Tyrosine Hydroxylase Cells Research Articles

Glial cell line-derived neurotrophic factor (GDNF) is required for differentiation of pontine noradrenergic neurons and patterning of central respiratory output

Genetic mutations affecting signaling by glial cell line-derived neurotrophic factor (GDNF) perturb development of breathing in mice and are associated with congenital central hypoventilation syndrome in humans. However, the role of GDNF in development of brainstem neurons that control breathing is largely unknown. The present study demonstrates that genetic loss of GDNF decreases the number of tyrosine hydroxylase (TH) neurons in the pontine A5 noradrenergic cell group, a major source of inhibitory input to the medullary respiratory pattern generator. This phenotype is associated with a significant increase in the frequency of central respiratory output recorded from the fetal medulla–spinal cord in vitro. In dissociate cultures of the A5 region from rat embryos, GDNF increases TH cell number and neurite growth without affecting total neuronal survival or proliferation of TH neurons. These effects of GDNF are inhibited by function blocking antibodies against endogenous brain-derived neurotrophic factor (BDNF), indicating that GDNF requires BDNF as a cofactor to stimulate differentiation of A5 neurons. Our findings demonstrate that GDNF is required for development of pontine noradrenergic neurons in vivo and indicate that defects in the A5 cell group may contribute to the effects of genetic disruption of GDNF signaling on respiratory control.

Neurotrophic effects of bone morphogenetic protein-7 in a rat model of Parkinson's disease

Previous studies have demonstrated that pretreatment with bone morphogenetic protein-7 (BMP7) reduces ischemic neuronal injury in vivo. Moreover, exogenous application of BMP7 increases both the number of tyrosine hydroxylase (+) cells and dopamine (DA) uptake in rat mesencephalic cell cultures. The purpose of this study was to investigate the in vivo effects of BMP7 on 6-hydroxydopamine (6-OHDA) induced lesioning of midbrain DA neurons. Adult Fischer 344 rats were anesthetized and injected with BMP7 or vehicle into the left substantia nigra, followed by local administration of 9 μg of 6-OHDA into the left medial forebrain bundle. The lesioned animals that received BMP7 pretreatment, as compared to vehicle/6-OHDA controls, had a significant reduction in methamphetamine-induced rotation 1 month after the surgery. BMP7-pretreatment partially preserved KCl-induced dopamine release in the lesioned striatum and significantly increased TH immunoreactivity in the lesioned nigra and striatum. In summary, our data suggest that BMP7 has neuroprotective and/or neuroreparative effects against 6-OHDA lesioning of the nigrostriatal DA pathway in an animal model of Parkinson's disease (PD).

Combined Inhibition of Apoptosis and Complement Improves Neural Graft Survival of Embryonic Rat and Porcine Mesencephalon in the Rat Brain

To define potential mechanisms of cell death during neural cell transplantation, we investigated the role of intracellular caspase activation in combination with the activation of serum complement. We demonstrated that ventral mesencephalic (VM) cells are susceptible to complement-mediated cell lysis that can be blocked with an anti-C5 complement inhibitor (18A10). We also determined that incubating freshly isolated allogenic VM cells with the caspase inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF), followed by immediate striatal implantation, led to a 2.5-fold increase in tyrosine hydroxylase (TH) cell survival 12 weeks postimplantation ( P < 0.05). In contrast, overnight incubation with BAF followed by striatal implantation led to a 2-fold reduction in TH cell survival at 12 weeks ( P < 0.05). Using the optimal BAF treatment and complement inhibition, we tested the hypothesis that these treatments would lead to increased cell survival in both allogeneic and xenogeneic transplantation models. We transplanted cell suspensions of (a) rat E14 VM or VM treated with (b) BAF alone, (c) anti-C5, or (d) a combination of BAF and anti-C5. There was a significant increase in the relative number of TH-positive cells in the BAF/anti-C5 group versus control at 12 weeks posttransplantation. Similar results were achieved in a pig to rat xenotransplant paradigm. A neuronal xenograft marker (70-kDa neurofilament) also demonstrated relative increases in graft volume in the BAF/anti-C5 treatment group. These studies indicate that more than one mechanism can mediate cell death during neural cell transplantation and that a combined treatment using caspase and complement inhibition can significantly improve cell survival.

