I. Introduction

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The existence of an endogenous vasoconstrictor in blood serum (Stevens and Lee, 1984; Brodie, 1900) and the presence in the gut of a substance that increases intestinal motility (Vialli and Ersparmer, 1933) had been known to scientists since the beginning of the century. However, it was not until the serum vasoconstrictor was identified as 5-hydroxytryptamine (5-HT)² (Rapport et al., 1948) that it became clear that this amine was also present in the mammalian central nervous system (CNS) (Twarog and Page, 1953). Shortly after its discovery in the CNS, and based on the observation that it was heterogeneously distributed in the dog brain (Bogdansky et al., 1956), 5-HT was considered for the first time a putative neurotransmitter in the CNS. A major turning point in 5-HT neurotransmission research then came about when Fuxe and Dahlström, using Falck-Hillarp histochemical fluorescence, provided the first description of 5-HT neurons (Dahlström and Fuxe, 1964) and their projections (Fuxe, 1965). Today, it is an established fact that no region in the mammalian CNS lacks 5-HT innervation (Dahlström and Fuxe, 1964; Steinbusch, 1981, 1984; see Azmitia, 1986; Jacobs and Azmitia, 1992; Azmitia-Whitaker and Azmitia, 1995).

As a neurotransmitter, the ubiquity of 5-HT is not only anatomical but also phylogenetic. Having been identified in neurons of the cnidarian Renilla koellikeri (Umbriaco et al., 1990), it could be one of the most ancient of currently known transmitters. From primates (see Azmitia and Gannon, 1986; Törk, 1990) to Chondrichtyes (Stuesse and Cruce, 1992), the adult 5-HT system is organized into two subsystems, i.e., a rostral division with cell bodies localized in the midbrain and rostral pons, providing projections to the forebrain, and a caudal division located primarily in the medulla oblongata with descending projections to the spinal cord and brainstem nuclei. Similarities found among such divergent vertebrate brains indicate that the major nuclear organization and the 5-HT projection network have remained remarkably stable across phylogeny. In spite of its remarkable evolutionary stability (Jacobowitz and MacLean, 1978), careful scrutiny of comparative anatomical evidence indicates that differences are as important as similarities. These phylogenetic differences may be summarized as follows: in higher mammals the system has evolved toward a fast, precise type of neurotransmission in which 5-HT neurons give rise to few collaterals (Fallon and Loughlin, 1982), have a high proportion of myelinated axons, and, due to the existence of a high proportion of junctional synapses, terminal field innervation is localized. In lower mammals the system is diffuse, highly branched, unmyelinated, and nonjunctional innervation predominates in terminal fields (see Azmitia 1986; Descarries et al., 1990; Törk, 1990; Jacobs and Azmitia, 1992; Azmitia and Whitaker-Azmitia, 1995).

Because 5-HT neurons are among the first neuroblasts to differentiate, the 5-HT system is also ontogenically ancient. Indeed, in the rat brain 5-HT-immunoreactivity appears as early as gestational day 12 (Olsen and Seiger, 1972; Lidov and Molliver, 1982), tryptophan hydroxylase activity is present at fertilization, and nearly every embryonic cell contains 5-HT until the gastrula stage (Harris, 1981). The first 5-HT neurons appear in early development and are located rostrally, within the mesencephalon (Lidov and Molliver, 1982) Two days later, a more caudal rhombencephalic group appears (Wallace and Lauder, 1983). In the next sections, focus will be on the rostral group.

	II. Morphological Aspects of the 5-HT Rostral System

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Dahlström and Fuxe (1964) divided 5-HT cell clusters into nine groups (B_1-9), B₁ being the most caudal group of cells. Today, however, the nomenclature most frequently used refers to 5-HT cells contained within cytoarchitectonic brainstem entities known as the raphe nuclei. Rostral 5-HT neurons are not confined to midline (raphe) nuclei, they are also present in more lateralized sites of the reticular formation, especially 1) dorsal to the medial lemniscus (rat: Dahlström and Fuxe, 1964; Baker et al., 1990) and 2) dorsal to the nucleus raphe pontis oralis (Lidov and Molliver, 1982; Baker et al., 1991).

1. The Caudal Linear Nucleus. The caudal linear nucleus (CLN), described in primates by Törk (1990) and Azmitia (see Azmitia, 1986; Azmitia and Gannon, 1986; Jacobs and Azmitia, 1992; Azmitia and Whitaker-Azmitia, 1995), is the most rostral group of serotonergic neurons in the mesencephalic midline, extending along the rostral boundary of the superior cerebellar decussation. Its ventral limit is defined by the interpeduncular nucleus and dorsally it contacts the dorsal raphe nucleus (DRN) through the gap left between the two medial longitudinal fasciculi. In rats, 5-HT neurons located rostral to the decussation of the superior cerebellar peduncle and above the interpeduncular nucleus have been considered in some cases a rostral extension of the median raphe nucleus (MRN) (Lorez et al., 1978; Parent et al., 1981). However, CLN and MRN neurons do not share common terminal projection fields (Imai et al., 1986) and display dissimilar dendritic morphology (Hornung and Fristchy, 1988). On the other hand, because DRN and CLN 5-HT neurons innervate similar terminal fields (e.g., caudate-putamen in adult rat brain; Imai et al., 1986) and share a common developmental origin (Wallace and Lauder, 1983), an alternative approach proposes that CLN neurons should be considered similar to those in DRN (see Jacobs and Azmitia, 1992).

2. The Dorsal Raphe Nucleus. The DRN is the largest of the brainstem serotonergic nuclei containing about 50% of 5-HT neurons in the rat CNS (Wiklund and Björklund, 1980; Descarries et al., 1982), 40% in the cat CNS (Wiklund et al., 1981), and 50 to 60% in the human CNS (Baker et al., 1990). Rostrally, the dorsal raphe is bound by the Edingher Westphal nucleus (III) and, caudally, it extends just ventral to the confluence of the fourth ventricle and the cerebral aqueduct (Steinbusch, 1981; Descarries et al., 1982; Imai et al., 1986; Törk, 1990; Jacobs and Azmitia; 1992). In most species, the DRN is composed of several subregions distinguished by their different cell density, morphology, and projections (Azmitia and Gannon, 1986; Baker et al., 1991; Johnson and Ma, 1993): 1) a medial portion, subdivided in turn into dorsomedial and ventromedial components, just below the cerebral aqueduct and surrounding the medial longitudinal fasciculus (MLF), respectively; 2) lateral portions or wings (much more prominent in primates than other mammals due to a lateralization process through phylogeny; Descarries et al., 1982; Azmitia and Gannon, 1986; Baker et al., 1991); and 3) a caudal component. During development (15 days of gestation), 5-HT-immunoreactive cells group themselves into two different clusters, dorsolateral and ventrolateral to the MLF (Wallace and Lauder, 1983). The dorsolateral portion will give rise to the lateral wings of the DRN, whereas the ventrolateral group will split to form the interfascicular portion of DRN and MRN. For this reason, it has been proposed that, in the primate CNS (see Jacobs and Azmitia, 1992; Azmitia and Whitaker-Azmitia, 1995), the interfascicular subregion of the DRN should be best considered an entity along with the MRN. Olzewski and Baxter (1954) had previously defined an anatomical entity, nucleus centralis superior, consisting of three groups of cells: 1) a dorsalis component situated between MLF, 2) a medialis component or MRN, and 3) a lateralis component which includes the 5-HT cells that form the ring around the nucleus reticularis pontis oralis (see Table 1). The absence of anatomical boundaries between the main rostral portion of the DRN (B₇) and the main rostral portion of the MRN (B₈) has also been observed in rats (Descarries et al., 1982) and cats (Wiklund et al., 1981). The main rostral component of the DRN (B₇) also merges, in its rear end, with the caudal component (B₆) of this same nucleus (see Jacobs and Azmitia, 1992; Azmitia and Whitaker-Azmitia, 1995). Furthermore, the latter caudal component is in continuity with the dorsocaudal portion of the MRN (see Jacobs and Azmitia, 1992; Azmitia and Whitaker-Azmitia, 1995) and the lateral wings of the DRN (Lidov and Molliver, 1982). Hence, anatomical proximity between different 5-HT cell clusters provides the ground for a network of homotypic interconnections among 5-HT neurons. Such interconnections constitute in turn the morphological ground for the high degree of cross-talk existing among 5-HT neurons at the somatodendritic level. The major implication underlying this arrangement is that the more closely knit and the larger the number of cells in a functional cluster, the more powerful will be the effect of the group on the target structure toward which it projects (Azmitia, 1986).

3. The Median Raphe Nucleus. The MRN consists of two distinct parts, i.e., a group of densely packed cells, vertically oriented and concentrated in the midline, and a second group, the paramedian columns, consisting of cells scattered in the periphery of the midline cluster (Köhler and Steinbusch, 1982). The nucleus is situated ventral to the MLF and has a rostrocaudal oblique orientation (Azmitia, 1981; Lidov and Molliver, 1982). In its rostral end, beyond the MLF, it is separated from the DRN in its rostral end by the superior cerebellar peduncle decussation. Its medial portion extends from the mesencephalic interpeduncular nucleus to the trapezoid body in the pons. Laterally, the limits of the nucleus are poorly defined toward the reticular formation (Köhler and Steinbusch, 1982), and in the rat the MRN merges laterally with the 5-HT group in nucleus raphe pontis oralis (Lidov and Molliver, 1982). The MRN forms the second largest cluster of 5-HT neurons in the mammalian CNS (Baker et al., 1990).

4. The Supralemniscal Region. The last group of 5-HT neurons to be considered corresponds to 5-HT cells in the supralemniscal region. In the rat, 5-HT cells belonging to this group are scattered along the dorsal border of the medial lemniscus and extend their dendrites in between the fiber bundles of the latter (Parent et al., 1981; Steinbusch, 1981). A similarity between supralemniscal 5-HT cells and those of the MRN was first noted by Dahlström and Fuxe (1964) in the rat CNS and has been later confirmed in primates (Hubbard and Di Carlo, 1974). In fact, the cells in the supralemniscal region may be continuous with those of the paramedian columns of the MRN (Azmitia and Gannon, 1986). Unlike rodents in which the supralemniscal cell cluster is predominantly mesencephalic, in humans it is entirely located in the pons (Baker et al., 1991).

In all species studied thus far, neurons containing 5-HT consist of a morphologically heterogeneous population (Steinbusch, 1981; Jacobs et al., 1984; Azmitia and Gannon, 1986; Törk and Hornung, 1990). The average cell diameter varies between 15 and 25 µm (Azmitia, 1978; Descarries et al., 1982), and, ultrastructurally, "5-HT specificity does not closely correlate with any particular neuronal configuration and/or intracellular build-up" (Descarries et al., 1982). No unique element allows to distinguish 5-HT cell bodies from non-5-HT surrounding neurons.

