Dopamine neuron systems in the brain: an update

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1 Review TRENDS in Neurosciences Vol.30 No.5 Dopamine neuron systems in the brain: an update Anders Björklund 1 and Stephen B. Dunnett 2 1 Neurobiology Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund SE-22184, Sweden 2 School of Biosciences, Cardiff University, Cardiff CF10 3US, Wales, UK The basic organization of the catecholamine-containing neuronal systems and their axonal projections in the brain was initially worked out using classical histofluorescence techniques during the 1960s and 1970s. The introduction of more versatile immunohistochemical methods, along with a range of highly sensitive tracttracing techniques, has provided a progressively more detailed picture, making the dopamine system one of the best known, and most completely mapped, neurotransmitter systems in the brain. The purpose of the present review is to summarize our current knowledge of the diversity and neurochemical features of the nine dopamine-containing neuronal cell groups in the mammalian brain, their distinctive cellular properties, and their ability to regulate their dopaminergic transmitter machinery in response to altered functional demands and aging. Introduction The study of catecholamine (CA) neurons in the brain goes back to the early 1960s. Using the newly introduced formaldehyde histofluorescence method [1] (Box 1), Carlsson, Falck and Hillarp [2], were the first to identify the two primary CAs, noradrenaline (NA) and dopamine (DA), in discrete neuronal systems in the brain. Two years later, Dahlström and Fuxe [3] published the first detailed account of the distribution of CA and serotonin-containing neurons in the rat brain. They identified twelve groups of CA cells (designated A1 A12) distributed from the medulla oblongata to the hypothalamus. Five additional cell groups, A13 A17, located in the diencephalon, olfactory bulb and retina, as well as three adrenaline-containing cell groups, C1 C3, were added later [4]. This nomenclature has been retained and has proved advantageous for two reasons. First, the CA cell groups are in most cases not confined to single, defined anatomical structures. Second, the distribution of cell bodies within each cell group varies markedly between different mammalian species (e.g. between rodents, primates and human), and is even more variable between different vertebrates [5,6]. The use of the A1 A17 nomenclature is thus convenient when comparing data obtained in different animal species. With the introduction of immunohistochemistry for the CA-synthesizing enzymes, tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC) and Corresponding author: Björklund, A. (anders.bjorklund@med.lu.se). Available online 3 April dopamine-b-hydroxylase (DBH) in the 1970s, it became possible to map the CA systems in greater detail, and these new tools made it possible to distinguish more accurately between different CAs. In Figure 1, the distribution of the nine major dopaminergic cell groups (excluding retina), as revealed by TH immunohistochemistry, is schematically illustrated in sagittal view of the rat brain, both in embryonic development (Figure 1a) and in adults (Figure 1b and Box 2). TH immunohistochemistry has largely confirmed the original mapping made with the histofluorescence technique, but with some notable discrepancies. In hypothalamus and adjacent areas of the basal forebrain, in particular, TH-positive cells far outnumber the DA-containing neurons detectable with the histofluorescence method. Additional systems of TH-immunoreactive neurons not containing detectable levels of DA or NA have been identified in the rostral hypothalamus and preoptic area (designated A15 by Hökfelt et al. [4]), and further abundant TH-positive cells were detected in primates and human in cortical and striatal areas [7,8]. The failure to detect the decarboxylating enzyme, AADC, or any CA in these cells raises the question as to whether all THexpressing cells are indeed catecholaminergic, that is, do they use DA or NA as their neurotransmitter? Immunohistochemistry has also revealed somewhat surprisingly that some DA neurons, particularly in the basal hypothalamus and the olfactory bulb, coexpress DA and g-aminobutyric acid (GABA) (or the GABA-synthesizing enzyme, glutamic acid decarboxylase, GAD), and might thus operate with more than one transmitter. In the hypothalamus, the DA neurons in the arcuate nucleus (group A12) have been shown to colocalize various neuropeptides, such as growth hormone-releasing hormone (GHRH), neurotensin, galanin, enkephalin and dynorphin, suggesting a broader neuroendocrine role of these neuron subtypes [9]. Moreover, in the mesencephalon, access to more refined tract-tracing techniques has gradually come to reveal a more complex picture of the anatomical organization and projection patterns of the DA neurons in the A8 (retrorubral area), A9 (substantia nigra, SN) and A10 (ventral tegmental area, VTA) cell groups than was originally conceived (see below). Are all TH-positive cells catecholaminergic? TH-positive cells that are undetectable with the histofluorescence technique occur in rodents in the hypothalamus (particularly in the rostral A15 cell group); in primates and /$ see front matter ß 2007 Published by Elsevier Ltd. doi: /j.tins

