Novel tropane-based irreversible ligands for the dopamine transporter.

3alpha-(diphenylmethoxy)tropane (benztropine) and its analogues are tropane ring-containing dopamine uptake inhibitors that display binding and behavioral profiles that are distinct from cocaine. We previously prepared a benztropine-based photoaffinity label [125I]-(N-[4-(4'-azido-3'-iodophenyl)butyl]-3alpha-[bis(4'-fluorophenyl)methoxy]tropane, [125I]1, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] 2 (RTI 82). To characterize further these different binding domains, photoaffinity ligands that had the 4'-azido-3'-iodophenyl substituent extended from the same position on the tropane ring were desirable. Thus, identification of the optimal alkyl linker between this substituent and the tropane nitrogen in the benztropine series was investigated to ultimately prepare the identical N-substituted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propyl] analogue of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthesized as well as two isothiocyanate analogues that do not require photoactivation (10a,b) for irreversible binding. The synthesis of these target compounds was achieved using a modification of the strategy developed for 1. Evaluation of these compounds for displacing [3H]WIN 35 428 binding at DAT in rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substituent, found in 1, provided optimal binding affinity and was chosen to replace the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothiocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesized. Both products bound to DAT with comparable potency (IC(50) = 30 nM) to RTI 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [3H]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These ligands will provide important tools for further characterizing the binding domains for tropane-based dopamine uptake inhibitors at the DAT.

Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter.

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.

Kappa-opioid receptor activation modifies dopamine uptake in the nucleus accumbens and opposes the effects of cocaine.

Coadministration of kappa-opioid receptor agonists (kappa-agonists) with cocaine prevents alterations in dialysate dopamine (DA) concentration in the nucleus accumbens (Acb) that occur during abstinence from repeated cocaine treatment. Quantitative microdialysis was used to determine the mechanism producing these effects. Rats were injected with cocaine (20 mg/kg, i.p.), or saline, and the selective kappa-agonist U-69593 (0.32 mg/kg, s.c.), or vehicle, once daily for 5 d. Extracellular DA concentration (DA(ext)) and extraction fraction (E(d)), an indirect measure of DA uptake, were determined 3 d later. Repeated cocaine treatment increased E(d), whereas repeated U-69593 treatment decreased E(d), relative to controls. Coadministration of both drugs yielded intermediate E(d) values not different from controls. In vitro DA uptake assays confirmed that repeated U-69593 treatment produces a dose-related, region-specific decrease in DA uptake and showed that acute U-69593 administration increases DA uptake in a nor-binaltorphimine reversible manner. Repeated U-69593 also led to a decrease in [(125)I]RTI-55 binding to the DA transporter (DAT), but did not decrease total DAT protein. These results demonstrate that kappa-opioid receptor activation modulates DA uptake in the Acb in a manner opposite to that of cocaine: repeated U-69593 administration decreases the basal rate of DA uptake, and acute U-69593 administration transiently increases DA uptake. kappa-agonist treatment also alters DAT function. The action of kappa-agonists on DA uptake or DAT binding, or both, may be the mechanism(s) mediating the previously reported "cocaine-antagonist" effect of kappa-opioid receptor agonists.

Differential effect of structural modification of human dopamine transporter on the inward and outward transport of dopamine.

The effect of structural modification of the human dopamine transporter protein on bi-directional transport was explored using site-directed mutagenesis and rotating disk electrode voltammetry. The substrate-induced DA efflux, as inferred from the K(m) or K(i), was dependent on common structural features for uptake of the substrate inducer: reduced by beta-hydroxylation, stereoselective to alpha-methylation, and relatively insensitive to a switch of a single phenolic hydroxyl group between m- and p-positions. The potencies for substrates to compete with external DA for uptake and to induce DA efflux were similar and highly correlated. Despite these similarities, the efflux of internal DA was substantially slower than the uptake of its inducers. Mutation of serine-528 of the hDAT to alanine (S528A) did not change the structure-activity relationships, maximal uptake rates, and the cation dependence for the uptake of external substrates, although it modestly reduced K(m) or K(i) of most tested substrates. In contrast, it substantially enhanced substrate-induced DA efflux, with maximal efflux rates doubled for all tested inducers. Simultaneous monitoring of tyramine uptake and resulting DA efflux revealed that S528A accelerated the DA efflux relative to tyramine uptake. Saturation analysis suggested that the mutation significantly enhanced the efflux kinetics of internal DA but it exerted little effect on the uptake kinetics of external DA. These findings suggest that Ser-528 may play a role in stabilizing a hDAT conformation unfavorable for outward transport of internal DA, thereby contributing to the efficiency of the transporter.

