Development and function of the midbrain dopamine system: what we know and what we need to

The past two decades have seen an explosion in our understanding of the origin and development of the midbrain dopamine system. Much of this work has been focused on the aspects of dopamine neuron development related to the onset of movement disorders such as Parkinson's disease, with the intent of hopefully delaying, preventing or fixing symptoms. While midbrain dopamine degeneration is a major focus for treatment and research, many other human disorders are impacted by abnormal dopamine, including drug addiction, autism and schizophrenia. Understanding dopamine neuron ontogeny and how dopamine connections and circuitry develops may provide us with key insights into potentially important avenues of research for other dopamine‐related disorders. This review will provide a brief overview of the major molecular and genetic players throughout the development of midbrain dopamine neurons and what we know about the behavioral‐ and disease‐related implications associated with perturbations to midbrain dopamine neuron development. We intend to combine the knowledge of two broad fields of neuroscience, both developmental and behavioral, with the intent on fostering greater discussion between branches of neuroscience in the service of addressing complex cognitive questions from a developmental perspective and identifying important gaps in our knowledge for future study.     

The lesion of the rat substantia nigra pars compacta dopaminergic neurons as a model for Parkinson's disease memory disabilities

1. In this article we review the studies of memory disabilities in a rat model o Parkinson's disease (PD).2. Intranigral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to rats causes a partial lesion in the substantia nigra, compact part (SNc) and a specific loss of dopamine and its metabolites in the striatum of rats.3. These animals present learning and memory deficits but no sensorimotor impairments, thus modeling the early phase of PD when cognitive impairments are observed but the motor symptoms of the disease are barely present.4. The cognitive deficits observed in these animals affect memory tasks proposed to model habit learning (the cued version of the water maze task and the two-way active avoidance task) and working memory (a working memory version of the water maze), but spare long-term spatial memory (the spatial reference version of the Morris water maze).5. The treatment of these animals with levodopa in a dose that restores the striatal level of dopamine does not reverse these memory impairments, probably because this treatment promotes a high level of dopamine in extrastriatal brain regions, such as the prefrontal cortex and the hippocampus.6. On the other hand, the adenosine receptor antagonist, caffeine, partly reverse the memory impairment effect of SNc lesion in these rats. This effect may be due to caffeine action on nigrostriatal neurons, since it induces dopamine release and modulates the interaction between adenosine and dopamine receptor activity.7. These results suggest that the MPTP SNc-lesioned rats are a good model to study memory disabilities related to PD and that caffeine and other selective A(2A) adenosine receptor antagonists are promising drugs to treat this symptoms in PD patients.     

Functional diversity of ventral midbrain dopamine and GABAergic neurons

Recent findings indicate that VTA and SN dopaminergic (DA) and GABAergic neurons form subpopulations that are divergent in their electrophysiological features, vulnerability to neurodegeneration, and regulation by neuropeptides. This diversity can be correlated with the anatomical organization of the VTA and SN and their inputs and outputs. In this review we describe the heterogeneity in ion channels and firing patterns, especially burst firing, in subpopulations of dopamine neurons. We go on to describe variations in vulnerability to neurotoxic damage in models of Parkinson’s disease in subgroups of DA neurons and its possible relationship to developmental gene regulation, the expression of different ion channels, and the expression of different protein markers, such as the neuroprotective marker calbindin. The electrophysiological properties of subgroups of GABAergic midbrain neurons, patterns of expression of protein markers and receptors, possible involvement of GABAergic neurons in a number of processes that are usually attributed exclusively to dopaminergic neurons, and the characteristics of a subgroup of neurons that contains both dopamine and GABA are also discussed.     

