Effects of Manganese on Extracellular Levels of Dopamine in Rat Striatum: An Analysis <Emphasis Type="Italic">In vivo</Emphasis> by Brain Microdialysis

The aim of this study is to determine the effects of intrastriatal administration of MnCl_2, on the extracellular levels of dopamine (DA) and metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in basal conditions and stimulated by depolarization with KCl and pargyline administration. Also, we studied the effect of MnCl₂ on extracellular levels of l-Dopa in the presence of aromatic amino acid decarboxylase (AADC) inhibitor 3-hydroxybencilhydracine-HCl (NSD 1015). This study concluded that MnCl₂, reduced the basal and K⁺-stimulated DA-release in striatum, without notably affecting the DOPAC and HVA levels. Intraperitoneal injection of pargyline increased striatal DA levels, decreasing DOPAC and HVA levels. The infusion of MnCl₂ removed the increase in DA levels, without affecting DOPAC and HVA levels. Perfusion of NSD 1015 increased the extracellular levels of l-DOPA in striatum, and MnCl₂ increased the effect of NSD1015 on l-Dopa.     

Semichronic Inhibition of Glutathione Reductase Promotes Oxidative Damage to Proteins and Induces both Transcription and Translation of Tyrosine Hydroxylase in the Nigrostriatal System

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

Is Dopamine-Induced Inhibition of Adenylate Cyclase Involved in the Autoreceptor-Mediated Negative Control of Tyrosine Hydroxylase in Striatal Dopaminergic Terminals?

Abstract The mechanism of the negative control of tyrosine hydroxylase (TH) activity induced by the stimulation of presynaptic 3,4-dihydroxyphenylethylamine (dopamine, DA) autoreceptors was investigated using rat striatal slices and synaptosomes incubated under control ([K⁺] = 4.8 mM) or depolarizing ([K⁺] = 60 mM) conditions. The stimulation of DA autoreceptors by 7-hydroxy-2-(di-n-propylamino) tetralin (1 μM 7-OH-DPAT) produced a significant decrease in TH activity extracted from striatal slices maintained under control conditions. This effect was associated with the complete conversion of TH into an enzyme form with a low affinity for its pterin cofactor (K_m～0.80 mM). Furthermore, compared to TH extracted from control tissues, that from 7-OH-DPAT-exposed striatal slices was more sensitive to the stimulatdry effects of exogenous heparin and cyclic AMP-dependent phosphorylation. Such changes were opposite to those induced by incubating striatal slices with the adenylate cyclase activator forskolin. Indeed, forskolin treatment completely converted TH into an enzyme form with a high affinity for its pterin cofactor (K_m～0.16 mM). Such conversion was associated with a shift in the optimal pH for TH activity from 5.8 (control) to 7.2 (forskolin). Under depolarizing conditions, the blockade by (—)-sulpiride of the stimulation of DA autoreceptors by endogenous DA was associated with a marked activation of TH. Modifications of enzymatic characteristics triggered by (—)-sulpiride were then similar to those induced by forskolin treatment. These data suggest that presynaptic DA autoreceptors modulate the activity of TH by controlling the degree of cyclic AMP-dependent phosphorylation of the enzyme. The blockade by Pertussis toxin of the 7-OH-DPAT-induced inhibition of TH activity is coherent with a possible negative coupling of presynaptic DA autoreceptors (closely related to the D₂ type) with adenylate cyclase. Such negative coupling would account for the reduction of TH activity when presynaptic DA autoreceptors are stimulated.     

Effects of intrastriatal kainic acid injection on [3H]dopamine metabolism in rat striatal slices: evidence for postsynaptic glial cell metabolism by both the type A and B forms of monoamine oxidase

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ-aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8-day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [³H]DA (10^?7 M) was examined in 8-day KA-lesioned rat striatal slices, an increase in [³H]dihydroxyphenylacetic acid (DOPAC) and [³H]homovanillic acid (HVA) formation was observed. The KA-induced elevation of [³H]DOPAC formation (but not [³H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [³H]DA (10^?5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA-lesioned striata. These results suggest that KA-sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.     

