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First Trimester Development of the Human Nigrostriatal Dopamine System
EXPERIMENTAL NEUROLOGY ARTICLE NO.
139, 227–237 (1996)
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First Trimester Development of the Human Nigrostriatal Dopamine System PER M. ALMQVIST, ELISABET ÅKESSON,* LARS U. WAHLBERG, HELMUT PSCHERA,† ÅKE SEIGER,* AND ERIK SUNDSTRO¨ M* Department of Clinical Neuroscience, Section of Neurosurgery, *Department of Clinical Neuroscience and Family Medicine, Section of Geriatric Medicine, and †Department of Obstetrics and Gynecology, Karolinska Institute, Stockholm, Sweden
INTRODUCTION The aim of the present study was to characterize the morphological and neurochemical differentiation of mesencephalic dopaminergic neurons in human embryos, derived from elective first trimester abortions. Embryonic brain tissue was taken for analysis of tyrosine hydroxylase (TH) by immunohistochemistry and Western blot, and for analysis of endogenous dopamine (DA) content using HPLC-ED. TH expression was first detected at 3.5 weeks of gestational age (Carnegie stage 11) by immunohistochemical staining of the primordial sympathetic trunk along both sides of the neural tube. In sagittal sections of the intact 4.5-week-old embryo, a small, distinct population of rounded, densely packed TH-immunoreactive perikarya with short primary processes was seen in the midbrain. During the latter half of the first trimester, the number of TH-stained cells as well as the length and number of axonal processes projecting toward and into the developing neostriatum increased rapidly. At the end of the first trimester, varicose fibers could be detected in the striatal anlage. In order to verify that TH was the antigen recognized by the antibodies used for immunohistochemistry on human tissue specimens, mesencephalic tissue of 5–10 weeks gestation was analyzed by Western blot technique. A single, homogeneous band with the apparent molecular weight of approximately 60 kDa was clearly detected at 5 weeks of age. The amount of TH/mg total protein increased at least 10-fold between 5–10 weeks of gestation. For comparison, the mesencephalon and the forebrain/basal ganglia were analyzed for endogenous DA content using HPLC-ED. DA was first detected at 5.5 weeks of gestational age in both mid- and forebrain, and DA levels were found to increase exponentially from 7 to 7.5 weeks of age, reaching 4–5.5 ng DA/mesencephalon and 50–75 ng DA/g caudate nucleus–putamen anlage at the end of the first trimester. Together, morphological and biochemical data presented here constitute evidence for a very early appearance, migration, and differentiation as well as functional development of human mesencephalic dopaminergic neurons and their projections into target areas during the first trimester. r 1996 Academic Press, Inc.
Monoamine-synthesizing neurons appear in the brain stem early during embryogenesis and their fiber pathways are considered crucial for brain development (29). The earliest production sites in the human embryonic brain are the developing locus coeruleus complex and the raphe nuclei of the rhombencephalon synthesizing noradrenalin (NA) and serotonin (5-HT), respectively. Both transmitters are detected at 5–6 weeks of gestation, while tyrosine hydroxylase-immunoreactive neuroblasts are discerned at 4 weeks of age (53). Another region of early development is the midventral proliferative area of mesencephalon, which participates in the formation of the red nucleus and the substantia nigra (40). The latter, with its content of dopaminergic (DA) neurons and their relevance for the pathophysiology of the disorder paralysis agitans (Parkinson’s disease), has been subjected to a vast number of morphological and neurochemical studies in rodents (5, 8, 28, 31, 32, 45, 48, 51, 52, 57) and in nonhuman primates (12, 30, 50). Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the catecholamine biosynthesis (34), and the human TH gene has been assigned to chromosome 11p15.5 (20). Evidence for TH gene expression and/or endogenous DA synthesis in human fetal mesencephalon has been reported consistently (10, 25, 35, 37, 42, 43), whereas the exact time of the first appearance of mesencephalic DA neurons during ontogeny is a matter for discussion. Three separate immunohistochemical studies report TH-immunoreactive cells in the mesencephalic flexure at 5 weeks [Carnegie stage (CS) 15–16] (56), at 5.5 weeks (CS 16–17) (49), and at 6.5 weeks (CS 18–19) (10), respectively. To clarify the issue of embryonic development of mesencephalic DA neurons in vivo, we have performed a combined morphological and neurochemical study with an extended sample size and gestational age range, monitoring TH expression and endogenous DA synthesis during the first trimester.
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0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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MATERIALS AND METHODS
Tissue Retrieval Embryonic nervous tissue was recovered from elective first trimester routine abortions using the standard vacuum aspiration technique. The collection of residual tissue was approved by the Human Ethics Committee of the Huddinge University Hospital, Karolinska Institute, and was in accordance with the guidelines of the Swedish Society of Medicine and the U.S. Public Health Service, including an informed consent from the pregnant women seeking abortions (38). The gestational age of each specimen was determined by its size and by examination of anatomical landmarks according to the atlas of England (6). A total of 50 first trimester tissue specimens, ranging from 3.5 to 11.5 weeks of gestation, were included in this study. Recovered nervous tissue was microdissected within 2 h of surgery and either fixed in paraformaldehyde for immunohistochemistry or fresh frozen on dry ice and stored at 2135°C pending SDS–PAGE/Western blot or HPLC-ED analyses.
