Number Of Tyrosine Hydroxylase-positive Neurons Research Articles

BMPs, FGF8 and Wnts regulate the differentiation of locus coeruleus noradrenergic neuronal precursors

In the present study, we investigated the involvement of rhombomere 1 patterning proteins in the regulation of the major noradrenergic centre of the brain, the locus coeruleus. Primary cultures of rat embryonic day 13.5 locus coeruleus were treated with fibroblast growth factor-8, noggin and members of the bone morphogenetic and Wnt protein families. We show that bone morphogenetic proteins 2, 5 and 7 increase and noggin decreases the number of tyrosine hydroxylase-positive locus coeruleus neurons. Interestingly, from all Wnts expressed in the first rhombomere by embryonic day 12.5 in the mice, we only found expression of wnt5a mRNA in the vicinity of the locus coeruleus. In agreement with this finding, from all Wnts studied in vitro, only Wnt5a increased the number of tyrosine hydroxylase-positive neurons in locus coeruleus cultures. Finally, we also found that fibroblast growth factor-8 increased the number of tyrosine hydroxylase-positive cells in locus coeruleus cultures. Neither of the identified factors affected the survival of tyrosine hydroxylase-positive locus coeruleus noradrenergic neurons or the proliferation of their progenitors or neurogenesis. Instead, our results suggest that these patterning signals of rhombomere 1 may work to promote the differentiation of noradrenergic progenitors at later stages of development.

Potential neuroprotective effect of low dose whole-body γ-irradiation against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic toxicity in C57 mice

Low dose whole-body γ-irradiation is recently reported to confer neuroprotection against optic nerve crush and contusive spinal cord injury. Here, we extended the study and investigated whether the pretreatment of a single low dose whole-body γ-irradiation may have a preventive effect in MPTP-induced model of PD. One week after the last MPTP treatment, HPLC determination of striatal dopamine and immunostaining for tyrosine hydroxylase (TH), CD11b and GFAP to detect dopamine neurons and associated glial reaction in the substantia nigra pars compacta (SNpc) were performed. MPTP treatment reduced striatal DA levels significantly; nigral TH immunoreactivity was reduced to a lower extent; robust gliosis was also observed in SNpc. We found that 3.5 Gy irradiation but not 5.5 Gy restores the level of DA and its metabolites decreased by MPTP. However, there was no difference in the number of TH positive neurons between 3.5 Gy irradiated and saline treated mice after MPTP treatment. Irradiation also did not have obvious influence on microgliosis and astroglial reaction induced by MPTP treatment. In conclusion, the results presented here demonstrated that low dose whole-body γ-irradiation renders neuroprotection against MPTP-mediated damage of striatal dopaminergic nerve fibers, though it does not seem to influence the MPTP-induced reduction of SNpc dopaminergic neurons and associated glial responses.

Alterations of the expression and activity of midbrain nitric oxide synthase and soluble guanylyl cyclase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice

The study was aimed at investigating the expression and the activity of neuronal nitric oxide synthase, and of soluble guanylyl cyclase and phosphodiesterase activities that regulate guanosine 3′,5′-cyclic monophosphate level in the midbrain, in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections. Adult male mice of the C57/BL strain were given three i.p. injections of physiological saline or three i.p. injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine solution in physiological saline at 2 h intervals (summary 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine dose: 40 mg/kg), and were killed 3, 7, or 14 days later. mRNA, protein level, and/or activities of neuronal nitric oxide synthase, soluble guanylyl cyclase, phosphodiesterase and guanosine 3′,5′-cyclic monophosphate were determined. Immunohistochemistry showed about 75% decrease in the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed increased midbrain guanylyl cyclase and total nitric oxide synthase activities at 3, 7, and 14 days post-treatment. The specific neuronal nitric oxide synthase inhibitor 7-nitroindazole (10 μM) and the specific inducible nitric oxide synthase inhibitor 1400W (10 μM) inhibited the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced excess in nitric oxide synthase activity by 63–70 and 13–25%, respectively. The increases in total midbrain nitric oxide synthase activity were accompanied by elevated guanosine 3′,5′-cyclic monophosphate, enhanced expression of neuronal nitric oxide synthase and of the β1 subunit of guanylyl cyclase at both mRNA and protein levels that persisted up to the end of the observation period, and by enhanced neuronal nitric oxide synthase and guanylyl cyclase β1 immunoreactivities in substantia nigra pars compacta 7 and 14 days after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The increases in guanylyl cyclase activity were found to occur exclusively due to increased maximal enzyme activity. No 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced change in phosphodiesterase activity has been detected in any brain region studied. 7-Nitroindazole prevented a significant increase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced midbrain guanosine 3′,5′-cyclic monophosphate level and neurodegeneration of dopaminergic neurons. These results raise the possibility that the nitric oxide/guanylyl cyclase/guanosine 3′,5′-cyclic monophosphate signaling pathway may play a role in maintaining dopaminergic neurons function in substantia nigra pars compacta.