Neurotensin interacts with dopaminergic neurons in rat brain

Neurotensin (NT) injected intracerebroventricularly in rat increases dopamine (DA) turnover in the corpus striatum and nucleus accumbens. Significant increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels occurred within 15 minutes after injection with peak levels at 60 minutes. The effect on NT on DOPAC and homovanillic acid (HVA) accumulation was dose-dependent at 3–100 μg. NT, like haloperidol, stimulated 3,4-dihydroxyphenylalanine (DOPA) accumulation in striatal neurons, in the presence of DOPA decarboxylase inhibitor, after injection of gamma-butyrolactone (GBL). NT had a similar stimulatory effect on DOPA levels in the accumbens while haloperidol (0.25 mg·kg^?1) had no significant effect in this brain region. NT did not block the inhibitory effect of apomorphine on DOPA accumulation in both the striatum and accumbens, while haloperidol inhibited apomorphine effect in both regions. NT also failed to displace ³H-spiperone from DA receptors and the presence of NT in the binding assay did not alter the ability of DA to displace ³H-spiperone in either brain region. These experiments demonstrate that NT increases DA turnover in both the nigrostriatal and mesolimbic pathways.     

YM-09151-2 but Not l-Sulpiride Induces Transient Dopamine Release in Rat Striatum via a Tetrodotoxin-Insensitive Mechanism

Abstract: The effects of the selective dopamine D₂ receptor antagonists YM-09151-2 and l -sulpiride on the in vivo release of dopamine (DA), l -3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat striatum were investigated. The drugs were injected into the striatum through a microinjection needle attached to a dialysis probe. YM-09151-2 (0.1 or 1.0 μg/0.5 μl) injected into the striatum produced a dramatic rapid-onset transient increase in striatal DA release in a dose-dependent manner. However, the DA increase induced by l -sulpiride (15 or 75 ng/0.5 μl) was small and of slower onset. An increase of DOPAC levels by YM-09151-2 was biphasic: The first peak occurred at 40 min, followed by a delayed-onset gradual increase. Slower-onset gradual increases were also found in DOPAC levels after l -sulpiride injection and in HVA levels after injections of both YM-09151-2 and l -sulpiride. The infusion of tetrodotoxin (TTX; 2 μM) revealed two different types of DA release mechanisms: The rapid-onset transient DA release induced by YM-09151-2 was TTX insensitive, whereas the slower-onset DA release induced by l -sulpiride was TTX sensitive. Moreover, the rapid-onset transient DA release was Ca²⁺ independent and was not affected by pre-treatment with l -sulpiride or nomifensine. Therefore, it is concluded that YM-09151-2 injected into the striatum produced a transient striatal DA release that is independent of D₂ receptors and the action potential.     

Changes in Neuronal Dopamine Homeostasis following 1-Methyl-4-phenylpyridinium (MPP+) Exposure

1-Methyl-4-phenylpyridinium (MPP⁺), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP⁺ exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DA_cyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP⁺ exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP⁺ concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP⁺ depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DA_cyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP⁺-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DA_cyt levels in MPP⁺-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in ∼30% reduction of MPP⁺-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP⁺ on neuronal DA homeostasis and neurotoxicity.     

Efficacy of Brain-Derived Neurotrophic Factor and Neurotrophin-3 on Neurochemical and Behavioral Deficits Associated with Partial Nigrostriatal Dopamine Lesions

