5-Iodo-A-85380 binds to alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors (nAChRs) as well as alpha4beta2* subtypes

Recent work suggests that 5-iodo-A-85380, a radioiodinated analog of the 3-pyridyl ether A-85380, represents a promising imaging agent for non-invasive, in vivo studies of alphaAbeta2* nicotinic acetylcholine receptors (nAChRs; *denotes receptors containing the indicated subunits), because of its low non-specific binding, low in vivo toxicity and high selectivity for alpha4beta2* nAChRs. As an approach to elucidate nAChR subtypes expressed in striatum, we carried out competitive autoradiography in monkey and rat brain using 5-[125I]iodo-A-85380 ([125I]A-85380) and [125I]alpha-conotoxin MII, a ligand that binds with high affinity to alpha6* and alpha3* nAChRs, but not to alpha4beta2* nAChRs. Although A-85380 is reported to be selective for alpha4beta2* nAChRs, we observed that A-85380 completely inhibited [125I]alpha-conotoxin MII binding in rat striatum and that A-85380 blocked >90% of [125I] alpha-conotoxin MII sites in monkey caudate and putamen. These results suggest that A-85380 binds to non-alpha4beta2* nAChRs, including putative alpha6* nAChRs. Experiments to determine the percentage of [125I]A-85380 sites that contain alpha-conotoxin MII-sensitive (alpha6beta2*) nAChRs indicate that they represent about 10% of [125I]A-85380 sites in rodent striatum and about 30% of sites in monkey caudate and putamen. These data are important for identifying alterations in nicotinic receptor subtypes in Parkinson's disease and other basal ganglia disorders both in in vitro and in in vivo imaging studies.     

Compensation in pre-synaptic dopaminergic function following nigrostriatal damage in primates

Clinical symptoms of Parkinson's disease only become evident after 70-80% reductions in striatal dopamine. To investigate the importance of pre-synaptic dopaminergic mechanisms in this compensation, we determined the effect of nigrostriatal damage on dopaminergic markers and function in primates. MPTP treatment resulted in a graded dopamine loss with moderate to severe declines in ventromedial striatum (approximately 60-95%) and the greatest reductions (approximately 95-99%) in dorsolateral striatum. A somewhat less severe pattern of loss was observed for striatal nicotinic receptor, tyrosine hydroxylase and vesicular monoamine transporter expression. Declines in striatal dopamine uptake and transporter sites were also less severe than the reduction in dopamine levels, with enhanced dopamine turnover in the dorsolateral striatum after lesioning. The greatest degree of adaptation occurred for nicotine-evoked [(3)H]dopamine release from striatal synaptosomes, which was relatively intact in ventromedial striatum after lesioning, despite > 50% declines in dopamine. This maintenance of evoked release was not due to compensatory alterations in nicotinic receptor characteristics. Rather, there appeared to be a generalized preservation of release processes in ventromedial striatum, with K(+)-evoked release also near control levels after lesioning. These combined compensatory mechanisms help explain the finding that Parkinson's disease symptomatology develops only with major losses of striatal dopamine.     

[3H]WIN 35,065–2: A Ligand for Cocaine Receptors in Striatum

[3H]WIN 35,065-2 binding to striatal membranes was characterized, primarily by centrifugation assay. Like [3H]cocaine, [3H]WIN 35,065-2 binds to both high- and low-affinity sites. [3H]WIN 35,065-2, however, exhibits consistently higher affinities than [3H]cocaine. Saturation experiments indicate a low-affinity binding site with an apparent KD of approximately 160 nM and a Bmax of 135 fmol/mg of tissue. A high-affinity site has also been identified with an apparent KD of 5.6 nM and a Bmax of 5.2 fmol/mg of tissue. The specific-to-nonspecific binding ratios with [3H]WIN 35,065-2 were higher than with [3H]cocaine in both centrifugation and filtration assays. Pharmacological characterization suggests that [3H]WIN 35,065-2 binds to the dopamine transporter. Mazindol, GBR 12909, nomifensine, and (-)-cocaine are potent inhibitors of [3H]WIN 35,065-2 binding. In contrast, the norepinephrine transporter ligand desipramine is a weak inhibitor, and the serotonin transporter ligand citalopram does not inhibit binding. The effect of sodium on binding was examined under conditions in which (a) the low-affinity site was primarily (87%) occupied and (b) approximately 50% of both sites were occupied. The results indicate that both sites are sodium dependent. Injection of 6-hydroxydopamine into the striatum results in a significant loss of both high- and low-affinity sites, a finding suggesting that both sites are on dopaminergic nerve terminals. Taken together, these data are consistent with the presence of multiple cocaine binding sites associated with the dopamine transporter.     

