Serotonergic modulation of the limbic system

Neuroscience and Biobehavioral Reviews 30 (2006) 203–214 www.elsevier.com/locate/neubiorev

Review

Serotonergic modulation of the limbic system Julie G. Hensler* Department of Pharmacology, MC 7764, University of Texas Health Science Center-San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA

Abstract The limbic system is composed of cortical as well as subcortical structures, which are intimately interconnected. The resulting macrostructure is responsible for the generation and expression of motivational and affective states. Especially high levels of serotonin are found in limbic forebrain structures. Serotonin projections to these structures, which arise from serotonergic cell body groups in the midbrain, form a dense plexus of axonal processes. In many areas of the limbic system, serotonergic neurotransmission can best be described as paracrine or volume transmission, and thus serotonin is believed to play a neuromodulatory role in the brain. Serotonergic projections to limbic structures, arising primarily from the dorsal and median raphe nuclei, compose two distinct serotonergic systems differing in their topographic organization, electrophysiological characteristics, morphology, as well as sensitivity to neurotoxins and perhaps psychoactive or therapeutic agents. These differences may be extremely important in understanding the role of these two serotonergic systems in normal brain function and in mental illness. Central serotonergic neurons or receptors are targets for a variety of therapeutic agents used in the treatment of disorders of the limbic system. q 2005 Elsevier Ltd. All rights reserved. Keywords: Serotonergic innervation; Dorsal raphe nucleus; Median raphe nucleus; Hippocampus; Cortex; Amygdala; Nucleus accumbens

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Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two distinct central serotonergic systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Electrophysiological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Morphological characteristics of serotonergic axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Differential expression of the serotonin transporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ascending serotonergic projections to limbic structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Serotonergic innervation of frontal cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Serotonergic innervation of hippocampus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Serotonergic innervation of entorhinal cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Serotonergic innervation of nucleus accumbens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Serotonergic innervation of the amygdaloid complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Serotonergic innervation of the anterior hypothalamus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volume transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Synaptic specializations, or lack there of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Extrasynaptic location of serotonin receptors and the serotonin transporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disorders of limbic system and treatment strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Major depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Anxiety disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: C1 210 567 4236; fax: C1 210 567 4303. E-mail address: [email protected]

0149-7634/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neubiorev.2005.06.007

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5.3. Schizophrenia . . . . . . . . . . . . . . . . . . . . . . Some considerations in the treatment of disorders of the limbic system . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Overview The limbic system is composed of cortical as well as subcortical structures, which are intimately interconnected. The major structures of the limbic system include prefrontal cortex, cingulate cortex, entorhinal cortex, hippocampus, nucleus accumbens (ventral striatum), ventral pallidum, amygdala, and anterior hypothalamus (see Swanson and Petrovich, 1998; Kandel et al., 2000; Heimer, 2003). Connections between these structures form complex circuits. Furthermore, projections between structures are organized in a strict topographical manner (e.g. van Groen et al., 2002; Heidbreder and Groenewegen, 2003). The resulting macrostructure is primarily responsible for the generation and expression of motivational and affective states. As emphasized by Morgane et al. (2005), such complex psychological states specifically pertaining to motivation and emotion warrant consideration of the corresponding anatomical components as a whole, in that these psychological processes are not performed by single structures but by complexes of interacting systems in the brain. The functional consequences of complex circuits interconnecting these structures include memory processing, emotional association with memory, judgment, affect, and motivation or the organization of planned actions. In humans, disorders associated with dysfunction of the limbic system include schizophrenia, obsessive–compulsive disorder, major depression, and drug abuse. The present review focuses on the role of the neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) in the limbic system. Especially high levels of serotonin are found in limbic forebrain structures. Serotonin projections to these forebrain structures, which arise from serotonergic cell body groups in the midbrain, form a dense plexus of axonal processes. These serotonergic projections, arising primarily from the dorsal and median raphe nuclei, compose two distinct serotonergic systems differing in their electrophysiological characteristics, topographic organization, morphology, as well as sensitivity to neurotoxins and perhaps psychoactive or therapeutic agents. These differences may be extremely important for understanding the role of these two serotonergic systems in normal brain function and in mental illness. In many areas of the limbic system, serotonergic neurotransmission can best be described as paracrine or volume transmission, and thus serotonin is believed to play a neuromodulatory role in the brain. Central serotonergic neurons or receptors are targets for a variety of therapeutic agents used in the treatment of disorders of the limbic system. In many

instances, review articles are cited, in addition to original papers, in the hope that the interested reader will pursue these topics further.