Abstract: Brain-derived neurotrophic factor (BDNF) promotes the survival of dopamine (DA) neurons, enhances expression of DA neuron characteristics, and protects these cells from 6-hydroxydopamine (6-OHDA) toxicity in vitro. We tested the ability of BDNF or neurotrophin-3 (NT-3) to exert similar protective effects in vivo during chronic delivery of 6-OHDA to the rat neostriatum. Chronic infusions of BDNF or NT-3 (12 µg/day) above the substantia nigra were started 6 days before and continued during an 8-day chronic intrastriatal infusion of 6-OHDA. In control and neurotrophin-treated animals, 6-OHDA treatment selectively depleted 50–60% of nigrostriatal DA nerve terminals but produced little if any loss of pars compacta DA cell bodies. This partial DA lesion resulted in three rotations per minute toward the lesioned hemisphere after treatment with the DA release-inducing drug d-amphetamine. Compared with supranigral infusions of vehicle, BDNF and NT-3 decreased the number of these ipsiversive rotations by 70 and 48% and increased by 20- and 10-fold, respectively, the number of contraversive rotations observed after amphetamine injection. When challenged with the DA receptor agonist apomorphine, BDNF- and NT-3-treated animals also exhibited a seven- and 3.5-fold increase in the number of contraversive rotations relative to the vehicle group, respectively. Compared with vehicle, BDNF increased striatal levels of homovanillic acid (HVA; 86%), 3,4-dihydroxyphenylacetic acid (DOPAC; 42%), and 5-hydroxyindoleacetic acid (5-HIAA; 32%) and the HVA/DA (43%) and 5-HIAA/serotonin (34%) ratios in the DA-denervated striatum. NT-3 augmented only striatal 5-HIAA levels (24%). Neither factor altered the 6-OHDA-induced decrease in striatal DA levels or high-affinity DA uptake and thus did not protect against the destruction of DA terminals and did not alter striatal D₁ or D₂ ligand binding. Choline, GABA, and glutamate uptake in the striatum were not altered by the lesion or neurotrophin treatment. Thus, BDNF and to a lesser extent NT-3 reverse rotational behavioral deficits and augment striatal DA and 5-HT metabolism in a partial DA lesion model.     

Effect of Long-Term Treatment with Selective Monoamine Oxidase A and B Inhibitors on Dopamine Release from Rat Striatum In Vivo

Abstract: Acute inhibition of monoamine oxidase B (MAO-B) in the rat does not affect striatal dopamine (DA) metabolism, but chronic MAO-B inhibition with deprenyl has been reported to increase the release of striatal DA, as shown using in vitro techniques. To see whether chronic MAO-B inhibition also causes an increase in DA release in vivo, rats were treated for 21 days with either deprenyl (0.25 mg/kg), TVP-1012 [R(+)-N-propargyl-1-aminoindan mesylate; 0.05 mg/kg), an irreversible inhibitor of MAO-B that is not metabolized to amphetamines, clorgyline (0.2 mg/kg), or saline (all doses once daily by subcutaneous injection). Concentric 4-mm-long microdialysis probes were implanted in the left striatum under pentobarbital/chloral hydrate anesthesia on day 21, and microdialysate DA, 3,4-dihydroxyacetic acid (DOPAC), and 4-hydroxy-3-methoxyphenyl acetic acid (HVA) were determined in the conscious animals on day 22. Baseline levels of DA were as follows: control, 0.34 ± 0.04 (n = 13); deprenyl, 0.88 ± 0.10 (n = 8, p < 0.01); TVP-1012, 0.94 ± 0.20 (n = 7, p < 0.01); clorgyline, 0.90 ± 0.12 (n = 7, p < 0.01) pmol/20 min. Levels of DOPAC and HVA were reduced only in the clorgyline-treated group. The incremental release of DA induced by depolarizing concentration of K⁺ (100 mM bolus of KCl in perfusate) was significantly greater in clorgyline- and deprenyl-treated rats and elevated (nonsignificantly) in TVP-1012-treated rats. Chronic treatment with the MAO-B inhibitors reduced striatal MAO-B activity by 90%, with 15% (TVP-1012) or 40% (deprenyl) inhibition of MAO-A. Clorgyline inhibited MAO-A by 95%, with 30% inhibition of MAO-B. A single dose of deprenyl (0.25 mg/kg, 24 h before microdialysis) had no significant effect on striatal efflux of DA. The results show that DA metabolism was reduced only by clorgyline, whereas neuronal release of DA was enhanced by both MAO-A and MAO-B inhibitors on chronic administration. The enhanced DA release by chronic MAO-B inhibition does not appear to be dependent on production of amphetamine-like metabolites of the inhibitor. Possible mechanisms for the release-enhancing effect of the MAO-B inhibitors include elevation in levels of endogenous β-phenylethylamine, or an inhibition of DA reuptake, which develops only on chronic administration, because both deprenyl and TVP-1012 have only very weak effects on amine uptake in acute experiments.     

