Adachi, M. and E. J. Lewis (2002). "The paired-like homeodomain protein, arix, mediates protein kinase A-stimulated dopamine {beta}-hydroxylase gene transcription through its phosphorylation status." J Biol Chem.
The homeodomain transcription factor Arix/Phox2a plays a critical role in the specification of noradrenergic neurons by inducing the expression of dopamine -hydroxylase (DBH), the terminal enzyme for noradrenaline biosynthesis. In reporter assays, Arix together with activation of cyclic AMP (cAMP)-dependent kinase (PKA) potentiates DBH gene transcription. We have evaluated whether post-translational modification of Arix regulates PKA-mediated DBH gene transcription. We found that Arix is constitutively phosphorylated in vivo at the basal level and that the phosphorylation level is substantially decreased upon stimulation of the PKA pathway. The change in the Arix phosphorylation state coincides with DNA-binding activity of Arix. Treatment of cells with forskolin results in a robust enhancement of Arixs DNA-binding, which is reversed by treatment with serine/threonine and tyrosine phosphatase inhibitors. Consistent with the DNA-binding activity of Arix, treatment of cultured cells with phosphatase inhibitors diminishes transcriptional activation with Arix plus forskolin. Amino acid analysis demonstrates the presence of phosphoserine within Arix. The results collectively suggest that dephosphorylation of Arix is a necessary event to fully activate PKA-mediated DBH transcription. Thus, the present study demonstrates that Arix can integrate extrinsic signals through post-translational modification, regulating DBH gene transcription in response to activation of the PKA pathway.

Adibhatla, R. M., J. F. Hatcher, et al. (2002). "Citicoline: neuroprotective mechanisms in cerebral ischemia." J Neurochem 80(1): 12-23.
Cytidine-5'-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in a number of CNS injury models and pathological conditions of the brain. Citicoline improved the outcome in several phase-III clinical trials of stroke, but provided inconclusive results in recent clinical trials. The therapeutic action of citicoline is thought to be caused by stimulation of PtdCho synthesis in the injured brain, although the experimental evidence for this is limited. This review attempts to shed some light on the properties of citicoline that are responsible for its effectiveness. Our studies in transient cerebral ischemia suggest that citicoline might enhance reconstruction (synthesis) of PtdCho and sphingomyelin, but could act by inhibiting the destructive processes (activation of phospholipases). Citicoline neuroprotection may include: (i) preserving cardiolipin (an exclusive inner mitochondrial membrane component) and sphingomyelin; (ii) preserving the arachidonic acid content of PtdCho and phosphatidylethanolamine; (iii) partially restoring PtdCho levels; (iv) stimulating glutathione synthesis and glutathione reductase activity; (v) attenuating lipid peroxidation; and (vi) restoring Na(+)/K(+)-ATPase activity. These observed effects of citicoline could be explained by the attenuation of phospholipase A(2) activation. Based on these findings, a singular unifying mechanism has been hypothesized. Citicoline also provides choline for synthesis of neurotransmitter acetylcholine, stimulation of tyrosine hydroxylase activity and dopamine release.

An, M., R. Luo, et al. (2002). "Differentiation and maturation of zebrafish dorsal root and sympathetic ganglion neurons." J Comp Neurol 446(3): 267-75.
The trunk neural crest of vertebrate embryos gives rise to dorsal root ganglion (DRG) sensory neurons and autonomic sympathetic neurons, among other derivatives. We have examined the development of DRG and sympathetic neurons during development in the zebrafish. We found that sensory neurons differentiate rapidly and that their overt neuronal differentiation significantly precedes that of sympathetic neurons in the trunk. Sympathetic neurons in different regions differentiate at different times. The most rostral population, which we call the cervical ganglion, differentiates several days before trunk sympathetic neurons. After undergoing overt neuronal differentiation, sympathetic neurons subsequently express the adrenergic differentiation markers dopamine beta-hydroxylase and tyrosine hydroxylase. A second population of adrenergic nonneuronal cells initially localized with cervical sympathetic neurons appears to represent adrenal chromaffin cells. In more mature fish, these cells were present in clusters within the kidneys. Individual DRG and sympathetic ganglia initially contain few neurons. However, the number of neurons in DRG and sympathetic ganglia increases continuously at least up to 4 weeks of age. Analysis of phosphohistone H3 expression and bromodeoxyuridine incorporation studies suggests that the increases in DRG and sympathetic ganglion neuronal cell number are due wholly or in part to the division of neuronal cells within the ganglia.

Azam, L., U. H. Winzer-Serhan, et al. (2002). "Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons." J Comp Neurol 444(3): 260-74.
Many behavioral effects of nicotine result from activation of nigrostriatal and mesolimbic dopaminergic systems. Nicotine regulates dopamine release not only by stimulation of nicotinic acetylcholine receptors (nAChRs) on dopamine cell bodies within the substantia nigra and ventral tegmental area (SN/VTA), but also on presynaptic nAChRs located on striatal terminals. The nAChR subtype(s) present on both cell bodies and terminals is still a matter of controversy. The purpose of this study was to use double-labeling in situ hybridization to identify nAChR subunit mRNAs expressed within dopamine neurons of the SN/VTA, by using a digoxigenin-labeled riboprobe for tyrosine hydroxylase as the dopamine cell marker and (35)S-labeled riboprobes for nAChR subunits. The results reveal a heterogeneous population of nAChR subunit mRNAs within midbrain dopamine neurons. Within the SN, almost all dopamine neurons express alpha2, alpha4, alpha5, alpha6, beta2, and beta3 nAChR mRNAs, with more than half also expressing alpha3 and alpha7 mRNAs. In contrast, less than 10% express beta4 mRNA. Within the VTA, a similar pattern of nAChR subunit mRNA expression is observed except that most subunits are expressed in a slightly lower percentage of dopamine neurons than in the SN. Within the SN, alpha4, beta2, alpha7, and beta4 mRNAs are also expressed in a significant number of nondopaminergic neurons, whereas within the VTA this only occurs for beta4. The heterogeneity in the expression of nAChR subunits within the SN/VTA may indicate the formation of a variety of different nAChR subtypes on cell bodies and terminals of the nigrostriatal and mesolimbic pathways.

Ballmaier, M., M. Zoli, et al. (2002). "Preferential alterations in the mesolimbic dopamine pathway of heterozygous reeler mice: an emerging animal-based model of schizophrenia." Eur J Neurosci 15(7): 1197-205.
Based on a number of neuroanatomical and behavioural similarities, recent evidence suggests that heterozygous reeler mice, haploinsufficient for reelin expression, represent a useful model of psychosis vulnerability. As brain mesolimbic dopamine pathways have been proposed to be associated with the pathophysiology of psychotic disorders, we thought it would be of interest to examine whether these animals present disturbances in the mesolimbic dopamine system. To this end we studied by immunocytochemical, in situ hybridization procedures and receptor autoradiography, several markers of the mesotelencephalic dopamine pathway in heterozygous reeler mice and controls. We report that heterozygous reeler mice exhibit a reduction in the number of tyrosine hydroxylase-immunoreactive cell bodies and tyrosine hydroxylase mRNA levels in the ventral tegmental area, as well as a reduction of tyrosine hydroxylase and dopamine transporter immunoreactivity in the dopamine terminal fields of the limbic striatum. In these areas we also observed a reduction of dopamine D2 receptor mRNA. Finally, a marked increase in D3 receptor mRNA levels was observed concomitant with a significant increase in D3 binding sites. On the contrary, the nigrostriatal pathway did not show any significant alteration in heterozygous reeler mice with regards to the dopaminergic markers examined in substantia nigra cell bodies and dorsal striatum dopamine terminal fields. These results suggest a specific link between reelin-related neuronal pathology and dopamine involvement in the pathophysiology of psychotic disorders.

Bandmann, O. and N. W. Wood (2002). "Dopa-Responsive Dystonia - The Story so Far." Neuropediatrics 33(1): 1-5.
Dopa-responsive dystonia (DRD) is an eminently treatable condition and its recognition is therefore of crucial importance. In classical cases, the disease manifests in early childhood with walking problems due to dystonia of the lower limbs. The dystonia is frequently accompanied by "parkinsonian" features such as reduced facial expression or slowing of fine finger movements. Biochemically, the disorder is typically characterized by low levels of the neurotransmitter metabolite homovanillic acid and reduced levels of neopterin and tetrahydrobiopterin (BH4) in the cerebrospinal fluid. This is due to heterozygote mutations of the GTP cyclohydrolase I gene, which is the rate-limiting enzyme in the synthesis of BH4. BH4 is an essential co-factor for tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine. Reduced levels of BH4 lead to the dopamine-deficit syndrome DRD because of reduced TH activity. Other genes implicated in the pathogenesis of this disorder are the TH gene itself and the parkin gene. This article summarizes all relevant aspects of DRD including recent advances in the genetics of this disorder and the widening phenotype. Particular emphasis is given to clinically relevant aspects such as diagnostic difficulties and atypical presentations in infancy and early childhood.

Battaglia, G., C. L. Busceti, et al. (2002). "Continuous subcutaneous infusion of apomorphine rescues nigro-striatal dopaminergic terminals following MPTP injection in mice." Neuropharmacology 42(3): 367-73.
Apomorphine has been introduced in the treatment of late-stage Parkinson's Disease (PD). The disadvantage of a short half-life of apomorphine is now overcome by the use of a continuous subcutaneous (s.c.) self-delivering system. We examined whether continuous s.c. infusion of apomorphine rescues nigro-striatal dopaminergic neurons from toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Apomorphine was continuously infused in mice by means of a s.c. minipump that delivered the drug at a rate of 0.5 or 3.15mg/kg/day. MPTP induced a >80% reduction in striatal dopamine (DA) after one day. DA levels were still substantially reduced one month following MPTP injection, in spite of a partial recovery. Similarly, striatal immunoreactivity for tyrosine hydroxylase and dopamine transporter was markedly reduced at this time interval. Continuous s.c. infusion of apomorphine starting 40h following MPTP injection rescued striatal dopaminergic terminals, as assessed by measurements of DA and its metabolites, as well as TH and DAT immunostaining after one month. The neurorescuing effect was more remarkable at a delivery rate of 3.15mg/kg/day of apomorphine. In contrast, no rescue was observed when apomorphine was administered as a single daily s.c. bolus of 1 or 5mg/kg starting 40h following MPTP. We conclude that apomorphine is able to rescue nigro-striatal dopaminergic neurons when continuously delivered at doses that are comparable to those delivered by minipumps in PD patients. These results suggest that continuous s.c. infusion of apomorphine not only relieves the symptoms, but also reduce the ongoing degeneration of nigro-striatal dopaminergic neurons in PD patients.

Battaglia, G., F. Fornai, et al. (2002). "Selective blockade of mGlu5 metabotropic glutamate receptors is protective against methamphetamine neurotoxicity." J Neurosci 22(6): 2135-41.
Methamphetamine (MA), a widely used drug of abuse, produces oxidative damage of nigrostriatal dopaminergic terminals. We examined the effect of subtype-selective ligands of metabotropic glutamate (mGlu) receptors on MA neurotoxicity in mice. MA (5 mg/kg, i.p.; injected three times, every 2 hr) induced, 5 d later, a substantial degeneration of striatal dopaminergic terminals associated with reactive gliosis. MA toxicity was primarily attenuated by the coinjection of the noncompetitive mGlu5 receptor antagonists 2-methyl-6-(phenylethynyl)pyridine and (E)-2-methyl-6-styrylpyridine both at 10 mg/kg, i.p.). In contrast, the mGlu1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (10 mg/kg, i.p.), and the mGlu2/3 receptor agonist (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (1 mg/kg, i.p.), failed to affect MA toxicity. mGlu5 receptor antagonists reduced the production of reactive oxygen species but did not reduce the acute stimulation of dopamine release induced by MA both in striatal synaptosomes and in the striatum of freely moving mice. We conclude that endogenous activation of mGlu5 receptors enables the development of MA neurotoxicity and that mGlu5 receptor antagonists are neuroprotective without interfering with the primary mechanism of action of MA.