2. Two distinct central serotonergic systems Serotonin-containing neuronal cell bodies are restricted to discrete groups of cells or nuclei located along the midline of the brainstem. Their axonal projections, however, innervate nearly every area of the central nervous system. Dahlstro¨m and Fuxe (1964), using the Falck–Hillarp technique of histofluorescence, observed that the majority of serotonergic soma were found in cell body groups previously designated by Taber et al. (1960) as the raphe nuclei based on cytoarchitectural criteria, i.e. on cell body structural characteristics and organization. Dahlstro¨m and Fuxe described nine groups of serotonin-containing cell bodies, designated B1–B9, which correspond for the most part with the raphe nuclei. The cell bodies of most serotonergic neurons are found largely within the boundaries of the raphe nuclei. However, it is important to note that although the serotonergic cell groups closely match the raphe nuclei, some serotonergic neuronal cell bodies are found outside the raphe nuclei, and not all the cell bodies within the raphe nuclei are serotonergic (Descarries et al., 1982; Kohler and Steinbusch, 1982; see also Molliver, 1987; Tork, 1990). The largest group of serotonergic cells, group B7 of Dahlstro¨m and Fuxe, is continuous with a smaller group of serotonergic cells, B6. Groups B6 and B7 are considered together as the dorsal raphe nucleus, with B6 being its caudal extension. Another prominent serotonergic cell body group is B8, which corresponds to the median raphe nucleus or nucleus centralis superior. Group B9, part of the ventrolateral tegmentum of the pons and midbrain, forms a lateral extension of the median raphe and therefore, is not considered one of the midline raphe nuclei. Ascending serotonergic projections innervating the forebrain, including limbic areas, arise primarily from the dorsal raphe, median raphe, and B9 cell group (Parent et al., 1981; Kohler and Steinbusch, 1982; see also Molliver, 1987). The other raphe nuclei, B1–B4, are more caudally situated (mid-pons to caudal medulla) and contain a smaller number of serotonergic cells, which give rise to serotonergic axons that project within the brainstem and to the spinal cord. Afferent connections to the raphe nuclei include connections between the dorsal and median raphe nuclei. Horseradish peroxidase (HRP) retrograde cell labeling

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techniques revealed intrinsic connections within the dorsal raphe nucleus, as well as input to the dorsal raphe from the median raphe nucleus (Mosko et al., 1977). Anterograde tracing studies with Phaseolus vulgaris leucoagglutinin (PHA-L) have shown moderately dense projections from the dorsal raphe nucleus to the median raphe nucleus (Vertes, 1991). The existence of serotonergic dendro-dendritic synaptic contacts and axon terminals suggests direct local interactions between serotonergic neurons within the dorsal raphe nucleus (Descarries et al., 1982; Kapadia et al., 1985; Chazal and Ralston, 1987). Such innervation may have considerable physiological and/or pharmacological relevance as serotonin released in the vicinity of serotonergic cell bodies regulates the firing of serotonergic neurons through the activation of somatodendritic autoreceptors. The raphe nuclei also receive input from other cell body groups in the brainstem such as the substantia nigra and ventral tegmental area (dopamine), superior vestibular nucleus (acetylcholine), locus coeruleus (norepinephrine), nucleus prepositus hypoglossi and nucleus of the solitary tract (epinephrine). Other afferents include neurons from the hypothalamus, cortex, and limbic forebrain structures such as the amygdala (see Jacobs and Azmitia, 1992). 2.1. Electrophysiological properties The serotonergic neurons of the median and dorsal raphe nucleus differ in their active and passive electrophysiological characteristics, and in their inhibition by somatodendritic autoreceptor activation (Kirby et al., 2003; Beck et al., 2004). In studies combining electrophysiological recording techniques with immunohistochemical identification of serotonin-containing neurons, Beck and co-workers have identified the membrane properties and receptor-mediated responses of both serotonergic and non-serotonergic neurons in the dorsal and median raphe nuclei. In the dorsal raphe, there are non-serotonergic neurons that have electrophysiological characteristics, which are similar to previously determined, classic properties of serotonergic neurons (i.e. slow rhythmic activity in spontaneous active cells, broad action potential and large afterhyperpolarization potential). However, in the median raphe, the non-serotonergic neurons have very different electrophysiological characteristics than those of serotonergic neurons (e.g. smaller after hyperpolarization amplitude and t1/2, shorter action potential duration, smaller membrane resistance). Furthermore, nonserotonergic as well as serotonergic neurons in the dorsal raphe are responsive to drugs activating 5-HT1A receptors. These data suggest that in the dorsal raphe nucleus 5-HT1A receptors function not only as somatodendritic autoreceptors, but also as heteroreceptors (i.e. 5-HT1A receptors on non-serotonergic neurons). In contrast, non-serotonergic neurons in the median raphe are not responsive to 5-HT1A receptor agonists, suggesting that in the median raphe, this receptor functions only as the somatodendritic autoreceptor

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(Kirby et al., 2003; Beck et al., 2004). These differences in the cellular electrophysiological characteristics and responsiveness to 5-HT1A receptor activation between the median and dorsal raphe neurons may be extremely important in understanding the role of the two serotonergic systems, which arise from these distinct cell body groups, in normal physiological processes and in the etiology and treatment of disorders of the limbic system. 2.2. Morphological characteristics of serotonergic axons Serotonergic axons and terminals appear to exhibit morphological differences related to the raphe nucleus of origin. The anterograde labeling of neurons following sitespecific injections of P. vulgaris leucoagglutinin (PHA-L) into the dorsal or median raphe has shown that axons from the median raphe nucleus are relatively coarse with large spherical varicosities (type M fibers). In contrast, axons from the dorsal raphe nucleus are very fine and typically have small, pleomorphic varicosities (type D fibers) (Kosofsky and Molliver, 1987). The serotonergic nature of these different classes of fibers arising from the dorsal and median raphe nucleus was determined in experiments combining serotonin immunocytochemistry with anterograde transport of PHA-L. Double-label analysis of fluorescence for PHA-L transport (rhodamine) and serotonin (FITC) allowed the direct comparison of individual fibers labeled by PHA-L and with serotonin

Fig. 1. The dual serotonergic system innervating the forebrain. The thin varicose axon system (D fibers) arises from the serotonergic cell bodies within the dorsal raphe (DR) with fibers that branch profusely in their target areas. The thick, non-varicose axon system (M fibers) arises from the serotonergic cell bodies within the median raphe (MnR) with fibers that branch with beaded, round or oval varicosities. PAG, periaqueductal gray matter; IC, inferior colliculus; ml, medial lemniscus (from Tork, 1990).