Semichronic inhibition of glutathione reductase promotes oxidative damage to proteins and induces both transcription and translation of tyrosine hydroxylase in the nigrostriatal system

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

Amperozide, a Novel Antipsychotic Drug, Inhibits the Ability of d-Amphetamine to Increase Dopamine Release In Vivo in Rat Striatum and Nucleus Accumbens

The in vivo effects of amperozide, a novel atypical antipsychotic drug, on the release of dopamine (DA) and the output of its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were investigated in the striatum and the nucleus accumbens of awake, freely moving rats using microdialysis. Amperozide (2-10 mg/kg, s.c.) significantly increased extracellular levels of DA in both the striatum and nucleus accumbens in a dose-dependent manner. It had a similar but lesser effect on extracellular DOPAC levels in both regions. d-Amphetamine (2 mg/kg, s.c.) alone produced a very large (43-fold) increase in DA release, together with a 70% decrease in DOPAC levels in both the striatum and the nucleus accumbens. Amperozide (1-5 mg/kg, s.c.) 30 min before d-amphetamine (2 mg/kg) dose-dependently attenuated d-amphetamine-induced DA release but had no effect on the d-amphetamine-induced decrease in extracellular DOPAC levels in both regions. The effect of amperozide on d-amphetamine-induced DA release in the nucleus accumbens may explain the inhibitory effect of amperozide on amphetamine-induced locomotor activity. However, the failure of amperozide to block amphetamine-induced stereotypy, despite marked inhibition of striatal DA release, suggests the need to reexamine the importance of striatal DA for amphetamine-induced stereotypy.     

Relationships of Dopamine, Cortical Oxygen Pressure, and Hydroxyl Radicals in Brain of Newborn Piglets During Hypoxia and Posthypoxic Recovery

Abstract: The present study describes the relationships of extracellular striatal dopamine, cortical oxygen pressure, and striatal hydroxyl radicals in brain of newborn piglets during hypoxia and posthypoxic reoxygenation. Hypoxia was induced by reducing the fraction of inspired oxygen (F_iO₂) from 22% (control) to 7% for 1 h. The F_iO₂ was then returned to the control value and measurements were continued for 2 h. Cerebral oxygen pressure was measured by the oxygen dependent quenching of phosphorescence and extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and hydroxy radicals in the striatum were determined by in vivo microdialysis. Hypoxia decreased the cortical oxygen pressure from 47 ± 2 to 9 ± 1.3 torr (p < 0.001); the levels of extracellular dopamine in the striatum increased to 16,000 ± 3,270% of control (p < 0.01), whereas the levels of DOPAC and HVA decreased to 25.3 ± 6% (p < 0.001) and 36 ± 5% (p < 0.01) of control, respectively. Compared with control, the hydroxyl radical levels at each time point were not significantly increased during hypoxia, although the sum of the measured values was significantly increased (p < 0.05). During the first 5 min after F_iO₂ was returned to 22%, the cortical oxygen pressure increased to control values and stayed at this level for the remainder of the measurement period. The extracellular level of dopamine declined to values not statistically different from control during 40 min of reoxygenation. During the first 10 min of reoxygenation, DOPAC and HVA further decreased and then began to slowly increase. By 70 min of reoxygenation, the values were not significantly different from control. Hydroxyl radicals were above control during the entire period of reoxygenation, with maximal values observed after 100 min of reoxygenation. This increase was largely abolished by injecting the animals with α-methyl-p-tyrosine 5 h before hypoxia, a procedure that depleted the brain of dopamine. Our results suggest that oxidation of striatal dopamine during posthypoxic reoxygenation is at least partly responsible for the observed increase in striatal level of hydroxyl radicals that may exacerbate posthypoxic cerebral injury.     

Prolonged Alterations in Canine Striatal Dopamine Metabolism Following Subtoxic Doses of l-Methyl-4-Phenyl- 1,2,3,6-Tetrahydropyridine (MPTP) and 4''-Amino-MPTP Are Linked to the Persistence of Pyridinium Metabolites

