MICROSCOPY RESEARCH AND TECHNIQUE 46:24–47 (1999) Distribution of the Catecholaminergic Neurons in the Central Nervous System of Human Embryos and Fetuses CATHERINE VERNEY* INSERM U.106, Bâtiment Pédiatrie, Hôpital Salpêtrière, 75651-Paris Cedex 13, France KEY WORDS development; dopamine; cerebral cortex; immunocytochemistry; noradrenaline; tyrosine-hydroxylase ABSTRACT The catecholaminergic cell groups in the human brain, denominated from A1 to A17, display some striking anatomical differences with those described in the rodent. These differences are essentially observed in the extent of the dopaminergic neurons and especially their axonal fields in the telencephalon. Immunocytochemistry for tyrosine-hydroxylase and dopamine-ßhydroxylase allowed the visualization of the precocious human catecholaminergic groups as early as 4.5 postovulatory weeks. Maps of tyrosine-hydroxylase positive neurons generated in the different rhombomeres, midbrain, and prosomeres are shown following the prosomeric model introduced by Puelles and Rubenstein [(1993) Trends Neurosci. 16:472–476]. Such a description is convenient to compare catecholaminergic systems in different mammalian species and provide clear anatomical landmarks of the embryonic substantia nigra (midbrain and prosomeres 1 and 2), that are necessary for transplantation of neural tissue in Parkinson’s disease. The development and early specification of the dopaminergic neurons expressing calbindin D28K phenotype in the substantia nigra and in the ventral tegmental area are described. The catecholaminergic axons enter the anlage of the cerebral cortex just after the formation of the cortical plate, from 7 postovulatory weeks on. They invade the subplate layer where they wait for 4 weeks before penetrating the cortical plate. At midgestation, the different areas and layers of the frontal cerebral wall are invaded by the catecholaminergic axons, before the layering of the cortex is completed, in a pattern of fiber distribution similar to that described in the adult human brain. The early pattern of development of the catecholamine systems appeared to be phylogenetically well preserved in mammals, but specific features emerging during the differentiation period are unique to humans. Microsc. Res. Tech. 46:24–47, 1999. r 1999 Wiley-Liss, Inc. INTRODUCTION The discovery of the technique of histofluorescence of catecholamines, more than 30 years ago allowed the description of the different catecholaminergic (CA) cell groups distributed along the caudorostral axis of the CNS and innervating numerous areas and nuclei (Fig. 1) (Björklund and Lindvall, 1984; Dahlström and Fuxe, 1964; Falck et al., 1962; Lindvall and Björklund, 1978, 1983). The central dopamine-, noradrenaline-, and adrenaline-containing neurons have a neuromodulatory function on various neuronal fields involved in autonomic, motor, or cognitive functions. The CA neuronal groups emerge and develop axonal fibers very early during development (Lauder and Bloom, 1974; Olson et al., 1973; Olson and Seiger, 1972; Specht et al., 1981; Verney et al., 1982, 1984). This led to the hypothesis of a regulatory or trophic role of the precocious CA fibers on their target neurons in the cerebral cortex during development in the rat or cat (Bear and Singer, 1986; Blue and Parnavelas, 1982; Kasamatsu and Pettigrew, 1976; Kasamatsu et al., 1981). For humans, a developmental dopaminergic (DA) dysfunction has been suggested in neurological and psychiatric disorders such as Parkinson’s disease and schizophrenia (Deutch, 1993; Grace, 1993; Lewis, 1997; Weinberger et al., 1988). The recent advent of r 1999 WILEY-LISS, INC. transplantation strategy in the treatment of Parkinson’s disease makes it essential to study the DA neurons of embryonic human specimens appropriate for grafting (Lindvall et al., 1992). Phylogenetic studies in mammals have shown striking particularities of the DA systems in primates vs. rodents in the distribution of cell bodies and in the extension of the axonal fibers especially in the telencephalon (Berger et al., 1991; Dubach, 1994; Kitahama et al.,1994; Panayotacopoulou and Swaab, 1993; Tillet, 1994). As most of the descriptive and developmental studies on the CA systems have been performed in the rat, it becomes essential to get more information in primates and especially in humans. The first section of this account presents an anatomical overview of the CA neuronal groups in the adult human brain compared to that described in monkey and rodent brain, with special emphasis on the CA innervation of the cerebral cortex. The second section reports our data on the development of these cell groups and of their efferences entering the cerebral cortex Contract grant sponsor: INSERM; Contract grant sponsor: ECC; Contract grant number:CI1 CT90 O848. *Correspondence to: Catherine Verney, INSERM U.106, Bâtiment Pédiatrie, Hôpital Salpêtrière, 47 Bd de l’Hôpital, 75651-Paris, France. E-mail: email@example.com Received 10 October 1998; accepted in revised form 4 January 1999 Fig. 1. Schematic drawing of the main CA groups named A1 to A15 and C1, C2 distributed along the caudorostral axis of the adult human brain and their predominant efferences (for details see Table 1). Dopaminergic groups are located in the midbrain and forebrain whereas noradrenergic and adrenergic are distributed in the hindbrain The scheme ‘‘development’’ presents the prenatal ages (in weeks or days) when the first neurons of the locus coeruleus (LC) and substantia nigra (SN) are generated in the human, monkey and rat. The fetal age at which the first DA and noradrenergic axons are visualized as they penetrate the cortical anlage are indicated for the human, monkey, and rat. 26 C. VERNEY during the first half of gestation in humans. We compare our data to those obtained by other teams and, since the literature on these topics is abundant, we will cite many review papers that the reader is referred to for more details. DISTRIBUTION OF THE CENTRAL CATECHOLAMINE NEURONS IN THE ADULT HUMAN BRAIN Methodological Remarks The pioneer histofluorescence technique of CA has now been replaced by immunocytochemical labeling, which allows the study of CA neurons in experimental animals as well as in human postmortem specimens (Dahlström and Fuxe, 1964; Falck et al., 1962; Hökfelt et al., 1974, 1984). The use of antisera directed against the enzymes of the synthetic pathway of CA, as well as against dopamine and noradrenaline themselves or their transporters, allow us to discriminate between the dopaminergic, noradrenergic, and adrenergic neuronal groups (Fig. 2A) (Blackely et al., 1994; Geffard et al., 1984; Giros et al., 1992; Hökfelt et al., 1974, 1984; Smeets and Steinbusch, 1990; Smiley et al., 1992; Verney et al., 1990). The first enzyme is tyrosinehydroxylase (TH), the rate-limiting enzyme, which converts tyrosine to DOPA, which is then converted to dopamine by a nonspecific decarboxylase, the L-aromatic aminoacid-decarboxylase (AADC). DOPA has been considered as a precursor for dopamine but its recent immunocytochemical visualization in different cell populations even in humans suggests that it could also have a neurotransmitter function (Ikemoto et al., 1997; Kitahama et al. 1988; Smeets and Steinbusch, 1990). TH-immunoreactivity is the phenotypic marker most commonly used for simultaneous characterization of dopaminergic, noradrenergic, and adrenergic cell bodies (Fig. 2A). Dopamine-ß-hydroxylase (DBH)immunoreactivity is specific to noradrenergic and adrenergic neurons and phenylethanolamineN-methyl-transferase (PNMT)-immunoreactivity is present only in adrenergic neurons (Kitahama et al., 1994). But, there have been puzzling results showing the presence of the amine without the synthetic enzyme or vice versa (Foster, 1994; Kitahama et al., 1994; Smeets and Reiner, 1994; Zecevic and Verney, 1995). For example, THimmunoreactivity could label cell populations in which no other CA traits have been identified as in the coliculli during mammal development (Jeager and Joh, 1983; Puelles and Verney, 1998; Zecevic and Verney, 1995). Also, most antisera directed against TH label preferentially DA rather than noradrenergic axons in the cerebral cortex during development (Verney et al., 1982, 1993) and in adult primates (Noack and Lewis, 1992; Gaspar et al., 1989). In situ hybridization technique allows to ascertain the presence of messengers for TH in cells where the protein is detected and, interestingly, human