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Development of Mesencephalic Dopamine Neurons in the Nonhuman Primate

Relationship to Survival and Growth Following Neural Transplantation

John R. Sladek, Jr., Barbara Blanchard, T. J. Collier, John D. Elsworth, Jane R. Taylor, Robert H. Roth, and D. Eugene Redmond, Jr.

DEVELOPMENT OF MESENCEPHALIC DOPAMINE NEURONS IN MAMMALS

Independent studies have characterized the appearance of dopamine (DA) biochemically and histochemically in mesencephalic neurons in the mouse and rat during early ontogenesis (4, 9, 11, 12, 13, 18, 20, 26, 27, 33, 34, 40). Similar studies have not been performed in comparable detail in nonhuman primates, but considerable data do exist for human brain (5, 19, 21, 23, 24, 29). Our understanding of the temporal events in the development of the mesencephalic DA neurons consequently is based primarily on analysis of rodents. While this information is useful, it may prove difficult to apply to experimental designs involving primates because of major differences in the timing of neurogenesis and the expression of transmitters. For example, DA histofluorescence was first noted during the latter half of gestation in the rat at approximately embryonic day 12.5 (i.e., E 12.5), which corresponds to a surge in the number of labeled cells seen during neurogenesis of the ventral mesencephalic neuron population (10). The presence of tyrosine hydroxylase was first detected in these neurons also at E 12.5, which suggests a temporal relationship that is consistent with the production of dopamine soon after the onset of neurogenesis. DA exists in neurons as they migrate through the deeper layers of the mesencephalon after division in the germinal zone. In fact, dopamine has been reported in neurons while still in the germinal zone, which reflects biosynthesis immediately after mitosis. Thus, dopamine is present during the latter half of gestation in the rat, whereas it is seen during the first trimester of primate development as described in more detail below. The early presence of dopamine in the primate brain may reflect a role for this neurotransmitter in neurogenesis. For example, interruption of the biosynthesis of a monoamine transmitter, serotonin, by prenatal administration of parachlorphenylalanine (PCPA) results in the delay of differentiation of target neurons of the serotonin system, particularly in the hippocampus (12). If similar mechanisms exist in primate brain, then the relatively early appearance of DA could be influential in the differentiation of critical targets such as the striatum and frontal cortex (see also Molecular and Cellular Mechanisms of Brain Development, The Development of Brain and Behavior, and Neurodevelopmental Perspectives on Schizophrenia).

DOPAMINE IN THE DEVELOPING PRIMATE BRAIN

The timing of the expression of DA in mesencephalic neurons in monkeys is understood incompletely and cannot be extrapolated from data generated from either the rat or human brain. DA is seen in mesencephalic neurons early in human gestation, but relatively later in the rat. Our examination of this question, although preliminary at present, is described below as a framework for comparison to other mammalian species, including human. A number of studies have been conducted on aborted human material as described below, but the utility of such information with respect to the design of experiments to test developmental hypotheses in primates is far too limited. Thus, knowledge of similar phenomena in nonhuman primates provides an important database for experimental manipulation of the developing DA systems in order to test theories on transmitter expression or differentiation (for example) which could not be examined in developing human brain.

