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In JoVE (1)
Other Publications (15)
- Neurobiology of Aging
- Journal of Neurochemistry
- Brain Research. Molecular Brain Research
- Experimental Neurology
- The Journal of Neuropsychiatry and Clinical Neurosciences
- Neurobiology of Aging
- Neurobiology of Aging
- The European Journal of Neuroscience
- Experimental Neurology
- PloS One
- Journal of Neurochemistry
- Psychopharmacology
- Neurobiology of Disease
- PloS One
- Experimental Neurology
Articles by Michael F. Salvatore in JoVE
JoVE Neuroscience
Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area
Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.
Other articles by Michael F. Salvatore on PubMed
Decreased Plasma Membrane Expression of Striatal Dopamine Transporter in Aging
Aging in rodents, monkeys, and man is correlated with a reduction in dopamine transporter (DAT) ligand binding and DAT function. Using Western blot techniques, we investigated whether the source of these age-related changes in DAT was correlated with decreases in DAT protein levels in the striatum, substantia nigra (SN), nucleus accumbens (NAc), and ventral tegmental area (VTA) of 6, 18, and 24-month-old male Fischer 344 rats. The relative levels of tyrosine hydroxylase (TH) were also determined in each region. In the striatum, we also assessed [3H]-DA uptake and DAT plasma membrane expression using a membrane-impermeant biotin analog in crude synaptosomes prepared from these age groups. There was no significant age-related difference in DAT immunoreactivity per total protein or per total TH in striatum, NAc, SN, or VTA. Significant age-related changes in TH were only seen in the VTA of the 24-month-old rats (approximately 60% decrease). However, [3H]-DA uptake and DAT protein recovered in the biotinylated fraction in 24-month-old rats were significantly decreased (approximately 30%) compared to 6-month-old animals in the striatal synaptosomes. These data suggest that age-related decreases in striatal DAT function and ligand binding are related to a decrease in plasma membrane expression of DAT and not a decrease in the steady-state levels of DAT protein or loss of dopaminergic neuropil.
Striatal GDNF Administration Increases Tyrosine Hydroxylase Phosphorylation in the Rat Striatum and Substantia Nigra
Glial cell line-derived neurotrophic factor (GDNF) improves motor dysfunction associated with aging in rats and non-human primates, in animal models of Parkinson's disease, and may improve motoric function in patients with advanced Parkinson's disease. These improvements are associated with increased dopamine function in the nigrostriatal system, but the molecular events associated with this increase are unknown. In these studies, 100 micro g of GDNF was injected into the striatum of normal aged (24-month-old) male Fischer 344 rats. The protein levels and phosphorylation of TH, ERK1/2, and related proteins were determined by blot-immunolabeling of striatum and substantia nigra harvested 30 days after injection. In GDNF-treated rats, TH phosphorylation at Ser31 increased approximately 40% in striatum and approximately 250% in the substantia nigra. In the substantia nigra, there was a significant increase in ERK1 phosphorylation. In striatum, there was a significant increase in ERK2 phosphorylation. Microdialysis studies in striatum showed that both amphetamine- and potassium-evoked dopamine release in GDNF recipients were significantly increased. These data show that GDNF-induced increases in dopamine function are associated with a sustained increase in TH phosphorylation at Ser31, which is greatest in the substantia nigra and maintained for at least one month following a single striatal administration of GDNF. These findings, taken from the nigrostriatal system of normal aged rats, may help explain the long lasting effects of GDNF on dopamine function and prior studies supporting that a major effect of GDNF involves its effects on dopamine storage and somatodendritic release of dopamine in the substantia nigra.
