Differential contribution of storage pools to the extracellular amount of accumbal dopamine in high and low responders to novelty: effects of reserpine

The present study examined the effects of reserpine on the extracellular concentration of accumbal dopamine in high responders (HR) and low responders (LR) to novelty rats. Reserpine reduced the baseline concentration of extracellular accumbal dopamine more in HR than in LR, indicating that the dopamine release is more dependent on reserpine-sensitive storage vesicles in non-challenged HR than in non-challenged LR. In addition, reserpine reduced the novelty-induced increase of the extracellular concentration of accumbal dopamine in LR, but not in HR, indicating that the dopamine release in response to novelty depends on reserpine-sensitive storage vesicles only in LR, not in HR. Our data clearly demonstrate that HR and LR differ in the characteristics of those monoaminergic storage vesicles that mediate accumbal dopamine release.     

Modification of dopamine neurotransmission in the nucleus accumbens of rats deficient in n-3 polyunsaturated fatty acids

We studied the effects of a diet chronically deficient in alpha-linolenic acid, the precursor of long-chain n-3 polyunsaturated fatty acids, on dopaminergic neurotransmission in the shell region of the nucleus accumbens of rats. In vivo microdialysis experiments showed increased basal levels of dopamine and decreased basal levels of metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in awake rats from the deficient group compared to controls. The release of dopamine under KCl stimulation was similar in both dietary groups. By contrast, the release of dopamine from the vesicular storage pool under tyramine stimulation was 90% lower in the deficient than in the control rats. Autoradiographic studies in the same cerebral region revealed a 60% reduction in the vesicular monoamine transporter sites in the deficient group. Dopamine D(2) receptors were 35% increased in these rats compared to controls, whereas no change occurred for D(1) receptors and membrane dopamine transporters. These results demonstrated that chronic n-3 polyunsaturated fatty acid deficiency modifies several factors of dopaminergic neurotransmission in the nucleus accumbens. These findings are in agreement with the changes in dopaminergic neurotransmission already observed in the frontal cortex, and with the behavioral disturbances described in these deficient rats.     

Effects of repeated morphine on locomotion, place preference and dopamine in heterozygous glial cell line-derived neurotrophic factor knockout mice

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be involved in the maintenance of striatal dopaminergic neurons. Neurotrophic factors are crucial for the plasticity of central nervous system and may be involved in long-term responses to drug exposure. To study the effects of reduced GDNF on dopaminergic behaviour related to addiction, we compared the effects of morphine on locomotor activity, conditioned place preference (CPP) and extracellular accumbal dopamine in heterozygous GDNF knockout mice (GDNF+/-) with those in their wild-type (Wt) littermates. When morphine 30 mg/kg was administered daily for 4 days, tolerance developed towards its locomotor stimulatory action only in the GDNF+/- mice. A morphine 5 mg/kg challenge dose stimulated locomotor activity only in the GDNF+/- mice withdrawn for 96 h from repeated morphine treatment, whereas clear and similar sensitization of the locomotor response was seen after a 10 mg/kg challenge dose in mice of both genotypes. Morphine-induced CPP developed initially similarly in Wt and GDNF+/- mice, but it lasted longer in the Wt mice. The small challenge dose of morphine increased accumbal dopamine output slightly more in the GDNF+/- mice than in the Wt mice, but doubling the challenge dose caused a dose-dependent response only in the Wt mice. In addition, repeated morphine treatment counteracted the increase in the accumbal extracellular dopamine concentration we previously found in drug-naive GDNF+/- mice. Thus, reduced endogenous GDNF level alters the dopaminergic behavioural effects to repeatedly administered morphine, emphasizing the involvement of GDNF in the neuroplastic changes related to long-term effects of drugs of abuse.     

Absence of synapsin I and II is accompanied by decreases in vesicular transport of specific neurotransmitters

Studies of synapsin-deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild-type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double-labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.     