The Mesencephalic Groups at E 40 in Nonhuman Primate

According to Levitt and Rakic (14), neurogenesis of the substantia nigra occurs in the rhesus monkey between E 36 and E 43. The expression of the DA phenotype in nigral neurons in a closely related Old World macaque, the African green monkey, occurs at a similar time as demonstrated by the presence of the synthetic enzyme tyrosine hydroxylase and shown at day E 40 in FIG. 1. This dark-field photomontage of an E 40–41 African green monkey shows the full extent of brain development from the forebrain (right) to the hindbrain (left). The ventricular system (V) forms a prominent cavity which can serve as a useful neuroanatomical landmark. The mesencephalic flexure (arrows) demarcates the developing ventral mesencephalon. This region is illustrated in greater detail in Fig. 2. , FIG. 2. The low-power insert illustrates the region of the mesencephalic flexure through its full dorsal (D) to ventral (V) extent. This section is adjacent to the midline; therefore the aqueduct of the mesencephalon is not visible. Nevertheless, newly born DA neuroblasts migrate from the germinal epithelium ventrally, and in so doing they provide the appearance seen in the high-power photomontage of small, bipolar neurons with elongated neurites that extend in a dorsoventral plane. The migrating neurons (arrows) are stained for tyrosine hydroxylase and demonstrate the early onset of transmitter enzyme in these neurons as depicted at E 40–41. , FIG. 3. The three panels illustrate the growth of DA neurons (arrows) of the ventral mesencephalon at three developmental ages: (A) E 40–41, (B) E 55, (C) E 90. The dramatic perikaryal growth and neuritic complexity is particularly evident because these three micrographs were taken at identical magnifications. , FIG. 4. The brainstem and basal forebrain are illustrated at E 55 following staining for tyrosine hydroxylase; rostral is to the left. In A, it is apparent that the ventral mesencephalon is filled with DA neurons (arrows) that have completed their migration from the germinal epithelium. In B, they appear densely packed and have minimal neuritic arborization at this level. ). This is consistent with a generalized "mammalian plan" which results in the biosynthesis of monoamine transmitters early in development, shortly after the initial wave of neurogenesis. The newly formed DA neurons are small and generally rounded, with an average perikaryal length of 9.2 mm (range: 7.4–12.2 mm). Many possess short, dorsally projecting neurites that are about the same length as the perikarya of the mesencephalic neurons (FIG. 2. The low-power insert illustrates the region of the mesencephalic flexure through its full dorsal (D) to ventral (V) extent. This section is adjacent to the midline; therefore the aqueduct of the mesencephalon is not visible. Nevertheless, newly born DA neuroblasts migrate from the germinal epithelium ventrally, and in so doing they provide the appearance seen in the high-power photomontage of small, bipolar neurons with elongated neurites that extend in a dorsoventral plane. The migrating neurons (arrows) are stained for tyrosine hydroxylase and demonstrate the early onset of transmitter enzyme in these neurons as depicted at E 40–41. ). Others have dorsal neurites that can be traced for distances of 4–5 times (29.6–55.0 mm) the length of the DA perikarya in a single histological section through the mesencephalon. These neurites, at this point in ontogeny, are not recognizable as a rostrally projecting pathway into or toward the striatum or basal forebrain. These migratory neurons occur throughout the depth of the embryonic mesencephalon (FIG. 2. The low-power insert illustrates the region of the mesencephalic flexure through its full dorsal (D) to ventral (V) extent. This section is adjacent to the midline; therefore the aqueduct of the mesencephalon is not visible. Nevertheless, newly born DA neuroblasts migrate from the germinal epithelium ventrally, and in so doing they provide the appearance seen in the high-power photomontage of small, bipolar neurons with elongated neurites that extend in a dorsoventral plane. The migrating neurons (arrows) are stained for tyrosine hydroxylase and demonstrate the early onset of transmitter enzyme in these neurons as depicted at E 40–41. ) with a high concentration dorsally at the germinal zone, as well as ventrally within the developing mesencephalic tegmentum. The density of neurons is exceptionally high, which provides a predominant appearance of cellularity in the E 40 mesencephalon.