Neurochemical Investigations of Dopamine Neuronal Systems in Iron-regulatory Protein 2 (IRP-2) Knockout Mice
Abnormal iron accumulations are frequently observed in the brains of patients with Parkinson's disease and in normal aging. Iron metabolism is regulated in the CNS by iron regulatory proteins (IRP-1 and IRP-2). Mice engineered to lack IRP-2 develop abnormal motoric behaviors including tremors at rest, abnormal gait, and bradykinesia at middle to late age (18 to 24 months). To further characterize the dopamine (DA) systems of IRP-2 -/- mice, we harvested CNS tissue from age-matched wild type and IRP-2 -/- (16-19 months) and analyzed the protein levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter (VMAT2), and DA levels in dorsal striatum, ventral striatum (including the core and shell of nucleus accumbens), and midbrain. We further analyzed the phosphorylation of TH in striatum at serine 40, serine 31, and serine 19. In both dorsal and ventral striatum of IRP-2 knockout mice, there was a 20-25% loss of TH protein and accompanied by a approximately 50% increase in serine 40 phosphorylation above wild-type levels. No change in serine 31 phosphorylation was observed. In the ventral striatum, there was also a significant loss (approximately 40%) of DAT and VMAT2. Levels of DA were decreased (approximately 20%) in dorsal striatum, but turnover of DA was also elevated ( approximately 30%) in dorsal striatum of IRP-2 -/- mice. We conclude that iron misregulation associated with the loss of IRP-2 protein affects DA regulation in the striatum. However, the modest loss of DA and DA-regulating proteins does not reflect the pathology of PD or animal models of PD. Instead, these observations support that the IRP-2 -/- genotype may enable neurobiological events associated with aging.
Point Source Concentration of GDNF May Explain Failure of Phase II Clinical Trial
Significant differences have been reported in results from three clinical trials evaluating intraputamenal infusion of glial cell line-derived neurotrophic factor (GDNF) for the treatment of Parkinson's disease. To determine if problems in drug bioavailability could have contributed to the discrepancies between studies, we have analyzed the distribution of intraputamenally infused GDNF in the rhesus monkey brain using the delivery system and infusion protocol followed in a phase 2 clinical trial that failed to achieve its primary endpoint. I125-GDNF was unilaterally infused into the putamen of three adult rhesus monkeys for 7 days. Three age- and sex-matched animals received vehicle infusions following identical procedures. GDNF levels in the brain, peripheral organs, blood and CSF were quantified and mapped by GDNF immunocytochemistry, GDNF ELISAs and I125 measurements. Infused GDNF was found to be unevenly concentrated around the catheter, with tissue levels dropping exponentially with increasing distance from the point source of the single opening in the catheter tip. The volume of distribution of GDNF around the catheter, as determined by immunocytochemistry, varied over four-fold between animals ranging from 87 to 369 mm3. The concentration of GDNF around the catheter tip and limited diffusion into surrounding brain parenchyma support the hypothesis that drug bioavailability was limited to a small portion (2-9%) of the human putamen in the clinical trial using this catheter and infusion protocol.
Low Dose Alpha-methyl-para-tyrosine (AMPT) in the Treatment of Dystonia and Dyskinesia
AMPT (alpha-methyl-para-tyrosine) is an inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis. In clinical settings, AMPT is approved to treat pheochromocytoma. Dystonias and dyskinesias seem to have their origin in inconsistent regulation of dopamine function in dopamine pathways. This paper presents case histories of the clinical efficacy of AMPT for treating certain individuals with neuroleptic-induced dystonia or dyskinesia. The authors propose that a special utility of AMPT in tardive disorders may be related to a downregulation of tyrosine hydroxylase activity that may be increased by neuroleptic-induced effects on tyrosine hydroxylase phosphorylation.
