Assessing the contribution of the chemical exposome to neurodegenerative disease.

Over the past few decades, numerous environmental chemicals from solvents to pesticides have been suggested to be involved in the development and progression of neurodegenerative diseases. Most of the evidence has accumulated from occupational or cohort studies in humans or laboratory research in animal models, with a range of chemicals being implicated. What has been missing is a systematic approach analogous to genome-wide association studies, which have identified dozens of genes involved in Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. Fortunately, it is now possible to study hundreds to thousands of chemical features under the exposome framework. This Perspective explores how advances in mass spectrometry make it possible to generate exposomic data to complement genomic data and thereby better understand neurodegenerative diseases.

Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits.

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

New insights into lung diseases using hyperpolarized gas MRI.

Hyperpolarized (HP) gases are a new class of contrast agents that permit to obtain high temporal and spatial resolution magnetic resonance images (MRI) of the lung airspaces. HP gas MRI has become important research tool not only for morphological and functional evaluation of normal pulmonary physiology but also for regional quantification of pathologic changes occurring in several lung diseases. The purpose of this work is to provide an introduction to MRI using HP noble gases, describing both the basic principles of the technique and the new information about lung disease provided by clinical studies with this method. The applications of the technique in normal subjects, smoking related lung disease, asthma, and cystic fibrosis are reviewed.

Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits.

The dopamine transporter is a key protein responsible for regulating dopamine homeostasis. Its function is to transport dopamine from the extracellular space into the presynaptic neuron. Studies have suggested that accumulation of dopamine in the cytosol can trigger oxidative stress and neurotoxicity. Previously, ectopic expression of the dopamine transporter was shown to cause damage in non-dopaminergic neurons due to their inability to handle cytosolic dopamine. However, it is unknown whether increasing dopamine transporter activity will be detrimental to dopamine neurons that are inherently capable of storing and degrading dopamine. To address this issue, we characterized transgenic mice that over-express the dopamine transporter selectively in dopamine neurons. We report that dopamine transporter over-expressing (DAT-tg) mice display spontaneous loss of midbrain dopamine neurons that is accompanied by increases in oxidative stress markers, 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC. In addition, metabolite-to-dopamine ratios are increased and VMAT2 protein expression is decreased in the striatum of these animals. Furthermore, DAT-tg mice also show fine motor deficits on challenging beam traversal that are reversed with l-DOPA treatment. Collectively, our findings demonstrate that even in neurons that routinely handle dopamine, increased uptake of this neurotransmitter through the dopamine transporter results in oxidative damage, neuronal loss and l-DOPA reversible motor deficits. In addition, DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity. The effects of increased dopamine uptake in these transgenic mice could shed light on the unique vulnerability of dopamine neurons in Parkinson's disease.

Computational modeling of synaptic neurotransmission as a tool for assessing dopamine hypotheses of schizophrenia.

Schizophrenia is a severe and complex mental disorder that causes an enormous societal and financial burden. Following the identification of dopamine as a neurotransmitter and the invention of antipsychotic drugs, the dopamine hypothesis was formulated to suggest hyperdopaminergia as the cause of schizophrenia. Over time there have been modifications and improvements to the dopamine-based model of schizophrenia, as well as models that do not implicate dopamine dysregulation as a primary cause of the disease. It seems clear by now that disruption of dopamine homeostasis occurs in schizophrenia and likely plays a major contributory role to its symptoms. Three primary versions of the dopamine hypothesis of schizophrenia have been proposed. In this article, we review these hypotheses and subject their assumptions to a computational model of dopamine signaling. Based on this review and analysis, we propose slight revisions to the existing hypotheses. Although we are still at the beginning of a comprehensive modeling effort to capture relevant phenomena associated with schizophrenia, our preliminary models have already yielded intriguing results and identified the systems biological approach as a beneficial complement to clinical and experimental research and a powerful method for exploring human diseases like schizophrenia. It is hoped that the past, present and future models will support and guide refined experimentation and lead to a deeper understanding of schizophrenia.

Steps of modeling complex biological systems.

