Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catecholamines

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine to l-dopa, which is the initial and rate-limiting step in the biosynthesis of catecholamines [CA; dopamine (DA), noradrenaline, and adrenaline], and plays a central role in the neurotransmission and hormonal actions of CA. Thus, TH is related to various neuro-psychiatric diseases such as TH deficiency, Parkinson’s disease (PD), and schizophrenia. Four isoforms of human TH (hTH1–hTH4) are produced from a single gene by alternative mRNA splicing in the N-terminal region, whereas two isoforms exist in monkeys and only a single protein exist in all non-primate mammals. A catalytic domain is located within the C-terminal two-thirds of molecule, whereas the part of the enzyme controlling enzyme activity is assigned to the N-terminal end as the regulatory domain. The catalytic activity of TH is end product inhibited by CA, and the phosphorylation of Ser residues (Ser¹⁹, Ser³¹, and especially Ser⁴⁰ of hTH1) in the N-terminus relieves the CA-mediated inhibition. Ota and Nakashima et al. have investigated the role of the N-terminus of TH enzyme in the regulation of both the catalytic activity and the intracellular stability by producing various mutants of the N-terminus of hTH1. The expression of the following three enzymes, TH, GTP cyclohydrolase I, which synthesizes the tetrahydrobiopterin cofactor of TH, and aromatic-l-amino acid decarboxylase, which produces DA from l-dopa, were induced in the monkey striatum using harmless adeno-associated virus vectors, resulting in a remarkable improvement in the symptoms affecting PD model monkeys Muramatsu (Hum Gene Ther 13:345–354, 2002). Increased knowledge concerning the amino acid sequences of the N-terminus of TH that control enzyme activity and stability will extend the spectrum of the gene-therapy approach for PD.     

The assays of activities and function of TH, AADC, and GCH1 and their potential use in ex vivo gene therapy of PD

In the past decades, there have been numerous studies in the gene therapy for Parkinson's disease (PD), especially in delivering genes of enzymes for dopamine (DA) synthesis. Gene therapy in PD appears to be at the brink of the clinical study phase. However, there are many questions that need to be solved before this approach can be contemplated clinically, especially the question about the control of DA production because too much DA could cause toxicity. Until recently, few studies have investigated the relation between DA production and PD improvement and respective expressed human tyrosine hydroxylase (hTH), human GTP-cyclohydrolase 1 (hGCH1), and human aromatic acid decarboxylase (hAADC) in ex vivo gene therapy for PD. Now, we have developed a simple, fast, and reliable method to assay the activities of TH and AADC and have provided the possibility of ex vivo gene therapy for PD by genetically modifying cells with separate hTH, hGCH1, and hAADC genes. Using the method, we found though hTH, hGCH1, and hAADC genes were expressed, respectively, they could fulfil the function of DA synthesis by incubating together in vitro, and more DA was synthesized in vitro when hTH, hGCH1, and hAADC genes were expressed together rather than hTH and hAADC genes expressed or hTH expressed. The result suggests that we could easily control DA production in ex vivo gene therapy before transplantation. By combining this method and microdialysis, we also could further investigate the DA production in vitro and in vivo and then decide the optimal number and ratio of different transduced cells to improve the therapy of PD. Thus, the method has potential use in ex vivo gene therapy of PD.     

Expression of human tyrosine hydroxylase type I in Escherichia coli as a protease-cleavable fusion protein

Wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants of human tyrosine hydroxylase type 1 (hTH1) fused to maltose-binding protein via the target sequence for a restriction protease were expressed in Escherichia coli and purified. The fused protein was treated with the restriction protease factor Xa or enterokinase to isolate hTH1 from the fused form. The treatment of fused wild-type and 35-amino acid-deleted mutant with factor Xa and enterokinase caused non-specific cleavages in the vicinity of the phosphorylation sites, Ser19 and Ser40, due to the flexible conformation of the N-terminus of hTH1.     