Nicotine partially protects against paraquat-induced nigrostriatal damage in mice; link to alpha6beta2* nAChRs

Epidemiological studies indicate that smoking is a negative, and exposure to pesticides, a positive risk factor for Parkinson's disease (PD). The purpose of this study was to assess the interplay between these two factors in a rodent model of nigrostriatal damage. To approach this, mice were administered nicotine, the agent in smoke implicated in neuroprotection. They were then treated for 3 weeks with the pesticide, paraquat, while nicotine was continued. Paraquat treatment decreased (25%) nigral dopaminergic neurons, consistent with previous results. Chronic nicotine administration significantly protected against nigral cell damage, with only a 16% decline in mice treated with both nicotine and paraquat. Paraquat treatment also decreased (14%) the striatal dopamine transporter, an effect that was partially prevented by nicotine. These changes in the striatal dopamine transporter paralleled those in a select striatal alpha6beta2* nicotinic receptor (nAChR) subtype. In contrast, striatal alpha4beta2* nAChRs were not decreased with paraquat treatment, suggesting they are on a differential subset of dopaminergic terminals. The results show that nicotine treatment partially protects against paraquat-induced declines in nigrostriatal dopaminergic neurons to which a select population of alpha6beta2* nAChRs are localized. Moreover, these data support epidemiological findings that environmental influences can elicit opposing effects on nigrostriatal dopaminergic integrity.     

Decline in Response to Nicotine in Aged Rat Striatum: Correlation with a Decrease in a Subpopulation of Nicotinic Receptors

Specific and reproducible changes involving the cholinergic and dopaminergic systems have been described in both the aging rodent and the human nervous system. Nevertheless, relatively little information is available on changes in nicotinic cholinergic receptors occurring in normal aging, and there have been few attempts to correlate alterations in receptor densities with changes in nicotinic actions. We have utilized the nicotine-mediated stimulation of endogenous dopamine efflux in a striatal slice preparation as a functional index of responsiveness to nicotine in aging. Following incubation with nicotine, this efflux was significantly lower in 25-month-old (aged) as opposed to 4-month-old (young) rats. In contrast, the release of striatal dopamine following a high-potassium stimulus was similar at both ages. Binding studies in young and aged animals did not reveal any significant change with age in the total number of striatal nicotinic receptors recognized by either [³H]nicotine or the neuronal nicotinic antagonist ¹²⁵l-neuronal bungarotoxin. However, there was a nearly 80% decline in the subpopulation of striatal nicotinic receptors jointly recognized by both nicotine and neuronal bungarotoxin, but not by α-bungarotoxin. Quantitative autoradiography demonstrated declines with age in this receptor subtype in several brain regions examined. Decrements in this specific subpopulation of nicotinic receptors or in the nerve cells expressing these receptors may contribute to the functional declines that take place in the aging motor and visual systems.     

