Role of high-affinity dopamine uptake and impulse activity in the appearance of extracellular dopamine in striatum after administration of exogenous L-DOPA: studies in intact and 6-hydroxydopamine-treated rats

The differential behavioral and neurochemical effects of exogenous L-DOPA in animals with intact versus dopamine (DA)-denervated striata raise questions regarding the role of DA terminals in the regulation of dopaminergic neurotransmission after administration of exogenous L-DOPA. In vivo microdialysis was used to monitor the effect of exogenous L-DOPA on extracellular DA in intact and DA-denervated striata of awake rats. In intact striatum, a small increase in extracellular DA was observed after administration of L-DOPA (50 mg/kg i.p.) but in DA-denervated striatum a much larger increase in extracellular DA was elicited. Additional experiments assessed the role of high-affinity DA uptake and impulse-dependent neurotransmitter release in the effect of exogenous L-DOPA on extracellular DA in striatum. Pretreatment with GBR-12909 (20 mg/kg i.p.), a selective DA uptake inhibitor, enhanced the ability of L-DOPA to increase extracellular DA in intact striatum. However, in DA-denervated striatum, inhibition of DA uptake did not alter the extracellular DA response to L-DOPA. Impulse-dependent neurotransmitter release was blocked by the infusion of tetrodotoxin (TTX; 1 microM), an inhibitor of fast sodium channels, through the dialysis probe. Application of TTX significantly attenuated the L-DOPA-induced increase in extracellular DA observed in striatum of intact rats pretreated with GBR-12909. In a similar manner, TTX infusion significantly attenuated the increase in extracellular DA typically observed in striatum of 6-OHDA-lesioned rats after the administration of L-DOPA. The present results indicate that DA terminals, via high-affinity uptake, play a crucial role in the clearance of extracellular DA formed from exogenous L-DOPA in intact striatum. This regulatory mechanism is absent in the DA-denervated striatum. In addition, this study has shown that DA synthesized from exogenous L-DOPA primarily is released by an impulse-dependent mechanism in both intact and DA-denervated striatum. The latter result suggests an important role for a nondopaminergic neuronal element in striatum that serves as the primary source of extracellular DA formed from exogenous L-DOPA.     

Dopamine transporters are involved in the onset of hypoxia-induced dopamine efflux in striatum as revealed by in vivo microdialysis

Although many studies have revealed alterations in neurotransmission during ischaemia, few works have been devoted to the neurochemical effects of mild hypoxia, a situation encountered during life in altitude or in several pathologies. In that context, the present work was undertaken to determine the in vivo mechanisms underlying the striatal dopamine efflux induced by mild hypoxaemic hypoxia. For that purpose, the extracellular concentrations of dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid were simultaneously measured using brain microdialysis during acute hypoxic exposure (10% O₂, 1 h) in awake rats. Hypoxia induced a +80% increase in dopamine. Application of the dopamine transporters inhibitor, nomifensine (10 μM), just before the hypoxia prevented the rise in dopamine during the early part of hypoxia; in contrast the application of nomifensine after the beginning of hypoxia, failed to alter the increase in dopamine. Application of the voltage-dependent Na⁺ channel blocker tetrodotoxin abolished the increase in dopamine, whether administered just before or after the beginning of hypoxia. These data show that the neurochemical mechanisms of the dopamine efflux may change over the course of the hypoxic exposure, dopamine transporters being involved only at the beginning of hypoxia.     

