Nicotine increases dopamine clearance in medial prefrontal cortex in rats raised in an enriched environment

Environmental enrichment results in differential behavioral and neurochemical responsiveness to nicotine. The present study investigates dopamine clearance (CL_DA) in striatum and medial prefrontal cortex (mPFC) using in vivo voltammetry in rats raised in enriched (EC) or impoverished conditions (IC) and administered nicotine (0.4 mg/kg) or saline. Baseline CL_DA in striatum or mPFC was not different between EC and IC. Across repeated DA application, striatal CL_DA increased in saline-control EC and IC. CL_DA increased in mPFC in saline-control IC; CL_DA did not change in saline-control EC. Thus, enrichment differentially alters dynamic responses of the dopamine transporter (DAT) to repeated DA application in mPFC, but not in striatum. In EC, nicotine increased mPFC CL_DA compared to saline-control, but had no effect on CL_DA in IC; nicotine had no effect in striatum in EC or IC. Compared to respective saline-controls, nicotine increased dihydroxyphenylacetic acid content in striatum and mPFC in EC, but not in IC. Nicotine also had no effect on DA content in striatum or mPFC in EC or IC. Results indicate that enrichment eliminated the dynamic response of mPFC DAT to repeated DA application in saline-control and augmented the nicotine-induced increase in DAT function in mPFC, but not in striatum.     

Dopamine D2 Receptor-Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum

Abstract : Presynaptic D₂ dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D₂ DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D₂^+/+), one (D₂^+/-), or no (D₂^-/-) functional copies of the gene coding for the D₂ DA receptor. In vivo microdialysis studies demonstrated that basal and K⁺-evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D₂^-/- mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D₂^+/+ mice. In D₂^+/+ mice, but not D₂^-/- mice, local application of the D₂-like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [³H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D₂ DA receptor subtype modulates activity of the striatal DAT.     

Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis

Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 microM treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K(m), in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three-fivefold in both cell types exposed to 50 microM DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.     

The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells

The effects of 17β-estradiol (E₂) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E₂ at physiological concentrations on DA efflux in nerve growth factor-differentiated rat pheochromocytoma cells that express estrogen receptors (ER) α, ERβ, and G-protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of ³H-DA from pre-loaded cells; a 9–15 min 10⁻⁹ M E₂ treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non-genomic response, and an E₂-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 min. Efflux dose–responses for E₂ were non-monotonic, also characteristic of non-genomic estrogenic actions. ERα siRNA knockdown abolished E₂-mediated DA efflux, while ERβ knockdown did not, and GPR30 knockdown increased E₂-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ERα is the predominant mediator of E₂-mediated DA efflux, with inhibitory contributions from GPR30 and ERβ. E₂ also caused trafficking of ERα to the plasma membrane, trafficking of ERβ away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERα is largely responsible for non-genomic estrogenic effects on DAT activity.     

Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic l-Amino Acid Decarboxylase: Neurochemical Effects

Abstract: To study the possibility that increasing striatal activity of aromatic l -amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to l -3,4-dihydroxyphenylalanine ( l -DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos- haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of ¹⁴CO₂ from l -[¹⁴C]DOPA. The K _m value for l -DOPA in intact and disrupted cells was 0.60 and 0.56 m M , respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 m M . The pH optimum for enzyme activity was 6.8. Grafting Cos- haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of l -DOPA. When measured 2 h after l -DOPA administration, the mean dopamine level in the striata of Cos- haadc -grafted animals was 2 µg/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 µg/g. At 6–8 h after l -DOPA administration, striatal dopamine content in the Cos- haadc -grafted animals was 0.67 µg/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered l -DOPA.     

Conjugates of Catecholamines with Cysteine and GSH in Parkinson's Disease: Possible Mechanisms of Formation Involving Reactive Oxygen Species

Abstract: Oxidation of l -3,4-dihydroxyphenylalanine ( l -DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinson's disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of l -DOPA, DA, and other catecholamines to a system generating O₂^•− radical led to O₂^•−-dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol-DA or -DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and l -DOPA, especially if H₂O₂ was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of l -DOPA, DA, and 3,4-dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of l -DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of l -DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with l -DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.     

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.     

Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by l-DOPA treatment

Dysregulation of dopamine (DA) receptors is believed to underlie Parkinson's disease pathology and l -DOPA-induced motor complications. DA receptors are subject to regulation by G protein-coupled receptor kinases (GRKs) and arrestins. DA lesion with 6-hydroxydopamine caused multiple protein- and brain region-specific changes in the expression of GRKs. In the globus pallidus, all four GRK isoforms (GRK2, 3, 5, 6) were reduced in the lesioned hemisphere. In the caudal caudate-putamen (cCPu) three GRK isoforms (GRK2, 3, 6) were decreased by DA depletion. The decrease in GRK proteins in globus pallidus, but not cCPu, was mirrored by reduction in mRNA. GRK3 protein was reduced in the rostral caudate-putamen (rCPu), whereas other isoforms were either unchanged or up-regulated. GRK6 protein and mRNA were up-regulated in rCPu and nucleus accumbens. l -DOPA (25 mg/kg, twice daily for 10 days) failed to reverse changes caused by DA depletion, whereas D₂/D₃ agonist pergolide (0.25 mg/kg daily for 10 days) restored normal levels of expression of GRK5 and 6. In rCPu, GRK2 protein was increased in most subcellular fractions by l -DOPA but not by DA depletion alone. Similarly, l -DOPA up-regulated arrestin3 in membrane fractions in both regions. GRK5 was down-regulated by l -DOPA in cCPu in the light membrane fraction, where this isoform is the most abundant. The data suggest that alterations in the expression and subcellular distribution of arrestins and GRKs contribute to pathophysiology of Parkinson's disease. Thus, these proteins may be targets for antiparkinsonian therapy.     

Monoamine Interactions Measured by Microdialysis in the Ventral Tegmental Area of Rats Treated Systemically with (±)-8-Hydroxy-2-(Di-n-Propylamino)tetralin

Abstract: The effect of (±)-8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT), a selective serotonin 5-HT_1A agonist, on levels of extracellular norepinephrine (NE), dopamine (DA), and 5-HT (measured simultaneously) was investigated by microdialysis in the ventral tegmental area (VTA) of freely moving rats, and their behavioral activity was monitored. At 50 µg/kg s.c., 8-OH-DPAT reduced 5-HT levels but enhanced NE and DA levels in VTA dialysate. These effects were not altered by pretreatment with systemic idazoxan (5 mg/kg i.p.), a selective α₂ antagonist, or local sulpiride (10 µ M ), a selective D₂/D₃ antagonist. At 500 µg/kg s.c., 8-OH-DPAT further enhanced or more persistently reduced dialysate NE or 5-HT content but had little effect on dialysate DA content. Its DA level-increasing effect could be seen dramatically with local infusion of cocaine (30 µ M ) and, to a lesser extent, sulpiride (10 µ M ). Depletion of endogenous 5-HT with p -chlorophenylalanine attenuated both the 5-HT level-reducing and DA level-enhancing effects of 8-OH-DPAT without affecting its maximal NE effect and the locomotor-stimulatory effect. Partial depletion of endogenous NE with N -(2-chloroethyl)- N -ethyl-2-bromobenzylamine failed to change the monoamine response but diminished the locomotion induced by 8-OH-DPAT. These results suggested that (a) the low dose of 8-OH-DPAT may act at presynaptic 5-HT_1A receptors to modulate 5-HT and DA release, while acting at postsynaptic 5-HT_1A receptors to modulate NE release; (b) the high dose of 8-OH-DPAT may activate D₂ receptors to offset its DA level-increasing effect; and (c) the locomotor-stimulatory effect of 8-OH-DPAT might be mediated primarily by postsynaptic 5-HT_1A receptors and the NE system.     

l-3,4-Dihydroxyphenylalanine Increases the Quantal Size of Exocytotic Dopamine Release In Vitro

Abstract: The catecholamine precursor l -3,4-dihydroxyphenylalanine ( l -DOPA) is used to augment striatal dopamine (DA), although its mechanism of altering neurotransmission is not well understood. We observed the effects of l -DOPA on catecholamine release in ventral midbrain neuron and PC12 pheochromocytoma cell line cultures. In ventral midbrain neuron cultures exposed to 40 m M potassium-containing media, l -DOPA (100 µ M for 1 h) increased DA release by >10-fold. The elevated extracellular DA levels were not significantly blocked by the DA/norepinephrine transport inhibitor nomifensine, demonstrating that reverse transport through catecholamine-uptake carriers plays little role in this release. In PC12 cells, where DA release from individual secretory vesicles can be observed, l -DOPA (50 µ M for 1 h) elevated DA release in high-potassium media by 370%. Amperometric measurements demonstrated that l -DOPA (50 µ M for 40–70 min) did not raise the frequency of vesicular exocytosis but increased the average size of quantal release to at least 250% of control levels. Together, these findings suggest that l -DOPA can increase stimulation-dependent transmitter release from DA cells by augmenting cytosolic neurotransmitter, leading to increased quantal size.     