TH exists as at least four isoforms generated by alternative splicing of mRNA (Alterio et al.,1998; Dumas et al., 1996; Grima et al., 1987; Kaneda et al., 1987; Lewis et al., 1993). Another specific method used to visualize CA fibers and terminals is based on their selective uptake in slices maintained in vitro. This method allowed the distinction of one CA axonal terminal field from another (Berger et al., 1986, 1988). Distribution of the Central Catecholamine-Containing Cell Groups In their first description of the central CA systems, Dahlström and Fuxe (1964) recognized 12 groups of fluorescent neurons in the rat brain, which they labeled A1 to A12, from caudal to rostral. Immunocytochemical techniques allowed Hökfelt and his colleagues (1984) to confirm the presence of these noradrenaline- and dopamine-cell groups and also to identify additional groups, A13 to A17. PNMT-immunocytochemistry allowed the visualization of the adrenergic neuronal groups in the brainstem of the rat named C1-C2 (Hökfelt et al., 1974) (Figs. 1, 2A). Those nomenclatures are largely adopted for all mammals including humans. Most of the CA groups (except the adrenergic cells) in human adult, contain melanin pigment (Bogerts, 1981; Gaspar et al., 1983; Saper and Petito, 1982) and could thus be recognized without immunohistochemical detection. Table 1 summarizes the locations of the CA neuronal groups detected in human (Dubach, 1994; Kitahama et al., 1994; Puelles and Verney, 1998; Tillet, 1994). Such a description is schematic since the CA neurons of each group are usually not confined to known cytoarchitectural boundaries, but intermingle with other cell types especially in humans (Kitahama et al., 1994; Pearson et al., 1990). Rhombencephalon. The noradrenergic neurons of A1-A2 groups associated with the adrenergic neurons of C1-C2 are scattered within a continuous longitudinal motor related band extending from the ventrolateral medulla (A1-C1) to the dorsomedial medulla (A2-C2) (Arango et al., 1988; Halliday et al., 1988; Kitahama et al,. 1988, 1994, 1996; Pearson et al., 1983, 1990; Robert et al., 1984). A3 group is not mentioned in Table 1 since its existence is questionable as no immunocytochemical studies mention it in the adult mammalian brain (Hökfelt et al., 1984; Smeets and Reiner, 1994). In humans, the pigmented nucleus of the cerebellar tegmentum (A4) is located near the locus coeruleus (A6), which extends dorsal to the locus subcoeruleus (A7). The rostral noradrenergic neurons of the A5 group are distributed near the Kölliker-Fuse nucleus ventral to the brachium conjunctivum whereas the caudal neurons of this group are near the lateral paragigantocellular nucleus in the medulla oblongata (Kemper et al., 1987; Kitahama et al,. 1994, 1996; Nobin and Björklund, 1973; Pearson et al., 1983, 1990; Robert et al., 1984). Mesencephalon. The DA groups, the retrorubral area A8, the substantia nigra A9, and the ventral tegmental area A10 are often called ‘‘midbrain’’ groups whereas in mammals including humans, the A9-A10 groups extend in the diencephalic segment, rostral to the third nerve root (Gaspar et al., 1983; German et al., 1983; Halliday and Tork, 1986; Hirsch et al., 1992; Kitahama et al., 1994; Pearson et al., 1983, 1990; Van Domburg and Donkelaar, 1991). In humans, the A8 DA group is dispersed in the tegmentum caudal to the red nucleus, and caudodorsal to the substantia nigra. The substantia nigra is composed of the pars compacta containing DA neurons densely packed in an horizontal band (A9), which penetrates ventrally the pars reticulata comprising GABA containing neurons (Oertel et al., 1982) (Fig. 3). The lateral extension of the DA EMBRYONIC CATECHOLAMINERGIC NEURONS IN HUMANS 27 Fig. 2. A: A schema of the synthetic pathway of catecholamines: dopamine, noradrenaline and adrenaline. B: Topological model of the longitudinal and neuromeric domains of the embryonic brain, illustrating the relative positions of the diverse TH-IR populations (modified with permission from Puelles and Verney, 1998). The longitudinal zones, floor (FP), basal (BP), alar (AP), and roof plates (RP) are indicated at the left side and the neuromeric elements are shown at the bottom; larger morphological units are identified at the top. The circle drawn in p6 represents the eye stalk containg A17 group. The different TH-IR areas indicated by dark grey represent the permanent populations whereas the light grey areas show transient ones. Terminology by Dahlström and Fuxe (1964) is followed to a large extent, with a few exceptions (see Puelles and Verney, 1998). DR, dorsal raphe nucleus; hb, habenula; HL, lateral hypothalamic cell group; lc, locus coeruleus; lsc, locus subcoeruleus; mam, mammillary group; RM, retromammillary area; PR, pontine raphe; tect, tectum. neurons forms the substantia nigra pars lateralis. In their atlas of the human brain, Olszewski and Baxter (1954) divided the pars compacta into three horizontal ventrodorsal bands called alpha, beta, and gamma. The gamma band corresponds to the dorsal horizontal DA neuronal population, coexpressing calbindin D28K phe- notype and projecting rather to limbic and cortical areas. The ventral DA neurons are less numerous to express calbindin D28K and rather project to motorrelated structures (Gaspar et al., 1992, 1993; McRitchie et al., 1996). More recent studies considered that the DA neurons of the dorsal band of the substantia nigra 28 C. VERNEY TABLE 1. Central catecholamine (CA)-containing neuronal groups in humans* CA A Group C1 Brain segment Main localization in human brain Caudal rhombencephalon Ventrolateral reticular area (ventral to the ambiguus nucleus) (Arango et al., 1988; Halliday et al., 1988; Kitahama et al., 1985; 1988; 1994, 1996; Pearson et al., 1983; 1990; Robert et al., 1984) A C2 Caudal rhombencephalon Substantia gelatinosa subnucleus and nucleus of the solitary tract (Arango et al., 1988; Halliday et al., 1988; Kitahama et al., 1985; 1988; 1994; 1996; Pearson et al., 1983; 1990; Robert et al., 1984) NA A1 Caudal rhombencephalon Ventrolateral reticular area (including the ambiguus nucleus) (Arango et al., 1988; Halliday et al., 1988; Kitahama et al., 1988; 1994, 1996; Pearson et al., 1983; 1990; Robert et al., 1984) NA A2 Caudal rhombencephalon Nucleus of the solitary tract, dorsal nucleus of nerve X and adjacent reticular parvocellular area (Arango et al., 1988; Halliday et al., 1988; Kitahama et al., 1988; 1994; 1996; Pearson et al., 1983; 1990; Robert et al., 1984) NA A4 Rostral rhombencephalon Pigmented nucleus of the cerebellar tegmentum (Kitahama et al., 1994; Nobin and Björklund, 1973; Pearson et al., 1983) NA A5 Rhombencephalon Kölliker-Fuse nucleus and lateral paragigantocellular nucleus (Kitahama et al., 1994; Nobin and Björklund, 1973; Pearson et al., 1983; 1990) NA A6 Rostral rhombencephalon Locus coeruleus (Pearson et al., 1983; 1990; Kemper et al., 1987; Kitahama et al., 1994; 1996; Nobin and Bjöklund, 1973; Pearson et al., 1983; 1990; Robert et al., 1984) NA A7 Rostral rhombencephalon Locus subcoeruleus (A6 for some authors) & (Pearson et al., 1983; 1990; Kemper et al., 1987; Kitahama et al., 1994; 1996; Pearson et al., 1983; 1990; Robert et al., 1984) DA A8 Mesencephalon-isthmus Tegmental field caudal to the red nucleus and dorsal to A9 (Hirsch et al., 1992; Halliday and Tork, 1986; Kitahama et al., 1994; Pearson et al.; 1983; 1990; Van Domburg and Donkelaar, 1991) DA A9 Mesencephalon-diencephalon Substantia nigra pars compacta (Gaspar et al., 1983; German et al., 1983; Halliday and Tork, 1986; Hirsch et al., 1992; Kitahama et al., 1994; Pearson et al., 1983; 1990; Van Domburg and Donkelaar, 1991) DA A10 Mesencephalon-diencephalon Ventral tegmental area (the paranigral and parabrachialis pigmentosus nuclei, the central and rostral linearis nuclei of raphe, the interfascicularis and the Edinger-Westphal nuclei. around the mammillary nuclei) (German et al., 1983; Halliday and Tork, 1986; Hirsch et al., 1992; Kitahama et al., 1994; Pearson et al., 1983; 1990; Puelles and Verney, 1998; Su et al., 1987; Van Domburg and Donkelaar, 1991) DA A11 Caudal diencephalon The periaqueductal grey—along the caudal part of the IIIrd ventricle, (Pearson et al., 1990; Su et al., 1987; Tillet et al., 1994) DA A12 Intermediate diencephalon Tuberoinfundibular nucleus (Pearson et al., 1990, Spencer et al., 1985; Su et al., 1987; Tillet et al., 1994) DA A13 Intermediate diencephalon Dorsomedial hypothalamus and zona incerta (Pearson et al., 1990, Spencer et al., 1985; Su et al., 1987; Tillet et al., 1994) DA A14 Rostral diencephalon Periventricular area of the IIIrd ventricle lateral anterior hypothalamic area (Pearson et al., 1990, Spencer et al., 1985; Su et al., 1987; Tillet et al., 1994) DA A15 Rostral diencephalon Supraoptic nucleus—paraventricular hypothalamic nucleus (Li et al., 1988; Pearson et al., 1990; Panayotacopoulou et Swaab, 1993; Su et al., 1987; Tillet et al., 1994) DA A16 Telencephalon Periglomerular neurons of the olfactory bulb; (Halasz and Shepherd, 1983; Smith et al., 1991) TH-IR neurons Telencephalon Basal forebrain near the olfactory tract, in the ventral striatum and in the basal nucleus of Meynert (Dubach, 1987; Gaspar et al., 1985; Köhler et al., 1983; Gouras, 1992). The cerebral cortex (Gaspar et al., 1987; Hornung et al., 1989; Kuljis et al., 1989, Lewis et al., 1991; Trottier et al., 1989). DA A17 Amacrine cells in inner nuclear layer of the retina (Frederick et al., 1982; Nguyen-Legros et al., 1992) *A: adrenaline, NA: noradrenaline, DA: dopamine. The nomenclature is adapted from Dahlström and Fuxe (1964) and Hökfelt et al., (1974). TH-IR: tyrosine hydroxylase immunoreactive neurons. could correspond or overlap those of the parabrachialis pigmentosus nucleus, which extend more medially and are included in the ventral tegmental area (A10 group) (Kitahama et al., 1994; McRitchie et al.,1996; Porrino and Goldman-Rakic, 1982). The ventral tegmental area is composed of DA neurons dispersed in the reticular formation adjacent to the nucleus interpeduncularis, the red nucleus, and the substantia nigra (Fig. 3). It comprises the nuclei paranigralis and parabrachialis pigmentosus, the central and rostral linearis nuclei of the raphe, the interfascicularis and the EdingerWestphal nuclei. The TH-IR neurons located around the mammillary nuclei in humans are included in the ventral tegmental area as they have a similar pattern of development to the DA neurons of this group (Pearson et al., 1990; Puelles and Verney, 1998; Su et al., 1987). Conversely, DA neurons of the periaqueductal gray, which have been included in the ventral tegmen- tal area in most studies, are classified within A11 group (Kitahama et al., 1994; Puelles and Verney, 1998; Smeets and Reiner, 1994). Most of these DA neurons of A8–A10 groups and of subpopulations of A9 group (aforementioned) express the calbindin D28K phenotype. The phenotypic expression of calbindin D28K within the DA neurons is interesting since it defines a neuronal subpopulation that appears to be more resistant to cell death induced by Parkinson’s disease than the one containing only dopamine (German et al., 1992; Hirsch et al., 1992; Yamada et al., 1990; Yamada et al., 1990). Diencephalon. The DA groups A11 to14 groups spread within the diencephalon but also include some midbrain neurons (Table 1) (Pearson et al., 1990; Spencer et al., 1985; Su et al., 1987; Tillet, 1994). TH positive DA neurons are distributed in a dorsal band extending along the ventricle from the mesencephalon EMBRYONIC CATECHOLAMINERGIC NEURONS IN HUMANS 29 Fig. 3. Semischematic drawings of a half of the human midbrain stained for TH-immunoreactivity. A: Section at the level of the red nucleus. B: Section at the level of the decussation of the superior cerebellar peduncle. The limit between the DA neurons of the parabrachialis pigmentous nucleus (pbp), which is considered as a part of the ventral tegmental area (vta), and those of the dorsal part of the substantia nigra pars compacta (snc) is not clear (short arrows). The ventral clustered DA neurons of the pars compacta send dendrites and intermingle with the neurons of the pars reticulata (snr) (long arrow). The ventral tegmental area (vta) comprises DA neurons in the paranigralis nucleus (pn) and the pbp, the central and rostral linearis nuclei of the raphe (cli, rli). IIIn, oculomotor nerve; aq, aqueduc; ip, interpeduncularis nucleus; lm, medial lemiscus; mlf, medial longitudinal fasciculus; rn, red nucleus; pag, periaqueductal grey; scp, superior cerebellar peduncle; snl, substantia nigra pars lateralis. Reprinted from Kitahama K, Nagatsu I, Pearson J. 1994. Catecholamine systems in mammalian midbrain and hindbrain: theme and variation. In: Smeets WJAJ, Reiner A, editors. Phylogeny and development of catecholamine systems in the CNS of vertebrates. Cambridge: Cambridge University Press. p 183–206. with permission . to the diencephalon: as mentioned above, we classified the DA neurons of the periaqueductal gray as the caudal part of A11 group. The A11 group in humans comprises the DA neurons along the border of the IIIrd ventricle in the caudal diencephalon, which are not clearly separated from those of A13 group located more rostrally. Numerous TH-positive neurons are observed within the paraventricular and supraoptic and hypothalamic nuclei in humans, the A15 group, whereas only a few scattered neurons are detected in this latter group in rodents (Li et al., 1988; Pearson et al., 1990; Panayotacopoulou et Swaab, 1993; Tillet, 1994). Telencephalon. Several studies have reported the presence of TH-IR cells in the telencephalon of human and non-human primates whereas they are not detected in the rat, excepted during development (see review in Dubach, 1994). In humans, they consist of heterogeneous TH-IR cell populations mostly dispersed in the basal forebrain near the olfactory tract and the ventral striatum (Gaspar et al., 1985; Köhler et al., 1983), spreading caudally in the basal nucleus of Mey- nert (Dubach, 1994; Gouras et al., 1992). Some TH-IR neurons coexpressing the dopamine transporter DAT were mentioned recently in the monkey striatum. These presumably DA neurons also coexpress GABA as mentioned for TH positive neurons described in the primate cerebral cortex (Betarbet et al., 1997; Trottier et al., 1989). Indeed, in primates, TH containing neurons visualized by immunocytochemistry and by in situ hybridization of RNA probe constitute less than 0.1% of the cortical neuronal population (Gaspar et al., 1987; Hornung et al., 1989; Kuljis et al., 1989; Lewis et al., 1987, 1991; Trottier et al., 1989). These TH positive cortical neurons present a characteristic laminar and regional distribution and are restricted to association cortices (prefrontal or limbic related areas), rather than to primary cortical areas (Gaspar et al., 1987; Lewis et al., 1991). As for striatal neurons, they could be DA and could also use GABA for non-aminergic neurotransmission (Trottier et al., 1989). But as these neurons are lacking other CA traits, DOPA has been also suggested as a possible neurotransmitter (Gaspar et al., 1987). 30 C. VERNEY Noradrenergic and Dopaminergic Ascending Efferent Fiber Systems to the Telencephalon and Particularly the Cerebral Cortex In mammals, the ascending noradrenergic axons originate in the locus coeruleus (A6) and run dorsally in the dorsal tegmental bundle. They assemble with the DA efferences from the substantia nigra pars compacta (A9) and the ventral tegmental area (A10) to constitute the medial forebrain bundle that innervates the diencephalic and telencephalic areas (Lindvall and Björklund, 1978, 1983). The bulk of the DA axons goes to the basal ganglia, and the CA fibers destined to the certebral cortex enter the frontal pole from its rostroventral aspect (Berger and Verney, 1984; Lindvall and Björklund, 1978, 1983). The noradrenergic axons are widely distributed in all layers and areas of the cerebral cortex in rodents and primates. A slight regional difference is observed in the pattern of fiber distribution in rodents (review in Berger and Gaspar, 1994; Levitt and Moore, 1978; Morrison et al., 1978), whereas a clear heterogeneity in the distribution of these fibers is detected in primates. Innervation is denser in the somatosensory and motor cortical areas and decreases caudally in the occipital lobe and rostrally in the frontal lobe. In primates, variations in the density of innervation are also observed across the different cortical layers (Berger and Gaspar, 1994; Gaspar et al., 1989; Lewis and Morrison, 1989; Morrison et al., 1982). The DA mesotelencephalic system in mammals refers to the ascending projections from the A9–A10 cell groups, which are segregated into two components: (1) the nigrostriatal system that originates in the ventral and lateral part of A9 and innervates the caudateputamen and globus pallidus (Gerfen, 1992; Graybiel, 1990; Haber and