus the brain tissue is not directly exposed to moving fluids under differing pressure. Instead, substances released in brain diffuse across the dialysis membrane (typically with a mol wt cutoff of 5,000) at a rate proportional to their concentration gradient. They are then collected and an10 SEC alyzed by radioimmunoassay or sensitive high-pressure FIG. 19. Polygraph traces displaying single-unit activity of seroliquid chromatography coupled to a variety of detection tonergic neuron in DRN of cat during quiet waking, feeding, and actechniques, including electrochemical, coulometric, or tive waking. Note dramatic increase in activity during feeding and fluorimetric techniques. pauses that occur during active waking, often in association with eye movements. EMG, electromyogram; EOG, electrooculogram. To date, most studies employing in vivo dialysis have used pharmacological and basic biochemical manipulations to validate that the method does, in fact, transmitter. If that is untrue to any significant degree, measure neuronally released 5-HT that diffuses into the it undermines the value of single-unit activity as a re- extracellular space (31, 253, 501). More importantly, flection of the functioning of brain neurochemical sys- from the perspective of this review, a few studies have used this approach to examine release of 5-HT under tems. Theoretically, there are a number of different mech- physiological conditions. The data are consistent with anisms that could allow release to become uncoupled what has been found with single-unit analyses. Thus from neuronal activity: as neuronal activity increases, 5-HT release in the anterior hypothalamus of cats inrelease may be unable to follow in a linear fashion; di- creases from sleep to waking to arousal by ~50-100% rect axoaxonal connections may modulate release (pre- (513,513a). Perhaps more significantly, when cats were synaptic inhibition or facilitation); factors, such as pep- exposed to two different challenges (synthetic pyrogen, tides or small ions, circulating in the extracellular space which significantly increased body temperature, and inaround the axon terminal may influence release; and tense white noise) there was no change in 5-HT release mechanisms intrinsic to the axon terminal may vary in beyond that attributable to a change in behavioral state produced by the manipulation, i.e., the 50-100% ina manner unrelated to neuronal activity (e.g., circadian crease seen in going from sleep to waking to arousal variation in reuptake). One of the few ways to explore this issue is to exam- (513, 513a). These data directly parallel the single-unit ine release in vivo, especially under conditions similar to results from studies employing the same manipulations. Similar results have also been reported in studies using those in which single-unit activity has been studied. Historically, measurement of neurotransmitter release in in vivo dialysis in rats. From a basal level of hippocamvivo was approached through the use of a push-pull per- pal 5-HT release during sleep (defined by behavioral crifusion technique, where artificial CSF is pumped into a teria), there was a 36% increase during a drowsy state target site and then the extracellular fluid that it has and a 45% increase during an alert state (251). In another part of this same study, handling and tail pinch mixed with is pulled out. There are, however, significant drawbacks to this approach (for review see Ref. 262). both increased hippocampal 5-HT release by ~50% UNIT Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 January 1992 STRUCTURE AND FUNCTION from an unspecified baseline period in the light phase of the light-dark cycle. Thus studies in both cats and rats indicate that activating a sleeping or quiescent animal increases the release of 5-HT by -5O-100%) closely paralleling the electrophysiological data on serotonergic neurons. 8. Summary BRAIN SEROTONIN SYSTEM 209 nergic neurons are coactivated in association with rhythmic central pattern generator-mediated behaviors and by the afferent inputs that normally influence such behaviors. The motoneurons that would mediate at least some of these oral-buccal behaviors in mammals receive very dense 5-HT inputs (e.g., MoV and MoVII). It is also interesting to note that 5-HT plays an important role in feeding (287, 412) and in other rhythmic behaviors (for review see Ref. 376a) in several invertebrate species. Finally, a further elaboration of this hypothesis is that serotonergic neurons in more caudal groups (e.g., NRM or NRO/NRPa) might display activation in association with other central pattern generators, such as locomotion, scratching, or respiration, that are controlled by more caudal portions of the neuraxis. Because this increase in serotonergic neuronal activity has been seen exclusively in association with oralbuccal movements, it raises a number of interesting questions. Is this relation peculiar to cats; would rats or monkeys manifest a different type of movement-related activity? Is this an emergent property of more complex vertebrate species or a remnant of serotonergic neurons related to feeding in invertebrates (412). Will other movement-related subgroups of serotonergic neurons be found in the DRN and MRN? Will all such neuronal increases be related exclusively to central pattern generator-mediated responses? Finally, what are the efferent projections of these serotonergic neurons and from what sources do their afferents derive? VII. POSTSYNAPTIC ACTIONS A. Introduction The early literature on the postsynaptic effects of 5-HT in the CNS is frequently contradictory. Investigators initially were expecting to find a consistent and unitary action, i.e., that 5-HT would universally be excitatory (increasing the activity of