TH基因修饰细胞脑内移植治疗猴帕金森病的实验研究

目的 评价包囊化酪氨酸羟化酶（tyrosine hydroylase,TH）基因修饰的基因工程细胞脑内移植治疗帕金森病的疗效。方法 将pcDNA3/hTH质粒转染人神经母细胞瘤细胞系SYTY细胞，筛选出阳性克隆，微包囊化处理后的含有TH基因修饰细胞植入PD猴模型脑内，观察其行为、CSF中DA含量的变化，用免疫组化法检查移植细胞的存活情况。结果 （1）pcDNA3/hTH基因经亚克隆，提取纯化的质粒，经ECORI酶切后产生1.9Kb和5.5Kb的片段。转基因后的SY5Y细胞免疫细胞化学染色显示TH染色强阳性；（2）移植后PD猴症状明显改善，脑脊液中DA含量升高；（3）SABC免疫组化发现移植区存在大量TH阳／性细胞。结论 构建的TH基因工程细胞体外和体内均表达人类TH基因；微包囊化处理后的基因工程细胞在PD猴脑内存活并发挥治疗作用。     

“抗帕颗粒”对帕金森病模型鼠的神经保护作用

目的探讨"抗帕颗粒"对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)帕金森病(PD)模型小鼠黑质纹状体区TH阳性神经元及多巴胺(DA)的影响。方法 90只健康雄性C57BL/6小鼠,鼠龄8~12w,随机分为3组:正常对照组30只、PD模型对照组30只、PD模型干预组30只;MPTP腹腔注射(40mg·kg~(-1)·d~(-1)×7)制备小鼠PD模型;正常对照组及PD模型对照组予生理盐水1m L·d~(-1)灌胃,PD模型干预组给予"抗帕颗粒"40mg·kg~(-1)·d~(-1)灌胃,连续喂养4个月。比较分析各组4月时黑质纹状体区TH阳性神经元及DA情况。结果 1正常对照组30只(30/30只)最终均存活,PD模型对照组4个月时存活27只(27/30只),PD模型干预组4个月时存活28只(28/30只);2PD模型对照组、PD模型干预组小鼠每次注射MPTP后,先有短暂兴奋(持续7.61±2.17min),表现为四处窜跳;随即出现全身中重度震颤,皮毛及尾巴时有竖立,活动减少,持续24.23±3.89min后震颤消失;随后出现活动减少;3经Imagepro~Plus 5.1系统分析,正常对照组TH阳性细胞面积为64145μm~2,高倍镜下可见大量胞质为褐色颗粒的TH阳性细胞;PD模型对照组TH阳性细胞染色面积为40012μm~2,高倍镜下见TH细胞数量明显减少;PD模型干预组TH阳性细胞染色面积为60952μm~2,高倍镜下见TH阳性细胞数量较PD模型对照组增加;4正常对照组DA含量为2.18±0.31μg·m L~(-1),与PD模型对照组1.57±0.22μg·m L~(-1)比较,P0.01;正常对照组与PD模型干预组2.04±0.18μg·m L~(-1)比较。P0.05;PD模型对照组与PD模型干预组比较,P0.01。结论 "抗帕颗粒"可使PD小鼠黑质纹状体中多巴胺能神经元一定程度地减少丢失,并改善DA含量的下降,对多巴胺能神经元的数量、形态及功能具有一定的保护作用,有望改善PD治疗现状并在临床进一步推广应用。     

Methamphetamine-induced loss of striatal dopamine innervation in BDNF heterozygote mice does not further reduce D3 receptor concentrations

Depletion of dopamine (DA) reduces D(3) receptor number, but D(3) receptor expression is also regulated by brain-derived neurotrophic factor (BDNF). We took advantage of transgenic heterozygous BDNF mutant mice (+/-) to determine if reduced BDNF and loss of DA fibers produced by methamphetamine were additive in their impact on D(3) receptor number. We assessed selective markers of the dopaminergic system including caudate-putamen DA concentrations and quantitative autoradiographic measurement of tyrosine hydroxylase (TH) levels, DA transporter (DAT), and DA D(3) receptor binding between vehicle and methamphetamine-treated BDNF +/- and their wildtype (WT) littermate control mice. Caudate-putamen DA concentrations, TH and DAT levels were significantly reduced following methamphetamine treatment in both WT and BDNF +/- mice. The extent of methamphetamine-induced reduction in TH and DAT was greater for the WT than BDNF +/- mice and DAT levels were also decreased to a greater extent in nucleus accumbens of WT as compared to BDNF +/- mice. Lower D(3) receptor existed in caudate-putamen and nucleus accumbens in BDNF +/- mice and these differences were not affected by methamphetamine treatment. Taken together, these results not only substantiate the importance of BDNF in controlling D(3) receptor expression, but also indicate that a methamphetamine-induced depletion of DA fibers fails to produce an additive effect with lowered BDNF for control of D(3) receptor expression. In addition, the reduction of D(3) receptor expression is associated with a decreased neurotoxic response to methamphetamine in BDNF +/- mice.     