Bilsland, J., S. Roy, et al. (2002). "Caspase inhibitors attenuate 1-methyl-4-phenylpyridinium toxicity in primary cultures of mesencephalic dopaminergic neurons." J Neurosci 22(7): 2637-49.
Parkinson's disease is characterized by a loss of dopaminergic nigrostriatal neurons. This neuronal loss is mimicked by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). MPP+ toxicity is mediated through inhibition of mitochondrial complex I, decreasing ATP production, and upregulation of oxygen radicals. There is evidence that the cell death induced by MPP+ is apoptotic and that inhibition of caspases may be neuroprotective. In primary cultures of rat mesencephalic dopaminergic neurons, MPP+ treatment decreased the number of surviving dopaminergic neurons in the cultures and the ability of the neurons to take up [3H]dopamine ([3H]DA). Caspase inhibition using the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) spared MPP+-treated dopaminergic neurons and increased somatic size. There was a partial restoration of neurite length in zVAD-fmk-treated cultures, but little restoration of [3H]DA uptake. Peptide inhibitors of caspases 2, 3, and 9, but not of caspase 1, caused significant neuroprotection. Two novel caspase inhibitors were tested for neuroprotection, a broad spectrum inhibitor and a selective caspase 3 inhibitor; both inhibitors increased survival to >90% of control. No neuroprotection was observed with an inactive control compound. MPP+ treatment caused chromatin condensation in dopaminergic neurons and increased expression of activated caspase 3. Inhibition of caspases with either zVAD-fmk or a selective caspase 3 inhibitor decreased the number of apoptotic profiles, but not expression of the active caspase. We conclude that MPP+ toxicity in primary dopaminergic neurons involves activation of a pathway terminating in caspase 3 activation, but that other mechanisms may underlie the neurite loss.

Bitner, R. S. and A. L. Nikkel (2002). "Alpha-7 nicotinic receptor expression by two distinct cell types in the dorsal raphe nucleus and locus coeruleus of rat." Brain Res 938(1-2): 45-54.
The alpha7 nicotinic acetylcholine receptor (nAChR) subunit can be assembled to form a homomeric-pentamer with high permeability to calcium. Although the expression of the alpha7-nAChR has been demonstrated throughout the CNS, the neurochemical phenotype of neurons expressing alpha7 remains to a large extent unknown. Using an antibody against the alpha7 nAChR subunit, immunohistochemical staining was observed in rat dorsal raphe nucleus (DRN) and locus coeruleus (LC), serotonergic and noradrenergic brainstem nuclei, respectively. In both the DRN and LC, there appeared to be two histologically distinct alpha7-expressing cell types as distinguished by size, i.e. large versus small diameter. In rats treated with either a serotonergic (5,7-dihydroxytryptamine) or noradrenergic (anti-dopamine-beta-hydroxylase saporin) neurotoxin, tryptophan hydroxylase and tyrosine hydroxylase immunostaining was abolished, respectively. Similarly, the alpha7-positive large-diameter cells were no longer detectable, suggesting that these cells were serotonergic DRN and noradrenergic LC neurons. Indeed, double-labeling experiments revealed in the large cell types coexpression of alpha7 with tryptophan hydroxylase in the DRN and with tyrosine hydroxylase in the LC of saline-treated rats. In contrast to the large-diameter cells, the alpha7-positive small-diameter cells were neither serotonergic nor adrenergic, and were still detected in both the DRN and LC of lesioned rats. Moreover, cell counts revealed an increase number of these cells in lesioned rats with expression of alpha7 in somal processes not seen in non-lesioned controls. Double labeling revealed coexpression of alpha7 and GABA within the majority, but not all, of the toxin-resistant cells. The results of these studies suggest that both serotonergic and noradrenergic neurons express alpha7 nAChRs. In addition, there appears to be a small-diameter cell-type in both the DRN and LC, possibly a GABAergic interneuron, expressing alpha7 that may be regulated by neurotoxic injury.

Bringmann, G., D. Feineis, et al. (2002). "1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) and related derivatives: chemistry and biochemical effects on catecholamine biosynthesis." Bioorg Med Chem 10(7): 2207-2214.
1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo, 2) is a mammalian alkaloid that readily originates in the human organism, by Pictet-Spengler condensation of endogenously present tryptamine (Ta) and the non-natural hypnotic agent trichloroacetaldehyde (chloral, Clo). Due to its structural analogy to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 1), TaClo is discussed to possibly contribute to the pathogenesis of Parkinson's disease acting as an environmental toxin. Previous investigations on rats and neuronal cell cultures revealed 2 to be capable of inducing severe disturbances on the dopamine metabolism. In this paper, we report on the effects of 2 on the activity of tyrosine hydroxylase [L-tyrosine, tetrayhydropteridine/oxygen oxidoreductase (3-hydroxylating), EC 1.14,16.2; TH] in vitro using rat brain homogenates prepared from the TH-rich nucleus accumbens. TaClo (2) dose-dependently inhibited basal TH activity (IC(50)=3&mgr;M), and after enzyme activation by pituitary adenylate cyclase-activating polypeptide (PACAP-27), it also reduced L-DOPA formation (IC(50)=15&mgr;M). Moreover, two presumable TaClo metabolites, 2-methyl-TaClo (N-Me-TaClo, 3) and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2)-THbetaC, 4), which were synthesized in good yields, also proved to be potent inhibitors of TH, with the strongest effect on basal activity (similar to TaClo) being observed for 3 (IC(50)=3&mgr;M). In contrast to TaClo, however, 3 and 4 showed biphasic effects after TH activation with PACAP-27, inducing a marked increase of enzyme activity in the nanomolar range (<0.1&mgr;M), while TH activity was nearly completely blocked at high concentrations (IC(100)=0.1mM). An X-ray diffraction investigation on the 3-dimensional structure of the 1-CCl(2)-THbetaC-derived trifluoroacetamide 7 revealed the voluminous and quite rigid dichloromethylene substituent to be only moderately twisted out of the beta-carboline ring 'plane', thus resulting in an inreased ring strain of the partially hydrogenated pyrido moiety accompanied by a strong steric hindrance of Cl(1), Cl(2), C(13), and N(2), which pushes the N-trifluoroacetyl group upwards to an even higher extent than for the TaClo-related trifluoroacetamide 8.

Callier, S., M. Le Saux, et al. (2002). "Evaluation of the protective effect of oestradiol against toxicity induced by 6-hydroxydopamine and 1-methyl-4-phenylpyridinium ion (Mpp+) towards dopaminergic mesencephalic neurones in primary culture." J Neurochem 80(2): 307-16.
Recent findings suggest that gonadal steroid hormones are neuroprotective and may provide clinical benefits in delaying the development of Parkinson's disease. In this report we investigated the ability of oestradiol to protect mesencephalic dopaminergic neurones cultured in serum-free or serum-supplemented medium from toxicity induced by 6-hydroxydopamine or 1-methyl-4-phenylpyridinium ion (MPP+). The efficiency of both toxins and oestradiol was evaluated by tyrosine hydroxylase (TH) immunocytochemistry, [3H]dopamine ([3H]DA) uptake, length of dopaminergic processes and lactate dehydrogenase (LDH) release measurement. In cultures grown in serum-supplemented medium, a 2-h pre-treatment with high concentrations (10-100 microM) of 17beta-oestradiol or 17alpha-oestradiol, the stereoisomer with weak oestrogenic activity, protected both dopaminergic and non-dopaminergic neurones from toxicity induced by 6-hydroxydopamine (6-OHDA; 40 or 100 microM) and by the high MPP+ concentrations (50 microM) necessary to obtain significant neuronal death under those culture conditions. At these concentrations, MPP+ was no longer selective for dopaminergic neurones but affected all cells present in the culture. In contrast, the hormonal treatments did not protect against selective degeneration of dopaminergic neurones induced by lower MPP+ concentrations (below 10 microM), related to inhibition of complex I of respiratory chain. In cultures grown in serum-free medium, oestradiol concentrations higher than 1 microM induced neuronal degeneration and no protection against 6-OHDA or MPP+ toxicity was observed at lower concentrations of the steroid. The neuroprotective effects of 17alpha- or 17beta-oestradiol evidenced in this model might be due to the antioxidant properties of these compounds. However, other non-genomic effects of the steroids cannot be excluded.

Carson, R. P. and D. Robertson (2002). "Genetic manipulation of noradrenergic neurons." J Pharmacol Exp Ther 301(2): 410-7.
The neurotransmitter norepinephrine has been the focus of intense investigation for nearly a century. With advances in technology come novel approaches for testing hypotheses about the physiological roles of norepinephrine and the genes involved in norepinephrine (NE) biosynthesis, metabolism, and noradrenergic signaling. Homologous recombination techniques, which generate mice deficient in specific gene products, aid the integrated physiologist and pharmacologist in the evaluation of protein function. Mouse models lacking proteins involved in NE biosynthesis or metabolism provide tools to expand the knowledge previously gleaned from pharmacologic studies. Removal of the biosynthetic enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase yield animals deficient in norepinephrine and have been used to further examine the role of NE in diverse physiologic roles. Complete removal of the vesicular monoamine transporter has demonstrated that mobilizing neurotransmitters to vesicles is required for animal survival. Lastly, the generation of animals in which the ability to remove NE from the synapse is impaired (norepinephrine transporter deficiency and extraneuronal monoamine transporter deficiency) and in which the enzymes responsible for the metabolism of NE have been removed (catechol-O-methyltransferase and monoamine oxidase) has facilitated the study of the long-term physiological consequences of altered NE homeostasis.

Casu, M. A., G. Colombo, et al. (2002). "Reduced TH-immunoreactive fibers in the limbic system of Sardinian alcohol-preferring rats." Brain Res 924(2): 242-51.
The mesolimbic dopamine (DA) system has long been known to be involved in reward behaviors. As with other substances of abuse, it has been extensively reported that ethanol influences the dopaminergic system. The present study examined whether selectively bred Sardinian-alcohol-preferring (sP) and Sardinian alcohol non-preferring rats (sNP), differ in the DA innervation in structures of the forebrain that are related to rewarding behaviors. To this aim, we performed an immunohistochemistry study with an antibody raised against tyrosine hydroxylase (TH), the rate-limited step enzyme in the biosynthesis of monoamines. The TH-positive innervation density was found to be significantly lower in the cingulate cortex and in the shell of the nucleus accumbens of the sP when compared with the sNP and unselected Wistar rats. These anatomical structures both cluster in the medial aspect of the mesolimbic system. No differences in other major DA brain regions, such as the nigro-striatal pathway were found. The analysis of cell-body area revealed no differences between sP, sNP and Wistar rats in the ventral tegmental area and substantia nigra (pars compacta and reticulata) and the density of the TH-positive fibers was not different in the caudate-putamen. These results indicate a selective reduction of terminal innervation in the medial portion of the mesocorticolimbic DA system in sP rats and suggest that the latter may consume larger amounts of ethanol, when compared with sNP rats, to compensate for the deficiency of dopamine to produce an adequate level of reward.

Chun, H. S., M. S. Yoo, et al. (2002). "Marked dopaminergic cell loss subsequent to developmental, intranigral expression of glial cell line-derived neurotrophic factor." Exp Neurol 173(2): 235-44.
Glial cell line-derived neurotrophic factor (GDNF) shows potent neuroprotective as well as neurorestorative actions on the adult neurons impacted in animal models of Parkinson's disease (PD). Long-term pharmaco-physiological effects of GDNF on developing dopaminergic (DA) neurons have not yet been explored because of technical difficulties in producing prolonged cell type-specific delivery of this neurotrophic factor in mammalian embryonic brain. The current studies used our previously characterized 9.0-kb tyrosine hydroxylase promoter to produce transgenic mice with neuronal cell type-specific expression of GDNF in substantia nigra pars compacta (SNc) and locus coeruleus (LC). These mice were used to test the parsimonious hypothesis that increased developmental expression of GDNF in SNc and LC would significantly enhance the number of postmitotic adult neurons. To our surprise, adult transgenic mice carrying the TH9.0kb-GDNF hybrid gene showed dramatic reductions in both the numbers and the volumes of SNc-DA and LC-noradrenergic (NA) neurons by quantitative morphometric analysis. The decrease in the number of DA neurons was apparent as early as postnatal day 2, the period before the major naturally occurring apoptotic cell death in midbrain. Aged transgenic mice exhibited no further significant deficits in motor behaviors. These data suggest that continuous, early developmental GDNF expression exerts physiological effects on newly differentiated, immature dopamine neurons that differ from those observed on more mature and adult DA neurons. Further elucidation of the mechanisms underlying differential GDNF actions will greatly improve the pharmacological efficacy of GDNF in fetal neural transplantation as well as adult neuronal gene therapy in PD patients.