postsynaptic target neurons) or inhibitory (decreasing target neuron activity). It is now clear that this was simplistic. A given neurotransmitter can exert multiple, sometimes opposing, actions, and this is determined by the characteristics of the receptor with which it interacts and the membrane and intracellular biochemical mechanisms coupled to the receptor. During the past 10 years it has become evident that there are multiple 5-HT receptors in the mammalian CNS (at least three families of receptors with six different membrane binding sites3 have now been reported for 5-HT). In this section we describe some of the more salient and well-established postsynaptic actions of 5-HT and the particular receptors 3 Establishing the existence of a true neurotransmitter receptor is much more conservative than the demonstration of a binding site. A receptor implies a demonstrable physiological function mediated by it, whereas a binding site merely implies attachment of the ligand to a membrane. Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 A large literature implicates brain serotonergic neurons in the control of physiological processes (such as cardiovascular function and thermoregulation) and the response to environmental conditions (such as noise, restraint, and pain). However, the experiments described here imply that at least the raphe groups of serotonergic neurons are not primarily involved in these activities, since no effect on serotonergic single-unit activity in behaving animals was seen independent of changes in behavioral arousal. This was true despite the fact that many of these studies employed stimuli chosen explicitly for the strength of their impact on the organism. The tonic activity of serotonergic neurons appears in general to vary in a stereotypical manner in association with motoric activity and behavioral state and not specifically in association with any process. The discharge of these neurons appears to reach a “ceiling” during active waking beyond which they cannot be activated, regardless of the impact of the evoking stimulus on the organism. Therefore the primary role of these neurons in physiology and behavior may be to coordinate the activity of target structures (or set the tone) in conjunction with the organism’s level of motor activity/ behavioral arousal. This is a major task for the nervous system because not only behavior but the output of all physiological systems in the organism is strongly modulated across the sleep-wake-arousal cycle (368, 525). Consistent with this view is the fact that serotonergic neurons discharge with an extraordinary tonic regularity, suggesting that little neuronal information is conveyed by this steady-state activity. Thus the message to target structures from serotonergic neurons may be an analog signal that indicates that the organism is in REM sleep (-0.0-0.3 spikes/s), in slow-wave sleep (-0.3-1.5 spikes/s), drowsy (- 1.5-2.0 spikes/s), in quiet waking (-2.0-3.0 spikes/s), active waking (-3.O5.0 spikes/s), or physically aroused (-4.0-7.0 spikes/s). In this schema, within a given behavioral state, the degree of influence that these neurons exert on their target sites is determined primarily by the density of their innervation of particular CNS regions and whether there is conjunctive neuronal activity in that target site. The necessity for coordinated neuronal activity is what transforms the serotonergic system from one exerting an indiscriminate or ubiquitous influence on the CNS to one that confers a specificity of action. Such specificity can also be imparted by the activation of at least some DRN and MRN neurons in association with particular complex behaviors. One hypothesis to account for these data is the DRN and MRN seroto- OF 210 BARRY L. JACOBS AND thought to mediate these effects (for review see Refs. 12, 78, 387, 479). The molecular biology of 5-HT receptors and the intracellular second-messenger systems and ion channels to which 5-HT receptors are coupled are beyond the scope of this paper (for review see Refs. 169a, 357a, 507). B. Inhibition/Modulation C. AZMITIA Volume 72 these data is that they are derived from several species. Unfortunately, little definitive information is available regarding the receptor subtype involved in these actions. However, a recent study indicates that the 5-HT, receptors may be more effective than the 5-HT, receptor in regulating nociception. When 5-HT was iontophoretitally applied to dorsal horn neurons in the spinal cord of anesthetized rats, 5-HTlB agonists mimicked the antinociceptive effects of 5-HT, while a 5-HT, agonist was without effect (5-HT,* agonists had nonspecific actions) (143). As described, 5-HT also exerts a strong inhibitory action on serotonergic neurons themselves (autoinhibition). This is the most studied and well-understood effect of 5-HT in the mammalian CNS and is attributable to an action at the 5-HT,* receptor (for review see Ref. 10). These receptors are found on the somatodendritic regions of serotonergic neurons (16), and the action of 5-HT here produces an increased conductance through the opening of potassium channels (9). Finally, we come to a postsynaptic action of 5-HT that cannot be described with unidimensional terms such as inhibition or excitation. If 5-HT is applied iontophoretically onto neurons in the somatosensory or visual cortex of anesthetized rats, it exerts little effect on the spontaneous activity of these cells (497a, 499). However, when 5-HT is applied in conjunction with afferentinduced excitation of these cells, it preferentially suppresses this activity. The same effect of 5-HT was observed in conjunction with afferent-induced inhibition of these neurons. (Unfortunately, no attempt was made in these studies to determine the 5-HT