Reserpine pretreatment prevents increases in extracellular striatal dopamine following L-DOPA administration in rats with nigrostriatal denervation

The influence of L-DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L-DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine-pretreated rats. In rats with 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L-DOPA (50 mg/kg i.p.). In 6-hydroxydopamine-lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L-DOPA (50 mg/kg i.p.)-induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p<0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L-DOPA-derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.     

l-3,4-Dihydroxyphenylalanine-Induced Dopamine Release in the Striatum of Intact and 6-Hydroxydopamine-Treated Rats: Differential Effects of Monoamine Oxidase A and B Inhibitors

Abstract: Administration of l -DOPA (50 mg/kg) elicits a significant increase in extracellular dopamine in striata of rats treated with the catecholaminergic neurotoxin 6-hydroxydopamine but not in striata of intact rats. To assess the role of dopaminergic nerve terminals in determining the effects of exogenous l -DOPA on extracellular dopamine levels in striatum, we examined the relative contributions of monoamine oxidase A and monoamine oxidase B to the catabolism of dopamine synthesized from exogenous l -DOPA. Extracellular concentrations of dopamine and its catabolite, 3,4-dihydroxyphenylacetic acid, were monitored with in vivo dialysis in striata of intact rats and of rats with unilateral 6-hydroxydopamine lesions of striatal dopamine. Clorgyline (2 mg/kg), an inhibitor of monoamine oxidase A, significantly increased dopamine and decreased 3,4-dihydroxyphenylacetic acid in intact but not in dopamine-depleted striata. Inhibition of monoamine oxidase B with either l -deprenyl (1 mg/kg) or Ro 19-6327 (1 mg/kg) did not significantly affect dopamine or 3,4-dihydroxyphenylacetic acid in striata of intact or dopamine-depleted rats. In intact rats, administration of clorgyline in conjunction with l -DOPA produced a >20-fold increase in dopamine and prevented the l -DOPA-induced increase in 3,4-dihydroxyphenylacetic acid. Although both l -deprenyl and Ro 19-6327 administered in combination with l -DOPA elicited a small but significant increase in dopamine, levels of 3,4-dihydroxyphenylacetic acid were not affected. In rats pretreated with 6-hydroxydopamine, clorgyline had no significant effect on the increases in dopamine and 3,4-dihydroxyphenylacetic acid elicited by l -DOPA. Furthermore, neither l -deprenyl nor Ro 19-6327 affected l -DOPA-induced increases in dopamine and 3,4-dihydroxyphenylacetic acid in dopamine-depleted striata. The present findings indicate that deamination by monoamine oxidase A is the primary mechanism for catabolism of striatal dopamine, both under basal conditions and after administration of exogenous l -DOPA. Loss of dopaminergic terminals eliminates this action of monoamine oxidase A but does not enhance deamination by monoamine oxidase B. These data support a model in which exogenous l -DOPA elicits enhanced extracellular accumulation of dopamine in the dopamine-depleted striatum because some transmitter synthesis occurs at nondopaminergic sites and the dopamine terminals that normally take up and catabolize this pool of transmitter are absent.     

Sources contributing to the average extracellular concentration of dopamine in the nucleus accumbens

Mesolimbic dopamine neurons fire in both tonic and phasic modes resulting in detectable extracellular levels of dopamine in the nucleus accumbens (NAc). In the past, different techniques have targeted dopamine levels in the NAc to establish a basal concentration. In this study, we used in vivo fast scan cyclic voltammetry (FSCV) in the NAc of awake, freely moving rats. The experiments were primarily designed to capture changes in dopamine caused by phasic firing - that is, the measurement of dopamine 'transients'. These FSCV measurements revealed for the first time that spontaneous dopamine transients constitute a major component of extracellular dopamine levels in the NAc. A series of experiments were designed to probe regulation of extracellular dopamine. Lidocaine was infused into the ventral tegmental area, the site of dopamine cell bodies, to arrest neuronal firing. While there was virtually no instantaneous change in dopamine concentration, longer sampling revealed a decrease in dopamine transients and a time-averaged decrease in the extracellular level. Dopamine transporter inhibition using intravenous GBR12909 injections increased extracellular dopamine levels changing both frequency and size of dopamine transients in the NAc. To further unmask the mechanics governing extracellular dopamine levels we used intravenous injection of the vesicular monoamine transporter (VMAT2) inhibitor, tetrabenazine, to deplete dopamine storage and increase cytoplasmic dopamine in the nerve terminals. Tetrabenazine almost abolished phasic dopamine release but increased extracellular dopamine to ～500?nM, presumably by inducing reverse transport by dopamine transporter (DAT). Taken together, data presented here show that average extracellular dopamine in the NAc is low (20-30?nM) and largely arises from phasic dopamine transients.     

