Ventral Mesencephalic Dopamine Neurons Research Articles

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells.

SummaryDevelopment of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types.

A quantitative evaluation of a 2.5-kb rat tyrosine hydroxylase promoter to target expression in ventral mesencephalic dopamine neurons in vivo

Adeno-associated viruses (AAVs) have become powerful tools in neuroscience for both basic research and potential therapeutic use. They have become especially important tools for optogenetic experiments based on their ability to achieve transgene expression in postmitotic neurons with regional selectivity. With the use of appropriate promoter elements they can achieve cellular specificity as well. One population of neurons that plays a central role in human neurodegenerative and psychiatric diseases are the dopamine neurons of the midbrain. To study these neurons in vivo with advanced techniques it would be highly advantageous to characterize an appropriate specific promoter. To this end we have characterized a 2.5-kb sequence of the rat tyrosine hydroxylase (TH) promoter. The rTHp(2.5) promoter induced expression of the fluorescent reporter protein mCherry in SN dopamine neurons. Although it showed excellent specificity in cortex and striatum, where no reporter expression was observed, in the SN region many neurons expressed reporter but not TH. We show that some of the TH negativity is due to the suppression of its expression by the transgene. We conclude that rTHp(2.5) does preferentially label dopamine neurons but its specificity is not complete within the substantia nigra and caution must be used.

Gender differences of oxidative stress reactions in ventral mesencephalic dopamine neurons

Objective To investigate the gender differences of oxidative stress reactions in ventral mesencephalic(VM) dopamine neurons.Methods VM dopamine neurons of male and female Balb/c fetal mice were cultured,and were divided into control group(without treatment),rotenone group(treated with 25 nmol/L rotenone for 8 h) and estrogen+rotenone group(treated with 1×10-7 mol/L 17 β-estradiol for 1 h and 25 nmol/L rotenone for 8 h).Immunohistochemistry was employed to observe the neuron injury of mice of different genders,MTT assay was adopted to measure the cell viability,and Western blotting was used to detect the expression of tyrosine hydroxylase(TH) protein in cells of mice of different genders.Results Immunohistochemistry revealed that cell injury of male mice was more severe than that of female mice,and cells of male mice with positive TH were more significant than those of female mice with positive TH in rotenone group.MTT assay indicated that there were significant differences in cell viability between male mice(66%) and female mice(75%) in rotenone group(P0.05),and cell viability of male mice(81%) and female mice(86%) in estrogen+rotenone group was significantly higher than that of mice of corresponding genders in rotenone group(P0.05).Western blotting demonstrated that the expression of TH protein in cells of male mice was significantly lower than that in cells of female mice in rotenone group(P0.05),and the expression of TH protein in estrogen+rotenone group was significantly higher than that of corresponding genders in rotenone group(P0.05).Conclusion There exist gender differences of oxidative stress reactions in VM dopamine neurons.Male is more vulnerable than female in respond to rotenone,and estrogen has protective effects on both male and female neurons.

Modulation of locomotor activation by the rostromedial tegmental nucleus.

The rostromedial tegmental nucleus (RMTg) is a strong inhibitor of dopamine neurons in the ventral tegmental area (VTA) reported to influence neurobiological and behavioral responses to reward omission, aversive and fear-eliciting stimuli, and certain drugs of abuse. Insofar as previous studies implicate ventral mesencephalic dopamine neurons as an essential component of locomotor activation, we hypothesized that the RMTg also should modulate locomotion activation. We observed that bilateral infusions into the RMTg of the gamma-aminobutyric acid A (GABAA) agonist, muscimol, indeed activate locomotion. Alternatively, bilateral RMTg infusions of the GABAA receptor antagonist, bicuculline, suppress robust activations of locomotion elicited in two distinct ways: (1) by disinhibitory stimulation of neurons in the lateral preoptic area and (2) by return of rats to an environment previously paired with amphetamine administration. The possibility that suppressive locomotor effects of RMTg bicuculline infusions were due to unintended spread of drug to the nearby VTA was falsified by a control experiment showing that bilateral infusions of bicuculline into the VTA produce activation rather than suppression of locomotion. These results objectively implicate the RMTg in the regulation of locomotor activation. The effect is important because much evidence reported in the literature suggests that locomotor activation can be an involuntary behavioral expression of expectation and/or want without which the willingness to execute adaptive behaviors is impaired.