receptor subtype where these actions were mediated.) Recently, 5-HT has been shown to decrease glutamateand quisqualate-induced excitation of cerebellar neurons in tissue slices from young rats (215a). These actions have been variously referred to as a reduction in the signal-tonoise ratio, a disenabling effect, or synaptic modulation (76, 136, 519). A similar modulatory action of 5-HT in the brain stem is described in the next section. C. Excitation/Modulation The predominant postsynaptic action of 5-HT in the brain stem and spinal cord (other than the aforementioned inhibitory action associated with nociception) is direct excitation or potentiation of a heterosynaptic excitatory effect. These data derive primarily from studies employing extracellular recordings in conjunction with microiontophoresis of 5-HT or electrical stimulation of serotonergic neurons (54, 89, 183, 221, 223, 273a, 324, 508a, 509). Most of this work is directed at actions on motoneurons, e.g., in the rat facial nucleus or in the ventral horn of the spinal cord. In these studies, iontophoretic application of 5-HT alone produced little or no change in single-unit activity. However, when this is interacted with excitatory influences on motoneurons, produced either by direct anolication of excitatorv amino acids or bv electrical Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 The predominant postsynaptic action of 5-HT in the forebrain, especially in the neocortex, is inhibition (27, 78,1.21,170,201,245,246,257a, 366,403,409,420,427,428, 494). However, it is clear that excitation is also frequently seen (87, 244,245, 376,405,408). These data derive primarily from studies employing extracellular recordings in conjunction with either microiontophoresis of 5-HT or electrical stimulation of serotonergic neurons. In some cases, intracellular analyses have been carried out, and the inhibition is attributable to membrane hyperpolarization (23,428). There are several reasons why these data often seem confusing. First, they are frequently derived from studies employing different methodologies, e.g., in vivo versus in vitro preparations or iontophoretic drug application versus bath application versus electrical stimulation. Second, many of these studies were carried out before the introduction of antagonists specific to particular 5-HT receptor subtypes. These latter pharmacological tools now frequently permit the systematic parceling out of apparently complex groups of data. Finally, these studies may be comparing data gathered from different sets of neurons within a particular structure, e.g., specific regions of hippocampus or particular layers of cortex. Nonetheless, despite these limitations, a relatively consistent picture is beginning to emerge. At least some of the inhibitory effects of 5-HT seen in the dorsal hippocampus and the neocortex, the two areas most extensively examined, appear to be attributable to the activation of a 5-HT,* receptor. This conclusion is based on electrophysiological studies employing agonists and/or antagonists selective for particular 5-HT receptor subtypes (23,108,520). Whether this action accounts for all of the reports of inhibition by 5-HT in the forebrain is not known. The ionic basis or the hyperpolarization produced by 5-HT in the hippocampus appears to be an increased resting conductance for potassium ions that is not calcium dependent (23,108,428). When the postsynaptic effects of the 5-HT,* receptor are examined in combination with the activation of the 5-HT, receptor, their interaction is subtractive, i.e., a reduction in the degree of inhibition (87, 123, 274). Another site in the CNS where extensive evidence shows that 5-HT produces an inhibitory action is on somatosensory neurons in the spinal cord, especially dorsal horn cells involved in nociception (for review see Ref. 94). These data are based on both electrical stimulation of the cell bodies of descending serotonergic neurons (140, 185, 196, 249, 333, 407) and on the iontophoretic abnlication of 5-HT (63. 198. 247. 398). A strength of EFRAIN January 1992 STRUCTURE AND FUNCTION OF stimulation of the dorsal root or motor cortex, 5-HT produced a strong facilitation of neuronal activity (324, 509). These effects can also be characterized as enabling, increased signal-to-noise ratio, or modulation. Such an action of 5-HT would have the effect of greatly potentiating the responsiveness of target cells to incoming excitatory stimuli. Intracellular analyses indicate that this increased excitability is attributable to a depolarization, probably mediated by a decreased resting membrane conductance to potassium ions (480). Furthermore, pharmacological analyses indicate that an action at a 5-HT, receptor appears to mediate this effect BRAIN SEROTONIN SYSTEM 211 (508). In contrast, when applied in the absence of the excitatory amino acid effects, these three substances had little effect of their own. This implies that all three may exert a modulatory influence on motoneurons. Contrary to these results, in the dorsal horn, 5-HT and substance P appear to exert opposing synaptic effects, inhibition and excitation, respectively (122, 240). No data are available on the postsynaptic actions of the simultaneous administration of two or three of these substances. Neither are any data available regarding whether the substances are coreleased and, if so, under what conditions. (324, 510). D. Cotransmission As described in section III, 5-HT and other neurotransmitters are frequently found to coexist in axon terminals along with hormones or neuropeptides. This raises the interesting questions of whether and how cotransmission differs from more traditional, single neurotransmitter synaptic actions in the CNS. Medullary neurons that project to the ventral