Groennewegen, 1990); (2) the mesocorticolimbic system originating in the dorsal and medial A9 and the adjacent A10 and innervating the cerebral cortex , the olfactory bulb, the anterior olfactory nucleus, the olfactory tubercle, the piriform cortex, the septal area, the nucleus accumbens, and the amygdaloid complex (Lindvall and Björklund, 1983). The DA innervation of the cerebral cortex is strikingly different between primates and rodents. The major DA terminal fields are restricted to frontal, cingulate, and entorhinal cortices in rodents, whereas DA innervation is present in all cortical areas with major regional differences in density and laminar distribution in primates (Berger et al., 1986, 1988, 1991; Berger and Gaspar, 1994; Gaspar et al., 1989; Lewis et al., 1987; Maeda et al., 1995). In humans, the DA innervation is densest in agranular cortices (motor area 4–6, cingulate area 24 and insula) and lowest in granular cortical areas (prefrontal area 9, parietal area 3b or visual area 17). In the agranular cortices, TH-IR axons are present in all layers with accumulations in patches in layer II and III, whereas in other cortices such as the occipital cortex, a bilaminar pattern of distribution in layer I and layers V–VI is observed (Gaspar et al., 1982). This widespread distribution of the DA fibers and teminals within the human cerebral cortex is a major evolutionary specialization when compared to rodents. DEVELOPMENT OF CATECHOLAMINE-CONTAINING NEURONS IN HUMAN EMBRYOS AND FETUSES The pioneer Swedish teams have detected the presence of fluorescent monoaminergic cell bodies at 7 gestational weeks in humans (Olson et al., 1973) but the distinct CA groups and axonal pathways have been clearly described in 3–4-month-old fetuses and onwards (Choi et al., 1975; Nobin and Björklund, 1973; Pearson et al., 1980; Sailaja and Gopinath, 1994; Su et al., 1987). At this latter developmental stage, fluorescent CA fibers were observed penetrating the basal ganglia and a few of them were seen in the cerebral cortex. THimmunolabeling could identify CA neurons in the sympathetic ganglia by 5 gestational weeks (Pickel et al., 1980), and in the substantia nigra and locus coeruleus by 9–10 gestational weeks (Pearson et al., 1980). In Rhesus monkeys, the neurons of the locus coeruleus and of the substantia nigra are generated between the 4th and 6th gestational week corresponding to the first quarter of gestation (Levitt and Rakic, 1982) whereas CA efferents towards the cortical anlage are currently observed at the 10th gestational week (Berger et al., 1992) (Fig. 1). In the rat, the CA neuronal groups are generated during the second half of gestation, between embryonic days 12 and 15 (Altman and Bayer, 1981; Lauder and Bloom, 1974; Specht et al., 1981) and the CA efferences to the cortex penetrate the frontal areas at embryonic day 16 for the DA innervation and day 17 for the noradrenergic (Schlumpf et al., 1980; Verney et al., 1982, 1984) (Fig. 1). Comparing the developmental stages of the locus coeruleus and substantia nigra neurons in rats and humans, O’Rahilly and collaborators (1987) suggested that embryonic days 13–15 in the rat approximatively corresponded to Carnegie stages 15–18 (4.5 to 6 postovulatory weeks) in humans. Indeed, our results and those of other teams, show the presence of TH immunoreactive CA groups during the embryonic period in humans (Almqvist et al., 1996; Freeman et al., 1991; Puelles and Verney, 1998; Ugrumov et al., 1996; Verney et al., 1991; Zecevic and Verney, 1995). Specimens and Methods We had access to specimens from 10 human embryos that corresponded to 4.5 to 8 postovulatory weeks (provided by Dr. Zecevic) and 10 fetuses aged from 10 to 24 weeks (O’Rahilly and Gardner, 1971; Puelles and Verney, 1998; Verney et al., 1996; Zecevic and Verney, 1995). These specimens were obtained in Yugoslavia and France from medically indicated or spontaneous abortions following the recommendations of the French (CCNESVS, 90 294) and Yugoslavian Ethical Committees. The embryonic specimens were obtained with no postmortem delay, which gave an excellent preservation of the tissue. On the other hand, the fetal brains were obtained with a postmortem delay ranged from 2 to 15 hours, which brought some variations in the density of positive fibers detected in the fetal cerebral cortex. Embryos or brain samples were fixed in 4% paraformaldehyde, rinsed, frozen, and cut serially in the frontal or sagittal planes. Groups of adjacent sections were immunostained with primary antisera against TH (polyclonal and monoclonal antibodies) EMBRYONIC CATECHOLAMINERGIC NEURONS IN HUMANS Fig. 4. Camera lucida mappings of sagittal sections of human embryos. The TH-immunoreactive (IR) neurons are indicated by dots and the interneuromeric boundaries by radial lines connecting the ventricle (shaded in gray) with the brain surface. Microphotographs of some regions of the histological sections drawn on these figures are shown in Figure 5A,E for panel A and Figure 7A for panel B. A: Medial level of the specimen at 4.5 postovulatory weeks (Carnegie stage 15). Rostral is to the left, dorsal side is up. Cranial nerves or ganglia are marked in Roman numbers. Rhombomeres or prosomeres are identified by Arabic numbers. TH-immunolabeling of the telencephalon, midbrain and rostral brainstem is shown in Figure 5A and a high magnification of the locus coeruleus area in Figure 5E. The main TH-immunoreactivity is detected within the IIIrd cranial nerve and root (III), and the rare TH-IR neurons, which correspond to the anlage of the A9-A10 groups, are not present at this level. B: TH-IR elements in medial sagittal sections of a 6-week-old specimen (stage 18). The TH-IR neurons are mapped by dots roughly proportional to the 31 relative density. The main TH-IR neuronal groups are well defined in the hindbrain, midbrain, and diencephalon and are identified with conventional abbreviations (sn, lc, vta). The vta is shown at a higher magnification in Figure 7A; Note that some labeled cells are detected in the habenular area (hab) around the pineal stalk and in the inferior colliculus (ci), while they have lost their transient immunoreactivity in the superior colliculus. IV, trochlear nucleus and nerve; cb, cerebellar primordium; cs, superior colliculus; dth, dorsal thalamus; lc, locus coeruleus; mam, mammillary body and cell group; mes, mesencephalon; ob, olfactory bulb; p1-p6, prosomeres; pc, posterior commissure; po, preoptic area; r1-r8, rhombomeres; rm, retromammillary area; sc, spinal cord; sol, nucleus of the solitary tract; tel, telencephalon; tm, tuberomammillary nucleus; tr, retroflex tract; vta, ventral tegmental area; vth, ventral thalamus; zi, zona incerta. Reprinted from Puelles L, Verney C. 1998. Early neuromeric distribution of tyrosineimmunoreactive neurons in human embryos. J Comp Neurol 394:283– 308, with permission. 32 C. VERNEY (Vigny and Henry, 1981), DBH, PNMT, calbindin D28K and calretinin, and gonadotropin-releasing hormone (Puelles and Verney, 1998; Verney et al., 1991, 1992, 1993, 1996; Zecevic and Verney, 1995). For single immunocytochemistry, we used the streptavidin-biotinperoxidase staining procedure while the double immunolabeling was accomplished either by the simultaneous visualization of immunofluorescent markers obtained in different species (Verney et al., 1992, 1993) or by a sequential double immunostaining (Verney et al., 1996). Embryonic Neuromeric Description of the TH-IR Catecholaminergic Neuronal Groups in Hindbrain, Midbrain, Diencephalon, and Prosencephalon The segmental organization of the vertebrate neural tube, introduced by the anatomists at the turn of the century, is largely accepted nowadays (see review of Bergquist and Kallen, 1954; Vaage, 1969). Analysis of expression of homeobox-containing genes has provided new insights into the organization of the vertebrate embryos revealing basic developmental units organized in both longitudinal and transverse subdivisions. The hindbrain is composed of 7–8 rhombomeres whose boundaries are determined by fixed pattern of brain stem motor nuclei and nerve roots (Lumsden and Keynes, 1989). In accord with Puelles and Rubenstein (1993), the forebrain (prosencephalon) includes the diencephalon composed of three prosomeres, p1, p2, p3 and the secondary prosencephalon subdivided into three additional segments p4, p5, p6 (Figs. 2B, 4, 5) (Bulfone et al., 1993; Puelles and Rubenstein, 1993; Puelles, 1995; Shimamura et