A disease like schizophrenia results from the malfunctioning of a complex, multi-faceted biological system. As a consequence, the root causes of such a disease and the trajectories from health toward the disease are very difficult to comprehend with simple cause-and-effect reasoning. Similarly, reductionistic investigations are crucial for the discovery of specific disease mechanisms, but they are not sufficient for comprehensive assessments and explanations. A promising option for advancing the field is the utilization of mathematical models that can quantitatively account for hundreds of components and their interactions and thus have the potential of truly explaining complex diseases. While the potential of mathematical models is quite evident in principle, their practical implementation is a daunting task. On the one hand, many distinctly different approaches are possible. For instance, in the case of schizophrenia, models could focus on neurological aspects, physiological features, or the biochemical malfunctioning within some cell complexes in the brain, and each model would ultimately be very different. On the other hand, it seems that there are no rules or recommendations that guide the development of a new mathematical model from scratch. We discuss here that, even though mathematical models in biology and medicine may ultimately have a very different appearance, their development can be structured as a sequence of generic steps. Major drivers for many of the details of model development are the goals and objectives of the modeling task and the availability and quality of data that can be used for model design and validation.

A mathematical model of presynaptic dopamine homeostasis: implications for schizophrenia.

Several lines of evidence implicate altered dopamine neurotransmission in schizophrenia. Current drugs for schizophrenia focus on postsynaptic sites of the dopamine signaling pathways, but do not target presynaptic dopamine metabolism. We have begun to develop a mathematical model of dopamine homeostasis, which will aid our understanding of how genetic, environmental, and pharmacological factors alter the functioning of the presynaptic dopamine neuron. Formulated within the modeling framework of BIOCHEMICAL SYSTEMS THEORY, the mathematical model integrates relevant metabolites, enzymes, transporters, and regulators involved in the control of the biochemical environment within the dopamine neuron. In this report we use the model to assess several components and factors that affect the dopamine neuron and have been implicated in schizophrenia. These include the enzymes COMT, MAO, and TH, different dopamine transporters, as well as administration of amphetamine or cocaine. We also investigate scenarios that could increase (or decrease) dopamine neurotransmission and thus exacerbate (or alleviate) symptoms of schizophrenia. Our results indicate that the model predicts the effects of various factors related to schizophrenia on the homeostasis of the presynaptic dopamine neuron rather well. Upon further refinements and testing, the model has the potential of serving as a tool for screening novel therapeutics aimed at altering presynaptic dopamine function and thereby potentially ameliorating some of the symptomology of schizophrenia.

PACAP38 increases vesicular monoamine transporter 2 (VMAT2) expression and attenuates methamphetamine toxicity.

Pituitary adenylyl cyclase activating polypeptide, 38 amino acids (PACAP38) is a brain-gut peptide with diverse physiological functions and is neuroprotective in several models of neurological disease. In this study, we show that systemic administration of PACAP38, which is transported across the blood-brain barrier, greatly reduces the neurotoxicity of methamphetamine (METH). Mice treated with PACAP38 exhibited an attenuation of striatal dopamine loss after METH exposure as well as greatly reduced markers of oxidative stress. PACAP38 treatment also prevented striatal neuroinflammation after METH administration as measured by overexpression of glial fibrillary acidic protein (GFAP), an indicator of astrogliosis, and glucose transporter 5 (GLUT5), a marker of microgliosis. In PACAP38 treated mice, the observed protective effects were not due to an altered thermal response to METH. Since the mice were not challenged with METH until 28 days after PACAP38 treatment, this suggests the neuroprotective effects are mediated by regulation of gene expression. At the time of METH administration, PACAP38 treated animals exhibited a preferential increase in the expression and function of the vesicular monoamine transporter (VMAT2). Genetic reduction of VMAT2 has been shown to increase the neurotoxicity of METH, thus we propose that the increased expression of VMAT2 may underlie the protective actions of PACAP38 against METH. The ability of PACAP38 to increase VMAT2 expression suggests that PACAP38 signaling pathways may constitute a novel therapeutic approach to treat and prevent disorders of dopamine storage.

Norepinephrine loss produces more profound motor deficits than MPTP treatment in mice.