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immunoreactivity, and confirmed the morphological characteristics of serotonergic neurons arising from the dorsal and median raphe nucleus (Kosofsky and Molliver, 1987) (see Fig. 1). These morphologically distinct serotonergic projections to the forebrain exhibit differential vulnerability to neurotoxic amphetamine derivatives. Fine serotonergic axons with minute varicosities (type D fibers) appear to be more vulnerable to certain neurotoxic amphetamine derivatives, e.g. 3,4-methylenedioxymethamphetamine (MDMA, or ‘ecstasy’) and parachloroamphetamine (PCA). Beaded serotonergic axons characterized by large, spherical varicosities appear to be more resistant to the neurotoxic effects of these drugs (Mamounas and Molliver, 1988; Mamounas et al., 1991; see Molliver et al., 1990). The nuclei of origin of drug-sensitive versus drug-resistant neurons were confirmed in studies utilizing the retrograde transport of Fluoro-Gold injected into the cortex after PCA administration to rats. PCA treatment results in a marked loss of fine serotonergic axon terminals in cortex and a concomitant reduction in the number of retrogradely labeled neurons in the dorsal raphe nucleus; beaded axons are spared and the number of labeled neurons in the median raphe remains unchanged after PCA treatment (Mamounas and Molliver, 1988). Blockade of the serotonin transporter (SERT) prevents the neurotoxic effects of amphetamine derivatives, indicating that activity of this transporter is critical for the neurotoxic effects of these drugs (Schmidt and Gibb, 1985; Fuller and Snoddy, 1986; see also Molliver et al., 1990). 2.3. Differential expression of the serotonin transporter The differential vulnerability of serotonergic axons arising from the dorsal or median raphe has led to the suggestion that two axonal types may have different densities of the SERT, which would result in differences in the ratio of surface area to volume such that a compound taken up by large varicosities may be diluted in a greater volume (see Molliver et al., 1990). Indeed in an elegant study combining immunocytochemistry for serotonin and the SERT, Brown and Molliver (2000) found that in the nucleus accumbens, two distinct types of serotonergic axons differ in not only in their regional distribution and morphology, but also in SERT expression, as well. Most regions of the nucleus accumbens are innervated by fine serotonergic axons that express the SERT, but in the caudal nucleus accumbens shell, nearly all serotonergic axons lack SERT and have large spherical varicosities. It is these varicose axons in the shell that are spared following administration of neurotoxic amphetamine derivatives (Brown and Molliver, 1963). The differential expression of the SERT in serotonergic neurons arising from the dorsal raphe nucleus versus the median raphe nucleus suggests that these two distinct serotonergic systems are differentially modulated by drugs that block serotonin reuptake (e.g. tricyclic antidepressants and selective serotonin reuptake

inhibitors), and thus, in the treatment of affective disorders such as major depression, anxiety and panic disorder, it is tempting to speculate that it is the serotonergic neurons arising from the dorsal raphe nucleus that are the therapeutic target.

3. Ascending serotonergic projections to limbic structures The dorsal periventricular path and the ventral tegmental radiations are the two main ascending serotonergic pathways from the midbrain raphe nuclei to the forebrain. Both pathways converge in the caudal hypothalamus where they join the medial forebrain bundle and axons of dopaminergic (A8, A9, A10) and noradrenergic (A6) cell body groups (Moore et al., 1978; Parent et al., 1981; see also Molliver, 1987; Vertes, 1991). The dorsal and median raphe nuclei give rise to distinct serotonergic projections to structures of the limbic system. These serotonergic projections to the forebrain from the dorsal and median raphe form dissimilar patterns of innervation, which are partially overlapping. For example, serotonergic processes arising from the dorsal raphe heavily innervate the prefrontal cortex, lateral septum, amygdala, striatum and ventral hippocampus (see Molliver, 1987). Structures receiving predominant serotonergic innervation from the median raphe nucleus include the dorsal hippocampus, medial septum and hypothalamus. Most areas of cortex receive convergent projections from both the dorsal and median raphe, with regional differences in the number of cells projecting from each nucleus (see Molliver, 1987). Within the dorsal and median raphe, cells are organized in particular zones or groups that send axonal processes to specific areas of brain, e.g. the frontal cortex receives heavy innervation from the rostral and lateral subregions of the dorsal raphe nucleus (O’Hearn and Molliver, 1984; Vertes, 1991). The hippocampus receives moderately dense projections from the caudal dorsal raphe and essentially none from the rostral dorsal raphe (Vertes, 1991). Moreover, raphe neurons send collateral axons to areas of brain that are related in function such as the amygdala and hippocampus, hippocampus and entorhinal cortex, or substantia nigra and caudate putamen (Imai et al., 1986; Kohler and Steinbusch, 1982). The highly organized innervation of forebrain structures by serotonergic neurons of the raphe nuclei is quite interesting in that it implies independent functions of sets of serotonergic neurons dependent on their origin and terminal projections (see Molliver, 1987). 3.1. Serotonergic innervation of frontal cortex The frontal cortex is densely innervated by serotonergic terminals. After labeling by uptake of [3H]5-HT in brain slices, early studies estimate the mean density of the