al., 1995). In an embryo of 4.5 postovulatory weeks (Carnegie stage 15), TH-immunoreactive (IR) cells are present in all transverse sectors of the brain: prosomeres, midbrain, rhombomeres, and spinal cord (Figs. 2B, 5). The abundance of TH-IR neurons suggests that these neurons could already exist in slighly younger specimens, but the observed pattern is immature enough to support the assumption that most TH-IR neurons are found close to their respective neuroepithelial source. The CA neurons express TH-immunoreactivity as soon as they leave the ventricular zone where they are generated. Each segment shows a specific pattern, with a dorsoventral topology of the TH-IR neurons distributed in the floorplate, basal plate, and alar plate as described in detail in Puelles and Verney (1998). Rhombomeres. The embryo of 4.5 postovulatory weeks (Carnegie stage 15) displays TH-positive neurons of the presumable A1-C1 and A2-C2 groups distributed, respectively, in the basal and alar plates of rhombomeres 6 (r6) and 7 (r7). Different TH-IR subpopulations are generated in the distinct rhombomeres migrating radially within the segment where they are generated with no clear-cut evidence for interneuro- Fig. 5. Photomicrographs of sagittal TH-immunostained sections of the specimen at 4.5 postovulatory weeks. Rhombomeric boundaries are indicated by arrowheads at the limiting ventricular ridges. Rostral is to the left, dorsal is side up. A: medial sagittal level shown in drawing Figure 4A where TH-immunoreactivity is observed in the III oculomotor neurons and rootlets. Numerous TH-IR neurons are detected in the hypothalamic area (prosomeres, p3–4). A row of TH positive cells is observed in the superior colliculus (cs). B,C: illustrate the dorsolateral hindbrain with diverse, segmentally-restricted groupings of TH-IR neurons and fibers, Note changes in number and density of positive cells across the boundaries of different rhombomeres. D,E: Details of the locus coeruleus area on serial sagittal sections visualized with DBH-immunocytochemistry (D) and TH-immunocytochemistry (E). Note the DBH-immunoreactivity of the trochlear nerve fibers along their intraneural ventrodorsal course. For abbreviations see Figure 4; V, trigeminal ganglion; VII, facial ganglion; VIII, acoustic ganglion; IX, glossopharyngeal nerve root; X, pneumogastric ganglion; XII, hypoglossal ganglion; rh, rhombencephalon. Scale bars ⫽ 100 µm in A–C, 50 mm in D, E. Reproduced from Puelles L, Verney C. 1998. Early neuromeric distribution of tyrosine-immunoreactive neurons in human embryos. J Comp Neurol 394:283–308, with permission. EMBRYONIC CATECHOLAMINERGIC NEURONS IN HUMANS Fig. 6. Sagittal section showing TH-IR elements in the hindbrain at 6 postovulatory weeks (stage 18). The rhombomeric limits (indicated by arrowheads) are traced by reference to the whole section series, cranial nerve roots, and nuclear formations as seen in adjacent Nissl-stained sections. The caudal limit of the locus coeruleus (lc) coincides with the r1/r2 boundary, while its rostral end seems to lie caudal to the isthmus (is), below the A4 cell group and the cerebellum (cb). The caudal A5 group lies in r6. The caudalmost hindbrain shows 33 the A1, A2 basal part—marked A2—and C1 basal cell groups, as well as numerous alar TH-IR cells of conventional A2 within the solitary nuclear complex. Positive axons of the ascending and descending CA pathways are clearly observed. Note the TH-IR neurons of the A8 primordium just rostral to the isthmo-mesencephalic (mes) boundary. Scale bar ⫽ 200 µm. Reproduced from Puelles L, Verney C. 1998. Early neuromeric distribution of tyrosine-immunoreactive neurons in human embryos. J Comp Neurol 394:283–308, with permission. 34 C. VERNEY meric migration (Fig.5 A–C). Within r1 segment, the anlage of the locus coeruleus (A6) and its cerebellar extension (A4) are labeled by both TH- and DBHantisera (Fig. 5 D, E). The hindbrain of older embryos corresponding to 5 to 6 postovulatory weeks (Carnegie stage from 16 to18) exhibits a larger number of TH positive neurons (Fig. 6). These cells are observed close to the motor nuclei, located near the boundary between the basal and alar plates (Nobin and Björklund, 1973; Puelles and Verney, 1998; Robert et al., 1984). TH-IR neurons of A1/C1 are associated with the ambiguus motor nucleus, and those of A2/C2 with the dorsal motor nucleus of vagus. These cell groups are located in rhombomeres r7–r8. The caudal part of A5 is near the facial motor nucleus whereas its rostral part is contiguous to the trigeminal motor nucleus. In the human infant hindbrain, Robert and collaborators (1984) reported similar TH positive neurons but identified the former part as A5 group and the latter as A7 group. The locus coeruleus clearly extends from the isthmus to r1/r2 boundary in our material, whereas the subcoeruleus group appears to be restricted to r2–r3, typically formed by larger cells lacking DBH immunoreactivity at this stage (Puelles and Verney, 1998). Midbrain and Diencephalon. In the midbrain of an embryo of 4.5 postovulatory weeks (Carnegie stage 15), the major TH-IR basal plate population coincides topographically with the oculomotor neurons and nerve root (Figs. 4A, 5A). Almquist et al. (1996) have recently shown a similar labeling in a 4.5-week-old embryo and interpreted the latter cell group as the prospective dopaminergic A9–A10 groups. We do not share this interpretation and believe that only the contiguous rare TH-IR cells observed at the medial floor plate of this same embryo represent the earliest anlage of the A9–A10 dopaminergic populations. During the 5–6 gestational weeks, the medial ventral tegmentum of the midbrain displays an increase in the number of packed TH-IR neurons corresponding to the anlage of A8-A9-A10 groups, which are generated in the mesencephalon but also in the contiguous prosomeres up to the mammillary bodies (Figs. 2B, 4B, 7A). We have studied in detail this region since it has not been clearly described in embryological studies. The analysis of adjacent sagittal and frontal sections stained by Nissl technique and immunostained for TH and DBH as well as for the differentiation markers such as calretinin and calbindin-D28K, allowing for the characterization of the relevant transverse interneuromeric boundary of different neuromeres and the limits between roof, alar, basal and floor plates (Puelles and Verney, 1998; Verney et al., 1992) (Figs. 4B, 7). The isthmic fovea and the trochlear decussation represent the border between the isthmus and the mesencephalon, the posterior commissure marks the boundary between the mesencephalon and the prosomere 1. The fasciculus retroflexus marks the limit between the prosomeres p1–p2, the zona limitans intrathalamica is located between p2–p3, and the mammillary pouch is present at the boundary between p3 and p4 (Verney et al., unpublished data). Most of the TH positive neurons of A8-A9-A10 groups are generated during the 5–7 postovulatory weeks in the ventricular zone of the floor plate. They migrate at first radially and later laterally within the basal plate. A few DA neurons are generated in the medial basal plate close to the boundary with the floor plate (Fig. 7B). In the classical embryological studies, the extent of the floor plate was restricted to the hindbrain, but the recent reexamination of this region supports the idea of a rostral extension of the floor plate up to the median eminence (Kingsbury, 1920; Kuhlenbeck, 1973; see also discussion in Puelles, 1995). Different data support this idea, for example, the expression of annexin IV, which defines a floor plate region that extends from the caudal spinal cord all the way rostrally to the diencephalon (Hamre et al., 1996). In addition, the induction in vitro of the DA phenotype by the mesencephalic floor plate in the chick is an argument in favor of the genesis of these DA neurons in the floor plate of all vertebrates (Hynes et al., 1995; Wang et al., 1995). The DA A9 A10 neuronal groups are generated in the midbrain as well as in the diencephalic segments p1, p2. Neuropathologists had already pointed out that the rostral, diencephalic third of the adult human substantia nigra (located rostral to the emergence of the third cranial nerve) displays DA neurons that are more resistant to neuronal death in Parkinson’s disease than the ones located in the intermediate and caudal thirds located within the mesencephalon (Ruberg et al., 1997; see review in