Methamphetamine rapidly decreases vesicular dopamine uptake

Vesicular sequestration is important in the regulation of cytoplasmic concentrations of monoamines such as dopamine. Moreover, recent evidence suggests that increases in cytoplasmic dopamine levels, perhaps attributable to changes in vesicular monoamine transporter function, contribute to methamphetamine-induced dopaminergic deficits. Hence, we examined whether striatal vesicular uptake is altered following methamphetamine treatment. Multiple administrations of methamphetamine rapidly (within 1 h) decreased vesicular dopamine uptake and dihydrotetrabenazine binding, an effect that (a) persisted at least 24 h, (b) was associated with dopamine and not serotonin neurons, and (c) was unrelated to residual drug introduced by the original methamphetamine treatment. These data suggest that methamphetamine rapidly decreases vesicular monoamine transporter function in dopaminergic neurons, a phenomenon that may be associated with the long-term damage caused by this stimulant.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.     

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.     

Mitochondrial damage by nitric oxide is potentiated by dopamine in PC12 cells

Mitochondrial damage in PC12 cells, a model for dopaminergic cells, was examined in terms of the contribution of oxidative stress, nitric oxide (*NO), and dopamine to impairment of mitochondrial respiratory control (RC). A kinetic analysis suggested that the oxidative deamination of dopamine catalyzed by monoamine oxidase (MAO) was not a significant source of hydrogen peroxide, because of constrains imposed by the low cytosolic level of dopamine. *NO induced irreversible damage of mitochondrial complex I in PC12 cells: this damage followed a sigmoid response on *NO concentration with a well-defined threshold level. Dopamine did not elicit damage of mitochondria in PC12 cells; however, the amine potentiated the effects of *NO at or near the threshold level, thus leading to irreversible impairment of mitochondrial respiration. This synergism between *NO and dopamine was not observed at *NO concentrations below the threshold level. Depletion of dopamine from the storage vesicles by reserpine protected mitochondria from *NO damage. Dopamine oxidation by *NO increased with pH, and occurred at modest levels at pH 5.5. In spite of this, calculations showed that the oxidation of dopamine in the storage vesicles (pH 5.5) was higher than that in the cytosol (pH 7.4), due to the higher dopamine concentration in the storage vesicles (millimolar range) compared to that in the cytosol (micromolar range). It is suggested that storage vesicles may be the cellular sites where the potential for dopamine oxidation by *NO is higher.These data provide further support to the hypothesis that dopamine renders dopaminergic cells more susceptible to the mitochondrial damaging effects of *NO. In the early stages of Parkinson's disease, *NO production increases until reaching a point near the threshold level that induces neuronal damage. Dopamine stored in dopaminergic cells may cause these cells to be more susceptible to the deleterious effects of *NO, which involve irreversible impairment of mitochondrial respiration.     

A single exposure to novelty differentially affects the accumbal dopaminergic system of apomorphine-susceptible and apomorphine-unsusceptible rats