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innervation at 5.8 million varicosities per cubic millimeter of tissue (Audet et al., 1989). Although serotoninimmunoreactive axons are widely distributed throughout the cortex, the density of serotonergic axons is highest in dorsal frontal cortex and progressively decreases in more caudal regions (e.g. Steinbusch, 1981; Hornung et al., 1990; Mamounas et al., 1991). In addition to this rostral–caudal gradient of serotonergic innervation of the cortex, there is a strict topographical organization of serotonergic processes arising from the dorsal or median raphe nucleus in many cortical areas. Thus, the two classes of serotonergic axons, fine (D fibers) and beaded (M fibers), have different patterns of termination and therefore affect different sets of cortical neurons. The frontal cortex of the rat contains an extremely dense plexus of fine axons with minute varicosities, arising from the dorsal raphe nucleus. Beaded axons with large, spherical varicosities arising from the median raphe are found less frequently (Kosofsky and Molliver, 1987). In contrast to the frontal cortex, the serotonergic innervation of the parietal cortex has a distinct laminar characteristic. Fine serotonergic axons are distributed throughout all layers, but are especially dense in layer V (Mamounas et al., 1991; Jansson et al., 2001). Beaded axons are concentrated in small irregular patches primarily in layers II–III; they are less frequent in layers I, IV, and VI and rare in layer V (Kosofsky and Molliver, 1987; Mamounas et al., 1991). A similar pattern of serotonergic innervation has been characterized in the cortex of primates (Wilson and Molliver, 1991). In the cortex of cat and marmoset, serotonergic processes with large varicosities have been described to form pericellular arrays, or baskets, as they surround certain cell bodies and proximal dendrites. These morphological specializations are most frequently observed in the frontal and anterior parietal cortex, which are found around stellate and horizontal cells in layer I and around stellate and bipolar cells in layers II and III. The distribution and cellular morphology of the cell surrounded by the serotonergic basket fibers is suggestive of a subpopulation of interneurons, e.g. GABAergic (Hornung et al., 1990; Tork, 1990; De Felipe et al., 1991). 3.2. Serotonergic innervation of hippocampus Using the method of retrograde fluorescent tracing in combination with serotonin immunohistochemistry, Kohler and Steinbusch (1982) have characterized serotonergic and non-serotonergic projections from the dorsal and median raphe to the hippocampus of the rat. Following injection of Granular Blue into the dorsal hippocampus, a small number of cells, which are serotonin-immunoreactive, are found in the caudal portion of the dorsal raphe (i.e. B6 of Dahlstro¨m and Fuxe). These cells are localized primarily along the midline. In the median raphe, labeled serotonergic cells are

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found predominantly in the midline portion of this nucleus (Kohler and Steinbusch, 1982). The rich serotonergic innervation of the dorsal hippocampus of the rat has been described using radioautographic counting of [3H]5-HT labeled varicosities (Oleskevich and Descarries, 1990), as well as immunocytochemical techniques (Mamounas et al., 1991). The density of serotonergic axons is highest in CA3, lower in dentate gyrus and lowest in CA1. Fine serotonergic axons with small varicosities are widely distributed throughout the hippocampus (Mamounas et al., 1991). In comparison with cortical areas, beaded serotonergic axons with large, spherical varicosities are especially prevalent in the hippocampus (Mamounas et al., 1991). These observations suggest that the dorsal hippocampus is innervated predominantly by the median raphe nucleus. Data from neurochemical studies using selective lesions (e.g. Geyer et al., 1976; Kellar et al., 1977) or in vivo microdialysis (Kreiss and Lucki, 1994) support this idea. In dentate gyrus, a dense network offine serotonergic axons is present in the hilar region and throughout the molecular layer, being especially prevalent in the outer portion of the molecular layer. In contrast, beaded axons form a dense narrow band immediately below the granule cell layer; a less dense and discontinuous innervation is found between the granule and molecular layers. Beaded axons also extend across the layer of granule cell bodies (Mamounas et al., 1991). In dentate gyrus of the cat and marmoset, serotonergic processes with large varicosities have been observed to form pericellular arrays or baskets as they surround certain cell bodies (Hornung et al., 1990; Tork, 1990). The CA3 subregion of hippocampus (both stratum radiatum and stratum oriens) is characterized by a dense plexus of beaded serotonergic axons, intermingled with a dense accumulation of fine serotonergic axons. Serotonergic axons are absent from the layer of pyramidal cell bodies in CA3, and the stratum lucidum, where the mossy fibers terminate (Mamounas et al., 1991). In the CA2 subregion, the serotonergic innervation is similar to that observed in CA3, except that a moderate number of beaded serotonergic axons ramify within the pyramidal cell layer, and immediately below it. At the boundary between CA2 and CA1 subregions of hippocampus, the density of serotonergic axons abruptly decreases, reflecting a decrease in both types of serotonergic axons in CA1. A narrow band of beaded serotonergic axons is present in stratum lacunosum of CA1. The stratum oriens and stratum radiatum of CA1 are innervated almost exclusively by fine serotonergic axons (Mamounas et al., 1991). 3.3. Serotonergic innervation of entorhinal cortex Using the method of retrograde fluorescent tracing in combination with serotonin immunohistochemistry, Kohler and Steinbusch (1982) have characterized serotonergic and non-serotonergic projections from the dorsal and median

