Characterization of A11 Neurons Projecting to the Spinal Cord of Mice

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons.     

Influences of Chronic Mild Stress Exposure on Motor,Non-Motor Impairments and Neurochemical Variables in Specific Brain Areas of MPTP/Probenecid Induced Neurotoxicity in Mice

Parkinson''s disease (PD) is regarded as a movement disorder mainly affecting the elderly population and occurs due to progressive loss of dopaminergic (DAergic) neurons in nigrostriatal pathway. Patients suffer from non-motor symptoms (NMS) such as depression, anxiety, fatigue and sleep disorders, which are not well focussed in PD research. Depression in PD is a predominant /complex symptom and its pathology lies exterior to the nigrostriatal system. The main aim of this study is to explore the causative or progressive effect of chronic mild stress (CMS), a paradigm developed as an animal model of depression in1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg. body wt.) with probenecid (250 mg/kg, s.c.) (MPTP/p) induced mice model of PD. After ten i.p. injections (once in 3.5 days for 5 weeks) of MPTP/p or exposure to CMS for 4 weeks, the behavioural (motor and non-motor) impairments, levels and expressions of dopamine (DA), serotonin (5-HT), DAergic markers such as tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporters—2 (VMAT 2) and α-synuclein in nigrostriatal (striatum (ST) and substantia nigra (SN)) and extra-nigrostriatal (hippocampus, cortex and cerebellum) tissues were analysed. Significantly decreased DA and 5-HT levels, TH, DAT and VMAT 2 expressions and increased motor deficits, anhedonia-like behaviour and α-synuclein expression were found in MPTP/p treated mice. Pre and/or post exposure of CMS to MPTP/p mice further enhanced the MPTP/p induced DA and 5-HT depletion, behaviour abnormalities and protein expressions. Our results could strongly confirm that the exposure of stress after MPTP/p injections worsens the symptoms and neurochemicals status of PD.     

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.     

Dopamine transporter relation to levodopa-derived synaptic dopamine in a rat model of Parkinson's: an in vivo imaging study

Studies showed that the dopamine (DA) transporter (DAT) modulates changes in levodopa-derived synaptic dopamine levels (Δ(DA)) in Parkinson's disease (PD). Here we evaluate the relationship between DAT and Δ(DA) in the 6-hydroxydopamine model of Parkinson's disease to investigate these mechanisms as a function of dopaminergic denervation and in relation to other denervation-induced regulatory changes. 27 rats with a unilateral 6-hydroxydopamine lesion (denervation ∼20–97%) were imaged with ¹¹C-dihydrotetrabenazine (VMAT2 marker), ¹¹C-methylphenidate (DAT marker) and ¹¹C-raclopride (D2-type receptor marker). For denervation <75%Δ(DA) was significantly correlated with a combination of relatively preserved terminal density and lower DAT. For denervation <90%, Δ(DA) was significantly negatively correlated with DAT with a weaker dependence on VMAT2. For the entire data set, no dependence on pre-synaptic markers was observed; Δ(DA) was significantly positively correlated with ¹¹C-raclopride binding-derived estimates of DA loss. These findings parallel observations in humans, and show that (i) regulatory changes attempt to normalize synaptic DA levels (ii) a lesion-induced functional dependence of Δ(DA) on DAT occurs up to ∼ 90% denervation (iii) for denervation < 75% relative lower DAT levels may relate to effective compensation; for higher denervation, lower DAT levels likely contribute to oscillations in synaptic DA associated with dyskinesias.     

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.     

Increased MPTP Neurotoxicity in Vesicular Monoamine Transporter 2 Heterozygote Knockout Mice

Abstract: The neurotoxic action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/−) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/− mice were more sensitive than their wild-type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose-dependent reductions in striatal DA levels in both VMAT2 +/− and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/− mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/− and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/− mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/− mice than in the wild-type: 15.8- versus 7.8-fold increase at 7.5 mg/kg and 20.1- versus 9.6-fold at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/− mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2-mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.     