Single toxic doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).HCl (2.5 mg/kg i.v.) and 4'-amino-MPTP.2HCl (22.5 mg/kg) induce loss of striatal dopamine (DA) and tyrosine hydroxylase (TH) activity and of nigral DA neurons in the dog. To examine the subacute neurochemical changes induced by low doses of MPTP and 4'-amino-MPTP, dose-response studies of these compounds were carried out in the dog, using 6- and 3-week survival times for these two compounds, respectively. Low single doses of MPTP (1.0, 0.5, and 0.1 mg/kg i.v.) and 4'-amino-MPTP (15, 7.5, and 3.75 mg/kg i.v.) did not cause depletion of canine striatal DA or TH or a loss of nigral neurons. However, levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were decreased in a dose-related fashion, with significant loss of DOPAC being evident 6 weeks after the lowest administered dose of MPTP and 3 weeks after 4'-amino-MPTP. This selective loss of DA metabolites following nontoxic doses of MPTP and 4'-amino-MPTP led to a shift in the ratio of DA to DOPAC or HVA, which was characteristic for each compound. The measurement of striatal 1-methyl-4-phenylpyridinium (MPP+) and 4'-amino-MPP+ levels revealed that high concentrations (up to 150 microM) persist in the striatum for weeks following administration of a single nontoxic dose of MPTP or 4'-amino-MPTP. A causal relationship between the striatal concentration of MPP+ or 4'-amino-MPP+ and the change in DA metabolism as reflected in the DA/DOPAC ratio is suggested by a significant correlation between these measures. It is suggested that presynaptic sequestration and retention of MPP+ and 4'-amino-MPP+ by striatal DA terminals result in the inhibition of the monoamine oxidase contained within these terminals.     

Release of endogenous dopamine, 3,4-dihydroxyphenylacetic acid,and amino acid transmitters from rat striatal slices

The release of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was measured in superfused striatal slices of the rat and the results compared with data obtained for the release of endogenous (a) DA and DOPAC in the cerebral cortex, nucleus accumbens and thalamus; (b) 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), GABA, and glutamate in the striatum; and (c) GABA, glutamate and 5-HT in the cerebral cortex. In superfused slices of all four CNS regions, there appeared to be a Ca²⁺-dependent, K⁺-stimulated release of endogenous DA. In addition, in slices of the striatum and nucleus accumbens there also appeared to be a Ca²⁺-dependent, 60 mM K⁺ stimulated release of endogenous DOPAC. In the striatum, 16 mM Mg²⁺ was as effective as 2.5 mM Ca²⁺ in promoting the 60 mM K⁺-stimulated release of DOPAC. In addition, 16 mM Mg²⁺ appeared to function as a weak Ca²⁺ agonist since it also promoted the release of DA to approximately 40% of the level attained with Ca²⁺ in the presence of 60 mM K⁺. On the other hand, in the striatum, 16 mM Mg²⁺ inhibited the Ca²⁺-dependent, 60 mM K⁺-stimulated release of GABA and glutamate. Similar Mg²⁺-inhibition was observed in the cerebral cortex not only for GABA and glutamate but also for DA and 5-HT. With the use of -methyl -tyrosine (tyrosine hydroxylase inhibitor), cocaine (uptake inhibitor) and pargyline (monoamine oxidase inhibitor), it was determined that (a) most of the released DA and DOPAC was synthesized in the slices during the superfusion; (b) DOPAC was not formed from DA which had been released and taken up; and (c) DA and DOPAC were released from DA nerve terminals. In addition, the data indicate a difference in the release process between the amino acids and the monoamines from striatal slices since Mg²⁺ inhibited the Ca²⁺-dependent, K⁺-stimulated release of GABA and glutamate and appeared to promote the release of DA and 5-HT.     

Enhanced dopamine D<Subscript>2</Subscript> receptor function in hypothalamus and corpus striatum: their role in liver,plasma and in vitro hepatocyte ALDH regulation in ethahol treated rats