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raphe to the entorhinal cortex of the rat. Injection of Granular Blue into the medial entorhinal area results in retrograde labeling of cells, which are serotonin-immunoreactive in both the dorsal and median raphe, as well as B9. The majority of these serotonergic cells in the dorsal raphe are located in the middle to caudal portions of this nucleus; many labeled cells are found contralateral to the injected hemisphere. In the median raphe, labeled cells are found predominantly in the midline portion of this nucleus. Injection of Granular Blue into the lateral parts of the entorhinal cortex results in labeling of cells, which are serotonin-immunoreactive in patterns similar to those seen after injection into the medial entorhinal area; however, fewer cells are labeled contralateral to the injected hemisphere. When the injection site is moved more laterally, there is greater labeling of serotonergic cells in the rostral part of the dorsal raphe (Kohler and Steinbusch, 1982). Serotonergic axons densely innervate all layers of entorhinal cortex, with regional and laminar differences in axon density (Kohler et al., 1981; Mamounas et al., 1991). The morphology of fine and beaded serotonergic axons in lateral entorhinal cortex is similar to that described in cortex and hippocampus (Kosofsky and Molliver, 1987; Mamounas et al., 1991). Fine serotonergic axons with minute varicosities, which are distributed through all layers of the medial and lateral entorhinal cortex, are found in highest density in layer I. In contrast, beaded serotonergic axons with large, spherical varicosities form a dense band in layer III. This extremely dense accumulation of beaded serotonergic axons is found only in lateral, and not in medial entorhinal cortex (Kohler et al., 1980; 1981; Mamounas et al., 1991). 3.4. Serotonergic innervation of nucleus accumbens The dorsal striatum is densely innervated by serotonergic axons, which are uniformly very fine, suggesting that the dorsal striatum is innervated predominantly by the dorsal raphe nucleus (Soghomonian et al., 1989). Data from neurochemical studies using selective lesions (e.g. Geyer et al., 1976; Kellar et al., 1977) or in vivo microdialysis (Kreiss and Lucki, 1994) support this idea. Brown and Molliver (2000) have characterized in detail the serotonergic innervation of the nucleus accumbens (ventral striatum) of the rat. The nucleus accumbens core and shell differ from the dorsal striatum in that the serotonergic axon density in both accumbens subdivisions is greater than in dorsal striatum. Within the nucleus accumbens, the shell contains a higher density of serotonergic axons than the core, a difference more marked in caudal regions of the nucleus accumbens. The core and shell subregions differ in the morphology of serotonergic axons innervating these structures. The core is innervated exclusively by fine serotonergic axons, which are lost following administration of neurotoxic amphetamine derivatives (Brown and Molliver, 2000). In contrast, the

caudal shell is innervated predominantly by larger, highly varicose serotonergic axons, which are spared following treatment with neurotoxic amphetamine derivatives. The rostral shell is innervated predominantly by thin serotonergic axons and receives few varicose serotonergic axons (Brown and Molliver, 1963). Ultrastructural studies have also shown that the core of the nucleus accumbens is innervated predominantly by fine serotonergic axons, whereas the shell is innervated by varicose axons (van Bockstaele and Pickel, 1993). The raphe nucleus of origin for serotonergic projections to the nucleus accumbens is yet to be determined. 3.5. Serotonergic innervation of the amygdaloid complex The amygdala is composed of several nuclei, which receive rich serotonergic innervation (Steinbusch, 1981). The heterogeneity of individual components of the amygdaloid complex (see Swanson and Petrovich, 1998), and distinct differences in serotonergic innervation (Steinbusch, 1981; Freedman and Shi, 2001) raise the interesting possibility of different functions for these subregions of the amygdala. Within the basal amygdaloid nucleus, an extensive, very dense region of serotonergic innervation is found in the rostral and medial subregions. There is a decrease in the density of serotonergic innervation more caudally in the basal amygdaloid nucleus at the bifurcation of this nucleus into its medial and lateral parts. A high density of serotonergic innervation is observed in the lateral amygdaloid nucleus. In the rat, the central and medial amygdaloid nuclei appear to receive very little serotonergic innervation (Steinbusch, 1981). This contrasts with what has been described in the macaque (see below). However, in the most caudal part of the amygdala complex, a very high density of serotonergic processes is found in the posterior aspect of the medial amygdaloid nucleus, and in the medial and lateral portions of the posterior amygdaloid nucleus (Steinbusch, 1981). In immunocytochemical studies of the amygdala of the squirrel monkey, Sadikot and Parent have observed that the predominant type of serotonergic process is the fine fiber with fusiform or pleomorphic varicosities. The larger and beaded axons are less frequent, being noted in the central, lateral and basal nuclei (Sadikot and Parent, 1990). The extended amygdala, first described by Alheid and Heimer (1988), is a continuum of structure that includes the central and medial amygdaloid nuclei, bed nucleus of the stria terminalis and sublenticular substantia innominata. These structures are thought to be important in drug abuse, schizophrenia, and affective disorders (see Freedman and Shi, 2001; Heimer, 2003). Using immunohistochemical techniques, Freedman and Shi (2001) have characterized the serotonergic innervation of the extended amygdala in the macaque. In the bed nucleus of the stria terminalis, serotonin-immunoreactive processes are found in the highest density in the lateral dorsal and lateral ventral

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subnuclei. In the amygdala, the densest serotonergic innervation is found in the lateral subregions of the central amygdala. There is also a relatively heavy concentration of serotonergic processes in the basal amygdala. Moderate levels of serotonergic innervation are found in the medial subregions of the central amygdala and in the medial amygdaloid nuclei (Freedman and Shi, 2001). 3.6. Serotonergic innervation of the anterior hypothalamus The anterior hypothalamus receives light to moderate serotonergic innervation. This contrasts with the ventromedial aspect of the suprachiasmatic nucleus, which is very heavily innervated by serotonergic processes (Steinbusch, 1981).