The Projections of Early Enteric Neurons Are Influenced by the Direction of Neural Crest Cell Migration

The enteric nervous system arises from the neural crest. In embryonic mice, vagal neural crest cells enter the developing foregut at approximately embryonic day 9.5 (E9.5) and then migrate rostrocaudally to colonize the entire gastrointestinal tract by E14.5. This study showed that a subpopulation of vagal crest-derived cells, very close to the migratory wavefront, starts to differentiate into neurons early, as shown by the expression of neuron-specific proteins and the absence of Sox10. Many of the early differentiating neurons transiently exhibited tyrosine hydroxylase (TH) immunoreactivity. The TH cells were demonstrated to be the progenitors of nitric oxide synthase (NOS) neurons. Immunohistochemistry, lesions, and DiI tracing were used to examine the projections of developing enteric neurons. The axons of first neurons in the gut (the TH–NOS neurons) projected in the same direction (caudally), and traversed the same pathways through the mesenchyme, as the migrating, undifferentiated, vagal crest-derived cells. To examine if the direction of migration and direction of axon projection are linked, coculture experiments were set up in which vagal crest-derived cells migrated either rostrocaudally (as they doin vivo), or caudorostrally (which they do not normally do), to colonize explants of embryonic aneural hindgut. The direction in which neurons projected was correlated with the direction of cell migration, but migration direction appears to be not the only mechanism influencing axon projection. Peristaltic reflexes involve both orally (rostrally) projecting neurons and anally (caudally) projecting neurons. Because few rostrally projecting neurons could be detected before birth, the full circuitry for peristaltic reflexes appears to develop after birth.

The projections of early enteric neurons are influenced by the direction of neural crest cell migration.

The enteric nervous system arises from the neural crest. In embryonic mice, vagal neural crest cells enter the developing foregut at approximately embryonic day 9.5 (E9.5) and then migrate rostrocaudally to colonize the entire gastrointestinal tract by E14.5. This study showed that a subpopulation of vagal crest-derived cells, very close to the migratory wavefront, starts to differentiate into neurons early, as shown by the expression of neuron-specific proteins and the absence of Sox10. Many of the early differentiating neurons transiently exhibited tyrosine hydroxylase (TH) immunoreactivity. The TH cells were demonstrated to be the progenitors of nitric oxide synthase (NOS) neurons. Immunohistochemistry, lesions, and DiI tracing were used to examine the projections of developing enteric neurons. The axons of first neurons in the gut (the TH-NOS neurons) projected in the same direction (caudally), and traversed the same pathways through the mesenchyme, as the migrating, undifferentiated, vagal crest-derived cells. To examine if the direction of migration and direction of axon projection are linked, coculture experiments were set up in which vagal crest-derived cells migrated either rostrocaudally (as they do in vivo), or caudorostrally (which they do not normally do), to colonize explants of embryonic aneural hindgut. The direction in which neurons projected was correlated with the direction of cell migration, but migration direction appears to be not the only mechanism influencing axon projection. Peristaltic reflexes involve both orally (rostrally) projecting neurons and anally (caudally) projecting neurons. Because few rostrally projecting neurons could be detected before birth, the full circuitry for peristaltic reflexes appears to develop after birth.

Androgen Effects on Tyrosine Hydroxylase Cells in the Northern Leopard Frog, Rana pipiens

The interaction between gonadal steroids and dopamine neurons has been examined extensively in rodent model systems. However, there have been few investigations examining the functional relation between gonadal steroids and dopaminergic systems in nonmammalian taxa, and none in amphibians. We examined the effects of testosterone (T) and dihydrotestosterone (DHT) on changes in tyrosine hydroxylase immunoreactive (TH-ir) neuron number in the fore- and midbrain of male Rana pipiens, the Northern leopard frog, using a whole-mount immunohistochemical procedure. Gonadectomized males had significantly fewer TH-ir neurons in the medial preoptic area (POA), suprachiasmatic nucleus (SCN), and the caudal hypothalamus/posterior tubercular region (HY/TP) compared with T-implanted males. A follow-up study demonstrated that T- and DHT-implanted males had similar numbers of TH-ir neurons in these three regions compared with intact males and that all three of these groups possessed significantly more TH-ir neurons compared with gonadectomized males. These results suggest that circulating sex steroids have a significant impact on the activity of dopaminergic neurons in male R. pipiens. Although the function of these specific dopaminergic neurons is not yet known, the POA, SCN, and TP/DH are known to be integral brain regions underlying the neural control of reproductive behavior in frogs. These results suggest that dopamine may be important in controlling the behavior or neuroendocrine mechanisms of reproduction in these animals and that dopaminergic activity in these areas is regulated by gonadal steroids.

