Characterization of a depolarizing dopamine response in a vertebrate neuronal somatic cell hybrid

The physiology and pharmacology of a depolarizing dopamine response was studied in the vertebrate neuronal somatic cell hybrid TCX11. The average resting membrane potential was ?50 mV (S.D. = ±7) with a membrane resistance of 40.5 mOhms (S.D. = ±8) as determined from intracellular recordings. Depolarizing current pulses did not elicit an action potential. Cells displayed a linear current-voltage relationship when artificially depolarized up to +30 mV. Iontophoretically applied dopamine elicited a depolarizing response with a conductance increase and a reversal potential of ?15 mV (S.D. = ±4.7). Experiments altering medium ion concentrations demonstrated the conductance increase was to sodium and most likely potassium. The dopamine agonist ET495 (Piribedil) and the analogue epinine mimicked dopamine, while closely related biogenic amines, with the exception of noradrenaline, elicited no response. Apomorphine also elicited a depolarizing response but was much less efficacious than Piribedil. Noradrenaline was less potent than dopamine and appeared to act at the dopamine receptor. Methylation (3-methoxytyramine) or absence of the 3-hydroxy group (tyramine) of dopamine resulted in total loss of activity. The dopamine antagonists chlorpromazine, trifluoperazine, promazine, and bulbocapnine reversibly blocked the response to dopamine at medium concentrations less than 5 μM. The adrenergic antagonist phentolamine blocked the response while phenoxybenzamine only reduced the response at higher concentrations. The acetylcholine antagonists α-bungarotoxin, hexamethonium, and scopolamine did not block the dopamine response. Both d-tubocurarine and atropine acted as antagonists. Collectively, these results demonstrate the presence of a receptor on a cultured cell line that is specific for dopamine, mediates a depolarizing and conductance increase response to dopamine, and displays the pharmacology most closely associated with dopamine receptors.     

The weaver mutant mouse as a model of nigrostriatal dysfunction

The weaver mutant mouse has a genetic defect that results in the loss of dopamine neurons in the nigrostriatal pathway. Striatal tyrosine hydroxylase and dopamine content are reduced by 60–70%, and dopamine uptake is reduced by as much as 95%. Deficits in all three of these striatal dopamine markers are seen as early as postnatal d 3. The striatal dopamine systems in the weaver apparently have the ability to compensate for this dopamine deficit. Thus, in the weaver, in vitro resting release, as well as amphetamine-evoked fractional release of endogenous dopamine are increased. An additional change seen in the weaver striatum is an elevated serotonin content. These alterations may play an adaptive role in attempting to compensate for the dopamine loss. In summary, the weaver mutant mouse has dramatic deficits in the nigrostriatal pathway, but also seems to develop certain adaptive mechanisms in dopaminergic and other transmitter systems that may compensate functionally for the dopamine deficit. Thus, the weaver mouse provides a unique animal model for studying naturally induced neuronal degeneration that complements those models using surgical and pharmacological protocols.     

Characterization of a novel D2-like dopamine receptor with a truncated splice variant and a D1-like dopamine receptor unique to invertebrates from Caenorhabditis elegans

We have cloned two novel Caenorhabditis elegans dopamine receptors, DOP-3 and DOP-4. DOP-3 shows high sequence homology with other D2-like dopamine receptors. As a result of alternative splicing, a truncated splice variant of DOP-3, DOP-3nf, was produced. Because of the in-frame insertion of a stop codon in the third intracellular loop, DOP-3nf lacks the sixth and seventh transmembrane domains that are found in the full-length DOP-3 receptor. Reporter gene assay showed that DOP-3 attenuates forskolin-stimulated cAMP formation in response to dopamine stimulation, whereas DOP-3nf does not. When DOP-3 was coexpressed with DOP-3nf, the ability to inhibit forskolin-stimulated cAMP formation was reduced. DOP-4 shows high sequence homology with D1-like dopamine receptors unique to invertebrates, which are distinct from mammalian D1-like dopamine receptors. Reporter gene assay showed that DOP-4 stimulates cAMP accumulation in response to dopamine stimulation. These two receptors provide new opportunities to understand dopaminergic signaling at the molecular level.     