4. Volume transmission The action of neurotransmitters may be restricted to the synaptic cleft, specifically referred to as hard-wired neurotransmission, or may require that the neurotransmitter diffuse to remote receptor sites, referred to as diffuse, volume or paracrine transmission. Important factors determining the type of neurotransmission include the location of receptors with respect to release sites, the amount of neurotransmitter released, rate of diffusion away from the release site, and the removal or reuptake of the neurotransmitter by its transporter. 4.1. Synaptic specializations, or lack there of In the dorsal raphe nucleus, serotonergic cell bodies and dendrites accumulate and store serotonin in vesicles, where it appears to be in a releasable form (He´ry and Ternaux, 1981; Chazal and Ralston, 1987). For some of the serotonergic dendrites that contain vesicles in their dendritic shafts, the vesicles are densely packed in small clusters and are associated with a well-defined synaptic specialization. For others, the vesicles are observed near the membrane but do not appear to be associated with any synaptic membrane specialization (Chazal and Ralston, 1987). Serotonergic axon collaterals and terminals are also present (Mosko et al., 1977; Liposits et al., 1985; Chazal and Ralston, 1987). Ultrastructural studies indicate that some serotonergic axonal varicosities and terminals in the dorsal raphe nucleus are associated with well-defined synaptic specializations. However, those serotonergic axonal varicosities and terminals that are not associated with synaptic specializations predominate (Descarries et al., 1982; Chazal and Ralston, 1987). In studies using fast-scan voltammetry to measure evoked serotonin efflux from slices containing the dorsal raphe nucleus, released serotonin appears not to be diminished by binding to receptor or transporter sites, but appears to enter the extracellular space at a rate governed by diffusion (Bunin and Wightman, 1998). Although classical

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synaptic transmission may exist in the dorsal raphe, these observations are consistent with the existence of paracrine or volume neurotransmission in this serotonergic cell body area. Extensive ultrastructural studies by Descarries and colleagues (for review, see Descarries and Mechawar, 2000) have shown that in the cerebral cortex and hippocampus of the rat, the majority of serotonergic varicosities are not associated with synaptic specializations. The proportion of varicosities associated with synaptic junctions ranges from 28% in layer VI of the primary motor cortex (Fr1–Fr2) to 46% in layers I–II of primary somatosensory cortex. All serotonergic synaptic varicosities were found to form asymmetrical junctions (i.e. with a thick postsynaptic density) exclusively on dendritic spines and shafts (Se´gue´la et al., 1989). The synaptic incidence of serotonergic varicosities in the hippocampus is also low. In CA3 region and dentate gyrus of hippocampus, the proportion of serotonergic varicosities associated with synaptic junctions ranges from 18 to 33%, with no significant difference between these two hippocampal regions, or between layers within these hippocampal subregions (Oleskevich et al., 1991). These findings indicate that the serotonergic innervation of cortex and hippocampus is predominantly non-junctional, specifically high densities of small vesicles exist in varicosities lacking synaptic specializations and located far from any recognizable postsynaptic density. It has been proposed by Descarries that monoamine varicosities deprived of synaptic attachment might undergo translocation and reshaping along their parent fiber, and that in the absence of synaptic junctions, their released amine might diffuse in the intercellular space to act on relatively distant cellular targets (Descarries et al., 1975; Beaudet and Descarries, 1976). This raises intriguing possibilities for synaptic plasticity and the pliant or tractable nature of the functional architecture of serotonergic neurons innervating structures of the limbic system. 4.2. Extrasynaptic location of serotonin receptors and the serotonin transporter In the raphe nuclei, the 5-HT1A receptor is found on serotonergic soma and dendrites (Riad et al., 2000), where it functions as the somatodendritic autoreceptor, regulating the firing rate of serotonergic neurons (De Montigny et al., 1984; Aghajanian et al., 1990). In limbic structures of the forebrain, the 5-HT1A receptor is located postsynaptically to serotonergic neurons. These 5-HT1A heteroreceptors, found on non-serotonergic neurons, inhibit the neuronal firing of pyramidal neurons of the hippocampus and prefrontal cortex (e.g. Sprouse and Aghajanian, 1988; Dong et al., 1997; Dong et al., 1998). Ultrastructural studies of the dorsal raphe nucleus and hippocampus have shown that 5-HT1A immunoreactivity is found predominantly along the extra-synaptic portions of dendrites and cell bodies (Riad et al., 2000). The localization of the

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somatodendritic 5-HT1A autoreceptor, as well as 5-HT1A heteroreceptors, predominantly to extra-synaptic sites suggests that 5-HT1A receptor-mediated serotonergic neurotransmission is mainly a volume transmission process. 5-HT1B receptors have been shown to function as terminal autoreceptors, inhibiting serotonin release in various terminal field areas of serotonergic innervation (e.g. cortex and hippocampus) (e.g. Engel et al., 1986; Hjorth and Tao, 1991; Trillet et al., 1997). In nonserotonergic neurons, 5-HT1B receptors also function as heteroreceptors inhibiting the release of different neurotransmitters (e.g. acetylcholine and GABA) (Maura and Raiteri, 1986; Johnson et al., 1992; Bolanos-Jimenez et al., 1994). Ultrastructural studies of the substantia nigra and globus pallidus, however, found no indication of 5-HT1B immunoreactivity in axon terminals; 5-HT1B receptors appear to be predominantly located on the membrane of preterminal portions of axons (Riad et al., 2000). The localization of 5-HT1B auto- and hetero-receptors predominantly to non-synaptic sites indicates that as observed for the 5-HT1A receptor, 5-HT1B receptor-mediated serotonergic neurotransmission is a volume transmission process. 5-HT2A receptors are located postsynaptically to serotonergic neurons, and are found in high density in many forebrain regions, particularly in cortical areas, hippocampus and nucleus accumbens. In the frontal cortex of the rat, the laminar distribution of 5-HT2A receptor-binding sites seems to follow the serotonergic innervation of this brain region arising from the dorsal raphe nucleus (Blue et al., 1988). In cortical areas and hippocampus, 5-HT2A receptors are found in pyramidal cell bodies and apical dendrites, as well as axons (Cornea-He´bert et al., 1999 and references therein). Using double-labeling immunocytochemistry, Jansson et al. (2001) have found that in forebrain regions, including limbic structures (i.e. frontoparietal cortex, hippocampus), there are few close associations between serotonergic varicosities and 5-HT2A receptors. The lack of juxtaposition of 5-HT2A receptors with serotonergic release sites is an evidence for volume neurotransmission of serotonin mediated by this serotonin receptor, and further supports a paracrine role for serotonin in brain. The action of serotonin in the extracellular space is terminated by a single protein, the serotonin transporter (SERT). Immunogold electron microscopic studies of the dorsal and median raphe have revealed that the SERT is distributed predominantly in the cytoplasm of soma and dendrites, and not on the plasma membrane (Tau-Cheng and Zhou, 1999). The absence of the SERT on plasma membranes of soma and dendrites is consistent with the observation that cell bodies are insensitive to neurotoxic amphetamine derivatives, and functionally meaningful in that serotonin released into the extracellular space would be allowed to diffuse sufficient distances to activate autoreceptors. In contrast, Tau-Cheng and Zhou (1999) found in the hippocampus, frontal cortex, as well as dorsal and median