2 Review TRENDS in Neurosciences Vol.30 No Figure 1. Distribution of DA neuron cell groups in the developing (a) and adult (b) rodent brain. The dopamine neurons in the mammalian brain are localized in nine distinctive cell groups, distributed from the mesencephalon to the olfactory bulb, as illustrated schematically, in a sagittal view, in (a) the developing and (b) the adult rat brain. The numbering of the cell groups, from A8 to A16, was introduced in the classic study of Dahlström and Fuxe in 1964 [3], and is still valid at present. (Drawing in (a) is modified from Marin et al. [65]; in(b) the principal projections of the DA cell groups are illustrated by arrows). Abbreviations: lge, lateral ganglionic eminence; mge, medial ganglionic eminence; p1 p3, prosomeres 1 3. Box 1. Catecholamine histofluorescence The histofluorescence method for visualization of catecholamines and serotonin in tissues was developed by Falck, Hillarp and coworkers in [1,2]. This was the first microscopic method that allowed sensitive visualization of a neurotransmitter within nerve cells, and it dominated the field of catecholamine research for more than two decades until it was replaced in the early 1980s by the more versatile immunohistochemical methods. The original Falck Hillarp method was based on the exposure of freeze-dried tissue to formaldehyde vapor, allowing dopamine and noradrenaline to be converted to isoquinoline molecules that emit a yellowgreen fluorescence in the microscope. Later modifications, notably the glyoxylic acid method (where the fluorophore formation is carried out in an auto-catalysed reaction in aqueous solution [48]), provided an improved sensitivity and precision, which allowed the detection of the dopamine- and noradrenaline-containing axons and axon terminals in great detail (Figure I). The drawback of these methods was that they required special equipment, and specialized skills, to give optimal results. For this reason, full and effective use of the histofluorescence techniques remained a privilege of a few specialized laboratories, in contrast to the current widespread use of immunohistochemistry. Figure I. DA terminals in the rat brain as visualized with the glyoxylic acid method. human, they are abundant also in the basal forebrain, striatum and cortical areas. These neurons do not contain any detectable CA (DA or NA) and they also lack the decarboxylating enzyme, AADC, as well as the vesicular monoamine transporter, VMAT-2 [10 12] (see Figure Ib in Box 2). The vast majority of these neurons have morphological features of GABAergic interneurons, but in marmosets, at least TH is also coexpressed with choline acetyltransferase in magnocellular cholinergic neurons of the nucleus basalis of Meynert [13]. During development, the TH-encoding gene is expressed in a subset of interneuron precursors that migrate from the lateral and/or medial ganglionic eminences to the cortex, striatum and the olfactory bulb. In rodents, the cells located in the cortex and striatum express TH protein only transiently during the first postnatal month [14,15]. In adult mice and rats, they express TH mrna, but no detectable levels of the TH protein, whereas the cells settling in the glomerular layer of the olfactory bulb express both TH protein and DA, probably as a result of synaptic activation from the olfactory afferents [16]. In contrast to the cortical and striatal TH-positive cells, the olfactory bulb interneurons express both AADC and DA. Interestingly, these cells have been reported to lack detectable levels of VMAT-2 [12]. Because VMAT-2 is required for vesicular storage and release, this suggests that the periglomerular DA interneurons, which possess dendrites but no axon, might release their transmitter in an unconventional, non-vesicular manner. In the striatum, the TH + /AADC neurons are located predominantly in two areas: dorsally, near the corpus callosum, and ventrally in and close to the nucleus accumbens and the anterior commissure. These latter cells appear to be continuous with the TH-positive cell populations present in the basal forebrain [7,17]. The striatal TH-positive neurons are present in modest numbers in the intact striatum in monkeys (66 cells per 50 mm-thick section; [17]) and human (3.2 cells per 20 mm section; [18]), but not in rodents [17,19]. Interestingly, the number of striatal TH-positive neurons increases several-fold after