Manganese exposure is cytotoxic and alters dopaminergic and GABAergic neurons within the basal ganglia

Manganese is an essential nutrient, integral to proper metabolism of amino acids, proteins and lipids. Excessive environmental exposure to manganese can produce extrapyramidal symptoms similar to those observed in Parkinson's disease (PD). We used in vivo and in vitro models to examine cellular and circuitry alterations induced by manganese exposure. Primary mesencephalic cultures were treated with 10–800 μM manganese chloride which resulted in dramatic changes in the neuronal cytoskeleton even at subtoxic concentrations. Using cultures from mice with red fluorescent protein driven by the tyrosine hydroxylase (TH) promoter, we found that dopaminergic neurons were more susceptible to manganese toxicity. To understand the vulnerability of dopaminergic cells to chronic manganese exposure, mice were given i.p. injections of MnCl₂ for 30 days. We observed a 20% reduction in TH-positive neurons in the substantia nigra pars compacta (SNpc) following manganese treatment. Quantification of Nissl bodies revealed a widespread reduction in SNpc cell numbers. Other areas of the basal ganglia were also altered by manganese as evidenced by the loss of glutamic acid decarboxylase 67 in the striatum. These studies suggest that acute manganese exposure induces cytoskeletal dysfunction prior to degeneration and that chronic manganese exposure results in neurochemical dysfunction with overlapping features to PD.     

Voltage-operated Ca2+ channels regulate dopamine release from somata of dopamine neurons in the substantia nigra pars compacta

Dopamine (DA) neurons release DA not only from axon terminals at the striatum, but from their somata and dendrites at the substantia nigra pars compacta (SNc). Released DA may auto-regulate further DA release or modulate non-DA cells. However, the actual mechanism of somatodendritic DA release, especially the Ca²⁺ dependency of the process, remains controversial. In this study, we used amperometry to monitor DA release from somata of acutely isolated rat DA neurons. We found that DA neurons spontaneously released DA in the resting state. Removal of extracellular Ca²⁺ and application of blockers for voltage-operated Ca²⁺ channels (VOCCs) suppressed the frequency of secretion events. Activation of VOCCs by stimulation with K⁺-rich saline increased the frequency of secretion events, which were also sensitive to blockers for L- and T-type Ca²⁺ channels. These results suggest that Ca²⁺ influx through VOCCs regulates DA release from somata of DA neurons.     

The effects of antipsychotics on beta-catenin, glycogen synthase kinase-3 and dishevelled in the ventral midbrain of rats

Protein kinase B and glycogen synthase kinase-3 have been identified as susceptibility genes for schizophrenia and altered protein and mRNA levels have been detected in the brains of schizophrenics post-mortem. Recently, we reported that haloperidol, clozapine and risperidone alter glycogen synthase kinase-3 and beta-catenin protein expression and glycogen synthase kinase-3 phosphorylation levels in the rat prefrontal cortex and striatum. In the current study, beta-catenin, adenomatous polyposis coli, Wnt1, dishevelled and glycogen synthase kinase-3 were examined in the ventral midbrain and hippocampus using western blotting. In addition, beta-catenin and GSK-3 were examined in the substantia nigra and ventral tegmental area using confocal and fluorescence microscopy. The results indicate that repeated antipsychotic administration results in significant elevations in glycogen synthase kinase-3, beta-catenin and dishevelled-3 protein levels in the ventral midbrain and hippocampus. Raclopride causes similar changes in beta-catenin and GSK-3 in the ventral midbrain, suggesting that D2 dopamine receptor antagonism mediated the changes observed following antipsychotic administration. In contrast, amphetamine, a drug capable of inducing psychotic episodes, had the opposite effect on beta-catenin and GSK-3 in the ventral midbrain. Collectively, the results suggest that antipsychotics may exert their beneficial effects through modifications to proteins that are associated with the canonical Wnt pathway.     

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.     

Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice culture

Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent.     