Transport-dependent accessibility of a cytoplasmic loop cysteine in the human dopamine transporter.

The effect of covalent sulfhydryl modification on dopamine uptake by the human dopamine transporter was determined by rotating disc electrode voltammetry. A transporter construct, X5C, with five mutated cysteines (C90A, C135A, C306A, C319F, and C342A) and the constructs into which the wild-type cysteines were substituted back into X5C, one at a time, all showed nearly normal binding affinity for [(3)H]CFT and for cocaine, but they displayed significant reductions in K(m) and V(max) for DA uptake. Reaction of Cys-90 or Cys-306 with impermeant methanethiosulfonate derivatives enhanced dopamine uptake to a similar extent as the previously observed enhancement of [(3)H]CFT binding caused by the same reaction, suggesting that cocaine may bind preferentially to a conformation in the transport cycle. m-Tyramine increased the rate of reaction of (2-aminoethyl)methanethiosulfonate (MTSEA) with X-A342C, the construct with a cytoplasmic loop residue Cys-342 restored. This m-tyramine-induced increase in reactivity appeared to require the inward transport rather than the outward transport or external binding of m-tyramine, and it was prevented by cocaine. Thus, inward translocation of substrates may involve structural rearrangement of hDAT, which likely exposes Cys-342 to reaction with MTSEA, and Cys-342 may be located on a part of the transporter associated with cytoplasmic gating.

Cocaine-induced changes in extracellular dopamine determined by microdialysis in awake squirrel monkeys.

RATIONALE: The behavioral effects of cocaine have been linked to brain dopamine systems. Extending the findings to neurochemical studies in the squirrel monkey would enhance our understanding of the behavioral pharmacology of cocaine in nonhuman primates. OBJECTIVES: The present studies characterized the effects of cocaine and the selective dopamine uptake inhibitor GBR 12909 on extracellular dopamine in the caudate nucleus of awake squirrel monkeys through microdialysis experiments. METHODS: Guide cannulae were implanted in the caudate nucleus of four monkeys using a stereotaxic apparatus and coordinates obtained from a standard squirrel monkey brain atlas. Accurate probe placement was confirmed in all subjects with magnetic resonance imaging. RESULTS: Collectively, the results support the feasibility of a repeated-measures design. Stability of tissue integrity after repeated probe insertion was supported by measurement of consistent basal levels of dopamine and its metabolites across several experiments, observation of potassium-induced dopamine release and absence of significant glial proliferation as assessed by GFAP (glial fibrillary acidic protein) immunochemistry. Moreover, peak drug effects and time-course of action were similar when multiple probes were positioned in the same anatomical site over several experiments. Cocaine (1.0 mg/kg i.m.) and GBR 12909 (3.0 mg/kg i.m.) elevated extracellular dopamine to approximately 300% of basal levels, but GBR 12909 produced a slower, more sustained elevation than cocaine. CONCLUSIONS: The results validate the use of microdialysis in awake primates using repeated sampling of the same anatomical site and demonstrate orderly changes in extracellular dopamine following administration of dopamine uptake inhibitors.

Dopamine D1/D2 agonists injected into nucleus accumbens and ventral pallidum differentially affect locomotor activity depending on site.