Pharmacologic properties of a phosphate ester of dopamine

The 3 or 4 phosphate ester of dopamine (PD) was hydrolyzed by homogenates of rat tissues to give inorganic phosphate (Pi) and dopamine. The rate of hydrolysis of PD by kidney homogenates was increased by exogenous MgCl2 but not CaCl2 or KCl. The activity of brain, heart or liver homogenates was insensitive to the added salts. Several lines of evidence indicate that alkaline phosphatase activity contributes to the high rate of PD hydrolysis by the kidney but not brain homogenate. The intravenous infusion of PD at 12 mumole/kg in one hr to anesthetized rats increased the dopamine content of the plasma, kidney and heart without altering brain or liver dopamine. The results suggest that PD may be more effective than dopamine for increasing dopamine levels of the kidney. In addition, the hydrolysis of PD by brain homogenates, which is independent of alkaline phosphatase activity, suggests that specific enzymes exist for the metabolism of PD.     

Expression of a Human Cocaine-Sensitive Dopamine Transporter in Xenopus laevis Oocytes

The human dopamine transporter was expressed in Xenopus laevis oocytes following injection of mRNA isolated from human brain substantia nigra. The specific accumulation of [3H]dopamine into these oocytes was time and Na+ dependent. Furthermore, [3H]dopamine accumulation was prevented by coincubation of oocytes with dopamine (100 microM) or with the dopamine uptake inhibitors GBR 12909 (1 microM) or cocaine (3 microM). In contrast, oocyte injection of mRNA isolated from human globus pallidus, an area devoid of dopamine neuron perikarya, did not elicit expression of the dopamine transporter. Oocyte expression of the human dopamine transporter can be used for the further characterization and cloning of this low-abundance membrane protein.     

Homeostatic mechanisms in dopamine synthesis and release: a mathematical model

Background

Dopamine is a catecholamine that is used as a neurotransmitter both in the periphery and in the central nervous system. Dysfunction in various dopaminergic systems is known to be associated with various disorders, including schizophrenia, Parkinson's disease, and Tourette's syndrome. Furthermore, microdialysis studies have shown that addictive drugs increase extracellular dopamine and brain imaging has shown a correlation between euphoria and psycho-stimulant-induced increases in extracellular dopamine [1]. These consequences of dopamine dysfunction indicate the importance of maintaining dopamine functionality through homeostatic mechanisms that have been attributed to the delicate balance between synthesis, storage, release, metabolism, and reuptake.

Methods

We construct a mathematical model of dopamine synthesis, release, and reuptake and use it to study homeostasis in single dopaminergic neuron terminals. We investigate the substrate inhibition of tyrosine hydroxylase by tyrosine, the consequences of the rapid uptake of extracellular dopamine by the dopamine transporters, and the effects of the autoreceoptors on dopaminergic function. The main focus is to understand the regulation and control of synthesis and release and to explicate and interpret experimental findings.

Results

We show that the substrate inhibition of tyrosine hydroxylase by tyrosine stabilizes cytosolic and vesicular dopamine against changes in tyrosine availability due to meals. We find that the autoreceptors dampen the fluctuations in extracellular dopamine caused by changes in tyrosine hydroxylase expression and changes in the rate of firing. We show that short bursts of action potentials create significant dopamine signals against the background of tonic firing. We explain the observed time courses of extracellular dopamine responses to stimulation in wild type mice and mice that have genetically altered dopamine transporter densities and the observed half-lives of extracellular dopamine under various treatment protocols.

Conclusion

Dopaminergic systems must respond robustly to important biological signals such as bursts, while at the same time maintaining homeostasis in the face of normal biological fluctuations in inputs, expression levels, and firing rates. This is accomplished through the cooperative effect of many different homeostatic mechanisms including special properties of tyrosine hydroxylase, the dopamine transporters, and the dopamine autoreceptors.     

Methamphetamine-induced apoptosis in a CNS-derived catecholaminergic cell line

Methamphetamine (METH) causes neurotoxic damages to the dopaminergic system in mammals, but whether it exerts toxicity to dopamine cells in culture has not been fully explored. In order to develop an in vitro model of METH-induced dopamine neurotoxicity toward more systemical examination of the mechanism, we investigated METH toxicity in a clonal dopamine producing cell line (CATH.a). We show in the present study that METH produces a time- and dose-dependent increase in cell death via a process similar to apoptosis. The METH toxicity seems to be produced by oxidative stress, as it was attenuated by the antioxidant glutathione, and to involve dopamine because dopamine release and synthesis inhibitors attenuated the toxicity. This catecholaminergic cell line derived from the central nervous system may become a useful in vitro model to elucidate the mechanism underlying the METH-induced dopaminergic neuronal damage.     

Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line

Mutations in alpha-synuclein have been linked to rare, autosomal dominant forms of Parkinson's disease. Despite its ubiquitous expression, mutant alpha-synuclein primarily leads to the loss of dopamine-producing neurons in the substantia nigra. alpha-Synuclein is a presynaptic nerve terminal protein of unknown function, although several studies suggest it is important for synaptic plasticity and maintenance. The present study utilized a new human mesencephalic cell line, MESC2.10, to study the effect of A53T mutant alpha-synuclein on dopamine homeostasis. In addition to expressing markers of mature dopamine neurons, differentiated MESC2.10 cells are electrically active, produce dopamine, and express wild-type human alpha-synuclein. Lentivirus-induced overexpression of A53T mutant alpha-synuclein in differentiated MESC2.10 cells resulted in down-regulation of the vesicular dopamine transporter (VMAT2), decreased potassium-induced and increased amphetamine-induced dopamine release, enhanced cytoplasmic dopamine immunofluorescence, and increased intracellular levels of superoxide. These results suggest that mutant alpha-synuclein leads to an impairment in vesicular dopamine storage and consequent accumulation of dopamine in the cytosol, a pathogenic mechanism that underlies the toxicity of the psychostimulant amphetamine and the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium. Interestingly, cells expressing A53T mutant alpha-synuclein were resistant to amphetamine-induced toxicity. Because extravesicular, cytoplasmic dopamine can be easily oxidized into reactive oxygen species and other toxic metabolites, mutations in alpha-synuclein might lead to Parkinson's disease by triggering protracted, low grade dopamine toxicity resulting in terminal degeneration and ultimately cell death.     

Dopamine inhibits calcium flux in the 7315a prolactin-secreting pituitary tumour

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.     

A cognate dopamine transporter-like activity endogenously expressed in a COS-7 kidney-derived cell line.

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney-derived COS-7, COS-1 and HEK-293 but not in other mammalian cell lines (CHO, Y1, Ltk-), we have characterized a putative functional dopamine transporter displaying a high affinity (Km approximately 250 nM) and a low capacity (approximately 0.1 pmol/10(5) cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated Ki values of numerous substrates and inhibitors for the COS-dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium-sensitive and reduced by phorbol ester pre-treatment. Two mRNA species of 3.8 and 4.0 kb in COS-7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse-transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of 1.3 kb, isolated from a COS-1 cDNA library and encoding transmembrane domains 1-6, displayed a deduced amino acid sequence homology of approximately 96% to the human dopamine transporter. Taken together, the data suggest the existence of a non-neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.     

The dopamine of the rat mammotroph in cell culture as a model for drug action

Dopamine can act directly on pituitary cells to inhibit prolactin release. This action can be blocked by dopamine receptor blocking drugs such as haloperidol, sulpiride and other neuroleptic agents. Comparison of the properties of the mammotroph dopamine receptor with the adenylate cyclase linked dopamine receptor of the limbic forebrain reveals some obvious differences. For example, dopamine receptor stimulants such as S-584 and lergotrile mesylate are inactive in stimulating the adenylate cyclase preparations but are potent in inhibiting pituitary prolactin secretion. Such inhibition of prolactin secretion can be reversed by haloperidol or sulpiride. In contrast to these observations, sulpiride does not block dopamine stimulation of cAMP formation. In addition, dopamine, apomorphine or lergotrile mesylate have no effect on a pituitary adenylate cyclase preparation and dopamine fails to elevate cAMP in the intact cells in culture. Despite the similarity between these two dopamine sensitive systems with respect to a number of agonists and antagonists, the exceptions described suggest that the pituitary system with further study may offer some greater reliability as a predictive test for clinically useful agents. These results also suggest that the receptors for dopamine, like that for norepinephrine, are of two types, only one of which is coupled to adenylate cyclase.     