Increased levels of ubiquitin in the 6-OHDA-lesioned striatum of rats

Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar Parkinson's disease. We therefore examined the possible role of 6-hydroxydopamine, a dopaminergic neurotoxin used in Parkinson's disease experimental models, in causing protein degradation and its association with the ubiquitin proteasome system. Using unilaterally 6-hydroxydopamine-denervated rats and mass spectrometry profiling directly on brain tissue sections, we here report for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted hemisphere. No similar changes were found in the intact hemisphere or in the ventral striatum of the dopamine depleted hemisphere. The lesioning of the dopamine innervation to the striatum was confirmed by a strongly reduced dopamine transporter binding in the striatum, indicating an abundant loss of dopamine neurons. These results suggest that denervation of dopamine neurons per se is implicated in the regulation of ubiquitin pathways, at least in a classical animal model of Parkinson's disease. This study adds additional information regarding the involvement of the ubiquitin-proteasome system in Parkinson's disease.     

The Dopamine Transporter Is Absent in Parkinsonian Putamen and Reduced in the Caudate Nucleus

The neuronal dopamine transporter/uptake site can be covalently labeled with the photoaffinity probe 1-(2-[bis-(4-fluorophenyl) methoxy]ethyl)-4-[2-(4-azido-3-[125I]iodophenyl)ethyl]piperazine [( 125I]FAPP) and visualized following sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Upon photolysis, [125I]FAPP specifically incorporated into a polypeptide of apparent Mr = 62,000 in membranes from both the putamen and the caudate nucleus of control, Alzheimer's, schizophrenia, and Huntington's diseased brain, and following complete deglycosylation, migrated as an Mr approximately 48,000 polypeptide. In parkinsonian postmortem putamen, however, there was no detectable photoincorporation of [125I]FAPP into the ligand binding subunit of the dopamine transporter. [125I]FAPP did specifically label the Mr 62,000 polypeptide of parkinsonian caudate, although with efficiencies of 20-50% of control. The asymmetrical loss of the dopamine transporter in Parkinson's diseased striatum was confirmed in reversible receptor binding experiments using [3H]GBR-12935 (3H-labeled 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenylpropyl)piperazine). In parkinsonian putamen, mazindol competitively inhibited the binding of [3H]GBR-12935 with an estimated affinity (Ki approximately 2,000 nM) 10 times lower than in controls (Ki approximately 30 nM), while the affinity of maxindol for [3H]GBR-12935 binding in the caudate was equal to that seen with controls (Ki approximately 50 nM). The proportion of [3H]GBR-12935 binding sites recognized by mazindol with high affinity in Parkinson's diseased caudate was, however, reduced by 50-80%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

Passive and active stabilization of dopamine in the striatum

Parkinson's disease is a neurodegenerative disorder associated with cell loss from the substantia nigra pars compacta (SNc). The dopaminergic cells of the SNc project to the striatum where the loss of dopaminergic tone is thought to be the main cause of Parkinsonism symptoms. Animal models have shown that striatal tissue content of dopamine declines proportionally to cell death in the SNc but the extracellular concentration of dopamine (EDA) in the striatum remains near normal until more than 85% of SNc neurons have died. We investigate various explanations for the remarkable homeostasis of EDA with a mathematical model that has recently been constructed for dopamine synthesis, release, and reuptake, which includes the effects of the autoreceptors. We provide evidence and explanations for the passive stabilization hypothesis and show that the autoreceptors enhance stabilization of EDA only when fewer than 25% of the SNc cells remain.     

Complete Conversion of Brain D2 Dopamine Receptors from the High- to the Low-Affinity State for Dopamine Agonists, Using Sodium Ions and Guanine Nucleotide