Immunohistochemical Localization of nNOS in the Head Kidney of Larval and Juvenile Rainbow Trout, Oncorhynchus mykiss

The aim of this investigation was to assess whether in teleosts, as in mammals, nitric oxide (NO) is involved in the regulation of cellular activity in the adrenal homolog. Larval and juvenile stages of the rainbow trout, Oncorhynchus mykiss, were used, in which the adrenal homolog consists of chromaffin adrenergic and interrenal steroidogenic cells localized mainly in the head kidney where there are also ganglion cells and nerve fibres that innervate the gland. In 12-month-old juveniles, the immunohistochemical reaction for neuronal nitric oxide synthase (nNOS), which catalyzes the synthesis of NO, revealed the presence of this enzyme in some nerve fibres and ganglion cells and only rarely in chromaffin cells. The latter are identified by the immunohistochemical reaction for tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT). In larvae at 27 days postfertilization, numerous cells dispersed in the head kidney are nNOS positive, whereas the TH and PNMT positive cells are very rare. At hatching (31 days postfertilization), the positivity for nNOS in the cells of the head kidney disappears and reappears at 60 days posthatching in some nerve cells and fibres. These results suggest an involvement of NO in the regulation of adrenal function as in mammals and the nature of nNOS positive cells present in the head kidney of larvae of 27 days is discussed.

Comparative study of glutamate mediated γ-aminobutyric acid release from nitric oxide synthase and tyrosine hydroxylase immunoreactive cells of the Cebus apella retina

The effects of excitatory amino acids (EAAs) upon transporter-mediated γ-aminobutyric acid (GABA) release were investigated in cells containing tyrosine hydroxylase (TH) or nitric oxide synthase (NOS) in retina of the primate Cebus apella. Retinas were treated in vitro with 50 μM Kainate (KA) or 5 mM l-Glutamate ( l-Glu), for 30 min at 37°C, in an Mg 2+-free Locke's solution with or without Ca 2+. The effects of EAAs were measured immunocytochemically by determining the GABA content in TH or NOS-immunoreactive cells in the inner retina, after stimulation. l-Glu and KA induced a Ca 2+-independent GABA release from most GABA-immunoreactive cells of the inner retina. Double label experiments indicated that this release occurs in NOS +/GABA + cells, but not in TH +/GABA + cells suggesting that these cell subpopulations may be differentiated in some functional aspects.

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

Astroglial and Microglial Reaction After A Partial Nigrostriatal Degeneration Induced by The Striatal Injection of Different Doses of 6-Hydroxydopamine

Astroglial and microglial activation was analyzed in adult male Wistar rats after a unilateral striatal injection of different doses (8, 4 and 1 μg) of 6-hydroxydopamine (6-OHDA). Control animals received the injection of the same volume of the solvent. The rotational behavior was registered by a rotometer 24 and 72 hours, 7, 10, 14 and 22 days after lesion. Following, animals were sacrificed and the tyrosine hydroxylase (TH) positive dopamine cells, the glial fibrillary acidic protein (GFAP) immunolabeled astrocytes and the OX42 immunoreactive microglia were visualized by mean of immunohistochemistry and quantified by stereologic method employing the optical disector and the point intercepts. The apomorphine (0.5 mg/kg)-induced circling behavior was seen only after 8μg of 6-OHDA from 72 hours postlesion until sacrifice. Decreases of the TH immunoreactive terminals and cell bodies were found in the sampled fields of the striatum and pars compacta of the substantia nigra (SNc), respectively, after 8 and 4 ug of 6-OHDA. The GFAP immunohistochemistry revealed increases in the number/density of astroglial cells in the ipsilateral neostriatum (137% of control) and ipsilateral SNc (83% of control) and also in the volumeal fraction of the astroglial processes in the ipsilateral neostriatum (30% of control) and ipsilateral SNc (38% of control) in the rats with higher dose of the neurotoxin. Increases in the number of OX42 microglial labeled profiles and in the volumeal fraction of microglial processes were found in the ipsilateral neostriatum (67% and 27%, respectively, of control) and ipsilateral SNc (100% and 50%, respectively, of control) in the 8 ug 6-OHDA injected rats. These results suggest that the retrograde degeneration induced by a intrastriatal injection of a small dose of the 6-OHDA leads to an astroglial and microglial reaction in the nigrostrialal dopamine pathway. The interaction between activated glial cells may be involved in the wounding and repair events in the partial lesioned nigrostriatal system as well as in the paracrine responses to surviving dopamine neurons.