raphe that the SERT is located preferentially on the plasma membrane of axons, as opposed to the cytoplasm, and distributed along the axon. Moreover, the majority of SERTpositive axonal varicosities or terminals were not associated with well-defined postsynaptic densities (Tau-Cheng and Zhou, 1999). Thus, the majority of uptake sites appear to be located beyond synaptic junctions, consistent with the process of volume transmission for serotonin. Freedman and Shi (2001), in their neuroanatomical studies of the extended amygdala of the macaque, note a prominence of SERT-immunoreactivity versus serotoninimmunoreactivity in the medial amygdaloid nucleus. These investigators propose that this prominence of SERT over serotonin labeling may be an indication that serotonin neurotransmission in this structure is not a volume transmission process, but that serotonin may function more as a classical transmitter in this brain region (Freedman and Shi, 2001).

5. Disorders of limbic system and treatment strategies In humans, disorders associated with dysfunction within the limbic system include schizophrenia, major depression, and anxiety disorders. Drugs used in the treatment of these disorders modulate or alter serotonergic neurotransmission (see Jones and Blackburn, 2002). 5.1. Major depression Monoamine reuptake inhibitors and monoamine oxidase inhibitors, which have dominated the treatment of depression, were initially shown to have antidepressant activity by chance. The discovery of their mechanism of action was instrumental in the formulation of the monoamine theory of depression, which emphasized norepinephrine, over serotonin or dopamine (Schildkraut, 1965). The development of selective serotonin reuptake inhibitors (SSRIs) had a tremendous impact, not only on our thinking about serotonin’s potential role in affective disorders, but also clinically in the treatment of these disorders. The efficacy of SSRIs is similar to that achieved by traditional antidepressant drugs, but the SSRIs have superior side effect profiles (Goodnick and Goldstein, 1998; see Jones and Blackburn, 2002). Like the traditional antidepressants, SSRIs have a delayed onset of therapeutic effect of several weeks. It has been proposed that the increase in serotonin present in the extracellular space, and therefore increased activation of postsynaptic serotonin receptors, is what contributes to the therapeutic action of these antidepressants. This desired increase in serotonin release, however, is not achieved until the somatodendritic autoreceptors in the raphe nuclei, which strictly control the firing rate of serotonergic neurons, become desensitized. Thus, selective blockade of somatodendritic autoreceptors, which are the 5-HT1A receptor

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subtype, would be expected to shorten the delay of onset of antidepressant action of the SSRIs by avoiding the need for somatodendritic autoreceptor desensitization (Blier, 2001). Artigas and co-workers, who used pindolol as a 5-HT1A receptor antagonist as there are currently no selective 5HT1A receptor antagonists available for clinical use, pioneered this approach. Evidence from at least half of the clinical trials, in which pindolol was administered in combination with an SSRI, indicates that this combination provides a faster onset of action. However, there appears to be no benefit in treatment-resistant depression (Artigas et al., 1994; Artigas et al., 2001; Perez et al., 2001). Other receptors of interest as potential therapeutic targets include the 5-HT1B receptor, which functions as the terminal autoreceptor. Blockade of this receptor would be expected to increase serotonin release. This mechanism would not rely on the desensitization of somatodendritic autoreceptors. Thus, 5-HT1B receptor antagonists may potentially be antidepressants with rapid onset of action. For a more thorough discussion of these issues, the reader is referred to Jones and Blackburn (2002) for an excellent review. 5.2. Anxiety disorders The most prevalent anxiety disorders include generalized anxiety disorder, obsessive–compulsive disorder, posttraumatic stress disorder, social anxiety disorder and panic attacks. Historically, the benzodiazepines have been considered first-line therapy for the treatment of generalized anxiety disorder. However, chronic treatment with these drugs is associated with the development of tolerance and a withdrawal anxiogenesis. SSRIs and selective inhibitors of both serotonin and norepinephrine (SNRIs) are now recommended as first-line treatment. The partial 5-HT1A receptor agonist buspirone is also effective in the treatment of generalized anxiety disorder. Antagonists at 5-HT3 and 5-HT2A, and 5-HT2A receptors are of interest as potentially useful therapeutic agents in the treatment of generalized anxiety disorder (see Jones and Blackburn, 2002). For the treatment of obsessive–compulsive disorder, social phobia, posttraumatic stress disorder and panic disorder, the SSRIs have replaced tricyclic antidepressants and monoamine oxidase inhibitors as the drugs or choice. Because of the delay of therapeutic benefit associated with the SSRIs, benzodiazepines are often given in addition to the SSRIs in the early stages of treatment (see Jones and Blackburn, 2002). 5.3. Schizophrenia The role of serotonin in the pathophysiology of schizophrenia and in the mechanism of action of antipsychotic drugs is an area of much interest. The syndrome induced by the NMDA receptor antagonist phencyclidine, a model of schizophrenia, is blocked by 5-HT2A receptor