Bilateral Effects of Unilateral Intrastriatal GDNF on Locomotor-excited and Nonlocomotor-related Striatal Neurons in Aged F344 Rats
In order to determine its effects on locomotor-related striatal electrophysiology in aged rats, glial cell line-derived neurotrophic factor (GDNF) was infused (vehicle or 30mug) into the right striatum of 24-25-month-old Fischer 344 (F344) rats. Multi-wire electrode arrays were then chronically implanted in striatum bilaterally. Thirty days later, striatal electrophysiological activity was recorded during freely moving conditions. Individual neurons were classified as locomotor-excited if they exhibited significant increases in firing rates during locomotor bouts versus periods of nonmovement. GDNF produced a significant increase in overall firing rates in locomotor-excited striatal neurons. This effect was observed in both the infused and the contralateral striatum. GDNF also attenuated the bursting activity of nonlocomotor-related striatal neurons, an effect that was also present bilaterally. These results suggest that GDNF's antiparkinsonism effects are associated with increased excitability of motor-related striatal neurons and diminished activity of neurons that do not exhibit explicit motor-related changes in activity. Such studies may aid in understanding the mechanism of potential therapies for movement disorders seen in aging and Parkinson's disease.
Reduced Plasma Membrane Surface Expression of GLAST Mediates Decreased Glutamate Regulation in the Aged Striatum
Extracellular L-glutamate poses a severe excitotoxic threat to neurons and glia when unregulated, therefore low synaptic levels of this neurotransmitter must be maintained via a rapid and robust transport system. A recent study from our laboratory showed a reduced glutamate uptake rate in the striatum of the aged Fischer 344 (F344) rat, yet the mechanism underlying this phenomenon is unknown. The current study utilized in vivo electrochemical recordings, immunoblotting and biotinylation in young (6 months), late-middle aged (18 months) and aged (24 months) F344 rats to elucidate the potential role that glutamate transporters (GLT-1, GLAST, and EAAC1) may play in this mechanism. Here we show that the time necessary to clear glutamate from the late-middle aged and aged striatum is significantly prolonged in comparison to the young striatum. In addition, an analysis of various sub-regions of the striatum revealed a marked dorsoventral gradient in terms of glutamate clearance times in the aged striatum, a phenomenon which was not present in the striatum of the animals of the remaining age groups. We also found that the decreased glutamate clearance time observed in the late-middle aged and aged rats is not due to a decrease in the production of total transporter protein among these three transporters. Rather, a significant reduction in the amount of GLAST expressed on the plasma membrane surface in the aged animals (approximately 55% when compared to young rats) may contribute to this phenomenon. These age-related alterations in extracellular l-glutamate regulation may be key contributors to the increased susceptibility of the aged brain to excitotoxic insults such as stroke and hypoxia.
Tau Expression Levels from Various Adeno-associated Virus Vector Serotypes Produce Graded Neurodegenerative Disease States
Neurodegenerative diseases involving neurofibrillary tangle pathology are pernicious. By expressing the microtubule-associated protein tau, a major component of tangles, with a viral vector, we induce neuropathological sequelae in rats that are similar to those seen in human tauopathies. We tested several variants of the adeno-associated virus (AAV) vector for tau expression in the nigrostriatal system in order to develop models with graded onset and completeness. Whereas previous studies with AAV2 tau vectors produced partial lesions of the nigrostriatal system, AAV9 or AAV10 tau vectors were more robust. These vectors had formidable efficacy relative to 6-hydroxydopamine for dopamine loss in the striatum. Time-courses for tau transgene expression, dopamine loss and rotational behavior tracked the disease progression with the AAV9 tau vector. There was a nearly complete lesion over a delayed time-course relative to 6-hydroxydopamine, with a sequence of tau expression by 1 week, dopamine loss by 2 weeks and then behavior effect by 3-4 weeks. Relative to AAV2 or AAV8, tau expression from AAV9 or AAV10 peaked earlier and caused more dopamine loss. Varying vector efficiencies produced graded states of disease up to nearly complete. The disease models stemming from the AAV variants AAV9 or AAV10 may be useful for rapid drug screening, particularly for tau diseases that affect the nigrostriatal system, such as progressive supranuclear palsy.