The average number of axosomatic boutons received by 5-HT neuron perikarya in the DRN has been quantified by Descarries et al. (1982). Although 100 µm of somatic membrane receive seven axonic boutons, the average number of spines on the same membrane length is 2.7 (Park et al., 1982). On the dendrites, the number of axonic boutons per 100-µm membrane length is roughly 60% higher than on the soma (Descarries et al., 1982). In cats and rats, the fibers contributing to these axosomatic or axodendritic contacts are non-5-HT fibers (Descarries et al., 1982; Chazal and Ralston, 1987). In primates, Kapadia et al. (1985) have described 5-HT fibers impinging on 5-HT dendrites.

1. Sources of Extracellular 5-HT in Rostral Raphe Nuclei The existence of serotonergic axon terminals in the raphe nuclei of cats and rats has been repeatedly reported (Baraban and Aghajanian, 1981; Chan-Palay, 1982; Descarries et al., 1982; Chazal and Ralston, 1987), but the terminals endowed with synaptic specializations have been found to be consistently in low numbers, ranging from "none" (Baraban and Aghajanian, 1981), "exceedingly small number" (Descarries et al., 1982), to "a few" (Chazal and Ralston, 1987). Furthermore, Baraban and Aghajanian (1981) found 5-HT fibers exclusively in axon bundles rather than in proximity to dendrites or cell bodies, and when 5-HT terminals were observed to make somatodendritic synaptic contacts, these were on non-5-HT neurons (Descarries et al., 1982; Chazal and Ralston, 1987). In the rare cases in which a 5-HT axon terminal was observed in close apposition to a 5-HT cell body, no demonstrable synaptic contact was present (Chazal and Ralston, 1987). Nonsynaptic 5-HT axon terminals are not exclusive to the raphe nuclei; they also exist in projection areas such as the cortex, striatum, and hippocampus (Descarries et al., 1990; Törk, 1990). 5-HT release therefore occurs not only from junctional but also from nonjunctional sites (Descarries et al., 1975, 1982, 1990).

2. Cell Bodies Found in the 5-HT Nuclei. Quantitative studies of the total number of 5-HT neurons located in the ascending raphe nuclei indicate that there are about 288,000 in the human brain (Törk, 1990), 33,000 in the cat brain (Wiklund et al., 1981), and 15,200 in the rat brain (see Jacobs and Azmitia, 1992). Moreover, 5-HT neurons represent but a small percentage of the total neuronal population of the raphe nuclei. Using histofluorescence-imaging techniques, Wiklund et al. (1981) reported that in the cat 5-HT neurons constitute 70% of medium-sized cells in the dorsal raphe and 35% of medium-sized neurons of the MRN. This percentage could be lower, serotonergic cells constituting 25 to 50% of the total DRN neuronal population and 20 to 30% of the MRN's. In the supralemniscal 5-HT cell group (B₉), the percentage of 5-HT neurons appears to be much lower than in the other two nuclei (O'Hearn and Molliver, 1984).

Afferent connections to the raphe nuclei have been studied using multiple techniques such as lesion and axon degeneration, histofluorescence, anterograde/retrograde tracer injection, autoradiography, and immunohistochemistry (Brodal et al., 1960; Fuxe, 1965; Aghajanian and Wang, 1977; Mosko et al., 1977; Sakai et al., 1977; Baraban and Aghajanian, 1981; Kalen et al., 1985; Stratford and Wirtshafter, 1988; Marzienkiewicz et al., 1989; Behzadi et al., 1990). Results from such studies have been summarized in Table 2.

Interconnections among raphe nuclei using retrograde-tracing techniques suggest a moderate-to-high density of 5-HT input. These observations contrast with those from most of the immunocytochemical or radioautographic ultrastructural studies indicating a low number of 5-HT fibers in the rostral raphe. Mosko et al. (1977) have reported that the main source of 5-HT fibers reaching the DRN arise either from the DRN itself or the MRN. The latter input has been confirmed by other groups (Sakai et al., 1977; Aghajanian and Wang, 1977; Kalen et al., 1985; Vertes and Kocsis, 1994). However, given the proximity between injection and labeled sites, it cannot be ruled out that tissue damage and tracer diffusion may account for part of the retrograde staining observed in the MRN after tracer injection into the DRN. Furthermore, since retrograde transport of horseradish peroxidase by dendrites has been reported (Smith et al., 1974), this raises the possibility that the accumulation of tracer by dorsal and median raphe perikarya resulted from retrograde transport by dendrites, rather than the axon terminals of these neurons. On the other hand, tracer diffusion or retrograde dendrite transport cannot account for the projections arising from more distant nuclei (Sakai et al., 1977; Kalen et al., 1985; Marzienkiewicz et al., 1989; Behzadi et al., 1990). Histochemical confirmation of the neurotransmitter contained in connections among 5-HT nuclei has been performed on rare occasions (Stratford and Wirtshafter, 1988), and when done, numerous nonserotonergic cells were retrogradely labeled after tracer injection into the MRN. It is then possible that not all of the retrogradely labeled fibers are serotonergic. The general impression would be that 5-HT axons connecting 5-HT nuclei are "diluted" within the dorsal and median raphe neuropil.

An important afferent area to the raphe, both in terms of selectivity and density, is the lateral habenula (Aghajanian and Wang, 1977; Wang and Aghajanian, 1977b). This habenular circuit appears to be comprised of both a monosynaptic GABAergic pathway (Wang and Aghajanian, 1977b; Stern et al., 1981; Park, 1987) and a polysynaptic pathway in which GABA, Substance P (Neckers et al., 1979; Nishikawa and Scatton, 1985), and excitatory amino acids serve as components (Kalen et al., 1985, 1989).

Noradrenergic fibers impinge directly onto the dendrites of 5-HT neurons (Baraban and Aghajanian, 1981), producing an excitatory input on the firing activity of 5-HT neurons (Baraban and Aghajanian, 1980). The lateral hypothalamus also gives rise to a monosynaptic excitatory input to the DRN (Aghajanian et al., 1987) although the transmitter is unknown. Multiple afferent fibers immunoreactive for different neuropeptides, such as -endorphin (Bloom et al., 1978), Substance P (Ljungdahl et al., 1978; Shults et al., 1984), neurotensin fibers (Uhl et al., 1979), CCK fibers (Vanderhaeghen et al., 1980), CLIP/adrenocorticotrophic hormone (ACTH) fibers (Romagnano and Joseph, 1983; Zheng et al., 1991; Léger et al., 1994), and VIP fibers (El Kafi et al., 1994), have also been described.

D. Efferent Pathways and Terminal Projection Areas

1. Efferent Pathways. The rostral 5-HT nuclei are the main source of 5-HT fibers projecting to telencephalon and diencephalon (Fuxe, 1965; Azmitia and Segal, 1978; Parent et al., 1981; Villar et al., 1987). Although the main contingent of fibers from these nuclei is ascending, they also innervate more sparely numerous brainstem structures (Vertes and Kocsis, 1994), the cerebellar cortex (Waterhouse et al., 1986; Zimny et al., 1988), and the spinal cord (Skagerberg and Björklund, 1985). In the nonhuman primate brain, two main ascending bundles have been described, a dorsal bundle (immediately ventral to the MLF), which receives fibers mainly from lateral wings and the ventromedial portion of the DRN, and a ventral bundle, which receives fibers from the midline DRN and MRN (Schofield and Everitt, 1981, Azmitia and Gannon, 1986). In the human fetus, two ascending axon bundles have also been observed, in the central gray, ventral to the fourth ventricle and the aqueduct ependyma, and between ventromedial and ventrolateral 5-HT groups (Nobin and Bjorklund, 1973). In the rat CNS, two major projection systems to the forebrain have also been described, a transtegmental system, which probably corresponds to the above-mentioned ventral bundle, courses through the midbrain forebrain bundle (MFB) and is the most prominent of the two systems described by Descarries in the rat brain, and a periventricular system, dorsally located along the longitudinal fasciculus of Schütz (Parent et al., 1981). In primates, unlike in rats, the dorsal component (dorsal raphe cortical tract) is much more developed than the MFB system, presumably due to an increase in fibers projecting to the cortex through the dorsal pathway. Moreover, the percentage of myelinated 5-HT fibers in rat MFB is 0.7% of the total immunoreactive 5-HT fibers, whereas it is as much as 25% in primate MFB (Azmitia and Gannon, 1983).

2. Terminal Projection Areas. Dorsal and median raphe nuclei each innervate specific terminal areas (Bobillier et al., 1975; Azmitia and Segal, 1978; Jacobs et al., 1978), and, in turn, each terminal projection area has its unique topographic representation within the respective nuclei. Labeling studies using wheat germ agglutinin, horseradish peroxidase, or fluorescent dyes have been used to unveil the midbrain raphe projection network. The more rostral portions of the midbrain raphe relate to the basal-ganglia-motor system and caudal areas are more related to the limbic system. Neurons projecting to the striatum occupy the caudal linear nucleus and a rostral portion of the DRN (Jacobs et al., 1978; Imai et al., 1986), whereas those projecting to substantia nigra (Imai et al., 1986) and the motor cortex (O'Hearn and Molliver, 1984; Waterhouse et al., 1986) reside within the rostral portions of the dorsal raphe (Imai et al., 1986). Hippocampus-projecting neurons are situated caudally in the DRN (caudal ventromedial portion and B₆), the MRN, and B₉ (Jacobs et al., 1978; Köhler and Steinbusch, 1982; Imai et al., 1986), similar to neurons projecting to the locus ceruleus (Imai et al., 1986) and to the entorhinal cortex (Köhler and Steinbusch, 1982). The raphe representation of the amygdala, on the other hand, bridges the "basal-ganglia-motor system" and the "limbic system representation" (Jacobs et al., 1978; Imai et al., 1986), possibly as a reflection of the functional diversity of amygdaloid nuclei. The consequence of this topographic arrangement is that the selective activation of a given functional group would simultaneously influence interconnected brain circuits. Neurons projecting to interrelated brain circuits, such as the sensorimotor cortex and cerebellar crus II, the visual cortex and cerebellar paraflocculus (Waterhouse et al., 1986), or to the substantia nigra and caudate-putamen (Imai et al., 1986), the entorhinal cortex and hippocampus (Köhler and Steinbusch, 1982), the trigeminal sensory complex and nucleus accumbens or amygdala (Li et al., 1993), arise from overlapping areas within the different nuclei. Moreover, in these regions, a single neuron may provide a common input to two different but functionally interrelated terminal areas. In the ventromedial portion of the DRN, the same neuron was found to project to at least three different forebrain structures related with the limbic system: septum, medial thalamus, and olfactory cortex (de Olmos and Heimer, 1980). Colateralization provides not only a means for producing concurrent influences on numerous functionally related circuits, but it is also a way of achieving extensive serotonergic innervation from a small number of raphe neurons.

