Dopamine synthesis by non-dopaminergic neurons in the arcuate nucleus of rat fetuses.

The aim of the present work was to verify the hypothesis that non-dopaminergic neurons expressing individual complementary dopamine synthesis enzymes can perform the co-located synthesis of dopamine. According to this hypothesis, neurons expressing tyrosine hydroxylase use L-tyrosine for the synthesis of L-dihydroxyphenylalanine (L-DOPA), which then enters neurons expressing aromatic amino acid decarboxylase, which converts L-DOPA to dopamine. Experiments were performed using the mediobasal hypothalamus of rat fetuses, which mostly contains single-enzyme neurons (>99%) and occasional double-enzyme neurons (<1%). Controls were obtained from the fetal substantia nigra, which is enriched with dopaminergic neurons. High-performance liquid chromatography was used to measure levels of dopamine and L-DOPA in cell extracts and the incubation medium after incubation in the presence and absence of exogenous L-tyrosine. Addition of L-tyrosine to the medium led to increases in the level of synthesis and release of L-DOPA in the mediobasal hypothalamus and substantia nigra. In addition, L-tyrosine increased dopamine synthesis in the substantia nigra and decreased dopamine synthesis in the mediobasal hypothalamus. This regional difference in levels of dopamine synthesis is probably due to inhibition of the uptake of L-DOPA from the intercellular medium by neurons in the mediobasal hypothalamus containing aromatic amino acid decarboxylase, due to the competitive binding of the L-DOPA transporter by L-tyrosine. Thus, these results provide the first evidence for the co-located synthesis of dopamine by non-dopaminergic neurons expressing single complementary enzymes involved in the synthesis of this neurotransmitter.

Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections.

Transplants of fetal ventral mesencephalic tissue are known to contain a mixture of two major dopamine (DA) neuron types: the A9 neurons of the substantia nigra pars compacta (SNpc) and the A10 neurons of the ventral tegmental area (VTA). Previous studies have suggested that these two DA neuron types may differ in their growth characteristics, but, because of technical limitations, it has so far been difficult to identify the two subtypes in fetal ventral mesencephalon (VM) grafts and trace their axonal projections. Here, we have made use of a transgenic mouse expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. The expression of the GFP reporter allowed for visualization of the grafted DA neurons and their axonal projections within the host brain. We show that the SNpc and VTA neuron subtypes in VM grafts can be identified on the basis of their morphology and location within the graft, and their expression of a G-protein-gated inwardly rectifying K+ channel subunit (Girk2) and calbindin, respectively, and also that the axonal projections of the two DA neuron types are markedly different. By retrograde axonal tracing, we show that dopaminergic innervation of the striatum is derived almost exclusively from the Girk2-positive SNpc cells, whereas the calbindin-positive VTA neurons project to the frontal cortex and probably also other forebrain areas. The results suggest the presence of axon guidance and target recognition mechanisms in the DA-denervated forebrain that can guide the growing axons to their appropriate targets and indicate that cell preparations used for cell replacement in Parkinson's disease will be therapeutically useful only if they contain cells capable of generating the correct nigral DA neuron phenotype.

Dopamine synthesis by non-dopaminergic neurons expressing individual complementary enzymes of the dopamine synthetic pathway in the arcuate nucleus of fetal rats.

This study was aimed to test our hypothesis about dopamine (DA) synthesis by non-DAergic neurons expressing individual complementary enzymes of the DA synthetic pathway in cooperation, i.e. L-dihydroxyphenylalanine (L-DOPA) synthesized in tyrosine hydroxylase (TH)-expressing neurons is transported to aromatic L-amino acid decarboxylase (AADC)-expressing neurons for conversion to DA. The mediobasal hypothalamus of rats at the 21st embryonic day was used as an experimental model because it contains mainly monoenzymatic TH neurons and AADC neurons (>99%) whereas the fraction of bienzymatic (DAergic) neurons does not exceed 1%. The fetal substantia nigra containing DAergic neurons served as a control. DA and L-DOPA were measured by high performance liquid chromatography in: (1) cell extracts of the cell suspension prepared ex tempora; (2) cell extracts and incubation medium after the static incubation of the cell suspension with, or without exogenous L-tyrosine; (3) effluents of the incubation medium during perifusion of the cell suspension in the presence, or the absence of L-tyrosine. Total amounts of DA and L-DOPA in the incubation medium and cell extracts after the static incubation were considered as the indexes of the rates of their syntheses. L-Tyrosine administration caused the increased L-DOPA synthesis in the mediobasal hypothalamus and substantia nigra. Moreover, L-tyrosine provoked an increase of DA synthesis in the substantia nigra and its decrease in the mediobasal hypothalamus. This contradiction is most probably explained by the L-tyrosine-induced competitive inhibition of the L-DOPA transport to the monoenzymatic AADC-neurons after its release from the monoenzymatic TH neurons. Thus, this study provides convincing evidence of cooperative DA synthesis by non-DAergic neurons expressing TH or AADC in fetal rats at the end of the intrauterine development.