Western Blot Microdissected, fresh mesencephalic tissue pieces, stored frozen in 1.5-ml sealed microcentrifuge tubes, were thawed by the addition of 9 volumes ice-cold lysis buffer, containing 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.5% (w/v) Triton X-100, 2 mM EDTA in 10 mM Tris–HCl, pH 8.0. Tissue pieces were homogenized by vortexing followed by sonication on ice. Tissue homogenate (10% w/v) corresponding to 100 mg of total protein, as analyzed by the Lowry method modified according to Markwell (32a), was loaded in 8-mm wells for SDS–PAGE on a 1.5-mm-thick 9% isocratic polyacrylamide gel (27). The molecular weight marker proteins (Sigma) were stained by Coomassie brilliant blue while proteins of the tissue homogenate were transferred to a nitrocellulose filter (31) followed by immunochemical detection of TH using the same antiserum as for TH immunohistochemistry (see above), diluted 1/1000. The antibody immunocomplexes were visualized by photographic exposure using a chemiluminiscence detection system (ECL, Amersham). DA Analysis by HPLC–ED
Immunohistochemistry Selected first trimester specimens were immersionfixed in phosphate-buffered 4% paraformaldehyde, pH 7.4, at 4°C overnight, and depending on gestational age, either as intact or as microdissected specimens, to optimize the preservation of the mesencephalic area. The fixed tissue was rinsed in phosphate-buffered 10% sucrose with several changes for up to 2 weeks before being sectioned (14 µm) either sagittally or transversely on a Leitz cryostat. For the localization of TH, an indirect immunohistochemical staining technique was used with a horseradish-peroxidase enzyme reaction. Briefly, the frozen sections were thaw-mounted on chrome alum gelatincoated microscope slides and further processed using a commercial kit (Vectastain) for avidin–biotin staining procedure (18). A rabbit antiserum raised against purified rat brain tyrosine hydroxylase (Pel-Freez, Arkansas) was used diluted 1/00 in PBS with 0.3% Triton X-100. Sections were incubated with the primary antibody at 14°C overnight in a humid chamber. Antibodies bound were visualized using 0.1% 3,3-diaminobenzidine (Sigma), with 0.04% NiCl2 for enhancement. Slides were mounted in Entellan (Merck). Sections adjacent to those stained for TH were processed for cresyl violet staining. The specificity of the TH antiserum was verified by Western blot technique. Negative control sections incubated as described above but without the primary antiserum were blank. Bright-field microscopy and photography were performed on an Axiophot microscope (Zeiss).
For quantitative analysis of DA, an extraction technique described by Keller and co-workers (23) was used with slight modifications. Briefly, tissue pieces were weighed and 0.1 M perchloric acid and 2 ng dihydroxybenzylamine (internal standard) were added. Mesencephalic and forebrain tissue pieces from 5- to 6-week-old embryos were pooled three by three, respectively. After sonication, 0.4 M Tris buffer (pH 8.6) with NaHSO3 and approximately 10 mg of Al2O3 were added and after shaking and washing, catecholamines were eluted by adding 50 µl of perchloric acid. The analysis was performed on a reversed phase column (C18, 3u, 75 3 4.2 mm, Beckman) with a mobile phase consisting of 0.1 M phosphate buffer, 30 mg/liter sodiumoctylsulfate, 120 mg/liter EDTA, and 4% methanol, adjusted to pH 3.70 at a flow of 1 ml/min. The column was attached to an electrochemical detector (LC-4B, Bioanalytical Systems, West Lafayette, IN) with a glassy carbon electrode set at 10.7 V. The detection level was in the order of 10 pg DA [for detailed information see (53)]. RESULTS
For most tissue retrievals, intact embryos were obtained up to 5 weeks of age, whereas tissue specimens obtained from the later half of the first trimester all suffered from surgical trauma and were damaged to various degrees. In some late-stage embryos (Gestational Week 7–8), the brain parenchyma had fortunately been separated en bloc from the skull cavity and was preserved, whereas the spinal cord was always entrapped within the mesenchyme of the primitive
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vertebral column. The site of disruption would usually be located either at the isthmus rhombencephali or at the cervical flexure between the lower brain stem and the spinal cord. In the residual tissue from a 9.5-weekold fetus, the whole brain including the brain stem was preserved and was fixed in paraformaldehyde for further processing for TH immunohistochemistry. For identification and trimming of residual CNS tissue, the meninges were always completely removed by microdissection procedures. Remaining meninges could have otherwise seriously damaged the tissue sections during cryostat sectioning for immunohistochemistry as well as interfered with the preparation of tissue homogenates for SDS–PAGE. Morphological Differentiation of Nigral DA Neurons The earliest tissue specimen recovered was an intact 3.5-week-old embryo [24 days, 20 somites, CS 11 (40)] which was processed for TH immunohistochemistry. In sagittal sections TH-positive, primitive, rounded cells were observed along the neural tube, caudal to the mesecephalic flexure. At 4.5 weeks of gestation, small, rounded TH-positive cells were identified in the midbrain. These cells formed a semicircular, well-defined population of neuroblasts located in the mantle layer of the basal plate, parallel to the ventricular zone, and at the level of the mesencephalic flexure. A few THpositive, primary processes were identified at this early stage (Fig. 1). One week later, the number of visualized cells as well as the intensity of TH immunoreactivity had increased significantly. Stained cells were now distributed all the way from the germinal ventricular zone to the marginal layer of the ventral tegmentum, as well as in rostro– caudal direction, within the mesencephalon. Numerous TH-positive neurites projected toward the caudal part of the diencephalon (Fig. 2). At the early fetal stage (9.5 weeks), the ventral tegmental area was filled with TH-immunoreactive perikarya and fibers, with the highest density of cells in the ventral mantle layer. From cells in the ventral half of tegmentum, major bundles of stained axons could be followed into subcortical forebrain. Here, axonal processes diverged radially and terminated in the developing basal ganglia as well as in caudal diencephalic target areas of the primordial hypothalamus (Fig. 3). At the end of the first trimester, varicose fibers could be detected in the striatal anlage (Fig. 4). Molecular Characterization of TH For a qualitative analysis of the antigen(s) recognized by the rabbit anti-rat brain TH antiserum in embryonic tissue, unfractionated homogenates of embryonic mesencephalic tissue were applied to SDS– PAGE and subsequently blotted to nitrocellulose mem-
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branes. Chemiluminiscence detection of immunocomplexes revealed a single, uniform band with the apparent molecular weight of approximately 60 kDa (Fig. 5). No other bands or confounding background staining were seen in any of the lanes. The putative TH band displayed no sign of molecular heterogeneity or apparent multiple components (Fig. 5, lanes A–C), and neither was there any evidence for a shift in apparent molecular weight with increasing gestational age (lanes A–G). The immunoblot also allowed a semiquantitative assessment of TH expression. A weak band was identified at 5 weeks of gestational age, the earliest stage analyzed. As evident from Fig. 5, there was a rapid and progressive increase in the relative abundance of TH in mesencephalic tissue during Gestational Weeks 5–9, as judged by the staining intensity. Detection of Endogenous DA In pooled samples of mesencephalic tissue derived from three embryos of the same gestational age, endogenous DA could already be detected at 5.5 weeks of age. The DA content was approximately 0.2 ng of DA per mesencephalon. To avoid a ‘‘dilution effect’’ of the growing tissue, results were presented to reflect the total amount of DA in each mesencephalon. A distinct elevation of DA levels started to occur at 7–7.5 weeks of gestation, followed by an exponential increase of DA content in the mesencephalon during the later part of the first trimester. At the end of this period, 4–5.5 ng per mesencephalon was measured (Fig. 6). The development of tissue concentrations of DA in the forebrain was similar to that of the mesencephalon. It should be noted, however, that due to the ongoing developmental processes of the brain, the dissection procedure had to be adjusted according to anatomical structures appearing during the first trimester. At Gestational Weeks 5 and 6, the entire forebrain was recovered for DA analysis, whereas during the 7th and 8th weeks subcortical forebrain was separated from the cortical plate and analyzed for DA. Thereafter, the caudate nucleus–putamen complex could be identified and recovered separately. Detectable levels of DA were found in the forebrain at 5.5 weeks of gestation (5–7.5 ng DA/g wet weight tissue) and reached 50–75 ng DA/g wet weight tissue of the caudate nucleus–putamen complex during the 12th gestational week (Fig. 7). DISCUSSION
In the present study, we have examined the early development of dopaminergic neurons in the human embryonic midbrain by use of two neurochemical markers, TH and DA. Their studies make both morphological and neurochemical analyses possible. Previous studies have suffered from small and heterogeneous samples of first trimester brain tissue, making developmental
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FIG. 1. A bright-field photomicrograph of a sagittal tissue section, showing TH-positive neurons (e.g., at arrowheads) and fibers (arrows) in the tegmental area of a 4.5-week-old human embryo. Rostral direction to the left. Magnification: 1603. The inset shows a cresyl violet-stained adjacent section at lower magnification. Note the heavily stained neuroepithelial cell layer in dorsal tegmentum. The dotted line indicates the close-up area. Magnification: 403.