Endogenous brain-derived neurotrophic factor protects dopaminergic nigral neurons against transneuronal degeneration induced by striatal excitotoxic injury

Injury to the central nervous system causes atrophy or death of connecting neurons and can modify the expression of neurotrophic factors. We observed transneuronal upregulation of brain-derived neurotrophic factor (BDNF) expression in the rat ipsilateral substantia nigra pars compacta after a striatal lesion induced by kainate. This effect is developmentally regulated because the enhancement of nigral BDNF expression was only observed when striatal lesion was performed on postnatal day (P) 15 and in adulthood, but not at P7. Interestingly, the lack of regulation of BDNF was coincident with the transynaptic degeneration of nigral neurons after striatal excitotoxic injury. Hence, the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta decreased when the lesion was performed at P7, but not at P15 or at P30. The analysis of the functional significance of this BDNF upregulation was done using trkB-IgG fusion proteins. After striatal injury, blockade of endogenous BDNF by trkB fusion proteins induced an atrophy of the dopaminergic neurons of the pars compacta. The injection of trkB-IgG fusion proteins did not modify the effects of kainate in the substantia nigra pars reticulata. Thus, our results show that BDNF exerts an autocrine/paracrine protective effect selectively on dopaminergic neurons against the loss of trophic support from the target striatum.

The distribution and the modulation of tyrosine hydroxylase immunoreactivity in the lateral olivocochlear system of the guinea-pig

It was previously shown that tyrosine hydroxylase (TH) immunoreactivity in the terminals of the lateral efferents of the cochlea is decreased by acoustic trauma and that sound preconditioning counteracted this decrease [Hear Res 174 (2002) 124]. Here we identify those neurons in the lateral olivocochlear system (LOC) in the brainstem that regulates the peripheral expression of TH in the cochlea. By employing retrograde tracing techniques, dextran-labeled neurons were found predominantly in the ipsilateral LOC system including lateral superior olive (LSO), and the surrounding periolivary regions (dorsal periolivary nucleus [DPO], dorsolateral periolivary nucleus [DLPO], lateral nucleus of trapezoid body [LNTB]). Employing immunocytochemistry, it was found that a control group had 35% of the ipsilateral LOC neurons positively stained with TH. Of the total population of TH neurons, 77% were double-stained (TH and dextran) in the LOC system. Acoustic trauma decreased the number of TH positive neurons in the LSO and the surrounding DLPO, and caused a reduction of TH fiber immunolabeling in these regions. Changes were not found in the DPO or the LNTB after acoustic trauma. Sound conditioning protected against the decrease of TH immunolabeling by acoustic trauma and increased the fiber staining for TH in the LSO and DLPO, but not in the DPO or the LNTB. These results provide evidence that TH positive neurons are present in the LOC system in the guinea-pig. It is now demonstrated that protection against acoustic trauma by sound conditioning has a central component that is governed by TH in the LSO and the surrounding periolivary DLPO region.

Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a.

The Wnts are a family of glycoproteins that regulate cell proliferation, fate decisions, and differentiation. In our study, we examined the contribution of Wnts to the development of ventral midbrain (VM) dopaminergic (DA) neurons. Our results show that beta-catenin is expressed in DA precursor cells and that beta-catenin signaling takes place in these cells, as assessed in TOPGAL [Tcf optimal-promoter beta-galactosidase] reporter mice. We also found that Wnt-1, -3a, and -5a expression is differentially regulated during development and that partially purified Wnts distinctively regulate VM development. Wnt-3a promoted the proliferation of precursor cells expressing the orphan nuclear receptor-related factor 1 (Nurr1) but did not increase the number of tyrosine hydroxylase-positive neurons. Instead, Wnt-1 and -5a increased the number of rat midbrain DA neurons in rat embryonic day 14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of Nurr1+ precursors, up-regulated cyclins D1 and D3, and down-regulated p27 and p57 mRNAs. In contrast, Wnt-5a primarily increased the proportion of Nurr1+ precursors that acquired a neuronal DA phenotype and up-regulated the expression of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled-8 (a Wnt inhibitor) blocked endogenous Wnts and the effects of Wnt-1 and -5a on proliferation and the acquisition of a DA phenotype in precursor cultures. These findings indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and that different Wnts have specific and unique activity profiles.