3 196 Review TRENDS in Neurosciences Vol.30 No.5 Box 2. Dopamine neurons on the net The Allen Brain Atlas contains data on the expression patterns of over genes in the mouse brain [66] and is now accessible on line ( Three genes directly linked to DA neurotransmission tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC, named dopa decarboxylase, DDC, in the database), and vesicular monoamine transporter-2 (VMAT-2; named Slc18a2) are presented in the Atlas as in situ hybridization histograms in 1:8 series of coronal sections from the olfactory bulb to the lower brain stem (and also, for AADC and VMAT-2, in the sagittal plane). The DA transporter DAT, however, is notably missing. 3D images generated from these data by the Brain Explorer software provided on the ABA website provide a general overview of the distribution of TH-, AADCand VMAT-2-expressing neurons as detected in the in situ autoradiograms. Figure Ia shows the distribution of TH-expressing cells only (in yellow); Figure Ib shows all three genes combined, TH (in yellow), AADC (in green) and VMAT-2 (in red). The size of the diamonds reflects the level of expression at each site. So, how accurate are these maps? The general distribution of TH-expressing cells, as depicted in Figure Ia, matches well the published data. The shapes of individual nuclei (e.g. SN pars compacta, VTA, the hypothalamic arcuate and periventricular nuclei) are not easily discernable in the reconstructed 3D image, but are well visible in the actual coronal sections. AADC and VMAT-2 are, as expected, coexpressed with TH in the midbrain A8 A10 cell groups (note, however, that the yellow, green and red dots are displayed separated because they were generated from adjacent sections in the coronal 1:8 series). The expression of AADC and VMAT-2 in the hypothalamic cell groups is overall lower and more variable, and the TH-expressing neurons in the A15 cell group do not express either AADC or VMAT-2, which is consistent with the literature. In the habenula (HAB) TH is known to be expressed in a population of small, rounded cells in the medial nucleus (named A10dr [4]). These cells do not express either AADC or VMAT-2 [12]; the AADC-expressing neurons present in this region are located in the medial part of the lateral habenular nucleus (i.e. in neurons not expressing any TH [67]). In the adult mouse brain scattered cells in striatum and cortex are known to express low levels of TH mrna, but not TH protein [16]. These cells are not depicted in the 3D images shown here, but they are visible in actual autoradiograms in the coronal sections of the ABA series. The TH-positive cells located caudal to the midbrain, and in cerebellum, represent noradrenaline and adrenaline neurons in the A1 A6 cell groups of Dahlström and Fuxe [3]. It is somewhat puzzling that the ABA autoradiograms do not indicate any AADC expression in the TH-positive periglomerular cells in the olfactory bulb (A16 group). It is known that the periglomerular DA neurons lack VMAT-2 [12], but the absence of AADC in these cells is inconsistent with published data [12,16]. This might be a case of false negatives resulting from a relatively high background threshold set for this section series. It is known that both AADC and VMAT-2 are expressed in other locations, such as in serotonergic neurons (e.g. the dorsal raphé nucleus, DRN) and in neurons expressing AADC alone (the so-called D-cell groups of Jaeger et al. [67]). VMAT-2 is also expressed in histaminergic neurons in the posterior hypothalamus [68]. The Atlas also depicts VMAT-2-expressing cells rostrally: the coronal autoradiograms reveal that these cells are located in the caudal septum and intermingled with the fibers of the ventral hippocampal commissure. These cells, however, do not match any known monoaminergic or histaminergic neuronal system in this area. Figure I. Distribution of cells expressing mrna for TH, AADC and VMAT-2, as revealed by the Allen Brain Atlas. (a) Distribution of TH-expressing cells only (in yellow). (b) All three genes combined, TH (in yellow), AADC (in green) and VMAT-2 (in red). The size of the diamonds reflects the level of expression at each site. A8 A16 denote the nine DA neuron cell groups according to Dahlström and Fuxe [3]. Abbreviations: cb, cerebellum; cx, cortex; hpc, hippocampus; ob, olfactory bulb; str, striatum; tect, tectum; thal, thalamus. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in monkeys [17,20] and also in the striatum in patients with Parkinson s disease [18], suggesting a compensatory upregulation. In 6-hydroxydopaminelesioned rats and MPTP-treated mice, few TH-immunoreactive cells (1 3 cells/section) have been described to appear in ventral striatum, mostly within or close to the nucleus accumbens and the anterior commissure [19,21 23]. Although these cells express the DA transporter (DAT), the vast majority of them do not express any detectable levels of either AADC or DA [10,19,23,24]. In both rodents and primates, they have morphological features of GABAergic interneurons and express both GAD and the TH-regulating transcription factor Nurr1, similarly to the periglomerular interneurons in the olfactory bulb. It is an intriguing possibility, therefore, that the increase in the number of TH-positive cells seen after DAdenervating lesions in the striatum is due to the formation of new neurons, derived either from the subventricular zone, which is the source of adult neurogenesis in the olfactory bulb, or from proliferative progenitors within the striatal parenchyma. Experiments using bromodeoxyuridine (BrdU) to label the newly formed cells in MPTPlesioned mice and monkeys [22,25], however, have failed to detect any BrdU incorporation in the newly appearing THpositive cells, suggesting that the increase in TH-positive