Functional significance of aldehyde dehydrogenase ALDH1A1 to the nigrostriatal dopamine system

Aldehyde dehydrogenase 1A1 (ALDH1A1) is a member of a superfamily of detoxification enzymes found in various tissues that participate in the oxidation of both aliphatic and aromatic aldehydes. In the brain, ALDH1A1 participates in the metabolism of catecholamines including dopamine (DA) and norepinephrine, but is uniquely expressed in a subset of dopaminergic (DAergic) neurons in the ventral mesencephalon where it converts 3,4-dihydroxyphenylacetaldehyde, a potentially toxic aldehyde, to 3,4-dihydroxyphenylacetic acid, a non toxic metabolite. Therefore, loss of ALDH1A1 expression could be predicted to alter DA metabolism and potentially increase neurotoxicity in ventral mesencephalic DA neurons. Recent reports of reduced levels of expression of both Aldh1a1 mRNA and protein in the substantia nigra (SN) of Parkinson's disease patients suggest possible involvement of ALDH1A1 in this progressive neurodegenerative disease. The present study used an Aldh1a1 null mouse to assess the influence of ALDH1A1 on the function and maintenance of the DAergic system. Results indicate that the absence of Aldh1a1 did not negatively affect growth and development of SN DA neurons nor alter protein expression levels of tyrosine hydroxylase, the DA transporter or vesicular monoamine transporter 2. However, absence of Aldh1a1 significantly increased basal extracellular DA levels, decreased KCl and amphetamine stimulated DA release and decreased DA re-uptake and resulted in more tyrosine hydroxylase expressing neurons in the SN than in wildtype animals. These data suggest that in young adult animals with deletion of the Aldh1a1 gene there is altered DA metabolism and dysfunction of the DA transporter and DA release mechanisms.

Glial cell line-derived neurotrophic factor is crucial for long-term maintenance of the nigrostriatal system

Glial cell line-derived neurotrophic factor (GDNF) is a potent factor for the ventral mesencephalic dopamine neurons. However, studies on the Gdnf gene deleted ( Gdnf −/−) mouse have been limited to fetal tissue since these mice die prematurely. To evaluate long-term effects of Gdnf gene deletion, this study involves co-grafts of ventral mesencephalon (VM) and lateral ganglionic eminence (LGE) derived from different Gdnf genotypes. The VM/LGE co-grafts were evaluated at 3, 6, and 12 months for tyrosine hydroxylase (TH) -positive cell survival and nerve fiber formation in the LGE co-transplant, visualized by dopamine- and cyclic AMP-regulated phosphoprotein relative molecular mass 32,000 (DARPP-32) -immunoreactivity. Cell counts revealed no difference in TH-positive neurons between Gdnf genotypes at 3 months postgrafting. At 6 months, a significant reduction in cell number was observed in the Gdnf −/− grafts. In fact, in the majority of the Gdnf −/− VM/LGE transplant had degenerated. At 12 months, a reduction in cell number was seen in both Gdnf −/− and Gdnf +/− compared to wild type transplants. In the Gdnf −/− grafts, TH-negative inclusion-like structures were present in the cytoplasm of the TH-positive neurons at 3 months. These structures were also found in the Gdnf +/− transplants at 12 months, but not in Gdnf +/+ controls at any time point. In Gdnf +/+ grafts, TH-positive nerve fiber innervation of the striatal co-grafts was dense and patchy and overlapped with clusters of DARPP-32-positive neurons. This overlap did mismatch in the Gdnf +/− grafts, while the TH-positive innervation was sparse in the Gdnf −/− transplants and the DARPP-32-positive neurons were widespread distributed. In conclusion, GDNF is essential for long-term maintenance of both the VM TH-positive neurons and for the striatal tissue, and appears crucial for generation of a proper organization of the striatum.