staging based on the use of differentiation markers unreliable, especially since the tissue easily gets traumatized to various extents during surgery. In the present report, we emphasize the mapping of the first appearance of TH and DA in mesencephalic neurons and their subsequent expression pattern during early ontogeny. The central nervous system is the first organ to
manifest its presence during the third week of the first trimester. Although the neural groove is completely open at this gestational age, the major divisions of the brain can be discerned, and the neural axis has bent at the midbrain, forming the mesencephalic flexure. Ventral to the aqueduct, the basal plate forms the tegmentum. However, it is uncertain whether the red nucleus and the substantia nigra are derived from the basal
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FIG. 2. A bright-field photomicrograph of a sagittal tissue section, showing large aggregates of TH-positive neurons (e.g., at arrowheads) in the tegmental area of a 5.5-week-old human embryo. Rostral direction to the bottom right. M.F., mesencephalic flexure. Original magnification: 1253. Inset figure shows a cresyl violet-stained adjacent tissue section at lower magnification. Note the third ventricle (V). The dotted line shows the area of the close up. Original magnification: 303.
FIG. 3. A bright-field photomicrograph of a sagittal tissue section, showing densely TH-immunostained neurons in the mesencephalic flexure (M.F.) and large bundles of TH-positive fibers extending rostrally (left) in the intact brain of a 9.5-week-old human fetus. Note the fourth ventricle (V). Magnification: 203. The inset figure shows a cresyl violet-stained adjacent tissue section at lower magnification. The dotted line marks the area of the close up. Magnification: 53.
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FIG. 4. (a) A bright-field photomicrograph of TH-immunostained neurons in the ventral tegmental area of a 9.5-week-old human fetus. Note the perinuclear, cytoplasmic TH staining of morphologically differentiating neurons, mostly with one major axonal process. Magnification: 3303. (b) A bright-field photomicrograph of TH-positive varicose nerve fibers in the striatal anlage of a 9.5-week-old human fetus. Magnification: 3303.
plate which is essentially efferent in function, or by migration, from the alar plate which is fundamentally afferent (40). At the spinal level, neural crest cells participate early (CS 12) in the formation of the spinal ganglia and migrate ventrally to form the sympathetic trunk (39). In our study, a number of primitive THpositive cells can already be seen along the ventral side of the neural tube at the time of closure of the caudal neuropore. From these data, we infer that these cells make up the rudimentary sympathetic chain previ-
ously reported to be TH-positive at 5.5 weeks of gestation (42). Rapid and important development occurs in the brain stem during the embryonic period, including the appearance of several groups of monoaminergic neuroblasts, of which the major components are the locus coeruleus (NA), the dorsal and median raphe nuclei (5-HT), and the substantia nigra (DA) (36, 53). In the rat, the DA neurons of the ventral mesencephalon are born at Embryonic Days E11 to E15 (1, 14). They
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FIG. 5. Western blot analysis of TH in mesencephalic tissue derived from embryonic, human specimens of 5, 5.5, 6, 6.5, 7, 8, and 9 weeks of gestational age (lanes A–G). In each well tissue homogenate corresponding to 100 mg total protein was loaded. Note the rapid increase in TH content in mesencephalic tissue during the second month of gestation.
migrate ventrally and rostrally from their origin in the dorsal midbrain to arrive at their final location within the ventral tegmental area at about E18 (32, 52, 57). Along their migration path, they gradually attain phenotypic features, indicating differentiation and maturity. TH appears in the brain stem at Embryonic Day 12–13 (3, 51, 52, 54), and DA-containing neurons are
FIG. 6. Levels of DA in the developing mesencephalon during the first trimester expressed as total amount of DA in ng/tissue specimen. For the analyses of 5- to 6-week-old mesencephalic tissue, pooled samples from three embryos were used.
detected in the midbrain at E13 by histofluorescence technique (36). Our observation of TH-immunoreactive cells in the mesencephalic flexure at the gestational age of 4.5 weeks, or CS 14, when the number of somites exceeds 35 [as presented by O’Rahilly and Mu¨ller (39)], is consistent with the corresponding histogenesis of rat DA neurons, as described. However, previous reports on the ontogeny of TH in the human midbrain have indicated the first appearance of the enzyme at 5–6.5 weeks of gestation (10, 49, 56). In agreement with previous reports by Angevine and Keller et al. (2, 23), TH-immunoreactive cells were seen exclusively outside the ependymal layer, which is consistent with the cessation of mitoses in these cells. Although postmitotic, TH-immunoreactive cells remained rounded, migrating ventrally from the germinal ventricular zone and accumulating in the mantle layer. During this process, they did not form any primary processes until reaching the ventral half of the mantle layer. Here, axonal processes developed promptly, already projecting toward the caudal part of diencephalon at 5.5 weeks of gestation. The nigrostriatal pathway starts to develop during the sixth and seventh gestational weeks, forming major bundles of TH-stained axons projecting into more rostral telencephalic regions. Since immunohistochemistry indicated a first appearance of TH-positive cells in the midbrain at an earlier developmental stage than previously reported and since the immunological detection of TH was based on cross-
FIG. 7. Concentration of DA in the forebrain during the first trimester. For anatomical reasons, DA was detected in the entire forebrain (prosencephalon) at 5–6 weeks of age (U), and while at 7–8 weeks diencephalon/subcortical forebrain was dissected and analyzed (V). At later stages, the caudate nucleus–putamen complex could be identified and dissected for DA analysis (N).