The Mesencephalic Groups at E 55 and E 61

The picture of dopamine neurons changes substantially by E 55 in the African green monkey. At this time, the DA neurons form a recognizable cluster in the ventral mesencephalon, although the separation into three distinct groups is not complete. Distinct groups are recognizable at E 61 based on size, shape, and staining density. Also, neurite extension is more extensive at this later stage. At E 55, the individual neurons have increased in size to an average diameter of 14.3 mm (range: 9.8–24.6 mm) and the extension of axons rostrally has proceeded to the level of the striatum (FIG. 5. A: Some axons (arrows) of the ventral mesencephalon at E 55 appear to course through the internal capsule (IC) as they ascend through the putamen (PT) and caudate nucleus (CD). B: Components of the ascending monoaminergic systems are seen as they pass through the region of the medial forebrain bundle at a diencephalic level. Although identifiable, the fiber density is minimal in comparison to stage E 90 as illustrated in Fig. 6. ), approximately 2 mm rostral to the ventral mesencephalon. The density of tyrosine-hydroxylase-stained fibers is modest as seen in the ascending DA pathway associated with the medial forebrain bundle (FIG. 5. A: Some axons (arrows) of the ventral mesencephalon at E 55 appear to course through the internal capsule (IC) as they ascend through the putamen (PT) and caudate nucleus (CD). B: Components of the ascending monoaminergic systems are seen as they pass through the region of the medial forebrain bundle at a diencephalic level. Although identifiable, the fiber density is minimal in comparison to stage E 90 as illustrated in Fig. 6. ) and in the vicinity of the internal capsule where it separates the caudate nucleus from the putamen. However, the ascending monoamine pathways collectively form a prominent bundle at this age, and it is difficult to determine what component is attributable to nigrostriatal axons in the absence of tracer studies or the use of more specific antisera.

The DA perikarya at this age stain with a higher intensity than at E 40 (FIG. 3. The three panels illustrate the growth of DA neurons (arrows) of the ventral mesencephalon at three developmental ages: (A) E 40–41, (B) E 55, (C) E 90. The dramatic perikaryal growth and neuritic complexity is particularly evident because these three micrographs were taken at identical magnifications. ). DA neurons are not present in the germinal zone, and migration through the tegmentum appears complete. Some tyrosine-hydroxylase-stained neurons are present in the mesencephalic periaqueductal gray and within the region of nucleus raphe dorsalis and could be misinterpreted as components of the germinal zone. It is likely that they represent the small number of catecholominergic neurons that are present in the adult macaque in the dorsal raphe and periaqueductal gray of the mesencephalon (8).