Collier, T. J., C. E. Sortwell, et al. (2002). "Embryonic ventral mesencephalic grafts to the substantia nigra of MPTP-treated monkeys: feasibility relevant to multiple-target grafting as a therapy for Parkinson's disease." J Comp Neurol 442(4): 320-30.
Transplantation of embryonic dopamine (DA) neurons is being studied as an experimental replacement therapy for the DA-deficiency characteristic of Parkinson's disease. Some studies suggest that one of the limitations of this approach is that intrastriatal placement of implants fails to consistently restore completely normal movement. One potential cause of this suboptimal therapeutic outcome is that changes in the neural activity of several structures in the basal ganglia circuitry resulting from striatal DA depletion is not adequately normalized by graft-derived DA replacement in striatum alone. In the present study, we assessed the feasibility of grafting embryonic DA neurons into the substantia nigra (SN) of adult parkinsonian monkeys as an approach to restoration of the DA modulation of striatal-nigral afferents that is lost after degeneration of SN neurons. Sixteen St. Kitts African green monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) received implants of embryonic monkey ventral mesencephalon (VM), or sham implants, aimed at the rostral SN. At 6 months after grafting, staining for tyrosine hydroxylase (TH) indicated that grafted DA neurons survived at this site, albeit often in reduced numbers compared with VM grafts to striatum. Grafted neurons extended neurites into the parenchyma of the SN, but there was no evidence of lengthy extension of graft-derived neurites rostrally along the trajectory of the mesostriatal fiber system. A region-specific, modest increase in DA levels and TH-positive fiber density in the ventral-medial putamen was detected, accompanied by modest but significant decreases in parkinsonian behaviors at 5-6 months after grafting. Our findings support the view that grafting embryonic tissue to the SN is a feasible procedure in nonhuman primates that provides a modest but detectable benefit of its own. These results encourage the further development of multiple-target grafting strategies as a means of restoring modulation of anatomically widespread basal ganglia structures relevant to treatment of Parkinson's disease.

Creutz, L. M. and M. F. Kritzer (2002). "Estrogen receptor-beta immunoreactivity in the midbrain of adult rats: regional, subregional, and cellular localization in the A10, A9, and A8 dopamine cell groups." J Comp Neurol 446(3): 288-300.
Estrogen modulates dopamine synthesis, release, and metabolism in corticolimbic and striatal targets of midbrain dopamine neurons. The relevant sites of receptor-mediated action, however, had been elusive, because all available evidence suggested a paucity of intracellular estrogen receptors in the A8, A9, and A10 dopamine regions and their afferent targets. More recent evidence of a relative abundance of the beta isoform of the estrogen receptor (ER) in the substantia nigra and ventral tegmental area (VTA), however, suggests that this newly described receptor may be important in estrogen's stimulation of midbrain DA systems. It is unknown, however, precisely how ERbeta is distributed with respect to the functionally and neurochemically diverse cell populations of the ventral midbrain. To address these issues, this study used single- and double-label immunocytochemistry to detail the regional, subregional, and cellular distributions of ERbeta immunoreactivity in and around midbrain dopamine-containing cell groups in hormonally intact adult male and female rats. These analyses revealed that ERbeta-immunoreactive nuclei were found only in neurons, more specifically, within subsets of both dopaminergic and nondopaminergic neurons in the dorsal VTA, the parabrachial pigmented nucleus, the substantia nigra pars lateralis, the retrorubral fields, and to a lesser extent the linear midline nuclei. These regional and cellular receptor distributions thus place the ERbeta isoform in anatomical register with midbrain dopamine systems known to participate in a spectrum of motor, cognitive, and affective functions.

Crisp, K. M., K. A. Klukas, et al. (2002). "Distribution and development of dopamine- and octopamine-synthesizing neurons in the medicinal leech." J Comp Neurol 442(2): 115-29.
Although the medicinal leech is a well-studied system in which many neurons and circuits have been identified with precision, descriptions of the distributions of some of the major biogenic amines, such as dopamine (DA) and octopamine (OA), have yet to be completed. In the European medicinal leech Hirudo medicinalis and the American medicinal leech Macrobdella decora,we have presented the first immunohistochemical study of DA neurons in the entire central nervous system, and of OA-immunoreactive (ir) neurons in the head and tail brains. Dopaminergic neurons were identified using the glyoxylic acid method and antisera to DA and its rate-limiting synthetic enzyme tyrosine hydroxylase (TH). Octopaminergic neurons were recognized using a highly specific antiserum raised against OA. An antibody raised against DA-beta-hydroxylase (DbetaH), the mammalian enzyme that converts DA to norepinephrine (NE), was found to immunostain OA-ir neurons. This antibody appears to cross-react with the closely related invertebrate enzyme tyramine-beta-hydroxylase, which converts tyramine to OA, suggesting that the OA-ir cells are indeed octopaminergic, capable of synthesizing OA. Because the DbetaH antiserum selectively immunostained the OA-ir neurons, but not the DA-synthesizing cells, our results also indicate that the DA-ir neurons synthesize DA and not NE as their end product. The expression of TH immunoreactivity was found to emerge relatively early in development, on embryonic day 9 (47-48% of development). In contrast, OA expression remained absent as late as embryonic day 20. Higher order processes of some of the dopaminergic and octopaminergic neurons in the adult brain were observed to project to a region previously described as a neurohemal complex. Several TH-ir processes were also seen in the stomatogastric nerve ring, suggesting that DA may play a role in the regulation of biting behavior. By mapping the distributions and developmental expression pattern of DA and OA neurons in the leech, we aim to gain a better understanding of the functional roles of aminergic neurons and how they influence behavior.

Cunningham, L. A. and C. Su (2002). "Astrocyte delivery of glial cell line-derived neurotrophic factor in a mouse model of Parkinson's disease." Exp Neurol 174(2): 230-42.
Primary astrocytes were genetically modified ex vivo to express recombinant glial cell line-derived neurotrophic factor (GDNF) and subsequently were tested for their ability to provide neuroprotection to dopaminergic neurons in a 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease. A replication-defective retrovirus was constructed, which contained the rat GDNF sequence and a sequence encoding a beta-galactosidase (beta-gal)/neomycin phosphotransferase fusion protein, linked via an internal ribosomal entry site. Murine astrocytes transduced with this vector secreted GDNF into the culture media at the rate of 115 +/- 34 pg/24 h/10(5) cells and expressed cytoplasmic beta-gal, whereas control nontransduced astrocytes were negative for GDNF production and cytoplasmic beta-gal expression. Mice that received implants of GDNF-producing astrocytes into the striatum or nigra displayed elevated levels of GDNF compared to mice that received control nontransduced astrocytes. In addition, tissue content of GDNF was increased bilaterally and in brain regions both proximal and distal to the graft, even though astrocyte migration away from the graft site did not occur. Importantly, GDNF-producing astrocytes provided marked neuroprotection of nigral dopaminergic perikarya, and partial protection of striatal dopaminergic fibers, when implanted into the midbrain 6 days prior to a retrograde 6-OHDA lesion, as assessed by tyrosine hydroxylase immunohistochemistry. Similarly, GDNF-producing astrocytes prevented the acquisition of amphetamine-induced rotational behavior in 6-OHDA-treated mice and completely prevented dopamine depletion within the substantia nigra, as assessed by high-performance liquid chromatography. These results indicate that continuous exposure to low levels of GDNF provided by transgenic astrocytes provides marked neuroprotection of nigral dopaminergic neurons. (c)2002 Elsevier Science (USA).

Diaz-Rios, M., E. Oyola, et al. (2002). "Colocalization of gamma-aminobutyric acid-like immunoreactivity and catecholamines in the feeding network of Aplysia californica." J Comp Neurol 445(1): 29-46.
Functional consequences of neurotransmitter coexistence and cotransmission can be readily studied in certain experimentally favorable invertebrate motor systems. In this study, whole-mount histochemical methods were used to identify neurons in which gamma-aminobutyric acid (GABA)-like immunoreactivity (GABAli) was colocalized with catecholamine histofluorescence (CAh; FaGlu method) and tyrosine hydroxylase (TH)-like immunoreactivity (THli) in the feeding motor circuitry (buccal and cerebral ganglia) of the marine mollusc Aplysia californica. In agreement with previous reports, five neurons in the buccal ganglia were found to exhibit CAh. These included the paired B20 buccal-cerebral interneurons (BCIs), the paired B65 buccal interneurons, and an unpaired cell with projections to both cerebral-buccal connectives (CBCs). Experiments in which the FaGlu method was combined with the immunohistochemical detection of GABA revealed double labeling of all five of these neurons. An antibody generated against TH, the rate-limiting enzyme in the biosynthesis of catecholamines, was used to obtain an independent determination of GABA-CA colocalization. Biocytin backfills of the CBC performed in conjunction with TH immunohistochemistry revealed labeling of the rostral B20 cell pair and the unpaired CBI near the caudal surface of the right hemiganglion. THli was also present in a prominent bilateral pair of caudal neurons that were not stained with CBC backfills. On the basis of their position, size, shape, and lack of CBC projections, the lateral THli neurons were identified as B65. Double-labeling immunohistochemical experiments revealed GABAli in all five buccal THli neurons. Finally, GABAli was observed in individual B20 and B65 neurons that were identified using electrophysiological criteria and injected with a marker (neurobiotin). Similar methods were used to demonstrate that a previously identified catecholaminergic cerebral-buccal interneuron (CBI) designated CBI-1 contained THli but did not contain GABAli. Although numerous THli and GABAli neurons and fibers were present in the cerebral and buccal ganglia, additional instances of their colocalization were not observed. These findings indicate that GABA and a catecholamine (probably dopamine) are colocalized in a limited number of interneurons within the central pattern generator circuits that control feeding-related behaviors in Aplysia.

Eaton, M. J., J. P. Herman, et al. (2002). "Immortalized chromaffin cells disimmortalized with Cre/lox site-directed recombination for use in cell therapy for pain after partial nerve injury." Exp Neurol 175(1): 49-60.
To prepare immortalized adrenal chromaffin cells for eventual clinical use, the immortalizing oncogene must be removed. We have utilized a Cre-mediated excision of a loxP-flanked Tag sequence to test whether immortalized chromaffin cells could be disimmortalized by this method. Cultures of embryonic rat adrenal cells were immortalized with the tsA-TN retroviral vector encoding the loxP-flanked temperature-sensitive allele of SV40 large T antigen (tsA-TN) and a positive/negative neo/HSV-TK sequence for selection with either G418 or gancyclovir, respectively. These cells were then infected with the 1710-CrePR1 bicistronic retroviral vector coding for a form of Cre modulatable by the synthetic steroid RU486. These immortalized loxTsTag/CrePR1/RAD cells expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). After initial incubation at 37 degrees C with RU486 for 3 days, followed by the addition of gancyclovir for 7 days, Tag-ir was not detectable in most of the surviving chromaffin cells, compared to 100% expression in immortalized loxTsTag/CreR1/RAD cells not treated with RU486 and gancyclovir. The expression of TH, DbetaH, and PNMT was increased after disimmortalization and the ability of disimmortalized cells to synthesize norepinephrine was also significantly increased compared to immortalized cells. When both types of chromaffin cells were transplanted in a model of neuropathic pain and partial nerve injury, both cell grafts were equally able to reverse the behavioral hypersensitivity induced by the injury. The use of Cre/lox site-directed disimmortalization of chromaffin cells that are able to deliver neuroactive molecules offers a novel approach to cell therapy.

Ershov, P. V., M. V. Ugrumov, et al. (2002). "Differentiation of tyrosine hydroxylase-synthesizing and/or aromatic L-amino acid decarboxylase-synthesizing neurons in the rat mediobasal hypothalamus: quantitative double-immunofluorescence study." J Comp Neurol 446(2): 114-22.
In this double-immunofluorescence study, we first quantified the neurons of the arcuate nucleus as immunoreactive (+) for tyrosine hydroxylase (TH) and/or aromatic L-amino acid decarboxylase (AADC) in rats at embryonic day 21 (E21), at postnatal day 9 (P9), and in adulthood by using conventional fluorescent or confocal microscopy. On E21, monoenzymatic (TH(+)AADC immunonegative (-) and TH(-)AADC(+)) neurons and bienzymatic (TH(+)AADC(+)) neurons accounted for 99% and 1%, respectively, of the whole neuron population expressing enzymes of dopamine synthesis. Further development was characterized by the dramatic increase in TH(+)AADC(-) dorsomedial and TH(+)AADC(+) dorsomedial populations from E21 to P9 as well as by the increase in the TH(+)AADC(+) dorsomedial population (in females) and a drop in the TH(+)AADC(-) ventrolateral and TH(+)AADC(-) dorsomedial (in males) populations from P9 to adulthood. In contrast to TH(+)AADC(-) (in males) and TH(+)AADC(+) neurons, the TH(-)AADC(+) neurons did not change in number from E21 to adulthood. Thus, in rat fetuses, the neurons synthesizing TH and/or AADC were mainly monoenzymatic, whereas during postnatal life the fraction of bienzymatic neurons increased by up to 60%.