Marked disparity between age-related changes in dopamine and other presynaptic dopaminergic markers in human striatum

Because age-related changes in brain dopaminergic innervation are assumed to influence human disorders involving dopamine (DA), we measured the levels of several presynpatic DAergic markers [DA, homovanillic acid, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT)] in post-mortem human striatum (caudate and putamen) from 56 neurologically normal subjects aged 1 day to 103 years. Striatal DA levels exhibited pronounced (2- to 3-fold) post-natal increases through adolescence and then decreases during aging. Similarly, TH and AADC increased almost 100% during the first 2 post-natal years; however, the levels of TH and, to a lesser extent, AADC then declined to adult levels by approximately 30 years of age. Although VMAT2 and DAT levels closely paralleled those of TH, resulting in relatively constant TH to transporter ratios during development and aging, a modest but significant decline (13%) in DAT levels was observed in only caudate during aging. This biphasic post-natal pattern of the presynaptic markers suggests that striatal DAergic innervation/neuropil appears to continue to develop well past birth but appears to become overelaborated and undergo regressive remodeling during adolescence. However, during adulthood, a striking discrepancy was observed between the loss of DA and the relative preservation of proteins involved in its biosynthesis and compartmentation. This suggests that declines in DA-related function during adulthood and senescence may be explained by losses in DA per se as opposed to DAergic neuropil.     

Cooperative interaction between ascorbate and glutathione during mitochondrial impairment in mesencephalic cultures

A decrease in total glutathione, and aberrant mitochondrial bioenergetics have been implicated in the pathogenesis of Parkinson's disease. Our previous work exemplified the importance of glutathione (GSH) in the protection of mesencephalic neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. Additionally, reactive oxygen species (ROS) generation was an early, contributing event in malonate toxicity. Protection by ascorbate was found to correlate with a stimulated increase in protein-glutathione mixed disulfide (Pr-SSG) levels. The present study further examined ascorbate-glutathione interactions during mitochondrial impairment. Depletion of GSH in mesencephalic cells with buthionine sulfoximine potentiated both the malonate-induced toxicity and generation of ROS as monitored by dichlorofluorescein diacetate (DCF) fluorescence. Ascorbate completely ameliorated the increase in DCF fluorescence and toxicity in normal and GSH-depleted cultures, suggesting that protection by ascorbate was due in part to upstream removal of free radicals. Ascorbate stimulated Pr-SSG formation during mitochondrial impairment in normal and GSH-depleted cultures to a similar extent when expressed as a proportion of total GSH incorporated into mixed disulfides. Malonate increased the efflux of GSH and GSSG over time in cultures treated for 4, 6 or 8 h. The addition of ascorbate to malonate-treated cells prevented the efflux of GSH, attenuated the efflux of GSSG and regulated the intracellular GSSG/GSH ratio. Maintenance of GSSG/GSH with ascorbate plus malonate was accompanied by a stimulation of Pr-SSG formation. These findings indicate that ascorbate contributes to the maintenance of GSSG/GSH status during oxidative stress through scavenging of radical species, attenuation of GSH efflux and redistribution of GSSG to the formation of mixed disulfides. It is speculated that these events are linked by glutaredoxin, an enzyme shown to contain both dehydroascorbate reductase as well as glutathione thioltransferase activities.     

Role of striatal serotonin2A and serotonin2C receptor subtypes in the control of in vivo dopamine outflow in the rat striatum

This study investigated, using in vivo microdialysis in the striatum of freely moving rats, the role of striatal serotonin2A (5-HT2A) and 5-HT2C receptor subtypes in the modulation of dopamine (DA) and 3, 4-dihydroxyphenylacetic acid (DOPAC) outflow, both in basal conditions and under activation induced by subcutaneous administration of 0.01 mg/kg haloperidol. The different 5-HT2 agents used were applied intrastriatally at a 1 microM concentration through the microdialysis probe. Basal DA efflux was enhanced (27%) by the 5-HT2A/2B/2C agonist 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI) and reduced (-30%) by the 5-HT2B/2C antagonist SB 206553. It was unaffected by infusion of the 5-HT2A antagonist SR 46349B. The effect of DOI was abolished by SB 206553 but not modified by SR 46349B. Haloperidol-stimulated DA efflux (65-70%) was reduced by both SR 46349B (-32%) and the 5-HT2A/2B/2C antagonist ritanserin (-30%) but not affected by SB 206553. Conversely, the effect of haloperidol was potentiated (22%) when DOI was coperfused with SB 206553. Also, haloperidol-stimulated DOPAC outflow (40-45%) was reduced (-20%) by SR 46349B and potentiated (25%) by the combination of SB 206553 with DOI. These results indicate that striatal 5-HT2A receptors, probably through activation of DA synthesis, positively modulate DA outflow only under activated conditions. In contrast, striatal 5-HT2C receptors exert a facilitatory control on basal DA efflux, which appears to be both tonic and phasic.     