4 Review TRENDS in Neurosciences Vol.30 No cell number is due to an upregulation of TH in GABAergic interneurons that are already present rather than a consequence of neurogenesis. TH-positive neurons lacking the AADC enzyme occur not only in forebrain cortical and striatal areas but are also abundant in the A12, A14 and A15 cell groups in the hypothalamus and preoptic region (see Figure Ib in Box 2). TH + /AADC neurons have also been reported to occur elsewhere in the CNS, including the thoracic spinal cord, the dorsal vagal complex (nucleus tractus solitarii and dorsal vagal motor nucleus), area postrema, the parabrachial nucleus and the habenula [12], and in the human mesencephalon (albeit in very low numbers) [26]. The functional role of TH in these cells remains unclear. In the absence of any detectable CA production, it is a distinct possibility that the TH protein detected by immunohistochemical methods is not enzymatically active, or that the necessary cofactor tetrahydrobiopterin is lacking in these cells. It is notable, however, that some of the TH + /AADC neurons appear to form and store L-DOPA, whereas DA is either undetectable or present only in low and varying amounts [27,28]. It seems possible that these neurons could express AADC but at levels that are too low to be detectable immunohistochemically, resulting in a low and possibly varying level of DA production. Nevertheless, since the TH + /AADC neurons also lack the vesicular transporter VMAT-2 [12], it is unlikely that they release DA at any functionally significant level. The presence of L-DOPA-like immunoreactivity in TH + / AADC neurons in the hypothalamus and the dorsal vagal complex [27 29] has raised the possibility that these cells might synthesize and release L-DOPA, which, either alone or together with DA, might act as a transmitter in these cells [30]. In the arcuate nucleus, Ugrumov et al. [31] have proposed that the TH + /AADC neurons collaborate with adjacent neurons that express AADC only in the production and release of DA, such that L-DOPA produced in TH-only neurons is transferred to the AADC-only cells where it is converted to DA. If such a cooperative DA synthesis exists, it might be of particular importance in the arcuate nucleus during prenatal development when 99% of the neurons in the A12 cell group express either TH or AADC, but not both enzymes together. Thus, it is clear that expression of TH is not in itself sufficient to prove that a neuron is catecholaminergic, let alone dopaminergic. Although TH immunohistochemistry accurately identifies dopaminergic neurons in certain areas (e.g. the ventral mesencephalon), additional markers are essential to identify cells as functional DA-producing neurons in other areas of the nervous system. Do all DA neurons express TH? The fact that the expression of TH is substantially increased in striatal interneurons in response to DA-denervating lesions shows that the cellular levels of TH enzyme can be dynamically regulated in response to deafferentation or functional changes. In the olfactory bulb, TH expression in the periglomerular DA interneurons is activity dependent and substantially downregulated in odor-deprived mice below the level of detection in many cells [32]. In the hypothalamic arcuate nucleus, both TH expression and the number of neurons expressing detectable levels of TH change during postnatal maturation and are hormonally regulated [9]. In monkeys, the number of TH-positive neurons in the SN declines with age. This effect, which amounts to 40 50% in aged animals, is due, at least in part, to a downregulation of TH in surviving DA neurons (as detected by DAT immunostaining and neuromelanin content) [33 35]. Chu et al. [36] have shown that this downregulation of the DA phenotype is associated with an increased a-synuclein content. Moreover, a similar inverse relationship between a-synuclein level and TH expression is also seen in aged humans and in the surviving DA neurons of patients with Parkinson s disease. These data suggest that the age-related decline in dopaminergic function seen in the nigrostriatal system is linked to a downregulation of the TH enzyme, and that this decline occurs also in dysfunctional (but surviving) DA neurons in Parkinson s disease. Kordower and colleagues have linked this phenotypic switch to a reduction in expression of the orphan nuclear receptor Nurr1 [34]. This transcription factor is known to be essential for survival and function of