The Nigrostriatal Dopaminergic System as a Preferential Target of Repeated Exposures to Combined Paraquat and Maneb: Implications for Parkinson's Disease

Experimental evidence supporting 1,1'-dimethyl-4,4'-bipyridinium [paraquat (PQ)] as a risk factor for Parkinson's disease (PD) is equivocal. Other agricultural chemicals, including dithiocarbamate fungicides such as manganese ethylenebisdithiocarbamate [maneb (MB)], are widely used in the same geographical regions as paraquat and also impact dopamine systems, suggesting that mixtures may be more relevant etiological models. This study therefore proposed that combined PQ and MB exposures would produce greater effects on dopamine (DA) systems than would either compound administered alone. Male C57BL/6 mice were treated twice a week for 6 weeks with intraperitoneal saline, 10 mg/kg paraquat, 30 mg/kg maneb, or their combination (PQ + MB). MB, but not PQ, reduced motor activity immediately after treatment, and this effect was potentiated by combined PQ + MB treatment. As treatments progressed, only the combined PQ + MB group evidenced a failure of motor activity levels to recover within 24 hr. Striatal DA and dihydroxyphenylacetic acid increased 1-3 d and decreased 7 d after injections. Only PQ + MB reduced tyrosine hydroxylase (TH) and DA transporter immunoreactivity and did so in dorsal striatum but not nucleus accumbens. Correspondingly, striatal TH protein levels were decreased only by combined PQ + MB 5 d after injection. Reactive gliosis occurred only in response to combined PQ + MB in dorsal-medial but not ventral striatum. TH immunoreactivity and cell counts were reduced only by PQ + MB and in the substantia nigra but not ventral tegmental area. These synergistic effects of combined PQ + MB, preferentially expressed in the nigrostriatal DA system, suggest that such mixtures could play a role in the etiology of PD.

Involvement of the ventrolateral medulla in parkinsonism with autonomic failure.

To determine whether patients with PD and autonomic failure (AF), manifested primarily with orthostatic hypotension (OH), have a consistent loss of tyrosine hydroxylase (TH) neurons in the rostral ventrolateral medulla (RVLM), similar to that occurring in patients with multiple system atrophy (MSA) and AF, and to determine whether there is loss of nicotinamide, adenine dinucleotide phosphate (NADPH) diaphorase (NADPH-d) RVLM neurons in both groups of patients. The numbers of TH and NADPH-d neurons in the RVLM was assessed in brain sections obtained at autopsy from five patients with suspected PD and OH, six patients with MSA, two patients with corticobasal ganglionic degeneration and no AF, and 10 control subjects with no history of neurologic disease. Cell numbers were compared among groups and correlated with their final neuropathologic diagnosis. The number of TH neurons in the RVLM of patients with PD and OH were not significantly different from control subjects, and there were marked individual variations. The TH cell numbers in the RVLM were significantly higher (p < 0.06) in patients with PD than in patients with MSA, despite a similar degree of severity of OH. As a group, patients with PD and OH had reduced numbers of NADPH-d cells in the RVLM compared with control subjects, but again there were marked individual variations. NADPH-d cell numbers were reduced consistently and more markedly in patients with MSA. Unlike the case in patients with MSA, the number of TH neurons in the RVLM is highly variable in patients with PD and is unlikely to contribute significantly to the pathophysiology of OH. As a group, patients with PD have reduced numbers of NADPH-d neurons in the RVLM, but some patients had cell counts similar to control subjects. On the other hand, NADPH-d cell depletion in the RVLM is a consistent finding in MSA and may contribute to cardiorespiratory dysfunction in this disorder.

Xenografting human T2 sympathetic ganglion from hyperhidrotic patients provides short-term restoration of catecholaminergic functions in hemiparkinsonian athymic rats.