C-fos and tyrosine hydroxylase expression after an excitotoxic lesion on the nigrostriatal system: a study on the effects of hypoxia used as a preconditioning stimulus

Hypoxia–ischemia during the perinatal period causes excitotoxic lesions in sensitive brain areas, such as the striatum. The impact of hypoxia–ischemia on nigral neurons is less well known. Hypoxia alone, a less traumatic event without overt histological sequelae, has neuroprotective properties when used as a preconditioning stimulus. In some pathologies, injured neurons of the nigrostriatal system in the adult may be the result of neurodegenerative processes that originated at early stages of life. The effects of hypoxia on the immunoreactivity to tyrosine hydroxylase of the dopaminergic neurons of the substantia nigra pars compacta and the effects of a period of hypoxia previous to an excitotoxic lesion were examined by means of histological and Western blot methods, at immediate and late periods of the episode. By counting the number of tyrosine hydroxylase-stained neurons and c-fos-positive nuclei a short period after injection of quinolinic acid into the striatum, we observed that hypoxia induced a more marked decrease in the number of tyrosine hydroxylase-stained neurons. On the contrary, c-fos-positive profiles decreased in the substantia nigra pars reticulata of the quinolinic acid-injected animals after the preconditioning hypoxia. Hypoxia alone did not affect the number of tyrosine hydroxylase-positive neurons in the pars compacta nor did hypoxia induce c-fos expression in the pars reticulata. More sensitive Western blot analysis of tissue blocks that included the whole substantia nigra demonstrated the same trend as the immunohistochemical results. We conclude that the responses of the substantia nigra neurons to hypoxia are regionalized and potential neuroprotective effects may depend on the vulnerability of each neuronal type.

Evidence for Dysfunction of the Nigrostriatal Pathway in the R6/1 Line of Transgenic Huntington's Disease Mice

The present multidisciplinary study examined nigrostriatal dopamine and striatal amino acid transmission in the R6/1 line of transgenic Huntington's disease (HD) mice expressing exon 1 of the HD gene with 115 CAG repeats. Although the number of tyrosine hydroxylase-positive neurons was not reduced and nigrostriatal connectivity remained intact in 16-week-old R6/1 mice, the size of tyrosine hydroxylase-positive neurons in the substantia nigra was reduced by 15%, and approximately 30% of these cells exhibited aggregated huntingtin. In addition, using in vivo microdialysis, we found that basal extracellular striatal dopamine levels were reduced by 70% in R6/1 mice compared to their wild-type littermates. Intrastriatal perfusion with malonate in R6/1 mice resulted in a short-lasting, attenuated increase in local dopamine release compared to wild-type mice. Furthermore, the size of the malonate-induced striatal lesion was 80% smaller in these animals. Taken together, these findings suggest that a functional deficit in nigrostriatal dopamine transmission may contribute to the behavioral phenotype and the resistance to malonate-induced neurotoxicity characteristic of R6/1 HD mice.

Lipid-Mediated Glial Cell Line-Derived Neurotrophic Factor Gene Transfer to Cultured Porcine Ventral Mesencephalic Tissue

Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM tissue grafts to PD patients indicate that one reason for the poor outcome of neural transplantation is the low survival and differentiation of grafted dopaminergic neurons. To improve dopaminergic cell survival through a gene-therapeutic approach we have established and report here results of lipid-mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly increased transfection yields. Expression of human GDNF via an episomal vector could be detected by in situ hybridization and by the measuring of GDNF protein secreted into the culture medium. When compared to mock-transfected controls, VM tissue expressing recombinant GDNF contained significantly higher numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo- and xenotransplantation treatment in Parkinson's disease.