Although Parkinson's disease (PD) is characterized primarily by loss of nigrostriatal dopaminergic neurons, there is a concomitant loss of norepinephrine (NE) neurons in the locus coeruleus. Dopaminergic lesions induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are commonly used to model PD, and although MPTP effectively mimics the dopaminergic neuropathology of PD in mice, it fails to produce PD-like motor deficits. We hypothesized that MPTP is unable to recapitulate the motor abnormalities of PD either because the behavioral paradigms used to measure coordinated behavior in mice are not sensitive enough or because MPTP in the absence of NE loss is insufficient to impair motor control. We tested both possibilities by developing a battery of coordinated movement tests and examining motor deficits in dopamine beta-hydroxylase knockout (Dbh-/-) mice that lack NE altogether. We detected no motor abnormalities in MPTP-treated control mice, despite an 80% loss of striatal dopamine (DA) terminals. Dbh-/- mice, on the other hand, were impaired in most tests and also displayed spontaneous dyskinesias, despite their normal striatal DA content. A subset of these impairments was recapitulated in control mice with 80% NE lesions and reversed in Dbh-/- mice, either by restoration of NE or treatment with a DA agonist. MPTP did not exacerbate baseline motor deficits in Dbh-/- mice. Finally, striatal levels of phospho-ERK-1/2 and DeltaFosB/FosB, proteins which are associated with PD and dyskinesias, were elevated in Dbh-/- mice. These results suggest that loss of locus coeruleus neurons contributes to motor dysfunction in PD.

MR grid-tagging using hyperpolarized helium-3 for regional quantitative assessment of pulmonary biomechanics and ventilation.

A new technique is demonstrated in six healthy human subjects that combines grid-tagging and hyperpolarized helium-3 MRI to assess regional lung biomechanical function and quantitative ventilation. 2D grid-tagging, achieved by applying sinc-modulated RF-pulse trains along the frequency- and phase-encoding directions, was followed by a multislice fast low-angle shot (FLASH)-based acquisition at inspiration and expiration. The displacement vectors, first and second principal strains, and quantitative ventilation were computed, and mean values were calculated for the upper, middle, and lower lung regions. Displacements in the lower region were significantly greater than those in either the middle or upper region (P < 0.005), while there were no significant differences between the three regions for the two principal strains and quantitative ventilation (P = 0.11-0.92). Variations in principal strains and ventilation were greater between subjects than between lung zones within individual subjects. This technique has the potential to provide insight into regional biomechanical alterations of lung function in a variety of lung diseases.

Hyperpolarized 3He lung ventilation imaging with B1-inhomogeneity correction in a single breath-hold scan.

The signal-to-noise ratio (SNR) of hyperpolarized noble gas MR images is sensitive to the flip angle used. Variations in flip angle due to B1-inhomogeneity of the RF coil cause intensity variation artifacts in lung ventilation images which may mask or mimic disease. We show these artifacts can be minimized by using the optimal flip angle and corrected if the local flip angle is known. Hyperpolarized 3He lung images were obtained in ten healthy subjects using both a conventional gradient-echo sequence and a new hybrid pulse sequence designed to simultaneously acquire lung ventilation images and corresponding flip-angle maps in comparable imaging time. Flip-angle maps and corrected images were calculated from the hybrid scan and compared with conventional images. The qualitative theoretical dependence of flip angle on SNR was verified. Ventilation images and flip-angle maps were successfully obtained with the hybrid sequence. Corrections to image intensity calculated from the flip-angle maps appeared reasonable for images acquired using an average flip angle near optimal. Use of the optimal flip angle is crucial to the quality of lung ventilation images. Artifactual intensity variations due to RF-coil inhomogeneity may be identified and potentially corrected using our hybrid sequence.

Exercise induces behavioral recovery and attenuates neurochemical deficits in rodent models of Parkinson's disease.

Exercise is thought to improve motor function and emotional well-being in patients with Parkinson's disease (PD). However, it is not clear if the improvements are due to neurochemical alterations within the affected nigrostriatal region or result from a more general effect of exercise on affect and motivation. In this study we show that motorized treadmill running improves the neurochemical and behavioral outcomes in two rodent models of PD: the unilateral 6-hydroxydopamine (6-OHDA) rat model and bilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model in aged C57bl mice. Exposure to the dopamine (DA) toxins 6-OHDA or MPTP resulted in permanent behavioral and neurochemical loss. In contrast, when lesioned animals were exposed to treadmill activity two times a day for the first 10 days post-lesion they displayed no behavioral deficits across testing days and had significant sparing of striatal DA, its metabolites, tyrosine hydroxylase, vesicular monoamine transporter, and DA transporter levels compared to lesion sedentary animals. These results demonstrate that exercise following nigrostriatal damage ameliorates related motor symptoms and neurochemical deficits in rodent models of PD.

Episodic neonatal hypoxia evokes executive dysfunction and regionally specific alterations in markers of dopamine signaling.