horn of rats have been shown to contain 5-HT and substance P, or 5-HT and TRH, or all three substance (243; for review see Ref. 508). On a gross physiological level, 5HT, substance P, and TRH appear to interact synergistically (for review see Ref. 466). Their effects are additive or even multiplicative in facilitating motor output. Unfortunately, only limited neurophysiological data are available regarding the direct postsynaptic actions of these substances. Nonetheless, they support the gross behavioral and physiological data. When applied iontophoretically onto motoneurons in the ventral horn of rats, all three substances augment or facilitate the increased motoneuron excitability produced by glutamate E. Latency and Duration of Action An important dimension of the postsynaptic actions of 5-HT is the temporal characteristics of its actions, i.e., latency and duration. This not only describes the window in which activation or inhibition of serotonergic neurons is likely to influence its target cells but may also bear on the nature of important physiological properties of serotonergic neurons, such as nonsynaptic release and receptor-coupled messenger systems. Phasic electrical stimulation of serotonergic cell bodies or axons is one of the only ways of addressing these issues. It is assumed that this causes an action potential(s) to invade the axon terminal to release physiological stores of the neurotransmitter. Because electrical stimuli are discrete short-lived events, their sequelae can be timed with precision. In contrast, several difficulties are inherent with employing iontophoretic application of 5-HT to answer these questions: inability of knowing whether the amount released is of the same order of magnitude as physiological release, the possibility that the amount released may swamp the ability of the presynaptic neuron for removal by a reuptake mechanism, lack of knowledge regarding the somatodendritic domain of the target cell where 5-HT is acting, and inability to precisely time events relative to the time of release. Almost the entire literature on latency and duration of the postsynaptic actions of 5-HT derives from studies employing anesthetized rats. Stimulation of the DRN or MRN consistently produces a relatively longduration (100-400 ms) inhibition of target neurons in the cerebral cortex, hippocampus, thalamus, septal area, and amygdala (27,244,257a, 420,427,494). A major discrepancy arises in the literature regarding latency of action. Several studies report that the inhibitory effects have a relatively short latency of lo-20 ms (27,244,494), whereas most of the other studies cited report a considerably longer latency of ~100 ms. The factor(s) responsible for this difference has not been determined, although differential activation of small- and large-caliber axons remains a possibility. Keeping in mind the aforementioned caveats regarding the iontophoretic application of neurotransmitter, it is interesting to note that effects on motoneurons lasting up to two orders of magnitude longer (i.e., tens or Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 An interesting corollary of these motoneuron results is the finding that 5-HT also exerts an excitatory action on neurons in the caudate/putamen of the extrapyramidal motor system. This conclusion is based on studies in anesthetized rats employing intracellular recordings in conjunction with electrical stimulation of the DRN (376,482). It is also important to place these motoric effects of 5-HT in a broader context. First, if animals are given any of a variety of drugs known to increase synaptic 5-HT, they display a constellation of active motor signs that has been called the “5-HT syndrome” (231). This is believed to be a direct reflection of 5-HT-induced motoneuron excitation. Also, recall the relation of serotonergic neuronal activity to oral-buccal activity and their general relationship to motor tone. The motor effects of 5-HT are, at least in part, mediated by its ability to move motoneurons from a stable hyperpolarized state, with little or no neuronal activity, to a stable depolarized “plateau” state, with tonic neuronal activity (22233, 223). All of this is consistent with our theme that a strong tie exists between serotonergic neuronal activity and various aspects of motor function. 21.2 BARRY L. JACOBS AND hundreds of seconds) than those found with electrical stimulation have been reported (324, 480, 508a). Whether such effects, which typically depend on continuous application of 5-HT for tens of seconds, are physiologically relevant (and involve engaging more complex membrane and/or intracellular mechanisms) is not known. F. Summary C. AZMITIA Volume 72 hibitory, or modulatory) and temporal characteristics of these actions are provided by the specific type of 5-HT receptor subtype affected and the membrane and intracellular biochemical mechanisms that are coupled to them. The long latency and long duration of these synaptic actions are compatible with the modulatory nature of the brain 5-HT system that is a focus of this review.4 It is also interesting to note that a similar conclusion can be reached regarding the temporal aspects of the synaptic actions of norepinephrine. The effect on target neurons of activating brain noradrenergic neurons is generally reported to be a long-latency, longduration inhibition of activity (75, 152). Thus monoamine neurotransmitters may be fundamentally different than faster acting excitatory and inhibitory amino acid neurotransmitters, which act almost exclusively by means of simple receptor-mediated ionic conductance changes (439). The implications of such an arrangement are discussed next. VIII. DISCUSSION AND SPECULATION Serotonergic neurons comprise the most expansive