Immunocytochemical investigations on the presence of neuron-specific antibodies in the CSF of Parkinson's disease cases

The CSF and sera of 7 patients with Parkinson's disease were investigated immunocytochemically, in order to see if antibodies were present which recognized DA-ergic cell bodies in glutaraldehyde fixed rat brain. In 2 patients a marked labeling of DA-ergic neurons in the substantia nigra was observed, identified by anti-DA antiserum and by 6-OHDA induced degeneration, but also other neurons in the ventral mesencephalon were recognized. The other patients were weakly positive or negative. Sera gave unspecific labelling of all neurons. In one patient the sub-classes of IgG were investigated and found to be of IgG₃ (labeling nerve terminals) and of IgG_1–2, low affinity type (recognizing perikarya). The epitopes recognized have not been identified, but are unlikely to be DA-like, since blocking experiments and ELISA-tests gave negative results. The possible clinical importance of the results are discussed.Special Issue dedicated to Prof. Holger Hydén.     

Neurochemical characterization of the release and uptake of dopamine in ventral tegmental area and serotonin in substantia nigra of the mouse

In the present report, fast-scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) -pars compacta (SNc) and -pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 microm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 microm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 microm) had no effect in any brain region. Using dopamine transporter- and serotonin transporter-knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.     

Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.     

Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes

Information on the molecular distribution and ageing trend of brain iron in post‐mortem material from normal subjects is scarce. Because it is known that neuromelanin and ferritin form stable complexes with iron(III), in this study we measured the concentration of iron, ferritin and neuromelanin in substantia nigra from normal subjects, aged between 1 and 90 years, dissected post mortem. Iron levels in substantia nigra were 20 ng/mg in the first year of life, had increased to 200 ng/mg by the fourth decade and remained stable until 90 years of age. The H‐ferritin concentration was also very low (29 ng/mg) during the first year of life but increased rapidly to values of ≈ 200 ng/mg at 20 years of age, which then remained constant until the eighth decade of life. L ‐Ferritin also showed an increasing trend during life although the concentrations were ≈ 50% less than that of H‐ferritin at each age point. Neuromelanin was not detectable during the first year, increased to ≈ 1000 ng/mg in the second decade and then increased continuously to 3500 ng/mg in the 80th year. A Mössbauer study revealed that the high‐spin trivalent iron is probably arranged in a ferritin‐like iron?oxyhydroxide cluster form in the substantia nigra. Based on this data and on the low H‐ and L‐ferritin content in neurones it is concluded that neuromelanin is the major iron storage in substantia nigra neurones in normal individuals.     

Bioenergetic and oxidative effects of free 3-nitrotyrosine in culture: selective vulnerability of dopaminergic neurons and increased sensitivity of non-dopaminergic neurons to dopamine oxidation

Protein bound and free 3-nitrotyrosine (3NT) levels are elevated in neurodegenerative diseases and have been used as evidence for peroxynitrite generation. Intrastriatal injection of free 3NT causes dopaminergic neuron injury and represents a new mouse model of Parkinson's disease (PD). We are investigating the nature of free 3NT neurotoxicity. In primary ventral midbrain cultures, free 3NT damaged dopaminergic neurons, while adjacent non-dopaminergic neurons were unaffected. Combined treatment with free 3NT and subtoxic amounts of dopamine caused extensive death of non-dopaminergic forebrain neurons in culture. Free 3NT alone directly inhibited mitochondrial complex I, decreased ATP, sensitized neurons to mitochondrial depolarization, and increased superoxide production. Subtoxic concentrations of rotenone (instead of free 3NT) caused similar results. Additionally, free 3NT and dopamine combined increased extraneuronal hydrogen peroxide and decreased intraneuronal glutathione levels more than dopamine alone. Oxidative and bioenergetic processes have been proposed to contribute to neurodegeneration in PD. As free 3NT is a compound that is increased in PD, damages dopamine neurons in vivo and in vitro and has detrimental effects on neuronal bioenergetics, it is possible that free 3NT is an endogenous contributing factor to neuronal loss, in addition to being a marker of oxidative and nitrative processes.     

