Presynaptic control of striatal dopamine neurotransmission in adult vesicular monoamine transporter 2 (VMAT2) mutant mice

The vesicular monoamine transporter 2 (VMAT2) plays a pivotal role in regulating the size of vesicular and cytosolic dopamine (DA) storage pools within the CNS, and can thus influence extracellular DA neurotransmission. Transgenic mice have been generated with a dramatically reduced (by approximately 95%) expression of the VMAT2 gene which, unlike complete knockout lines, survive into adulthood. We compared the pre-synaptic regulation of both impulse-dependent (exocytotic) and carrier-mediated (via reversal of the DA transporter, DAT) DA release in the dorsolateral caudate putamen (CPu) of striatal slices derived from adult homozygous VMAT2 mutant and wild-type mice using fast cyclic voltammetry. Impulse-dependent DA release, evoked by a single electrical pulse, was lower in homozygous (116 nm) than wild-type mice (351 nm) indicating smaller vesicular DA stores, an observation supported by the evanescent effect of amfonelic acid (300 nm) in homozygous mice. Amphetamine (2 microm) increased extracellular DA via DAT reversal in both wild-type (by 459 nm) and VMAT2 mutant (by 168 nm, p < 0.01 vs. wild-type) mice. In both cases, the effect was blocked by the DAT inhibitor GBR12935 (1 microm). Simultaneously, amphetamine decreased impulse-dependent DA release, albeit less in homozygous (by 55%) than in wild-type (by 78%) mice. In wild-types, this decrement was largely reversed by GBR12935 but not by the D2/D3 autoreceptor antagonist (-)sulpiride (1 microm). Conversely, in homozygous VMAT2 mutant mice, it was attenuated by (-)sulpiride but not GBR12935. The D2/D3 receptor agonist quinpirole inhibited impulse-dependent DA release with a lower EC50 value in homozygous mice (12 nm) compared with wild-types (34 nm), indicating the compensatory presence of functionally supersensitive release-regulating autoreceptors. However, analysis of DA reuptake kinetics obtained in the absence and presence of DAT blockade (by cocaine and amfonelic acid) revealed only minor differences in DAT functionality. These results demonstrate that impaired vesicular DA storage constrains extracellular DA levels in the dorsolateral CPu whether induced by either impulse-dependent or carrier-mediated mechanisms and that the relative importance of the DAT and terminal autoreceptors as control mechanisms in the actions of amphetamine are reversed in VMAT2 mutant mice.     

Presynaptic dopaminergic properties of differentiated mouse embryonic stem cells

This study characterized the presynaptic dopaminergic properties of neuronally differentiated mouse embryonic stem (ES) cells. Approximately 30% of the ES cells expressed tyrosine hydroxylase (TH) immunoreactivity when co-cultured with PA6 cells. These cultures expressed high affinity, sodium-dependent dopamine uptake as well as depolarization-induced and calcium-dependent dopamine release of this transmitter. These and other important dopaminergic genes found expressed in these cultures by RT-PCR included Nurr1, vesicular monoamine transporter 2 (VMAT2), TH, dopamine transporter (DAT), and glial cell line-derived neurotrophic factor (GDNF) receptors c-Ret and GFRalpha1. These results demonstrate that differentiated ES cells have the presynaptic functions for maintaining dopaminergic homeostasis, which may be essential for their long-term use in restoring CNS levels of this transmitter.     

BRSK2 is regulated by ER stress in protein level and involved in ER stress-induced apoptosis

Loss-of-functional mutation in the DJ-1 gene causes a subset of familial Parkinson's disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. Dopamine is synthesized by two enzymes and then packed into synaptic vesicles by vesicular monoamine transporter 2 (VMAT2). In this study, we found that knockdown of DJ-1 expression reduced the levels of mRNA and protein of VMAT2, resulting in reduced VMAT2 activity. Co-immunoprecipitation and pull-down experiments revealed that DJ-1 directly bound to VMAT2, and DJ-1 was co-localized with VMAT2 in cells. Furthermore, ectopic expression of wild-type DJ-1, but not that of L166P, M26I and C106S mutants of DJ-1, increased mRNA and protein levels of VMAT2 and VMAT2 activity. Since VMAT2 and a portion of DJ-1 are localized in the synaptic membrane, these results suggest that DJ-1, but not pathogenically mutated DJ-1, stimulates VMAT2 activity in the synapse by transactivation of the VMAT gene and by direct binding to VMAT2 and that cysteine 106 is necessary for the stimulating activity of DJ-1 toward VMAT2.     