Immobilization stress causes increases in tetrahydrobiopterin, dopamine, and neuromelanin and oxidative damage in the nigrostriatal system

Oxidative stress is believed to contribute to the pathophysiology of Parkinson's disease, in which nigrostriatal dopaminergic (DA) neurons undergo degeneration. Identification of endogenous molecules that contribute to generation of oxidative stress and vulnerability of these cells is critical in understanding the etiology of this disease. Exposure to tetrahydrobiopterin (BH4), the obligatory cofactor for DA synthesis, was observed previously to cause oxidative damage in DA cells. To demonstrate the physiological relevance of this observation, we investigated whether an overproduction of BH4 and DA might actually occur in vivo, and, if it did, whether this might lead to oxidative damage to the nigrostriatal system. Immobilization stress (IMO) elevated BH4 and DA and their synthesizing enzymes, tyrosine hydroxylase and GTP cyclohydrolase I. This was accompanied by elevation of lipid peroxidation and protein-bound quinone, and activities of antioxidant enzymes. These increases in the indices of oxidative stress appeared to be due to increased BH4 synthesis because they were abolished following administration of the BH4 synthesis inhibitor, 2,4-diamino-6-hydroxy-pyrimidine. IMO also caused accumulation of neuromelanin and degeneration of the nigrostriatal system. These results demonstrate that a severe stress can increase BH4 and DA and cause oxidative damages to the DA neurons in vivo, suggesting relevance to Parkinson's disease.     

Role of extracellular signal-regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinson's disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal-enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose- and time-dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal-regulated kinases (ERK-1/2), 4 and 6 h after treatment. MEK-1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK-1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo-oxygenase (COX), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) does not have protective effects. Co-localization studies show that p-ERK-1/2 expression after BSO treatment increased in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glial cells with fluoroacetate decreased ERK activation. However, blockade of microglial activation with minocycline did not. Our results indicate that neuronal death induced by GSH depletion is due to ROS-dependent activation of the ERK-1/2 signalling pathway in glial cells. These data may be of relevance in Parkinson's disease, where GSH depletion and glial dysfunction have been documented.     

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.     

Interactions of glutamate and dopamine in a computational model of the striatum

A network model of simplified striatal principal neurons with mutual inhibition was used to investigate possible interactions between cortical glutamatergic and nigral dopaminergic afferents in the neostriatum. Glutamatergic and dopaminergic inputs were represented by an excitatory synaptic conductance and a slow membrane potassium conductance, respectively. Neuronal activity in the model was characterized by episodes of increased action potential firing rates of variable duration and frequency. Autocorrelation histograms constructed from the action potential activity of striatal model neurons showed that reducing peak excitatory conductance had the effect of increasing interspike intervals. On the other hand, the maximum value of the dopamine-sensitive potassium conductance was inversely related to the duration of firing episodes and the maximal firing rates. A smaller potassium conductance restored normal firing rates in the most active neurons at the expense of a larger proportion of neurons showing reduced activity. Thus, a homogeneous network with mutual inhibition can produce equally complex dynamics as have been proposed to occur in a striatal network with two neuron populations that are oppositely regulated by dopamine. Even without mutual inhibition it appears that increased dopamine concentrations could partially compensate for the effects of reduced glutamatergic input in individual neurons.     