mesencephalic DA neurons during development, and, therefore, might also play a key role in the regulation of both the phenotype and function of DA neurons in the adult nigrostriatal system. Taken together, these observations indicate that the level of TH expression in DA neurons can change over time and vary in response to changes in hormonal status and functional demand. Moreover, it seems clear that the presence of the TH enzyme at immunohistochemically detectable levels is not always a reliable (let alone necessary) condition for identifying DA neurons, even if it remains perhaps the most sensitive and consistent single marker currently available to us. Midbrain DA neurons and their projections It is often presumed, as a convenient heuristic, that the mesencephalon contains two major DA neuron subtypes: the nigral A9 neurons projecting to the striatum along the nigrostriatal pathway and the A10 neurons of the VTA projecting to limbic and cortical areas along mesolimbic and mesocortical pathways. This has long been recognized as an oversimplification. The SN contains not only neurons projecting to the striatum, but also neurons that innervate cortical and limbic areas; in addition, the DA neurons of the VTA project to the ventral striatum and the ventro-medial part of the head of the caudate-putamen in rodents (equivalent to nucleus caudatus in primates). The A8 cell group that forms a dorsal and caudal extension of the A9 cell group contains cells that project to both striatal, limbic and cortical areas (see Ref. [37] for a recent, comprehensive review). The system increases in size and complexity in primates. In rodents, the total number of TH-positive cells in all three cell groups bilaterally is in mice and in rats, with about half of the cells located in the SN. This number increases to between in monkeys and in humans, with >70% of the neurons located in the SN. The expansion is particularly evident in SN: from

5 198 Review TRENDS in Neurosciences Vol.30 No.5 Figure 2. Dorsal and ventral tier DA neurons of the ventral mesencephalon. The DA neurons of the SN and VTA are arranged in a dorsal (dt) and a ventral tier (vt), respectively, that can be distinguished on the basis of their expression of the ion channel protein GIRK2 (red) and calbindin (green). The ventral tier, cells [red in (a), (c), (d) and (e)] form a sheet of more densely packed, angular cells that are located in the ventral parts of the SNc and extends in to the VTA. These cells project, probably exclusively, to the sensorimotor part of the striatum. The dorsal tier cells [green in (b), (c), (d) and (e)] are located in the dorsal aspect of VTA and SN, as well as the A8 cell group, and project to the ventral striatal, limbic and cortical areas, as well as the matrix compartment of the dorsal striatum. (a c) are from the same double-stained section (GIRK2, red; calbindin, green; both combined in c); (d) shows a detail from (c) at higher magnification; (e) shows the arrangement of the cells in the SN from an adult rat. Courtesy of Lachlan Thompson DA neurons on each side in the rat to per side in monkeys, and over per side in young humans [33,34,37 39]. This matches well the expansion of the DA innervation territory, particularly in the neocortex, which is much more extensively innervated by midbrain DA afferents in primates and human than in rodents. In rodents, the cortical DA innervation is largely confined to areas of the frontal, cingulate and entorhinal cortex, whereas, in primates, the DA innervation territory covers the entire cortical mantle [37,40,41]. This extensive cortical projection is derived from all parts of the mesencephalic DA neuron complex, forming a continuous sheet of cells distributed in the dorsal aspect of the A8, A9 and A10 cell groups; the increase in number of nigral DA neurons projecting to the cortex is particularly striking [42]. Based on connectivity and morphological features, the midbrain DA neurons can be separated into a dorsal and a ventral tier. The dorsal tier includes cells located in the dorsal aspect of VTA and SN, and cells of the A8 cell group innervating ventral striatal, limbic and cortical areas, as well as the matrix compartment of the dorsal striatum [37,43,44]. These cells are round or fusiform in shape, are characteristically calbindin-positive, and express relatively low levels of the DAT transporter (green cells in Figure 2). The ventral tier comprises a sheet of more densely packed, angular cells located in the ventral parts of VTA and SN. These cells are calbindin-negative, express