Exogenous erythropoietin provides neuroprotection of grafted dopamine neurons in a rodent model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease marked by severe loss of dopamine (DA) neurons in the nigrostriatal system, which results in depletion of striatal DA. Transplantation of embryonic ventral mesencephalic (VM) DA neurons into the striatum is a currently explored experimental treatment aimed at replacing lost DA in the nigrostriatal system, but is plagued with poor survival (5–20%) of implanted neurons. Here, we tested the ability of erythropoietin (Epo) to provide neuroprotection for embryonic day 14 (E14) VM DA neurons. Epo was tested in vitro for the ability to augment tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival under normal cell culture conditions. In vitro, Epo did not increase the number of TH-ir neurons when administered at the time of plating the E14 VM cells in culture. We also tested the efficacy of Epo to enhance E14 VM transplants in vivo. Rats unilaterally lesioned with 6-hydroxydopamine received transplants that were incubated in Epo. Treatment with Epo produced significant increases in TH-ir neuron number, soma size, and staining intensity. Animals receiving Epo-treated grafts exhibited significantly accelerated functional improvements and significantly greater overall improvements from rotational asymmetry compared to control grafted rats. These data indicate that the survival of embryonic mesencephalic TH-ir neurons is increased when Epo is administered with grafted cells in a rodent model of PD. As direct neurotrophic effects of Epo were not observed in vitro, the mechanism of Epo neuroprotection remains to be elucidated.

Characterization of the Bex gene family in humans, mice, and rats

To better understand the development of ventral mesencephalic dopamine neurons, we performed subtractive hybridization screens to find ventral mesencephalic genes expressed at rat embryonic day 10 when these neurons begin to differentiate. The most commonly identified genes in these screens were members of the Bex (Brain expressed X-linked) gene family, rat Bex1 ( Rex3), and a novel gene, rat Bex4. After identifying these genes, we then sought to characterize the Bex gene family. Two additional novel Bex genes (human Bex5 and mouse Bex6) were discovered through genomic databases. Bex5 is present in humans and monkeys, but not rodents, while Bex6 exists in mice, but not humans. Bex4 and Bex5 are localized to the X chromosome, are expressed in brain, and are similar in sequence. Bex4 and Bex5 are 54% and 56% identical to human Bex3 ( pHGR74, NADE). Mouse Bex6 is on chromosome 16 and is 67% identical to mouse Bex4. Human Bex gene expression was studied with tissue expression arrays probed with specific oligonucleotides. Human Bex1 and Bex2 have similar expression patterns in the central nervous system with high levels in pituitary, cerebellum, and temporal lobe, and Bex1 is widely expressed outside of the central nervous system with high expression in the liver. Human Bex4 is highly expressed in heart, skeletal muscle, and liver, while Bex3 and Bex5 are more widely expressed. The subcellular localization of the Bex proteins varies from nuclear (rat Bex1) to cytoplasmic (rat Bex3, human Bex5, and mouse Bex6) and to both nuclear and cytoplasmic (rat Bex2 and rat Bex4). Rat Bex3, rat Bex4, human Bex5, and mouse Bex6 are degraded by the proteasome, while rat Bex1 or Bex2 are not. Rat Bex3 protein can likely bind transition metals through a histidine-rich domain. Because this gene family was originally named Bex and because these genes are unified by sequence similarity and gene structure, we believe the Bex nomenclature should prevail over nomenclature based on function (NADE) that has not been extended to the other Bex genes. We conclude that the Bex gene family members are highly homologous but differ in their expression patterns, subcellular localization, and degradation by the proteasome.

Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection

Molecular differences between dopamine (DA) neurons may explain why the mesostriatal DA neurons in the A9 region preferentially degenerate in Parkinson's disease (PD) and toxic models, whereas the adjacent A10 region mesolimbic and mesocortical DA neurons are relatively spared. To characterize innate physiological differences between A9 and A10 DA neurons, we determined gene expression profiles in these neurons in the adult mouse by laser capture microdissection, microarray analysis and real-time PCR. We found 42 genes relatively elevated in A9 DA neurons, whereas 61 genes were elevated in A10 DA neurons [> 2-fold; false discovery rate (FDR) < 1%]. Genes of interest for further functional analysis were selected by criteria of (i) fold differences in gene expression, (ii) real-time PCR validation and (iii) potential roles in neurotoxic or protective biochemical pathways. Three A9-elevated molecules [G-protein coupled inwardly rectifying K channel 2 (GIRK2), adenine nucleotide translocator 2 (ANT-2) and the growth factor IGF-1] and three A10-elevated peptides (GRP, CGRP and PACAP) were further examined in both alpha-synuclein overexpressing PC12 (PC12-alphaSyn) cells and rat primary ventral mesencephalic (VM) cultures exposed to MPP+ neurotoxicity. GIRK2-positive DA neurons were more vulnerable to MPP+ toxicity and overexpression of GIRK2 increased the vulnerability of PC12-alphaSyn cells to the toxin. Blocking of ANT decreased vulnerability to MPP+ in both cell culture systems. Exposing cells to IGF-1, GRP and PACAP decreased vulnerability of both cell types to MPP+, whereas CGRP protected PC12-alphaSyn cells but not primary VM DA neurons. These results indicate that certain differentially expressed molecules in A9 and A10 DA neurons may play key roles in their relative vulnerability to toxins and PD.

A Murine Dopamine Neuron-Specific cDNA Library and Microarray: Increased COXI Expression during Methamphetamine Neurotoxicity

Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter–lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity.

Evidence for target-specific outgrowth from subpopulations of grafted human dopamine neurons.

Clinical and experimental grafting in Parkinson's disease has shown the need for enhanced survival of dopamine neurons to obtain improved functional recovery. In addition, it has been suggested that a limited number of surviving dopamine neurons project to the dopamine-denervated host striatum. The aim of this study was to investigate if subpopulations of ventral mesencephalic dopamine neurons project to their normal targets, i.e., dorsal vs. ventral striatum. Following implantation of human ventral mesencepahlic tissue into the lateral ventricle of dopamine-depleted rats, human-derived dopamine reinnervation was achieved both in dorsal and ventral striatum. Treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in a degeneration of tyrosine hydroxylase (TH)-immunoreactive nerve fibers in dorsal striatum but not in ventral areas in some animals, while MPTP was without effect in other animals. TH-immunoreactive neurons were small and appeared shrunken in animals carrying grafts affected by the MPTP treatment. In conclusion, grafted dopamine neurons projected nerve fibers into areas that they normally innervate. Thus, when searching for factors that may enhance survival of grafted dopamine neurons it is important to study which subpopulation(s) of ventral mesencephalic dopamine neurons is affected, such that a proper reinnervation may be achieved.

Glial Cell Line-Derived Neurotrophic Factor Is Essential for Postnatal Survival of Midbrain Dopamine Neurons

Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent trophic factors that have been identified for midbrain dopamine (DA) neurons. Null mutations for trophic factor genes have been used frequently for studies of the role of these important proteins in brain development. One problem with these studies has been that often only prenatal development can be studied because many of the knockout strains, such as those with GDNF null mutations, will die shortly after birth. In this study, we looked at the continued fate of specific neuronal phenotypes from trophic factor knockout mice beyond the time that these animals die. By transplanting fetal neural tissues from GDNF -/-, GDNF +/-, and wild-type (WT) mice into the brain of adult wild-type mice, we demonstrate that the continued postnatal development of ventral midbrain dopamine neurons is severely disturbed as a result of the GDNF null mutation. Ventral midbrain grafts from -/- fetuses have markedly reduced DA neuron numbers and fiber outgrowth. Moreover, DA neurons in such transplants can be "rescued" by immersion in GDNF before grafting. These findings suggest that postnatal survival and/or phenotypic expression of ventral mesencephalic DA neurons is dependent on GDNF. In addition, we present here a strategy for studies of maturation and even aging of tissues from trophic factor and other knockout animals that do not survive past birth.