Dopamine D₂ receptors are involved in ethanol self- administration behavior and also suggested to mediate the onset and offset of ethanol drinking. In the present study, we investigated dopamine (DA) content and Dopamine D₂ (DA D₂) receptors in the hypothalamus and corpus striatum of ethanol treated rats and aldehyde dehydrogenase (ALDH) activity in the liver and plasma of ethanol treated rats and in vitro hepatocyte cultures. Hypothalamic and corpus striatal DA content decreased significantly (P < 0.05, P < 0.001 respectively) and homovanillic acid/dopamine (HVA/DA) ratio increased significantly (P < 0.001) in ethanol treated rats when compared to control. Scatchard analysis of [³H] YM-09151-2 binding to DA D₂ receptors in hypothalamus showed a significant increase (P < 0.001) in B_max without any change in K_d in ethanol treated rats compared to control. The K_d of DA D₂ receptors significantly decreased (P < 0.05) in the corpus striatum of ethanol treated rats when compared to control. DA D₂ receptor affinity in the hypothalamus and corpus striatum of control and ethanol treated rats fitted to a single site model with unity as Hill slope value. The in vitro studies on hepatocyte cultures showed that 10⁻⁵ M and 10⁻⁷ M DA can reverse the increased ALDH activity in 10% ethanol treated cells to near control level. Sulpiride, an antagonist of DA D₂, reversed the effect of dopamine on 10% ethanol induced ALDH activity in hepatocytes. Our results showed a decreased dopamine concentration with enhanced DA D₂ receptors in the hypothalamus and corpus striatum of ethanol treated rats. Also, increased ALDH was observed in the plasma and liver of ethanol treated rats and in vitro hepatocyte cultures with 10% ethanol as a compensatory mechanism for increased aldehyde production due to increased dopamine metabolism. A decrease in dopamine concentration in major brain regions is coupled with an increase in ALDH activity in liver and plasma, which contributes to the tendency for alcoholism. Since the administration of 10⁻⁵ M and 10⁻⁷ M DA can reverse the increased ALDH activity in ethanol treated cells to near control level, this has therapeutic application to correct ethanol addicts from addiction due to allergic reaction observed in aldehyde accumulation.     

Striatal and Urinary DOPAC/DA Ratio May Indicate a Long-Lasting DA Release Enhancement by MPP+ and MPTP

The DOPAC/DA ratio in mouse striatum, in striatal synaptosomes, and in rat urine after MPP⁺ and MPTP neurotoxin administrations to the animals was followed temporally. The neurotoxins were given intraperitoneally and, in some experiments, to enhance the sensitivity, the animals were subsequently reserpinized before either sacrifice or 24 hour urine collection. MPP⁺ treatment, followed by saline, weakly lowered mouse striatal DOPAC/DA ratio up to 6 hours; in reserpinized animals, however, the neurotoxin reduced striatal ratio potently and for longer periods. Similarly, MPP⁺ reduced rat (saline treated) urinary DOPAC level and DOPAC/DA ratio in the short term (1.0 hr) while the neurotoxin effects could still be detected following longer periods up to 27 days in reserpinized animals. A single MPTP treatment (90 min.), followed by preparation of striatal synaptosomal fraction and its incubation (37°C) with or without reserpine, also led to a reduced DOPAC/DA ratio. Although mainly the pooled peripheral effect is directly indicated by urinary DOPAC/DA ratio, MPP⁺ may reduce DA oxidation in the CNS and may similarly affect the amine oxidation in the peripheral tissues. The CNS and peripheral effects differ, however, in respect to dose-sensitivity and time course. The similarities between the CNS and peripheral effects suggest that a blunted rise of urinary DOPAC/DA ratio after reserpine challenge could be utilized as a peripheral marker of MPP⁺ action in the CNS, a marker that is not currently available.     

ser31 tyrosine hydroxylase phosphorylation parallels differences in dopamine recovery in nigrostriatal pathway following 6‐OHDA lesion

Compensatory mechanisms in dopamine (DA) signaling have long been proposed to delay onset of locomotor symptoms during Parkinson's disease progression until ~ 80% loss of striatal DA occurs. Increased striatal dopamine turnover has been proposed to be a part of this compensatory response, but may occur after locomotor symptoms. Increased tyrosine hydroxylase (TH) activity has also been proposed as a mechanism, but the impact of TH protein loss upon site‐specific TH phosphorylation in conjunction with the impact on DA tissue content is not known. The tissue content of DA was determined against TH protein loss in the striatum and substantia nigra (SN) following 6‐hydroxydopamine lesion in the medial forebrain bundle in young Sprague–Dawley male rats. Although DA predictably decreased in both regions following 6‐hydroxydopamine, there was a significant difference in DA loss between the striatum (75%) and SN (40%), despite similar TH protein loss. Paradoxically, there was a significant decrease in DA against remaining TH protein in striatum, but a significant increase in DA against remaining TH in SN. In the SN, increased DA per remaining TH protein was matched by increased ser31, but not ser40, TH phosphorylation. In striatum, both ser31 and ser40 phosphorylation decreased, reflecting decreased DA per TH. However, in control nigral and striatal tissue, only ser31 phosphorylation correlated with DA per TH protein. Combined, these results suggest that the phosphorylation of ser31 in the SN may be a mechanism to increase DA biosynthesis against TH protein loss in an in vivo model of Parkinson's disease.