Bilateral Effects of Unilateral GDNF Administration on Dopamine- and GABA-regulating Proteins in the Rat Nigrostriatal System
Dopamine (DA) affects GABA neuronal function in the striatum and together these neurotransmitters play a large role in locomotor function. We recently reported that unilateral striatal administration of GDNF, a growth factor that has neurotrophic effects on DA neurons and enhances DA release, bilaterally increased striatal neuron activity related to locomotion in aged rats. We hypothesized that the GDNF enhancement of DA function and resulting bilateral enhancement of striatal neuronal activity was due to prolonged bilateral changes in DA- and GABA-regulating proteins. Therefore in these studies we assessed dopamine- and GABA-regulating proteins in the striatum and substantia nigra (SN) of 24 month old Fischer 344 rats, 30 days after a single unilateral striatal delivery of GDNF. The nigrostriatal proteins investigated were the DA transporter (DAT), tyrosine hydroxylase (TH), and TH phosphorylation and were examined by blot-immunolabeling. The striatal GABA neuron-related proteins were examined by assay of the DA D1 receptor, DARPP-32, DARPP-32 Thr34 phosphorylation, and glutamic acid decarboxylase (GAD). Bilateral effects of GDNF on TH and DAT occurred only in the SN, as 30 microg GDNF increased ser19 phosphorylation, and 100 microg GDNF decreased DAT and TH protein levels. GDNF also produced bilateral changes in GAD protein in the striatum. A decrease in DARPP-32 occurred in the ipsilateral striatum, while increased D1 receptor and DARPP-32 phosphorylation occurred in the contralateral striatum. The 30 microg GDNF infusion into the lateral striatum was confined to the ipsilateral striatum and substantia nigra. Thus, long-lasting bilateral effects of GDNF on proteins regulating DA and GABA neuronal function likely alter physiological properties in neurons, some with bilateral projections, associated with locomotion. Enhanced nigrostriatal excitability and DA release by GDNF may trigger these bilateral effects.
Aging Reveals a Role for Nigral Tyrosine Hydroxylase Ser31 Phosphorylation in Locomotor Activity Generation
Tyrosine hydroxylase (TH) regulates dopamine (DA) bioavailability. Its product, L-DOPA, is an established treatment for Parkinson's disease (PD), suggesting that TH regulation influences locomotion. Site-specific phosphorylation of TH at ser31 and ser40 regulates activity. No direct evidence shows that ser40 phosphorylation is the dominating mechanism of regulating TH activity in vivo, and physiologically-relevant stimuli increase L-DOPA biosynthesis independent of ser40 phosphorylation. Significant loss of locomotor activity occurs in aging as in PD, despite less loss of striatal DA or TH in aging compared to the loss associated with symptomatic PD. However, in the substantia nigra (SN), there is equivalent loss of DA or TH in aging and at the onset of PD symptoms. Growth factors increase locomotor activity in both PD and aging models and increase DA bioavailability and ser31 TH phosphorylation in SN, suggesting that ser31 TH phosphorylation status in the SN, not striatum, regulates DA bioavailability necessary for locomotor activity.
GFR α-1 Receptor Expression in the Aging Nigrostriatal and Mesoaccumbens Pathways
We recently reported that age-related bradykinesia was associated with reduced dopamine (DA) tissue content, ser31 tyrosine hydroxylase (TH) phosphorylation, and total TH levels in substantia nigra (SN) only. In this study, we propose that these decreases result from reduced glial cell line-derived neurotrophic factor family receptor α-1 (GFR α-1) levels in the aged (30-month-old) cohort of rats. Analysis of GFR α-1 receptor protein in SN, striatum, ventral tegmental area, and nucleus accumbens from 12- and 30-month-old Brown-Norway/Fischer 344 F(1) hybrid rats revealed immunoreactivity at ∼48 and 52 kDa, bands previously characterized to correspond to soluble and glycosyl-phosphatidylinositol-linked forms of GFR α-1, respectively. The nigrostriatal pathway had significantly greater levels of the soluble GFR α-1 than the mesoaccumbens pathway. Aging significantly reduced soluble and total GFR α-1 in the SN. The levels of GFR α-1 significantly correlated with TH protein in SN, striatum, and nucleus accumbens, but only in the SN did GFR α-1 significantly correlate with DA levels. Based on these observations and findings from the literature, we speculate that (i) GFR α-1 receptor expression may regulate nigral DA bioavailability in vivo, (ii) age-related decreases in soluble GFR α-1 in SN may contribute to bradykinesia in aging, and (iii) differences in expression of the GFR α-1 forms between the nigrostriatal and mesoaccumbens pathways and allied tissue may indicate that glial cell line-derived neurotrophic factor-signaling differs between these DA pathways.