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reactivity of the rabbit anti-purified rat brain TH antibodies to the human isoform, we performed a qualitative analysis of the recognized antigen(s). Western blot technique confirmed the identity of an immunoreactive band of about 60 kDa. The appearance of the band indicated the lack of major size- or chargeheterogeneity in combination with an apparent molecular weight consistent with that of the translation products of full-length human TH cDNAs and genomic DNA (33). In contrast to mice (19) and rats (4), humans produce four different isoforms of TH mRNA via alternative splicing from a single primary transcript (13, 17, 21). Based on DNA sequencing, the predicted molecular weights for the human TH isoforms range from 55.6 to 58.5 kDa, by the inclusion, at amino acid 30, of 4, 27, or 31 amino acids, respectively. Selectively expressed in COS cells, the four TH isoforms gave immunoreactive bands at 61, 61, 65, and 65 kDa, as analyzed by Western blotting (24). Haycock has previously demonstrated the presence of all four TH isoform proteins in the human adrenal medulla and in neuroblastoma cell lines utilizing isoform-specific antibodies (15). The expression pattern of TH isozyme mRNA in human fetal (24 weeks of gestation) and neonate brain stem has been investigated by primer extension analysis and proved the existence of type 1 and 2 TH isoforms at equal expression levels (22). In the present study, the use of an anti-rat TH antiserum did not allow immunological discrimination between the different types of human TH isozymes. However, judged by the apparent molecular weight of TH detected by Western blotting, it is consistent with the expression of TH isozyme 1 or 2, or both. The Western blot technique also allows of a semiquantitative assessment of TH expression. This experiment clearly showed a rapid, continuous increase of TH protein following the detection of the enzyme at 5 weeks of gestational age. This was in good agreement with the immunohistochemical stainings, showing the number of TH-immunoreactive cells in the ventral mesencephalon progressively increasing in number during these developmental stages (Figs. 1–3). When comparing mesencephalic DA levels with the content of other monoamines in the brain stem during first trimester development, both NA and 5-HT were found in higher quantities than DA in the corresponding regions with monoaminergic cell bodies (53). However, the exponential increase in levels as a function of time was similar for all three monoamines. DA concentration in the caudate nucleus–putamen complex was, e.g., in the same range as that of NA in the spinal cord at the end of the first trimester. The very dense innervation of DA terminals in the adult caudate nucleus–putamen, with DA levels by far exceeding those of any other region of the CNS, obviously develops at a later stage (35, 37). Consequently, the concentration of DA in the basal ganglia at the 12th gesta-
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tional week is only about 1% of that found in the mature, adult brain (44). In the embryonic rat, detectable DA levels were found in striatum at E15–E16, 3–4 days after the appearance of TH-immunoreactive cells in mesencephalon (9, 41). From the analyses of endogenous DA content in the human embryonic midbrain, it is evident that mesencephalic neuroblasts synthesize DA within an approximately 1-week period following the onset of cytoplasmic TH expression in these cells. At this stage, dopaminergic neurons are still morphologically very immature with short primary processes reaching caudal parts of the hypothalamus. The early start of monoamine synthesis is interesting evidence of neurochemical differentiation of these cells even prior to their innervation of their target area in the basal ganglia anlage. Studies of the in vivo development of the nigrostriatal dopaminergic system in man is relevant to the field of human embryology, but also for guidance in the use of first trimester mesencephalic tissue as donor material in clinical trials and animal models for treatment of Parkinson’s disease by intracerebral implantation of dopaminergic neurons. The course of genetically programmed differentiation is different in human and rodent grafts and functional recovery of transplanted animals was reported markedly delayed when using human tissue versus rodent (55). The influences of donor gestational age, of tissue retrieval, storage, and processing prior to implantation, and of the intracerebral positioning of the grafts are still subjects of interest and controversy [reviewed by Fine (7)]. The long-term survival of grafted mesencephalic DA neurons varies in different animal models, but has been reported to be about 5–10% (16). Embryonic mesencephalic tissue (,9 weeks of gestation) is generally preferred to fetal donor material, since graft survival has been shown to be related to the ontogeny of the human nigrostriatal system (11). However, the absolute number of DA neurons contained within the embryonic mesencephalon is significantly less, as shown by the present study. Also important to consider is the early and extensive axo–dendritic differentiation of these cells during the first trimester. At gestational age exceeding 6–7 weeks, dopaminergic axonal trajectories extend into the primitive forebrain, which will result in a traumatic axotomy of DA neurons when retrieved for grafting. To approach this problem, multiple embryonic grafts have been used for each patient subjected to intracerebral implantation (58). Improved motor function, increase in fluorodopa uptake in the grafted area on PET scanning, and long-term survival and extensive dopaminergic reinnervation of grafted regions in a 59-year-old male Parkinson patient were recently reported (26). A novel strategy has emerged with the report of in
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vitro isolation of neural progenitor cells from adult and embryonic brain (46, 47). By isolating and expanding the population of mesencephalic stem cells in vitro, a possible source of relatively undifferentiated cells with the potential of developing a dopaminergic phenotype may be obtained for transplant studies. We are currently testing multipotent EGF-responsive cells in vitro in order to find conditions that may guide and support them in the differentiation process toward dopaminergic neurons.
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ACKNOWLEDGMENTS 15. This study was supported by the Swedish Medical Research Council, Grants 14X-06555 and B96-13X-11570-01A, The Miami Project Foundation, The Daniel Heumann Fund For Spinal Cord Research, The Foundations of the Karolinska Institute, The Magnus Bergvall Fund for Medical Research, The Foundations of Sven Jerring and of Tore Nilson, and the Spinalis Foundation. Expert technical assistance by Eva-Britt Samuelsson is gratefully acknowledged.