Reduced vesicular storage of dopamine exacerbates methamphetamine-induced neurodegeneration and astrogliosis

The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5–10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

p53-Knockout Mice Are Protected Against the Long-Term Effects of Methamphetamine on Dopaminergic Terminals and Cell Bodies

Abstract: p53-knockout mice provide a useful model to test the role of p53 in the neurotoxic effects of drugs in vivo. To test the involvement of p53 in methamphetamine (METH)-induced toxicity, wild-type mice, as well as heterozygous and homozygous p53-knockout male mice, were administered four injections of three different doses (2.5, 5.0, and 10.0 mg/kg) of the drug given at 2-h intervals within the space of 1 day. METH caused a marked dose-dependent loss of dopamine transporters in both the striatum and the nucleus accumbens of wild-type mice killed 2 weeks after drug administration. However, this METH-induced decrease in dopamine transporters was attenuated in both homozygous and heterozygous p53-knockout mice, with homozygous animals showing significantly greater protection. The possibility for p53 involvement in METH-induced toxicity was also supported by the observation that METH caused marked increases in p53-like immunoreactivity in the striata of wild-type mice and very little change in heterozygous p53-knockout mice, whereas no p53-like immunostaining was detected in the homozygous p53-knockout mice. Further support for p53 involvement was provided by the fact that METH treatment caused significant decreases in dopamine transporter mRNA and the number of tyrosine hydroxylase-positive cells in the substantia nigra pars compacta and the ventral tegmental area of wild-type but not homozygous p53-knockout mice killed 2 weeks after cessation of METH administration. These results provide concordant evidence for a role of the tumor suppressor, p53, in the long-term deleterious effects of a drug acting on brain dopamine systems.     

Age-Dependent Alterations in Dopamine Content, Tyrosine Hydroxylase Activity, and Dopamine Uptake in the Striatum of the Weaver Mutant Mouse

Abstract: Mice of different ages and homozygous or heterozygous for the weaver gene ( wv ) were used to study the time course for the effect of the weaver gene on several striatal dopaminergic parameters. Dopamine uptake was decreased in the homozygous weaver at all ages examined. The deficit in uptake at the earliest age studied, postnatal day 3, was approximately 50% and increased to greater than 70% at older ages. In control mice, dopamine uptake reached a maximum by postnatal day 22, but in homozygous weaver mice, development of uptake activity was curtailed by postnatal day 7. Dopamine content and tyrosine hydroxylase activity were significantly decreased in the homozygous weaver at all ages studied except postnatal days 7 and 10. The magnitude of the deficit in dopamine content ranged from approximately 40% at postnatal days 3 and 5 to about 70% in adults (6 months to 1 year of age). The magnitude of the deficit in tyrosine hydroxylase activity ranged from 40 to 70%. In general, no major differences between heterozygotes and controls were observed for any of the dopaminergic parameters investigated. The results of the present investigation indicate that neurochemical alterations can be observed in the striata of weaver mice as early as postnatal day 3 and raise the possibility that the striatal dopamine transporter may be an early target of the weaver mutation.     

INHIBITION OF CATALASE IN MESENCEPHALIC CULTURES BY l-DOPA AND DOPAMINE

Catalase activity in cell cultures of fetal rat mesencephalon was decreased by 42 and 50%, respectively, after exposure to l-3,4-dihydroxyphenylalanine (l-DOPA, 100 μM) or dopamine (100 μM) for 48 h. Catalase activity was also decreased 21% by 10 μM hydroquinone. Ascorbic acid (200 μM), an agent that suppresses the autoxidation of l-DOPA and dopamine, blocked the anti-catalase effect of l-DOPA, but not that of dopamine. Inhibitors of the A and B forms of monoamine oxidase (20 μM clorgyline plus 20 μM pargyline) had no effect on the anti-catalase action of either l-DOPA or dopamine. The latter results suggest that products of the oxidative deamination of dopamine by monoamine oxidase are not involved in the suppression of catalase activity. However, autoxidation reactions of l-DOPA may play a role since ascorbate suppressed the anti-catalase effect of l-DOPA. On the contrary, the basis for the failure of ascorbate to similarly block the anti-catalase effect of dopamine is uncertain. l-DOPA and dopamine (25 μM) also inhibited crystalline catalase in solution after incubation for 1 h at neutral pH (40–50% inhibition). Inhibition was blocked by 0.45 M ethanol, indicating a need for autoxidation and the formation of compound II, which is an enzymatically inactive form of catalase. The ability to model the enzyme inhibition in purely chemical experiments indicates a probable mechanism for loss of enzymatic activity in cell cultures. Inhibition of catalase may contribute to cell damage during incubation of cultures with l-DOPA, dopamine, or other autoxidizable compounds. Copyright © 1996 Elsevier Science Ltd     

Absence of synapsin I and II is accompanied by decreases in vesicular transport of specific neurotransmitters

Studies of synapsin-deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild-type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double-labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.     