L-DOPA does not enhance hydroxyl radical formation in the nigrostriatal dopamine system of rats with a unilateral 6-hydroxydopamine lesion

The debate about the toxicity of L-DOPA to dopaminergic neurons has not been resolved. Even though enzymatic and nonenzymatic metabolism of L-DOPA can produce hydrogen peroxide and oxygen free radicals, there has been controversy as to whether L-DOPA generates an oxidant stress in vivo. This study determined whether acute or repeated administration of L-DOPA caused in vivo production of hydroxyl radicals in striatum and other brain regions in rats with a unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal projections. Salicylate trapping combined with in vivo microdialysis provided measurements of extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA) in striatum following L-DOPA administration systemically (100 mg/kg, i.p.) or by intrastriatal perfusion (1 mM, via the microdialysis probe). Tissue concentrations of 2,3-DHBA and salicylate were also measured in striatum, ventral midbrain, and cerebellum following repeated administration of L-DOPA (50 mg/kg, i.p., once daily for 16 days). In each instance, treatment with L-DOPA did not increase 2,3-DHBA concentrations, regardless of the nigrostriatal dopamine system's integrity. When added to the microdialysis perfusion medium, L-DOPA resulted in a significant decrease in the striatal extracellular concentration of 2,3-DHBA. These results suggest that administration of L-DOPA, even at high doses, does not induce hydroxyl radical formation in vivo and under some conditions may actually diminish hydroxyl radical activity. Furthermore, prior damage to the nigrostriatal dopamine system does not appear to predispose surviving dopaminergic neurons to increased hydroxyl radical formation following L-DOPA administration. Unlike L-DOPA, systemic administration of methamphetamine (10 mg/kg, s.c.) produced a significant increase in the concentration of 2,3-DHBA in striatal dialysate, suggesting that increased formation of hydroxyl radicals may contribute to methamphetamine neurotoxicity.     

The role of the plasmalemmal dopamine and vesicular monoamine transporters in methamphetamine-induced dopaminergic deficits

Amphetamine (AMPH) and methamphetamine (METH) are members of a collection of phenethylamine psychostimulants that are commonly referred to collectively as "amphetamines." Amphetamines exert their effects, in part, by affecting neuronal dopamine transport. This review thus focuses on the effects of AMPH and METH on the plasmalemmal dopamine transporter and the vesicular monoamine transporter-2 in animal models with a particular emphasis on how these effects, which may vary for the different stereoisomers, contribute to persistent dopaminergic deficits.     

Energy Stress-Induced Dopamine Loss in Glutathione Peroxidase-Overexpressing Transgenic Mice and in Glutathione-Depleted Mesencephalic Cultures

Abstract: The role of the glutathione system in protecting dopamine neurons from a mild impairment of energy metabolism imposed by the competitive succinate dehydrogenase inhibitor, malonate, was investigated in vitro and in vivo. Treatment of mesencephalic cultures with 10 µ M buthionine sulfoxamine for 24 h reduced total glutathione levels in the cultures by 68%. Reduction of cellular glutathione per se was not toxic to the dopamine population, but potentiated toxicity when the cultures were exposed to malonate. In contrast, transgenic mice overexpressing glutathione peroxidase (hGPE) that received an intrastriatal infusion of malonate (3 µmol) into the left side had significantly less loss of striatal dopamine than their hGPE-negative littermates when assayed 1 week following infusion. These studies demonstrate that manipulation of the glutathione system influences susceptibility of dopamine neurons to damage due to energy impairment. The findings may provide insight into the loss of dopamine neurons in Parkinson's disease in which defects in both energy metabolism and the glutathione system have been identified.     

Brief,repeated exposure to substrates down-regulates dopamine transporter function in Xenopus oocytes in vitro and rat dorsal striatum in vivo