neurochemical network in the vertebrate CNS. These midline brain stem neurons innervate virtually all portions of the neuraxis. It is also a system that appears to be conserved across phylogeny. Developmentally, they are among the first neurons to appear, and the fact that they synthesize 5-HT before making contact with their target neurons is consistent with their serving important trophic functions. In turn, they also respond vigorously during development to a variety of growth regulatory factors. Similarly, when these neurons are damaged in the adult, they demonstrate a robust sprouting response that is also responsive to many of these factors. This vast anatomy is complemented by a basic physiological role for this system. Functionally, serotonergic neurons exert a gain control primarily through a modulatory action on behavioral and physiological processes by interacting with six or more 5-HT receptor subtypes. This influence on target neurons is expressed in direct association with the motor activity and behavioral state of the organism, i.e., highest during arousal and active waking, intermediate during quiet waking, and off during REM sleep. (Serotonergic neuronal activity is unperturbed by a variety of environmental and physiological stressors.) Finally, there is evidence from anatomic studies for an evolutionary trend toward greater specificity in the serotonergic system. Thus in higher mammals, serotonergic neurons are more likely to have myelinated 4 A recent paper provides a challenge to this generalization (129). In the peripheral nervous system of the guinea pig there is evidence that the excitatory effect of 5-HT at a 5-HT, receptor may be a “fast” one (short latency and short duration) mediated by a direct action at a ligand-gated cation channel. This remains to be demonstrated for the CNS. Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 It is becoming increasingly clear that neurotransmitters such as 5-HT exert multiple postsynaptic effects in the CNS and that these differential effects are often directly attributable to actions at specific receptor subtypes. An area of intense current interest is the identification of the heterogeneous membrane and intracellular biochemical mechanisms coupled to each receptor subtype, e.g., G proteins that either activate or inhibit formation of CAMP or that activate phospholipase C, those that act on a specific simple ion channel, and those that may influence a calcium-activated potassium conductance (10, 419). The issue regarding the duration of the postsynaptic effects of 5-HT will also certainly be shown to be dependent on which of these aforementioned mechanisms is activated. Recent evidence in rats also indicates that different receptor subtypes, 5-HT, and 5-HT,, may be related to different types of 5-HT axons, thin and thick, deriving from different nuclei, DRN and MRN, respectively (for review see Ref. 81; see sect. III@. It will be interesting to see whether such a relationship also holds for other species. The different caliber of axon, the specific receptor, and the type of release site, synaptic or nonsynaptic, are the major determinants of the latency of the postsynaptic action of 5-HT. One of the limiting factors in this research on 5-HT is the lack of cross-species data. For example, whereas most of the physiological studies of the postsynaptic actions of 5-HT have been carried out in rats (typically under anesthesia), all of the studies of the activity of serotonergic neurons under physiological conditions have been carried out in behaving cats. There are almost no data from primates on either of these issues. The finding that different 5-HT receptors in a given brain region may mediate reciprocal or antagonistic actions is deserving of further investigation. This may imply that the postsynaptic actions of 5-HT under normal physiological conditions are in a somewhat balanced state. This concept of dynamic balance may also apply to control at a more molar level. For example, there is increasing evidence that 5-HT, by its actions at two or more receptor sites, may exert opposing effects on the control of blood pressure (187). It may also explain why hallucinogenic drugs, acting preferentially at a 5-HT, receptor subtype, and thus creating an imbalance, can exert such strong organismic effects (232,234). In summary, 5-HT generally seems to exert a longlatency and long-duration action on its postsynaptic target neurons. Diversitv in the nature (excitatorv. in- EFRAIN January STRUCTURE 1992 AND FUNCTION axons, more discrete projection domains, and a higher percentage of synaptic release sites. We speculate that this anatomic trend may be associated with a similar degree of physiological specialization. In this final section, we elaborate on these major themes in this review. A. Activity and Actions of Serotonergic Neurons BRAIN SEROTONIN SYSTEM 213 of activity in association with the sleep-wake-arousal cycle, going from total inactivity during REM sleep to their highest discharge (3-5 spikes/s) during an aroused or active waking state. We believe that this reflects a primary role of serotonergic neurons in the mammalian CNS: to send information to their target neurons restricted to that regarding the level of motor activity/behavioral state of the organism. (Their slow and highly regular discharge pattern dictates that their activity can convey little in the way of complex information.) Then, in accordance with their conjunctive relation with the particular target structure, they may facilitate or suppress its activity. Aghajanian and colleagues (12, 479) have speculated that serotonergic neurons may act to facilitate motor systems while suppressing sensory systems. Thus when an organism is aroused or stressed, serotonergic neurons