Ventral pallidal dopamine has been recently shown to play an important role in psychostimulant reward and locomotor activation. The aim of the present study was to compare the roles of ventral pallidal D1 and D2 receptors in evoking locomotor activity with those in the nucleus accumbens. The D1 agonist SKF 38393 and the D2 agonist quinpirole hydrochloride (0.3-3 microg/ 0.5 microl) were bilaterally injected into ventral pallidum or nucleus accumbens through pre-implanted cannulae. In the ventral pallidum, 0.3-1 microg SKF 38393 increased locomotor activity while 3 microg had no effect; 3 microg quinpirole suppressed locomotion while 0.3-1 microg had no effect. Locomotor activity induced by an equigram (0.3 microg) mixture of SKF 38393 and quinpirole, while significantly higher than that induced by 0.3 microg quinpirole was not significantly higher than that induced by 0.3 microg SKF 38393 alone. At the 3 microg dose, SKF 38393 injections into anterior ventral pallidum increased activity; injections into posterior ventral pallidum decreased activity. In the nucleus accumbens, 0.3-3 microg SKF 38393 dramatically increased locomotor activity while quinpirole moderately increased locomotion. In the group that had previously received the full quinpirole dose range, injection of the equigram (0.3 microg) mixture of SKF 38393 and quinpirole induced locomotor activation which was higher than that induced by either drug alone or by the addition of the effect of each drug alone, i.e. synergy occurred. Moreover, rats that had previously received SKF 38393 developed a sensitized locomotor response to subsequent SKF 38393, quinpirole or the mixture of these two drugs. The difference in locomotor response to dopamine agonists between the ventral pallidum and nucleus accumbens is consistent with electrophysiological evidence collected at these two sites. These findings suggest that, unlike the nucleus accumbens, where D1 and D2 receptor activation may facilitate each other to induce a synergistic effect on locomotor activity, ventral pallidal D1 and D2 receptors may be located on different neurons and coupled with different, if not opposite, behavioral output.

Cationic modulation of human dopamine transporter: dopamine uptake and inhibition of uptake.

Effects of cations on dopamine (DA) uptake into cells expressing the human dopamine transporter and on inhibition of DA uptake by various substrates and inhibitors were investigated by using rotating disk electrode voltammetry. The Na(+) dependence of DA uptake varied with Na(+) substitutes, hyperbolic with Li(+), almost linear at 1 microM DA but hyperbolic at 8 microM DA with choline, and sigmoidal with K(+). With Na(+) substituted by Li(+), K([DA]) decreased and V(app) remained constant with increasing [Na(+)], whereas K([Na+]) decreased and V(app) increased with increasing [DA], suggesting an ordered sequence with Na(+) binding before DA. Similar trends for the Na(+)-DA interactions were observed in the presence of cocaine. Cocaine inhibited DA uptake solely by increasing K([DA]), with its K(i) not significantly different at 55 and 155 mM [Na(+)], whereas it inhibited Na(+) stimulation by reducing V(app) more than K([Na+]) at 1 microM DA, and V(app) only and less potently at 8 microM DA. Thus, cocaine may compete with DA, not with Na(+), for the transporter, and might not follow a strictly ordered reaction with Na(+). With Na(+) substituted by K(+), K([DA]) or K([Na+]) became insensitive to Na(+) or DA. K(+) impaired the DA uptake mainly by reducing V(app,) but affected cocaine inhibition by elevating K(i). Despite their different patterns for inhibiting DA uptake, nontransportable inhibitors cocaine, methylphenidate, mazindol, and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenyl-2-propyl)piperazi ne (GBR12909) showed similarly modest Na(+) dependence in their K(i) values. In contrast, substrates DA, m-tyramine, and amphetamine displayed a similarly stronger Na(+) requirement for their apparent affinities.

Effects of serine mutations in transmembrane domain 7 of the human norepinephrine transporter on substrate binding and transport.