An AAV-derived Apaf-1 dominant negative inhibitor prevents MPTP toxicity as antiapoptotic gene therapy for Parkinson's disease.

Adeno-associated virus (AAV) vector delivery of an Apaf-1-dominant negative inhibitor was tested for its antiapoptotic effect on degenerating nigrostriatal neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. The wild-type caspase recruitment domain of Apaf-1 was used as a dominant negative inhibitor of Apaf-1 (rAAV-Apaf-1-DN-EGFP). An AAV virus vector was used to deliver it into the striatum of C57 black mice, and the animals were treated with MPTP. The number of tyrosine hydroxylase-positive neurons in the substantia nigra was not changed on the rAAV-Apaf-1-DN-EGFP injected side compared with the noninjected side. We also examined the effect of a caspase 1 C285G mutant as a dominant negative inhibitor of caspase 1 (rAAV-caspase-1-DN-EGFP) in the same model. However, there was no difference in the number of tyrosine hydroxylase-positive neurons between the rAAV-caspase-1-DN-EGFP injected side and the noninjected side. These results indicate that delivery of Apaf-1-DN by using an AAV vector system can prevent nigrostriatal degeneration in MPTP mice, suggesting that it could be a promising therapeutic strategy for patients with Parkinson's disease. The major mechanism of dopaminergic neuronal death triggered by MPTP seems to be the mitochondrial apoptotic pathway.

Evidence for Target-Specific Nerve Fiber Outgrowth from Subpopulations of Grafted Dopaminergic Neurons: A Retrograde Tracing Study Using in Oculo and Intracranial Grafting

Efforts have been made to counteract the symptoms of Parkinson's disease by substituting the loss of dopaminergic neurons with fetal ventral mesencephalic grafts. One of the postulated limiting factors in this treatment is the relatively poor cell survival and limited graft-derived fiber outgrowth. Recent results documenting enhanced survival of grafted dopaminergic neurons showed no positive correlation to enhanced innervation of the striatal target. Therefore this study was undertaken to investigate whether all surviving grafted dopaminergic neurons projected to the striatal target. Hence, fetal ventral mesencephalic tissue was implanted adjacent to mature versus immature striatal tissue using in oculo and intraventricular grafting techniques. In in oculo grafting, fetal ventral mesencephalon was implanted simultaneously with fetal lateral ganglionic eminence (immature striatal target) or to already matured striatal in oculo grafts (mature striatal target). Furthermore, fetal ventral mesencephalon was implanted into the lateral ventricle adjacent to mature dopamine-depleted striatum. The retrograde tracer fluorogold was injected into the striatal portion of the in oculo cografts and into reinnervated areas of the adult brain. Immunohistochemistry revealed that a significantly larger proportion of tyrosine hydroxylase-positive neurons in the ventral mesencephalic graft was innervating in oculo immature striatal tissue, and hence was fluorogold-positive, in comparison with the number of tyrosine hydroxylase-positive neurons innervating mature striatal tissue. Moreover, intracranial transplantations showed that tyrosine hydroxylase-positive neurons were distributed within the grafts in dense clusters of cells. In most clusters tyrosine hydroxylase-positive cells were fluorogold-negative but calbindin-positive. In a few tyrosine hydroxylase-positive cell clusters, neurons were coexpressing fluorogold but were calbindin-negative. In conclusion, significantly more dopamine neurons projected to immature than to mature striatal tissue and thus, a subpopulation of grafted dopaminergic neurons was not projecting into adult striatum. Thus, the results from this study show that further attempts to enhance survival of grafted dopamine neurons in purpose to enhance graft-derived fiber outgrowth and efficacy should also consider different subtypes of dopamine neurons.

GDNF reverses priming for dyskinesia in MPTP-treated, L-DOPA-primed common marmosets.