will facilitate motor output and inhibit afferent input. Reciprocally, as the organism becomes drowsy or falls asleep, motor output will be disfacilitated, and afferent sensory input will be disinhibited. Serotonergic neurons also modulate the activity of the autonomic nervous system so that its output is appropriate to the level of motor activity being displayed by the organism. Several points are worth recalling in this context. First, the activity of serotonergic neurons anticipates the end of REM sleep, seemingly preparing the organism for responding to the external world. Second, serotonergic neurons do not become inactive during REM sleep in brain-lesioned animals displaying REM sleep without atonia. Finally, a group of serotonergic neurons in the DRN and MRN of cats are strongly activated during central pattern generator-mediated oralbuccal movements, such as feeding, lapping, and grooming. These latter results are consistent with a large body of pharmacological and physiological data showing that 5-HT facilitates a number of vertebrate rhythmic motor activities (207, 231, 341a, 488a, 492). One of the major factors determining how serotonergic neurons influence their target neurons is their duration of action. If the influence of 5-HT is a modulatory one, then the coincidence of its action with other afferent inputs is the critical event. If a serotonergic neuron is discharging at 2 spikes/s during waking and its postsynaptic action lasts for 500 ms, then it will exert an almost continuous influence on its target neurons during this behavioral state. On the other hand, if it exerts a “fast” action, lasting for only IO ms, then any heterosynaptic afferent inputs may have to be precisely timed to be coincident with (and thus modulated by) this serotonergic effect. Throughout this review we described much of the CNS serotonergic system as broad and expansive. It should be clear, however, that this concept should not be extended to imply that its influence is diffuse and nonselective. This point can best be made, perhaps, by examining the serotonergic input to the primary visual cortex (area 17) in monkeys. As discussed in section III, not only do serotonergic neurons display a preferential innervation of particular laminae in this cortical area, but this pattern implies a relationship to specific visual func- Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 Activity of brain serotonergic neurons is slow and often highly regular. There is evidence that this characteristic pattern of activity is generated by mechanisms intrinsic to individual serotonergic neurons. Some serotonergic neurons have also been described as having nonclocklike activity; however, this activity can become highly regular under specific conditions, such as their activation during oral-buccal movements and following pharmacological blockade of autoreceptor-mediated feedback inhibition. Thus all serotonergic neurons appear to have an intrinsic clocklike activity that can become nonclocklike when any of a variety of extrinsic factors act on them. The slow discharge of serotonergic neurons may also exert a significant influence on the “state” of the postsynaptic receptors on their target neurons. Leysen and Pauwels (302) have speculated that this may account for the ease with which many treatments can downregulate 5-HT receptors (decrease the apparent number of available receptor sites) but few, if any, can upregulate them. This would imply that any plasticity manifested in this system under physiological conditions is much more likely to be seen when serotonergic neurons are activated rather than deactivated. The slow and highly regular neuronal activity of serotonergic neurons appears to be necessary for the normal function of a number of complex processes. The ability of transplanted, isolated fetal serotonergic neurons to restore the lost adult serotonergic connection lends strong support to the concept that these neurons must convey a basic message to their postsynaptic target neurons. Several points are especially pertinent. First, the fetal serotonergic neurons can find their proper target sites and reestablish lost synaptic contacts. Trophic signals play a dominant role in achieving the appropriate innervation density that provides the source of 5-HT. Second, the neurons release 5-HT to the target cells in a physiologically relevant manner. The studies showing that raphe neurons have an intrinsic firing pattern provide the mechanism for release. The loss of the normal afferents to serotonergic neurons does not appear to be critical, possibly because some of these afferents may be reestablished at the transplant site. Finally, the information conveyed by serotonergic neurons is communicated by a slow and rhythmic firing rate. It is not known whether information about state will still be supplied by the transplanted neurons. This obviously depends on whether these neurons receive signals providing state-dependent information. Serotonergic neurons displav their complete range OF 214 BARRY L. JACOBS AND C. AZMITIA Volume 7.2 ergic system should dominate, whereas the serotonergic system should prevail under conditions where a stimulus is repeatedly presented on a continuing basis. Both systems are thought to exert their strongest and most specific influence when their activity is coordinate with that in particular target sites in the CNS. B. Growth/Plasticity and Physiology An implicit assumption throughout this review has been that the growth and plasticity of serotonergic neurons is linked to their physiology and function. In this section, we make explicit our ideas of how and why this synthesis might occur. The initial growth and development of serotonergic neurons in the fetus is under genetic control. Beyond