Two serine residues in the beta-adrenergic receptor (beta-AR) have been proposed to form hydrogen bonds with the catechol moiety of the ligand and contribute to the activation of the receptor. These conserved serine residues in the dopamine (DA) and norepinephrine transporters (DAT and NET, respectively) have also been shown to affect substrate transport in the rat DAT. In the present work, hydrogen bonding interactions between the corresponding serine residues in the human NET (hNET), 354 and 357, and the hydroxyl groups on the substrate were systematically evaluated by examining the transport and binding properties of DA and several single hydroxyl analogues of DA at wild-type and serine-to-alanine-substituted transporters. A comparison of [3H]nisoxetine binding at the serine 354 mutant, in which K(D) increased 70-fold from the wild-type value, with the binding of DA, m-tyramine (m-TYR), and p-tyramine (p-TYR) at mutant 354, where the increase in Ki was less dramatic, revealed that serine 354 is more influential in inhibitor than substrate binding. The binding of m-TYR and p-TYR at the serine 354 and serine 357 mutants did not show a direct interaction between one serine and one substrate catechol hydroxyl group. DA, m-TYR, and p-TYR binding affinity did not deviate from the wild-type value at the serine 357 and double mutant transporters. At these two transporters, however, the Km of DA uptake increased, suggesting that the roles of serine 357 and serine 354 in substrate transport are different from their roles in binding. The K'm for induced efflux of DA decreased at the serine 357 mutant compared with the wild-type, whereas the K'm at the serine 354 mutant was the same as that of the wild-type. Further investigation of the role of substrate hydroxyls in the transport process revealed no difference between the transport of m-TYR or p-TYR, as measured indirectly through their induced efflux of DA, at any of the mutants. Although these serines are influential in inhibitor and substrate binding to the transporter and substrate uptake and efflux, they do not appear to be involved in a direct hydrogen bond interaction with substrate, suggesting that the pattern of distinct hydrogen bonding interactions at the beta-AR does not exist at the hNET.

Cocaine acts as an apparent competitive inhibitor at the outward-facing conformation of the human norepinephrine transporter: kinetic analysis of inward and outward transport.

The inhibition by cocaine of inward and outward transport of dopamine (DA) at the cloned human norepinephrine transporter (hNET) and the relationship of the inhibitory patterns of cocaine to the conformational requirements of the transporter were investigated. This was done using rotating disk electrode voltammetry in transfected cells. The uphill uptake of external DA, the lack of inhibition by internal substrates on DA uptake, and the accelerated exchange of internal DA by external m-tyramine support a carrier model in which the hNET alternates between outward-facing and inward-facing conformations. Cocaine exhibited competitive inhibition of DA uptake, which was insensitive to intracellular substrates. In contrast, the inhibition by cocaine of the m-tyramine-induced DA efflux appeared noncompetitive relative to intracellular DA, but competitive relative to extracellular m-tyramine. Simultaneous measurement of m-tyramine uptake and accompanying DA efflux at various concentrations of intracellular DA showed that cocaine did not alter the ratio of DA efflux to m-tyramine uptake. Moreover, cocaine displayed similar potency for inhibiting DA uptake and efflux. Additionally, the inhibition profile of cocaine was unrelated to the addition time of cocaine, simultaneously with or earlier than a substrate. All of the findings are consonant with a competitive interaction between cocaine and substrates at the outward-facing conformation of the hNET. This action directly prevents the inward transport of external substrates, thereby inhibiting the outward transport of internal substrates by reducing the availability of the inward-facing conformation. Consequently, the experimental inhibition pattern of cocaine depends on the conformation of the hNET to which the transported substrate is exposed.

Application of cloud-point extraction-reversed-phase high-performance liquid chromatography. A preliminary study of the extraction and quantification of vitamins A and E in human serum and whole blood.