Three Types of Tyrosine Hydroxylase-Positive CNS Neurons Distinguished by Dopa Decarboxylase and VMAT2 Co-Expression

Sumary 1. We investigate here for the first time in primate brain the combinatorial expression of the three major functionally relevant proteins for catecholaminergic neurotransmission tyrosine hydroxylase (TH), aromatic acid acid decarboxylase (AADC), and the brain-specific isoform of the vesicular monoamine transporter, VMAT2, using highly specific antibodies and immunofluorescence with confocal microscopy to visualize combinatorial expression of these proteins.2. In addition to classical TH, AADC, and VMAT2-copositive catecholaminergic neurons, two unique kinds of TH-positive neurons were identified based on co-expression of AADC and VMAT2.3. TH and AADC co-positive, but VMAT2-negative neurons, are termed “nonexocytotic catecholaminergic TH neurons.” These were found in striatum, olfactory bulb, cerebral cortex, area postrema, nucleus tractus solitarius, and in the dorsal motor nucleus of the vagus.4. TH-positive neurons expressing neither AADC nor VMAT2 are termed “dopaergic TH neurons.” We identified these neurons in supraoptic, paraventricular and periventricular hypothalamic nuclei, thalamic paraventicular nucleus, habenula, parabrachial nucleus, cerebral cortex and spinal cord. We were unable to identify any dopaergic (TH-positive, AADC-negative) neurons that expressed VMAT2, suggesting that regulatory mechanisms exist for shutting off VMAT2 expression in neurons that fail to biosynthesize its substrates.5. In several cases, the corresponding TH phenotypes were identified in the adult rat, suggesting that this rodent is an appropriate experimental model for further investigation of these TH-positive neuronal cell groups in the adult central nervous system. Thus, no examples of TH and VMAT2 co-positive neurons lacking AADC expression were found in rodent adult nervous system.6. In conclusion, the adult mammalian nervous system contains in addition to classical catecholaminergic neurons, cells that can synthesize dopamine, but cannot transport and store it in synaptic vesicles, and neurons that can synthesize only L-dopa and lack VMAT2 expression. The presence of these additional populations of TH-positive neurons in the adult primate CNS has implications for functional catecholamine neurotransmission, its derangement in disease and drug abuse, and its rescue by gene therapeutic maneuvers in neurodegenerative diseases such as Parkinson's disease.     

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators'' choice.We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein ^1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA ^6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).     

Hyperactivity and alteration of the midbrain dopaminergic system in maternally stressed male mice offspring

We recently demonstrated that prolonged maternal stress produces profound and long-lasting deficits in brain functions by programming a subset of target genes. We have now examined the possible effects of prenatal stress on the motility of adult offspring and dopamine (DA)-related gene expression in their midbrains, one of the target brain regions of stress hormones. Maternally stressed adult male mice showed impaired response habituation to novelty, and increased wheel-running activity associated with altered responses to DA receptor and DA transporter (DAT) blockers. Along with the behavioral changes, the expression profiles of several genes of the midbrain DAergic system appeared to be altered. Expression of DAT was reduced and expression of DA receptors and striatal DA-regulated neuropeptide genes was also affected. Taken together, the present findings indicate that maternal stress can cause hyperactivity in adult offspring associated with alterations in the midbrain DAergic system suggestive of a functional hyperdopaminergic state.     

Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis

Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 microM treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K(m), in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three-fivefold in both cell types exposed to 50 microM DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.     

Attenuation of estradiol on the reduction of striatal dopamine by amphetamine in ovariectomized rats

Amphetamine (AMPH) is a highly addictive drug of abuse which exhibits toxicity to dopaminergic neurons in long‐term abusers. Estrogen seems to show neuroprotection in dopamine (DA) deficit caused by AMPH. The present study was to investigate the effects of estradiol on the levels of striatal DA in ovariectomized (Ovx) rats treated with or without AMPH. Female rats were Ovx for 2 weeks before administration of AMPH (5 mg/kg/day, i.p.) with or without 17β‐estradiol benzoate (EB) (25 µg/kg/day, s.c.) for 7 days. The striatal tissues were collected, homogenized with DA mobile phase, and centrifuged. The concentrations of DA in the supernatants were detected by HPLC. The protein expressions of dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT‐2), and tyrosine hydroxylase (TH) were analyzed by Western blotting. The results indicated that AMPH could attenuate DA level significantly in striatum (P < 0.01). Comparing to control groups, administration of either EB or EB plus AMPH increased DA level (P < 0.01). The protein expression of striatal DAT was significant greater (P < 0.01) in rats treated with AMPH plus EB than AMPH treated animals. These results suggest that the DA levels in striatum can be enhanced by EB via an increase of DAT expression following administration of AMPH. J. Cell. Biochem. 108: 1318–1324, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