Increased dopaminergic signaling impairs aversive olfactory memory retention in Drosophila

Dopamine is necessary for the aversive olfactory associative memory formation in Drosophila, but its effect on other stages of memory is not known. Herein, we studied the effect of enhanced dopaminergic signaling on aversive olfactory memory retention in flies. We used l-3,4-dihydroxyphenylalanine (l-DOPA) to elevate dopamine levels: l-DOPA-treated flies exhibited a normal learning performance, but a decrease in 1-h memory. Dopamine transporter (DAT) mutant flies or flies treated with the DAT inhibitor desipramine exhibited poor memory retention. Flies subjected to heat stress after training exhibited a decrease in memory. Memory was restored by blocking dopaminergic neuronal output during heat stress, suggesting that dopamine is involved in heat stress-induced memory impairment in flies. Taken together, our findings suggest that increased dopaminergic signaling impairs aversive olfactory memory retention in flies.     

Differential effects of l-DOPA on monoamine metabolism, cell survival and glutathione production in midbrain neuronal-enriched cultures from parkin knockout and wild-type mice

l-DOPA is the most effective treatment for Parkinson's disease but in isolated neuronal cultures it is neurotoxic for dopamine (DA) neurones. Experiments in vivo and clinical studies have failed to show toxicity of l-DOPA in animals or patients but that does not exclude the possibility of a toxic effect of l-DOPA on patients with certain genetic risk factors. Mutations of the parkin gene are the most frequent cause of hereditary parkinsonism. Parkin null mice have a mild phenotype that could be modified by different neurotoxins. The aim of this study was to investigate whether the toxic effects of l-DOPA on DA neurones are amplified in parkin null mice. We have measured the effects of l-DOPA on cell viability, tyrosine hydroxylase (TH) expression, DA metabolism and glutathione levels of parkin knockout (PK-KO) midbrain cultures. Neuronal-enriched cultures from PK-KO mice have similar proportions of the different cell types with the exception of a significant increment of microglial cells. l-DOPA (400 microm for 24 h) reduced the number of TH-immunoreactive cells to 50% of baseline and increased twofold the percentage of apoptotic cells in cultures of wild-type (WT) animals. The PK-KO mice, however, are not only resistant to the l-DOPA-induced pro-apoptotic effects but they have an increased number of TH-immunoreactive neurones after treatment with l-DOPA, suggesting that l-DOPA is toxic for neurones of WT mice but not those of parkin null mice. MAPK and phosphatidylinositol-3 kinase signalling pathways are not involved in the differential l-DOPA effects in WT and PK-KO cultures. Intracellular levels of l-DOPA were not different in WT and parkin null mice but the intracellular and extracellular levels of DA and 3-4-dihydroxyphenylacetic acid, however, were significantly increased in parkin null animals. Furthermore, monoamine oxidase activity was significantly increased in parkin null mice, suggesting that these animals have an increased metabolism of DA. The levels of glutathione were further increased in parkin null mice than in controls both with and without treatment with l-DOPA, suggesting that a compensatory mechanism may protect DA neurones from neuronal death. This study opens new avenues for understanding the mechanisms of action of l-DOPA on DA neurones in patients with Park-2 mutations.     

VMAT2 and dopamine neuron loss in a primate model of Parkinson's disease

We used positron emission tomography (PET) to measure the earliest change in dopaminergic synapses and glial cell markers in a chronic, low-dose MPTP non-human primate model of Parkinson's disease (PD). In vivo levels of dopamine transporters (DAT), vesicular monoamine transporter-type 2 (VMAT2), amphetamine-induced dopamine release (AMPH-DAR), D2-dopamine receptors (D2R) and translocator protein 18 kDa (TSPO) were measured longitudinally in the striatum of MPTP-treated animals. We report an early (2 months) decrease (46%) of striatal VMAT2 in asymptomatic MPTP animals that preceded changes in DAT, D2R, and AMPH-DAR and was associated with increased TSPO levels indicative of a glial response. Subsequent PET studies showed progressive loss of all pre-synaptic dopamine markers in the striatum with expression of parkinsonism. However, glial cell activation did not track disease progression. These findings indicate that decreased VMAT2 is a key pathogenic event that precedes nigrostriatal dopamine neuron degeneration. The loss of VMAT2 may result from an association with α-synuclein aggregation induced by oxidative stress. Disruption of dopamine sequestration by reducing VMAT2 is an early pathogenic event in the dopamine neuron degeneration that occurs in the MPTP non-human primate model of PD. Genetic or environmental factors that decrease VMAT2 function may be important determinants of PD.     