The Mesencephalic Groups at E 90

By E 90 the mesencephalic DA neurons have attained an adult-like appearance with respect to shape, size, neurite outgrowth, and position (FIG. 7. This photomontage illustrates the position of developing catecholamine systems in the brainstem, the ascending axons caudal to the putamen, and the dense patch-matrix appearance of the developing striatum. Three large clusters of perikarya are identified ventrally in the brainstem (right); these correspond to the ventral noradrenergic neuronal groups (A1, A2, A5, A7) that give rise to the ventral noradrenergic pathway, seen here immediately caudal to the second group, the locus coeruleus (A6). The third, and most prominent, cluster represents the DA neurons of the ventral mesencephalon which form an exceptionally dense cluster of intensely stained neurons (A8, A9). Ascending nigrostriatal axons (arrows) and the dense patch-matrix appearance of the caudate (CD) and putamen (PT) are prominent features. AC, anterior commissure; IC, internal capsule; IOC, inferior olivary complex; SC, superior colliculus. , FIG. 8. This higher-power view of the ventral mesencephalon at age E 90 illustrates the dense packing of DA neurons as well as the highly developed dendritic bundles (arrows) that course from the substantia nigra zona compacta (ZC) into the zona reticulata (ZR). Ascending axons en route to the striatum are seen in the upper left corner of this photograph. , FIG. 9. These four panels illustrate various cell types (arrows) seen within the developing mesencephalon at age E 90. In A, densely packed neurons reminiscent of zona compacta are seen, whereas in B the smaller, more oval neurons of the ventral tegmental area (A10) are illustrated. Panel C represents the more dorsally and caudally placed neurons of the retrorubal group (A8), while panel D illustrates dendritic bundles ventral to the zona compacta. ). Perikaryal diameters have enlarged to approximately 17.4 mm (range: 11.7–24.3 mm), and individual neurons resemble those seen in adults in all three mesencephalic cell groups (FIG. 9. These four panels illustrate various cell types (arrows) seen within the developing mesencephalon at age E 90. In A, densely packed neurons reminiscent of zona compacta are seen, whereas in B the smaller, more oval neurons of the ventral tegmental area (A10) are illustrated. Panel C represents the more dorsally and caudally placed neurons of the retrorubal group (A8), while panel D illustrates dendritic bundles ventral to the zona compacta. ). Neurite extension has progressed over 3.7 mm to reach the striatum where the initial innervation is seen in the form of early patch matrix, or tyrosine-hydroxylase-fiber-rich "islands" (FIG. 6. The ascending DA axons are highly developed at E 90 and are seen as a dense parallel array of fibers (B) as they approach the neostriatum. Their terminal fields in the caudate nucleus are seen in A, where the presence of numerous "patches" of DA fibers (arrows) begins to be manifested. This is also seen in the overview in Fig. 7. ) similar to those first described by Tennyson et al. (37, 38) in rabbit neostriatum and by Olson and Seiger (20) in the rat. A prominent bundle of tyrosine-hydroxylase-positive fibers is present rostral to the mesencephalon and appears as a continuous pathway into the striatum (FIG. 6. The ascending DA axons are highly developed at E 90 and are seen as a dense parallel array of fibers (B) as they approach the neostriatum. Their terminal fields in the caudate nucleus are seen in A, where the presence of numerous "patches" of DA fibers (arrows) begins to be manifested. This is also seen in the overview in Fig. 7. and FIG. 7. This photomontage illustrates the position of developing catecholamine systems in the brainstem, the ascending axons caudal to the putamen, and the dense patch-matrix appearance of the developing striatum. Three large clusters of perikarya are identified ventrally in the brainstem (right); these correspond to the ventral noradrenergic neuronal groups (A1, A2, A5, A7) that give rise to the ventral noradrenergic pathway, seen here immediately caudal to the second group, the locus coeruleus (A6). The third, and most prominent, cluster represents the DA neurons of the ventral mesencephalon which form an exceptionally dense cluster of intensely stained neurons (A8, A9). Ascending nigrostriatal axons (arrows) and the dense patch-matrix appearance of the caudate (CD) and putamen (PT) are prominent features. AC, anterior commissure; IC, internal capsule; IOC, inferior olivary complex; SC, superior colliculus. ). Dendrite bundles, another feature of the adult substantia nigra, also exist from the zona compacta to the zona reticulata of the substantia nigra (FIG. 8. This higher-power view of the ventral mesencephalon at age E 90 illustrates the dense packing of DA neurons as well as the highly developed dendritic bundles (arrows) that course from the substantia nigra zona compacta (ZC) into the zona reticulata (ZR). Ascending axons en route to the striatum are seen in the upper left corner of this photograph. and FIG. 9. These four panels illustrate various cell types (arrows) seen within the developing mesencephalon at age E 90. In A, densely packed neurons reminiscent of zona compacta are seen, whereas in B the smaller, more oval neurons of the ventral tegmental area (A10) are illustrated. Panel C represents the more dorsally and caudally placed neurons of the retrorubal group (A8), while panel D illustrates dendritic bundles ventral to the zona compacta. ). The neuropil of the ventral mesencephalon reflects highly developed DA systems with considerable interwoven fibers in the local environment (FIG. 9. These four panels illustrate various cell types (arrows) seen within the developing mesencephalon at age E 90. In A, densely packed neurons reminiscent of zona compacta are seen, whereas in B the smaller, more oval neurons of the ventral tegmental area (A10) are illustrated. Panel C represents the more dorsally and caudally placed neurons of the retrorubal group (A8), while panel D illustrates dendritic bundles ventral to the zona compacta. ).

Although preliminary at this time (one or two determinations at each time point), we have performed biochemical determinations of DA content in dissected striatum and ventral mesencephalon of fetal African green monkey brains at stages of development comparable to stages reported above. The concentration of homovanillic acid, the main end metabolite of DA in this species, parallels that of DA. At E 47, DA concentration, although relatively low, is higher in the ventral mesencephalon than in the striatum. Later, at about 70–90 days, DA levels in the striatum rise sharply, consistent with synaptogenesis in the striatum. At this time, striatal DA concentration is 53.5 ng/mg protein; by comparison this value is 5.05 at E 70 and 0.415 at E 47.