Perinatal ischemic-anoxic and prolonged anoxic insults lead to impaired dopaminergic signaling and are hypothesized to contribute, at least in part, to the pathogenesis of disorders of minimal brain dysfunction such as attention-deficit hyperactivity disorder. We hypothesized that subtle intermittent hypoxic insults, occurring during a period of critical brain development, are also pathogenic to dopaminergic signaling, thereby contributing to behavioral and executive dysfunction. Between postnatal days 7 and 11, rat pups were exposed to either 20-s bursts of isocapnic hypoxic gas, compressed air, or were left undisturbed with the dam. On postnatal days 23 pups were instrumented with electroencephalographic/electromyographic electrodes and sleep-wake architecture was characterized. Locomotor activity was assessed between postnatal days 35 and 38, learning, and working memory evaluated between postnatal days 53 and 64. Rats were killed on postnatal day 80 and tyrosine hydroxylase, vesicular monoamine transporter, dopamine transporter, and dopamine D1 receptors were quantified in the prefrontal cortex, primary sensorimotor cortex, and precommissural striatum by Western blot analyses. Post-hypoxic pups spent less time awake and more time in rapid-eye-movement sleep during the lights-on phase of the circadian cycle, were hyperlocomotive, and expressed impaired working memory. Striatal expression of vesicular monoamine transporter and D1 receptor proteins were increased in post-hypoxic rats, consistent with depressed dopaminergic signaling. These observations lead to the intriguing hypothesis that intermittent hypoxia occurring during a period of critical brain development evokes behavioral and neurochemical alterations that are long lasting, and consistent with disorders of minimal brain dysfunction.

Transgenic expression of a mutant glycine receptor decreases alcohol sensitivity of mice.

Glycine receptors (GlyRs) are pentameric ligand-gated ion channels that inhibit neurotransmission in the adult brainstem and spinal cord. GlyR function is potentiated by ethanol in vitro, and a mutant GlyR subunit alpha(1)(S267Q) is insensitive to the potentiating effects of ethanol. To test the importance of GlyR for the actions of ethanol in vivo, we constructed transgenic mice with this mutation. Under the control of synapsin I regulatory sequences, transgenic expression of S267Q mutant GlyR alpha(1) subunits in the nervous system was demonstrated using [(3)H]strychnine binding and immunoblotting. These mice showed decreased sensitivity to ethanol in three behavioral tests: ethanol inhibition of strychnine seizures, motor incoordination (rotarod), and loss of righting reflex. There was no change in ethanol sensitivity in tests of acute functional tolerance or body temperature, and there was no change in ethanol metabolism. Transgene effects were pharmacologically specific for ethanol, compared with pentobarbital, flurazepam, and ketamine. These results support the idea that glycine receptors contribute to some behavioral actions of ethanol and that ethanol sensitivity can be changed in vivo by transgenic expression of a single receptor subunit.

Forced limb-use effects on the behavioral and neurochemical effects of 6-hydroxydopamine.

Rats with unilateral depletion of striatal dopamine (DA) show marked preferential use of the ipsilateral forelimb. Previous studies have shown that implementation of motor therapy after stroke improves functional outcome (Taub et al., 1999). Thus, we have examined the impact of forced use of the impaired forelimb during or soon after unilateral exposure to the DA neurotoxin 6-hydroxydopamine (6-OHDA). In one group of animals, the nonimpaired forelimb was immobilized using a cast, which forced exclusive use of the impaired limb for the first 7 d after infusion. The animals that received a cast displayed no detectable impairment or asymmetry of limb use, could use the contralateral (impaired) forelimb independently for vertical and lateral weight shifting, and showed no contralateral turning to apomorphine. The behavioral effects were maintained throughout the 60 d of observation. In addition to the behavioral sparing, these animals showed remarkable sparing of striatal DA, its metabolites, and the expression of the vesicular monoamine transporter, suggesting a decrease in the extent of DA neuron degeneration. Behavioral and neurochemical sparing appeared to be complete when the 7 d period of immobilization was initiated immediately after 6-OHDA infusion, only partial sparing was evident when immobilization was initiated 3 d postoperatively, and no sparing was detected when immobilization was initiated 7 d after 6-OHDA treatment. These results suggest that physical therapy may be beneficial in Parkinson's disease.

Hyperactivity and impaired response habituation in hyperdopaminergic mice.