Funakoshi, K., M. Nakano, et al. (2002). "Catecholaminergic innervation of the sympathetic preganglionic cell column of the filefish Stephanolepis cirrhifer." J Comp Neurol 442(3): 204-16.
Nerve fibers immunoreactive for enzymes synthesizing catecholamines were examined in the central autonomic nucleus, a column of sympathetic preganglionic neurons, in the filefish Stephanolepis cirrhifer. Varicose nerve fibers immunoreactive for tyrosine hydroxylase were densely distributed in the rostral part, sometimes in contact with perikarya but were sparse in the caudal part of this nucleus. Fluorescent double labeling distinguished noradrenergic nerve fibers immunoreactive for both tyrosine hydroxylase and dopamine beta hydroxylase, and dopaminergic fibers immunoreactive only for tyrosine hydroxylase. In the brainstem, catecholaminergic neurons were observed in the locus coeruleus, the caudal dorsomedial reticular zone of the medulla, and the area postrema. Double labeling of tyrosine hydroxylase and dopamine beta hydroxylase showed that the neurons in the locus coeruleus were all noradrenergic, and those in the caudal dorsomedial medulla were mostly noradrenergic, whereas the area postrema contained both noradrenergic and dopaminergic neurons. No catecholaminergic neurons were found in the ventral region of the brainstem. After application of DiI to the central autonomic nucleus, retrogradely labeled neurons were seen in the caudal dorsomedial medulla but not in the locus coeruleus or the area postrema. These findings suggest that the sympathetic preganglionic neurons of the filefish may receive noradrenergic axonal projections from neurons in the caudal dorsomedial medulla. In the light of previous studies, inputs of these catecholaminergic fibers to the central autonomic nucleus may be involved in regulation of sympathetic activity of peripheral organs, together with serotoninergic and peptidergic inputs to this nucleus.

Gayle, D. A., Z. Ling, et al. (2002). "Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide." Brain Res Dev Brain Res 133(1): 27-35.
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons of the substantia nigra pars compacta (SNc). Although the exact mechanisms responsible for this cell loss are unclear, emerging evidence suggests the involvement of inflammatory events. In the present study, we characterized the effects of the proinflammatory bacteriotoxin lipopolysaccharide (LPS) on the number of tyrosine hydroxylase immunoreactive (THir) cells (used as an index for DA neurons) in primary mesencephalic cultures. LPS (10-80 microg/ml) selectively decreased THir cells and increased culture media levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as well as nitrite (an index of nitric oxide (NO) production). Cultures exposed to both LPS and neutralizing antibodies to IL-1beta or TNF-alpha showed an attenuation of the LPS-induced THir cell loss by at least 50% in both cases. Inhibition of the inducible form of nitric oxide synthase (iNOS) by L-NIL did not affect LPS toxicity, but increased the LPS-induced levels of both TNF-alpha and IL-1beta. These findings suggest that neuroinflammatory stimuli which lead to elevations in cytokines may induce DA neuron cell loss in a NO-independent manner and contribute to PD pathogenesis.

Gerhardt, G. A., W. A. Cass, et al. (2002). "Changes in somatodendritic but not terminal dopamine regulation in aged rhesus monkeys." J Neurochem 80(1): 168-77.
For these studies, young (8-9 years), middle-aged (14-17 years) and aged (23-28 years) rhesus monkeys were used as a model of normal aging in humans to investigate changes in dopamine (DA)-containing neurons in senescence. Aged monkeys exhibited significant age-related motoric declines as compared to the young animals. In vivo microdialysis studies showed that basal levels of the DA metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were diminished by 44% and 79%, respectively, in the substantia nigra (SN) of aged monkeys. In addition, d-amphetamine-evoked overflow of DA in the SN was diminished by 30% in the middle-aged animals and 67% in the aged monkeys. Post-mortem measures of DA and DA metabolites showed significant decreases in DA (20%), DOPAC (47%) and HVA (22%) levels in the putamen and a 25% decline in HVA tissue levels in the SN of the aged monkeys as compared to the young animals. Unbiased stereological cell counting of tyrosine hydroxylase (TH)-immunoreactive neurons in the SN showed a small (15-20%) but significant age-related decline in TH-positive neurons. In addition, there was a small (15-20%) but significant decline in TH-positive fiber density and TH-positive cell size. In comparison to the massive loss of DA neurons responsible for the movement dysfunctions seen in Parkinson's disease, pronounced functional changes in DA release in the SN and putamen may significantly contribute to the motoric dysfunctions characterizing normal aging in rhesus monkeys.

Glavan, G., D. Sket, et al. (2002). "Modulation of neuroleptic activity of 9,10-didehydro-N-methyl-(2-propynyl)-6-methyl-8-aminomethylergoline bimaleinate (LEK-8829) by D1 intrinsic activity in hemi-parkinsonian rats." Mol Pharmacol 61(2): 360-8.
Parkinsonism, a common unwanted side effect of typical antipsychotic (neuroleptic) drugs, is induced by the blockade of striatal dopamine D2 receptors. In rats with hemi-parkinsonism induced by unilateral lesion of dopaminergic nigrostriatal neurons with 6-hydroxydopamine, D2 antagonists inhibit contralateral turning induced by D2 agonists and augment the levels of neurotensin mRNA in dopaminergically intact striatum. By contrast, D1 agonists induce contralateral turning and augment neurotensin mRNA levels in dopamine-depleted striatum. These effects could be inhibited by D1 but not by D2 antagonists. Here we used a hemi-parkinsonian model to investigate the effects of putative D1 agonist/D2 antagonist LEK-8829 (9,10-didehydro-N-methyl-(2-propynyl)-6-methyl-8-aminomethylergoline bimaleinate), an experimental antipsychotic, on turning behavior and the expression of striatal neurotensin, preprotachykinin and neurotransmitter-induced early gene protein 4 (ania-4) mRNAs. We found that LEK-8829 inhibited contralateral turning induced by D2 agonist quinpirole, but only if the rats were cotreated with D1 antagonist SCH-23390. In situ hybridization showed that LEK-8829 induced the expression of neurotensin and ania-4 mRNAs in dopamine-intact striatum that could be completely blocked only by the combined treatment with SCH-23390 and quinpirole. In addition, LEK-8829 augmented the expression of neurotensin, preprotachykinin and ania-4 mRNAs in dopamine-depleted striatum that could be completely blocked by SCH-23390. This study clearly demonstrates that in hemi-parkinsonian rats D1 agonistic activity of LEK-8829 confers its anti-parkinsonian drug-like properties and modulates its neuroleptic drug-like properties, which are dependent on the blockade of dopamine D2 receptors. These findings imply that atypical antipsychotics with D1 intrinsic activity might have a reduced propensity for the induction of extrapyramidal syndrome.

Gouhier, C., S. Chalon, et al. (2002). "Protection of dopaminergic nigrostriatal afferents by GDNF delivered by microspheres in a rodent model of Parkinson's disease." Synapse 44(3): 124-31.
The use of glial cell line-derived neurotrophic factor (GDNF) appears to be a promising strategy to promote survival and function of the nigrostriatal dopaminergic pathway damaged in Parkinson's disease (PD). However, effective intracerebral administration is required for optimal therapeutic benefit and tools to evaluate such therapies must be developed. A rodent model of PD was therefore developed using striatal injection of 6-hydroxydopamine (6-OHDA) with simultaneous implantation of GDNF-delivering microspheres. The effects of GDNF released from microspheres were assessed by classical methods such as amphetamine-induced rotating behavior and tyrosine hydroxylase (TH) immunoreactivity, as well as by quantitative autoradiography using PE2I, a dopamine transporter (DAT) radiotracer, which is also suitable for SPET imaging in humans. 6-OHDA-lesioned animals that received microspheres without GDNF were used as controls. During the first 3 weeks after simultaneous lesion and implantation, the amphetamine-induced rotating behavior of GDNF-treated rats was improved compared to controls and an increase in TH expression (+26%) was measured in the striatum 6 weeks after lesion. In accordance with these results, an increase in striatal PE2I-labeled DAT density was obtained (+17%) after 3 and 6 weeks of treatment. In conclusion, this study demonstrates the neuroprotective action of GDNF delivered by microspheres and suggests that PE2I may be an appropriate radiotracer for use in SPET scintigraphy to follow up treatment of PD in humans.

Grattan-Smith, P. J., R. A. Wevers, et al. (2002). "Tyrosine hydroxylase deficiency: clinical manifestations of catecholamine insufficiency in infancy." Mov Disord 17(2): 354-9.
Inborn errors of catecholamine biosynthesis are rare but of great interest as they are genetic disorders, and in some, treatment may completely reverse severe neurological abnormalities. They also provide insights into the action of the biogenic amines in the developing brain. We describe the clinical course of an infant with tyrosine hydroxylase (TOH) deficiency over a 30-month period. The parents are consanguineous, and genetic analysis revealed the infant to be homozygous for the common G698A mutation in the TOH gene. TOH deficiency can be seen as a model of pure catecholamine deficiency. Experimental evidence, reports of other disorders of biogenic amines, and our experience with this infant suggest that the symptoms of catecholamine deficiency in infancy can be broadly subdivided. Signs of dopamine deficiency include tremor, hypersensitivity to levadopa (L-dopa) therapy, oculogyric crises, akinesia, rigidity, and dystonia. Manifestations of norepinephrine deficiency include ptosis, miosis, profuse oropharyngeal secretions, and postural hypotension. Hypersensitivity to L-dopa was a particular management problem in this infant.

Iravani, M. M., K. Kashefi, et al. (2002). "Involvement of inducible nitric oxide synthase in inflammation-induced dopaminergic neurodegeneration." Neuroscience 110(1): 49-58.
The loss of dopaminergic neurones in the substantia nigra with Parkinson's disease may result from inflammation-induced proliferation of microglia and reactive macrophages expressing inducible nitric oxide synthase (iNOS). We have investigated the effects of the supranigral administration of lipopolysaccharide on iNOS-immunoreactivity, 3-nitrotyrosine formation and tyrosine hydroxylase-immunoreactive neuronal number, and retrogradely labelled fluorogold-positive neurones in the ventral mesencephalon in male Wistar rats. Following supranigral lipopolysaccharide injection, 16-18 h previously, there was intense expression of NADPH-diaphorase and iNOS-immunoreactivity in non-neuronal, macrophage-like cells. This was accompanied by intense expression of glial fibrillary acidic protein-immunoreactive astrocytosis in the substantia nigra. There were also significant reductions in the number of tyrosine hydroxylase(50-60%)- and fluorogold (65-75%)-positive neurones in the substantia nigra. In contrast, tyrosine hydroxylase-immunoreactivity in the ventral tegmental area was not altered. Pre-treatment of animals with the iNOS inhibitor, S-methylisothiourea (10 mg kg(-1), i.p.), led to a significant reduction of lipopolysaccharide-induced cell death. Similar reduction of tyrosine hydroxylase-immunoreactivity and fluorogold-labelled neurones in the substantia nigra following lipopolysaccharide administration suggests dopaminergic cell death rather than down-regulation of tyrosine hydroxylase. We conclude that the expression of iNOS- and 3-nitrotyrosine-immunoreactivity and reduction of cell death by S-methylisothiourea suggest the effects of lipopolysaccharide may be nitric oxide-mediated, although other actions of lipopolysaccharide (independent of iNOS induction) cannot be ruled out.

Jeyarasasingam, G., L. Tompkins, et al. (2002). "Stimulation of non-alpha7 nicotinic receptors partially protects dopaminergic neurons from 1-methyl-4-phenylpyridinium-induced toxicity in culture." Neuroscience 109(2): 275-85.
Previous work has shown that nicotine treatment protects against nigrostriatal degeneration in rodents, findings that may be of relevance to the decreased incidence of Parkinson's disease in cigarette smokers. In the present studies, we investigated the effect of nicotine against 1-methyl-4-phenylpyridinium-induced toxicity in dopaminergic ventral mesencephalic cultures to identify the nicotinic receptor population that may be involved. [3H]Epibatidine, a ligand that binds to receptors containing alpha2-alpha6 subunits, bound to at least two populations of sites that were up-regulated by nicotine in a time and dose dependent manner. We next examined the effect of nicotine on cultures treated with 1-methyl-4-phenylpyridinium, a neurotoxin that selectively damages nigrostriatal dopaminergic neurons. Pre-treatment with nicotine, at 10(-7)-10(-4) M, partially prevented the toxin-induced decline in dopaminergic cells. Pre-exposure to nicotine for 24 h resulted in optimal protection, suggesting that receptor up-regulation may contribute to the observed neuroprotective effect. Nicotine-mediated protection was blocked by pre-incubation with the nicotinic receptor antagonist d-tubocurarine (10(-4) M), but not the alpha7 receptor-selective antagonist alpha-bungarotoxin (10(-7) M).Our results show that nicotinic receptor activation partially protects nigral dopaminergic neurons from 1-methyl-4-phenylpyridinium-induced toxicity in culture and that this appears to occur through an interaction at non-alpha7 containing receptors.