Plasmalogen Augmentation Reverses Striatal Dopamine Loss in MPTP Mice

Plasmalogens are a class of glycerophospholipids shown to play critical roles in membrane structure and function. Decreased plasmalogens are reported in the brain and blood of Parkinson’s disease (PD) patients. The present study investigated the hypothesis that augmenting plasmalogens could protect striatal dopamine neurons that degenerate in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in mice, a PD model. First, in a pre-treatment experiment male mice were treated for 10 days with the docosahexaenoic acid (DHA)-plasmalogen precursor PPI-1011 (10, 50 and 200 mg/kg). On day 5 mice received MPTP and were killed on day 11. Next, in a post-treatment study, male mice were treated with MPTP and then received daily for 5 days PPI-1011 (5, 10 and 50 mg/kg). MPTP treatment reduced serum plasmalogen levels, striatal contents of dopamine (DA) and its metabolites, serotonin, DA transporter (DAT) and vesicular monoamine transporter 2 (VMAT2). Pre-treatment with PPI-1011 (10 and 50 mg/kg) prevented all MPTP-induced effects. Positive correlations were measured between striatal DA contents and serum plasmalogen levels as well as striatal DAT and VMAT2 specific binding. Post-treatment with PPI-1011 prevented all MPTP-induced effects at 50 mg/kg but not at lower doses. Positive correlations were measured between striatal DA contents and serum plasmalogen levels as well as striatal DAT and VMAT2 specific binding in the post-treatment experiment. PPI-1011 treatment (10 days at 5, 10 and 50 mg/kg) of intact mice left unchanged striatal biogenic amine contents. These data demonstrate that treatment with a plasmalogen precursor is capable of protecting striatal dopamine markers in an animal model of PD.     

Dopamine turnover is upregulated in the caudate/putamen of asymptomatic MPTP-treated rhesus monkeys

In Parkinson's disease (PD) and experimental parkinsonism, losses of up to 60% and 80%, respectively, of dopaminergic neurons in substantia nigra, and dopamine (DA) in striatum remain asymptomatic. Several mechanisms have been suggested for this functional compensation, the DA-mediated being the most established one. Since this mechanism was recently challenged by striatal DA analysis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, we present data on several DAergic parameters in three groups of rhesus monkeys: MPTP-treated asymptomatic animals; symptomatic MPTP-treated animals with stable parkinsonism; and untreated sex and age matched controls. We determined ratios of striatal and nigral 3,4-dihydroxyphenyl acetic acid (DOPAC) to DA levels and tyrosine hydroxylase (TH) enzyme activity to DA levels, in addition to the commonly used homovanillic acid (HVA)/DA ratios which, as such, might be less reliable under the conditions of partial denervation. We found that in the asymptomatic MPTP monkeys the DOPAC/DA ratios in putamen and caudate nucleus were shifted with high statistical significance 1.9-5.8-fold, as compared to controls, the shifting of the ratios being in the same range as the 2.6-5.4-fold shifts in the symptomatic animals. Also TH/DA ratios were significantly increased in both, the asymptomatic and the symptomatic MPTP-treated monkeys, with shifts in the putamen and caudate nucleus of 3- and 2.7-7.0-fold, respectively. In the substantia nigra, DOPAC levels and TH activity were strongly decreased after MPTP (-77 to -97%), but the ratios DOPAC/DA and TH/DA were not changed in this brain region. Collectively, our findings support the concept of DAergic compensation of the progressive striatal DA loss in the presymptomatic stages of the parkinsonian disease process.     

Iron deficiency alters dopamine uptake and response to L-DOPA injection in Sprague-Dawley rats

Iron deficiency (ID) disrupts brain dopamine (DA) and norepinephrine (NE) metabolism including functioning of monoamine transporters and receptors. We employed caudate microdialysis and no net flux (NNF) in post-weaning rats to determine if ID decreased the extraction fraction ( E _d). Five micromolar quinpirole, a dopamine D₂ receptor agonist, resulted in 80% decrease in extracellular DA and 45% higher E _d in control animals. The D₂ agonist had no effect on E _d in ID animals despite a reduction in basal DA. DAT mRNA levels were reduced by 58% with ID, while DAT protein in ventral midbrain and caudate and membrane associated DAT were also reduced by ID. Carbidopa/ l -DOPA was administered to determine if elevated extracellular DA in ID was due to increased release. The DA response to l -DOPA in ID rats was 50% smaller and delayed, whereas the NE response was threefold higher. The caudate concentration of NE was also elevated in ID. Elevated dopamine-β-hydroxylase activity in ID provides a tentative explanation for the increased NE response to l -DOPA. These experiments provide new evidence that ID results in altered synthesis and functioning of DAT and perhaps suggests some compensatory changes in NE metabolism.     