Abnormal dopaminergic transmission is implicated in schizophrenia, attention deficit hyperactivity disorder, and drug addiction. In an attempt to model aspects of these disorders, we have generated hyperdopaminergic mutant mice by reducing expression of the dopamine transporter (DAT) to 10% of wild-type levels (DAT knockdown). Fast-scan cyclic voltammetry and in vivo microdialysis revealed that released dopamine was cleared at a slow rate in knockdown mice, which resulted in a higher extracellular dopamine concentration. Unlike the DAT knockout mice, the DAT knockdown mice do not display a growth retardation phenotype. They have normal home cage activity but display hyperactivity and impaired response habituation in novel environments. In addition, we show that both the indirect dopamine receptor agonist amphetamine and the direct agonists apomorphine and quinpirole inhibit locomotor activity in the DAT knockdown mice, leading to the hypothesis that a shift in the balance between dopamine auto and heteroreceptor function may contribute to the therapeutic effect of psychostimulants in attention deficit hyperactivity disorder.

Immunochemical analysis of dopamine transporters in Parkinson's disease.

The identification of specific and selective markers of the dopamine-producing neurons that are lost in Parkinson's disease has been a major research focus since Hornykiewicz first reported a dopamine deficiency in the disease (1). Antibodies to dopamine or tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, have been used to identify these neurons. Recently, considerable attention has been given to the plasma membrane dopamine tranporter (DAT) and the vesicular monoamine transporter (VMAT2), which are responsible for the transport, packaging, and release of dopamine (2). DAT acts to terminate dopamine transmission by rapid reuptake of dopamine from the synapse, and VMAT2 packages cytoplasmic dopamine into vesicles for storage and subsequent release. We have developed specific antibodies to these transporters and used them to characterize the distribution and expression of DAT and VMAT2 in brain from human idiopathic Parkinson's disease and animal models of the disease. The purpose of this chapter is to describe the immunochemical techniques involved in assessing damage to dopamine neurons in Parkinson's disease and experimental models of the disease.

Dopamine transporter and vesicular monoamine transporter knockout mice : implications for Parkinson's disease.

One of the most valuable methods for understanding the function of a particular protein is the generation of animals that have had the gene encoding for the protein of interest disrupted, commonly known as a "quo;knockout"quo; or null mutant. By incorporating a sequence of DNA (typically encoding antibiotic resistance to aid in the selection of the mutant gene) into embryonic stem cells by homologous recombination, the normal transcription of the gene is effectively blocked (Fig. 1). Since a particular protein is encoded by two copies of a gene, it is necessary to have the gene on both alleles "quo;knocked out."quo; This is performed by cross-breeding animals with one affected allele (heterozygote) to generate offspring that have inherited two mutant alleles (homozygote). This procedure has been used to generate animals lacking either the plasma membrane dopamine transporter (DAT; Fig. 2) or the vesicular monoamine transporter (VMAT2; Fig. 3). Both DAT and VMAT2 are essential for dopamine homeostasis and are thought to participate in the pathogenesis of Parkinson's disease (1-5). Fig. 1. Maps of the targeting vector and the mock construct. The mouse genomic fragment (clone 11) was isolated from a Stratagene 129 SvJ library by standard colony hybridization using a PCR probe from the 5' end of rat cDNA. The restriction site abbreviations are as follows: H, HindIII; N, NotI; Sc, SacI; Sn, SnaI; X, XbaI; and Xh, XhoI. The region between HindIII and SnaI on clone 11 containing the coding sequence from transmembrane domains 3 and 4 of VMAT2 was deleted and replaced with PGK-neo. The 3' fragment of clone 11 was reserved as an external probe for Southern analysis. To facilitate PCR screening of embryonic stem cell clones, a mock construct containing the SnaI/XbaI fragment and part of the Neo cassette was generated as a positive control. pPNT and pGEM4Z were used to construct knockout and mock vectors, respectively. (Reproduced with permission from ref. 1). Fig. 2. DAT and VMAT2 expression in wild-type and DAT knockout midbrain. DAT immunoreactivity in wild-type (A) and DAT knockout midbrain (B). VMAT2 immunoreactivity in wild-type (C) and DAT knockout midbrain (D). Robust immunoreactivity was observed in the ventral tegmental area and substantia nigra pars compacta and reticulata in the wild-type brain. Note absence of DAT immunoreactivity and modest reduction of VMAT2 immunoreactivity in the DAT knockout. Fig. 3. Characterization of VMAT2 gene disruption. (A) Southern blot analysis of mouse genomic DNA. The Southern blot was prepared with 15 µg of genomic DNA per lane and probed with a 1.4-kb 3' external genomic fragment. +/+, wild type littermates; +/-, heterozygote; -/-, homozygote. (B) RT-PCR analysis of mouse brain poly(A)+ RNA. For each reverse transcription assay, 0.5 µg of poly(A)+ RNA was used. Equal volumes of cDNA templates were used for each PCR assay. The PCR primers used flank the neomycin cassette for the purpose of detecting potential readthrough of the neomycin DNA. The heterozygote has a reduced amount of transcripts compared with the wild-type littermate; the homozygote is devoid of VMAT2 transcripts. G3PDH was used as internal control. (C) Western blot analysis of wholebrain synaptic vesicles. Samples (25 µg) of vesicles were solubilized and separated by SDS-PAGE, transferred to nitrocellulose, subjected to Western blot analysis with anti-VMAT2-Ct (top) or anti-a-tubulin (bottom) antibodies, and developed with chemiluminescence. Molecular mass markers (kDa) are shown to the left. To confirm equal loading and transfer of proteins, the blots were stripped and reprobed with an antibody to α-tubulin. (Reproduced with permission from ref. 1). The importance of DAT in neuronal function is highlighted in animals in which DAT has been genetically deleted (DAT KO) (3). In the homozygote DAT KO mice, released dopamine remains in the extracellular space up to 300 times longer than normal. As expected, these animals display behaviors consistent with persistent activation of dopamine receptors, such as hyperlocomotion. Genetic deletion of VMAT2 reveals the essential role of vesicular storage and release of monoamines. Homozygote VMAT2 knockout mice survive for only a few days, whereas heterozygotes appear normal. Studies performed in homozygote pups and heterozygote adults clearly show that the level of VMAT2 expression calibrates the level of vesicular filling (1,2,bi4). With only 50% of normal VMAT2, heterozygote animals have reduced vesicular filling and release. These alterations in presynaptic monoamine function in the heterozygotes are thought to be responsible for the observed sensitization to the psychostimulants cocaine and amphetamine and to ethanol (1). Knockout animals also appear to parallel the changes that occur in reserpinized animals, suggesting that the adverse actions of this drug are mediated by VMAT2.