Methods available for quantification of vitamins A and E in serum or blood requires preconcentration and clean-up by liquid-liquid extraction, evaporation of the extract, and reconstitution of the extract in a solvent of choice before analysis. This process not only involves the use of toxic organic solvents but also requires a long sample preparation time. The lipids and other non-polar coextractants often require additional steps for sample clean-up and evaporation, which may cause sample losses. The use of cloud-point extraction eliminates most of these sample clean-up problems. We recently demonstrated that cloud-point extraction (CPE) can be used for extraction and quantification of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) from human serum. We now demonstrate how CPE can be used with human serum and blood, at volumes as low as 50 microl, and report a methodology for extracting and quantifying two clinically important vitamins, (A and E) from human serum and blood. Vitamins A and E were extracted from human serum and blood by using Genapol X-80 as the cloud-point extractant under salting out conditions. Serum and blood samples were diluted in organic-free water to get sufficiently large sample volumes for CPE. The surfactant-rich phases were separated by centrifugation, and the samples were analyzed by HPLC-UV after deleterious coextractants were removed by precipitating them with acetonitrile. The recoveries of spiked vitamins A and E were found to be 85.6+/-0.4% and 82.6+/-5.2%, respectively. The average concentration of vitamins A and E in a serum pool after correction for recoveries were found to be 43.4+/-1.8 microg/dl (1.5+/-0.1 micromol/l) and 564.3+/-65.3 microg/dl (13.1+/-1.5 micromol/l), respectively. Vitamin A and E concentrations in whole blood were found to be 26.3+/-0.4 microg/dl (0.92+/-0.01 micromol/l) and 457.5+/-15.6 microg/dl (10.6+/-0.4 micromol/l), respectively. These values are comparable with those obtained by the reference method used at the Centers for Disease Control and Prevention. The success of the preliminary study will lead to a comprehensive validation of this method for vitamins A and E in serum and blood.

Voltammetric studies on mechanisms of dopamine efflux in the presence of substrates and cocaine from cells expressing human norepinephrine transporter.

The effects of substrates m-tyramine and beta-phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration-dependent manner. Their EC50 values for inducing DA efflux were similar to their IC50 values for inhibiting DA uptake. The substrate-induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na+] with Na+,K+-ATPase inhibition, but was insensitive to membrane potential-altering agents valinomycin, veratridine, and high [K+]. The initial rate of m-tyramine-induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis-Menten expression. In contrast, DA efflux in the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na+], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time course of the DA efflux showed that the apparent first-order rate constant for m-tyramine-induced DA efflux declined with increased preloaded [DA], whereas that for the DA efflux in the presence of cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET-dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine "uncovers" NET-independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.

GABAergic modulation of ventral pallidal dopamine release studied by in vivo microdialysis in the freely moving rat.

The mesopallidal dopamine system, which originates from the ventral tegmental area and projects to the ventral pallidum (VP), has been recently shown to play an important role in self-stimulation reward and cocaine reward. VP also receives a GABAergic projection from nucleus accumbens (NAS). The aim of the present study was to examine the involvement of this GABAergic projection in the modulation of VP dopamine release. Both the GABAA antagonist picrotoxin (2-200 microM) and the GABAB antagonist phaclofen (20-2,000 microM), perfused locally, dose-responsively increased VP extracellular dopamine 2-2.5-fold. Cocaine (10 microM) produced a 6.5-fold increase of VP dopamine. Neither picrotoxin (200 microM), phaclofen (2,000 microM), nor GABA (20-2,000 microM) altered the response of VP dopamine to locally applied cocaine. GBR 12909 (0.5 microM), a selective dopamine uptake blocker, induced a 3.5-fold increase of VP dopamine. The increase of VP dopamine in response to GBR 12909 was further augmented to 8.5-fold of baseline when picrotoxin (200 microM) was added to the perfusate. The data from the present study demonstrate that the GABAergic NAS-VP projection can modulate ventral pallidal dopamine release. However, the effect of GABA on the mesopallidal dopamine system's response to locally applied cocaine may be complicated by actions of cocaine other than dopamine uptake inhibition.

Dissociation of morphine-induced potentiation of turning and striatal dopamine release by amphetamine in the nigrally-lesioned rat.

Morphine has been reported to increase extracellular levels of dopamine in the brain of intact rats and to potentiate turning induced by amphetamine in nigrally-lesioned rats. The present study tested the hypothesis that there is a causal relationship between these two effects of morphine. We tested morphine alone, amphetamine alone, and the combination in separate groups of nigrally-lesioned rats for effects on turning and, by microdialysis, on extracellular dopamine levels. Morphine (3.0 or 10 mg/kg) did not produce significant turning but amphetamine (1.0 mg/kg) did. The lower dose, but not the higher dose, of morphine potentiated amphetamine-induced turning. Amphetamine, but not morphine, produced increases in extracellular dopamine levels. In contrast to what occurred with turning, 10 mg/kg but not 3.0 mg/kg morphine potentiated amphetamine-induced increases in extracellular dopamine levels. These results show that the potentiation of amphetamine-induced turning by morphine in nigrally-lesioned rats is not due to the potentiation of dopamine release in the intact striatum.