Parkinson's disease (PD) is associated with a progressive loss of dopamine neurons in the substantia nigra and degeneration of dopaminergic terminals in the striatum. Although L-DOPA treatment provides the most effective symptomatic relief for PD it does not prevent the progression of the disease, and its long-term use is associated with the onset of dyskinesia. In rodent and primate studies, glial cell line-derived neurotrophic factor (GDNF) may prevent 6-OHDA- or MPTP-induced nigral degeneration and so may be beneficial in the treatment of PD. In this study, we investigate the effects of GDNF on the expression of dyskinesia in L-DOPA-primed MPTP-treated common marmosets, exhibiting dyskinesia. GDNF or saline was administered by two intraventricular injections, 4 weeks apart, to MPTP-treated, L-DOPA-treated common marmosets primed to exhibit dyskinesia. Prior to GDNF or saline administration, all animals displayed marked dyskinesia when treated with L-DOPA. GDNF administration produced a significant improvement in motor disability and, following the second injection of GDNF, a significant improvement in the locomotor activity was observed. Following the administration of L-DOPA there was a greater reversal of disability and a reduction in the intensity of L-DOPA-induced dyskinesia in GDNF-treated animals compared to saline-treated controls. However, there was no significant difference in L-DOPA's ability to increase locomotor activity between GDNF-treated and saline-treated animals. GDNF treatment caused a significant increase in the number of tyrosine hydroxylase-positive neurons in the substantia nigra, but no change in [(3)H]mazindol binding to dopamine terminals was found in the striatum of GDNF-treated animals compared to saline-treated controls. In GDNF-treated animals a small but significant reduction in enkephalin mRNA was observed in the caudate nucleus but not in the putamen or the nucleus accumbens. Substance P mRNA expression was equally reduced in the caudate nucleus and the putamen of the GDNF-treated animals but not in the nucleus accumbens. Intraventricular administration of GDNF improved MPTP-induced disability and reversed dopamine cell loss in the substantia nigra. GDNF also diminished L-DOPA-induced dyskinesia, which may relate to its ability to partly restore nigral dopaminergic transmission or to modify the activity of striatal output pathways.

Differential Alterations in Nicotinic Receptor α6 and β3 Subunit messenger RNAs in Monkey Substantia nigra After Nigrostriatal Degeneration

Our previous studies showed that α4, α6, α7, β2, β3 and β4 nicotinic acetylcholine receptor messenger RNAs are present in monkey substantia nigra, with a particularly intense and localized labelling of the α6 and β3 subunit messenger RNAs to this brain region. Because loss of nigrostriatal neurons is a central feature of Parkinson’s disease and evidence suggests that nicotinic agonists potentiate antiparkinsonian effects of l-dopa, experiments were done to determine whether nicotinic receptor subunit messenger RNAs and binding sites were altered in the basal ganglia after nigrostriatal degeneration. Squirrel monkeys ( Saimiri sciureus) were rendered parkinsonian by systemic injection of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Behavioral studies showed that this treatment decreased baseline motor activity to 36±11% of control. One month after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, caudate and putamen dopamine levels were reduced to 51±15% and 43±6% of control, respectively, while the number of tyrosine hydroxylase-positive neurons in the substantia nigra was 75±6% of control. Despite the reduction in nigral cell number after nigrostriatal degeneration, there were no changes in α4, α7, β2 and β4 messenger RNA levels in the substantia nigra. In contrast, α6 mRNA levels were significantly increased (143±10%) and the β3 transcript decreased (62±6%) in the substantia nigra after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Declines were also observed in [ 125I]epibatidine binding in both the caudate–putamen and substantia nigra, with no change in α7 receptor binding. These results may suggest a dissociation in the regulation of receptor messenger RNA and binding sites, and/or that there are differential alterations in the different receptor subtypes measured using [ 125I]epibatidine. The changes in the two nicotinic receptor subunit messenger RNAs, α6 and β3, which exhibit a selective localization to the substantia nigra, may indicate that nicotinic receptors containing these subunits are altered after nigrostriatal degeneration.

Long-term nonpassaged EGF-responsive neural precursor cells are stem cells.

We have screened lines of nonpassaged epidermal growth factor-responsive neurospheres from embryonic striatum and brainstem. They have been maintained in defined medium with epidermal growth factor over a period of 2 years and remained in an undifferentiated state to this date. Since isolation from the brain 2 years ago, these nonpassaged epidermal growth factor responsive neurospheres have shown active proliferation and self-renewal capacity. When subplated on a poly-D-lysine coated surface, they resumed differentiation within 24 hours. The differentiation process of the nonpassaged epidermal growth factor responsive neurosphere appeared to recapitulate the neural development in the brain. Many cells migrated, extending radial processes while expressing nestin and S100 in the early 7-day subplating culture. They continued to differentiate into major neural types in 14-day subplating culture, including fibrous and cytoplasmic astrocytes, oligodendrocytes, and serotonin, gamma-aminobutyric acid, and a small number of tyrosine hydroxylase-positive neurons. The nonpassaged epidermal growth factor-responsive neurospheres in many ways resemble hemopoietic cells. Both are proliferative, possess the potential of indefinite self-renewal, yet multipotent, and are capable of resuming the differential pathway. The nonpassaged epidermal growth factor responsive neurospheres meet the criteria of stem cells and have been found to be a useful model to study the development in vitro.