Van Domburg and Donkelaar, 1991). Although this diencephalic DA neuronal population of A9 group is more restricted than the mesencephalic one, it could have acquired from its embryonic origin a characteristic that makes it more akin to other diencephalic hypothalamic DA populations, all of which resist cell death in this disease (Matzuk and Saper, 1985). Diencephalon and Secondary Prosomeres (Telencephalon). At 4.5 postovulatory weeks, prosomeres p3 to p6 display a series of TH-IR neuronal groups mostly located in the basal plate that correspond to the anlage of the A11–15 hypothalamic groups (Figs. 2B, 4A, 5A) (for details see in Puelles and Verney, 1998). Although the DA hypothalamic neurons are composed of heterogenous cell populations, the precocity detected in humans is not observed in rodents (Ugrumov, 1994). In humans, one or two weeks later, substantial TH-IR Fig. 7. The area of the anlage of the substantia nigra and ventral tegmental area at 6 postovulatory weeks (stage 18). A: On a midsagittal section, TH-IR neurons of the ventral tegmental area (vta) are packed in the mesencephalon (mes) and decrease in number in the prosomeres p1 and p2 (this section is schematized in Fig. 4B). Rostral is to the left, dorsal is side up. There is a sharp caudal limit at the isthmo-mesencephalic boundary. Note the numerous positive neurons in the retromammillary (rm) and mammillary areas (mam). Reproduced from Puelles L, Verney C. 1998. Early neuromeric distribution of tyrosine-immunoreactive neurons in human embryos. J Comp Neurol 394:283–308, with permission. B,C: Serial coronal sections at the level of the boundary between the prosomeric p1and p2 segments. Numerous neurons are immunostained for TH (B) and calbindin D28K (CABP) (C). The limit between these segments is the fasciculus retroflexus (fr) indicated by an arrow in B. The black lines indicate the limits between the floor (fp), basal (bp), and alar plates (ap). In B, the TH positive neurons of the substantia nigra (sn) generated in the ventricular zone (vz) of the fp and contiguous bp, migrate first superficially and then laterally within the superficial bp. In C, a restricted calbindin D28K positive neuronal population, is generated in the medial fp and follow the same pattern of migration as the TH-immunoreactive neurons. Note the calbindin D28K positive fibers of the posterior commissure (pc). IIIv: third ventricle. Scale bars ⫽ 50 µm. EMBRYONIC CATECHOLAMINERGIC NEURONS IN HUMANS Fig. 7. 35 Fig. 8. TH and DBH-immunostaining of frontal sections at the 13th gestational week. A, B: Sequential sections of the noradrenergic locus coeruleus (A6) labeled with TH- (A) and DBH-immunocytochemistry (B). The dorsal cell group is the locus coeruleus. Numerous positive terminals obscure the neurons of the ventral group, the locus subcoeruleus. C: Substantia nigra (SN): cluster of packed TH-IR neurons (arrow) of the pars compacta (c), which send their dendrites in the sizewise restricted pars reticulata (r). CPe, cerebral peduncle. Scale bars ⫽ 50 µm in A, B, 100 µm in C). Reproduced from Zecevic N, Verney C. 1995. Development of the catecholamine neurons in human embryos and fetuses, with special emphasis on the innervation of the cerebral cortex. J Comp Neurol 351:509–535, with permission. Fig. 9. Frontal sections at the 7th postovulatory week. A: TH positive cells are observed at two locations, in the ganglion terminale (long arrow) and in the retina (thick arrow). B: Higher magnification of the TH-IR neurons migrating along the medial part of the ganglion terminale (gt) and nervus terminalis towards the telencephalon (tel). op, olfactory placode; ns, nasal septum. Reproduced from Verney C, El Amraoui A, Zecevic N. 1996. Comigration of tyrosine hydroxylase- and gonadotropin-releasing hormone-immunoreactive neurons in the nasal area of human embryos. Dev Brain Res 97:251–259, with permission. C: Migrating retinoblasts exhibit TH-immunoreactivity in the peripheral retina. L, crystallin lens. D: TH-positive post-migratory round somata and fibers in the nerve fiber layer are observed in the posterior retina. E: An enlarged view of the TH-IR retinoblasts in the peripheral retina: the nuclei observed at different levels of the single neuroblastic layer exhibit attachment processes to both limiting membranes of the neural retina (arrows). In the more central retina, postmigratory cells which have lost their attachment processes are seen (arrowhead). Reproduced from Versaux-Botteri C, Verney C, Zecevic N, Nguyen-Legros J. 1992. Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos. Dev Brain Res 69:283–287, with permission. Scale bars ⫽ 50 µm (B–D), 10 mm (E). 38 C. VERNEY neuronal populations are observed in the basal plate, around the mammillary bodies, that we have included in A10 group, and in the arcuate region of p4, p5 segments as the anlage of A12 group. The alar TH-IR neuronal population located along the cerebral aqueduct in the prosomeres p1–p2 corresponds to the caudal extention of A11 group. Rostrally, the alar TH positive cells in the zona incerta correspond to A13 group. A band of TH positive cells are observed in the alar plate of p4, p5, p6; overlapping the periventricular nucleus is presumably the A14 group. TH-IR neurons migrate toward the supraoptic nucleus (A15) whereas a separate TH-IR population is present in the anterior preoptic area across p5–p6 segments (Fig. 2B, 4B) (Su et al., 1987; Tillet, 1994; Ugrumov, 1994). Differentiation Features of the Catecholaminergic Cell Bodies As already emphasized, TH-immunoreactivity is detectable very early in different CA groups. DBH phenotype is observed as early as TH positivity in the noradrenergic neurons of the locus coeruleus area but in the other hindbrain CA neurons it is detected only during the fetal period. PNMT phenotype could be detected in the hindbrain of human specimens we had, only from the 13th gestational week onward. From 8 gestational weeks on, no TH positivity is detected near the proliferative ventricular zone. This indicates that all CA neurons are generated during the embryonic period similarly to what has been described for the monoaminergic cells in the Rhesus monkey (Levitt and Rakic, 1982). Hindbrain. At 4 months old, the noradrenergic and adrenergic neurons of the fetal hindbrain have developed dendrites, and small fluorescent neurons are seen in the area postrema (Nobin and Björklund, 1973). At this age, we observed the locus coeruleus located dorsal to the locus subcoeruleus along a rostrocaudal extention of 500 µm. TH-IR and DBH-IR neurons of the locus coeruleus exhibited two or three long dendritic processes whereas in the the locus subcoeruleus area, a dense network of positive terminals overlaps the positive cells (Fig. 8A,B). Midbrain and Diencephalon. During the early fetal period, the lateral and dorsal TH-IR neurons of the substantia nigra and of the ventral tegmental area differentiate earlier than the ventral and medial ones. At 4 months old, TH-IR neurons of the A8-A9-A10 complex display the same overall distribution pattern as in adult humans (Gaspar et al., 1983; Halliday and Tork, 1986; Kitahama et al., 1994; Pearson et al., 1983, 1990; Van Domburg and Donkelaar, 1991; Zecevic and Verney, 1995) but their differentiation is not yet complete. Along the rostrocaudal extention of the A9–A10 groups, the DA neurons of the dorsal horizontal band show more differentiated features than the one located ventrally and medially. The ventral DA neurons of the pars compacta are clustered and develop dendrites ventrally towards the pars reticulata which is rather small at this stage (Figs. 2, 8C) (Zecevic and Verney, 1995). In the ventral tegmental area, TH positive neurons display a similar distribution to that described in adult human brain (Fig. 3) (Gaspar et al., 1983; Pearson et al., 1983, 1990; Zecevic and Verney, 1995). Caudal to the red nucleus and to the bulk of the substantia nigra, TH-IR neurons of the A8 group are dispersed within the reticular formation. Early Expression of the Calbindin D28 Kphenotype Within a Subpopulation of Dopaminergic Neurons of A8-A9-A10 Groups. The calbindin D28K phenotype is observed within DA neurons of the A8-A9A10 groups as early as 5 gestational weeks in human (Fig. 7B). Contrary to what is usually observed in many other brain regions during development, calbindin D28K-immunoreactivity is not transient within these TH-IR neurons as demonstrated by double immunocytochemical labeling