Mice lacking the norepinephrine transporter are supersensitive to psychostimulants.

The action of norepinephrine (NE) is terminated, in part, by its uptake into presynaptic noradrenergic neurons by the plasma-membrane NE transporter (NET), which is a target for antidepressants and psychostimulants. Disruption of the NET gene in mice prolonged the clearance of NE and elevated extracellular levels of this catecholamine. In a classical test for antidepressant drugs, the NET-deficient (NET-/-) animals behaved like antidepressant-treated wild-type mice. Mutants were hyper-responsive to locomotor stimulation by cocaine or amphetamine. These responses were accompanied by dopamine D2/D3 receptor supersensitivity. Thus altering NET expression significantly modulates midbrain dopaminergic function, an effect that may be an important component of the actions of antidepressants and psychostimulants.

Heptachlor alters expression and function of dopamine transporters.

Epidemiological data support a relationship between pesticide exposure and Parkinson's disease; however, no experimental evidence has been provided to support this association. Here we report that subchronic administration of the organochlorine insecticide heptachlor (0, 3, 6, 9, or 12 mg/kg given 3 times over a 2 week period) leads to a pronounced increase in both the plasma membrane transport of dopamine and the expression of the plasma membrane dopamine transporter (DAT), as well as the vesicular monoamine transporter (VMAT2) in the striatum of C57BL mice. To address possible mechanisms of increased DAT and VMAT2 expression, we performed transport studies in cell lines expressing the human forms of either DAT or VMAT2. In a DAT expressing cell line, acute treatment with the putative toxic species of heptachlor, heptachlor epoxide, did not alter plasma membrane dopamine uptake. In a VMAT2 expressing cell line, heptachlor epoxide significantly inhibited vesicular uptake of dopamine (45% reduction at 10 microM). Since DAT has been proposed to be the molecular gateway for dopaminergic toxins, such as the parkinsonism-inducing neurotoxin MPP, and VMAT2 has been proposed to protect cells from MPP and other toxins by sequestering the toxin into vesicles, the combined effects of heptachlor could increase the susceptibility of the nigrostriatal dopamine system to neurodegeneration. We further propose that altered dopamine transport by exposure to pesticides may provide a molecular basis for the increased incidence of Parkinson's disease.