Individual differences in responses to mild, acute stressors in laboratory animals have commonly been observed in behavioural tests and at the level of hypothalamic-pituitary-adrenal axis responses. These differences are associated with dopamine transmission in the nucleus accumbens. Although the effect of mild stressors on dopamine transmission has been studied with microdialysis, it has not been studied at the level of the catecholaminergic network in the nucleus accumbens. In this study we have used microdialysis to measure extracellular concentrations of dopamine in vivo and immunocytochemistry for the enzyme tyrosine hydroxylase to assess the effect of a single exposure to novelty on the neurochemistry of the nucleus acc umbens in apomorphine-susceptible and apomorphine-unsusceptible rats. These rats are a valid animal model for studying individual differences in responses to environmental stressors and drugs of abuse. We demonstrated that a mild stressor like novelty increased the extracellular concentration of dopamine in the nucleus accumbens in apomorphine-susceptible rats to a larger and longer-lasting degree than in apomorphine-unsusceptible rats. Furthermore we demonstrated that novelty increased the tyrosine hydroxylase-immunoreactive fibre network in the nucleus accumbens shell of apomorphine-susceptible rats, which are rats that are particularly reactive to stressors, but not in the shell of apomorphine-unsusceptible rats, which are rats that are relatively stress-resistant. In conclusion, we have shown that the accumbal dopaminergic system of apomorphine-susceptible rats is more sensitive to an environmental stressor than that of apomorphine-unsusceptible rats. Combined with the fact that these animals also differ in their sensitivity to drugs of abuse, which are known to affect the dopaminergic system, these data provide a solid basis for further studying the differences in the dopaminergic responsiveness to drugs of abuse between apomorphine-susceptible and apomorphine-unsusceptible rats.     

Changes in Sympathetic Nerve Terminals in the Heart of Cold-Exposed Rats

Abstract: Changes in sympathetic nerve terminals of the heart after varying periods of exposure of rats to 4°C were investigated. Two indices were used for changes in the number of noradrenaline storage vesicles, i.e., vesicular dopamine β-hydroxylase (DBH) activity and noradrenaline storage capacity. The latter was obtained after uptake of [³H]noradrenaline; endogenous content, uptake of exogenous noradrenaline, and degree of saturation of the vesicles were calculated using the specific activity of the [³H]noradrenaline. As a measure of tyrosine hydroxylase activity, whole ventricular noradrenaline, dopamine, and dihydroxyphenylacetic acid content were used. After 4 h of cold exposure there was an increase in vesicular endogenous noradrenaline content, uptake, storage capacity, and DBH activity as well as a large increase in whole ventricular dopamine. After 6 h in the cold, vesicular endogenous noradrenaline content, storage capacity, and DBH activity were decreased. The results suggest that during cold exposure there is an initial increase followed by a decrease in the number of functional vesicles in the nerve terminal, which could explain the fluctuations in the rate of noradrenaline release.     

Loaded dopamine is preferentially stored in the halo portion of PC12 cell dense core vesicles

Large dense core vesicles in rat pheochromocytoma cells are morphologically distinct from dense core vesicles in mast and chromaffin cells in that the dense core occupies a much smaller fraction of the vesicular volume, allowing for a much larger vesicular clear space, or halo. In this work, we present evidence indicating that upon treatment with L-DOPA the majority of the dopamine loaded into these vesicles is preferentially compartmentalized into the halo portion of the vesicle. Amperometry was used to monitor release of loaded neurotransmitter from cells in both isotonic and hypertonic extracellular conditions, with the latter condition causing inhibition of dense core dissociation. In combination with this we have used transmission electron microscopy to determine the morphological characteristics of dense core vesicles before and after treatment with L-DOPA in solutions of varied osmolarity. The results provide a more complete understanding of the complex interaction of molecules within dense core vesicles, suggesting that newly loaded dopamine is located in the halo of the vesicle. This finding has fundamental significance for studies of neurotransmitter release from dense core vesicles, as the core appears to have a function involving more than simple storage of neurotransmitter and associated molecules, and the often overlooked vesicular halo appears to be an important storage compartment for neurotransmitter.     