at different stages (Verney et al., 1992, Verney et al., unpublished data). At the 13th gestational week, the pattern of distribution of the DA neuronal population expressing calbindin D28K phenotype is similar to that described in the adult: these cells are located in a dorsal band extending from the lateral substantia nigra to the medial ventral tegmental area and are disseminated within the A10-A8 groups. The presence of calbindin D28K has been hypothesized to play a neuroprotective role in the selective neuronal vunerability of DA neurons in Parkinson’s disease (German et al., 1992; Hirsch et al., 1992; Lavoie and Parent, 1991; Yamada et al., 1990). The function of this early calbindin D28K expression is not known but, by analogy, it could also protect these neurons from developmental cell death occurring in the substantia nigra (Oo and Burke, 1997). Transient Embryonic Expression of Tyrosine Hydroxylase Immunoreactivity in Discrete Neuronal Populations in Humans As already mentioned for the oculomotor neurons and root, additional TH positive cells are noticed during early development in several areas where no CA neurons are detected in the adult. Transient expression of cellular TH- and DBH-immunoreactivity has been detected in different discrete regions of the embryos such as the spinal cord, the alar plate of the rhombomeres r4–6 (Puelles and Verney, 1998; Zecevic and Verney, 1995). A transient TH positive cell population is present in the colliculi of human embryos similarly to observations made in the colliculi of the postnatal rat (Jaeger and Joh, 1983). Fig. 10. Lateral sagittal sections at the 7th postovulatory week. A: Low magnification photomicrograph of the ascending CA pathways originating in the locus coeruleus (lc) and in the substantia nigra (sn). TH positive axons run rostrally within the medial forebrain bundle (mfb) to reach the ganglionic eminence (ge). B,C: TH-IR fibers penetrate ventrally the ganglionic eminence (ge) and the lateral telencephalic anlage. The cortical plate (cp) composed of a few rows of neurons is indicated by short arrows. In darkfield illumination of the same section, TH-IR axons are seen to enter the lateral anlage of the cerebral cortex by the intermediate zone (long white arrow) below the cp. Note the rare TH positive axons in the marginal zone above the cortical plate. D: Dark field illumination of a sagittal dorsal section at the 11th gestational week: in the cerebral cortex. TH-IR fibers are located in the intermediate zone (iz), subplate layer (sp) and sparsely in the marginal zone (mz). No positive fibers are observed in the cortical plate (cp) or ventricular zone. cb, cerebellum; ci, inferior colliculus; lv, lateral ventricle; sol, nucleus of the solitary tract; tel, telencephalon. Scale bars ⫽ 250 µm in A, 100 µm in B,C, 50 µm in D. B,C,D reproduced from Zecevic N, Verney C. 1995. Development of the catecholamine neurons in human embryos and fetuses, with special emphasis on the innervation of the cerebral cortex. J Comp Neurol 351:509–535, with permission. EMBRYONIC CATECHOLAMINERGIC NEURONS IN HUMANS Fig. 10. 39 40 C. VERNEY Two main regions of TH-immunoreactivity are noticed in humans (Fig. 9). One is the anlage of the eye where TH-immunoreactive retinoblasts and ganglionlike cells are observed sending labeled axons into the optic nerve of embryos of 6–7 postovulatory weeks (Fig. 9C–E). Since in the adult retina, the only TH positive cells are a subpopulation of amacrine cells, the expression of this enzyme in some ganglion-like cells represents either a transient developmental event or indicates that these cells subsequently undergo transformation through axonal degeneration (Frederick et al., 1982; Nguyen-Legros et al., 1992; Versaux-Botteri et al., 1992). The second area is the nasal region where TH-IR cells generated in the olfactory placode migrate towards the medial and rostral telencephalon (Fig. 9A,B). In fact, they follow the same migratory stream as the gonadotropin-releasing-hormone (GnRH)-containing hypothalamic neurons that are generated in the olfactory placode and migrate towards the preoptic area (Schwanzel-Fukuda et al., 1996). Some neurons exhibit only the TH or GnRH phenotype and others express both phenotypes. These neurons migrate towards the basal forebrain and we do not know if they could correspond to the TH-IR neuronal populations observed in the human basal telencephalon of the adult described in a former paragraph (Verney et al., 1996). Development of the Catecholaminergic Axons in the Human Telencephalon The TH positive axons are observed as soon as the neurons are generated and migrate superficially at 4.5 postovulatory weeks (Puelles and Verney, 1998). At the end of the embryonic period, the different pathways described in rodents are well recognizable in humans (Lindvall and Björklund, 1974; Nobin and Björklund, 1973; Puelles and Verney, 1998; Zecevic and Verney, 1995). Ascending and descending axons run through the central tegmental tract (Fig. 6) from the medulla oblongata to the mesencephalon. The noradrenergic fibers, arising from locus coeruleus neurons, run rostrally in the dorsal tegmental bundle. They join the ascending mesotelencephalic tract emerging from the DA cell groups A8–A10 and form the medial forebrain bundle (Fig. 10A). This TH-positive bundle provides efferent fibers to the hypothalamic areas and to the basal ganglia. Positive axons are observed in the ganglionic eminence at the end of the embryonic period (Fig. 10B). TH positive patches are noticed in the anlage of the basal ganglia, at 13 gestational weeks (Verney, personal observation). At this stage, and later during prenatal period, the nigrostriatal system also expresses several markers of DA transmission such as D1 and D2 receptors and dopamine transporter (Aubert et al., 1997; Brana et al., 1997). Penetration of the Noradrenergic and Dopaminergic Axons to the Anlage of the Cerebral Cortex. No positive fibers penetrate the anlage of the cerebral cortex at 6 gestational week in the primordial plexiform layer, before the formation of the cortical plate. When the first cortical plate neurons migrate at 7–8 gestational weeks in humans, the first TH positive fibers penetrate the lateral frontal cortex. This takes place below the cortical plate in the intermediate zone with rare positive axons running in the marginal zone (Fig. 10B,C) (Marin-Padilla, 1970; Sidman and Rakic, 1973; Zecevic, 1993). So, the arrival of CA axons coincides with the formation of the cortical plate, and no THimmunoreactive axons are detected in cortical regions where the cortical plate is absent. Such an invasion of CA axons after the formation of the cortical plate has also been observed in rodents by us and others (Schlumpf et al., 1980; Verney et al., 1982, 1984). In humans, sparse DBH-IR noradrenergic axons penetrate the telencephalic wall in a pattern similar to that described for TH-IR fibers and following the same timing. During the following three to four weeks, the invasion of CA fibers does not follow the lateral to medial gradient of formation of the cortical plate (Zecevic, 1993), because the fronto-medial cortical areas are penetrated before the dorsal cortex. The ‘‘waiting period,’’ when the TH-IR axons are restricted to the subplate layer and intermediate zone without penetrating the cortical plate, lasts for approximately 4 weeks. The subplate layer, that is particularly thick in humans, is known to represent a transitional layer containing differentiated neurons, afferent fibers (first thalamic fibers) and the first synapses (Kostovic and Rakic, 1980, 1990; Shatz et al., 1988). In our results, the CA fibers arrive in this ‘‘waiting compartment’’ from the 8 postovulatory weeks on, before the other afferents currently identified in humans. The callosal and corticocortical connections start to develop at 12–13 gestational weeks (Rakic and Yakovlev, 1968) in parallel with the subcortical (mostly thalamic) afferences currently visualized by their acetylcholinesterase positivity (Candy et al., 1985; Kostovic and Goldman-Rakic, 1983). In fact, in rodents, the first thalamic afferents are known to penetrate the subplate layer of cortical anlage in similar timing as the CA axons (Molnar and Blackmore, 1995; Verney et al., 1982, 1984). Therefore, one could expect the first human thalamic afferents to penetrate the cortical anlage as early as 8 gestational weeks. The first synapses in the developing human cortex are