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0096
First Trimester Development of the Human Nigrostriatal Dopamine System PER M. ALMQVIST, ELISABET ÅKESSON,* LARS U. WAHLBERG, HELMUT PSCHERA,† ÅKE SEIGER,* AND ERIK SUNDSTRO¨ M* Department of Clinical Neuroscience, Section of Neurosurgery, *Department of Clinical Neuroscience and Family Medicine, Section of Geriatric Medicine, and †Department of Obstetrics and Gynecology, Karolinska Institute, Stockholm, Sweden
INTRODUCTION The aim of the present study was to characterize the morphological and neurochemical differentiation of mesencephalic dopaminergic neurons in human embryos, derived from elective first trimester abortions. Embryonic brain tissue was taken for analysis of tyrosine hydroxylase (TH) by immunohistochemistry and Western blot, and for analysis of endogenous dopamine (DA) content using HPLC-ED. TH expression was first detected at 3.5 weeks of gestational age (Carnegie stage 11) by immunohistochemical staining of the primordial sympathetic trunk along both sides of the neural tube. In sagittal sections of the intact 4.5-week-old embryo, a small, distinct population of rounded, densely packed TH-immunoreactive perikarya with short primary processes was seen in the midbrain. During the latter half of the first trimester, the number of TH-stained cells as well as the length and number of axonal processes projecting toward and into the developing neostriatum increased rapidly. At the end of the first trimester, varicose fibers could be detected in the striatal anlage. In order to verify that TH was the antigen recognized by the antibodies used for immunohistochemistry on human tissue specimens, mesencephalic tissue of 5–10 weeks gestation was analyzed by Western blot technique. A single, homogeneous band with the apparent molecular weight of approximately 60 kDa was clearly detected at 5 weeks of age. The amount of TH/mg total protein increased at least 10-fold between 5–10 weeks of gestation. For comparison, the mesencephalon and the forebrain/basal ganglia were analyzed for endogenous DA content using HPLC-ED. DA was first detected at 5.5 weeks of gestational age in both mid- and forebrain, and DA levels were found to increase exponentially from 7 to 7.5 weeks of age, reaching 4–5.5 ng DA/mesencephalon and 50–75 ng DA/g caudate nucleus–putamen anlage at the end of the first trimester. Together, morphological and biochemical data presented here constitute evidence for a very early appearance, migration, and differentiation as well as functional development of human mesencephalic dopaminergic neurons and their projections into target areas during the first trimester. r 1996 Academic Press, Inc.
Monoamine-synthesizing neurons appear in the brain stem early during embryogenesis and their fiber pathways are considered crucial for brain development (29). The earliest production sites in the human embryonic brain are the developing locus coeruleus complex and the raphe nuclei of the rhombencephalon synthesizing noradrenalin (NA) and serotonin (5-HT), respectively. Both transmitters are detected at 5–6 weeks of gestation, while tyrosine hydroxylase-immunoreactive neuroblasts are discerned at 4 weeks of age (53). Another region of early development is the midventral proliferative area of mesencephalon, which participates in the formation of the red nucleus and the substantia nigra (40). The latter, with its content of dopaminergic (DA) neurons and their relevance for the pathophysiology of the disorder paralysis agitans (Parkinson’s disease), has been subjected to a vast number of morphological and neurochemical studies in rodents (5, 8, 28, 31, 32, 45, 48, 51, 52, 57) and in nonhuman primates (12, 30, 50). Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the catecholamine biosynthesis (34), and the human TH gene has been assigned to chromosome 11p15.5 (20). Evidence for TH gene expression and/or endogenous DA synthesis in human fetal mesencephalon has been reported consistently (10, 25, 35, 37, 42, 43), whereas the exact time of the first appearance of mesencephalic DA neurons during ontogeny is a matter for discussion. Three separate immunohistochemical studies report TH-immunoreactive cells in the mesencephalic flexure at 5 weeks [Carnegie stage (CS) 15–16] (56), at 5.5 weeks (CS 16–17) (49), and at 6.5 weeks (CS 18–19) (10), respectively. To clarify the issue of embryonic development of mesencephalic DA neurons in vivo, we have performed a combined morphological and neurochemical study with an extended sample size and gestational age range, monitoring TH expression and endogenous DA synthesis during the first trimester.
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0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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MATERIALS AND METHODS
Tissue Retrieval Embryonic nervous tissue was recovered from elective first trimester routine abortions using the standard vacuum aspiration technique. The collection of residual tissue was approved by the Human Ethics Committee of the Huddinge University Hospital, Karolinska Institute, and was in accordance with the guidelines of the Swedish Society of Medicine and the U.S. Public Health Service, including an informed consent from the pregnant women seeking abortions (38). The gestational age of each specimen was determined by its size and by examination of anatomical landmarks according to the atlas of England (6). A total of 50 first trimester tissue specimens, ranging from 3.5 to 11.5 weeks of gestation, were included in this study. Recovered nervous tissue was microdissected within 2 h of surgery and either fixed in paraformaldehyde for immunohistochemistry or fresh frozen on dry ice and stored at 2135°C pending SDS–PAGE/Western blot or HPLC-ED analyses.
Western Blot Microdissected, fresh mesencephalic tissue pieces, stored frozen in 1.5-ml sealed microcentrifuge tubes, were thawed by the addition of 9 volumes ice-cold lysis buffer, containing 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.5% (w/v) Triton X-100, 2 mM EDTA in 10 mM Tris–HCl, pH 8.0. Tissue pieces were homogenized by vortexing followed by sonication on ice. Tissue homogenate (10% w/v) corresponding to 100 mg of total protein, as analyzed by the Lowry method modified according to Markwell (32a), was loaded in 8-mm wells for SDS–PAGE on a 1.5-mm-thick 9% isocratic polyacrylamide gel (27). The molecular weight marker proteins (Sigma) were stained by Coomassie brilliant blue while proteins of the tissue homogenate were transferred to a nitrocellulose filter (31) followed by immunochemical detection of TH using the same antiserum as for TH immunohistochemistry (see above), diluted 1/1000. The antibody immunocomplexes were visualized by photographic exposure using a chemiluminiscence detection system (ECL, Amersham). DA Analysis by HPLC–ED
Immunohistochemistry Selected first trimester specimens were immersionfixed in phosphate-buffered 4% paraformaldehyde, pH 7.4, at 4°C overnight, and depending on gestational age, either as intact or as microdissected specimens, to optimize the preservation of the mesencephalic area. The fixed tissue was rinsed in phosphate-buffered 10% sucrose with several changes for up to 2 weeks before being sectioned (14 µm) either sagittally or transversely on a Leitz cryostat. For the localization of TH, an indirect immunohistochemical staining technique was used with a horseradish-peroxidase enzyme reaction. Briefly, the frozen sections were thaw-mounted on chrome alum gelatincoated microscope slides and further processed using a commercial kit (Vectastain) for avidin–biotin staining procedure (18). A rabbit antiserum raised against purified rat brain tyrosine hydroxylase (Pel-Freez, Arkansas) was used diluted 1/00 in PBS with 0.3% Triton X-100. Sections were incubated with the primary antibody at 14°C overnight in a humid chamber. Antibodies bound were visualized using 0.1% 3,3-diaminobenzidine (Sigma), with 0.04% NiCl2 for enhancement. Slides were mounted in Entellan (Merck). Sections adjacent to those stained for TH were processed for cresyl violet staining. The specificity of the TH antiserum was verified by Western blot technique. Negative control sections incubated as described above but without the primary antiserum were blank. Bright-field microscopy and photography were performed on an Axiophot microscope (Zeiss).