	III. Physiological and Pharmacological Aspects of the 5-HT System

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Midbrain raphe 5-HT neurons exhibit a spontaneous, slow (1-5 spikes/s), regular discharge pattern (Aghajanian and Vandermaelen, 1982; Vandermaelen and Aghajanian, 1983). Intracellular recordings from dorsal raphe neurons reveal that 5-HT cells undergo repetitive cycles of interspike hyperpolarization and depolarization, spikes arising from depolarizing ramps rather than from excitatory postsynaptic potentials (Aghajanian and Vandermaelen, 1982; Vandermaelen and Aghajanian, 1983). The ionic basis for this electrical activity is summarized in Fig. 1.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Representative voltage tracings obtained from acutely isolated DRN neurons under current clamp (modified from Penington et al., 1991). A, spontaneous activity exhibited by the cell at resting membrane potential. Typical action potentials consist of an initiating ramp of depolarization, spike, shoulder upon repolarization, and an after-hyperpolarization. B, depolarization of a cell bathed with the Na+ channel blocker TTX. TTX abolished the fast component of the action potential and uncovered low- and high-threshold Ca2+ components of the action potential. Although T channels seem to be responsible for the low-threshold current, at least three different channel types (including N- and L-type) underlie the high-threshold current (Penington and Kelly, 1990; Penington et al., 1991). The after-hyperpolarization that follows, as in many other vertebrae and invertebrae neurons, is mediated by a Ca2+-activated K+ outward current (Vandermaelen and Aghajanian, 1982; Aghajanian, 1985; Aghajanian et al., 1987). This after-hyperpolarization is responsible for a long-lasting refractory period and the slow firing rate of 5-HT neurons. As Ca2+ that entered during the action potential is sequestered/extruded, the Ca2+-dependent K+ current and the after-hyperpolarization decrease. When the membrane potential reaches again the value for the low-threshold Ca2+ current (approximately 60 mV), a new spike will be triggered. As repolarization from membrane potentials below the resting potential takes place, a voltage-dependent outward K+ current that slows the rate of depolarization is simultaneously activated, the so-called IA current (Aghajanian, 1985).

In freely moving cats, regular stereotyped intrinsic activity of 5-HT neurons remains unchanged over exposure to a hot environment or administration of a pyrogen, increase in blood pressure, insulin-induced hypoglycemia, administration of painful stimuli, physical restraint, or exposure to powerful aversive stimuli (see Jacobs and Fornal, 1993). However, the basic pattern of activity is not constantly the same, and it has been shown to change dramatically during the sleep-wake-arousal cycle. Firing activity progressively slows down from an aroused state through quiet waking and slow wave sleep (SWS), to become almost silent during rapid eye movement (REM) sleep (for review, see McGinty and Harper, 1976; Jacobs and Fornal, 1993). The suppression of firing of 5-HT neuron during REM sleep correlates well with the production of muscle atonia secondary to inhibition of motoneurons controlling antigravity muscles (Trulson et al., 1981; Steinfels et al., 1983). More recently, a relationship between motor output and 5-HT neuron activity has been observed. During quiet wakefulness, when cats engage in various types of stereotyped oral-buccal activities such as chewing and biting, licking, or grooming with the tongue, approximately 25% of DRN and MRN increase their firing activity 2- to 5-fold (see Jacobs and Azmitia, 1992; Jacobs and Fornal, 1993). This increased neuronal activity precedes the onset of stereotyped motor behaviors and ends with its offset; it does not occur during purposive episodic movements, but some of the neurons may be activated by somatosensory and proprioceptive stimulations of the head and neck area. These data have given support to the current motor hypothesis of 5-HT function in which the primary role of the 5-HT system would be facilitation of motor output and concurrent inhibition of sensory information processing (Jacobs and Fornal, 1993).

The firing activity of midbrain 5-HT neurons is controlled by two main mechanisms, i.e., autoregulatory influences arising from 5-HT neurons themselves and heteroregulation by local neurons or afferents to the raphe nuclei.

1. Autoregulation of 5-HT Neuron Firing Activity. The firing rate of 5-HT neurons is decreased by 5-HT, and this effect is mediated by somatodendritic 5-HT_1A autoreceptors (Aghajanian et al., 1972; Vandermaelen et al., 1986; Blier and de Montigny, 1987). 5-HT and 5-HT_1A agonists inhibit 5-HT firing activity by inducing membrane hyperpolarization which is brought about by a 2-fold mechanism, i.e., by increasing conductance to potassium ions (Aghajanian and Lakoski, 1984; Yoshimura and Higashi, 1985) and by reducing a high-threshold Ca²⁺ current (Fig. 1; Penington and Kelly, 1990; Penington and Fox, 1994). In both cases, the response to 5-HT is G protein-mediated via a direct interaction between G proteins and the respective ion channel (Innis and Aghajanian, 1987; Penington et al., 1991; Penington et al., 1993; Penington and Fox, 1994).

2. Role of Non-5-HT Receptors in the Regulation of 5-HT Neuron Firing Activity. Autoradiographic and binding studies have documented the presence of different 5-HT-binding sites in the rat raphe nuclei (Waeber et al., 1988; Herrick-Davis and Titeler, 1988; Waeber et al., 1989a; Laporte et al., 1992). However, the role of 5-HT receptors, other than 5-HT_1A in modulating 5-HT neuron firing activity at the cell body level, has not been confirmed. The lack of effect of 1-[3-(trifluoromethyl)phenylpiperazine (TFMPP) and m-chlorophenylpiperazine on the firing activity of 5-HT neurons led Sprouse and Aghajanian (1986, 1987) to conclude that 5-HT_1B receptors were not involved in regulating 5-HT neuron firing activity in the rat brain. The systemic injection and the microiontophoretic application of the preferential 5-HT_2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) reduce 5-HT neuron firing frequency. However, this effect could not be blocked by the 5-HT_2A/2C antagonists ketanserin and ritanserin (Wright et al., 1990; Garratt et al., 1991). Moreover, the microiontophoretic application of ketanserin does not block the inhibition of 5-HT neuron firing induced by the neurotransmitter itself. It does, however, reduce the basal firing rates in the majority of 5-HT cells tested (Lakoski and Aghajanian, 1985). These observations, along with the fact that the effectiveness of systemic 8-OH-DPAT but not that of DOI to inhibit the firing activity of 5-HT neurons is decreased following repeated DOI administration (Kidd et al., 1991), suggest that 5-HT₂ receptors are not directly involved in the regulation of 5-HT neuron firing. The role of 5-HT₃ receptors in regulating 5-HT neuron firing activity has also been investigated. The fact that systemic administration of the 5-HT₃ antagonist BRL 46470A does not block the reduction of the dorsal raphe 5-HT neuron firing rate induced by the microiontophoretic application of the 5-HT₃ agonist 2-methyl-5-HT (Haddjeri and Blier, 1995), and that 5-HT neuron firing activity remains unchanged after systemic administration of three different 5-HT₃ receptor antagonists, MDL 72222, ICS-205-930, and ondansetron (Adrien et al., 1992), indicate that 5-HT₃ receptors do not contribute to the regulation of 5-HT neuron firing activity. 8-OH-DPAT has often been used as the "gold standard" for defining 5-HT_1A receptors, but there is evidence that it also has reasonable good affinity at the rat (Shen et al., 1993) and guinea pig (To et al., 1995) 5-HT₇ receptor. Moreover, 8-OH-DPAT has also been identified as the most potent agonist at a cyclase-linked receptor not yet fully characterized (Becker et al., 1992), and it also binds to the serotonin transporter (SERT) (Schoemaker and Langer, 1988; Alexander and Wood, 1988; Ieni and Meyerson, 1988). However, the fact that methiothepin blocks 5-HT₇-mediated responses (Terron, 1997; Kitazawa et al., 1998) but not the effect of i.v. 8-OH-DPAT (Blier et al., 1989b) suggests that 5-HT₇ receptors are not involved in 8-OH-DPAT actions on 5-HT neuron firing activity. Furthermore, the dose at which 8-OH-DPAT interferes with 5-HT uptake is two to three orders of magnitude higher than the one currently used in electrophysiological experiments to assess drug effect on neuronal firing activity. Also, without excluding the role of other 5-HT receptors, the extensive list of 5-HT_1A compounds in Table 3 confirms the role of the latter receptor in regulating 5-HT neuron firing.

3. Heteroregulation of 5-HT Neuron Firing Activity. N-methyl-D-aspartate (NMDA) receptors may elicit excitatory postsynaptic potentials (EPSPs) (Pan and Williams 1989; Pinnock, 1992; Johnson, 1994a) and increase the firing activity of 5-HT neurons (Alojado et al., 1994) in vitro, but they do not seem to maintain a tonic activation of 5-HT neuron firing in vivo (Levine and Jacobs, 1992). However, glutamate does mediate the increase in firing activity observed following presentation of phasic auditory stimuli (Levine and Jacobs, 1992). In turn, excitatory amino acid (EAA) release in the dorsal raphe is negatively regulated by -opioid receptors (Pinnock, 1992). Rather unexpectedly, the systemic administration of the NMDA channel blocker (+)-MK-801 has been shown to facilitate the electrical activity of 5-HT neurons in the DRN. Such an observation could be explained by assuming that the channel blocker reduces the facilitation of an inhibitory influence. Microiontophoretic application of GABA onto dorsal raphe 5-HT neurons produces an inhibition of their firing rate (Gallager and Aghajanian, 1976; Levine and Jacobs, 1992). Also, the GABA blocker picrotoxin reduces the suppressant effect on 5-HT neuron activity caused by habenula and pontine reticular formation stimulation (Wang et al., 1976; Wang and Aghajanian, 1977; Stern et al., 1981). In freely moving animals, microiontophoretic application of bicuculline produces a significant increase of 5-HT neuron firing activity during SWS, but not during REM or quiet waking, indicating that GABAergic input is state-dependent and not tonic (Levine and Jacobs, 1992). In anesthetized rats, microiontophoretic and systemic administration of  adrenoceptor antagonists as well as 6-hydroxydopamine pretreatment suppress 5-HT neuron firing activity, suggesting a tonic facilitatory role for noradrenaline on dorsal raphe 5-HT neurons (Baraban and Aghajanian, 1980). Furthermore, activation of ₂-adrenergic autoreceptors by clonidine decreases norepinephrine (NE) output and suppresses the firing activity of 5-HT neurons (Clement et al., 1992; Haddjeri et al., 1996a). In contrast, systemic administration of  adrenoceptor antagonists in the awake freely moving animals produced no change in 5-HT neuron firing activity (Heym et al., 1981). Unlike EAAs, NE does not elicit EPSPs on 5-HT neurons, rather it suppresses the voltage-dependent K⁺ current I_A (Fig. 1; Aghajanian, 1985), leading to a more rapid activation of the low-threshold inward calcium current that triggers the spike at the end of the pacemaker cycle of these neurons. Activation of CCK_A receptors located on 5-HT neurons also stimulates their firing activity (Boden et al., 1991), as does bombesin via the stimulation of neuromedin B receptors (Pinnock et al., 1994).