that, both genetic and epigenetic factors serve to guide and sculpt serotonergic neurons until they reach their adult form. A number of the neurotrophic factors controlling that growth are described in this review. *4dditionally, we believe that the characteristic neuronal activity of serotonergic neurons contributes to their expansive axon terminal domain. Recall that an endogenously generated, slow, and highly regular discharge pattern of neuronal activity is first observed in the late fetal stage or in the early postnatal period. Thus during development, serotonergic neurons are physiologically either silent or highly rhythmic in their activity. This general lack of episodic or pulsatile activity increases the likelihood that serotonergic neurons will be active in phase with their target neurons. This may contribute to their ability to establish connections with neurons throughout the neuraxis. It has further been proposed that inactive neurons may make more indiscriminate connections (95). Thus the final adultlike anatomic pattern may be attained when this slow and regular activity is concordant with the pattern of activity of postsynaptic target neurons. This idea is supported by the developmental studies of Changeux and Danchin (94) and Constantine-Paton (log), who showed, both physiologically and morphologically, that synaptic formation is made between neurons that are simultaneously active and is eliminated for neurons whose activity is asynchronous. Thus this basic rhythmic activity stabilizes the extensive connections made by the rapidly growing and branching fetal serotonergic neurons. Once formed, these morphological connections can be strengthened by physiological activity (208). Consistent with this general line of thought regarding the interaction between anatomy and physiology, we also described evidence that serotonergic neurons may grow to the periphery of a particular CNS target site and then await some signal before the final infiltration takes place. The type of neuronal activity displayed by these neurons may also be relevant to their regrowth following damage to serotonergic neurons in the adult. The regenerating sprouts in the adult brain will most likely encounter many neurons displaying a variety of discharge patterns. The serotonergic fibers, by virtue of Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 tion. Thus 5-HT more heavily innervates the magnocellular stream over the parvocellular stream, which implies that 5-HT may be more importantly involved in the processing of visual information regarding movement over that regarding form (485). Finally, the topic of the role of CNS 5-HT frequently includes a discussion of CNS norepinephrine as a parallel or interacting system. In fact, these two neurotransmitter systems are often seen as being strongly interrelated. Recall our initial discussion of Brodie and Shore’s (90) hypothesis that they might serve coordinated functions as the central representation of the two branches of the autonomic nervous system. From an electrophysiological perspective it is interesting to note that the norepinephrine neurons of the locus coeruleus are the only other group of neurons in the CNS (other than cu-motoneurons) that display the same pattern of activity across the sleep-wake-arousal cycle as do serotonergic neurons. They are silent in REM sleep, display a low level of activity during slow-wave sleep, become moderately active during waking, and increase their activity still further during active waking or arousal (152, 234a). There are, however, several important differences in the response properties of serotonergic and noradrenergic neurons. First, whereas serotonergic neurons display no habituation in their response to sensory stimuli, the response of noradrenergic neurons rapidly habituates so that they often become unresponsive within a few repeated trials. Second, we described a wide variety of situations that evoked physiological activation of the organism (e.g., the sympathoadrenal system) but that failed to activate serotonergic neuronal activity above an undisturbed active waking baseline level. However, when the discharge of noradrenergic neurons in the area of the locus coeruleus was examined under the same conditions, it invariably displayed a doubling or tripling of activity above an active waking baseline (1,339-341). Thus we may characterize the serotonergic system as a stable or conservative system whose activity is tied strongly to the tonic level of motor activity/sleep-wake-arousal state of the organism. Beyond that, it is essentially unperturbed by challenging or stressful conditions. In contrast, the brain noradrenergic system is seen as dynamic and as a system whose full range of activity goes beyond a linear relationship to the behavioral state of the organism. This system responds strongly to phasically arousing stimuli and to tonic stimuli that represent a real or apparent danger to the organism. Third, a group of serotonergic neurons are strongly activated during rhythmic vegetative activities, such as feeding, licking, and grooming, and are inhibited during orientation. Reciprocally, locus coeruleus neurons are phasically activated during orientation and either display a decrease or no change in discharge during the aforementioned vegetative activities (27a). Shifts in the predominance of the activity of one or the other of these two neurochemical systems will have profound implications for their target neurons. For example, in emergency situations, such as hemorrhage or restraint, the influence of the noradren- EFRAIN January 1992 STRUCTURE AND FUNCTION BRAIN SEROTONIN SYSTEM 215 liminary evidence indicates that albino rats reared from birth for 30 days in either constant light or constant dark display