外源EGFP和hTH基因在恒河猴骨髓源神经干细胞内的稳定表达

目的构建携带人酪氨酸羟化酶(hTH)的荧光真核表达质粒-pEGFP-C2-hTH,转染骨髓基质细胞源神经干细胞(BMSCs-D-NSCs),观察外源EGFP和hTH基因在BMSCs-D-NSCs中的表达情况。方法应用基因重组技术,将pWAV2-TH中的TH目的基因亚克隆到荧光真核表达载体 pEGFP-C2,以酶切和测序鉴定重组质粒pEGFP-C2-hTH的正确性:pEGFP-C2-hTH经NucleofectorTM 核转染仪转染培养的恒河猴BMSCs-D-NSCs,24 h后观察绿色荧光蛋白的瞬时表达情况,10 d后行 TH单克隆抗体的免疫组化和TH基因的RT-PCR。结果 (1)酶切、PCR和DNA序列鉴定均证实插入片段的正确性;(2)细胞转染24 h后,荧光显微镜下可观察到绿色荧光蛋白(GFP)的表达,观察到 80％的转染细胞发出绿色荧光;转染10 d后细胞的RT-PCR检测到hTH基因的表达,TH单克隆抗体免疫组化结果显示转染细胞呈阳性染色,同时在荧光显微镜下观察到绿色荧光。结论构建的 hTH荧光真核表达重组质粒pEGFP-C2-hTH,经电转染方法转染至BMSCs-D-NSCs内,成功表达hTH 和EGFP,为BMSCs-D-NSCs基因治疗提供了实验基础。     

In VivoL-DOPA Production by Genetically Modified Primary Rat Fibroblast or 9L Gliosarcoma Cell Grafts via Coexpression of GTPcyclohydrolase I with Tyrosine Hydroxylase

To investigate the biochemical requirements forin vivoL-DOPA production by cells genetically modifiedex vivoin a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH₄), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH₄. As striatal BH₄levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH₄is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH₄in this rat model of PD.     

Tyrosine hydroxylase, aromatic L-amino acid decarboxylase and dopamine metabolism after chronic treatment with dopaminergic drugs.

Mice were treated with dopamine (DA) receptor agonist and antagonist drugs: Agonists: (+/-)-SKF 38393 ((+/-)-1-phenyl-2,3,4, 5-tetrahydro-(1H)-3-benzazepine-7,8-diol) [DA D1-like]; bromocriptine, [DA D2 selective]; quinpirole, [DA D2/D3 preferring]; (+/-)-7-hydroxy-dipropylamino-tetralin (7-OH-DPAT), [DA D3/D2 preferring], Antagonists: R(+)-SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine), [DA D1-like]; and haloperidol, [DA D2-like]. All drugs were administered intraperitoneally, two injections daily 8 h apart for 30 days. Aromatic L-amino acid decarboxylase (AAAD) and tyrosine hydroxylase (TH) activity, protein and mRNA, as well as DA metabolism were followed with time thereafter in the nigrostriatal neurons. We observed that chronic administration of D1-like agonists had no effect on TH or AAAD activity, while D2-like agonists decreased AAAD, but not TH activity. Additionally, chronic blockade of DA D2-like receptors resulted in prolonged induction of TH and AAAD, while chronic blockade of DA D1-like receptors induced changes of AAAD only. Compared to TH the induction of AAAD was longer lasting. DA metabolism was altered by chronic administration of drugs acting on DA D2-like, but not DA D1-like receptors, and in general the patterns of change did not follow those for TH or AAAD. When studied 48 h after the last dose of the chronic haloperidol schedule TH displayed tolerance to acute drug challenge. At the same time interval, there was tolerance to the enhancing effects of haloperidol and SCH 23390 on DA metabolism. The induction of AAAD by haloperidol or SCH 23990 did not appear to develop tolerance after chronic administration. These observations complement existing knowledge, and provide novel information about AAAD that may have practical importance for Parkinson's patients on L-DOPA therapy.     