The firing activity of 5-HT neurons is not inhibited only by administration of 5-HT_1A agonists (Vandermaelen et al., 1986; Blier and de Montigny, 1987; Godbout et al., 1990; Schechter et al., 1990; Hadrava et al., 1995). This effect is also produced by drugs such as SSRIs (Blier and de Montigny, 1983; Blier et al., 1984; Chaput et al., 1986b; Jolas et al., 1994; Arborelius et al., 1995; Hajós et al., 1995) and monoamine oxidase inhibitors (MAOIs) (Blier and de Montigny, 1985; Blier et al., 1986a,b), which induce an activation of 5-HT_1A autoreceptors due to an immediate increase in extracellular 5-HT at the somatodendritic level (see Sharp and Hjorth, 1990; Artigas, 1993). However, following sustained administration of SSRIs, MAOIs, and 5-HT_1A agonists, a progressive desensitization of somatodendritic 5-HT_1A autoreceptors takes place, and the effectiveness of 5-HT_1A agonists and antidepressant drugs to inhibit 5-HT neuron firing activity decreases. After 14 to 21 days of treatment, 5-HT neurons recover their pretreatment firing frequency (Blier and de Montigny, 1983, 1985, 1987; Blier et al., 1984, 1986; Chaput et al., 1986b; Chaput et al., 1991; Godbout et al., 1990; Schechter et al., 1990; Jolas et al., 1994; Arborelius et al., 1995; Hadrava et al., 1995; Dong et al., 1997, 1998). These multiple in vivo and in vitro electrophysiological studies have suggested that desensitization of somatodendritic 5-HT_1A autoreceptors could be a possible explanation for the recovery of 5-HT neuron firing activity. However, despite the fact that considerable functional evidence supports the occurrence of 5-HT_1A autoreceptor desensitization, the mechanism underlying this adaptative process remains unclear. The possibility that down-regulation of 5-HT_1A receptors in the midbrain may mediate the observed electrophysiological desensitization has not been confirmed. Although long-term treatment with gepirone (Welner et al., 1989), buspirone (Gobbi et al., 1991), and fluoxetine (Li et al., 1994) reduce the total number of [³H]8-OH-DPAT-binding sites in midbrain raphe nuclei, sustained administration of citalopram, sertraline (Hensler et al., 1991), paroxetine, fluoxetine (Le Poul et al., 1995), and cericlamine (Jolas et al., 1994), as well as that of clorgyline, phenelzine, tranylcypromine (Hensler et al., 1991), and ipsapirone (Schechter et al., 1990), did not modify [³H]8-OH-DPAT-binding parameters in the same region (all drugs were administered for a 14- to 21-day period at doses that induce functional desensitization). Desensitization of 5-HT_1A-mediated inhibition of adenylyl cyclase has also been documented after the administration of clorgyline, tranylcypromine, fluoxetine, and buspirone (see Newman et al., 1993), and the use of molecular biology techniques has allowed us to determine that agonist-induced desensitization of adenylyl cyclase inhibition correlates well with 5-HT_1A receptor down-regulation in Swiss 3T3 cells (Van Huizen et al., 1993). However, patterns of desensitization may differ depending on the host cell used to express the receptor, as well as on the response being investigated. Indeed, desensitization of the inhibitory effect of 5-HT_1A receptor activation on cAMP production is linked to receptor phosphorylation by PKC in Chinese hamster ovary cells (Raymond, 1991) and by G protein-coupled receptor kinases in insect Sf9 cells (Nebigil et al., 1995). It is unlikely then that results obtained in non-neuronal cell lines may be directly extrapolated to 5-HT neurons. Furthermore, since 5-HT_1A receptors that control the firing activity of these neurons are linked (via a G protein) to K⁺ and Ca²⁺ channels (Innis and Aghajanian, 1987; Penington et al., 1991, 1993, Penington and Fox, 1994), it is not certain that they possess the same desensitization mechanisms as receptors linked to adenylate cyclase. Alternatively, it has been proposed that antidepressant-induced desensitization of 5-HT_1A-mediated responses could be mediated at the signal-transducing (G protein) level (Lesch et al., 1991, 1992; Lesch and Manji, 1992; Chen and Rasenick, 1995). Sustained fluoxetine and clorgyline administration have been found to respectively decrease G_s and increase G₁₂ mRNA in rat midbrain (Lesch et al., 1992; Lesch and Manji, 1992). Finally, another possible target for antidepressant-induced desensitization of somatodendritic 5-HT_1A receptor-mediated control of 5-HT neuron firing are the effector channels to which the receptor is linked by the G protein. The effect of sustained 5-HT_1A receptor activation on K⁺ channels has been studied on p-neurons of the leech CNS, where they induce phosphorylation of two different types of K⁺ channels, increasing their open-state probability (Goldermann et al., 1994). Similar to the above-mentioned restrictions, it may not be concluded that such a mechanism might account for desensitization of 5-HT_1A-mediated responses in mammalian 5-HT neurons. However, this as well as the previous observations open new research avenues that would be worth exploring.

C. 5-HT Release

1. Neurotransmitter Release and Its Regulation: Cellular and Molecular Aspects. Exocytosis is the main mechanism used by neuronal cells for releasing neurotransmitter molecules. By this process, synaptic vesicles fuse with the plasma membrane and the neurotransmitter(s) contained within them reach(es) the synaptic cleft. Exocytosis is triggered by cell depolarization. Depolarization induces opening of voltage-sensitive calcium channels and subsequent Ca²⁺ entry. Achieving localized concentrations of 10 to 100 µM around the open channel (Smith and Augustine, 1988), the intracellular Ca²⁺ increase constitutes the major coupling signal that links depolarization and exocytotic secretion (reviewed by Burgoyne and Cheek, 1995). One of the main characteristics of neurotransmitter release is its high speed, the complete cycle being achieved in hundreds of milliseconds. This is apparently due to the fact that secretory vesicles are already docked to the plasma membrane so that when Ca²⁺ entry takes place, vesicles in a close vicinity of activated calcium channels will immediately void their content into the synaptic cleft by formation of a fusion pore (see Burgoyne and Cheek, 1995). Docking and fusing of the vesicles to the plasmalemma is achieved by Ca²⁺-sensitive vesicle membrane proteins (Augustine et al., 1985; Smith and Augustine, 1988; Leveque et al., 1992). One of these proteins, synapsin I, in an unphosphorylated state, fixes secretory granules to the cytoskeleton. Once it undergoes Ca²⁺-calmodulin/cAMP-dependent phosphorylation, it releases the vesicles from the cytoskeletal network and allows them to move to the presynaptic membrane (Valtorta et al., 1992) where they will be ultimately docked and voided to the extracellular space. The cytoskeleton is not likely to be involved in a first burst of release, which usually empties already docked vesicles, yet releasing the bound granules from the actin network will facilitate their subsequent recruitment by the plasma membrane in preparation for the arrival of the next axon potential (see Burgoyne and Cheek, 1995).

2. Physiological Role of Extracellular 5-HT Bioavailability in Midbrain Raphe Nuclei. It is important to bear in mind that all of the methods presently used to determine neurotransmitter release, such as synaptosomal or slice superfusion, in vitro or in vivo voltametry and microdialysis, measure not only the release but also the summation of neurotransmitter release, uptake, diffusion, and metabolism, generally referred to as neurotransmitter output. Hence, whenever the term release is used, it is understood that it does not strictly refer to 5-HT release but rather to what is detected by the method used. With this in mind, the extracellular concentration of 5-HT in the DRN has been estimated to be between 3 and 10 nM using in vivo voltametry and microdialysis experiments (Crespi et al., 1988; Bel and Artigas, 1992; Adell et al., 1993). There seems to be general agreement on the fact that extracellular 5-HT levels may vary according to behavioral state changes (Cespuglio et al., 1990; Houdouin et al., 1991; Portas and McCarley; 1994). However, there is still controversy with regard to the direction of these changes. On the one hand, Jouvet's group has observed the highest 5-hydroxindole peak during sleep (Cespuglio et al., 1990; Houdouin et al., 1991), thus supporting the idea that enhanced 5-HT_1A autoreceptor activation secondary to dendritic 5-HT release triggered by hypnogenic factors such as CLIP or VIP (El Kafi et al., 1994) might be responsible for determining the decrease in 5-HT neuron firing activity observed during SWS and REM (see Jacobs and Azmitia, 1992; Jacobs and Fornal, 1993). On the other hand, Portas and McCarley (1994) argue that extracellular 5-HT in the DRN is highest during wakefulness, lowest during REM, and that extracellular somatodendritic 5-HT availability depends directly on the serotonergic action potential activity. The latter observations are in agreement with results from electrophysiological studies in which 5-HT_1A antagonists were more effective in increasing 5-HT neuron firing activity during wakefulness than during sleep (Fornal et al., 1994a,b). Furthermore, in vitro neurochemical studies indicate that within a range of 5 to 100 Hz, higher stimulation frequencies elicit increasingly higher extracellular concentrations of 5-HT, as measured by fast cyclic voltametry in rat DRN slices (O'Connor and Kruk, 1991).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   A, effect of systemic administration of the 5-HT1A/1B antagonist ()-penbutolol on spontaneous hippocampal 5-HT release in anesthesized rats. B, effect of hippocamal perfusion of the 5-HT1A/1B antagonist ()-penbutolol and systemic administration of the 5-HT1A antagonist S-UH-301 on spontaneous 5-HT release in rat hippocampus. C, effect of the systemic administration of the 5-HT1B/1D antagonist GR 127935 on cortical 5-HT release of freely moving guinea pigs. D, effect of the combined administration of a 5-HT1A (WAY 100135) and the 5-HT1D antagonist on cortical 5-HT release in freely moving guinea pigs (see text for details).

