The respective scales of morphological approaches in the brain are called in . Thus, imaging of the human living brain provides nowadays an incredible amount of information on functionally linked regions and, according to the availability of selective radiotracers, on millimetric clusters of binding sites. Morphological approaches including immunohistochemistry, in situ hybridization histochemistry and autoradiography allow to visualize a nucleus like the dorsal raphe, as well as a single labeled neuronal element of approximately one micrometer in diameter (eg, an axon varicosity) in brain tissue sections ( ). Electron microscopy studies in the human brain and, more often, in other mammalian species give ultrastructural details (eg, junctions between neuronal elements or 5-HT1A receptor internalization). 117

Cellular mapping of 5-HT-producing neurons in the CNS

Due to the postmortem instability of 5-HT118 and other possible methodological bias,119 quantitative biochemical estimation of 5-HT in the human brain subdivisions should be interpreted with caution, as illustrated by the numerous discrepant data reported since the 1950s. For the same reason, morphological approaches by formaldehyde-induced fluorescence or immunohistochemistry using antibodies against 5-HT are limited to biopsies and fetal brain tissues. Most of the anatomical studies in human are based on regional autoradiography of SERT binding sites to selective radioligands and immunohistochemical studies using antibodies against TPOH, which represent more stable postmortem markers. Therefore, from these studies and those performed in much detail in other species including rodents,120 cat,121 and nonhuman primates,122 it appears that the anatomy of the serotonergic system has remained somewhat similar between different species of mammals.

The 5-HT systems belong to the neuronal systems composed of a restricted number of neurons emitting extensively branched, non- or poorly myelinated axons that innervate almost all brain nuclei. As first described in human fetuses123,124 and later in adults by several authors,125-129 the distribution of the 5-HT cell bodies (approximately 350 000 cells) in the human brain is restricted to the brain stem. As illustrated in , a large majority of them is concentrated along the midline in the raphe nuclei, extending from the caudalmost level of the medulla oblongata to mid-level of mesencephalon, but a substantial number is located in the reticular formation lateral to these nuclei. The 5-HT neurons form a continuum of cells with loosely defined boundaries along the raphe nuclei. On the basis of studies of cell body localization and their respective projections, the 5-HT neurons can be separated into two groups: a rostral group located in the mesencephalic and rostral pons, sending axons to the forebrain, and a caudal group lying in the rostral pons and medulla oblongata, sending axons in the brain stem and spinal cord (refs in ref 128) In humans, the rostral group contains approximately 85% of the 5-HT neurons. It is composed of neurons located in four nuclei and one area, namely the interpeduncular, the caudal linear, the dorsal raphe (DRN with 165 000 neurons) and the median raphe (MRN with 64 000 neurons) nuclei. The additional area corresponds to the caudal mesencephalic and rostral pontine reticular formation. 5-HT neurons spread in this area were already observed in the rat and cat species and their large number estimated in human (60 000 neurons).

The caudal group accounts for 15% of all the 5-HT neurons. It is composed of 5-HT neurons located in three raphe nuclei, namely the raphe magnus (30 000 neurons), the raphe obscurus, and the raphe pallidus (1000 neurons), and in the ventral medullary reticular formation lying lateral to the raphe magnus and the pyramids. As noted earlier, the rostral and caudal groups have separate afferent projections, with, however, some overlapping in the brain stem and as far down as the spinal cord. The trajectories of the efferent pathways have been studied in laboratory animals, often combining retrograde tracing with immunohistochemistry. Thus, a rostral and a ventral pathway emerge from the rostral group, rapidly join ventrally and split again into a lateral projection running in the internal capsule to innervate the lateral cortex and a longitudinal rostral projection running in the medial forebrain bundle to innervate the hypothalamus, basal forebrain, septum, basal ganglia, and amygdala. This rostral projection extends into the cingulum and innervates the medial cortex and the hippocampus.

The density of innervation in terminal areas reported in certain human brain areas has been extensively studied in cat and rodents. This density greatly varies from one region to the other and also within a region ( ). In the cerebral cortex, the superficial layer receives more axons than the other layers. A dense innervation is observed in the ventromedial part of the caudate-putamen and in the globus pallidus. Ventral to them, the subtantia innominata is also richly supplied in 5-HT terminals. In the amygdala, the basal nucleus stands out for its very high number of 5-HT axons. In humans, like in animals, the 5-HT axons innervating the cortex and the hippocampus display two different morphologies.130 One category of axons bears spaced small and elongated varicosities while the other category displays closely spaced, large, and round varicosities. It can be noted that the two populations of axons show several interesting properties. First, they are respectively issued from two different raphe nuclei, the DRN and the MRN. Second, the small varicose axons correspond to the numerous 5-HT axons not engaged in true synaptic contacts. For example, it is remarkable that only 5% of the varicosities display synapses in the rat frontoparietal cortex.48 Thirdly, and of special clinical interest, the small varicose axons are more susceptible to degeneration caused by amphetamine derivatives, like ecstasy.131 The caudal group of 5HT neurons sends axons both laterally in the reticular formation and downwards in the spinal cord. In the reticular formation, the 5-HT axons are particularly abundant in the cranial motor nuclei (trigeminal, facial and hypoglossal). In the spinal cord, the 5-HT axons terminate in all subdivisions and along the whole length of the cord. In the dorsal horn, the superficial layers are densely innervated. In the intermediate gray, the preganglionic sympathetic neurons of the intermediolateral column are densely surrounded by 5-HT axons. In the ventral horn, the 5-HT axons are in close apposition to the motor neurons, especially in primates.132