Development of a new immunosensor for the detection of dopamine

Graphite immunoelectrodes as immunosensors using indirect immobilization of a hapten were investigated for their applicability to detect dopamine hydrochloride at low levels. Conditions were optimized to achieve the highest sensitivity using the indirect immobilization of dopamine hydrochloride through a polymerized glutaraldehyde network on microtiter plates using ELISA technique. The conditions were later transferred to the graphite rods (phi 0.8 m x 20 mm) and a comparison between the two different sensitivities (IC50 midpoint of test) was carried out. Graphite electrodes showed higher sensitivity towards dopamine than ELISA, since they were able to detect dopamine with a midpoint of test of 0.2 mmol/l while using ELISA they were able to detect dopamine hydrochloride at 2 mmol/l.     

Increased renal dopamine and acute renal adaptation to a high-phosphate diet

The current experiments explore the role of dopamine in facilitating the acute increase in renal phosphate excretion in response to a high-phosphate diet. Compared with a low-phosphate (0.1%) diet for 24 h, mice fed a high-phosphate (1.2%) diet had significantly higher rates of phosphate excretion in the urine associated with a two- to threefold increase in the dopamine content of the kidney and in the urinary excretion of dopamine. Animals fed a high-phosphate diet had a significant increase in the abundance and activity of renal DOPA (l-dihydroxyphenylalanine) decarboxylase and significant reductions in renalase, monoamine oxidase A, and monoamine oxidase B. The activity of protein kinase A and protein kinase C, markers of activation of renal dopamine receptors, were significantly higher in animals fed a high-phosphate vs. a low-phosphate diet. Treatment of rats with carbidopa, an inhibitor of DOPA decarboxylase, impaired adaptation to a high-phosphate diet. These experiments indicate that the rapid adaptation to a high-phosphate diet involves alterations in key enzymes involved in dopamine synthesis and degradation, resulting in increased renal dopamine content and activation of the signaling cascade used by dopamine to inhibit the renal tubular reabsorption of phosphate.     

The decrease of NAD(P)H has a prominent role in dopamine toxicity

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.     

Tissue and plasma levels of a teratogenic dose of dopamine in the chick embryo following pretreatment with metoprolol or phosphate buffered saline

Metoprolol pretreatment has been shown to reduce the cardiovascular malformation rate produced by topical doses of dopamine in the stage 24 chick embryo. We report on the tissue and plasma levels and teratogenic effect of dopamine hydrochloride following topical application of a teratogenic dose in stage 24 chick embryos pretreated with either metoprolol tartrate or phosphate buffered saline (PBS). Pretreatment with either metoprolol or PBS resulted in similar patterns of dopamine distribution in the head, body, and heart, with peak levels occurring at 12 hours after dopamine treatment. Plasma concentrations of dopamine were similar for both PBS and metoprolol pretreated embryos, with plasma levels exceeding tissue concentrations, but also peaking at 12 hours after dopamine treatment. Pretreatment with PBS followed by a teratogenic dose of dopamine resulted in a decrease in the teratogenic effect of dopamine similar to that found in previous work in our lab with pretreatment with metoprolol. The developing chick cardiovascular system experiences peak susceptibility to the teratogenic effects of dopamine at stage 24 during development, which represents a time frame of about 12 hours. A delay in the peak levels of dopamine to 12 hours after dopamine treatment as compared to previous work in our lab reporting peak levels of dopamine at 1 hour, suggests that the previously reported antiteratogenic effects of metoprolol may be due, at least in part, to a delayed absorption of dopamine past the time of peak susceptibility of the embryo to the teratogen.     

Dopamine-independent locomotor actions of amphetamines in a novel acute mouse model of Parkinson disease

Brain dopamine is critically involved in movement control, and its deficiency is the primary cause of motor symptoms in Parkinson disease. Here we report development of an animal model of acute severe dopamine deficiency by using mice lacking the dopamine transporter. In the absence of transporter-mediated recycling mechanisms, dopamine levels become entirely dependent on de novo synthesis. Acute pharmacological inhibition of dopamine synthesis in these mice induces transient elimination of striatal dopamine accompanied by the development of a striking behavioral phenotype manifested as severe akinesia, rigidity, tremor, and ptosis. This phenotype can be reversed by administration of the dopamine precursor, L-DOPA, or by nonselective dopamine agonists. Surprisingly, several amphetamine derivatives were also effective in reversing these behavioral abnormalities in a dopamine-independent manner. Identification of dopamine transporter- and dopamine-independent locomotor actions of amphetamines suggests a novel paradigm in the search for prospective anti-Parkinsonian drugs.     