6 Review TRENDS in Neurosciences Vol.30 No Figure 3. Cells of origin of the mesostriatal, mesolimbic and mesocortical pathways in the rat. The DA neurons projecting to (a) striatal, (b) limbic and (c) cortical areas are partly intermixed: The cells located in the ventral tier of the SNc [red dots in (a)] innervate, probably exclusively, the sensorimotor part of the caudate-putamen [red area in the inset in (a)], whereas the cells of the dorsal tier comprise neurons that project widely to both limbic and cortical forebrain regions, as illustrated in (b) and (c), respectively. Abbreviations: SNc, substantia nigra pars compacta; SNl, substantia nigra pars lateralis; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. Adapted from Fallon and Loughlin in Ref. [46]. higher levels of DAT and are mostly immunopositive for the ion channel protein GIRK2 (red cells in Figure 2). The ventral tier neurons extend (probably exclusively) to the striatum where they innervate, above all, the patch compartment; many of these cells possess prominent dendrites that project ventrally into the SN pars reticulata (SNr) [43,45]. The calbindin-positive cells of the dorsal tier constitute a more mixed population, comprising cells that project not only to limbic and cortical areas, but also to the matrix compartment of the striatum [37,43 46]. Thus, the mesencephalic DA neurons, in particular, exhibit a rich and complex organization, with subcomponents that differ in their morphology, but also in several molecular markers and patterns of forebrain projections. Forebrain projections Early histofluorescence studies suggested three distinct ascending DA projection systems from the SN VTA complex, the nigrostriatal (or, more accurately, mesostriatal ), mesolimbic and mesocortical pathways, with widespread projections to forebrain targets [47 49]. Subsequent studies support the view that those three pathways are both anatomically and functionally distinct, but that their cells of origin in the SN VTA complex are intermixed. Retrograde, double-labeling studies have shown that DA neurons projecting to the striatum only rarely (1 5% of cases) send collaterals to extrastriatal areas, and that collaterals from the mesolimbic or mesocortical DA neurons to striatum, or other forebrain areas, are equally sparse; this indicates that the projections of the mesolimbic and mesocortical pathways are largely confined to their limbic or cortical targets [50 53]. The frequently expressed perspective of anatomically separate nigral and ventral tegmental projection systems (with SN neurons exclusively projecting to the striatum and the VTA neurons giving rise to the mesolimbic and mesostriatal pathways) does not, however, hold up. In both rat (Figure 3) and monkey (Figure 4), the striatal DA innervation is derived not only from the SN pars compacta (SNc), but also from cells located in the lateral VTA and the A8 cell group; moreover, the DA neurons projecting to limbic and cortical areas are located not only in the VTA, but also in the dorsal tier of the SN and the A8 cell group. This intermixing is particularly prominent in primates where the cells of origin of the mesolimbic and mesostriatal pathways are widely distributed throughout the dorsal tier, interspersed among the cells projecting to the striatum (Figure 4c,d; [42,54]). The nigrostriatal DA pathway, in a restricted sense, is derived from neurons located in both the dorsal and ventral tiers of the SNc, and is the predominant source of DA innervation of the sensorimotor striatum. The anteromedial and ventral parts of the striatum (the limbic part, including the nucleus accumbens rostrally and the central nucleus of the amygdala and adjacent parts of the caudal striatum, caudally, in rats) derive their innervations from a much wider sector, including the lateral part of the VTA and the A8 cell group. Thus, the more inclusive term mesostriatal DA pathway might conveniently be used to include all components of the midbrain DA system projecting to the striatum. DA projections to downstream striatal targets Interestingly, not only the caudate-putamen but also several other basal ganglia structures are innervated by midbrain DA neurons. This includes the external and

7 200 Review TRENDS in Neurosciences Vol.30 No.5 Figure 4. Distribution of midbrain DA neurons projecting to striatal, limbic and cortical areas in the primate. The intermingling of the cells of origin of the mesostriatal, mesolimbic and mesocortical pathways in primates is even more pronounced than that in the rat (compare with Figure 3). The extensive cortical projection, in particular, is derived from all parts of the mesencephalic DA neuron complex, forming a continuous sheet of cells distributed in the dorsal aspect of the A8, A9 and A10 cell groups, and the increase in number of nigral DA neurons projecting to the cortex is particularly striking. Data compiled from Lewis and Sessack [41] for (a) and Williams and Goldman- Rakic [42] for (b). Abbreviations: CP, cerebral peduncle; DSCP, decussation of the superior cerebellar peduncle; dt, dorsal tier; IL, infralimbic area of the frontal cortex; ip, interpeduncular nucleus; ML, medial lemniscus; NIII, occulomotor nerve exit; PL, prelinbic area of the frontal cortex; RN, red nucleus; vt, ventral tier. internal segments of the globus pallidus (entopeduncular nucleus in rodents), parts of the ventral pallidum and the subthalamic nucleus [55 59]. In the SN