In vivo characterization of somatodendritic dopamine release in the substantia nigra of 6-hydroxydopamine-lesioned rats

We investigated the effect of an injection of 6-hydroxydopamine (6-OHDA) into the rat medial forebrain bundle (MFB) on the degeneration and the function of the dopaminergic cell bodies in the substantia nigra (SN) 3 and 5 weeks after lesioning. After injection of 6-OHDA into the MFB a complete loss of dopamine content was apparent in the striatum 3 weeks after lesioning. In the SN the amount of tyrosine hydroxylase-immunoreactive dopamine cells decreased gradually, with a near-complete lesion (> 90%) obtained only after 5 weeks, indicating that neurodegeneration of the nigral cells was still ongoing when total dopamine denervation of the striatum had already been achieved. Baseline dialysate and extracellular dopamine levels in the SN, as determined by in vivo microdialysis, were not altered by the lesion. A combination of compensatory changes of the remaining neurones and dopamine originating from the ventral tegmental area may maintain extracellular dopamine at near-normal levels. In both intact and lesioned rats, the somatodendritic release was about 60% tetrodotoxin (TTX) dependent. Possibly two pools contribute to the basal dopamine levels in the SN: a fast sodium channel-dependent portion and a TTX-insensitive one originating from diffusion of dopamine. Amphetamine-evoked dopamine release and release after injection of the selective dopamine reuptake blocker GBR 12909 were attenuated after a near-complete denervation of the SN (5 weeks after lesioning). So, despite a 90% dopamine cell loss in the SN 5 weeks after an MFB lesion, extracellular dopamine levels in the SN are kept at near-normal levels. However, the response to a pharmacological challenge is severely disrupted.     

多巴胺D3受体(D3R)的神经科学新进展

多巴胺(DA)是脑内一种重要的神经递质,通过不同DA受体亚型调控运动功能、认知活动和药物成瘾等生理、病理过程。多巴胺D3受体(D3R)属于D2样受体,但其功能长期不明。近年来,人们对它在神经科学中的意义有了新的认识。首先,D3R的信号通路独特,它被激活后显示细胞增殖效应,但cAMP信号传导途径不明显。其次,D3R基因敲除小鼠研究提示,正常生理状态下D3R仅表现辅助功能：在特定病理条件下,D3R显示出重要的“平衡缓冲作用”,在精神分裂症、帕金森病(PD)治疗中运动障碍副作用LID的发生和毒品复吸等病理过程扮演了重要角色。因此,D3R是一个重要的药物靶标。D3R拮抗剂在精神分裂症治疗中显示了临床前景,D3R激动剂则对PD治疗和毒品复吸防治展示了应用价值。     

Differential protection against MPTP or methamphetamine toxicity in dopamine neurons by deletion of ppN/OFQ expression

Nociceptin (N/OFQ) is an endogenous neuropeptide that plays a role in the behavioral deficits associated with Parkinson's disease (PD). The purpose of the present study was to characterize the protective effects of prepro (pp)N/OFQ gene deletion against two dopamine toxins, MPTP and methamphetamine (METH). Results demonstrate that ppN/OFQ gene deletion attenuates the loss of both the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc) and loss of TH and vesicular monoamine transporter-2 (VMAT) immunoreactivity in the caudate putamen (CPu) of MPTP-treated mice. This protection was unaffected by age or gender, although, when loss of TH exceeded 90% in 5-6 month-old mice, the protective effect was greatly diminished. In contrast, METH administration preferentially damaged dopaminergic terminals in the CPu with little effect on dopamine neurons in the SNpc, an effect not reversed by ppN/OFQ gene deletion. To determine if N/OFQ and MPP+ act directly and synergistically on dopamine neurons, differentiated SH-SY5Y cells were incubated with N/OFQ and/or MPP+. N/OFQ did not increase MPP+-mediated cell loss, suggesting an indirect action of N/OFQ. These studies demonstrate that inhibition of the endogenous N/OFQ system may represent a new therapeutic target for prevention of neuronal loss associated with PD.