l-DOPA activates ERK signaling and phosphorylates histone H3 in the striatonigral medium spiny neurons of hemiparkinsonian mice

In the dopamine-depleted striatum, extracellular signal-regulated kinase (ERK) signaling is implicated in the development of l -DOPA-induced dyskinesia. To gain insights on its role in this disorder, we examined the effects of l -DOPA on the state of phosphorylation of ERK and downstream target proteins in striatopallidal and striatonigral medium spiny neurons (MSNs). For this purpose, we employed mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoters for the dopamine D₂ receptor ( Drd2 -EGFP mice) or the dopamine D₁ receptor ( Drd1a -EGFP mice), which are expressed in striatopallidal and striatonigral MSNs, respectively. In 6-hydroxydopamine-lesioned Drd2 -EGFP mice, l -DOPA increased the phosphorylation of ERK, mitogen- and stress-activated kinase 1 and histone H3, selectively in EGFP-negative MSNs. Conversely, a complete co-localization between EGFP and these phosphoproteins was observed in Drd1a -EGFP mice. The effect of l -DOPA was prevented by blockade of dopamine D₁ receptors. The same pattern of activation of ERK signaling was observed in dyskinetic mice, after repeated administration of l -DOPA. Our results demonstrate that in the dopamine-depleted striatum, l -DOPA activates ERK signaling specifically in striatonigral MSNs. This regulation may result in ERK-dependent changes in striatal plasticity leading to dyskinesia.     

Presynaptic control of striatal dopamine neurotransmission in adult vesicular monoamine transporter 2 (VMAT2) mutant mice

The vesicular monoamine transporter 2 (VMAT2) plays a pivotal role in regulating the size of vesicular and cytosolic dopamine (DA) storage pools within the CNS, and can thus influence extracellular DA neurotransmission. Transgenic mice have been generated with a dramatically reduced (by approximately 95%) expression of the VMAT2 gene which, unlike complete knockout lines, survive into adulthood. We compared the pre-synaptic regulation of both impulse-dependent (exocytotic) and carrier-mediated (via reversal of the DA transporter, DAT) DA release in the dorsolateral caudate putamen (CPu) of striatal slices derived from adult homozygous VMAT2 mutant and wild-type mice using fast cyclic voltammetry. Impulse-dependent DA release, evoked by a single electrical pulse, was lower in homozygous (116 nm) than wild-type mice (351 nm) indicating smaller vesicular DA stores, an observation supported by the evanescent effect of amfonelic acid (300 nm) in homozygous mice. Amphetamine (2 microm) increased extracellular DA via DAT reversal in both wild-type (by 459 nm) and VMAT2 mutant (by 168 nm, p < 0.01 vs. wild-type) mice. In both cases, the effect was blocked by the DAT inhibitor GBR12935 (1 microm). Simultaneously, amphetamine decreased impulse-dependent DA release, albeit less in homozygous (by 55%) than in wild-type (by 78%) mice. In wild-types, this decrement was largely reversed by GBR12935 but not by the D2/D3 autoreceptor antagonist (-)sulpiride (1 microm). Conversely, in homozygous VMAT2 mutant mice, it was attenuated by (-)sulpiride but not GBR12935. The D2/D3 receptor agonist quinpirole inhibited impulse-dependent DA release with a lower EC50 value in homozygous mice (12 nm) compared with wild-types (34 nm), indicating the compensatory presence of functionally supersensitive release-regulating autoreceptors. However, analysis of DA reuptake kinetics obtained in the absence and presence of DAT blockade (by cocaine and amfonelic acid) revealed only minor differences in DAT functionality. These results demonstrate that impaired vesicular DA storage constrains extracellular DA levels in the dorsolateral CPu whether induced by either impulse-dependent or carrier-mediated mechanisms and that the relative importance of the DAT and terminal autoreceptors as control mechanisms in the actions of amphetamine are reversed in VMAT2 mutant mice.     