serotonergic innervation of the nucleus suprachiasmaticus of the hypothalamus that is significantly different from that of rats reared under standard light-dark laboratory conditions (259a). Animals reared under these constant conditions have a sparser innervation of the nucleus suprachiasmaticus of the hypothalamus, and, in addition, those reared in constant light display innervation that is more restricted to the ventral portion of the nucleus. Nearby nonvisual regions of the preoptic area of the hypothalamus did not display these differences. C. Two Serotonergic Systems One of the most interesting developments in this field in recent years has been the emergence of the concept that there are two types of serotonergic systems. The strongest evidence for this comes from anatomic studies; however, this appears to be paralleled by evidence from physiological studies. Anatomically, there is strong evidence for two types of nuclear organization of serotonergic neurons. In the rat brain stem these cells are densely packed in midline nuclei (type I), whereas in higher mammals the cells are often seen in smaller, less densely packed clusters located off the midline (type II). The morphological description of the serotonergic neurons can also be grouped in two general types. In rats, the neurons are fine in caliber, highly branched, largely unmyelinated, and with large nonsynaptic terminal fields (type I), whereas in higher mammals the axons are thicker, have fewer branches, are often myelinated, and have more restricted synaptic terminal fields (type II) (see Ref. 83a for a discussion of the phylogenetic trend toward less collateralization of descending serotonergic neurons). Although this classification into two types of nuclear organizations and two types of neuronal morphology has not been firmly correlated, it has been proposed that laterally placed serotonergic neurons have fewer collaterals (148). Physiologically, in behaving animals, the typical serotonergic neuron displays a slow and regular activity that is relatively unperturbable across a wide variety of behavioral, environmental, and physiological conditions. On the other hand, we have also seen evidence for a group of serotonergic neurons whose activity is significantly altered in association with specific conditions: increased during repetitive oral-buccal related movements and decreased during orientation. These anatomic and physiological data raise the interesting question of whether these two data sets are related, i.e., whether functionally specific serotonergic neurons are of the type II class. If they are, then a phylogenetic trend toward type II neurons would have profound implications for serotonergic function in the human brain. Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017 their continuous rhythmic activity, would be able to interact more easily with this ongoing activity. Thus their growth would not be arrested, as would be expected if they showed episodic activity out of phase with the neurons encountered. Another aspect of this issue is related to the evidence that functionally related nuclei can be innervated by the same group of serotonergic neurons and in some cases by the same serotonergic neuron (33,229,235,334, 489,498). For example, the MRN innervates the cingulate cortex, septal nuclei, and hippocampus while the DRN innervates the substantia nigra, corpus striatum, amygdala, and nucleus accumbens. Individual neurons in the DRN and MRN of rats may project to sensorimotor portions of both cerebral and cerebellar cortex or to visual portions of both, but they do not seem to project to two different functional systems in these structures (498). Similar examples can be found in the inferior groups where the NRO innervates the motor cranial nuclei of X and XII and the ventral horn of the spinal cord while the NRM innervates the substantia gelatinosum of the spinal cord. As discussed, the connections made by a synaptically linked area could be stabilized by the collaterals of a single serotonergic neuron or groups of serotonergic neurons. This suggests that when serotonergic neurons are active (e.g., during motor activity or arousal), the interactions of preformed connections would become stabilized, but when the neurons are silent (e.g., during sleep), the interconnections would become labile. Consistent with this line of thought, during development 5-HT terminals may be one of the last extrinsic afferents to complete their innervation of the cortex (304). It was proposed that the 5-HT innervation signals the completion of various neuronal circuits and is involved in verification and consolidation of interneuronal contacts (304). This extends the idea that functionally interconnected nuclei are innervated by the same group of serotonergic nuclei (33, 36). The final point in this section concerns the relationship between developmental growth factors and adult function. The responsiveness of the sprouting serotonergic neurons to a variety of growth signals contrasts with the known physiology of the adult system. Attracted by a number of neuronotrophic and guidance factors, the fibers actively send out processes to innervate the CNS. Many of the same factors that produce substantial changes in growth rate (e.g., steroids, peptides, and glucose) produce no change in the firing rate of the neurons in the adult animal. We believe that the role of these trophic factors is to determine the serotonergic innervation density during periods of growth. Thus perturbations occurring during normal development may influence the ability of serotonergic neurons to reach and innervate their appropriate targets and may have long-lasting effects on the physiology of the system. 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