In heterologous expression systems, dopamine transporter (DAT) cell-surface localization is reduced after relatively prolonged exposure to d-amphetamine (AMPH) or dopamine (DA), suggesting a role for substrate-mediated regulation of transporter function. Here, we investigated whether brief, repeated periods of substrate exposure modulated transporter function, first, in an in vitro model system and, second, in intact rat brain. In human DAT-expressing Xenopus laevis oocytes, repeated exposure to low micromolar concentrations of DA, AMPH or tyramine markedly reduced transport-mediated currents. This functional down-regulation was attenuated by inclusion of a protein kinase C (PKC) inhibitor and probably reflects DAT redistribution, as cell-surface [3H]WIN 35 428 binding was significantly lower following DA exposure. High-speed chronoamperometry was used to measure clearance of exogenously applied DA in dorsal striatum (STR) and nucleus accumbens (NAc) of anesthetized rats. In STR, frequent (every 2 min) applications of DA altered DA clearance parameters in a manner consistent with profound down-regulation of DAT function. Similar changes were not observed in NAc or after repeated vehicle (ascorbic acid) application. Together, our results suggest that brief, repeated periods of substrate exposure lead to rapid down-regulation of DAT activity and that this type of regulation can occur in vivo in STR, but not NAc.     

Modulation of dopaminergic neurotransmission in rat striatum upon in vitro and in vivo diclofenac treatment

Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory drug, which also act as a mitochondrial toxin. As it is known that selective mitochondrial complex I inhibition combined with mild oxidative stress causes striatal dopaminergic dysfunction, we tested whether DCF also compromise dopaminergic function in the striatum. [3H]Dopamine ([3H]DA) release was measured from rat striatal slices after in vitro (2 h, 10-25 micromol/L) or in vivo (3 mg/kg i.v. for 28 days) DCF treatment. In vitro treatment significantly decreased [3H]DA uptake and dopamine (DA) content of the slices. H2O2 (0.1 mmol/L)-evoked DA release was enhanced. Intracellular reactive oxygen species production was not significantly changed in the presence of DCF. After in vivo DCF treatment no apparent decrease in striatal DA content was observed and the uptake of [3H]DA into slices was increased. The intensity of tyrosine hydroxylase immunoreactivity in the striatum was highly variable, and both decrease and increase were observed in individual rats. The H2O2-evoked [3H]DA release was significantly decreased and the effluent contained a significant amount of [3H]octopamine, [3H]tyramine, and [3H]beta-phenylethylamine. The ATP content and adenylate energy charge were decreased. In conclusion, whereas in vitro DCF pre-treatment resembles the effect of the mitochondrial toxin rotenone, in vivo it rather counteracts than aggravates dopaminergic dysfunction.     

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.     

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases. In vivo selective brain monoamine oxidase inhibition and prevention of MPTP-induced striatal dopamine depletion

Several multifunctional iron chelators have been synthesized from hydroxyquinoline pharmacophore of the iron chelator, VK-28, possessing the monoamine oxidase (MAO) and neuroprotective N-propargylamine moiety. They have iron chelating potency similar to desferal. M30 is a potent irreversible rat brain mitochondrial MAO-A and -B inhibitor in vitro (IC50, MAO-A, 0.037 +/- 0.02; MAO-B, 0.057 +/- 0.01). Acute (1-5 mg/kg) and chronic [5-10 mg/kg intraperitoneally (i.p.) or orally (p.o.) once daily for 14 days]in vivo studies have shown M30 to be a potent brain selective (striatum, hippocampus and cerebellum) MAO-A and -B inhibitor. It has little effects on the enzyme activities of the liver and small intestine. Its N-desmethylated derivative, M30A is significantly less active. Acute and chronic treatment with M30 results in increased levels of dopamine (DA), serotonin(5-HT), noradrenaline (NA) and decreases in DOPAC (dihydroxyphenylacetic acid), HVA (homovanillic acid) and 5-HIAA (5-hydroxyindole acetic acid) as determined in striatum and hypothalamus. In the mouse MPTP (N-methy-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease (PD) it attenuates the DA depleting action of the neurotoxin and increases striatal levels of DA, 5-HT and NA, while decreasing their metabolites. As DA is equally well metabolized by MAO-A and -B, it is expected that M30 would have a greater DA neurotransmission potentiation in PD than selective MAO-B inhibitors, for which it is being developed, as MAO-B inhibitors do not alter brain dopamine.     