Effect of Aging on Tyrosine Hydroxylase Protein Content and the Relative Number of Dopamine Nerve Terminals in Human Caudate

This study examined the effect of aging on the relative number of dopamine (DA) nerve terminals in human caudate nucleus, their content of tyrosine hydroxylase (TH) protein, and the relative abundance of TH monomers with different molecular weights. Preliminary studies on brain tissue cryopreservation, performed with rat striatum, indicated that intact synaptosomes can be prepared from fresh tissue slowly frozen in 0.32 M sucrose with 5% dimethyl sulfoxide and then thawed rapidly prior to synaptosome preparation. Synaptosomes were prepared in this manner from postmortem caudate nucleus tissue obtained from normal humans 1 month to 63 years of age. To determine the relative number of DA nerve terminals for each individual, dopaminergic synaptosomes were selectively labeled with a monoclonal antibody to TH and quantified by fluorescence-activated cell sorting. To determine the relative amount of TH protein for each individual, the concentration of TH protein in the same synaptosomal preparations was determined using immunoblots. Our results suggest that caudate TH levels plateau soon after birth and tend to remain relatively stable during aging, since no changes in either the relative number of TH-containing nerve terminals or the concentration of TH protein were found in subjects 15-63 years of age. In light of previous studies showing an age-related loss of DA cell bodies, these findings suggest that remaining DA neurons compensate to maintain caudate levels of TH protein and TH-containing nerve terminals. Immunoblot studies identified three forms of TH monomer (60.6, 61.7, and 65.1 kDa), indicating that mRNAs coding for high molecular mass forms of TH may be actively translated in human brain. No age-related differences in the relative abundance of these forms were found.     

Effects of cypermethrin on monoamine transporters,xenobiotic metabolizing enzymes and lipid peroxidation in the rat nigrostriatal system

Abstract

Long-term exposure to cypermethrin induces the nigrostriatal dopaminergic neurodegeneration in adult rats and its pre-exposure in the critical periods of brain development enhances the susceptibility during adulthood. Monoamine transporters, xenobiotic metabolizing enzymes and oxidative stress play critical roles in the nigrostriatal dopaminergic neurodegeneration. The study was undertaken to investigate the effects of cypermethrin on DAT, VMAT 2, CYP2E1, GST Ya, GST Yc and GSTA4-4 expressions, CYP2E1 and GST activities and lipid peroxidation in the nigrostriatal system of adult rats with/without post-natal exposure to cypermethrin. Cypermethrin reduced VMAT 2 and increased CYP2E1 expressions without causing significant change in DAT. Although GSTA4-4 mRNA expression and lipid peroxidation were increased, no significant changes were observed in GST Ya and GST Yc expressions and total GST activity. The results obtained demonstrate that long-term exposure to cypermethrin modulates VMAT 2, CYP2E1, GSTA4-4 expressions and lipid peroxidation, which could contribute to the nigrostriatal dopaminergic neurodegeneration.     

Altered expression of key dopaminergic regulatory proteins in the postnatal brain following perinatal n-3 fatty acid dietary deficiency

The consequences of maternal linolenic acid (LNA, 18:3n-3) dietary deficiency on key dopamine (DA)-associated regulatory proteins in mesolimbic and mesocortical structures of the postnatal rat brain have been investigated. A marked (4.5-fold) decrease of the DA-synthesizing enzyme tyrosine hydroxylase accompanied by a down-regulation (approx 7.5-fold) of the vesicular monoamine transporter (VMAT-2) and a depletion of VMAT-associated vesicles in the hippocampus were observed in deficient offspring compared with adequately fed controls. The DA transporter (DAT) was not affected by the LNA deficiency indicative of a DAT/VMAT-2 ratio increase that may enhance the risk of damage of the dopaminergic (DAergic) terminal. A robust increase in DA receptor (DAR1 and DAR2) levels was noticed in the cortex and striatum structures possibly to compensate for the low levels of DA in synaptic clefts. Microglia activation characterized by enhanced levels of ED1 antibody and nuclear internalization of p65 NFκB was noticed following LNA deficiency. Diminished levels of 22:6n-3 docosahexaenoic acid ( Schiefermeier and Yavin 2002 ), the most ubiquitous metabolite generated by LNA is proposed to reduce the anti-oxidant arsenal in the developing brain and cause microglia activation and enhanced oxidative stress to increase the risk of certain DA-associated neurological disorders.