Biphasic Dopamine Regulation in Mesoaccumbens Pathway in Response to Non-contingent Binge and Escalating Methamphetamine Regimens in the Wistar Rat
Methamphetamine (MA) increases extracellular dopamine (DA) and at chronic high doses induces toxicity as indicated by decreased expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT). Notably, rats will self-administer MA in escalating quantities producing such toxicity. However, the impact of MA at sub-toxic doses on DA regulation is not well established.
Social Enrichment Attenuates Nigrostriatal Lesioning and Reverses Motor Impairment in a Progressive 1-methyl-2-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Mouse Model of Parkinson's Disease
Environmental enrichment has been shown to be both neuroprotective and neurorestorative in 1-methyl-2-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models of Parkinson's disease (PD). However, whether social interaction or novel physical stimulation is responsible for this recovery is controversial. In the current study, we have investigated the effects of only social enrichment (SocE) in progressively MPTP-lesioned mice. After mice were lesioned using a progressively increased dose (4 mg/kg, 8 mg/kg, 16 mg/kg and 32 mg/kg; each dose daily for 5 days), the MPTP-induced behavioral deficits, after the 32 mg/kg dose, were reversed with acute L-DOPA. This acute behavioral recovery suggests that this progressive MPTP-induced neurodegeneration is an appropriate murine model of PD. Mice were housed four per cage for the first 2 weeks of progressive lesioning or vehicle treatment. After the 8 mg/kg MPTP dose (prior to SocE intervention) mice showed a significant decrease in rearing and foot fault behaviors (FF/BB) compared to the vehicle group. Additionally, there was a 38% decrease in mean number of tyrosine hydroxylase immunoreactive (TH-ir) substantia nigra pars compacta (SNpc) neurons/section, and a 50% decrease in the optical density of TH-ir dorsolateral caudate putamen (CPu) terminals compared to the vehicle group. Mice were then housed either two (socially limited environment; SLE) or twelve (SocE) mice per cage during continued MPTP lesioning for the next 2 weeks at 16 mg/kg and 32 mg/kg MPTP. MPTP treatment was then discontinued, while mice remained in the SLE or SocE cages for an additional week. Rearing behavior was further impaired in SLE-MPTP mice following progressive MPTP, accompanied by additional decreases in the mean number of TH-ir SNpc neurons/section and CPu TH-ir terminals. CPu TH and dopamine transporter (DAT) protein expression, as well as dopamine tissue and TH protein levels was significantly decreased compared to either vehicle group. However, the deficit in rearing behavior in SLE-MPTP mice was reversed with acute L-DOPA following the intervention period. SocE-MPTP mice showed rearing and FF/BB behaviors similar to vehicle levels, although FF/BB was not significantly different from pre-intervention levels. The reversal from pre-intervention rearing deficits was correlated with an attenuated decrease in the mean number of SNpc TH-ir neurons/section and CPu TH and DAT protein, and with a blocked decrease in CPu TH-ir terminals compared to pre-intervention levels. Our findings show that SocE mice not only resist further nigrostriatal lesioning and FF/BB deficit, but rearing behavior is recovered to the level of the vehicle group despite continued MPTP treatment. In contrast, SLE mice showed continued loss of nigrostriatal TH-ir and decline of motor behaviors with progressive MPTP. The data suggest that non-pharmacological intervention that started at an early stage of dopamine loss is effective at slowing or blocking further nigrostriatal degeneration.