4. Heteroregulation of Neurotransmitter Release in Midbrain Raphe Nuclei. Numerous neurotransmitters and neuromodulators from afferent terminals or intrinsic non-5-HT neurons influence 5-HT release in the raphe nuclei. For example, low-frequency (1.5 Hz) stimulation of the habenulo-raphe pathway elicits a decrease in 5-HT release in the DRN (Reisine et al., 1982). Similar results are obtained by intraraphe or systemic administration of GABA or GABA agonists (Scatton et al., 1985; Becquet et al., 1990). This pharmacological inhibitory effect on DRN 5-HT release is abolished by transection of the habenulo-raphe pathway (Nishikawa and Scatton, 1985). The need for the integrity of the habenulo-raphe connection has thus been interpreted in two alternative ways: 1) the effect of GABA or GABA agonists is an indirect one, and GABA receptors are located on habenulo-raphe fibers or 2) the inhibitory effect of GABA is made evident only if 5-HT neurons have a certain degree of tonic activation, provided in turn by the habenulo-raphe pathway. It is worth noting that the most consistently described tonic excitatory input originates in locus ceruleus (Baraban and Aghajanian, 1981). The common feature shared by both of the interpretations above is that they assume that the habenulo-raphe pathway must contain an excitatory neurotransmitter, an assumption which is in disagreement with the fact that the main observed effect of habenulo-raphe pathway stimulation is depression of 5-HT neuron firing (72-88% of the 5-HT neurons are inhibited; Stern et al., 1981). However, when a high stimulation frequency (15 Hz) is used, the habenulo-raphe pathway induces a marked increase in 5-HT release in projection areas, an effect that is blocked by injection of kynurenic acid into the DRN (Kalen et al., 1989). Such an observation has led Kalen et al. (1989) to suggest that EAAs are the main neurotransmitters in the habenulo-raphe pathway, and that glutamatergic fibers could have a double effect on 5-HT neuron, i.e., direct phasic stimulation and indirect inhibition by stimulating GABAergic interneurons. Such an observation is in keeping with in vitro findings in which stimulation of midbrain slices with an electrode in the DRN causes fast IPSPs and EPSPs that are respectively blocked by bicuculline or picrotoxin and the NMDA antagonists CNQX and APV (Pan et al., 1989; Pinnock 1992). The EPSPs due to electrically evoked release of EAA from afferent fibers onto 5-HT neurons may be reduced by activation of presynaptic inhibitory -opioid receptors located on the glutamatergic fibers (Pinnock, 1992). However, the predominant effect of systemically administered morphine is a stimulation of 5-HT release, an effect contrary to the one expected from activating inhibitory receptors on excitatory fibers impinging onto 5-HT neurons. A possible explanation for such a contradiction could be that the effect of activation of -receptors on EAA terminals may be overcome by activation of other opioid receptors, probably also located within the raphe nucleus, as suggested by Tao and Auerbach (1994). Apart from eliciting postsynaptic potentials on 5-HT neurons recorded from midbrain raphe slices, GABA and glutamate have been shown to modulate 5-HT release in rostral rhombencephalic raphe cells in primary cultures (Becquet et al., 1993b). GABA produces its negative modulation predominantly via GABA_A but also GABA_B receptors, whereas EAA induce 5-HT release by stimulating NMDA receptors (Becquet et al., 1993a). Furthermore, in vivo application of the GABA_A antagonist picrotoxin into the DRN locally increased 5-HT release in unanesthetized rats (Becquet et al., 1990), suggesting that the latter receptors may induce a tonic inhibition of 5-HT release in the dorsal raphe. The release of 5-HT in the DRN is also modulated by tachykinins and catecholamines. Indeed, Substance P has been shown to increase 5-HT release in vitro in midbrain raphe slices (Kerwin and Pycock, 1979) and in vivo following its intraraphe injection (Reisine et al., 1982). The local infusion of amphetamine, apomorphine, and the selective D₂ receptor agonist quinpirole also increases 5-HT release in DRN, the effect of apomorphine being blocked by the selective D₂ receptor antagonist raclopride but not the D₁ antagonist SCH 23390 (Ferré and Artigas, 1993; Ferré et al., 1994). The latter observations confirm the role of D₂ receptors in modulating extracellular availability of 5-HT in the DRN, but whether the source of DA are dopaminergic afferents or DA neurons within the nucleus is still unknown. In the case of NA, this catecholamine inhibited K⁺-evoked release of [³H]5-HT from raphe slices. This effect was mimicked by ₂ adrenoceptor agonists clonidine and oxymetazoline, but not by the ₁ adrenoceptor agonists phenylephrine and methoxamine. Furthermore, yohimbine and rauwolscine not only blocked the effect of clonidine, but, when administered alone, they both increased the K⁺-induced release of [³H]5-HT. A possible interpretation of the auto- and heteroregulatory connections of 5-HT release in the DRN is given in Fig. 3.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Graphic representation of auto- and heteroregulatory connections of midbrain 5-HT neurons: somatodendritic 5-HT1A as well as 5-HT1D/1D-like receptors are principally influenced by extracellular 5-HT of somatodendritic origin. They respectively inhibit 5-HT neuron firing or local release of neurotransmitter. Extracellular 5-HT also modulates terminal 5-HT1B autoreceptors located either on axon collaterals from efferent axons of local 5-HT neurons or those located on axons impinging from neighboring 5-HT nuclei. Activation of 1 adrenoceptors increases 5-HT neuron firing activity and that of 2 adrenoceptors inhibits 5-HT release. Both receptors are stimulated by NA released from axons originating in locus ceruleus. Pharmacological stimulation of dopaminergic D2 receptors increases local 5-HT release. It is not clear whether these receptors are physiologically stimulated by DA released from intraraphe DA neurons or dopaminergic projections originating in the VTA. The stimulation of the habenulo-raphe pathway is believed to induce glutamate release, which, via NMDA receptors, evokes a direct EPSP on 5-HT neurons. Glutamate also stimulates interneurons which may contain either Substance P or GABA. Although GABA inhibits 5-HT neuron firing activity (mainly via GABAA receptors), Substance P may produce an increase in local 5-HT release. Finally, GABAergic and enkephalinergic interneurons both inhibit glutamate release. Dashed lines, afferent nuclei boundaries.

5. Autoregulation of 5-HT Release in Terminal Projection Areas: Cortex and Hippocampus. Evidence indicating that activation of 5-HT_1B terminal autoreceptors inhibit 5-HT release is extensive and convincing (rat, mouse, guinea pig, pig, rabbit, human brain; see Table 4 for pharmacological profiles of terminal autoreceptors; Hoyer and Middlemiss, 1989; Starke et al., 1989; Göthert, 1990; Middlemiss and Hutson, 1990). Apart from this well established fact, there is now considerable evidence indicating that more than one receptor subtype could be involved in controlling release in the same region, and that some of these receptors might not be of the 5-HT_1B subtype. In rat cortical slices, 8-OH-DPAT has been shown to inhibit electrically or K⁺-evoked 5-HT release (Hamon et al., 1984; Limberger et al., 1991). Because such an effect was not blocked by 300 nM of the 5-HT_1B antagonist isamoltane (Waldmeier et al., 1988; Schoeffter and Hoyer, 1989), it was suggested that 5-HT_1D isamoltane-resistant sites might contribute to regulate 5-HT release in the rat frontal cortex (Limberger et al., 1991). Today we know that 8-OH-DPAT has considerable affinity not only for 5-HT_1D, but also 5-HT₅ and 5-HT₇ receptors (Table 5), and also that mRNAs for all three receptor subtypes are present in the DRN (Table 5). This suggests that, similar to the 5-HT_1B receptor subtype, 5-HT_1D, 5-HT₅, as well as 5-HT₇ receptors could be expressed as autoreceptors on 5-HT neurons. The concentrations at which 8-OH-DPAT effectively inhibited evoked 5-HT release in the above-mentioned studies were higher than 100 nM. Thus, it is tempting to speculate that the probability for 5-HT_1D or 5-HT₅ receptors to be involved in this effect is greater than for 5-HT₇ receptors, since the affinity for the latter is very high (Table 5) and 8-OH-DPAT would have been expected to be effective at lower concentrations. Moreover, it should be noted that at concentrations of up to 1 µM 8-OH-DPAT, some groups were unable to demonstrate an inhibition of cortical evoked release of 5-HT (Middlemiss, 1984b; Engel et al., 1986; Maura et al., 1986). Additional evidence supporting the fact that 5-HT receptors controlling 5-HT release in rat hippocampus are heterogeneous has been obtained by our group. In superfusion experiments, sumatriptan (1-1000 nM) and CP 93129 (1-300 nM) induced a dose-dependent inhibition of electrically evoked [³H]5-HT release from rat hippocampal slices. Although the effects of CP 93129 and sumatriptan were blocked by (±)-cyanopindolol (1 µM), only that of sumatriptan was blocked by mianserin (0.3 and 1 µM). On the other hand, only the effect of CP 93129 was blocked by ()-propranolol (0.3 µM). Moreover, incubation of rat hippocampal slices with the alkylating agent N-ethylmaleimide (NEM), abolished the effect of CP 93129 but not that of sumatriptan (Piñeyro and Blier, 1996). Furthermore, the inhibitory effect of 5-MeOT in rat hippocampus has been shown to remain unaffected by NEM, pertussis, or cholera toxin pretreatment, further indicating that, in the rat, there is an hippocampal 5-HT receptor subpopulation which is indeed resistant to G_i/o protein inactivation (Blier, 1991). Results from superfusion studies in 5-HT_1B knockout mice provide further evidence that a nonhomogeneous terminal autoreceptor population exists: non-5-HT_1B receptors continue to control 5-HT release in terminal projection areas in 5-HT_1B knockout mice (Piñeyro et al., 1995a). Moreover, it is possible that the combination of receptor subtypes in terminal autoreceptor populations may differ among specific projection areas (e.g., cortex and hippocampus). This would not be surprising given that each of the 5-HT nuclei contribute in a different manner to the innervation of distinct terminal fields. In vivo experiments in the frontal cortex and hippocampus of 5-HT_1B knockout mice also support the idea that non-5-HT_1D, non-5-HT_1B receptors regulate 5-HT release in the mouse frontal cortex and that terminal 5-HT autoreceptor populations in the cortex and hippocampus may be different (Trillat et al., 1996).