For quantitative analysis of DA, an extraction technique described by Keller and co-workers (23) was used with slight modifications. Briefly, tissue pieces were weighed and 0.1 M perchloric acid and 2 ng dihydroxybenzylamine (internal standard) were added. Mesencephalic and forebrain tissue pieces from 5- to 6-week-old embryos were pooled three by three, respectively. After sonication, 0.4 M Tris buffer (pH 8.6) with NaHSO3 and approximately 10 mg of Al2O3 were added and after shaking and washing, catecholamines were eluted by adding 50 µl of perchloric acid. The analysis was performed on a reversed phase column (C18, 3u, 75 3 4.2 mm, Beckman) with a mobile phase consisting of 0.1 M phosphate buffer, 30 mg/liter sodiumoctylsulfate, 120 mg/liter EDTA, and 4% methanol, adjusted to pH 3.70 at a flow of 1 ml/min. The column was attached to an electrochemical detector (LC-4B, Bioanalytical Systems, West Lafayette, IN) with a glassy carbon electrode set at 10.7 V. The detection level was in the order of 10 pg DA [for detailed information see (53)]. RESULTS
For most tissue retrievals, intact embryos were obtained up to 5 weeks of age, whereas tissue specimens obtained from the later half of the first trimester all suffered from surgical trauma and were damaged to various degrees. In some late-stage embryos (Gestational Week 7–8), the brain parenchyma had fortunately been separated en bloc from the skull cavity and was preserved, whereas the spinal cord was always entrapped within the mesenchyme of the primitive
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vertebral column. The site of disruption would usually be located either at the isthmus rhombencephali or at the cervical flexure between the lower brain stem and the spinal cord. In the residual tissue from a 9.5-weekold fetus, the whole brain including the brain stem was preserved and was fixed in paraformaldehyde for further processing for TH immunohistochemistry. For identification and trimming of residual CNS tissue, the meninges were always completely removed by microdissection procedures. Remaining meninges could have otherwise seriously damaged the tissue sections during cryostat sectioning for immunohistochemistry as well as interfered with the preparation of tissue homogenates for SDS–PAGE. Morphological Differentiation of Nigral DA Neurons The earliest tissue specimen recovered was an intact 3.5-week-old embryo [24 days, 20 somites, CS 11 (40)] which was processed for TH immunohistochemistry. In sagittal sections TH-positive, primitive, rounded cells were observed along the neural tube, caudal to the mesecephalic flexure. At 4.5 weeks of gestation, small, rounded TH-positive cells were identified in the midbrain. These cells formed a semicircular, well-defined population of neuroblasts located in the mantle layer of the basal plate, parallel to the ventricular zone, and at the level of the mesencephalic flexure. A few THpositive, primary processes were identified at this early stage (Fig. 1). One week later, the number of visualized cells as well as the intensity of TH immunoreactivity had increased significantly. Stained cells were now distributed all the way from the germinal ventricular zone to the marginal layer of the ventral tegmentum, as well as in rostro– caudal direction, within the mesencephalon. Numerous TH-positive neurites projected toward the caudal part of the diencephalon (Fig. 2). At the early fetal stage (9.5 weeks), the ventral tegmental area was filled with TH-immunoreactive perikarya and fibers, with the highest density of cells in the ventral mantle layer. From cells in the ventral half of tegmentum, major bundles of stained axons could be followed into subcortical forebrain. Here, axonal processes diverged radially and terminated in the developing basal ganglia as well as in caudal diencephalic target areas of the primordial hypothalamus (Fig. 3). At the end of the first trimester, varicose fibers could be detected in the striatal anlage (Fig. 4). Molecular Characterization of TH For a qualitative analysis of the antigen(s) recognized by the rabbit anti-rat brain TH antiserum in embryonic tissue, unfractionated homogenates of embryonic mesencephalic tissue were applied to SDS– PAGE and subsequently blotted to nitrocellulose mem-
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branes. Chemiluminiscence detection of immunocomplexes revealed a single, uniform band with the apparent molecular weight of approximately 60 kDa (Fig. 5). No other bands or confounding background staining were seen in any of the lanes. The putative TH band displayed no sign of molecular heterogeneity or apparent multiple components (Fig. 5, lanes A–C), and neither was there any evidence for a shift in apparent molecular weight with increasing gestational age (lanes A–G). The immunoblot also allowed a semiquantitative assessment of TH expression. A weak band was identified at 5 weeks of gestational age, the earliest stage analyzed. As evident from Fig. 5, there was a rapid and progressive increase in the relative abundance of TH in mesencephalic tissue during Gestational Weeks 5–9, as judged by the staining intensity. Detection of Endogenous DA In pooled samples of mesencephalic tissue derived from three embryos of the same gestational age, endogenous DA could already be detected at 5.5 weeks of age. The DA content was approximately 0.2 ng of DA per mesencephalon. To avoid a ‘‘dilution effect’’ of the growing tissue, results were presented to reflect the total amount of DA in each mesencephalon. A distinct elevation of DA levels started to occur at 7–7.5 weeks of gestation, followed by an exponential increase of DA content in the mesencephalon during the later part of the first trimester. At the end of this period, 4–5.5 ng per mesencephalon was measured (Fig. 6). The development of tissue concentrations of DA in the forebrain was similar to that of the mesencephalon. It should be noted, however, that due to the ongoing developmental processes of the brain, the dissection procedure had to be adjusted according to anatomical structures appearing during the first trimester. At Gestational Weeks 5 and 6, the entire forebrain was recovered for DA analysis, whereas during the 7th and 8th weeks subcortical forebrain was separated from the cortical plate and analyzed for DA. Thereafter, the caudate nucleus–putamen complex could be identified and recovered separately. Detectable levels of DA were found in the forebrain at 5.5 weeks of gestation (5–7.5 ng DA/g wet weight tissue) and reached 50–75 ng DA/g wet weight tissue of the caudate nucleus–putamen complex during the 12th gestational week (Fig. 7). DISCUSSION
In the present study, we have examined the early development of dopaminergic neurons in the human embryonic midbrain by use of two neurochemical markers, TH and DA. Their studies make both morphological and neurochemical analyses possible. Previous studies have suffered from small and heterogeneous samples of first trimester brain tissue, making developmental
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FIG. 1. A bright-field photomicrograph of a sagittal tissue section, showing TH-positive neurons (e.g., at arrowheads) and fibers (arrows) in the tegmental area of a 4.5-week-old human embryo. Rostral direction to the left. Magnification: 1603. The inset shows a cresyl violet-stained adjacent section at lower magnification. Note the heavily stained neuroepithelial cell layer in dorsal tegmentum. The dotted line indicates the close-up area. Magnification: 403.