Johansson, S. and I. Stromberg (2002). "Guidance of dopaminergic neuritic growth by immature astrocytes in organotypic cultures of rat fetal ventral mesencephalon." J Comp Neurol 443(3): 237-49.
Astrocytes, with their many functions in producing and controlling the environment in the brain, are of great interest when it comes to studying regeneration after injury and neurodegenerative diseases such as in grafting in Parkinson's disease. This study was performed to investigate astrocytic guidance of growth derived from dopaminergic neurons using organotypic cultures of rat fetal ventral mesencephalon. Primary cultures were studied at different time points starting from 3 days up to 28 days. Cultures were treated with either interleukin-1 beta (IL-1 beta), which has stimulating effects on astrocytic proliferation, or the astrocytic inhibitor cytosine arabinoside (Ara-C). Tyrosine hydroxylase (TH)-immunohistochemistry was used to visualize dopaminergic neurons, and antibodies against glial fibrillary acidic protein (GFAP) and S100 beta were used to label astrocytes. The results revealed that a robust TH-positive nerve fiber production was seen already at 3 days in vitro. These neurites had disappeared by 5 days. This early nerve fiber outgrowth was not guided by direct interactions with glial cells. Later, at 7 days in vitro, a second wave of TH-positive neuritic outgrowth was clearly observed. GFAP-positive astrocytic processes guided these neurites. TH-positive neurites arborized overlying S100 beta-positive astrocytes in an area distal to the GFAP-positive astrocytic processes. Treatment with IL-1 beta resulted in an increased area of TH-positive nerve fiber network. In cultures treated with Ara-C, neither astrocytes nor outgrowth of dopaminergic neurites were observed. In conclusion, this study shows that astrocytes play a major role in long-term dopaminergic outgrowth, both in axonal elongation and branching of neurites. The long-term nerve fiber growth is preceded by an early transient outgrowth of dopamine neurites.

Joseph, V., J. Soliz, et al. (2002). "Dopaminergic metabolism in carotid bodies and high-altitude acclimatization in female rats." Am J Physiol Regul Integr Comp Physiol 282(3): R765-73.
We tested the hypothesis that ovarian steroids stimulate breathing through a dopaminergic mechanism in the carotid bodies. In ovariectomized female rats raised at sea level, domperidone, a peripheral D2-receptor antagonist, increased ventilation in normoxia (minute ventilation = +55%) and acute hypoxia (+32%). This effect disappeared after 10 daily injections of ovarian steroids (progesterone + estradiol). At high altitude (3,600 m, Bolivian Institute for High-Altitude Biology-IBBA, La Paz, Bolivia), neutered females had higher carotid body tyrosine hydroxylase activity (the rate-limiting enzyme for catecholamine synthesis: +129%) and dopamine utilization (+150%), lower minute ventilation (-30%) and hypoxic ventilatory response (-57%), and higher hematocrit (+18%) and Hb concentration (+21%) than intact female rats. Consistent signs of arterial pulmonary hypertension (right ventricular hypertrophy) also appeared in ovariectomized females. None of these parameters was affected by gonadectomy in males. Our results show that ovarian steroids stimulate breathing by lowering a peripheral dopaminergic inhibitory drive. This process may partially explain the deacclimatization of postmenopausal women at high altitude.

Kawasaki, H., H. Suemori, et al. (2002). "Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity." Proc Natl Acad Sci U S A 99(3): 1580-5.
We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH(+)) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH(+) neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH(+) neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (approximately 5 weeks). In addition, 8% of the colonies contain large patches of Pax6(+)-pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinson's disease and retinitis pigmentosa.

Kirik, D., C. Rosenblad, et al. (2002). "Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system." J Neurosci 22(7): 2780-91.
Recombinant adeno-associated viral vectors display efficient tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated human alpha-synuclein in the nigrostriatal dopamine neurons of adult rats for up to 6 months. Cellular and axonal pathology, including alpha-synuclein-positive cytoplasmic inclusions and swollen, dystrophic neurites similar to those seen in brains from patients with Parkinson's disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine neurons and were not observed in other nondopaminergic neurons transduced by the same vectors. The degenerative changes were accompanied by a loss of 30-80% of the nigral dopamine neurons, a 40-50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell loss exceeded a critical threshold of 50-60%. At 6 months, signs of cell body and axonal pathology had subsided, suggesting that the surviving neurons had recovered from the initial insult, despite the fact that alpha-synuclein expression was maintained at a high level. These results show that nigral dopamine neurons are selectively vulnerable to high levels of either wild-type or mutant alpha-synuclein, pointing to a key role for alpha-synuclein in the pathogenesis of Parkinson's disease. Targeted overexpression of alpha-synuclein in the nigrostriatal system may provide a new animal model of Parkinson's disease that reproduces some of the cardinal pathological, neurochemical, and behavioral features of the human disease.

Kirik, D., B. Georgievska, et al. (2002). "Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer." Proc Natl Acad Sci U S A 99(7): 4708-13.
Intrastriatal delivery of the tyrosine hydroxylase gene by viral vectors is being explored as a tool for local delivery of L-dopa in animals with lesions of the nigrostriatal pathway. The functional effects reported using this approach have been disappointing, probably because the striatal L-dopa levels attained have been too low. In the present study, we have defined a critical threshold level of L-dopa, 1.5 pmol/mg of tissue, that has to be reached to induce any significant functional effects. Using new generation high-titer recombinant adeno-associated virus vectors, we show that levels of striatal L-dopa production exceeding this threshold can be obtained provided that tyrosine hydroxylase is coexpressed with the cofactor synthetic enzyme, GTP-cyclohydrolase-1. After striatal transduction with this combination of vectors, substantial functional improvement in both drug-induced and spontaneous behavior was observed in rats with either complete or partial 6-hydroxydopamine lesions of the nigrostriatal pathway. However, complete reversal of motor deficits occurred only in animals in which part of the striatal dopamine innervation was left intact. Spared nigrostriatal fibers thus may convert L-dopa to dopamine and store and release dopamine in a more physiologically relevant manner in the denervated striatum to mediate better striatal output-dependent motor function. We conclude that intrastriatal L-dopa delivery may be a viable strategy for treatment and control of adverse side effects associated with oral L-dopa therapy such as on-off fluctuations and drug-induced dyskinesias in patients with Parkinson's disease.

Kloberg, A. J. and R. Fritsche (2002). "Catecholamines are present in larval Xenopus laevis: a potential source for cardiac control." J Exp Zool 292(3): 293-303.
Changes in noradrenaline (NA), adrenaline (A), and dopamine (DA) levels in the heart, kidneys, and whole body (without heart and kidneys) during embryonic development were investigated in the frog, Xenopus laevis using high-performance liquid chromatography (HPLC). In addition, the presence of cells immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and/or phenylethanolamine-N-methyltransferase (PNMT) in the heart of Xenopus larvae was investigated using immunohistochemical techniques. The presence of nerve fibers was visualized using antibodies against acetylated tubulin (AcT). NA and DA concentrations in the heart were low and steady in NF 40-56, showed an increased value at NF 57, and decreased again in froglets. A trend toward higher concentrations of A was observed at NF 43-49 and NF 57. Cells immunoreactive to TH, DBH, and PNMT were found in the heart from NF 40, and the TH immunoreactive cells became more abundant in the whole heart at later stages. The presence of catecholamines in the non-innervated larval heart together with the finding of TH/DBH/PNMT immunoreactive cells suggests that catecholamines are synthesized and stored in the heart and could therefore have a paracrine role in cardiac control in Xenopus larvae. Detectable concentrations of catecholamines were also found in kidneys and whole bodies (except heart and kidneys). Therefore, catecholamine-producing cells outside the heart can be an important source of circulating catecholamines involved in adrenergic cardiac control in Xenopus larvae.

Kuhn, D. M. and T. J. Geddes (2002). "Reduced nicotinamide nucleotides prevent nitration of tyrosine hydroxylase by peroxynitrite." Brain Res 933(1): 85-9.
Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine (DA). TH activity is inhibited by peroxynitrite (ONOO(-)) by a mechanism that involves nitration of tyrosine residues and oxidation of cysteine residues in the enzyme. Reduced forms of the nicotinamide adenine dinucleotide cofactors, NADH and NADPH, protect TH from inhibition by ONOO(-) and prevent nitration of tyrosine residues. NAD, the oxidized form of the cofactors, neither protects TH from ONOO(-)-induced inhibition nor prevents the nitration of tyrosine residues in the enzyme. These results suggest that the redox status of the nicotinamide nucleotide cofactors could influence the ability of ONOO(-) to modify proteins that are important to the function of DA neurons.

Kuhn, D. M., M. Sadidi, et al. (2002). "Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis." J Biol Chem 277(16): 14336-42.
Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues.

Lastres-Becker, I., H. H. Hansen, et al. (2002). "Alleviation of motor hyperactivity and neurochemical deficits by endocannabinoid uptake inhibition in a rat model of Huntington's disease." Synapse 44(1): 23-35.
Recent studies have demonstrated a loss of cannabinoid CB1 receptors in the postmortem basal ganglia of patients affected by Huntington's disease (HD) and in transgenic mouse models for this disease. These studies have led to the notion that substances that increase the endocannabinoid activity, such as receptor agonists or inhibitors of endocannabinoid uptake and/or metabolism, might be useful in the treatment of hyperkinetic symptoms of this disease. In the present study, we employed a rat model of HD generated by bilateral intrastriatal injections of 3-nitropropionic acid (3-NP), a toxin that selectively damages striatal GABAergic efferent neurons. These rats exhibited biphasic motor disturbances, with an early (1-2 weeks) hyperactivity followed by a late (3-4 weeks) motor depression. Analysis of GABA, dopamine, and their related enzymes, glutamic acid decarboxylase and tyrosine hydroxylase, in the basal ganglia proved marked decreases compatible with the motor hyperkinesia. In addition, mRNA levels for CB1 receptor, neuronal-specific enolase, proenkephalin, and substance P decreased in the caudate-putamen of 3-NP-injected rats. There were also reductions in CB1 receptor binding in the caudate putamen, the globus pallidus, and, to a lesser extent, the substantia nigra. By contrast, mRNA levels for tyrosine hydroxylase in the substantia nigra remained unaffected. Interestingly, the administration of AM404, an inhibitor of endocannabinoid uptake, to 3-NP-injected rats attenuated motor disturbances observed in the early phase of hyperactivity. Administration of AM404 also tended to induce recovery from the neurochemical deficits caused by the toxin in GABA and dopamine indices in the basal ganglia. In summary, morphological, behavioral, and biochemical changes observed in rats intrastriatally lesioned with 3-NP acid were compatible with a profound degeneration of striatal efferent GABAergic neurons, similar to that occurring in the brain of HD patients. As expected, a loss of CB1 receptors was evident in the basal ganglia of these rats. However, the administration of substances that increase endocannabinoid activity, by inhibiting the uptake process, allowed an activation of the remaining population of CB1 receptors, resulting in a significant improvement of motor disturbances and neurochemical deficits. These observations might be relevant to the treatment of hyperkinetic symptoms in HD, a human disorder with unsatisfactory symptomatic treatment for most patients.

Li, X. J., J. Gu, et al. (2002). "Melatonin attenuates MPTP-induced dopaminergic neuronal injury associated with scavenging hydroxyl radical." J Pineal Res 32(1): 47-52.
To clarify the relationship between melatonin's hydroxyl radical (*OH) scavenging ability and its protective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal injury, in the present study, the salicylate trapping method combined with high-performance liquid chromatography (HPLC)-electrochemical detection were used to measure the contents of dihydroxybenzoic acid (DHBA) and dopamine (DA) in brain tissues of C57BL/6 mice. Immunocytohistochemistry was used to detect tyrosine hydroxylase (TH)-like positive staining neurons. Results show that MPTP treatment induced an increase in the content of DHBA and decrease in the level of DA as well as the number of TH positive stained neurons in the mouse brain. However, melatonin dose-dependently inhibited the increase of DHBA levels in ventral midbrain tissues, the decrease of DA content and the loss of dopaminergic neurons. Moreover, the relationship between the changes of DHBA and DA levels in the brain of mice following MPTP and melatonin treatment showed a statistically significant negative correlation. Present results suggest that melatonin can ameliorate MPTP-induced dopaminergic neuronal lesions probably, at least partially, because of its inhibition of *OH generation.