APPLICATION TO TRANSPLANTATION STUDIES IN PRIMATES

Transplantation of neurons into the central nervous system has been studied for over 100 years (39). This technique provides a useful tool for the analysis of neuronal development and can be used to test the specificity of connections between transmitter-defined neural systems. For the past decade, neural grafts have been tested as a therapeutic intervention in neurodegenerative disorders, particularly Parkinson's disease (7), 15, 31; see also Parkinson’s Disease). Considerable work in nonhuman primates (1, 25, 32) and with xenografting of human neurons (2, 3, 22, 36) has demonstrated the feasibility of neural transplants in highly ordered primate brain to produce functional, DA-producing grafts that are capable of reversing motor deficits in animal models of Parkinson's disease. Recent reports from several centers suggest that the implantation of embryonic DA neuroblasts can result in some degree of improvement in advanced parkinson patients (6, 16, 17, 35), particularly if the symptoms were caused by the (unknowing) intake of a DA-specific neurotoxin (41). Approximately 300 parkinson patients worldwide have received neural grafts of embryonic mesencephalic neurons, and the recent lifting of a federal restriction on the use of federal support for this experimental surgery will prompt additional attempts at this experimental clinical approach. Yet a number of critical characteristics of the developing DA neurons are incompletely understood such as the optimal embryonic age for survival of grafted neurons in primates, including human.

Knowledge gained from basic developmental studies can be applied to questions of neuronal survival following transplantation. For example, if the outgrowth of neurites is considered as a potential determinant of viability following the extirpation of DA neurons from embryonic brain, then the observation of neurite extension from the region of the developing substantia nigra to the striatum in the E 55 monkey described above suggests that neurons from this age are too well developed to survive the obligatory axotomy that accompanies the extirpation. In fact, we have observed between one-tenth and one-twentieth as many DA neurons following grafting of mesencephalon from later-stage embryos such as E 55 in monkey in comparison to younger donor grafts (FIG. 10. Representative transplants (arrows) of embryonic DA neurons are seen in two adult African green monkeys at the same magnification. This comparison of grafted neurons collected from an optimally aged donor (B) versus those of an older donor (A) illustrates the dramatic increase in viability seen following grafting of the younger tissue. These illustrations are representative of grafts that contain approximately 500 (older donor) and 5000 (younger donor) tyrosine-hydroxylase-positive neurons as reflected by the density of perikarya. This enhanced survival undoubtedly relates to the immature stage of neuritic development at the earlier time, but may be attributed to other factors as well. ). Moreover, the "window" for the best survival probably is quite narrow. Ten times as many DA neurons were seen following grafting of E 44 tissue in comparison to E 49 in African green monkey (30), and our recent observations suggest that maximal DA neuron survival is achieved from donor tissue during a 48-hr period, at E 41–42. If we consider that gestation in the monkey is approximately 6 months, as compared to 9 months for the human, then the window for human tissue might be 50% longer and perhaps span a 3-day period at a critical point in neurogenesis or neuronal differentiation. The determination of that critical point is an essential next step for neural grafting experiments in humans. This is particularly crucial because a relatively wide range of embryonic ages has been used in the extant human experiments in parkinson patients (6, 16, 17, 35)). The predictive nature of developmental studies in human material is underscored by the observations described below that attempt to characterize transmitter expression, neurite extension, and the growth of terminal plexuses of DA neurons as a template for transplantation. Thus, the presumption is that survival is influenced by the degree to which axons have extended from the perikarya; that is, extension past a specific distance renders neurons incapable of surviving grafting if axons are severed during the dissection. In this case, only those neurons or neuroblasts that appear as rounded cells with no or short processes will survive grafting. This pool would include that percentage of neuroblasts that are "born" immediately prior to grafting. Neurogenesis takes place over at least 1 week in monkeys, and Freeman et al. (5) reported that tyrosine-hydroxylase-positive neuroblasts were present in the germinal zone in humans as late as 10 weeks, which is 3.5 weeks after they were first seen in this region. This suggests that neurogenesis of the human substantia nigra may occur over a considerably longer period than that in monkeys and that a certain percentage of neurons are capable of surviving grafting at any point during this 3.5-week period. This has been verified in our studies in monkeys where survival of DA neurons was observed in late-stage tissue (27, 32), and predictably the number of grafted tyrosine-hydroxylase-positive neurons was exceptionally low (i.e., often fewer than 100/animal).