     

Role of excitatory amino acids in the regulation of dopamine synthesis and release in the neostriatum

Summary We have explored the role of excitatory amino acids in the increased dopamine (DA) release that occurs in the neostriatum during stress-induced behavioral activation. Studies were performed in awake, freely moving rats, usingin vivo microdialysis. Extracellular DA was used as a measure of DA release; extracellular 3,4-dihydroxyphenylalanine (DOPA) after inhibition of DOPA decarboxylase provided a measure of apparent DA synthesis. Mild stress increased the synthesis and release of DA in striatum. DA synthesis and release also were enhanced by the intra-striatal infusion of N-methyl-D-aspartate (NMDA), an agonist at NMDA receptors, and kainic acid, an agonist at the DL-a-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA)/kainate site. Stress-induced increase in DAsynthesis was attenuated by co-infusion of 2-amino-5-phosphonovalerate (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), antagonists of NMDA and AMPA/kainate receptors, respectively. In contrast, intrastriatal APV, CNQX, or kynurenic acid (a non-selective ionotropic glutamate receptor antagonist) did not block the stress-induced increase in DArelease. Stress-induced increase in DA release was, however, blocked by administration of tetrodotoxin along the nigrostriatal DA projection. It also was attenuated when APV was infused into substantia nigra. Thus, glutamate may act via ionotropic receptors within striatum to regulate DA synthesis, whereas glutamate may influence DA release via an action on receptors in substantia nigra. However, our method for monitoring DA synthesis lowers extracellular DA and this may permit the appearance of an intra-striatal glutamatergic influence by reducing a local inhibitory influence of DA. If so, under conditions of low extracellular DA glutamate may influence DA release, as well as DA synthesis, by an intrastriatal action. Such conditions might occur during prolonged severe stress and/or DA neuron degeneration. These results may have implications for the impact of glutamate antagonists on the ability of patients with Parkinson's disease to tolerate stress.     

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.     

Maternal Lead Exposure Produces Long-Term Enhancement of Dopaminergic Reactivity in Rat Offspring

To determine the effect of prenatal lead exposure on brain monoaminergic systems, pregnant rats were given tap water containing 250 ppm lead acetate, for the duration of pregnancy, while tap water without lead (Pb²⁺) was substituted at birth. Control rats were derived from dams that consumed tap water during pregnancy, and had no exposure to lead afterwards. At 12 weeks after birth, Pb²⁺ content of brain cortex was increased 3- to 4-fold (P < 0.05). At this time the endogenous striatal levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid were 19% lower in Pb²⁺ exposed rats (P < 0.05), while there was no change in the striatal level of dopamine (DA), noradrenaline, 3,4-dihydroxyphenylglycol, serotonin (5-HT) and 5-hydroxyindoleacetic acid (HPLC/ED). Also there was no change in these monoamines and metabolites in the prefrontal cortex of Pb²⁺ exposed rats. However, turnover of 5-HT in prefrontal cortex, as indicated by 5-hydroxytryptophan accumulation 30 min after acute treatment with the decarboxylase inhibitor NSD-1015 (100 mg/kg IP), was lower in the Pb²⁺ exposed rats. In the striatum AMPH-induced (1 mg/kg IP) turnover of DA, evidenced as L-DOPA accumulation after NSD-1015, was increased to a lesser extent in the Pb²⁺ exposed rats (P < 0.05). The nitric oxide synthase inhibitor 7-nitroindazole (10 mg/kg IP) attenuated the latter effect, indicating that neuronal NO mediates this AMPH effect, at least in part. Moreover, DA D₂ receptor sensitivity developed in Pb²⁺ exposed rats, as evidenced by enhanced quinpirole-induced yawning activity and enhanced quinpirole-induced locomotor activity (each, P < 0.05). These findings indicate that ontogenetic exposure to lead can have consequences on monoaminergic neuronal function at an adult stage of life, generally promoting accentuated behavioral effects of direct and indirect monoaminergic agonists, and related to increased dopamine turnover in basal ganglia. Special issue dedicated to Dr. Moussa Youdim.