Tauroursodeoxycholic acid improves the survival and function of nigral transplants in a rat model of Parkinson's disease.

There is accumulating evidence showing that the majority of cell death in neural grafts results from apoptosis when cells are implanted into the brain. Tauroursodeoxycholic acid (TUDCA), a taurine-conjugated hydrophilic bile acid, has been found to possess antiapoptotic properties. In the present study we have examined whether the supplementation of TUDCA to cell suspensions prior to transplantation can lead to enhanced survival of nigral grafts. We first conducted an in vitro study to examine the effects of TUDCA on the survival of dopamine neurons in serum-free conditions. The number of tyrosine hydroxylase (TH)-positive neurons in the TUDCA-treated cultures was significantly greater than that of control cultures 7 days in vitro. In addition, a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay showed that the number of apoptotic cells in the TUDCA-treated cultures was dramatically smaller than that in the control cultures. In the transplantation study, a 50 microM concentration of TUDCA was added to the media when nigral tissue from Sprague-Dawley (SD) rats was trypsinized and dissociated. Two microliters of cell suspension containing TUDCA was then stereotaxically injected into the striatum of adult SD rats subjected to an extensive unilateral 6-hydroxydopamine lesion of the nigrastriatal dopamine pathway. At 2 weeks after transplantation, the rats that received a cell suspension with TUDCA exhibited a significant reduction in amphetamine-induced rotation scores when compared with pretransplantation value. There was a significant increase (approximately threefold) in the number of TH-positive cells in the neural grafts for the TUDCA-treated group when compared with the controls 6 weeks postgrafting. The number of apoptotic cells was much smaller in the graft areas in the TUDCA-treated groups than in the control group 4 days after transplantation. These data demonstrate that pretreatment of the cell suspension with TUDCA can reduce apoptosis and increase the survival of grafted cells, resulting in an improvement of behavioral recovery.

Altered distribution of dopaminergic neurons in the brain of L1 null mice.

Dopaminergic neurons of the mouse mesencephalon originate in the ventricular zone and migrate radially along radial glia then tangentially along nerve fibers that express the neural cell adhesion molecule L1 to form the substantia nigra (A9 group) and ventral tegmental area (VTA) (A10 group). The role of L1 in migration of dopaminergic neuronal precursors was investigated in L1 knockout mice by tyrosine hydroxylase (TH) immunostaining. An altered rostrocaudal distribution of dopaminergic neurons was observed within the substantia nigra and VTA of L1-minus mice. In L1-minus mice at postnatal day 0, TH-positive cells were present abnormally in the dorsomedial mesencephalon, suggesting impaired migration. Axons projecting from the substantia nigra to the caudate putamen also exhibited an abnormal targeting pattern. There was no evidence of dopaminergic cell loss in the mutant SN. Abnormal localization of dopaminergic neurons in L1-minus mice was also evident in the zona incerta of the thalamus (A13 group), and the arcuate (A12) and periventricular nucleus (A14) of the hypothalamus. Cell bodies and axons in the substantia nigra, VTA, and hypothalamus of wild type mouse embryos expressed L1. These results suggested that L1 plays an important developmental role in the organization of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.

The growth of the feline brain from fetal into adult life. II. A morphometric study of subcortical nuclei.