6. Heteroregulation of Neurotransmitter Release from 5-HT Fibers in Cortex and Hippocampus. Several in vivo and in vitro studies using brain slices or synaptosomes have provided evidence for the involvement of multiple neurotransmitters in the local regulation of 5-HT release in terminal projection areas. ₂-Adrenergic heteroreceptors on 5-HT terminals in the brain of different species have long been known to inhibit 5-HT release (Gobbi et al., 1990; Raiteri et al., 1990; see Göthert and Schlicker, 1991; Maura et al., 1992b; Blier et al., 1993c). Several findings support the idea that the ₂-adrenergic heteroreceptors have different properties as compared with ₂ autoreceptors regulating NA release in terminal projection areas: 1) they have different pharmacological profiles (Raiteri et al., 1983b; Maura et al., 1992b; Mongeau et al., 1993), 2) ₂ auto- and heteroreceptors are differentially modulated by neuropeptides such as NPY (Martire et al., 1989), and 3) only the former desensitize following long-term treatment with befloxatone, a selective MAO-A inhibitor (see Blier et al., 1993c). The idea of distinct functional properties of adrenoceptors regulating 5-HT and NE release in terminal projection areas has been exploited in the development of new antidepressant drugs. Interestingly, selective ₂ heteroreceptor antagonists, even given acutely, may have the capacity to enhance 5-HT neurotransmission (Haddjeri et al., 1996). Like NE, histamine also exerts a negative regulation of 5-HT release, probably via activation of H₃ heteroreceptors (Fink et al., 1990). On the other hand, acetylcholine (ACh) enhances forebrain 5-HT release by activating nicotinic receptors (Toth et al., 1992; Summers and Giacobini, 1995). The fact that kynurenic acid almost completely prevents the enhancing effect on neurotransmitter release induced by the local administration of nicotine suggests that its effect on 5-HT release is indirect, mediated via glutamic acid release (Toth et al., 1992). Inhibitory amino acids are also involved in the regulation of 5-HT release in cortex and hippocampus. Following systemic administration of benzodiazepine agonists, spontaneous or evoked release of 5-HT in either region has been shown to decrease (Hitchcott et al., 1990; Broderick, 1991; Cheng et al., 1993). Local effects are more ambiguous. In the hippocampal formation, for example, the activation of the GABA_A receptor complex has been shown to produce either a decrease, no effect, or an increase in 5-HT release (Pei et al., 1989; Lista et al., 1990). The fact that the local application of the Cl channel blocker picrotoxin induced a robust increase in hippocampal 5-HT of freely moving rats supports the idea that GABA exerts a tonic inhibition of 5-HT release in this region (Pei et al., 1989). Numerous polypeptides have also been found to locally modulate 5-HT release in projection areas. For example, PYY and pancreatic polypeptide inhibit cortical 5-HT release via the activation of the same presynaptic G protein-coupled receptor as NPY (Schlicker et al., 1991). On the other hand, Substance P and neurokinin A, two coexisting neuropeptides of the tachykinin family, stimulate 5-HT release in this same area (Iverfeldt et al., 1990). Pharmacological activation of local opioid receptors (, , and µ) suggests that endogenous hippocampal opiates may also be involved in the negative regulation of 5-HT release in this area (Passarelli and Costa, 1989; Cui et al., 1994).

7. 5-HT_1B versus 5-HT_1D Receptors. 5-HT_1B-binding sites, as opposed to 5-HT_1A sites, were initially pharmacologically described. They represented the sites that bind spiperone and 8-OH-DPAT with low affinity (Pedigo et al., 1981; Middlemiss and Fozard, 1983) but present high affinity for ¹²⁵I-cyanopindolol (Hoyer et al., 1985a,b). They have also been defined by exclusion as [³H]5-HT-binding sites which are neither of the 5-HT_1A nor of the 5-HT_2C subtype (Blurton and Wood, 1986; Peroutka, 1986; Alexander and Wood, 1987). Originally, these pharmacologically described 5-HT_1B sites were described in rodents (Hoyer et al., 1985a; Waeber et al., 1989a; Waeber and Palacios, 1992) but not in guinea pig, pig, cow, or human brain (Heuring et al., 1986; see Bruinvels et al., 1993 and references within). In nonrodent species, the sites visualized in the presence of saturating concentrations of 8-OHDPAT and mesulergine (Heuring and Peroutka, 1987; Waeber et al., 1988, 1989b) were originally named 5-HT_1D and more recently reclassified as 5-HT_1B. Hence, in spite of their pharmacological differences, sites defined by exclusion in rodent and nonrodent brain would fall under the present 5-HT_1B category. However, it has been established that the sites labeled in this way represent an heterogeneous receptor population composed of at least 5-HT_1B/1D (5-CT-sensitive) and 5-HT_1E (5-CT-insensitive) receptors (Leonhardt et al., 1989; Sumner and Humphrey, 1989; Beer et al., 1992; Lowther et al., 1992; Miller and Teitler, 1992). Furthermore, [³H]5-CT has been shown to label two different subpopulations of 5-HT₁ as high-affinity sites in guinea pig cortex and striatum (Mahle et al., 1991). It was the development of an iodinated radioligand, serotonin-5-O-carboxymethyl-glycyl-¹²⁵I-tyrosinamide (¹²⁵I-GTI; Boulenguez et al., 1991, 1992), that allowed 5-HT_1B binding sites to be directly labeled in human, nonhuman primate, and guinea pig brain, among others (Bruinvels et al., 1991, 1992).

1D

1D

1D

D. Effect of Antidepressant Drug Administration on 5-HT Release

1. Administration of 5-HT Reuptake Blockers. The reported effect of acute 5-HT reuptake blockade depends on the dose used and the region examined. At high doses (10 mg/kg i.p or s.c), the acute administration of fluoxetine, citalopram, or sertraline has been shown to induce an increase in extracellular 5-HT in terminal projection areas such as cortex, striatum, or diencephalon (Dailey et al., 1992; Invernizzi et al., 1992a; Perry and Fuller, 1992; Rutter and Auerbach, 1993, see Fuller, 1994). The increase in extracellular 5-HT is dependent on neuronal firing because it is blocked by TTX (Perry and Fuller, 1992) or 8-OH-DPAT (Rutter and Auerbach, 1993). The latter observation is somewhat puzzling given the fact that ED₅₀ of i.v. doses of different SSRIs to inhibit 5-HT neuron firing are within the 0.1 and 0.5 mg/kg range (Blier and de Montigny, 1980; Blier et al., 1984; Chaput et al., 1986b; Gartside et al., 1995; Hajós et al., 1995; Maudhuit et al., 1995; Kasamo et al., 1996), suggesting that at 10 mg/kg, no matter what SSRI is given, the result would be total shut-down of 5-HT neuron firing activity. Even if rats are much more rapid metabolizers than humans, a dose of 10 mg/kg is 15 to 35 times the therapeutic dose. Hence, other studies have been performed using much lower doses of SSRIs. In such cases, the systemic administration of 1 mg/kg citalopram or 32 µmol/kg sertraline (Invernizzi et al., 1991, 1992a) produced no increase in extracellular 5-HT in cortical projection areas, and experiments in which extracellular 5-HT was simultaneously measured in cortex and raphe nuclei reveal that, following systemic administration of these reuptake inhibitors, there is a preferential increase in extracellular 5-HT in the raphe region (Adell and Artigas, 1991; Bel and Artigas, 1992). It is this increase in somatodendritic extracellular 5-HT that activates the powerful 5-HT_1A autoreceptor feedback loop leading 5-HT neurons to establish their own "ceiling" on the extent to which uptake inhibitors increase extracellular 5-HT in terminal projection areas. Reasoning that long-term reuptake blockade induces a desensitization of somatodendritic 5-HT_1A autoreceptors, Bel and Artigas (1993) proposed the possibility to overcome negative feedback and increase extracellular availability of 5-HT in terminal projection areas even when using low doses of SSRIs. Indeed, they treated rats with 1 mg/kg fluvoxamine (s.c.) for 2 weeks, and at the end of this time period the increase in extracellular concentration of 5-HT in frontal cortex of treated rats, still carrying the osmotic minipump, was similar to that observed following a 10-mg/kg acute i.v. dose (Bel and Artigas, 1992, 1993). These results have been confirmed in a study by Invernizzi et al. (1994), in which sustained treatment with citalopram (10 mg/kg/day for 14 days) facilitated the enhancing effect on terminal 5-HT release produced by a dose of 1 mg/kg of the SSRI administered 24 h after the end of the treatment. Moreover, this study also showed that the reducing effect of a systemic dose of 25 µg/kg 8-OH-DPAT on terminal 5-HT release was abolished following long-term citalopram administration, thus confirming a desensitization of 5-HT_1A autoreceptors (Invernizzi et al., 1994). On the other hand, Bosker et al. (1995a,b) reported that a 14- or 21-day treatment with oral or s.c. fluvoxamine (3 mg/kg or 6.5 mg/kg at study outset, respectively) did not produce desensitization of 5-HT_1A autoreceptors, facilitation of another dose of fluvoxamine, nor increase in hippocampal 5-HT output. If indeed 5-HT_1A autoreceptor desensitization may in part account for the antidepressant effect of prolonged SSRI administration, an acute reduction of the activation of the feedback mechanism should produce an effect similar to that of sustained SSRI treatment. This assumption has been verified in seven of eight placebo-controlled clinical trials showing that in depressed patients the combination of pindolol and an SSRI produced either a significant acceleration of the antidepressant response or a greater proportion of patients presenting a response at the end of the trial (Maes et al., 1996, 1999; Berman et al., 1997; Pérez et al., 1997; Tome et al., 1997; Zanardi et al., 1997, 1998; Bordet et al., 1998). These results are in agreement with results referred to in a previous section, in which blockade of 5-HT_1A autoreceptors was shown to increase extracellular availability of 5-HT in terminal projection areas after the acute administration of an SSRI.