staging based on the use of differentiation markers unreliable, especially since the tissue easily gets traumatized to various extents during surgery. In the present report, we emphasize the mapping of the first appearance of TH and DA in mesencephalic neurons and their subsequent expression pattern during early ontogeny. The central nervous system is the first organ to
manifest its presence during the third week of the first trimester. Although the neural groove is completely open at this gestational age, the major divisions of the brain can be discerned, and the neural axis has bent at the midbrain, forming the mesencephalic flexure. Ventral to the aqueduct, the basal plate forms the tegmentum. However, it is uncertain whether the red nucleus and the substantia nigra are derived from the basal
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FIG. 2. A bright-field photomicrograph of a sagittal tissue section, showing large aggregates of TH-positive neurons (e.g., at arrowheads) in the tegmental area of a 5.5-week-old human embryo. Rostral direction to the bottom right. M.F., mesencephalic flexure. Original magnification: 1253. Inset figure shows a cresyl violet-stained adjacent tissue section at lower magnification. Note the third ventricle (V). The dotted line shows the area of the close up. Original magnification: 303.
FIG. 3. A bright-field photomicrograph of a sagittal tissue section, showing densely TH-immunostained neurons in the mesencephalic flexure (M.F.) and large bundles of TH-positive fibers extending rostrally (left) in the intact brain of a 9.5-week-old human fetus. Note the fourth ventricle (V). Magnification: 203. The inset figure shows a cresyl violet-stained adjacent tissue section at lower magnification. The dotted line marks the area of the close up. Magnification: 53.
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FIG. 4. (a) A bright-field photomicrograph of TH-immunostained neurons in the ventral tegmental area of a 9.5-week-old human fetus. Note the perinuclear, cytoplasmic TH staining of morphologically differentiating neurons, mostly with one major axonal process. Magnification: 3303. (b) A bright-field photomicrograph of TH-positive varicose nerve fibers in the striatal anlage of a 9.5-week-old human fetus. Magnification: 3303.
plate which is essentially efferent in function, or by migration, from the alar plate which is fundamentally afferent (40). At the spinal level, neural crest cells participate early (CS 12) in the formation of the spinal ganglia and migrate ventrally to form the sympathetic trunk (39). In our study, a number of primitive THpositive cells can already be seen along the ventral side of the neural tube at the time of closure of the caudal neuropore. From these data, we infer that these cells make up the rudimentary sympathetic chain previ-
ously reported to be TH-positive at 5.5 weeks of gestation (42). Rapid and important development occurs in the brain stem during the embryonic period, including the appearance of several groups of monoaminergic neuroblasts, of which the major components are the locus coeruleus (NA), the dorsal and median raphe nuclei (5-HT), and the substantia nigra (DA) (36, 53). In the rat, the DA neurons of the ventral mesencephalon are born at Embryonic Days E11 to E15 (1, 14). They
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FIG. 5. Western blot analysis of TH in mesencephalic tissue derived from embryonic, human specimens of 5, 5.5, 6, 6.5, 7, 8, and 9 weeks of gestational age (lanes A–G). In each well tissue homogenate corresponding to 100 mg total protein was loaded. Note the rapid increase in TH content in mesencephalic tissue during the second month of gestation.
migrate ventrally and rostrally from their origin in the dorsal midbrain to arrive at their final location within the ventral tegmental area at about E18 (32, 52, 57). Along their migration path, they gradually attain phenotypic features, indicating differentiation and maturity. TH appears in the brain stem at Embryonic Day 12–13 (3, 51, 52, 54), and DA-containing neurons are
FIG. 6. Levels of DA in the developing mesencephalon during the first trimester expressed as total amount of DA in ng/tissue specimen. For the analyses of 5- to 6-week-old mesencephalic tissue, pooled samples from three embryos were used.
detected in the midbrain at E13 by histofluorescence technique (36). Our observation of TH-immunoreactive cells in the mesencephalic flexure at the gestational age of 4.5 weeks, or CS 14, when the number of somites exceeds 35 [as presented by O’Rahilly and Mu¨ller (39)], is consistent with the corresponding histogenesis of rat DA neurons, as described. However, previous reports on the ontogeny of TH in the human midbrain have indicated the first appearance of the enzyme at 5–6.5 weeks of gestation (10, 49, 56). In agreement with previous reports by Angevine and Keller et al. (2, 23), TH-immunoreactive cells were seen exclusively outside the ependymal layer, which is consistent with the cessation of mitoses in these cells. Although postmitotic, TH-immunoreactive cells remained rounded, migrating ventrally from the germinal ventricular zone and accumulating in the mantle layer. During this process, they did not form any primary processes until reaching the ventral half of the mantle layer. Here, axonal processes developed promptly, already projecting toward the caudal part of diencephalon at 5.5 weeks of gestation. The nigrostriatal pathway starts to develop during the sixth and seventh gestational weeks, forming major bundles of TH-stained axons projecting into more rostral telencephalic regions. Since immunohistochemistry indicated a first appearance of TH-positive cells in the midbrain at an earlier developmental stage than previously reported and since the immunological detection of TH was based on cross-
FIG. 7. Concentration of DA in the forebrain during the first trimester. For anatomical reasons, DA was detected in the entire forebrain (prosencephalon) at 5–6 weeks of age (U), and while at 7–8 weeks diencephalon/subcortical forebrain was dissected and analyzed (V). At later stages, the caudate nucleus–putamen complex could be identified and dissected for DA analysis (N).