Involvement of protease-activated receptor-1 in the in vitro development of mesencephalic dopaminergic neurons

In situ hybridization studies have revealed high levels of protease (thrombin)-activated receptor-1 messenger RNA in the mesencephalon of rats, suggesting that dopaminergic neurons are a target for thrombin's actions. We have evaluated the effect of thrombin receptor activation, either by thrombin or by thrombin receptor agonist peptide, a 14 amino acid agonist of protease-activated receptor-1, on tyrosine hydroxylase-positive neurons. Pure cultures of rat mesencephalic neurons or co-cultures of mesencephalic neurons and glial cells were treated with either thrombin or thrombin receptor agonist peptide the day after plating. Tyrosine hydroxylase-positive cell counting, [ 3H]dopamine uptake and morphometric analysis were performed on day 5. Thrombin and thrombin receptor agonist peptide influenced neurite elongation, branching and the number of primary, secondary and tertiary neurites of tyrosine hydroxylase-positive neurons. In pure cultures, the most significant effects of thrombin and thrombin receptor agonist peptide were to delay branching and to increase the centrifugal growth of neurites without affecting the total neuritic length. Thrombin (up to 10 nM) and thrombin receptor agonist peptide did not affect the number of tyrosine hydroxylase-positive neurons or [ 3H]dopamine uptake. Neurotrophin-4 also influenced the morphology of tyrosine hydroxylase-positive neurons. The increase of neuritic length initiated by this neurotrophin is complementary to the radial elongation induced by protease-activated receptor-1 activation. When neurons were cultured in the presence of glial cells, the effects of thrombin and thrombin receptor agonist peptide on most of these parameters were larger than those observed with pure cultures. Thus, thrombin is able to initiate a complex remodelling of the architecture of tyrosine hydroxylase-positive neurons through the activation of protease-activated receptor-1. These results provide further support for the involvement of protease-activated receptor-1 activation in the development and differentiation of the central nervous system.

Chronic exposure to Ro20-1724 protects dopaminergic neurons in vitro against the neurotoxic action of N-methyl- d-aspartate and 1-methyl-4-phenylpyridinium

Using organotypic cultures of rat ventral mesencephalon, the effects of chronic (12–15 day) exposure to the type IV cAMP phosphodiesterase inhibitor, Ro20-1724, were examined. At concentrations of 10 −8–10 −5 M, Ro20-1724 alone had no effect upon the number of tyrosine hydroxylase-positive neurons or upon neurite outgrowth. However, the drug offered significant protection, with maximum effect at 10 −6 M, against subsequent acute (48 h) exposure to the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP +) and N-methyl- d-aspartate (NMDA).

Immunohistochemical studies of mesolimbic dopaminergic neurons in Fischer 344 and Lewis rats

Previous work has shown that the inbred Lewis and Fischer 344 rat strains differ in several behavioral measures related to mesolimbic dopamine function. Moreover, the ventral tegmental area (VTA) of the Lewis rat has been shown to contain higher levels of tyrosine hydroxylase compared to that of the Fischer rat by blot immunolabeling procedures. To investigate structural correlates of this biochemical difference, an immunohistochemical study of VTA dopaminergic neurons in these two strains was undertaken. Results show that the density and total number of tyrosine hydroxylase-positive neurons in the VTA of the Lewis rat is about 50% of that found in the Fischer rat. In contrast, examination of the substantia nigra in the same sections revealed no differences in the density and number of tyrosine hydroxylase-positive cells between these strains. Fischer-Lewis strain differences were also evident for cholecystokinin immunoreactivity in the VTA, with much lower levels seen in the Lewis rat, consistent with the known colocalization of this neuropeptide in many VTA dopamine neurons. The finding of 50%v fewer tyrosine hydroxylase-positive neurons in the VTA of the Lewis rat, along with our earlier results showing 45% higher levels of tyrosine hydroxylase by blot immunolabeling, would suggest much higher levels of tyrosine hydroxylase per VTA neuron in this strain. However, no obvious strain difference in the cellular intensity of tyrosine hydroxylase immunoreactivity could be detected by immunohistochemistry. Finally, the density of VTA dopamine neurons was assessed in 1-week-old Fischer and Lewis rats. In contrast to the results obtained for adult animals, no difference in the number of tyrosine hydroxylase-positive neurons was apparent in these young animals, indicating that the Fischer-Lewis strain difference in VTA dopamine neurons appears later in postnatal development. These anatomical findings shed new light on the differences in the mesolimbic dopamine system between Fischer and Lewis rats that may contribute to the behavioral differences exhibited by these animals.