Acid-sensing ion channels contribute to the increase in vesicular release from SH-SY5Y cells stimulated by extracellular protons

Acid-sensing ion channels (ASICs) have been reported to play a role in the neuronal dopamine pathway, but the exact role in neurotransmitter release remains elusive. Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line, which can release monoamine neurotransmitters. In this study, the expression of ASICs was identified in SH-SY5Y cells to further explore the role of ASICs in vesicular release stimulated by acid. We gathered evidence that ASICs could be detected in SH-SY5Y cells. In whole cell patch-clamp recording, a rapid decrease in extracellular pH evoked inward currents, which were reversibly inhibited by 100 μM amiloride. The currents were pH dependent, with a pH of half-maximal activation (pH(0.5)) of 6.01 ± 0.04. Furthermore, in calcium imaging and FM 1-43 dye labeling, it was shown that extracellular protons increased intracellular calcium levels and vesicular release in SH-SY5Y cells, which was attenuated by PcTx1 and amiloride. Interestingly, N-type calcium channel blockers inhibited the vesicular release induced by acidification. In conclusion, ASICs are functionally expressed in SH-SY5Y cells and involved in vesicular release stimulated by acidification. N-type calcium channels may be involved in the increase in vesicular release induced by acid. Our results provide a preliminary study on ASICs in SH-SY5Y cells and neurotransmitter release, which helps to further investigate the relationship between ASICs and dopaminergic neurons.     

Inhibition of vesicular uptake of monoamines by hyperforin

Hyperforin is the major active ingredient of Hypericum perforatum (St John's Wort), a traditional antidepressant medication. This study evaluated its inhibitory effects on the synaptic uptake of monoamines in rat forebrain homogenates, comparing the nature of the inhibition at synaptic and vesicular monoamine transporters. A hyperforin-rich extract inhibited with equal potencies the sodium-dependent uptake of the monoamine neurotransmitters serotonin [5-HT], dopamine [DA] and norepinephrine [NE] into rat brain synaptosomes. Hyperforin inhibited the uptake of all three monoamines noncompetitively, in marked contrast with the competitive inhibition exerted by fluoxetine, GBR12909 or desipramine on the uptake of these monoamines. Hyperforin had no inhibitory effect on the binding of [3H]paroxetine, [3H]GBR12935 and [3H]nisoxetine to membrane presynaptic transporters for 5-HT, DA and NE, respectively. The apparent presynaptic inhibition of monoamine uptake could reflect a "reserpine-like mechanism" by which hyperforin induced release of neurotransmitters from synaptic vesicles into the cytoplasm. Thus, we assessed the effects of hyperforin on the vesicular monoamine transporter. Hyperforin inhibited with equal potencies the uptake of the three tritiated monoamines to rat brain synaptic vesicles. Similarly to the synaptosomal uptake, the vesicular uptake was also noncompetitively inhibited by hyperforin. Notably, hyperforin did not affect the direct binding on [3H]dihydrotetrabenazine, a selective vesicular monoamine transporter ligand, to rat forebrain membranes. Our results support the notion that hyperforin interferes with the storage of monoamines in synaptic vesicles, rather than being a selective inhibitor of either synaptic membrane or vesicular monoamine transporters.     

Effect of adrenalectomy and corticosterone on cocaine-induced sensitization in rats.

Effects of adrenalectomy (ADX) and corticosterone (CORT) on the development and expression of sensitization to the locomotor effect of cocaine (COC) were studied in rats. Sensitization was evoked by 5 daily injections of COC (10 mg/kg) and measured after a challenge dose of the drug (10 mg/kg) after a 5-day withdrawal (on day 10 of the experiment). ADX, performed before the start of COC administration, completely blocked the manifestation of COC-induced sensitization. In contrast, ADX performed on animals already sensitized to COC did not affect the sensitized locomotor activity response to a challenge dose of COC (on day 18). Pretreatment with CORT, 10 mg/kg, but not 5 mg/kg, before each of the 5 daily COC injections facilitated the development of COC sensitization, tested after a 5-day withdrawal. When pretreated with CORT alone (10 mg/kg), the challenge dose of COC administered on day 10 induced cross-sensitization to CORT. CORT (10 mg/kg) injected acutely before COC on day 10, potentiated the expression of COC sensitization. When given alone, on day 10 CORT (5-10 mg/kg) induced an increase in the locomotor activity of rats pretreated daily (5 injections) with COC. No drug treatment induced conditioned locomotion, as measured after saline challenge on day 8. Our results indicate that CORT facilitates the development and expression of COC sensitization, while ADX blocks the initiation of the behavioral phenomenon only. Moreover, there takes place cross-sensitization between CORT and COC, which indicates a close relationship between the drug-related mechanism and behavioral sensitization.     

Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness

The aim for this study was to examine whether the F4 generation of two strains of rainbow trout divergent in their plasma cortisol response to confinement stress (HR: high responder or LR: low responder) would also differ in stress-induced effects on forebrain concentrations of mRNA for corticotropin-releasing factor (CRF), arginine vasotocin (AVT), CRF receptor type 1 (CRF-R1), CRF receptor type 2 (CRF-R2) and AVT receptor (AVT-R). In addition, plasma cortisol concentrations, brainstem levels of monoamines and monoamine metabolites, and behaviour during confinement were monitored. The results confirm that HR and LR trout differ in their cortisol response to confinement and show that fish of these strains also differ in their behavioural response to confinement. The HR trout displayed significantly higher locomotor activity while in confinement than LR trout. Moreover, following 180 min of confinement HR fish showed significantly higher forebrain concentrations of CRF mRNA than LR fish. Also, when subjected to 30 min of confinement HR fish showed significantly lower CRF-R2 mRNA concentrations than LR fish, whereas there were no differences in CRF-R1, AVT or AVT-R mRNA expression between LR and HR fish either at 30 or 180 min of confinement. Differences in the expression of CRF and CRF-R2 mRNA may be related to the divergence in stress coping displayed by these rainbow trout strains.     

Inhibition of vesicular monoamine transporter enhances vulnerability of dopaminergic cells: relevance to Parkinson's disease

Parkinson's disease is a neurodegenerative disorder associated with progressive loss of dopaminergic cells in the substantia nigra. Oxidative stress has been implicated in the pathogenesis of the disease, and dopamine has been suggested as a contributing factor that generates reactive oxygen species due to its unstable catechol moiety. We have previously shown that tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine synthesis, also contributes to the vulnerability of dopamine-producing cells by generating oxidative stress. This study shows that the presence of dopamine in the cytosol enhances the cell's vulnerability to BH4. Upon exposure to ketanserin, a vesicular monoamine transporter inhibitor, BH4-induced dopaminergic cell death is exacerbated, accompanied by increased lipid peroxidation and protein bound quinone. While intracellular amount of DOPAC is elevated by ketanserin, the monoamine oxidase inhibitor pargyline showed no significant protection. Instead, the thiol agent N-acetylcysteine and quinone reductase inducer dimethyl fumarate abolish BH4/ketanserin-induced cell death, suggesting that quinone production plays an important role. Therefore, it can be concluded that the presence of dopamine in the cytosol seems to contribute to the cells' vulnerability to BH4 and that vesicular monoamine transporter plays a protective role in dopaminergic cells by sequestering dopamine not only from monoamine oxidase but also from BH4-induced oxidative stress.     

Inhibition of Vesicular Monoamine Transporter-2 Activity in α-Synuclein Stably Transfected SH-SY5Y Cells

α-Synuclein plays a key role in the pathological neurodegeneration in Parkinson’s disease. Although its contribution to normal physiology remains elusive, the selective degeneration of α-synuclein-containing dopaminergic neurons in Parkinson’s disease may be linked to abnormal α-synuclein induced toxicity. In the present study, a complex of α-synuclein and vesicular monoamine transporter-2 was identified by GST-Pull Down experiment. In wild-type α-synuclein stably transfected SH-SY5Y cell lines, the activity of vesicular monoamine transporter-2 decreased by 31% as determined by [³H] dopamine uptake, and its expression also decreased in both protein and mRNA levels using western and northern blot analysis. Overexpression of wild-type α-synuclein did not induce cell death or apoptosis, but significantly enhanced the intracellular reactive oxygen species level as assayed by flow cytometry. These data suggest that Up-regulated α-synuclein expression inhibits the activity of vesicular monoamine transporter-2, thereby interrupting dopamine homeostasis and resulting in dopaminergic neuron injury in Parkinson’s disease.