2. Prolonged Administration of MAOIs. In this case the situation is reversed, brain 5-HT being actually increased after sustained blockade of MAO-A (Blier et al., 1986a,b; Celada and Artigas, 1993; Ferrer and Artigas, 1994). On the other hand, similar to SSRIs, the acute systemic administration of selective MAO-A or nonselective MAO-A/MAO-B inhibitors produces an immediate inhibition of 5-HT metabolism and a reduction in 5-HT neuron firing activity (Blier and de Montigny, 1985; Blier et al., 1986a,b). It is not surprising then that, when given acutely, MAOIs and SSRIs, produce a preferential increase in extracellular 5-HT in midbrain raphe nuclei as compared to terminal projection areas. In contrast, prolonged MAOI administration increases extracellular availability of 5-HT to a similar extent in pre- and postsynaptic projection areas (Celada and Artigas, 1993; Ferrer and Artigas, 1994; Bel and Artigas, 1995) with a time course similar to that of the desensitization of somatodendritic 5-HT_1A autoreceptors (Blier and de Montigny, 1985; Blier et al., 1986a,b; Piñeyro and Blier, 1996). In long-term experiments, tranylcypromine given at a dose that had no effect in acute experiments (0.5 mg/kg/day s.c. for 14 days) produced a greater increase in extracellular cortical availability of 5-HT than the acute administration of a dose six times higher, even if the increases in tissue 5-HT concentrations were of 40 and 700%, following the long-term/low-dose and acute/high-dose treatments, respectively. These observations suggest that even if the increase in intracellular 5-HT is almost 20-fold higher following an acute high dose of tranylcypromine, the neurotransmitter is trapped within 5-HT terminals, only a small part of it being available for release (intracellular:extracellular ratio of 5-HT increase in frontal cortex: 11.6 and 5.5 for acute/high-dose and prolonged/low-dose treatments, respectively). In part, increased extracellular availability of 5-HT after long-term MAOI administration is due to desensitization of 5-HT_1A autoreceptors and recovery of 5-HT neuron firing frequency. However, an increase in terminal 5-HT release (hippocampus, cortex, and hypothalamus) is seen not only in vivo in the whole animal, but also in vitro in slices containing only 5-HT terminals (Blier and de Montigny, 1985; Blier et al., 1986a,b; Blier and Bouchard, 1994; Mongeau et al., 1994), indicating a 5-HT_1A-independent enhancement of neurotransmitter release. Unlike long-term treatment with SSRIs, the sensitivity of 5-HT autoreceptors remains unchanged (Blier et al., 1986a,b; Blier and Bouchard, 1994). The question arising then is, what is the mechanism involved in increasing the releasable amount of 5-HT after long-term MAOI administration? It has long been known that the A form of MAO catalyzes the oxidative deamination not only of 5-HT but also of NE (Hall et al., 1969; Yang and Neff, 1973), and that 5-HT terminals are endowed with inhibitory ₂-adrenergic heteroreceptors (Göthert et al., 1980; Göthert et al., 1981; Maura et al., 1982). More recently, our laboratory has shown that enhanced 5-HT release after sustained MAO-A inhibition correlates with the production of ₂-adrenergic heteroreceptor desensitization by these treatments (Blier et al., 1986a,b; Blier and Bouchard, 1994; Mongeau et al., 1994). Moreover, destruction of the NE system impairs the heteroreceptor desensitization caused by the prolonged administration of the reversible MAO-A inhibitor befloxatone (Mongeau et al., 1994).

3. Antidepressants with ₂ Adrenoceptor Antagonistic Properties. Antidepressants with ₂ antagonists like mianserin and (±)-mirtazapine, whose long-term administration increases the duration of suppression of firing of CA₃ pyramidal neurons produced by 5-HT pathway stimulation, have also been shown to desensitize ₂-adrenergic heteroreceptors on 5-HT neurons (Mongeau et al., 1994; Haddjeri et al., 1997). Other studies of the neurochemical effects of long-term mianserin administration (Raiteri et al., 1983a; Schoups and De Potter, 1988) reported no change in the sensitivity of these receptors. A likely explanation for this discrepancy could be that in the latter studies mianserin was injected i.p., whereas in the studies in which they induced ₂-adrenergic heteroreceptor desensitization, antidepressant drugs were delivered continuously via osmotic minipumps implanted s.c.

4. Activation of 5-HT_1A Receptors by 5-HT_1A Agonists. Direct activation of presynaptic 5-HT_1A receptors by 5-HT_1A agonists causes inhibition of 5-HT cell firing, synthesis, and release in forebrain areas (Blier and de Montigny, 1987; Hjorth and Magnusson, 1988; Sharp et al., 1989a; Schechter et al., 1990; Godbout et al., 1991). At postsynaptic receptors, biochemical and electrophysiological experiments in the hippocampus (Yocca and Maayani, 1985; Yocca et al., 1986; Andrade and Nicoll, 1987a,b) have shown that clinically available 5-HT_1A agonists such as buspirone (Glitz and Pohl, 1991) act as partial agonists and may inhibit cAMP formation pyramidal neuron firing activity. Since upon acute administration there is net decrease in forebrain extracellular availability of 5-HT and their beneficial clinical antidepressant and anxiolytic effects are usually not observed until a few weeks of administration (see Charney et al., 1990; Glitz and Pohl, 1991), it appears that the presynaptic effects of 5-HT_1A agonists override their postsynaptic actions. Based on electrophysiological results, it has been proposed that tolerance develops to the autoreceptor-mediated effects (Blier and de Montigny, 1987; Schechter et al., 1990; Godbout et al., 1991), and a combination of normal 5-HT firing activity along with simultaneous activation of postsynaptic normosensitive receptors by the drug and endogenous 5-HT may account for their therapeutic actions (see Haddjeri et al., 1998). Neurochemical studies demonstrating 5-HT_1A autoreceptor desensitization after sustained 5-HT_1A agonist administration have been less consistent, with positive (Kreiss and Lucki, 1992) and negative results (Sharp et al., 1993a; Söderpalm et al., 1993). A possible explanation for the discrepancy between electrophysiological and neurochemical studies could be that the sensitivity of 5-HT_1A autoreceptors was tested directly on 5-HT neurons in the former and systemically in the latter. As previously discussed, systemic administration of 5-HT_1A agonists may modify 5-HT neuron electrophysiological and neurochemical activities acting not only at presynaptic but also postsynaptic 5-HT_1A receptors. Furthermore, the hypothesis generated by electrophysiological data predicts that concurrent administration of a 5-HT_1A agonist with a selective 5-HT_1A autoreceptor blocker should produce rapid enhancement of 5-HT neurotransmission (Blier and de Montigny, 1994), and indeed the administration of buspirone (20 mg/day) with pindolol (2.5 mg/kg thrice daily) produced quick reductions of depressive symptomatology in patients treated with this drug combination (Blier et al., 1997).

Following release, 5-HT is actively cleared from the synaptic cleft by a high-affinity transporter located on presynaptic neuronal membranes (Kuhar et al., 1972; see Kanner and Schuldiner, 1987; O'Reilly and Reith, 1988), which functions in series with another type of carrier, the vesicular transporter, that sequesters intracellular 5-HT within secretory vesicles. The carriers taking up neurotransmitter from the extracellular space into the neuron are integral membrane proteins with 12 transmembrane spanning domains, they couple reuptake to Na⁺ and Cl displacement across the plasma membrane, and are encoded by a closely related gene family, the type I or plasma membrane Na⁺/Cl-coupled transporter family, which includes GABA, catecholamine, and 5-HT transporters. The vesicular transporter belongs to a different gene superfamily and will not be considered further (for reviews, see Uhl and Hartig, 1992; Amara and Kuhar, 1993; Lester et al., 1994).

1. Molecular Characteristics of the 5-HT Transporter (SERT). Two main strategies have been used in an attempt to identify the molecular characteristics of the SERT, i.e., biochemical purification and cloning of cDNA coding for the protein. Using digitonin as a detergent, the SERT in rat brain and human platelets has been solubilized and purified in a conformational state that retained a pharmacological profile almost identical with that observed in native membrane preparations (Biessen et al., 1990; Graham et al., 1991; Launay et al., 1992). In a further step, the human placental SERT has been reconstituted after purification, displaying not only the same antidepressant-binding profile as the native carrier but also NaCl-dependent 5-HT transport (Ramamoorthy et al., 1993b). The molecular weight of this functional protein isolated from human placenta is 300,000 (Ramamoorthy et al., 1993b). On the other hand, the other purified proteins have a molecular weight ranging between 55,000 and 78,000 (Biessen et al., 1990; Launay et al., 1992) and that of the cloned SERT itself is about 70,000 (Blakely et al., 1991; Lesch et al., 1993; Ramamoorthy et al., 1993b). Thus, in analogy with the Na⁺/glucose transporter (Stevens et al., 1990), it has been proposed that the SERT may exist as an homotetramer (Ramamoorthy et al., 1993b). However, although dimeric concatenated constructs of the transporter have been shown to possess 5-HT transport activity similar to the monomer's, concatenated tetramers have substantially lower activity (Chang et al., 1994). The latter observation does not preclude the existence of functional tetramers since simple functional monomers or dimers could associate and efficiently transport 5-HT.

2. The Mechanism of 5-HT Uptake. The mechanism of 5-HT uptake has been thoroughly studied in platelets (Rudnick, 1977; Nelson and Rudnick, 1979, 1982), mouse brain plasma membrane vesicles (O'Reilly and Reith, 1988; Reith et al., 1989), human placenta brush-border membrane vesicles (Cool et al., 1990; Ramamoorthy et al., 1993b), and, more recently, after stable expression of cloned SERTs, in different expression systems (Blakely et al., 1991; Hoffman et al., 1991; Ramamoorthy et al., 1993a; Corey et al., 1994; Demchyshyn et al., 1994; Gu et al., 1994; Mager et al., 1994). 5-HT is the specific substrate for the transporter, and K_m values reported across different studies are summarized in Table 8. Reported values for the turnover number (maximal number of 5-HT molecules carried by one transporter in 1 min) in different systems have also been variable: 500 5-HT molecules/porcine SERT in platelet membrane vesicles/min (Talvenheimo et al., 1979), 110 5-HT molecules/rSERT expressed in parental LLC-PK₁ cells/min (Gu et al., 1994), or 30 5-HT molecules/rSERT expressed in Xenopus oocytes/min (Mager et al., 1994). Tryptamine and its derivatives, as well as 5-HT derivatives and phenylethyamines such as (+)-amphetamine and PCA, are additional substrates for the SERT (Segonzac et al., 1984; Wölfel and Graefe 1992; Mager et al., 1994). On the other hand, tryptophan, 5-hydroxytryptophan, 5-HIAA, histamine, and the catecholamines NE and DA, at concentrations as high as 10 µM, do not significantly bind to this carrier (Ramamoorthy et al., 1993; Hoffman et al., 1991; Wölfel and Graefe, 1992; Corey et al., 1994; Barker and Blakely, 1995). However, higher concentrations of DA, 20 to 40 µM, have been reported not only to bind to the transporter but to exchange with [³H]5-HT (Ramamoorthy et al., 1993; Wölfel and Graefe, 1992; Corey et al., 1994).

3. Anatomical and Cellular Localization of the SERT. In the periphery, the SERT is expressed in enteric 5-HT neurons (Wade et al., 1996) and non-neuronal cells such as mast cells (Gripenberg, 1976), crypt epithelial cells, and very discretely in enterochromaffin cells (Wade et al., 1996). It is also found in platelets (Rudnick, 1977; Qian et al., 1995), lung membranes (Qian et al., 1995), and maternal brush-border of syncytiotrophoblasts (Cool et al., 1990; Ramamoorthy et al., 1993b).

4. Pharmacological Properties of the SERT. The SERT is the pharmacological target for various therapeutic and abused substances. Compounds that block 5-HT reuptake such as tricyclic antidepressants and SSRIs are in the first group, whereas stimulants such as amphetamine and its derivatives, which block 5-HT uptake and promote release, are part of the latter category. Cocaine, although it binds and blocks the 5-HT carrier, is believed to exert most of its behavioral effects by blocking DA rather than 5-HT uptake (Woolverton and Kleven, 1992; Barker and Blakely, 1995; Caron, 1996).