An inverse correlation between the apparent rate of dopamine clearance and tonic autoinhibition in subdomains of the rat striatum: a possible role of transporter-mediated dopamine efflux

The dopaminergic terminal field in the rat striatum is compartmentalized into sub-domains that exhibit distinct dynamics of electrically evoked dopamine release. The fast striatal domains, where dopamine release is predominantly vesicular, exhibit conventional dopaminergic activity. However, vesicular dopamine release is tonically autoinhibited in the slow domains, which suggests that dopamine reaches the autoreceptors via a non-vesicular route. Hence, it appears that the domains use distinct mechanisms to regulate the basal dopamine concentration available to activate, or not, pre-synaptic autoinhibitory receptors. However, direct detection of local variations in tonic extracellular dopamine concentrations is not yet possible. So, the present study employed voltammetry to test the hypothesis that the apparent rate of dopamine clearance from the extracellular space should be domain-dependent. The apparent rate of dopamine clearance is equal to the difference in the rates of dopamine release and uptake that determine extracellular dopamine concentrations. This study confirms that the apparent rate of dopamine clearance is slower in the slow striatal domains where vesicular dopamine release is tonically autoinhibited. These findings support the view that the basal concentration in slow domains is maintained by a non-vesicular release process, possibly transporter-mediated efflux.     

Dopamine inhibits pulmonary edema through the VEGF-VEGFR2 axis in a murine model of acute lung injury

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0-75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR(-/-) mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.     

Evidence for a functional coupling between dopamine reuptake and tyrosine hydroxylation in striatal nerve terminals

In rat striatal synaptosomes incubated with [¹⁴C]tyrosine, the evolution of ¹⁴CO₂, taken as a measure of dopamine synthesis, was inhibited by exogenous dopamine and by the dopaminergic receptor agonist ADTN. The inhibition was not counteracted by dopaminergic receptor antagonists (haloperidol, sulpiride, pimozide or domperidone). Instead, it was prevented by dopamine uptake blockers, suggesting that dopamine and ADTN (a substrate of the dopamine carrier) acted once inside the nerve endings and not through activation of autoreceptors on their external membrane. The dopamine uptake inhibitors nomifensine, benztropine and cocaine increased ¹⁴CO₂ evolution from incubated striatal synaptosomes. Depolarization with KCl also increased dopamine synthesis and this action was potentiated when the reuptake of the released catecholamine was prevented by carrier blockers. The rate of dopamine synthesis was lowered when synaptosomal dopamine was raised upon incubation with monoamine oxidase inhibitors or with l-DOPA. The inhibition was counteracted by dopamine reuptake blockers. The data suggest that dopamine synthesis in striatal nerve endings is under the inhibitory control of the transmitter recaptured following release.     

Loss of mitochondrial complex I activity potentiates dopamine neuron death induced by microtubule dysfunction in a Parkinson's disease model

Mitochondrial complex I dysfunction is regarded as underlying dopamine neuron death in Parkinson's disease models. However, inactivation of the Ndufs4 gene, which compromises complex I activity, does not affect the survival of dopamine neurons in culture or in the substantia nigra pars compacta of 5-wk-old mice. Treatment with piericidin A, a complex I inhibitor, does not induce selective dopamine neuron death in either Ndufs4(+/+) or Ndufs4(-/-) mesencephalic cultures. In contrast, rotenone, another complex I inhibitor, causes selective toxicity to dopamine neurons, and Ndufs4 inactivation potentiates this toxicity. We identify microtubule depolymerization and the accumulation of cytosolic dopamine and reactive oxygen species as alternative mechanisms underlying rotenone-induced dopamine neuron death. Enhanced rotenone toxicity to dopamine neurons from Ndufs4 knockout mice may involve enhanced dopamine synthesis caused by the accumulation of nicotinamide adenine dinucleotide reduced. Our results suggest that the combination of disrupting microtubule dynamics and inhibiting complex I, either by mutations or exposure to toxicants, may be a risk factor for Parkinson's disease.