Lindgren, N., M. Goiny, et al. (2002). "Activation of extracellular signal-regulated kinases 1 and 2 by depolarization stimulates tyrosine hydroxylase phosphorylation and dopamine synthesis in rat brain." Eur J Neurosci 15(4): 769-73.
Production of dopamine is regulated via phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines. Here we have used a preparation of rat striatal slices to examine the involvement of two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), in the depolarization-dependent regulation of TH phosphorylation and dopamine synthesis. Depolarization with elevated KCl (45 mm) caused an increase in the phosphorylation state and, thereby, activation of ERK1/2. The same stimulus also increased TH phosphorylation at Ser19, Ser31 and Ser40 (measured using site- and phospho-specific antibodies) and TH activity [measured as 3,4-dihydroxyphenylalanine (DOPA) accumulation]. A MAPK/ERK kinase inhibitor, PD098059, decreased the basal levels of phospho-ERK1/2 and prevented the increase in ERK1/2 phosphorylation induced by depolarization. PD098059 also decreased both basal and depolarization-induced phosphorylation of TH at Ser31 and reduced the increase in Ser40 phosphorylation induced by high potassium, but did not affect Ser19 phosphorylation. PD098059 alone inhibited basal TH activity and decreased the accumulation of DOPA induced by depolarization. These data provide evidence for the involvement of ERK1/2 in the regulation of the state of phosphorylation of TH at Ser31 and Ser40 and a correlation between ERK1/2-dependent phosphorylation of TH and stimulation of dopamine synthesis in the brain.

Ling, Z., D. A. Gayle, et al. (2002). "In utero bacterial endotoxin exposure causes loss of tyrosine hydroxylase neurons in the postnatal rat midbrain." Mov Disord 17(1): 116-24.
We investigated whether in utero exposure to the Gram(-) bacteriotoxin lipopolysaccharide (LPS) induces dopamine (DA) neuron loss in rats. The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) kills DA neurons and is elevated in the brains of patients with Parkinson's disease (PD). LPS is a potent inducer of TNF-alpha, and both are increased in the chorioamniotic environment of women who have bacterial vaginosis (BV) during pregnancy, suggesting that BV might interfere with the normal development of fetal DA neurons. Gravid female rats were injected intraperitoneally with either LPS or normal saline at embryonic day 10.5 and their pups were killed at postnatal day 21. The brains of the pups were assessed for DA and TNF-alpha levels and DA cell counts in the mesencephalon using tyrosine hydroxylase immunoreactive (THir) cells as a DA neuron marker. Prenatal LPS exposure significantly reduced striatal DA (29%) and increased DA activity (72%) as well as TNF-alpha (101%). Stereological cell counts in the mesencephalon were also significantly reduced (27%) by prenatal LPS exposure. Prenatal exposure to LPS, as might occur in humans with BV, produces a significant loss of THir cells in rats that is still present 33 days following a single injection of LPS. Since this cell loss is well past the normal phase of DA neuron apoptosis that occurs in early postnatal life, rats so exposed may have a permanent loss of DA neurons, suggesting that prenatal infections may represent risk factors for PD.

Maloney, K. J., L. Mainville, et al. (2002). "c-Fos expression in dopaminergic and GABAergic neurons of the ventral mesencephalic tegmentum after paradoxical sleep deprivation and recovery." Eur J Neurosci 15(4): 774-8.
Evidence suggests that dopaminergic neurons of the ventral mesencephalic tegmentum (VMT) could be important for paradoxical sleep (PS). Here, we examined whether dopamine (DA) and adjacent gamma-aminobutyric acid (GABA)-synthesizing neurons are active in association with PS recovery as compared to PS deprivation or control conditions in different groups of rats by using c-Fos expression as a reflection of neural activity, combined with dual immunostaining for tyrosine hydroxylase (TH) or glutamic acid decarboxylase (GAD). Numbers of TH+/c-Fos+ neurons in the substantia nigra (SN) were not significantly different across groups, whereas those in the ventral tegmental area (VTA) were significantly different and greatest in PS recovery. Numbers of GAD+/c-Fos+ neurons in both VTA and SN were greatest in PS recovery. Thus, DA neuronal activity does not appear to be suppressed by local GABAergic neuronal activity during PS but might be altered in pattern by this inhibitory as well as other excitatory, particularly cholinergic, inputs such as to allow DA VTA neurons to become maximally active during PS and thereby contribute to the unique physiological and cognitive aspects of that state.

Mengual, E. and V. M. Pickel (2002). "Ultrastructural immunocytochemical localization of the dopamine D2 receptor and tyrosine hydroxylase in the rat ventral pallidum." Synapse 43(3): 151-62.
The mesopallidal dopamine system plays a role in locomotor activity and reward. To understand the potential contribution of the dopamine D2 receptor (D2R) to the action of dopamine in the ventral pallidum (VP), we used electron microscopic immunocytochemistry to examine the cellular and subcellular localization of an antipeptide antiserum against the D2R in both ventromedial and dorsolateral VP compartments. In each region the majority of the total D2R-labeled profiles (n = 1,132) were axon terminals (55%) and small unmyelinated axons (27%). These terminals were often apposed to other axon terminals or dendrites and formed almost exclusively symmetric, inhibitory-type axodendritic synapses. Immunogold D2R labeling in axon terminals was seen on the plasmalemma and membranes of nearby synaptic vesicles. In ventral pallidal sections processed for dual detection of D2R peptide and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH), D2R labeling was detected in a few axons and axon terminals containing TH immunoreactivity as well as in axons contacted by TH-labeled terminals. In most cases, however, the D2R-labeled profiles were located at a distance from small axons and terminals containing TH. Our results provide the first ultrastructural evidence that D2Rs in the two VP subterritories are strategically located for primary involvement in modulation of the presynaptic release of nondopaminergic inhibitory transmitters. They also suggest that in this region the presynaptic D2 receptors are 1) minimally involved in autoregulation of dopaminergic transmission, and 2) differentially activated by dopamine, depending in part on levels and distance from release sites.

Metzger, M., S. Jiang, et al. (2002). "A quantitative immuno-electron microscopic study of dopamine terminals in forebrain regions of the domestic chick involved in filial imprinting." Neuroscience 111(3): 611-623.
The mediorostral neostriatum/hyperstriatum ventrale and neostriatum dorsocaudale of the domestic chick are crucially involved in filial imprinting and are major targets of mesotelencephalic dopaminergic projections. To better understand the functional role of dopamine in these forebrain regions, the ultrastructure of dopamine terminals was studied by serial section electron microscopy using immunohistochemical labeling with antibodies to tyrosine hydroxylase and dopamine.At light as well as electron microscopic level, dopamine and tyrosine hydroxylase-immunoreactive fibers were present at moderate densities in the mediorostral neostriatum/hyperstriatum ventrale and high densities in the neostriatum dorsocaudale. The frequency of tyrosine hydroxylase-immunoreactive profiles per unit area was significantly higher in the neostriatum dorsocaudale than in the mediorostral neostriatum/hyperstriatum ventrale. In both regions, tyrosine hydroxylase-immunoreactive terminals were relatively small, with mean areas of 0.55 &mgr;m(2) in the mediorostral neostriatum/hyperstriatum ventrale and 0.48 &mgr;m(2) in the neostriatum dorsocaudale. The majority of tyrosine hydroxylase-immunoreactive synapses were symmetrical (83% in the mediorostral neostriatum/hyperstriatum ventrale, 75% in the neostriatum dorsocaudale) as opposed to asymmetrical (17 and 25%, respectively), but there were also tyrosine hydroxylase-immunoreactive terminals which lacked clear synaptic specializations. The preferred targets of the synaptic tyrosine hydroxylase-immunoreactive terminals were dendritic shafts (64% in the mediorostral neostriatum/hyperstriatum ventrale, 63% in the neostriatum dorsocaudale) and less frequently dendritic spines (17 and 23%, respectively) or perikarya (19 and 14%, respectively). In both forebrain regions, immunoreactive terminals were often found in close apposition to unstained terminals making asymmetrical synapses.In conclusion, these results indicate that the ultrastructural features of dopamine terminals in the avian telencephalon are very similar to those described in mammals and that dopamine may exert its effects primarily by modulating excitatory inputs.

Morimoto, K., R. Miyatake, et al. (2002). "Delusional disorder. Molecular genetic evidence for dopamine psychosis." Neuropsychopharmacology 26(6): 794-801.
Since delusional disorder is characterized by mono-symptomatic paranoid symptoms, it can be a good clinical model for investigating the dopaminergic mechanism responsible for paranoid symptoms. We examined neuroleptic responses, plasma homovanillic acid (pHVA) and genes of the dopamine receptor (DR) and its synthesizing enzyme (tyrosine hydroxylase: TH) in patients with delusional disorder and compared them with those of schizophrenic patients and healthy controls. RESULTS: (1) A relatively small dose of haloperidol was more effective for delusional disorder than for schizophrenia. (2) The pretreatment level of pHVA was higher in patients with persecution-type, but not in those with jealousy-type delusional disorder, compared with age- and sex-matched controls. This increased pHVA level was decreased eight weeks after successful haloperidol treatment. (3) The genotype frequency of the DRD2 gene Ser311Cys was significantly higher in patients with persecution-type delusional disorder (21%), compared with schizophrenic patients (6%) or controls (6%). (4) Patients homozygous for the DRD3 gene Ser9Ser had higher pretreatment levels of pHVA than those heterozygous for Ser9Gly. (v) A significant positive correlation was found between the polymorphic (TCAT)(n) repeat in the first intron of the TH gene and pretreatment levels of pHVA in delusional disorder. We suggest that delusional disorder, especially the persecution-type, includes a "dopamine psychosis," and that polymorphism of the DRD2, DRD3 and/or TH gene is part of the genetic basis underlying the hyperdopaminergic state that produces paranoid symptoms. Further studies on a large sample size are required.

Muramatsu, S., K. Fujimoto, et al. (2002). "Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes." Hum Gene Ther 13(3): 345-54.
One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.

Nagy, L. and L. Hiripi (2002). "Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust." Neurochem Int 41(1): 9-16.
The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120pmol/100&mgr;l haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12&mgr;M and V(max)=24pmol per ganglion/10min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.

Nocjar, C., B. L. Roth, et al. (2002). "Localization of 5-HT(2A) receptors on dopamine cells in subnuclei of the midbrain A10 cell group." Neuroscience 111(1): 163-76.
Considerable evidence suggests that a dysfunction of the dopamine and serotonin (5-hydroxytryptamine or 5-HT) neurotransmitter systems contributes to a diverse range of pathological conditions including schizophrenia, depression and drug abuse. Recent electrophysiological and behavioral studies suggest that 5-HT modulates dopaminergic neurons in the ventral tegmental area via activation of 5-HT(2A) receptors. It is currently unknown if 5-HT(2A) receptors mediate their actions on dopaminergic neurons in the ventral tegmental area via direct or indirect mechanisms. This study investigated whether 5-HT(2A) receptors were localized on dopamine cells within the A10 dopamine subnuclei of the rat, including the ventral tegmental area. We discovered that 5-HT(2A) receptor-like immunoreactivity colocalized with tyrosine hydroxylase, a marker for dopamine neurons, throughout the A10 dopamine cell population. Colocalization was most prominent in rostral and mid A10 regions, including the paranigral, parabrachial, and interfascicular subnuclei. Though more rare, non-dopaminergic neurons also expressed 5-HT(2A) receptor immunoreactivity in the ventral tegmental area. Additionally, although a dense population of 5-HT(2A) immunoreactive cells was observed in the rostral dorsal raphe nucleus, rarely were these cells immunoreactive for tyrosine hydroxylase. The linear raphe A10 dopamine subdivisions also displayed a low degree of 5-HT(2A) receptor and tyrosine hydroxylase colocalization.These findings provide an anatomical basis for the physiological modulation of dopamine neurons in the rostral ventral tegmental area either directly, by 5-HT(2A) receptors localized on dopamine cells, or indirectly, through a non-dopaminergic mechanism. Interestingly, 5-HT(2A) receptors were expressed on dopamine neurons in several A10 subnuclei that project to mesolimbic forebrain regions implicated in drug addiction, and recent evidence indicates that ventral tegmental area 5-HT(2A) receptor activation may modulate reward-related behavior in rodents. 5-HT(2A) receptors were also expressed on dopamine cells in A10 subnuclei that project to forebrain areas that have been implicated in schizophrenia, and atypical antipsychotic drugs have high affinities for 5-HT(2A) receptors. Thus, findings in this study could have important implications for understanding 5-HT and dopamine circuitry dysfunction in schizophrenia.