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study.

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms.

Intrastriatal Grafts From Multiple Donors Do Not Result in a Proportional Increase in Survival of Dopamine Neurons in Nonhuman Primates

We examined the potential for “double grafts,” i.e., grafts from two donors in each recipient, to enhance the total number of ventral mesencephalic dopamine neurons that survive grafting in adult African green monkeys. Because dopamine cell survival in grafts represents a small percentage of the total number of neurons grafted, several human clinical trials recently have employed grafts of tissue from multiple donors (e.g., from two to eight embryos per host recipient) in attempts to increase the total number of dopamine neurons that survive in grafts. Presumably, this is intended to elevate dopamine levels by providing more dopamine neurons to the damaged brain to alleviate the symptoms of parkinsonism. While well-developed grafts with several thousand dopamine neurons were found in most recipient animals, we observed a reduced total number of tyrosine hydroxylase positive neurons in the grafts in spite of the presence of some double grafts that were larger than normal. The overall growth of the grafts was impressive; some grafts were so large that they spanned the full dorsoventral extent of the caudate nucleus, probably reflecting the fact that twice as much tissue was implanted in each drop site in comparison to our standard protocol. However, some animals revealed atypical patterns of neurite outgrowth that appeared limited to the grafted tissue, and at least one monkey revealed “amorphous” grafts generally lacking in cellular structure, which suggests a possible rejection phenomenon. These findings raise questions about the use of multiple donors and suggest that the likelihood of rejection and/or cell death may be enhanced, which is of potential importance in the designof grafting strategies for clinical applications.

Intrastriatal grafts from multiple donors do not result in a proportional increase in survival of dopamine neurons in nonhuman primates.

We examined the potential for "double grafts," i.e., grafts from two donors in each recipient, to enhance the total number of ventral mesencephalic dopamine neurons that survive grafting in adult African green monkeys. Because dopamine cell survival in grafts represents a small percentage of the total number of neurons grafted, several human clinical trials recently have employed grafts of tissue from multiple donors (e.g., from two to eight embryos per host recipient) in attempts to increase the total number of dopamine neurons that survive in grafts. Presumably, this is intended to elevate dopamine levels by providing more dopamine neurons to the damaged brain to alleviate the symptoms of parkinsonism. While well-developed grafts with several thousand dopamine neurons were found in most recipient animals, we observed a reduced total number of tyrosine hydroxylase positive neurons in the grafts in spite of the presence of some double grafts that were larger than normal. The overall growth of the grafts was impressive; some grafts were so large that they spanned the full dorsoventral extent of the caudate nucleus, probably reflecting the fact that twice as much tissue was implanted in each drop site in comparison to our standard protocol. However, some animals revealed atypical patterns of neurite outgrowth that appeared limited to the grafted tissue, and at least one monkey revealed "amorphous" grafts generally lacking in cellular structure, which suggests a possible rejection phenomenon. These findings raise questions about the use of multiple donors and suggest that the likelihood of rejection and/or cell death may be enhanced, which is of potential importance in the design of grafting strategies for clinical applications.

A microtransplantation approach for cell suspension grafting in the rat parkinson model: A detailed account of the methodology