NIGROSTRIATAL DA NEURON FIRST TRIMESTER DEVELOPMENT
reactivity of the rabbit anti-purified rat brain TH antibodies to the human isoform, we performed a qualitative analysis of the recognized antigen(s). Western blot technique confirmed the identity of an immunoreactive band of about 60 kDa. The appearance of the band indicated the lack of major size- or chargeheterogeneity in combination with an apparent molecular weight consistent with that of the translation products of full-length human TH cDNAs and genomic DNA (33). In contrast to mice (19) and rats (4), humans produce four different isoforms of TH mRNA via alternative splicing from a single primary transcript (13, 17, 21). Based on DNA sequencing, the predicted molecular weights for the human TH isoforms range from 55.6 to 58.5 kDa, by the inclusion, at amino acid 30, of 4, 27, or 31 amino acids, respectively. Selectively expressed in COS cells, the four TH isoforms gave immunoreactive bands at 61, 61, 65, and 65 kDa, as analyzed by Western blotting (24). Haycock has previously demonstrated the presence of all four TH isoform proteins in the human adrenal medulla and in neuroblastoma cell lines utilizing isoform-specific antibodies (15). The expression pattern of TH isozyme mRNA in human fetal (24 weeks of gestation) and neonate brain stem has been investigated by primer extension analysis and proved the existence of type 1 and 2 TH isoforms at equal expression levels (22). In the present study, the use of an anti-rat TH antiserum did not allow immunological discrimination between the different types of human TH isozymes. However, judged by the apparent molecular weight of TH detected by Western blotting, it is consistent with the expression of TH isozyme 1 or 2, or both. The Western blot technique also allows of a semiquantitative assessment of TH expression. This experiment clearly showed a rapid, continuous increase of TH protein following the detection of the enzyme at 5 weeks of gestational age. This was in good agreement with the immunohistochemical stainings, showing the number of TH-immunoreactive cells in the ventral mesencephalon progressively increasing in number during these developmental stages (Figs. 1–3). When comparing mesencephalic DA levels with the content of other monoamines in the brain stem during first trimester development, both NA and 5-HT were found in higher quantities than DA in the corresponding regions with monoaminergic cell bodies (53). However, the exponential increase in levels as a function of time was similar for all three monoamines. DA concentration in the caudate nucleus–putamen complex was, e.g., in the same range as that of NA in the spinal cord at the end of the first trimester. The very dense innervation of DA terminals in the adult caudate nucleus–putamen, with DA levels by far exceeding those of any other region of the CNS, obviously develops at a later stage (35, 37). Consequently, the concentration of DA in the basal ganglia at the 12th gesta-
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tional week is only about 1% of that found in the mature, adult brain (44). In the embryonic rat, detectable DA levels were found in striatum at E15–E16, 3–4 days after the appearance of TH-immunoreactive cells in mesencephalon (9, 41). From the analyses of endogenous DA content in the human embryonic midbrain, it is evident that mesencephalic neuroblasts synthesize DA within an approximately 1-week period following the onset of cytoplasmic TH expression in these cells. At this stage, dopaminergic neurons are still morphologically very immature with short primary processes reaching caudal parts of the hypothalamus. The early start of monoamine synthesis is interesting evidence of neurochemical differentiation of these cells even prior to their innervation of their target area in the basal ganglia anlage. Studies of the in vivo development of the nigrostriatal dopaminergic system in man is relevant to the field of human embryology, but also for guidance in the use of first trimester mesencephalic tissue as donor material in clinical trials and animal models for treatment of Parkinson’s disease by intracerebral implantation of dopaminergic neurons. The course of genetically programmed differentiation is different in human and rodent grafts and functional recovery of transplanted animals was reported markedly delayed when using human tissue versus rodent (55). The influences of donor gestational age, of tissue retrieval, storage, and processing prior to implantation, and of the intracerebral positioning of the grafts are still subjects of interest and controversy [reviewed by Fine (7)]. The long-term survival of grafted mesencephalic DA neurons varies in different animal models, but has been reported to be about 5–10% (16). Embryonic mesencephalic tissue (,9 weeks of gestation) is generally preferred to fetal donor material, since graft survival has been shown to be related to the ontogeny of the human nigrostriatal system (11). However, the absolute number of DA neurons contained within the embryonic mesencephalon is significantly less, as shown by the present study. Also important to consider is the early and extensive axo–dendritic differentiation of these cells during the first trimester. At gestational age exceeding 6–7 weeks, dopaminergic axonal trajectories extend into the primitive forebrain, which will result in a traumatic axotomy of DA neurons when retrieved for grafting. To approach this problem, multiple embryonic grafts have been used for each patient subjected to intracerebral implantation (58). Improved motor function, increase in fluorodopa uptake in the grafted area on PET scanning, and long-term survival and extensive dopaminergic reinnervation of grafted regions in a 59-year-old male Parkinson patient were recently reported (26). A novel strategy has emerged with the report of in
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vitro isolation of neural progenitor cells from adult and embryonic brain (46, 47). By isolating and expanding the population of mesencephalic stem cells in vitro, a possible source of relatively undifferentiated cells with the potential of developing a dopaminergic phenotype may be obtained for transplant studies. We are currently testing multipotent EGF-responsive cells in vitro in order to find conditions that may guide and support them in the differentiation process toward dopaminergic neurons.
12.
13.
14.
ACKNOWLEDGMENTS 15. This study was supported by the Swedish Medical Research Council, Grants 14X-06555 and B96-13X-11570-01A, The Miami Project Foundation, The Daniel Heumann Fund For Spinal Cord Research, The Foundations of the Karolinska Institute, The Magnus Bergvall Fund for Medical Research, The Foundations of Sven Jerring and of Tore Nilson, and the Spinalis Foundation. Expert technical assistance by Eva-Britt Samuelsson is gratefully acknowledged.
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