itself, DA is known to be released from a plexus of dendritic terminals that is derived from the DA neurons located in the ventral tier of the SNc, and extends throughout large parts of the SNr [60]. Although the density of their terminals in these nuclei is much less than in caudate or putamen, the ventral tier neurons are strategically placed to regulate some crucial aspects of neurotransmission in the basal ganglia circuitry. Thus, in addition to their potent influence at the level of the caudate nucleus and putamen, the midbrain DA neurons can directly modulate the activity of basal ganglia output neurons at both the pallidal, subthalamic and nigral levels. In the SN, dendritic DA release has been shown to provide a mechanism by which the nigral DA neurons can regulate not only the activity of the DA neurons themselves, but also the release of GABA within the SNr and the activity of its efferent projections [60,61]. Parent and coworkers [45,55,62] have shown that at least a part of the DA innervation in the globus pallidus and subthalamic nucleus is derived from collateral branches of DA axons that pass through these nuclei on their way to the caudate-putamen. In primates, the internal segment of the globus pallidus receives a particularly dense DA innervation, which seems to be derived from a set of neurons located in both SNc and VTA, which is substantially separate from those innervating the caudateputamen [57,59]. This projection is relatively spared in MPTP-treated monkeys and in early cases of Parkinson s disease [63]. Moreover, Whone et al. [64] have observed a compensatory increase in 18 F-fluorodopa uptake in the internal segment of the globus pallidus in early stage Parkinson s disease patients, which is lost at advanced stages of the disease. This suggests that enhanced activity in the DA projection to the internal pallidum can help maintain normal motor function when the nigrostriatal system starts to fail. Thus, interdigitated neurons dispersed throughout the midbrain project widely to diverse forebrain targets. However, they typically exhibit only a limited degree of collateralization that is certainly far less than one might assume from the extent of overlap reported in retrograde labeling studies involving injection of single tracers into different forebrain areas. Concluding remarks Over the 50 years since their discovery, DA neurons have been among the most widely studied systems of the brain, in part owing to various tools available for their study and analysis. Yet, the more we have learned, the less clear-cut have the principles of their organization become. The defining feature of a CA neuron, that is, the expression of the essential synthetic enzyme TH has been complicated by the discovery of neuronal systems (above all in hypothalamus, striatum and cortex) that express TH but neither synthesize DA or NA, nor store or release either transmitter in functional quantities. The coexpression of DA with amino-acid transmitters and neuropeptides, and the apparent lack of vesicular storage (i.e. VMAT-2) in interneurons of, for example, the hypothalamus and the olfactory bulb have revealed unconventional features that

8 Review TRENDS in Neurosciences Vol.30 No do not readily fit our standard views of a classical dopaminergic neuron. Moreover, the ability of the dopaminergic transmitter machinery in particular the expression of the DA synthetic enzymes TH and AADC to change in response to altered functional demands, damage or aging, suggests interesting dynamic and adaptive properties of DA neurotransmission in the brain that deserve to be pursued in greater depth. A solid foundation in the organization and dynamic properties of the dopaminergic systems is fundamental to our understanding of their role in regulating essential functions of the organism (spanning motor control, motivation and emotion, neuroendocrine regulation and cognition), as well as of their involvement in profound disorders of human neurology and psychiatry. The separation of the midbrain DA projection systems into three anatomically and functionally distinctive components, that is, the mesostriatal, mesolimbic and mesocortical pathways, as defined three decades ago, remains valid today. Over the past decades, however, our view of the midbrain DA projection system has changed from a simple organization of anatomically separate nigral and ventral tegmental neurons projecting to discrete forebrain targets to a more intricately organized complex of interdigitated DA neuron subtypes that express different morphological features, cotransmitters and other distinctive marker proteins. For a full understanding of DA neuron diversity in the SN VTA complex, we need to gain deeper insights into the developmental and phenotypic characteristics of defined DA neuron subtypes in the SN VTA complex, based on their axonal projection patterns, synaptic connectivity and functional properties. References 1 Falck, B. et al. 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