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antagonists (Millan et al., 1999). This observation has made the interaction between serotonergic and glutamatergic systems in the pathophysiology of schizophrenia prominent (see Aghajanian and Marek, 2000). There has been much attention focused on the 5-HT2A/2C receptors. The ‘atypical’ antipsychotic agents, of which clozapine in the prototype, are antagonists at 5-HT2A/2C receptors. These drugs have improved efficacy on the negative symptoms of schizophrenia (i.e. social and emotional withdrawal, poverty of speech, inability to initiate goal-directed behavior). They also have a lower incidence of extrapyramidal side effects, the very serious parkinsonian motor abnormalities associated with the chronic dopamine D2 receptor blockade, than the ‘typical’ antipsychotic drugs (see Jones and Blackburn, 2002; Meltzer et al., 2003). Very recently, there has been much excitement about the 5-HT1A receptor as a potential therapeutic target in the treatment of schizophrenia (Millan, 2000; Meltzer et al., 2003). 5-HT1A receptors, localized to the dorsal and median raphe nuclei, function as the somatodendritic autoreceptor, and therefore play a key role in the regulation and control of serotonergic neurotransmission in forebrain structures. In addition, postsynaptic 5-HT1A receptors are found in high density in the frontal cortex, hippocampus, and other corticolimbic structures implicated in the pathogenesis of schizophrenia and in the therapeutic action of antipsychotic drugs (Barnes and Sharpe, 1999). Several lines of evidence implicate 5-HT1A receptors in the etiology of schizophrenia and in the action of antipsychotic drugs: (i) increased density of 5-HT1A receptors in frontal cortex, hippocampus and nucleus accumbens; (ii) functional actions of selective 5-HT1A receptor agonists in modulating dopaminergic neurotransmission; (iii) partial agonist properties of clozapine at 5-HT1A receptors. For a more thorough presentation of these points, see the excellent review by Millan (2000).

6. Some considerations in the treatment of disorders of the limbic system Serotonergic projections from the dorsal and median raphe differ in the topographical organization and density of their respective innervation of forebrain structures. With regard to the treatment of affective disorders, i.e. major depression and anxiety disorders, it is an intriguing possibility that these two distinct serotonergic systems are differentially modulated by drugs that block serotonin reuptake (e.g. tricyclic antidepressants and selective serotonin reuptake inhibitors). Indeed, Brown and Molliver (2000) have shown in the nucleus accumbens that the fine serotonergic axons express SERT, whereas as beaded axons with large spherical varicosities do not. In the treatment of affective disorders, such as major depression, anxiety and panic disorder, it is tempting to speculate that it is the serotonergic neurons arising from the dorsal raphe nucleus

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that are the therapeutic targets for drugs that block serotonin reuptake. Regional differences in the level of SERT expression may also make specific areas of the limbic system particular targets of the action of drugs that block serotonin reuptake. For example, the relatively high levels of SERT expression in the lateral extended amygdala (Freedman and Shi, 2001) may in part explain the activation of this region by antidepressants such as the SSRIs (Morelli et al., 1999). As mentioned above, the partial 5-HT1A receptor agonist buspirone is effective in the treatment of generalized anxiety disorder (Jones and Blackburn, 2002). Partial 5-HT1A receptor agonists are also of interest as potential therapeutic agents in the treatment of schizophrenia (Millan, 2000). It is important to note that acutely, drugs that appear to be partial agonists at postsynaptic 5-HT1A receptors act as full agonists at somatodendritic 5-HT1A autoreceptors to inhibit serotonergic neuronal firing due to the presence of receptor reserve in the raphe nuclei (see Barnes and Sharpe, 1999 and references therein). Thus, it is proposed that certain drugs of intermediate efficacy simultaneously activate somatodendritic 5-HT1A autoreceptors, and block postsynaptic 5-HT1A receptors (see Millan, 2000). Of particular interest with regard to chronic drug administration, whether for the treatment of affective disorders or schizophrenia, are the observations of Kreiss and Lucki (1997). In studies using in vivo microdialysis, they found that 5-HT1A autoreceptors in the dorsal raphe are more readily desensitized by chronic agonist administration than those in the median raphe nuclei (Kreiss and Lucki, 1997). As discussed earlier in this review, serotonergic neurons arising from the dorsal and median raphe nuclei differentially innervate the structures of the limbic system. The desensitization of somatodendritic autoreceptors preferentially in the dorsal raphe nucleus would be expected to result in a loss of autoinhibition and an increase in serotonergic neurotransmission in the areas of brain innervated primarily by dorsal raphe, e.g. the frontal cortex. Much work is still required to further our understanding of the differential innervation of limbic structures by the distinct serotonergic systems arising from the dorsal and median raphe, and how these systems are modulated by therapeutic agents, such as 5-HT1A receptor agonists and the SSRIs. A greater appreciation of the interaction of these drugs with distinct types of serotonergic neurons in discrete brain regions may further our understanding of the etiology of these disorders of the limbic system, as well as advance the development of more efficacious and selective therapeutic agents.

Acknowledgements This work was supported by US PHS grants MH 52369 and funds from the National Association for Research on Schizophrenia and Depression (NARSAD).

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