Accumulation of phosphorylated tyrosine hydroxylase into insoluble protein aggregates by inhibition of an ubiquitin–proteasome system in PC12D cells

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamines including dopamine. The relationship between proteasomal dysfunction and the etiology of Parkinson’s disease has been suggested, but it is unknown if TH protein is affected by proteasomal dysfunctions. Here, we examined the effect of inhibition of ubiquitin–proteasomal pathway on biochemical characteristics of TH protein in the neuronal cells. Inhibition of 20S or 26S proteasome by proteasome inhibitor I, or MG-132 in NGF-differentiated PC12D cells induced dot-like immunoreactivities with the anti-⁴⁰Ser-phosphorylated TH (p40-TH) antibody. These dots were tightly co-localized with ubiquitin and positive to Thioflavine-S staining. These dot-like immunoreactivities were not obvious when immunostaining was performed against total-TH or choline acetyltransferase. Western blotting analysis showed time-dependent increase of p40-TH in the Triton-insoluble fractions. We also examined the effect of okadaic acid, an inhibitor of protein phosphatase 2A, which is a phosphatase acting on p40-TH. Okadaic acid increased the amount of insoluble p40-TH. These data suggest that p40-TH is prone to be insolubilized and aggregated by dysfunction of an ubiquitin–proteasome system in PC12D cells.     

基因修饰骨髓源性神经元样干细胞治疗帕金森大鼠的研究

目的 观察大鼠酪氨酸羟化酶(tyrosinehydroxylase,TH)修饰的骨髓基质源性神经元样干细胞(neuronoid stem cells derived from bone marrow stem cells,NdSCs-D-BMSCs)在脑室移植途径下对帕金森病(Parkinson disease,PD)大鼠的治疗作用.方法 将酶切鉴定后的新构建质粒pEGFP-C2-TH经电穿孔法转染培养第8天NdSCs-D-BMSCs,注射到PD大鼠模型右侧脑室,观察大鼠行为学变化,移植细胞在大鼠脑组织内的迁移,以及高效液相方法检测脑内DA含量.结果 质粒pEGFP-C2-TH转染NdSCs-D-BMSCs移植后10周,PD大鼠症状显著改善,DA恢复至正常水平33.0%,移植细胞可以在PD大鼠脑内存活,并出现远处迁移.结论 TH修饰的大鼠NdSCs-D-BMSCs经脑室移植对PD大鼠具有明显的治疗作用,为临床中腰椎穿刺干细胞移植的应用提供实验依据.     

Co-existence of tyrosine hydroxylase and dopamine beta-hydroxylase immunoreactivity in glomus cells of the cat carotid body.

Catecholamines are thought to play an important role in sensory transduction in the arterial chemoreceptors of the mammalian carotid body, and classical cytochemical techniques have demonstrated their presence in the type I (glomus) cells of this organ. However, it remains controversial whether dopamine (DA) and norepinephrine (NE) occur in the same or in different subtypes of glomus cells. In the present study, we have addressed this issue using immunocytochemistry to compare the localization of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (D beta H) in the cat carotid body. Both pre- and post-embedding double-labelling immunohistochemical techniques were employed. TH and D beta H were found to co-exist in over 90% of the glomus cells, and they were co-localized at equivalent levels in almost 80% of the cells; less than 5% contained only TH. The results suggest that DA and NE are synthesized and stored in a common cell population in the cat carotid body.     

Differential regulation of short and long dopamine D2 receptor mRNA levels by hypoxia in the adrenals of 1-day-old and adult rabbits

We tested the hypothesis that hypoxic exposure exerts distinct modulation on the mRNA level of the short and long isoform of the D2 dopamine (DA) receptor (D(2)r) and on tyrosine hydroxylase (TH) in the adrenals and superior cervical ganglion (SCG) of 1-day-old and adult rabbits. One-day-old and adult rabbits have been exposed to moderate (FiO(2): 0.15) or severe (FiO(2): 0.08) hypoxia for 0 (control), 6 or 24 h. At the end of the hypoxic exposure, the adrenals and SCG were rapidly dissected and kept frozen. Standard procedure for Northern blot and RT-PCR for evaluation of the short (D(2S)) or long (D(2L)) isoform of D(2)r and TH have been used. In the adrenals of adult rabbits, moderate and severe hypoxia decreased the D(2S) and D(2L) mRNA with a more prominent effect on the short isoform and increased TH mRNA. In 1-day-old rabbits, changes of D(2)r and TH mRNA levels were observed only after 24 h of severe or moderate hypoxic exposure and only the D(2S) receptor mRNA was significantly reduced. In the SCG, the expression level of both TH and D(2)r mRNA were not affected by hypoxic exposure in adult and newborn rabbits. Hence, sustained hypoxic exposure exerts distinct changes on mRNA level of D(2)r isoform and TH in an age- and tissue-dependent pattern.     