Norepinephrine Microinjections in the Hypothalamic Paraventricular Nucleus Increase Extracellular Dopamine and Decrease Acetylcholine in the Nucleus Accumbens: Relevance to Feeding Reinforcement

Abstract: Norepinephrine (NE) was microinjected into the paraventricular nucleus (PVN), while microdialysis was used to monitor extracellular dopamine (DA) and acetylcholine (ACh) in the nucleus accumbens (NAc). The PVN is a site where exogenously administered NE can act through α₂ receptors to elicit eating behavior and preference for carbohydrates. It was hypothesized that NE in the PVN acts on a behavior reinforcement system by altering the DA/ACh balance in the NAc. NE microinjections (80 nmol in 0.3 µl), which effectively elicited feeding in satiated rats in a separate test, caused a significant increase in extracellular DA (109%) and decrease in ACh (−27%) when the same animals were tested in the absence of food. In contrast when the food was available and ingested, ACh increased (51%) instead of decreasing. These results support the hypothesis that a functional link exists between the PVN and the NAc in which DA helps initiate and ACh helps stop appetitive behavior involved in the reinforcement of eating.     

In Vivo Microdialysis Evidence for Transient Dopamine Release by Benzazepines in Rat Striatum

Abstract: The effects of benzazepine derivatives on extracellular levels of dopamine (DA) and l -3,4-dihydroxyphenylacetic acid (DOPAC) in the dorsal striatum of freely moving rats were studied using in vivo microdialysis. Direct injection of SKF-38393 (0.5 or 1.5 µg/0.5 µl), a selective D₁ receptor agonist, into the striatum through a cannula secured alongside a microdialysis probe produced a rapid dose-dependent transient increase in striatal DA efflux and a more gradual reduction in efflux of DOPAC. The rapid increase in DA efflux was not affected by infusion of tetrodotoxin (TTX; 2 µ M ) or Ca²⁺-free Ringer's solution and occurred after either enantiomer of SKF-38393. A TTX-insensitive increase in DA level similar to that induced by SKF-38393 was also seen after other benzazepines acting as agonists (SKF-75670 and SKF-82958, each 1.5 µg in 0.5 µl) and antagonists (SCH-23390, 1.5 µg in 0.5 µl) at the D₁ receptor and after (+)-amphetamine. These effects were inhibited by infusion of nomifensine (100 µ M ). It is concluded that the transient increases in striatal DA efflux seen after intrastriatal injection of SKF-38393 and other benzazepines are not mediated by presynaptic D₁ receptors but by an amphetamine-like action on the dopamine transporter.     

Dopamine release mediated by the dopamine transporter, facts and consequences

Spontaneous and/or stimulated neural activity of the nigrostriatal dopamine (DA) pathway makes amines run out from the neurons. This DA dynamic follows a rather complex path, running in or out the terminals, and flushing or diffusing into the extracellular space. The location of this leakage is not limited to the axon terminals; it also occurs from the cell bodies and dendrites. This molecular release mechanism was, for a long time, considered as being produced, in part, by the exocytosis of previously stored vesicles. The DA carrier protein (DAT, DA transporter) embedded in the DA cell membrane is known to clear previously released amines through an inward DA influx. The DAT also appears to be an active vector of amine release. Particular local conditions and the presence of numerous psychostimulant substances are able to trigger an outward efflux of DA through the DAT. This process, delivering slowly large amounts of amine could play a major regulatory role in extracellular DA homeostasis.     

Development of Polyclonal Anti-D2 Dopamine Receptor Antibodies to Fusion Proteins: Inhibition of D2 Receptor–G Protein Interaction

Abstract: Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera. The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay. Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist [¹²⁵I]NCQ 298 and digitonin-solubilized extracts of canine and rat caudate. [¹²⁵I]-NCQ 298 bound reversibly and with high affinity (K_D= 0.14 n M ) to receptors in solubilized extracts enriched by chromatography on heparin-agarose. The anti-UBI-D2i3_L and anti-UBI-D2i3_s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with [¹²⁵I]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins in digitonin-solubilized rat caudate. Both anti-UBI-D2i3_L and anti-UBI-D2i3_s antisera were able to inhibit the high-affinity binding of the agonist N -propylnorapomorphine to digitonin-solubilized rat caudate. These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.     