Since previous work had shown that brain D2 3,4-dihydroxyphenylethylamine (dopamine) receptors were only partly converted from their high-affinity state to their low-affinity state, we here tested whether it was possible to obtain a complete 100% conversion of these receptors into their low-affinity state. It was first essential to resolve the components of [3H]spiperone binding to dopaminergic sites and nondopaminergic sites in rat striatal homogenates. In the presence of 50 microM S-sulpiride (to occlude the dopaminergic sites), therefore, we first determined that the residual binding of [3H]spiperone (approximately 20%) was inhibited by serotonergic agonists much more effectively than dopamine or noradrenaline, thus identifying the serotonergic component of [3H]spiperone binding. Thus, dopamine (or ADTN) inhibited the binding of [3H]spiperone at a high-affinity site (with dissociation constant of 10 nM dopamine), at a low-affinity site (with dissociation constant of 2,000 nM dopamine), and at the serotonergic site (with dissociation constant of 50,000 nM dopamine). In the absence of sodium ions, the high-affinity site was about 50% occupied by [3H]spiperone, and guanine nucleotide had no effect on this proportion. In the presence of 120 mM NaCl, however, the high-affinity site was reduced to 15% and guanine nucleotide completely eliminated this high-affinity site, 100% of the sites having been completely converted to their low-affinity state. Using [3H]N-propyl-norapomorphine to label the high-affinity state of the dopamine receptor, 50% conversion into the low-affinity state occurred at 45 mM LiCl, 69 mM NaCl, and 202 mM KCl. We conclude that it is possible to convert brain D2 dopamine receptors completely into their low-affinity state, in the presence of NaCl and a guanine nucleotide, providing that appropriate allowance is made for the serotonergic component of [3H]spiperone binding.     

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5-[125I]iodo-3-(2-azetidinylmethoxy)pyridine ([125I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 receptors. Although the predominant subtype in rat brain was alpha4beta2, non-alpha4beta2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha3beta4-like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha3beta4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha3beta2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.     

P25α / TPPP expression increases plasma membrane presentation of the dopamine transporter and enhances cellular sensitivity to dopamine toxicity

Parkinson's disease is characterized by preferential degeneration of the dopamine-producing neurons of the brain stem substantia nigra. Imbalances between mechanisms governing dopamine transport across the plasma membrane and cellular storage vesicles increase the level of toxic pro-oxidative cytosolic dopamine. The microtubule-stabilizing protein p25α accumulates in dopaminergic neurons in Parkinson's disease. We hypothesized that p25α modulates the subcellular localization of the dopamine transporter via effects on sorting vesicles, and thereby indirectly affects its cellular activity. Here we show that co-expression of the dopamine transporter with p25α in HEK-293-MSR cells increases dopamine uptake via increased plasma membrane presentation of the transporter. No direct interaction between p25α and the dopamine transporter was demonstrated, but they co-fractionated during subcellular fractionation of brain tissue from striatum, and direct binding of p25α peptides to brain vesicles was demonstrated. Truncations of the p25α peptide revealed that the requirement for stimulating dopamine uptake is located in the central core and were similar to those required for vesicle binding. Co-expression of p25α and the dopamine transporter in HEK-293-MSR cells sensitized them to the toxicity of extracellular dopamine. Neuronal expression of p25α thus holds the potential to sensitize the cells toward dopamine and toxins carried by the dopamine transporter.     

Chronic oral nicotine treatment protects against striatal degeneration in MPTP-treated primates

The present studies were done to investigate the effect of long-term nicotine treatment against nigrostriatal damage in non-human primates. Monkeys were administered nicotine in drinking water for 6 months to provide chronic but intermittent delivery as with smoking. Plasma nicotine levels ranged from 10 to 15 ng/mL, which were within the range in cigarette smokers. Animals were then lesioned with low doses of the dopaminergic neurotoxin MPTP for several months while nicotine was continued. The results showed that levels of striatal tyrosine hydroxylase, dopamine transporter, vesicular monoamine transporter, dopamine and nicotinic receptors were greater in nicotine-treated MPTP-lesioned primates than in lesioned animals not receiving nicotine. Nicotine had no effect in unlesioned animals. Monoamine oxidase activity was similar in unlesioned and lesioned animals treated with or without nicotine, suggesting that nicotine did not exert its effects through changes in MPTP or dopamine metabolism. MPTP-induced cell loss in the substantia nigra was unaffected by nicotine treatment, indicating that nicotine acts at the striatal level to restore/maintain dopaminergic function. These data further support the possibility that nicotine contributes to the lower incidence of Parkinson's disease in smokers.     

Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse

BackgroundThe acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels.HypothesisThe hypothesis was tested that the dopaminergic neuronal system had also adapted.MethodsRadioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity.ResultsDopamine receptor levels were decreased 28-fold for the D₁-like, and more than 37-fold for the D₂-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity.ConclusionSurvival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.     

Presynaptic Nicotinic Modulation of Dopamine Release in the Three Ascending Pathways Studied by In Vivo Microdialysis: Comparison of Naive and Chronic Nicotine-Treated Rats

Abstract: The modulation of dopamine release by presynaptic nicotinic receptors in vitro is well established, but the significance of this effect in vivo is unclear. We have characterised the effect of nicotine, locally applied via a microdialysis probe, on dopamine release from the terminal regions of three ascending dopaminergic pathways in conscious, freely moving rats. Nicotine caused a dose-dependent increase in dopamine release in the striatum, the nucleus accumbens, and, to a lesser extent, the frontal cortex. Metabolite levels were unaltered by any concentration of nicotine. Prior administration of mecamylamine via the probe abolished the nicotine-evoked increase in dopamine release, confirming the mediation of nicotinic receptors. The dose dependence of mecamylamine-sensitive, nicotine-evoked dopamine release was similar in all three brain regions. However, 10⁻⁵ M tetrodotoxin totally blocked nicotine-stimulated dopamine release in the striatum and the accumbens but not the cortex. Daily subcutaneous injections of nicotine (0.4 mg kg⁻¹ for 7 days) increased the response to a subsequent local application of nicotine in the striatum, and a similar trend was found in the other brain areas. The same daily dose of nicotine given as a continuous infusion had no effect, whereas infusion of 4 mg kg⁻¹ day⁻¹ increased the response to a subsequent nicotine challenge. The localisation and regulation of nicotinic receptors in the terminal fields of dopaminergic pathways are discussed.     

Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease

Fourteen years after transplantation into the striatum of an individual with Parkinson's disease, grafted nigral neurons were found to have Lewy body-like inclusions that stained positively for alpha-synuclein and ubiquitin and to have reduced immunostaining for dopamine transporter. These pathological changes suggest that Parkinson's disease is an ongoing process that can affect grafted cells in the striatum in a manner similar to host dopamine neurons in the substantia nigra. These findings have implications for cell-based therapies and for understanding the cause of Parkinson's disease.     

Rapid alterations in corticostriatal ensemble coordination during acute dopamine-dependent motor dysfunction

Dopaminergic dysregulation can cause motor dysfunction, but the mechanisms underlying dopamine-related motor disorders remain under debate. We used an inducible and reversible pharmacogenetic approach in dopamine transporter knockout mice to investigate the simultaneous activity of neuronal ensembles in the dorsolateral striatum and primary motor cortex during hyperdopaminergia ( approximately 500% of controls) with hyperkinesia, and after rapid and profound dopamine depletion (<0.2%) with akinesia in the same animal. Surprisingly, although most cortical and striatal neurons ( approximately 70%) changed firing rate during the transition between dopamine-related hyperkinesia and akinesia, the overall cortical firing rate remained unchanged. Conversely, neuronal oscillations and ensemble activity coordination within and between cortex and striatum did change rapidly between these periods. During hyperkinesia, corticostriatal activity became largely asynchronous, while during dopamine-depletion the synchronicity increased. Thus, dopamine-related disorders like Parkinson's disease may not stem from changes in the overall levels of cortical activity, but from dysfunctional activity coordination in corticostriatal circuits.     