observed above and below the cortical plate at 7 gestational weeks (Molliver et al., 1973; Larroche and Houcine, 1982). Involvement of the CA fibers in the earliest, transient synapses in the subplate layer has not been directly demonstrated, but in the monkey cortex the earliest synapses often contain dense core vesicles, suggesting that they can be CA (Zecevic et al., 1989). A rostrocaudal gradient of penetration of CA fibers is observed with a clear invasion of the subplate layer of the occipital pole at the 13th gestational week. At that time, the TH-IR axons penetrate the cortical plate in the rostral areas, mostly ascending from fibers in the subplate layer, rarely descending from the marginal zone. Further Development of the Noradrenergic and Dopaminergic Fibers and Terminals in the Cerebral Cortex. In 20–24-week-old human fetuses, a widespread TH-IR and DBH-IR innervation is observed in a regional and laminar specific pattern of distribution in the frontal cortex (Fig.11) (Verney et al., 1993). The densest dopaminergic TH positive innervation is observed in the anlage of the motor, cingulate and insular cortices whereas a lower density is detected in the rostral prefrontal cortical anlage. DBH-IR noradrenergic afferents are less numerous than dopaminergic Fig. 11. Fetus of 24 gestational weeks. Contiguous 10-mm-thick cryostat sections of the dorsofrontal cortex in the presumable motor area. A: Dark-field illumination of TH-IR fibers distributed in the whole thickness of the cortex . B: Nissl stained section. The neurons of the cortical plate (cp) are not densely packed (except in layer II) as they started to fifferentiate, but the cortical layering is yet not visible. C: dark field illumination of DBH-IR fibers at the same level. Scale bar ⫽ 100 µm. Reproduced from Verney C, Milosevic A, Alvarez C, Berger B. 1993. Immunocytochemical evidence of well-developed dopaminergic and noradrenergic innervations in the frontal cerebral cortex of human fetuses at midgestation. J Comp Neurol 336:331–344, with permission of the publisher. 42 C. VERNEY Fig. 12. TH-labeled axons in the frontal medial area 9 at different postnatal stages in Rhesus monkey observed on 40-µm-thick cryostat sections. To compare the density of TH-positive axons of these postnatal stages to that observed at a prenatal stage shown in Figure 11, the different thickness of the sections made in both cases must be taken into account. A–C: Darkfield photomicrographs of TH immunoreactivity. The density of labeled axons is increasing especially in layer III to reach its maximal value in the adolescent animals (2.75-yearold) before declining to adult levels (5.7-year-old). Scale bars ⫽ 200 µm. D,G: Examples of recontructions, using the Eutectic Neuron Tracing System, of TH-labeled axons and varicosities in deep layer III from animals of the following ages: 8 days (D), 37 days (E), 2.8 years (F), and 5.7 years (G). Reproduced from Rosenberg DR, Lewis DA. 1995. Postnatal maturation of the dopaminergic innervation of monkey prefrontal and motor cortices: a tyrosine hydroxylase immunohistochemical analysis. J Comp Neurol 358:383–400, with permission of the publisher. TH positive ones in all the cortical areas studied (Fig. 11). In all areas, the upper subplate and the lower part of the cortical plate exhibit a dense TH and DBH-IR axonal innervation whereas fewer axons are present in the molecular layer and intermediate zone. Surprisingly, the pattern of distribution and even the density of fibers are already comparable to those described previously in the adult human cerebral cortex using similar techniques (Gaspar et al., 1989; Verney et al., 1993). At the time when the CA afferents invade the different areas and layers of the frontal cortex, all cortical neurons have been generated but their migration has not ended and the differenciation process is far from completion (Rakic, 1988). It is difficult to compare the pattern of distribution of CA axons at midgestation with that found in the adult since the cortical layering EMBRYONIC CATECHOLAMINERGIC NEURONS IN HUMANS is not visible at midgestation. The more obvious difference between these two stages is the low density of DA axons in layer I of the fetal frontal cortex while this layer is one of the most densely innervated layer at birth (Lewis and Harris, 1991) and in adult primates (Fig. 12) (Berger et al., 1988; Gaspar et al., 1989; Lewis et al., 1987). In the adult monkey cerebral cortex, the majority of the DA synapses are located on spines of pyramidal cells dendrites (Smiley and Goldman-Rakic, 1993; Smiley et al., 1992), which are not yet present early in gestation. The bulk of synaptogenesis in monkeys occurs during the second half of gestation and during the first postnatal weeks (Bourgeois et al., 1994; Zecevic et al., 1989), in parrallel with the appearance of binding sites for DA receptors (Lidow et al., 1991). During the protracted prenatal and postnatal periods, a reorganization in the distribution of DA fibers and terminals is likely to occur in humans as it has been described for monkeys (Foote and Morrison, 1984; Rosenberg and Lewis, 1995). Rosenberg and Lewis (1995) have observed a transient sprouting of DA fibers and terminal fields in layer III of the prefrontal monkey cortex during postnatal development with a peak at adolescence and a decrease in adulthood (Fig. 12). One might expect a similar discontinuity in the growth of CA terminal field in the developing human cerebral cortex. Conclusions Our results on early development reveal important information for the use of the first trimester mesencephalic tissue as donor material in clinical trials for treatment of Parkinson’s disease (Lindvall et al., 1992). First, the sampling of the anlage of the substantia nigra should be made at the mesencephalic level as well as anteriorly in the prosomeres p1–p2 and, second, this sampling should be done early in development, around 6–7 gestational weeks, when all the DA neurons are being generated but before the development of the extended ascending axonal pathways. In primates, the development of CA neurons occurs earlier than in rodents when normalized to the length of gestation. In fact, the overall pattern of early development of the CA systems appears to be phylogenetically well preserved in mammals. This is particularly true for the development of the noradrenergic and adrenergic cell bodies located in the rhombencephalon, and for the DA neurons of the midbrain. However, the DA neuronal populations distributed in the diencephalon and telencephalon cell groups display specific features unique to humans when compared to rodents: (1) in humans, numerous DA neurons are detected in mammillary areas early in development whereas only rare neurons are detected in rodents, (2) the hypothalamic DA cell groups are particularly precocious in humans and there is an important DA neuronal subpopulation in the supraoptic nucleus which is specific to humans, (3) numerous TH-IR, presumably DA neurons, are detected in the human basal telencephalon, and (4) there is a widespread DA innervation in all the different areas and layers of the human cerebral cortex as compared to the innervation in restricted cortical areas in rodents. The precocity of development of the DA innervation in early fetal life raises once again the 43 difficult question on their role during the protracted prenatal and early postnatal period in man. Recently, the presence of a monoamine, serotonin, within the developing thalamic efferents towards the cerebral cortex in mice has been shown to be essential for the cytoarchitectonic differentiation of the barrel fields in the somatosensory cortex. Moreover, monoaminergic transporters have also been found in the thalamocortical system (Cases et al., 1996; Lebrand et al., 1996, 1998). It would be interesting to see if similar expression of monoamine transporters is present during the early thalamic development in humans. ACKNOWLEDGMENTS The author is indebted to Prof. L. Puelles for his great help brought in the understanding of the organization of the human embryos and Dr. N. Zecevic for providing the embryonic specimens. We thank A.Vigny and J.P. Henry, who gave us the anti-TH and anti-DBH antibodies, respectively, Drs. P. Gaspar, E. Bloch-Gallego, P. Gressens, and K. Kultas-Ilinsky for critical reading of the manuscript, and Chantal Alvarez and Aude Muzerelle for technical work. 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