Norton, C. S., C. R. Neal, et al. (2002). "Nociceptin/orphanin FQ and opioid receptor-like receptor mRNA expression in dopamine systems." J Comp Neurol 444(4): 358-68.
Although nociceptin/orphanin FQ (N/OFQ) influences dopamine (DA) neuronal activity, it is not known whether N/OFQ acts directly on DA neurons, indirectly by means of local circuitry, or both. We used two parallel approaches, dual in situ hybridization (ISH) and neurotoxic lesions of DA neurons by using 6-hydroxydopamine (6-OHDA), to ascertain whether N/OFQ and the N/OFQ receptor (NOP) mRNA are expressed in DA neurons in the ventral tegmental area (VTA) and substantia nigra compacta (SNc). In the VTA and SNc, small populations (approximately 6-10%) of N/OFQ-containing neurons coexpressed mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Similarly, very few (1-2%) TH-positive neurons contained N/OFQ mRNA signal. A majority of NOP-positive neurons (approximately 75%) expressed TH mRNA and roughly half of the TH-containing neurons expressed NOP mRNA. Many N/OFQ neurons (approximately 50-60%) expressed glutamic acid decarboxylase 65 and 67 mRNAs, markers for gamma-aminobutyric acid (GABA) neurons. In the 6-OHDA lesion studies, NOP mRNA levels were nearly 80 and 85% lower in the VTA and SNc, respectively, on the lesioned side. These lesions appear to lead to compensatory changes, with N/OFQ mRNA levels approximately 60% and 300% higher in the VTA and SNc, respectively, after 6-OHDA lesions. Finally, N/OFQ-stimulated [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate levels were decreased in the VTA and SNc but not the prefrontal cortex after 6-OHDA lesions. Accordingly, it appears that N/OFQ mRNA was found largely on nondopaminergic (i.e., GABA) neurons, whereas NOP mRNA was located on DA neurons. N/OFQ is in a position to influence DA neuronal activity by means of the NOP located on DA neurons.

Oo, T. F., R. Siman, et al. (2002). "Distinct nuclear and cytoplasmic localization of caspase cleavage products in two models of induced apoptotic death in dopamine neurons of the substantia nigra." Exp Neurol 175(1): 1-9.
An emerging theme in programmed cell death (PCD) of neurons is that the mechanisms involved depend on the cellular context and the death-inducing stimulus. One particular class of neurons for which it is important to identify the mechanisms of PCD are the dopamine neurons of the substantia nigra, the neurons which degenerate in Parkinson's disease. PCD has been shown to occur in these neurons during normal development and to be induced in neurotoxin models of parkinsonism. Conventional histologic stains and TUNEL labeling have not revealed morphologic differences in the apoptosis observed in these neurons in any context. We now show that in two models of induced PCD in postmitotic dopamine neurons, one induced by early striatal target injury and another induced by the neurotoxin 6-hydroxydopamine (6OHDA), there are differences in the cellular localization and type of caspase cleavage products. Using two antibodies to caspase cleavage products (fractin and AB127), we show that in the target lesion model immunostaining is localized to the nucleus, whereas in the 6OHDA model intense cytoplasmic as well as nuclear staining is observed. Another antibody, AB246, to a caspase cleavage product of spectrin, immunostains apoptotic profiles only in the 6OHDA model. These findings suggest that the cellular compartment and therefore the role of the caspases may differ in apoptosis induced in pathologic settings, such as that due to neurotoxins, from that observed in models of natural or induced natural cell death. It will be important to recognize these differences in the consideration of caspase inhibitors in the treatment of degenerative neurologic disease.

Pendleton, R. G., A. Rasheed, et al. (2002). "Effects of tyrosine hydroxylase mutants on locomotor activity in Drosophila: a study in functional genomics." Behav Genet 32(2): 89-94.
The brain of the adult fruit fly, Drosophila melanogaster, contains tyrosine hydroxylase, the rate-limiting enzyme required for catecholamine biosynthesis, as well as dopa decarboxylase. Catecholamines, principally dopamine, are also present. We have previously shown that pharmacological inhibition of tyrosine hydroxylase with alpha-methyl-p-tyrosine results in a dose-related inhibition of locomotor activity in adult organisms. Similar results were found with reserpine, a well-known inhibitor of catecholamine uptake into storage granules. The drug-induced inhibition could be prevented in each case by the concomitant administration of L-dopa. The single-copy gene coding for tyrosine hydroxylase in Drosophila is pale (ple). Both null and temperature-sensitive loss of function mutant alleles of ple are recessive embryonic lethals. Heterozygous null mutant flies have normal locomotor activity demonstrating that only a single dose of the wild type form of ple is required to support normal function. Both hemizygous and homozygous temperature-sensitive ple mutants (ple(ts1)) also show normal locomotor activity at the permissive temperature for this mutant allele (18 degrees C), which progressively declines as the temperature is increased to its restrictive level (29 degrees C). These abnormal locomotor effects are reversible by L-dopa. Thus the effects on locomotor activity resulting from the pharmacological inhibition of catecholamine synthesis or storage are the same as those resulting from lack of tyrosine hydroxylase expression. These findings indicate that brain catecholamine loss decreases locomotor activity in the fly, as it does in mammals, and demonstrate the ability of functional genomic studies to mimic that of pharmacological inhibition of enzyme function or other similar processes.

Pendleton, R. G., F. Parvez, et al. (2002). "Effects of pharmacological agents upon a transgenic model of Parkinson's disease in Drosophila melanogaster." J Pharmacol Exp Ther 300(1): 91-6.
The human gene that codes for the protein alpha-synuclein has been transferred into the Drosophila melanogaster genome. The transgenic flies recapitulate some of the essential features of Parkinson's disease. These include the degeneration of certain dopaminergic neurons in the brain accompanied by the appearance of age-dependent abnormalities in locomotor activity. In the present study, we tested the locomotor response of these transgenic flies to prototypes of the major classes of drugs currently used to treat this disorder. A time course study was first conducted to determine when impaired locomotor activity appeared relative to normal "wild-type" flies. A climbing or negative geotaxis assay measuring the ability of the organisms to climb up the walls of a plastic vial was used. Based on the results obtained, normal and transgenic flies were treated with each of the drugs in their food for 13 days and then assayed. The activity of transgenic flies treated with L-DOPA was restored to normal. Similarly, the dopamine agonists pergolide, bromocriptine, and 2,3,4,5-tetrahydro-7,8-dihydroxy- 1-phenyl-1H-3-benzazepine (SK&F 38393) were substantially effective. Atropine, the prototypical muscarinic cholinergic receptor antagonist, was also effective but to a lesser extent than the other antiparkinson compounds. p-Chlorophenylalanine, an inhibitor of serotonin synthesis, was without beneficial effect as was alpha-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, the rate-limiting step in catecholamine biosynthesis. This behavioral study further demonstrates the utility of this model in studying Parkinson's disease and reinforces the concept that inhibition of the action of alpha-synuclein may be useful in its treatment as may dopamine D(1) receptor agonists.

Perez, R. G., J. C. Waymire, et al. (2002). "A role for alpha-synuclein in the regulation of dopamine biosynthesis." J Neurosci 22(8): 3090-9.
The alpha-synuclein gene is implicated in the pathogenesis of Parkinson's disease. Although alpha-synuclein function is uncertain, the protein has homology to the chaperone molecule 14-3-3. In addition, alpha-synuclein can bind to 14-3-3, and both alpha-synuclein and 14-3-3 bind to many of the same proteins. Because 14-3-3 binds to and activates tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) biosynthesis, we explored whether alpha-synuclein also bound to tyrosine hydroxylase and influenced its activity. Immunoprecipitation revealed an interaction between alpha-synuclein and tyrosine hydroxylase in brain homogenates and MN9D dopaminergic cells. Colocalization of alpha-synuclein with tyrosine hydroxylase was confirmed by immunoelectron microscopy. To explore the consequences of the interaction, we measured the effect of recombinant alpha-synuclein on tyrosine hydroxylase activity in a cell-free system and observed a dose-dependent inhibition of tyrosine hydroxylase by alpha-synuclein. To measure the impact of alpha-synuclein on tyrosine hydroxylase in dopaminergic cells, we stably transfected MN9D cells with wild-type or A53T mutant alpha-synuclein. Overexpression of wild-type or A53T mutant alpha-synuclein did not significantly alter tyrosine hydroxylase protein levels in our stably transfected cells. However, overexpressing cell lines had significantly reduced tyrosine hydroxylase activity and a corresponding reduction in dopamine synthesis. The reduction in cellular dopamine levels was not caused by increased dopamine catabolism or dopamine efflux. These data suggest that alpha-synuclein plays a role in the regulation of dopamine biosynthesis, acting to reduce the activity of tyrosine hydroxylase. If so, a loss of soluble alpha-synuclein, by reduced expression or aggregation, could increase dopamine synthesis with an accompanying increase in reactive dopamine metabolites.

Philippe, A., M. Guilloud-Bataille, et al. (2002). "Analysis of ten candidate genes in autism by association and linkage." Am J Med Genet 114(2): 125-8.
We studied the possible involvement of ten candidate genes in autism: proenkephalin, prodynorphin, and proprotein convertase subtilisin/kexin type 2 (opioid metabolism); tyrosine hydroxylase, dopamine receptors D2 and D5, monoamine oxidases A and B (monoaminergic system); brain-derived neurotrophic factor, and neural cell adhesion molecule (involved in neurodevelopment). Thirty-eight families with two affected siblings and one family with two affected half-siblings, recruited by the Paris Autism Research International Sibpair Study (PARIS), were tested using the transmission disequilibrium test and two-point affected sib-pair linkage analysis. We found no evidence for association or linkage with intragenic or linked markers. Our family sample has good power for detecting a linkage disequilibrium of 0.80. Thus, these genes are unlikely to play a major role in the families studied, but further studies in a much larger sample would be needed to highlight weaker genetic effects.

Riaz, S. S., E. Jauniaux, et al. (2002). "The controlled conversion of human neural progenitor cells derived from foetal ventral mesencephalon into dopaminergic neurons in vitro." Brain Res Dev Brain Res 136(1): 27-34.
The expansion and differentiation of neural progenitor cells in vitro provides an approach to study the development and differentiation of neurons. The ventral mesencephalic area of the brain is an important source of neural progenitor cells and the differentiated neural progenitor cell has paramount potential for use in transplant therapies such as those used in the treatment of neurodegenerative diseases. Here, the controlled conversion of human foetal progenitor cells derived from ventral mesencephalon into dopaminergic neurons is reported. The immunoreactivity to tyrosine hydroxylase (TH) and levels of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), secreted into culture medium, were used to assess dopaminergic neuronal phenotype. Expansion of the neural progenitor cells for 3 weeks in the presence of basic fibroblast growth factor (2 ng/ml) followed by its withdrawal resulted in approximately 60% of cells staining positive for TH, when challenged in concert with brain-derived neurotrophic factor (50 ng/ml), DA (10 &mgr;M) and forskolin (10 &mgr;M) for a further 3 weeks. A corresponding 41-fold increase in DA and DOPAC was measured in the incubation medium by HPLC. Therefore, the successful conversion of human foetal progenitor cells in vitro resulting in the desired dopaminergic neuronal phenotype, could provide a solution to the problem of limited availability of human foetuses for clinical surgical transplantation therapies, which are currently in progress for the treatment of neurodegenerative diseases such as Parkinson's disease.

Ricci, A., F. Amenta, et al. (2002). "Age-related changes of dopamine receptor protein immunoreactivity in the rat mesenteric vascular tree." Mech Ageing Dev 123(5): 537-46.
Dopamine D1-D5 receptor protein immunoreactivity and tyrosine hydroxylase (TH) immunoreactivity were investigated on the mesenteric arterial tree by immunohistochemistry. The density of various dopamine receptors and TH immunoreactivity was compared between young (6-month-old), adult (15-month-old) and senescent (24-month-old) Fischer 344 rats by computer-assisted microdensitometry. The dopamine D1-like (D1 and D5) receptors were localized on the tunica media of different sized mesenteric artery branches. The D2-like (D2, D3 and D4) receptors as well as TH immunoreactivity were localized only on the adventitia-media transitional zone of mesenteric arterial tree. Expression of the D1 and D5 receptors was decreased in both adult and senescent rats compared to the young rats, suggesting an age-related decline in these receptors. Of the D2-like receptors, the expression of the D2 receptor was decreased as a function of age, while the D3 receptor was unchanged in the senescent rats compared to the young rats. Expression of the D4 receptor was increased in adult, but was unchanged in the senescent rats compared to young animals. TH immunoreactivity was increased as a function of age. The above data suggest that reduction in the D1, D2 and D5 receptor expression may contribute to the deficiency in the dopamine-mediated vasorelaxation and hence blood flow in the mesenteric vascular tree in aging. The different sensitivity to aging of sympathetic neuroeffector junctions labeled by TH and of dopamine D2-like receptors that are known to be prejunctional, suggests that age-related changes of dopamine receptor expression in the mesenteric vasculature reflect more complicated mechanisms than simple up- or down-regulation phenomena.