5. Tianeptine, a Class by Itself? Tianeptine is a tricyclic agent (dibenzothiazepine nucleus) with a long (aminoheptanoic acid) lateral chain (Labrid et al., 1988). In France, it is indicated for the treatment of "neurotic and reactional depressive conditions" and has been claimed to be a unique type of antidepressant that produces its effect by enhancing 5-HT uptake.

The results from radioligand and functional studies following prolonged antidepressant treatments have often been found to be controversial (Table 9), and the reasons for this controversy have been attributed to three main limitations: 1) the use of [³H]imipramine as a radioligand which, due to its binding to heterogeneous sites, may confound interpretation of results (it is worth noting that when the effect of long-term antidepressant administration was assessed on high- and low-affinity [³H]imipramine sites, treatment-induced decreases in affinity for imipramine were observed at both sites; Hrdina, 1987); 2) examination of the effects of long-term antidepressants being limited in most cases to cortical or hippocampus homogenates which, unlike autoradiographic analysis, prevents modest changes in discrete brain areas to be assessed; and 3) the use of low doses and mainly administration routes (i.p. and s.c. injections; p.o. administration) that cause important fluctuations (peak and trough) in the plasmatic concentration of antidepressant drugs may also account for a high proportion of negative results. Nevertheless, if these limitations are taken into account, certain general conclusions may be drawn.

When selective ligands are used to label the SERT, repeated electroconvulsive shocks (ECS) and long-term MAOI administration were the only types of treatment that consistently showed an increase in the number of SERTs (Kovachich et al., 1992; Hayakawa et al., 1995). The use of [³H]imipramine as a radioligand has shown increases in SERT sites after ECS or deprenyl administration (Zsilla et al., 1983; Barkai, 1986). The studies that yielded negative results were either performed in homogenates, used smaller number of ECS, or p.o./i.p. treatments (Zsilla et al., 1983; Graham et al., 1987; Gleiter and Nutt, 1988; Cheetham et al., 1993). In hybridization as in binding studies either no change or increased mRNA hybridization was found in the midbrain raphe complex after prolonged clorgyline administration (Lesch et al., 1993; Lopéz et al., 1994). Interestingly, in studies yielding positive results, treatment was given i.p. (López et al., 1994), whereas in those in which no change was observed, steady drug plasma levels were achieved by using osmotic minipumps (Lesch et al., 1993). The fact that the dose used in the latter (Lesch et al., 1993) was four times smaller than the one used in the former (López et al., 1994) may explain the results obtained. In the case of SSRIs, functional studies show a decrease in V_max in cortical and amygdala synaptosomes of rats that had received prolonged fluoxetine or sertraline treatment, as well a decrease in maximal [³H]5-HT uptake in cortical and hippocampal slices obtained from rats that had received paroxetine for 21 days (Hrdina, 1987; Butler et al., 1988; Piñeyro et al., 1994). In rats that had received citalopram administered in their diets, no functional changes were observed when assessed in whole-brain synaptosomes (Hyttel et al., 1984). Sustained administration of sertraline and paroxetine decreases the number of SERTs in amygdala, perirhinal cortex, hippocampus, and rat frontal cortex (Kovachich et al., 1992; Piñeyro et al., 1994), but fluoxetine induced an increase in [³H]paroxetine binding in the two latter areas (Hrdina and Vu, 1993). However, at the same dose as in the last study and using the same route of administration, fluoxetine was seen to decrease V_max in cortical synaptosomes (Hrdina, 1987). Since synaptosomes in which V_max was decreased were prepared from frontal cortex, and the increased numbers in SERT sites was observed in frontoparietal, striatal, and hippocampal cortices, a possible explanation for these two sets of divergent observations following fluoxetine treatment could be accounted for by regional differences in the adaptative properties of the SERTs. Hybridization studies indicate a decrease in SERT mRNA in midbrain raphe nuclei after prolonged fluoxetine administration via osmotic minipumps (Lesch et al., 1993). Whether this decrease in transcriptional activity is secondary to a decrease in SERT protein turnover (in agreement with at least a transitory increase in the number of uptake sites), or it is the cause for a reduction in SERT number, cannot be deduced from these results. Once again, studies in which prolonged SSRI administration induced no changes in SERT activity or binding sites were performed in homogenates and/or drugs were administered i.p. (Graham et al., 1987; Kovachich et al., 1992; Dewar et al., 1993; Spurlock et al., 1994). The fact that, in spite of both sertraline and citalopram being administered i.p., only the first produced a reduction in [³H]cyanoimipramine binding (Kovachich et al., 1992), and cortical 5-HT V_max (Butler et al., 1988) is probably due to the fact that, of these two SSRIs, only sertraline has an active metabolite whereas citalopram is rapidly inactivated (see Piñeyro et al., 1994). Finally, results from prolonged tricyclic administration may not be systematized: imipramine and desipramine have been shown to decrease [³H]imipramine but not [³H]paroxetine binding whereas chlomipramine, not only a tricyclic but also a potent and selective 5-HT uptake blocker, did not produce any significant change in SERT sites or its mRNA (Table 9). In a recent study by our group, three main lines of evidence indicate a reduction in 5-HT uptake activity in the rat hippocampus after prolonged administration of the SSRI paroxetine: 1) a decrease in the density of SERT sites, 2) a tolerance to the in vivo electrophysiological effects of the drug, and 3) a decrease of the in vitro [³H]5-HT uptake capacity. A reduction in the total number of [³H]paroxetine-binding sites in cortical and hippocampal membranes and a decrease in the amount of [³H]5-HT taken up by dorsal raphe slices indicate that the plasticity of the SERT occurs not only in multiple projection areas, but also in the cell body and dendrites of 5-HT neurons (Piñeyro et al., 1994). Superfusion experiments in midbrain raphe slices provide additional evidence of the functional consequences of somatodendritic SERT down-regulation: after prolonged administration of paroxetine, the electrically evoked release of [³H]5-HT in rat and guinea pig midbrain raphe slices is much higher than that observed after sustained befloxatone treatment (El Mansari and Blier, 1996; Piñeyro and Blier, 1996). That the enhanced somatodendritic output of [³H]5-HT is due to SERT desensitization was demonstrated by the fact that superfusion of midbrain raphe slices with medium containing 1 µM paroxetine (introduced 20 min before the stimulations) produced a 50% increase in slices obtained from saline- or befloxatone-treated rats but remained unchanged in slices obtained from rats that had received paroxetine for 21 days. Furthermore, in the frontal cortex of long-term paroxetine-treated guinea-pigs, there is no 5-HT_1D autoreceptor desensitization (unlike in the hippocampus and the hypothalamus), and electrically evoked [³H]5-HT release is still enhanced (Blier and Bouchard, 1994; El Mansari and Blier, 1996). This is due to a desensitization of the SERT that was demonstrated by the reduced effectiveness of the same dose of paroxetine in enhancing [³H]5-HT uptake in frontal cortex slices of guinea pigs treated with the SSRI for 21 days, as compared to 2 days (Blier and Bouchard, 1994).

Based on the previous analysis, the observations in Table 9 could be summarized by saying that an increase in 5-HT uptake may be expected after repeated ECS or sustained MAOI administration, whereas a decrease in this function is more likely to occur after prolonged SSRI treatment. Hence, it seems that the antidepressant effect is not correlated with a specific adaptative response of the 5-HT carrier.

Apart from drugs, numerous physiological processes regulate 5-HT uptake activity. Understanding them and finding the similarities they might have with any given pharmacological treatment may help us to understand how antidepressant drugs or ECS regulate SERT activity. ACTH and ACTH fragments up-regulate SERT expression during 5-HT neuron differentiation (Azmitia and De Kloet, 1987; Eaton and Whittemore, 1990). A transcription increase also occurs in raphe neurons of aged rats, an effect that may compensate for 5-HT leakage from degenerating terminals and/or increased release induced by age-related decline in terminal autoreceptor regulation (Meister et al., 1995). Similarly, 5-HT neuron sprouting in raphe nuclei following a 5,7-DHT lesion has been correlated with an increase in [³H]paroxetine B_max values in the brainstem (Pranzatelli and Martens, 1992). Up-regulation of the SERT secondary to transcriptional activation may occur via an increase in cAMP (Cool et al., 1991; King et al., 1992; Ramamoorthy et al., 1993a) which has been recently proposed to induce the activation of the hSERT gene promoter via immediate early gene products such as transcription factors of the c-fos/c-jun family (Heils et al., 1995). cAMP-independent mechanisms activated by interleukin-1 (Ramamoorthy et al., 1995b) or the PKC inhibitor staurosporine (Ramamoorthy et al., 1995a) may also activate SERT mRNA production. Alternatively, 5-HT uptake activity may be rapidly enhanced without altering the transporter density by a mechanism involving transporter phosphorylation/dephosphorylation. Nitric oxide-cGMP pathway activation has been shown to have this effect (Miller and Hoffman, 1994). Phosphorylation/dephosphorylation of the transporter via other signaling pathways such as PKC and calmodulin induce an opposite effect, reducing 5-HT uptake (Myers et al., 1989; Anderson and Horne, 1992; Jayanthi et al., 1994). It is unlikely, however, that these rapid regulatory responses may account for the decrease in 5-HT uptake observed after prolonged SSRI administration, since the former take place within 1 h or less after treatment, and in the case of paroxetine, tolerance to the drug was not expressed within a 48-h period but rather after a various number of days of treatment (Piñeyro et al., 1994). Furthermore, a recent study indicating that reduction in SERT mRNA does not always result in a decrease in the number of SERT in 5-HT neurons (Yu et al., 1994) suggests that a decrease in transcription might not always serve as an explanation for a long-latency down-regulation in the SERT. On the other hand, a dysregulated expression of the SERT gene (Heils et al., 1995) has been suggested as a possible explanation for one of the most consistent findings in biological psychiatry: the disease-associated decrease in brain and platelet 5-HT uptake sites observed in patients with affective disorders (see Lesch and Bengel, 1995).

	IV. Concluding Remarks

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This article provided an overview of the morphological aspects of the 5-HT system, analyzed the autoregulatory mechanisms with which 5-HT neurons are endowed and how they contribute to the regulation of firing activity, neurotransmitter release, and reuptake, along with the drugs that modify these functions. Special attention has been given to antidepressant drug treatments, their long-term effects, and possible strategies that may allow one to bypass this complex autoregulatory machinery to achieve quicker and/or more effective antidepressant responses. Autoregulatory processes not previously considered as targets for antidepressant drugs, such as regulation of somatodendritic 5-HT release by non-5-HT_1A receptors, have also been considered. Although many of the mechanisms described may still be a matter of debate, there is no doubt that a comprehensive approach to the physiology of the 5-HT system is paving the road to a better pharmacological management of affective and anxiety disorders.