Identification and kainic acid-induced up-regulation of low-affinity p75 neurotrophin receptor (p75NTR) in the nigral dopamine neurons of adult rats

Parkinson's disease is a common and severe debilitating neurological disease that results from massive and progressive degenerative death of dopamine neurons in the substantia nigra, but the mechanisms of neuronal degeneration and disease progression remains largely obscure. We are interested in possible implications of low-affinity p75 neurotrophin receptor (p75NTR), which may mediate neuronal apoptosis in the central nervous system, in triggering cell death of the nigral dopamine neurons. The RT-PCR and immunohistochemistry were carried out to detect if p75NTR is expressed in these nigral neurons and up-regulated by kainic acid (KA) insult in adult rats. It revealed p75NTR-positive immunoreactivity in the substantia nigra, and co-localization of p75NTR and tyrosine hydroxylase (TH) was found in a large number of substantia nigra neurons beside confirmation of p75NTR in the choline acetyltransferase (ChAT)-positive forebrain neurons. Cell count data further indicated that about 47-100% of TH-positive nigral neurons and 98-100% of ChAT-positive forebrain neurons express p75NTR. More interestingly, significant increasing in both p75NTR mRNA and p75NTR-positive neurons occurred rapidly following KA insult in the substantia nigra of animal model. The present study has provided first evidence on p75NTR expression and KA-inducing p75NTR up-regulation in substantia nigra neurons in rodent animals. Taken together with previous data on p75NTR functions in neuronal apoptosis, this study also suggests that p75NTR may play important roles in neuronal cell survival or excitotoxic degeneration of dopamine neurons in the substantia nigra in pathogenesis of Parkinson's disease in human beings.     

New dual electrochemical detector for microbore liquid chromatography determination of dopamine and serotonin in rat striatum dialysates

A new type of liquid chromatographic (LC) dual thin-layer amperometric detector for the simultaneous measurement of trace levels of dopamine and serotonin in microdialysates is described. The concentrations of these analytes in rat dialysates are usually in the sub-nanomolar concentration range (typically, 0.10–5.00 pg in 5-μl dialysates). With this dual electrode, a glass-lined microbore column provides excellent sensitivity, selectivity, and separation. In addition, a three- to five-fold improvement in anodic current or cathodic responses over conventional dual electrodes in microbore LC can be achieved. Due to the irreversible electrochemical properties of some interference peaks, this dual electrode provides reliable measurement of dopamine based on the cathodic signal. The detection limit (signal-to-noise RATIO=3) of this assay is 0.02 pg per injection for dopamine or serotonin. This new dual electrode allows the simultaneous measurements of basal dopamine and serotonin in rat striatum dialysates without the use of re-uptake inhibitors in perfusion medium.     

Simultaneous determination of cholecystokinin,dopamine, glutamate and aspartate in cortex and striatum of the rat using in vivo microdialysis

Summary Extracellular levels of cholecystokinin (CCK), dopamine (DA), glutamate (Glu) and aspartate (Asp) were simultaneously monitored in the frontoparietal cortex and the striatum of halothane-anaesthetized rats using in vivo microdialysis. Under basal conditions, cortical and striatal CCK levels were 3.11 ± 0.39 pM and 2.76 ± 0.15 pM, respectively. Local KCl (10^–1 M) and bicuculline (10^–4 M) co-application in cortex or striatum increased the CCK levels 18-fold and 26-fold, respectively. The DA level in striatum was 3.78 ± 0.28 nM and the local perfusion with KCl + bicuculline led to a 45-fold increase. The cortical and striatal outputs of Glu were of the order of 2 · 10^–6 M and Asp levels were around 6 · 10^–7 M. Local stimulation with KCl (10^–1 M) and bicuculline (10^–4 M) caused a small increase (2 fold) in cortical and striatal levels of Glu and Asp. The addition of KCl (10^–1 M) and bicuculline (10^–4 M) to the cortical perfusion medium did not modify CCK, DA or Glu concentrations in striatum. These results demonstrate that CCK, DA, Glu and Asp may be simultaneously monitored in vivo and support the idea that their extracellular levels recovered in the microdialysis perfusates could be derived from neuronal pools.