Factors other than axon elongation may play a critical role in determining cell survival following transplantation. The development of intracellular messengers, DA autoreceptors, transporter mechanisms, availability of trophic factors, and gene regulation of transmitters and associated enzymes all could influence the ability of embryonic neuroblasts to survive neural transplantation. Systematic analysis of these, as well as other, potential variables is needed to provide a clear understanding of this question in human as well as nonhuman primate brain.

COMPARISON TO HUMAN DEVELOPMENT

A number of investigators have examined events in the development of human monoaminergic neuron systems, particularly with histofluorescence and immunohistochemical techniques as indices of transmitter expression. The first description by Olson et al. (21) noted (a) the relatively early appearance of monoamines in human neuroblasts of the mesencephalon and (b) the gradual growth of fluorescent ascending axons and striatal patches of catecholamine histofluorescence. At 7 weeks, fluorescence attributed to serotonin and catecholamines was seen within small, round neuroblasts that generally possessed few processes (0–2/cell). By 10 weeks of gestation the neurons were situated in the ventral mesencephalon in groups, but were not subdivided into the A8–A10 subgroups. Some fluorescent axons were present in the striatum, although the development of islands was not seen until 12.5 weeks. The more caudally placed putamen received a DA innervation prior to that of the caudate, which is consistent with the directional course of the ascending DA fiber bundle which approaches the striatal complex in a caudal-to-rostral direction. The mesencephalic complex of DA neurons was "heavily subdivided" by 15.5 weeks, and the striatum was more heavily supplied with a dense patchy pattern of terminals of nigrostriatal origin. Thus, over the relatively short period of 10 weeks the mesencephalic DA neurons are born, grow into the host striatum, and develop a terminal pattern reminiscent of adults. By comparison, the mesocortical pathways were reported by these investigators to follow a somewhat later time course with respect to terminal innervation, although the histofluorescence technique was less capable of demonstrating cortical monoamine fibers than were later versions of the method as described below. A parallel study by Nobin and Björklund (19) basically confirmed these findings and noted that the overall stage of development of the monoamine systems in the human fetus was comparable to that of the rat during the second week after birth, which raises questions about the potential role of the relatively early appearing DA in the human brain.

More recent investigations of human gestation by Freeman et al. (5) using immunohistochemistry noted the presence of tyrosine hydroxylase in mesencephalic neuroblasts at 6.5 weeks and neurite extension at 8 weeks. Tyrosine hydroxylase fibers were reported first in the putamen at 9 weeks, and, remarkably, putative DA neurons were seen adjacent to the ventricle, presumably in the germinal zone, at 10 weeks of gestation. A similar study by Silani et al. (28) reported a few tyrosine hydroxylase cells in the germinal zone of the mesencephalon as early as 5.5 weeks in human material. Thus, the systems apparently begin to produce synthetic enzyme in advance of the first reported demonstration of DA fluorescence, and the presence of DA is an early event that begins during and shortly after neurogenesis. The role of DA at these early stages has been the subject of investigation as described earlier and will undoubtedly continue to be examined carefully, for example, with molecular techniques as interest on the development of monoamine neurons, particularly those that utilize DA, continues to focus on neurologic and psychiatric disorders that may in part be due to abnormal development.

ACKNOWLEDGMENTS

This research was made possible by the contributions of the staff of St. Kitt's Biomedical Research Institute. This study was funded by NIH grants PO1 NS 24032 and RSA MH 00643 (D.E.R.) and by the Axion Research Foundation.

published 2000