Previous studies have suggested that allografting peripheral sympathetic ganglia, such as superior cervical ganglia, partially relieves clinical or behavioral deficits in parkinsonian patients and animals. However, removal of these ganglia can cause Homer's syndrome, which limits the utilization of this approach. Hyperhidrosis, a disease of excessive sweating, is commonly seen in young Orientals. Treatment of hyperhidrosis often involves surgical removal of the second thoracic sympathetic ganglia (T2G), which contain catecholaminergic neurons. The purpose of our study was to investigate behavioral responses and tyrosine hydroxylase (TH) immunoreactivity in hemiparkinsonian rats at different time points after transplantation of human T2G from hyperhidrotic patients. Athymic Fisher 344 rats were injected unilaterally with 6-hydroxydopamine into the medial forebrain bundle to destroy the nigrostriatal dopaminergic (DA) pathway. The effectiveness of lesions was tested by measuring methamphetamine (MA)-induced rotations. These unilaterally lesioned rats were later transplanted with T2G or T2 fiber tract (T2F) obtained from adult hyperhidrotic patients. Animals grafted with T2G showed a reduction in MA-induced rotation by 2 weeks; however, rotation returned to the pregrafting levels by 3 months. Animals receiving T2F grafts did not show any reduction of rotation over a 3-month period. Animals were later sacrificed for TH immunostaining at different time points. Tyrosine hydroxylase-positive [TH(+)] cell bodies and fibers were found in the lesioned striatum 2-4 weeks after T2G grafting, suggesting the survival of transplants. Two to 3 months after grafting, TH(+) fibers were still found in almost all the recipients. However, TH(+) cell bodies were found in only three of seven rats studied. Animals receiving T2F grafting did not show any TH immunoreactivity in the lesioned striatum over the 3-month period. These data indicate that T2G transplants from adult hyperhidrotic patients can survive and provide transient normalization of the motor behavior in the hemiparkinsonian athymic rats. Because of the short-term improvement in behavior after grafting, the use of T2G in human trials should be cautious at the present time. Further laboratory research is required.

Ovarian steroids differentially regulate the expression of PRL-R in neuroendocrine dopaminergic neuron populations: a double label confocal microscopic study

The aims of this study were (1) to identify the possible hypothalamic targets for a short prolactin (PRL) feedback in the adult female rat by identifying DAergic neuron populations expressing PRL receptor (PRL-R); (2) to describe the effect of ovarian steroids on the expression of PRL-R and (3) to compare the distribution of both the extracellular (EC) and ligand binding (LB) domains of the PRL-R on the hypothalamic dopaminergic neurons by applying double label immunocytochemistry for the different domains of PRL-R and for tyrosine hydroxylase (TH). Five- to six-month-old female rats were ovariectomized (OVX) and implanted with either 17β-estradiol (E 2), progesterone (P 4) or received an E 2 and a P 4 implant (E 2+P 4) at the same time. In the periventricular nucleus and in the dorsomedial portion of the middle arcuate nucleus, a dramatic increase in PRL-R EC immunoreactivity was observed in E 2 implanted rats. This increase was attenuated in E 2+P 4 rats, but P 4 treatment alone had no effect. Changes in PRL-R EC expression were paralleled by changes in serum PRL levels. Interestingly, PRL-R EC expression in the rostral arcuate nucleus decreased in P 4 implanted rats, however, P 4 did not attenuate the E 2-induced increase in PRL-R EC density. PRL-R EC immunostaining was observed on the membrane, in the cytoplasm and in the nucleus. PRL-R LB immunoreactivity was also detectable in the TH positive neurons, but no nuclear staining was observed with this antibody. However, we found a strong PRL-R LB immunostaining in the ependymal lining of the 3rd ventricle and in the processes of tanycytes projecting to the median eminence. These data indicate that (1) all neuroendocrine DAergic cells can be targets for PRL, (2) expression of PRL-R is differentially affected by ovarian steroids in the different TH cell populations, (3) PRL-R LB domain may be involved in trafficking PRL in the median eminence.

Inhibition of immune cell proliferation with haloperidol and relationship of tyrosine hydroxylase expression to immune cell growth

Previous studies indicated that exogenous dopamine and its agonists directly regulated mitogen-induced immune cell proliferation. In this study, we further investigated role of endogenous dopamine in immune cell growth. Haloperidol, a general antagonist for dopamine receptors, could reduce the cell growth rate of T cell hybridoma (101) and rat nervous pheochromocytoma cells (PC12). Tyrosine hydroxylase (TH) catalyzes the initial rate-limiting step of catecholamine biosynthesis in the nervous system. Flow cytometric analysis indicated the expression of TH in various immune cells. The presence of TH in PC12 cells was used as a control. Temporal studies indicated that the expression of TH increased during 101 cell growth. Both α-methyl-p-tyrosine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine reduced TH expression and cell growth in a dose-dependent manner. These results suggest that immune T cells express TH which is correlated to cell growth, and that dopamine released from these cells may bind to the receptors to act in an autocrine or paracrine way.