D2S, D2L, D3, and D4 dopamine receptors couple to a voltage-dependent potassium current in N18TG2 x mesencephalon hybrid cell (MES-23.5) via distinct G proteins

We utilized the approach of stably expressing different dopamine (DA) receptors into identified cell lines in an attempt to better understand the coupling of these receptors to membrane ion channels via second messenger systems. Recently, we examined the N18TG2 x mesencephalon (MES-23.5) cell line that is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-dependent K+ current present in these cells. Untransfected MES-23.5 cells displayed a voltage-dependent slow-onset, slowly inactivating outward current which was not altered by bath application of either the D2 DA receptor agonist quinpirole (QUIN; 10-100 microM) or the D1 DA receptor agonist SKF38393, indicating that these cells were devoid of DA receptors. The K+ current studied was activated upon depolarization from a holding potential of -60 mV to a level more positive than -20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected to stably express the D2S, D2L, D3, and D4 receptors, the same current was observed. In cells expressing D2L, D2S, and D3 receptors, application of the DA receptor agonists QUIN (1-80 microM), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1-80 microM), and dopamine (DA, 1-80 microM), increased the peak outward current by 35-40%. In marked contrast, cells stably expressing the D4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D2-like receptor antagonist sulpiride (SUL 5 microM), when coapplied with QUIN (10 microM), totally abolished the change in K+ current normally observed, while coapplication of the D1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D2L, D3, and D4 receptor stimulation was prevented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml for 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes Goalpha completely blocked the ability of D2L, D3, and D4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against Goalpha was without effect, whereas intracellular application of an antibody recognizing Gsalpha abolished the ability of the D2S receptor to enhance K+ current. These findings demonstrate that different members of the D2 DA receptor family may couple in a given cell to a common effector in dramatically different ways.     

Synaptic input of ON‐bipolar cells onto the dopaminergic neurons of the mouse retina

In the retina, dopamine fulfills a crucial role in neural adaptation to photopic illumination, but the pathway that carries cone signals to the dopaminergic amacrine (DA) cells was controversial. We identified the site of ON‐cone bipolar input onto DA cells in transgenic mice in which both types of catecholaminergic amacrine (CA) cells were labeled with green fluorescent protein or human placental alkaline phosphatase (PLAP). In confocal Z series of retinal whole mounts stained with antibodies to tyrosine hydroxylase (TH), DA cells gave rise to varicose processes that descended obliquely through the scleral half of the inner plexiform layer (IPL) and formed a loose, tangential plexus in the middle of this layer. Comparison with the distribution of the dendrites of type 2 CA cells and examination of neurobiotin‐injected DA cells proved that their vitreal processes were situated in stratum S3 of the IPL. Electron microscope demonstration of PLAP activity showed that bipolar cell endings in S3 established ribbon synapses onto a postsynaptic dyad in which one or both processes were labeled by a precipitate of lead phosphate and therefore belonged to DA cells. In places, the postsynaptic DA cell processes returned a reciprocal synapse onto the bipolar endings. Confocal images of sections stained with antibodies to TH, kinesin Kif3a, which labels synaptic ribbons, and glutamate or GABA_A receptors, confirmed that ribbon‐containing endings made glutamatergic synapses onto DA cells processes in S3 and received from them GABAergic synapses. The presynaptic ON‐bipolar cells most likely belonged to the CB3 (type 5) variety. J. Comp. Neurol. 518:2035–2050, 2010. © 2010 Wiley‐Liss, Inc.     