As a continuation of the morphometric studies on the preceding paper, here we report on the rate of growth of the caudate nucleus (n.), thalamus, red n., and the substantia (s.) nigra using, with few exceptions, the same cohort of cats. The same previously used brains (n=64 cats) were allocated to the following age groups: fetal (E) 59 days, postnatal (P) days 1, 7, 15, 30, 45, 60, 90, 120, and 180. Sixteen additional cats, interspersed within the groups, were substituted for the red n. and s. nigra studies. There were six subjects per group (except for E59, n=4). Using a projection microscope and cytochrome oxidase-stained coronal sections, a combined (left plus right sides) total of 4693, 3822, 1636, and 1180 sections were drawn for the caudate, thalamus, s. nigra, and red n., respectively. With computer assistance, the drawings were digitized to calculate mean cross-sectional areas and then the mean volume of each structure per group. The growth time tables for the caudate n., thalamus and s. nigra were fairly synchronous. In terms of percentage of the adult volume, for the left side (both sides grew at a similar rate), the three structures grew at a fast pace between E59 and P30. Thus, at E59 their respective percentages relative to adult volume were 23.7, 29.8 and 22.6% and by P30 the percentages were within adult range (85.2, 115.1 and 87.5%, respectively). Starting at P30, for the thalamus and at P45 for the caudate n., there was a consistent tendency to an overgrow which ranged between 4.3 and 30.9% (at P180, P<0.5) for the caudate and between 0.3 and 15.1% for the thalamus. In addition, starting at P30, the right thalamus tended to be consistently larger than the left by a margin ranging between 0.5 and 11.2% (P120, P<0.05). The red n. grew at a different, slower pace. Starting from a fetal volume equivalent to an 18.6% of adult size, its volume was only a 61.0% of the adult value at P30 and came within range of adulthood size only by P60 (81. 3%). Neither the s. nigra nor the red n. showed any consistent tendency to overgrow or to asymmetry. These findings are discussed in the context of the literature. Furthermore, we discuss general conclusions and considerations pertaining to both papers as well as draw comparisons with the maturational time tables of other developmental landmarks in cats. Finally, in a comparison with growth of human brain structures, we point at the limitations and complexities involved in studying human material and, noting interspecies similarities, we propose that the present data from an advanced gyrencephalic mammal may form the bases for a model of structures maturation in humans.

[Pathophysiological bases, clinical results and indications for surgical treatment in Parkinson disease]. / Bases fisiopatológicas, resultados clínicos e indicaciones del tratamiento quirúrgico de la enfermedad de Parkinson.

We review the present status of surgery for Parkinson's disease. Surgical options for Parkinson's disease are rapidly spanding. The main objectives of surgical techniques are to restore the dopaminergic deficit in the striatum (transplantation) and to normalize the neuronal activity of the subthalamic-pallidal circuit (pallidotomy and deep brain stimulation). Whereas cell transplantation is still considered an experimental procedure, ablative procedures and deep brain stimulation are widely used. Both types of surgical procedures are supported by strong scientific data. However, much work remains to be done in order to understand several aspects not clearly elucidated at present. The results and current indications for pallidotomy and deep brain stimulation are analyzed.

[Differentiation of dopaminergic neurones in situ, in vitro, and in the transplant]. / Differentsirovka dofaminergicheskikh neironov in situ, in vitro i v transplantate.

Four stages were distinguished in differentiation of the hypothalamic dopaminergic (DA) neurones: a) origin of the neurone, b) expression of enzymes and dopamine specific synthesis and of the dopamine transmembrane transportation mechanisms, c) development of permanent and transient efferent connections, d) forming of afferent innervation and synaptogenesis. The differentiating DA neurones revealed sexual dimorphism in the neurone origin dynamics and in expression of the enzyme synthesis. The maternal and placenta factors did not affect the differentiation of the DA neurones. The period from the 6th to 10th foetal week was found to be optimal for transplantation of the neurones to the striatum of parkinsonian patients. Grafted DA neurones seem to get involved in regulation of the target neurones in the striatum.

The combined method of transplantation of foetal substantia nigra and stereotactic thalamotomy for Parkinson's disease.

Five cases with late Parkinson's disease improved following treatment with a combination of foetal substantia nigra grafts and stereotactic thalamotomy. The average Webster's score decreased from 21 to 11 points. This modified method is simple, safe and effective.