High-and Low-Affinity States of Striatal D2 Receptors Are Not Affected by 6-Hydroxydopamine or Chronic Haloperidol Treatment

Specific D2 binding in rat striatum was characterized and then the effects of chronic disruption of dopaminergic activity on antagonist and agonist binding to these sites were studied. D2 receptors were defined as those sites capable of binding [3H]spiperone in the presence of cinanserin, a 5-HT2 antagonist, but not in the presence of (+)-butaclamol, a D2 and 5-HT2 blocker. Saturation, competition, and kinetic analyses suggested that D2 receptors are a homogeneous population exhibiting more complex interactions with agonists than antagonists. Antagonist binding was monophasic and guanine nucleotide-insensitive whereas agonist binding was biphasic and guanine nucleotide-sensitive. D2 receptor density was elevated by more than 40% following dopamine depletion by 6-hydroxydopamine or chronic receptor blockade by haloperidol. However neither treatment altered the affinities or magnitudes of the high- and low-affinity components associated with agonist binding to the D2 receptor.     

Partial recovery of dopaminergic pathway after graft of adult mesenchymal stem cells in a rat model of Parkinson's disease

Cellular therapy with adult stem cells appears as an opportunity for treatment of Parkinson's disease. To validate this approach, we studied the effects of transplantation of rat adult bone-marrow mesenchymal stem cells in a rat model of Parkinson's disease. Animals were unilaterally lesioned in the striatum with 6-hydroxydopamine. Two weeks later, group I did not undergo grafting, group II underwent sham grafting, group III was intra-striatal grafted with cells cultured in an enriched medium and group IV was intra-striatal grafted with cells cultured in a standard medium. Rotational amphetamine-induced behavior was measured weekly until animals were killed 6 weeks later. One week after graft, the number of rotations/min was stably decreased by 50% in groups III and IV as compared with groups I and II. At 8 weeks post-lesion, the density of dopaminergic markers in the nerve terminals and cell bodies, i.e. immunoreactive tyrosine hydroxylase, membrane dopamine transporter and vesicular monoamine transporter-2 was significantly higher in group III as compared with group I. Moreover, using microdialysis studies, we observed that while the rate of pharmacologically induced release of dopamine was significantly reduced in lesioned versus intact striatum in no grafted rats, it was similar in both sides in animals transplanted with mesemchymal stem cells. These data demonstrate that graft of adult mesemchymal stem cells reduces behavioral effects induced by 6-hydroxydopamine lesion and partially restores the dopaminergic markers and vesicular striatal pool of dopamine. This cellular approach might be a restorative therapy in Parkinson's disease.     

Neuroadaptations to hyperdopaminergia in dopamine D3 receptor-deficient mice

The dopamine D3 receptor (D3R) has been implicated in schizophrenia, drug addiction, depression and Parkinson's disease. The D3R is localized post-synaptically on nucleus accumbens neurons, but is also an autoreceptor on dopaminergic neurons in the mesencephalon. Its functional role as autoreceptor is highly debated, but supported by the elevated basal extracellular dopamine levels found in D3R-deficient mice. To investigate the functional role of the D3R in vivo, we used mice with a targeted disruption of the D3R gene. We found a higher basal level of grooming in D3R-deficient mice, compared to their wild-type littermates. This behavior, which is under the control of D1R stimulation, may be related to an increased dopaminergic tone, since no changes in the gene expression of dopamine D1 and D2 receptors were noticed in the striatum of these mice. D3R-deficient mice displayed other neuroadaptive changes, including decreased tyrosine hydroxylase, increased dopamine transporter mRNAs and increased dopamine reuptake in striatum. The level of tyrosine hydroxylase protein was unchanged in the striatum, as preprodynorphin and preproenkephalin gene expressions. All the changes identified in D3R-deficient mice cannot explain hyperdopaminergia, but, on the contrary, tend to attenuate this phenotype. These results support a distinct role for D2R and D3R as autoreceptors: the D2R is the release-regulating and firing rate-regulating autoreceptor, whereas the D3R may control basal dopamine levels in the striatum, by an unknown mechanism, which does not involve regulation of dopamine transporters or tyrosine hydroxylase. This hyperdopaminergia phenotype of D3R-deficient mice may explain their hyperactivity to drug-paired environmental cues.