Richfield, E. K., M. J. Thiruchelvam, et al. (2002). "Behavioral and neurochemical effects of wild-type and mutated human alpha-synuclein in transgenic mice." Exp Neurol 175(1): 35-48.
Human alpha-synuclein (halpha-SYN) is implicated in the Parkinson's disease phenotype (PDP) based on a variety of studies in man, animal models, and in vitro studies. The normal function of halpha-SYN and the mechanism by which it contributes to the PDP remains unclear. We created transgenic mice expressing either wild-type (hwalpha-SYN) or a doubly mutated (hm2alpha-SYN) form of halpha-SYN under control of the 9-kb rat tyrosine hydroxylase promoter. These mice expressed halpha-SYN in cell bodies, axons, and terminals of the nigrostriatal system. The expression of halpha-SYN in nigrostriatal terminals produced effects in both constructs resulting in increased density of the dopamine transporter and enhanced toxicity to the neurotoxin MPTP. Expression of hm2alpha-SYN reduced locomotor responses to repeated doses of amphetamine and blocked the development of sensitization. Adult hwalpha-SYN-5 transgenic mice had unremarkable dopaminergic axons and terminals, normal age-related measures on two motor coordination screens, and normal age-related measures of dopamine (DA) and its metabolites. Adult hm2alpha-SYN-39 transgenic mice had abnormal axons and terminals, age-related impairments in motor coordination, and age-related reductions in DA and its metabolites. Expression of hm2alpha-SYN adversely affects the integrity of dopaminergic terminals and leads to age-related declines in motor coordination and dopaminergic markers.

Rink, E. and M. F. Wullimann (2002). "Connections of the ventral telencephalon and tyrosine hydroxylase distribution in the zebrafish brain (Danio rerio) lead to identification of an ascending dopaminergic system in a teleost." Brain Res Bull 57(3-4): 385-7.
We studied the connections and catecholaminergic organization of the subpallium in the zebrafish, in particular to demonstrate the origin of the ascending dopaminergic system of teleosts, by using the tracers DiI or biocytin in combination with tyrosine hydroxylase (TH) immunohistochemistry. Retrogradely labeled cells were found in the olfactory bulb, the area dorsalis telencephali, the preoptic region, the dorsal and ventral thalamus, the posterior tubercle, the preglomerular region, and the medulla oblongata. Moreover, the zebrafish subpallium has strong reciprocal connections with the tuberal hypothalamus. Double-labeled cells (for TH and tracer) were identified in two locations of the rostral posterior tubercle: small round neurons in its periventricular nucleus and large pear-shaped cells adjacent to it. These double-labeled cells of the posterior tubercle presumably represent the teleostean dopaminergic system ascending to the striatum.

Sakata, M., H. Sei, et al. (2002). "Mesolimbic dopaminergic system is involved in diurnal blood pressure regulation." Brain Res 928(1-2): 194-201.
Parkinson's disease (PD) patients with autonomic failure show no nocturnal decrease in blood pressure (BP). At present, it is not clear if this symptom is attributable to the disturbance of the dopaminergic (DA) system that is responsible for PD. In the present study, we determined that the mesolimbic DA system is involved in diurnal profiles of the mean BP (MBP) by destroying the A10 DA system in rats with 6-hydroxydopamine. In control rats, a clear dip in the MBP and heart rate (HR) occurs during the light, that is, resting period, analogous to the nocturnal dip in normal humans. This normal daytime decrease in MBP and HR was disturbed by inducing a lesion of the ventral tegmental area (VTA) DA neurons, although the rhythms of wake-sleep duration and behavioral activity remained relatively intact. On the basis of this evidence, the absence of a nocturnal dip in BP in PD patients is attributed to impairment of the mesolimbic DA system.

Sanchez, J. J., P. Abreu, et al. (2002). "Sodium nitroprusside stimulates L-DOPA release from striatal tissue through nitric oxide and cGMP." Eur J Pharmacol 438(1-2): 79-83.
The effects of the nitric oxide (NO) donor, sodium nitroprusside, on L-DOPA and dopamine release from striatal tissue were evaluated using a static incubation system in which the striatal tissue released between three and six times more L-DOPA than DA, although the DA content was four times higher than that of L-DOPA. Sodium nitroprusside stimulated L-DOPA release in a time- and concentration-dependent (25, 50 and 100 microM) manner. This effect was not due to an increase in L-DOPA synthesis because sodium nitroprusside did not modify the tyrosine hydroxylase activity of striatal tissue. DA release was also stimulated by sodium nitroprusside but it required a higher concentration (500 microM) and longer incubation (60 min). Neither basal nor sodium nitroprusside-stimulated L-DOPA release was influenced by Ca(2+) deprivation (EGTA 5 mM) and/or the presence of nitrendipine (1 microM), a blocker Ca(2+) channel, in the incubation medium. However, cGMP (1 mM) increased L-DOPA release, and the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) (5 microM), partially blunted the stimulatory effect of sodium nitroprusside 100 microM. In addition, the presence of certain scavengers of free radicals, such as uric acid (300 microM) or melatonin (300 microM) but not of superoxide dismutase (1000 UI/ml) or salicylic acid (300 microM), completely blocked sodium nitroprusside (100 microM)-induced L-DOPA release. These results show that NO stimulates L-DOPA release from striatal tissue by an apparently Ca(2+)-independent mechanism, mediated by cGMP but also by peroxynitrite.

Schmidt, R. E. (2002). "Age-related sympathetic ganglionic neuropathology: human pathology and animal models." Auton Neurosci 96(1): 63-72.
Systematic studies of the autonomic nervous system of human subjects and development of well-defined animal models have begun to substantially improve our understanding of the pathogenesis of autonomic dysfunction in aging and may eventually provide strategies for intervention. Neuropathological studies of the sympathetic ganglia of aged human subjects and rodent models have demonstrated that neuroaxonal dystrophy involving intraganglionic terminal axons and synapses is a robust, unequivocal and consistent neuropathological finding in the aged sympathetic nervous system of man and animals. Quantitative studies have demonstrated that markedly swollen argyrophilic dystrophic axon terminals develop in the prevertebral superior mesenteric (SMG) and coeliac, but to a much lesser degree in the superior cervical ganglia (SCG) as a function of age, sex (males more than females) and diabetes. Dystrophic axons were immunoreactive for neuropeptide Y, tyrosine hydroxylase, dopamine-beta-hydroxylase, trkA and p75NTR, an immunophenotype consistent with their origin from postganglionic sympathetic neurons, and contained large numbers of highly phosphorylated neurofilaments or tubulovesicular elements. The sympathetic ganglia of aged rodents also showed the hallmark changes of neuroaxonal dystrophy as a function of age and location (many more in the SMG than in the SCG). Plasticity-related synaptic remodeling could represent a highly vulnerable target of the aging process. The fidelity of animal models to the neuropathology of aged humans suggests that similar pathogenetic mechanisms may be involved in both and that therapeutic advances in animal studies may have human application.

Serova, L., M. Rivkin, et al. (2002). "Estradiol stimulates gene expression of norepinephrine biosynthetic enzymes in rat locus coeruleus." Neuroendocrinology 75(3): 193-200.
Gender-specific differences in susceptibility to a number of disorders related to catecholaminergic systems, including depression and hypertension, have been postulated to be mediated, at least in part, by estrogens. In this study, we examined if estrogens may regulate gene expression of norepinephrine biosynthetic enzymes. Administration of five injections of 15 or 40 microg/kg estradiol benzoate to ovariectomized (OVX) female rats elicited a dose-dependent elevation in mRNA levels of tyrosine hydroxylase (TH) in locus coeruleus, to as great as 3-fold over control. Dopamine beta-hydroxylase (DBH) mRNA levels were also similarly increased. To examine the mechanism, PC12 cells were cotransfected with luciferase reporter constructs under control of DBH or TH promoters [pDBH/Luc(-2,236/+21) or pTH/Luc(-272/+27 or -773/+27)] with an expression vector for estradiol receptor alpha. The cells were treated with 17beta-estradiol (E(2)) for 12-36 h. E(2) triggered a several fold increase in luciferase activity under control of the DBH promoter in a dose-dependent fashion. Omission of estrogen receptor alpha or addition of the estrogen receptor antagonist ICI 182,780 prevented the DBH promoter-driven increase in luciferase. When E(2) was given with 0.2 mM CPT-cAMP, reporter activity with pDBH/Luc(-2,236/+21) was increased greater than with either treatment alone. In contrast, addition of E(2) to cells transfected with pTH/Luc(-272/+27) elicited no change in basal luciferase activity nor in the response to 0.2 mM CPT-cAMP. These findings are the first to reveal that estrogen can stimulate DBH gene expression. Differing mechanisms may underlie the regulation of TH and DBH gene expression by estrogens.

Stefani, A., F. Spadoni, et al. (2002). "D2-mediated modulation of N-type calcium currents in rat globus pallidus neurons following dopamine denervation." Eur J Neurosci 15(5): 815-25.
We have studied the effects of dopamine and the D2-like agonist quinpirole on calcium currents of neurons isolated from the striatum and the globus pallidus (GP). Experiments were performed in young adult rats, either in control conditions or following lesion of the nigrostriatal pathway by the unilateral injection of 6-hydroxydopamine (6-OHDA) in the substantia nigra. Apomorphine-driven contralateral turning, 15 days after lesioning, assessed the severity of the dopamine denervation. In addition, the loss of tyrosine hydroxylase immunohistochemistry confirmed the extent of the toxin-induced damage. In both striatal medium spiny (MS) and GP neurons of control animals dopamine and quinpirole promoted a very modest inhibition of calcium conductance. Following 6-OHDA, the inhibition was unaltered in MS (from 10 to 12%), but significantly augmented in GP neurons (21% vs. 9%). Interestingly, analogous inhibition was observed in GP neurons dissociated 20 h after reserpine treatment. Further features of the D2 response were thus studied only in neurons isolated from 6-OHDA-lesioned GP. The D2 modulation was G-protein-mediated but not strictly voltage-dependent. omega-Conotoxin-GVIA occluded the response implying the involvement of N-type calcium channels. The effect of quinpirole developed fast and was insensitive to alterations of cytosolic cAMP. The incubation in phorbol esters or OAG blocked the D2 effect, supporting the involvement of PKC. These findings suggest that postsynaptic D2-like receptors are functionally expressed on GP cell bodies and may supersensitize following dopamine-denervation. A direct D2 modulation of calcium conductance in GP may alter GP firing properties and GABA release onto pallidofugal targets.

Sutoo, D., K. Akiyama, et al. (2002). "Comparison analysis of distributions of tyrosine hydroxylase, calmodulin and calcium/calmodulin-dependent protein kinase II in a triple stained slice of rat brain." Brain Res 933(1): 1-11.
The immunohistochemical distributions of tyrosine hydroxylase (TH), calmodulin (CaM) and calcium/CaM-dependent protein kinase II (CaMKII) in the rat forebrain were compared quantitatively to confirm our previous finding that TH activity and dopamine synthesis in the brain are regulated by a calcium/CaM-dependent system. The same slice was triply stained and the above substances were detected immunohistochemically. Their distributions in the slice were measured using a brain mapping analyzer which is a microphotometry system for the analysis of the distribution of neurochemicals in a large tissue slice. Each coronal section was divided into approximately 250000 to 310000 microareas at 20-&mgr;m intervals, and the immunohistochemical fluorescence intensities of the three substances in these microareas were analyzed independently. Quantitative images of the distributions were reconstructed from the data, and the distribution of each substance was investigated. Immunoreactive staining of TH, CaM and CaMKII was observed in almost all areas of the brain, but the intensities varied. Markedly intense TH-, CaM- and CaMKII-like immunoreactivities were distributed in the anterior dorsolateral and posterior areas of the neostriatum, nucleus accumbens and olfactory tubercle. In the previous study, the amount of dopamine was increased by the intracerebroventricular administration of calcium chloride in the neostriatum and nucleus accumbens. Combining these results with those previously reported, it is suggested that TH activity and dopamine synthesis in these regions are regulated by calcium ions via CaM and CaMKII. This method is a powerful technique for quantitative and comparative analysis of the distributions of various neurochemicals in the same slice, and we believe that it will facilitate investigation of the functions of the central nervous system and disorders thereof in various diseases.

Themann, C., D. Alvarez Fischer, et al. (2002). "Effect of repeated treatment with high doses of selegiline on behaviour, striatal dopaminergic transmission and tyrosine hydroxylase mRNA levels." Naunyn Schmiedebergs Arch Pharmacol 365(1): 22-8.
The anti-parkinsonian drug selegiline is a monoamine oxidase B (MAO-B) inhibitor and a potential neuroprotective agent which facilitates dopaminergic transmission. Its metabolites (-)-amphetamine and (-)-metamphetamine might contribute to the pharmacological effects as they are also able to increase dopaminergic transmission and in addition might lead to behavioural sensitization after repeated administration. We investigated the effects of acute and repeated treatment with a high dose of selegiline on dopamine overflow in the striatum as well as on behaviour and on tyrosine hydroxylase (TH) mRNA levels in midbrain. Two experimen