Distribution of glucocorticoid receptors in the brain of rainbow trout (Oncorhynchus mykiss): possible relationships with the neuroendocrine systems controlling reproduction

The distribution of the glucocorticoid receptor (GR)within the forebrain of rainbow trout has been studied by means of in situ hybridization (ISH) and immunohistochemistry (ICC) using probes and antibodies directed against the AB-domain of the rainbow trout GR (rtGR). Both rtGR mRNA and protein exhibited the same pattern of distribution. A strong labelling was observed in all subdivisions of the preoptic nucleus, in the thalamic region and in the mediobasal hypothalamus (nucleus recessus lateralis, nucleus lateralis tuberis).It was notably found that all magnocellular neurons of the nucleus preopticus pars magnocellularis (NPOmc) were positive with both ISH and ICC. Furthermore, numerous cells of the anterior lobe of the pituitary were positively stained. Double staining studies allowed to identify part of the rtGR expressing cells. At the telencephalic level, it was found that GnRH neurons frequently express rtGR in vitellogenic females. Virtually all tyrosine hydroxylase (TH) cells in the anteroventral part of the preoptic nucleus (NPOav) were rtGR-positive. In this same area, a colocalization between rainbow trout estradiol receptor (rtER) and TH has already been documented, indirectly demonstrating that rtER and rtGR are colocalized. The same holds true for the GTH-II cells of the anterior pituitary which express both types of receptors. These results provide information on the potential sites of cortisol action on the reproductive axis directly at the pituitary level or indirectly on neuroendocrine Systems controllingGTH-II secretion, possibly via GR-ER interactions.

GTP-cyclohydrolase-I like immunoreactivity in rat brain

GTPCH-I immunoreactive structures in the rat brain were studied using a polyclonal antibody raised in the chick. General mapping was made using the avidin–biotin–peroxidase technique and compared with the distribution of tyrosine hydroxylase and serotonin immunoreactivities. Double immunofluorescence was performed in order to establish real intracellular colocalization. GTPCH-I immunoreactivity was generally found to be low. Immunostained neurons were present in all the serotonin cell groups. In catecholaminergic neurons, although tyrosine hydroxylase immunoreactivity was always very high, GTPCH-I immunoreactivity was extremely variable, from relatively strong (substantia nigra, ventral tegmental area) to low (locus coeruleus, caudal part of the hypothalamus), extremely low (rostral hypothalamus, ventral brainstem) or almost absent (dorsal brainstem, some hypothalamic nuclei). When feasible, double immunolabeling revealed that all the serotonin cells and most of the tyrosine hydroxylase cells were also expressing GTPCH-I. Our results argue in favor of a regulation of tyrosine hydroxylase activity by the intracellular synthesis of BH4.

Differential dissection of the rat E16 ventral mesencephalon and survival and reinnervation of the 6-OHDA-lesioned striatum by a subset of aldehyde dehydrogenase-positive TH neurons

The retinoic acid-generating enzyme, aldehyde dehydrogenase (AHD), is expressed in a subpopulation of dopaminergic neurons found in the substantia nigra. Using AHD and tyrosine hydroxylase (TH) as immunohistochemical markers, we determined whether differential dissection of the embryonic (E16) ventral mesencephalon (VM) into its lateral and medial portions contributed equally to the number of TH cells surviving transplantation, if grafted AHD/TH neurons reinnervate the host striatum according to their normal projection patterns, and examined the functional recovery caused by the implanted cells as assessed by amphetamine-induced rotation in a 6-OHDA-lesioned model of Parkinson's disease. The embryonic tissue was transplanted as solid pieces injected via a 20-gauge lumbar puncture needle into the center of the deafferented striatum. Groups received either one complete ventral mesencephalic piece (VM), two medial pieces of ventral mesencephalic tissue (MVM), or two lateral pieces of ventral mesencephalic tissue (LVM). Both VM and MVM groups showed a significant decrease in amphetamine-induced rotation over time and, there was no difference in the degree of reduction observed between the two groups. Histological evaluation of the transplants revealed a much larger total number of surviving TH cells in grafts from the VM and MVM groups compared to the LVM group. Surviving AHD/TH neurons were found in all groups. Whereas TH staining of the transplanted striatum displayed a halo of graft-derived fibers all around the transplant and integration of these fibers into the host neuropil, AHD staining showed a preferential reinnervation of the dorsolateral striatum corresponding to the normal projection pattern of AHD/TH neurons. In summary, selective dissection of the embryonic ventral mesencephalon is possible, functional recovery as assessed by amphetamineinduced rotation in animals transplanted with MVM is similar to that seen in animals grafted with VM, and AHD/TH neurons have a selective reinnervation pattern in the PD transplantation paradigm. These findings may have implications for the grafting of fetal mesencephalic tissue in PD patients.