Shortcomings of current techniques used for the intracerebral transplantation of ventral mesencephalic dopamine neurons include low graft survival, high variability, considerable implantation trauma and suboptimal graft integration. In order to overcome these limitations, we have adopted a microtransplantation approach which allows precise and reproducible implantation of ventral mesen-cephalon cell suspensions at single or multiple sites with minimal trauma and improved survival and integration of the grafted neurons [ Nikkhah et al. (1994) Brain Res. 633, 133–143]. The present study was undertaken to determine the influence of different grafting parameters as well as the time-course of development of micrografted dopaminergic neurons and to devise an optimal microtransplantation procedure in the rat Parkinson model. Rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway received four graft deposits of either 0.25, 0.5, 1.0 or 2.0 μl along four injection tracts (150,000 cells/μl) using either a glass capillary (o.d. 50–70 μm) or a regular cannula (o.d. 0.50 mm, metal cannula grafts). At one, two and 12 weeks postgrafting (capillary grafts) and at 12 weeks postgrafting (metal cannula grafts) dopamine neuron survival and graft volumes were measured and the implantation trauma assessed by glial fibrillary acidic protein expression. The results demonstrate that single deposits of 50,000–75,000 cells in 0.5 μl, implanted with a glass capillary, provide the best environment both for dopaminergic and non-dopaminergic neuron survival. Grafts implanted with the glass capillary showed much weaker long-term glial fibrillary acidic protein expression along the injection tract and around the implants than was the case in grafts implanted with the thicker metal cannula. Optimal graft integration and minimal disturbances of host brain structures can reliably be achieved by small-sized implants (20,000–35,000 cells/deposit). Tyrosine hydroxylase-positive fiber outgrowth from micrografted dopaminergic neurons was seen not only in the surrounding caudate-putamen, but also along white matter tracts into the nucleus accumbens and the overlying cerebral cortex. Spreading of dopaminergic micrografts over multiple small deposits rather than increasing the volume of single grafts gave more extensive reinnervation of the entire host striatum. The micrografting technique provides a useful tool to improve graft-host interactions in the rat Parkinson model, and it allows more precise and reproducible quantitative studies on dopamine neuron survival and growth in intrastriatal ventral mesencephalon transplants. This technique should also be highly useful for the intracerebral implantation of cells derived from primary cultures or cell lines [ Gage and Fisher (1991) Neuron 6, 1–12].

Effect of Prior Dopamine Denervation on Survival and Fiber Outgrowth from Intrastriatal Fetal Mesencephalic Grafts.

[3H]Dopamine (DA) uptake radioautography and tyrosine hydroxylase (TH) immunocytochemistry were used to assess quantitatively the effects of the presence or absence of host mesostriatal DA afferents on the survival and fiber outgrowth from fetal ventral mesencephalic DA neurons grafted into the neostriatum of adult recipient rats. Rats received bilateral intrastriatal transplants of fetal ventral mesencephalic tissue 1 month after a unilateral injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle (denervated side). Five to six months later, some of the grafted rats received a second 6-OHDA injection in the left nigrostriatal bundle (acutely denervated or 'intact' side). After a further 7 days, slices of each hemisphere from the latter rats were incubated with [3H]DA and processed for film and high resolution radioautography. The density of the film radioautographs was measured with a computerized image analysis system and calibrated by silver grain cluster (i.e. DA terminal) counting over selected areas of the same sections in light microscope radioautographs. The brains of the remaining grafted rats were processed for TH immunoreactivity 6 - 12 months after graft surgery. Neither the size of the grafts, nor the number of surviving TH-positive graft neurons showed any significant difference between the nondenervated and the denervated sides. However, the size of the TH-positive cell bodies was significantly greater in the grafts on the denervated side. In the [3H]DA uptake radioautographs, considerable outgrowth of DA fibers was evident in the neostriatum on the 'intact' side in spite of the presence of an intact host DA innervation until 7 days before sacrifice. The overall DA fiber outgrowth was nevertheless almost two-fold greater on the denervated side, and extended deeper into the host neostriatum than on the 'intact' side; only 7% of the total neostriatal area, on average, was at background level compared to 30% on the 'intact' side, and the overall density of neostriatal DA innervation amounted to 36% of normal as compared to 20% on the 'intact' side. The correlation between the overall density of graft-derived DA innervation and the size of the grafts was linear on the 'intact' side, but reached a plateau with relatively small grafts on the denervated side. However, the ventral striatum on both sides was very poorly innervated by these grafts. These findings demonstrate that the mature neostriatal tissue can support axonal growth and innervation from grafted fetal DA neurons even in the presence of a normal complement of endogenous DA fibers. Prior removal of the host striatal DA innervation does not influence the overall size of the grafts nor the number of surviving DA neurons, but induces an increase in the cell body size and fiber outgrowth of the grafted DA neurons.