Glial cell line-derived neurotrophic factor exerts neurotrophic effects on dopaminergic neurons in vitro and promotes their survival and regrowth after damage by 1-methyl-4-phenylpyridinium.

The effect of glial cell line-derived neurotrophic factor (GDNF) on the growth of mesencephalic dopaminergic neurons and on their survival following exposure to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) was examined in vitro. In cultures developing under normal conditions, GDNF at 1 ng/ml optimally improved the survival and stimulated the growth of dopaminergic neurons without affecting glial growth. In cultures treated with MPP+, GDNF could not prevent toxicity to dopaminergic neurons. The uptake of [3H]dopamine and the number of tyrosine hydroxylase-positive neurons were similarly reduced by MPP+ in the presence or absence of GDNF. However, after removal of MPP+, GDNF protected dopaminergic neurons from the continuous cell death and stimulated the regrowth of dopaminergic fibers damaged by MPP+. We conclude that GDNF supports the growth of normally developing dopaminergic neurons and stimulates their survival and recovery after damage. These findings suggest that GDNF could be useful in the development of therapeutic approaches to Parkinson's disease, which is characterized by dopaminergic cell loss.

Differential effects of platelet-derived growth factor isoforms on dopamine neurons in vivo: -BB supports cell survival, -AA enhances fiber formation

Trophic effects of platelet-derived growth factor -AA and -BB on developing (embryonic day 14) ventral mesencephalon were studied using the in vivo model of intraocular transplantation to sympathetically denervated host eyes. This model enabled studies of survival and growth of grafted brain tissue, dopaminergic fiber outgrowth from the grafts onto the host iris as well as morphological effects of platelet-derived growth factor on grafted tissue using markers for tyrosine hydroxylase and glial fibrillary acid protein. Growth of grafts was followed by repeated observations directly through the cornea of the host using a stereomicroscope. This revealed that there was no apparent effect on volume increase of mesencephalic grafts after treatments with either platelet-derived growth factor-AA (100 ng/ml buffer), platelet-derived growth factor-BB (100 ng/ml buffer) or vehicle solution (high salt buffer) alone. Growth factor treatments were administered immediately prior to grafting by incubating the grafts in the appropriate factor as well as on days 5, 10 and 15 postgrafting by administration of 5-μl intraocular injections of similiar solutions as used for incubation. Platelet-derived growth factor-AA significantly enhanced dopaminergic fiber outgrowth from mesencephalic grafts when compared to both plateletderived growth factor-BB and controls, without an accompanying rise in the number of tyrosine hydroxylase-positive neurons. In contrast, a significantly greater number of tyrosine hydroxylase-positive neurons was seen in grafts treated with platelet-derived growth factor-BB but without an accompanying increase in outgrowth of dopamine-containing fibers. Both platelet-derived growth factor-AA and -BB treatments caused a slight but significant decrease (= normalization) in the GFAP-like immunoreactive elements within the grafts as compared to grafts treated with vehicle alone. These findings suggest that in vivo, the two platelet-derived growth factor isoforms both act on the ventral mesencephalic grafts, each in a distinct manner, -AA enhancing the formation of dopamine fibers and -BB acting as a maintenance factor for tyrosine hydroxylase-positive cells in this area. This leads to the conclusion that these two platelet-derived growth factor isoforms could act as trophic factors for mesencephalic dopamine neurons.

Toxic and protective effects of L-dopa on mesencephalic cell cultures.

The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 microM) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [3H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions. Ascorbate (200 microM), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [3H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, L-DOPA prevented cell death by L-BSO.