Differential dissection of the rat E16 ventral mesencephalon and survival and reinnervation of the 6-OHDA-lesioned striatum by a subset of aldehyde dehydrogenase-positive TH neurons.

The retinoic acid-generating enzyme, aldehyde dehydrogenase (AHD), is expressed in a subpopulation of dopaminergic neurons found in the substantia nigra. Using AHD and tyrosine hydroxylase (TH) as immunohistochemical markers, we determined whether differential dissection of the embryonic (E16) ventral mesencephalon (VM) into its lateral and medial portions contributed equally to the number of TH cells surviving transplantation, if grafted AHD/TH neurons reinnervate the host striatum according to their normal projection patterns, and examined the functional recovery caused by the implanted cells as assessed by amphetamine-induced rotation in a 6-OHDA-lesioned model of Parkinson's disease. The embryonic tissue was transplanted as solid pieces injected via a 20-gauge lumbar puncture needle into the center of the deafferented striatum. Groups received either one complete ventral mesencephalic piece (VM), two medial pieces of ventral mesencephalic tissue (MVM), or two lateral pieces of ventral mesencephalic tissue (LVM). Both VM and MVM groups showed a significant decrease in amphetamine-induced rotation over time and, there was no difference in the degree of reduction observed between the two groups. Histological evaluation of the transplants revealed a much larger total number of surviving TH cells in grafts from the VM and MVM groups compared to the LVM group. Surviving AHD/TH neurons were found in all groups. Whereas TH staining of the transplanted striatum displayed a halo of graft-derived fibers all around the transplant and integration of these fibers into the host neuropil, AHD staining showed a preferential reinnervation of the dorsolateral striatum corresponding to the normal projection pattern of AHD/TH neurons. In summary, selective dissection of the embryonic ventral mesencephalon is possible, functional recovery as assessed by amphetamine-induced rotation in animals transplanted with MVM is similar to that seen in animals grafted with VM, and AHD/TH neurons have a selective reinnervation pattern in the PD transplantation paradigm. These findings may have implications for the grafting of fetal mesencephalic tissue in PD patients.

An adenovirus vector encoding tyrosine hydroxylase activity may enter human cns cells in primary dissociated cultures

An adenovirus encoding tyrosine hydroxylase (TH) activity was inserted in neuronal and glial cultured cells obtained from human fetal central nervous system (CNS) tissue. Using a double fluorescence immunostaining, we characterized inoculated CNS cells, with a TH antiserum and one of the following antibodies: microtubule-associated protein (MAP2) and GABA for neuronal cells, vimentin (Vim) for glial cells and glial fibrillary acidic protein (GFAP) for astrocytes. The characterization of inoculated neuronal cells was established by the detection of TH-MAP2-stained neurons in cultures obtained from the thoracic and lumbar parts of the spinal cord where no intrinsic TH cells are described. Inoculated glial cells were characterized by the detection of TH-Vim and TH-GFAP-stained CNS cultured cells. We also observed GABA neurons expressing TH immunoreactivity which could be considered as inoculated neurons expressing the GABA phenotype. Whatever the time of inoculation, transfection was observed in both neuronal and glial cells, after up to 4 months of culture. Although no precise quantitation was performed, the percentage of inoculation was found on microscopic inspection to be greater in glia than in neurons, as previously reported. We concluded that a gene coding for a key neuronal enzyme can be incorporated in embryonic human glial and neuronal cells through the use of a recombinant adenovirus.