Chronic intermittent L-DOPA treatment induces changes in dopamine release

3,4-Dihydroxyphenyl- l -alanine (l- DOPA)-induced dyskinesia often develops as a side effect of chronic l -DOPA therapy. This study was undertaken to investigate dopamine (DA) release upon l -DOPA treatment. Chronoamperometric measurements were performed in unilaterally DA-depleted rats, chronically treated with l -DOPA, resulting in dyskinetic and non-dyskinetic animals. Normal and lesioned l -DOPA naïve animals were used as controls. Potassium-evoked DA releases were significantly reduced in intact sides of animals undertaken chronic l -DOPA treatment, independent on dyskinetic behavior. Acute l -DOPA further attenuated the amplitude of the DA release in the control sides. In DA-depleted striata, no difference was found in potassium-evoked DA releases, and acute l -DOPA did not affect the amplitude. While immunoreactivity to serotonin uptake transporter was higher in lesioned striata of animals displaying dyskinetic behavior, no correlation could be documented between serotonin transporter-positive nerve fiber density and the amplitude of released DA. In conclusions, the amplitude of potassium-evoked DA release is attenuated in intact striatum after chronic intermittent l -DOPA treatment. No change in amplitude was found in DA-denervated sides of either dyskinetic or non-dyskinetic animals, while release kinetics were changed. This indicates the importance of studying DA release dynamics for the understanding of both beneficial and adverse effects of l -DOPA replacement therapy.     

GABAA Receptor Blockade in the Anterior Ventral Tegmental Area Increases Extracellular Levels of Dopamine in the Nucleus Accumbens of Rats

Abstract: Previously, it was shown that microinfusion of the GABA_A antagonist picrotoxin into the anterior ventral tegmental area (VTA) is reinforcing. It was hypothesized that this reinforcing effect of picrotoxin in the anterior VTA is mediated, at least in part, by the activation of the mesoaccumbens dopamine (DA) system. The objective of the present study was to determine if blockade of GABA_A receptors in the anterior VTA can increase extracellular levels of DA in the nucleus accumbens (ACB), using an in vivo microdialysis technique in freely moving rats. Concentrations of picrotoxin (40, 80, and 160 µ M ) that had previously been shown to produce a reinforcing effect increased the extracellular levels of DA and its major metabolites in the ACB. The increased extracellular DA levels induced by intra-VTA injection of picrotoxin was markedly attenuated by coadministration with the GABA_A agonist muscimol, whereas intra-VTA injection of muscimol alone did not have an apparent effect on extracellular DA levels in the ACB. Microinjection of another GABA_A antagonist, bicuculline, into the anterior VTA also increased the extracellular release of DA in the ACB. These results suggest that DA neurons projecting from the anterior VTA to the ACB are tonically inhibited by GABA through its actions at the GABA_A receptors.     

Antagonism of Serotonin 5-HT1A Receptors Potentiates the Increases in Extracellular Monoamines Induced by Duloxetine in Rat Hypothalamus

Abstract: In the current study we examined the effects of coadministration of a serotonin 5-HT_1A antagonist, (±)-1-(1 H -indol-4-yloxy)-3-(cyclohexylamino)-2-propanol maleate (LY 206130), and a dual 5-HT and norepinephrine (NE) uptake inhibitor, duloxetine, on extracellular levels of NE, 5-HT, dopamine (DA), 5-hydroxyindoleacetic acid, and 3,4-dihydroxyphenylacetic acid in rat hypothalamus microdialysates. LY 206130 (3.0 mg/kg, s.c.) alone significantly increased NE and DA levels by 60 and 34%, respectively, without affecting 5-HT levels. Duloxetine administration at 4.0 mg/kg, i.p. alone produced no significant changes in levels of 5-HT, NE, or DA. In contrast, when LY 206130 and duloxetine were coadministered at 3.0 mg/kg, s.c. and 4.0 mg/kg, i.p., respectively, 5-HT, NE, and DA levels increased to 5.7-, 4.8-, and threefold over their respective basal levels. These data demonstrate that antagonism of somatodendritic 5-HT_1A autoreceptors and concomitant inhibition of 5-HT and NE uptake with duloxetine may promote synergistic increases in levels of extracellular 5-HT, NE, and DA in hypothalamus of conscious, freely moving rats.