Dichotomy of Tyrosine Hydroxylase and Dopamine Regulation Between Somatodendritic and Terminal Field Areas of Nigrostriatal and Mesoaccumbens Pathways
Measures of dopamine-regulating proteins in somatodendritic regions are often used only as static indicators of neuron viability, overlooking the possible impact of somatodendritic dopamine (DA) signaling on behavior and the potential autonomy of DA regulation between somatodendritic and terminal field compartments. DA reuptake capacity is less in somatodendritic regions, possibly placing a greater burden on de novo DA biosynthesis within this compartment to maintain DA signaling. Therefore, regulation of tyrosine hydroxylase (TH) activity may be particularly critical for somatodendritic DA signaling. Phosphorylation of TH at ser31 or ser40 can increase activity, but their impact on L-DOPA biosynthesis in vivo is unknown. Thus, determining their relationship with L-DOPA tissue content could reveal a mechanism by which DA signaling is normally maintained. In Brown-Norway Fischer 344 F₁ hybrid rats, we quantified TH phosphorylation versus L-DOPA accumulation. After inhibition of aromatic acid decarboxylase, L-DOPA tissue content per recovered TH protein was greatest in NAc, matched by differences in ser31, but not ser40, phosphorylation. The L-DOPA per catecholamine and DA turnover ratios were significantly greater in SN and VTA, suggesting greater reliance on de novo DA biosynthesis therein. These compartmental differences reflected an overall autonomy of DA regulation, as seen by decreased DA content in SN and VTA, but not in striatum or NAc, following short-term DA biosynthesis inhibition from local infusion of the TH inhibitor α-methyl-p-tyrosine, as well as in the long-term process of aging. Such data suggest ser31 phosphorylation plays a significant role in regulating TH activity in vivo, particularly in somatodendritic regions, which may have a greater reliance on de novo DA biosynthesis. Thus, to the extent that somatodendritic DA release affects behavior, TH regulation in the midbrain may be critical for DA bioavailability to influence behavior.
Transient Striatal GLT-1 Blockade Increases EAAC1 Expression, Glutamate Reuptake, and Decreases Tyrosine Hydroxylase Phosphorylation at Ser(19)
Three glutamate transporters, GLT-1, GLAST, and EAAC1, are expressed in striatum. GLT-1 and, to a lesser extent, GLAST are thought to play a primary role in glutamate reuptake and mitigate excitoxicity. Progressive tyrosine hydroxylase (TH) loss seen in Parkinson's disease (PD) is associated with increased extracellular glutamate. Glutamate receptor antagonists reduce nigrostriatal loss in PD models. These observations suggest that excess synaptic glutamate contributes to nigrostriatal neuron loss seen in PD. Decreased GLT-1 expression occurs in neurodegenerative disease and PD models, suggesting decreased GLT-1-mediated glutamate reuptake contributes to excitotoxicity. To determine how transient GLT-1 blockade affects glutamate reuptake dynamics and a Ca(2+)-dependent process in nigrostriatal terminals, ser(19) phosphorylation of TH, the GLT-1 inhibitor dihydrokainic acid (DHK) was delivered unilaterally to striatum in vivo and glutamate reuptake was quantified ex vivo in crude synaptosomes 3h later. Ca(2+)-influx is associated with excitotoxic conditions. The phosphorylation of TH at ser(19) is Ca(2+)-dependent, and a change resulting from GLT-1 blockade may signify the potential for excitotoxicity to nigrostriatal neurons. Synaptosomes from DHK infused striatum had a 43% increase in glutamate reuptake in conjunction with decreased ser(19) TH phosphorylation. Using a novel GLAST inhibitor and DHK, we determined that the GLAST-mediated component of increased glutamate reuptake increased 3-fold with no change in GLAST or GLT-1 protein expression. However, GLT-1 blockade increased EAAC1 protein expression ~20%. Taken together, these results suggest that GLT-1 blockade produces a transient increase in GLAST-mediated reuptake and EAAC1 expression coupled with reduced ser(19) TH phosphorylation. These responses could represent an endogenous defense against excitotoxicity to the nigrostriatal pathway.