Protein kinase A affects trafficking of the vesicular monoamine transporters in PC12 cells

Previous studies have shown that the vesicular monoamine transporter 2 (VMAT2) is localized to both large dense core vesicles and synaptic vesicles in vivo. However, when exogenously expressed in PC12 cells, VMAT2 localizes only to large dense core vesicles. This distribution is similar to that of the endogenous vesicular monoamine transporter 1 (VMAT1) in PC12 cells. When VMAT2 was expressed in a protein kinase A (PKA)-deficient PC12 cell line it localized to synaptic-like microvesicles. Expression of recombinant VMAT1 in the same cell line showed a heterogeneous distribution to both large dense core vesicles and synaptic-like microvesicles. Coexpression of the PKA catalytic subunit partially restored trafficking of both VMAT2 and VMAT1 to large dense core vesicles; treatment of wild-type PC12 cells with the PKA inhibitor H89 increased VMAT2 on synaptic-like microvesicles. The VMAT1 and VMAT2 in large dense core vesicles exhibit a larger molecular size than those located on synaptic-like microvesicles. This difference is due to differential N-linked glycosylation. In vitro phosphorylation experiments show that PKA does not phosphorylate VMAT2. A chimera containing the VMAT2 cytoplasmic C-terminus fused to vesicular acetylcholine transporter (VAChT) shows mislocalization to synaptic-like microvesicles and VAChT-like glycosylation in the PKA-deficient cell line. However, coexpression with PKA changes the chimera's trafficking to large dense core vesicles and increases the molecular size. These results suggest that protein kinase A affects the formation and/or composition of VMAT trafficking complexes.     

GDNF modulates HO-1 expression in substantia nigra postnatal cell cultures

Heme oxygenase-1 (HO-1) has been strongly highlighted because of its induction in many cell types by toxic stimuli, including oxidative stress. The intense HO-1 immunostaining in the substantia nigra of Parkinson disease (PD) patients suggests its involvement in the pathogenesis of this neurodegenerative disease. In this work we investigated HO-1 expression in rat substantia nigra postnatal cell cultures under conditions mimicking dopamine toxicity and its modulation by glial cell line-derived neurotrophic factor (GDNF), a potent neuroprotective factor for dopaminergic neurons. In neuron–glia cultures, we found that H₂O₂, a product of dopamine metabolism, or l-3,4-dihydroxyphenylalanine (l-DOPA), the dopamine precursor used in the therapy of PD, induced a fast up-regulation of HO-1 mRNA and protein levels, followed by a secondary down-regulation. H₂O₂ and l-DOPA also increased HO-1 expression in astrocyte cultures, but with a delayed time course in H₂O₂-treated cultures. HO-1 expression was decreased in neuron–glia cultures under conditions under which GDNF up-regulation was observed. Because exogenously applied GDNF prevented HO-1 up-regulation in cultures treated with H₂O₂ or l-DOPA, and antibody neutralization of GDNF prevented the secondary HO-1 down-regulation observed in neuron–glia cultures, we propose that GDNF negatively modulates HO-1 expression induced by oxidative stress. To our knowledge, this is the first report showing the modulation of HO-1 expression by GDNF.     

The vesicular monoamine transporter 2 contains trafficking signals in both its N-glycosylation and C-terminal domains

The vesicular acetylcholine transporter (VAChT) and the vesicular monoamine transporter (VMAT) belong to the same transporter family that packages acetylcholine into synaptic vesicles (SVs) and biogenic amines into large dense core vesicles (LDCVs) and/or SVs, respectively. These transporters share similarities in sequence and structure with their N- and C-terminal domains located in the cytoplasm. When expressed in PC12 cells, VMAT2 localizes to LDCV, whereas VAChT is found mainly on synaptic-like microvesicles. Previous studies have shown that the cytoplasmic C-terminal domain of VAChT contains signals targeting this transporter to SVs. However, the targeting signals for VMAT have not been completely elucidated. To identify signals targeting VMAT2 to LDCV, the subcellular localization of VMAT2-VAChT chimeras was analyzed in PC12 cells. Chimeras having either the N-terminal region through transmembrane domain 2 of VMAT2 or the C-terminal domain of VMAT2 do not traffic to LDCV efficiently. In contrast, chimeras having both of these regions, or the luminal glycosylated loop in conjunction with transmembrane domains 1 and 2 and the C-terminal domain of VMAT2, traffic to LDCV. Treatment of PC12 cells with 1-deoxymannojirimycin, a specific alpha-mannosidase I inhibitor, causes VMAT2 to localize to synaptic-like microvesicles. The results indicate that both mature N-linked glycosylation and the C-terminus are important for proper trafficking of VMAT2 and that the locations of trafficking signals in VMAT2 and VAChT are surprisingly different.     