Generation of dopamine neurons from embryonic stem cells in the presence of the neuralizing activity of bone marrow stromal cells derived from adult mice

Stromal cell lines such as PA6 and MS5 have been employed for generating dopamine (DA) neurons from embryonic stem (ES) cells. The present study was designed to test whether bone marrow stromal cells (BMSC) derived from adult mice might be available as a feeder layer to produce DA cells efficiently from ES cells. When ES cells were grown on BMSC in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), about 40% of TuJ1-positive neurons expressed tyrosine hydroxylase (TH). Because these cells labeled with TH were negative for dopamine-beta-hydroxylasae (DBH), the marker for noradrenergic and adrenergic neurons, the TH-positive cells were most likely DA neurons. They indeed expressed midbrain DA neuron markers such as Nurr 1, Ptx-3, and c-ret and were capable of synthesizing and releasing DA in vitro. Furthermore, DA neurons differentiated from ES cells in this differentiation protocol survived transplantation in rats with 6-hydroxydopamine lesions and reversed the lesion-induced circling behavior. The data indicate that BMSC can facilitate an efficient induction of DA neurons from ES cells and that the generated DA neurons are biologically functional both in vitro and in vivo. Insofar as BMSC have recently been employed in autologous cell therapy for ischemic heart and arteriosclerotic limb diseases, the present study raises the possibility that autologous BMSC can be applied in future cell transplantation therapy in Parkinson's disease.     

Induction of Dopaminergic Neurotransmitter Phenotype in Rat Embryonic -Cerebrocortex by the Synergistic Action of Neurotrophins and Dopamine

Neurotrophins, including nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/51 (NT-4/5), have been shown to enhance survival and differentiation of a variety of central neuronal populations, such as those with the dopaminergic, cholinergic, GABAergic phenotype during development. In this paper we present evidence that BDNF, NT-3 and NT-4/5 acting synergistically with dopamine (DA) can artificially induce the full dopaminergic phenotype in rat fetal cerebral cortex which normally has very few dopaminergic neurons in adulthood. Thus, BDNF/DA, NT-3/DA, NT-4/DA elicited a great increase in the number of tyrosine hydroxylase (TH)-immunoreactive cells, which was up to 57% of total neuronal population in cultures of fetal rat cortical cells. This stimulatory effect was not dependent on glial proliferation, or on addition of serum to the culture. Pharmacological studies showed that dopamine receptors D, and D2 were involved in this effect. The TH’cortical cells possessed other biochemical phenotypic features of dopaminergic neurons. Thus, high-affinity DA uptake was elevated in cortical cultures treated with neurotrophin/DA. Also DA and 3,4–dihydroxyphenlacetic acid production was detected (5.42 ± 1.24 and 13.72 ? 2.84 pmol/dish respectively, zero in controls). This shows the presence of functionally active TH, aromatic acid decarboxylase and monoamine oxidase. Neurotrophins/DA had no effect on noradrenergic phenotype expression by cortical fetal cells. Taken together, these results support the long-standing view that development of the central nervous system is determined not only by intrinsic genetic programmes, but also involves environmental influences such as the action of growth factors and extracellular neurotransmitters. In this case we report the effect of specific DA phenotype-inducing agents.     

Mouse strain- and age-dependent effects of binge methamphetamine on dopaminergic signaling

We have shown that a single "binge" dose of methamphetamine (Meth) in mice has long-lasting effects on open-field behavior dependent on mouse strain and age. Here we further investigated the impact of genotype and age on tyrosine hydroxylase (TH) loss and dopamine (DA) metabolism due to a high binge dose of Meth (4 × 5 mg/kg × 2 h × 2 days). Administration of high dose Meth or saline (Sal) to adolescent (PND 40) and adult (PND 80) C57BL/6 (B6), DBA/2 (DBA), and 129S6SvEv/Tac (129) mice was followed by a 1mg/kg Meth or Sal (control) challenge 40 days later. Striatal and prefrontal cortex tissues were collected 1h following the challenge. Meth-pretreated adolescent B6 and DBA mice exhibited losses in striatal DA concentrations; DBA adolescents also showed losses in striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and increased DA turnover. Pre-exposed B6 and 129 adults demonstrated significant decreases in striatal DA, DOPAC, and increased DA turnover; DBA adults showed significant losses in striatal DA and increased DA turnover. 129 and DBA adults exhibited increases and decreases, respectively, in prefrontal cortex DA. Adult pretreated B6 mice produced significant losses in striatal TH. The results again show age and genotype dependent differences in Meth-induced DA alterations.