Neurotoxic mechanisms of 2,9-dimethyl-beta-carbolinium ion in primary dopaminergic culture

beta-Carbolines are potential endogenous and exogenous neurotoxicants that may contribute to the pathogenesis of Parkinson's disease. The 2,9-dimethyl-beta-carbolinium ion (either 2,9-dimethyl-beta-norharmanium or 2,9-Me(2)NH(+)) was found to be neurotoxic in primary mesencephalic cultures and to be a potent inhibitor of mitochondrial complex I. However, the precise mechanisms of cell death remained obscure. Here, we investigated the mechanism of cell death in primary dopaminergic cultures of the mouse mesencephalon mediated by 2,9-Me(2)NH(+). The beta-carboline caused preferential death of dopaminergic neurones, which could not be attributed to cellular uptake via the dopamine transporter. Transient incubation with 2,9-Me(2)NH(+) for 48 h caused a progressive deterioration in the morphology of dopaminergic neurones during a 5-day recovery period and persistent damage to the overall culture. An increase in free radical production and caspase-3 activity, as well as a decrease of respiratory activity, mitochondrial membrane potential and ATP content, contributed to toxicity and pointed to an apoptotic mode of cell death, although a significant quantity of cells dying via necrosis were present simultaneously. These data underline the preferential susceptibility of dopaminergic neurones to 2,9-Me(2)NH(+) as a potent, oxidative stress generating neurotoxin.     

The vesicular monoamine content regulates VMAT2 activity through Galphaq in mouse platelets. Evidence for autoregulation of vesicular transmitter uptake

Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content may be modulated by vesicle-associated Go(2), which down-regulates the activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin O-permeabilized platelets, VMAT2 activity is also down-regulated by the G protein activator guanosine 5'-(beta(i)gamma-imido)triphosphate (GMppNp). Using serotonin-depleted platelets from peripheral tryptophan hydroxylase knockout (Tph1-/-) mice, we show here that the vesicular filling initiates the G protein-mediated down-regulation of VMAT2 activity. GMppNp did not influence VMAT2 activity in naive platelets from Tph1-/- mice. GMppNp-mediated inhibition could be reconstituted, however, when preloading Tph1-/- platelets with serotonin or noradrenaline. Galpha(q) mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from Galpha(q) deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A(2) receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle itself regulates transmitter transporter activity via its content and vesicle-associated Galpha(q).     

Characterization of the striatal dopaminergic neurotransmission in MEN2B mice with elevated cerebral tissue dopamine

The Ret receptor tyrosine kinase is the common signaling receptor for the glial cell line-derived neurotrophic factor (GDNF) family ligands. The Met918Thr mutation leads to constitutive activation of Ret and is responsible for dominantly inherited cancer syndrome MEN2B. Previously, we found that the mice carrying the mutation (MEN2B mice) have profoundly increased tissue dopamine (DA) concentrations in the striatum as well as increased striatal levels of tyrosine hydroxylase (TH) and dopamine transporter. The aim of this study was to characterize the striatal dopaminergic neurotransmission in MEN2B mice and to clarify the mechanisms by which they compensate their over-production of DA. We found that tyrosine hydroxylase activity and DA synthesis are increased in MEN2B mice. Augmented effects of α-methyl-para-tyrosine (αMT, an inhibitor of TH) and tetrabenazine (VMAT2 blocker) on DA levels suggest that also storage of DA is increased in MEN2B mice. There was no difference in the basal extracellular DA concentrations or potassium-evoked DA release between the genotypes. The effects of cocaine and haloperidol were also similar between the genotypes as assessed by in vivo microdialysis. However, with in vivo voltammetry we found increase in stimulated DA release in MEN2B mice and detailed analysis of DA overflow showed that uptake of DA was also enhanced in MEN2B mice. Thus, our data show that enhanced synthesis of DA leading to increased storage and releasable pools in pre-synaptic terminals in MEN2B mice apparently also leads to increased DA release, which in turn is compensated by higher dopamine transporter activity.