Naloxone does not alter amphetamine-induced rotational behavior or striatal dopamine levels of nigrally-lesioned rats.

Previous studies on rats have shown that the opioid antagonist naloxone attenuates amphetamine-induced stimulation of locomotor activity and increases in extracellular dopamine in the brain. However, in this study, naloxone did not attenuate amphetamine-induced rotational behavior or increases of extracellular dopamine in the intact striatum of nigrally-lesioned rats. These results suggests differences in the way in which endogenous opioids contribute to the behavioral and neurochemical effects of amphetamine in nigrally-lesioned compared to intact rats.

Dissociation of locomotor and conditioned place preference responses following manipulation of GABA-A and AMPA receptors in ventral pallidum.

1. This study examined the roles of GABAergic and glutamatergic neurotransmission in ventral pallidum (VP) in conditioned place preference and locomotor activity. 2. Picrotoxin (0.1 microgram), a GABA antagonist, and (+/-)alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 0.14 microgram), a non-NMDA glutamatergic agonist, were injected bilaterally into VP through implanted cannulae. 3. Both drugs produced a robust increase in locomotion, but neither produced conditioned place preference. 4. These results suggest a dissociation of locomotor activity and reward at the level of ventral pallidum. In addition, it was argued that the GABAergic projection from nucleus accumbens to ventral pallidum may not be involved in the processing of reward initiated from dopaminergic activation in nucleus accumbens.

6-Hydroxydopamine lesion of ventral pallidum blocks acquisition of place preference conditioning to cocaine.

In parallel with nucleus accumbens (NAS), ventral pallidum (VP) also receives a dopaminergic projection from the ventral tegmental area (VTA). The present study examined the involvement of this mesopallidal dopaminergic system in the action of cocaine. In the first experiment, the effect of cocaine injections on VP dopamine was examined by microdialysis. Intraperitoneal (i.p.) injections of cocaine 5-20 mg/kg dose-dependently increased the extracellular dopamine level in VP 2.5-4.5-fold. In addition, intra-VP perfusion of 20 microM cocaine induced a 12-fold increase of dopamine locally. The second experiment examined the role of VP dopamine in cocaine-induced conditioned place preference (CPP) and locomotor activation. Rats received bilateral intra-VP injections of 3-4 microg 6-OHDA or ascorbic acid vehicle in 0.5 microl volume. Tissue assays indicated that the 6-OHDA-lesioned rats had significantly lowered dopamine concentration in VP, but not in NAS or striatum. As a group, 6-OHDA lesions blocked the development of CPP to 5 mg/kg cocaine but not to 10 mg/kg cocaine. However, rats with more than 60% depletion in VP dopamine did not develop CPP to cocaine at either dose. Preference for the cocaine-paired side correlated significantly with dopamine concentration in VP, but not in NAS or striatum. It was concluded that VP dopamine may play a critical role in the initial rewarding effect of cocaine. 6-OHDA lesions also blocked locomotor activation induced by 5 mg/kg cocaine but had no effect on 10 mg/kg cocaine-induced locomotion. Dopamine concentration in VP did not correlate with the locomotor activation response to cocaine at either dose. These findings further establish the involvement of the mesopallidal dopaminergic system in the action of cocaine.

Effect of neostigmine on concentration and extraction fraction of acetylcholine using quantitative microdialysis.

A quantitative microdialysis method was used to determine the effect of local perfusion of 0, 100, 200, and 300 nM neostigmine (NEO) on acetylcholine (ACh) extracellular concentration and microdialysis extraction fraction (E(d)) in the striatum of the rat. Because of the efficiency of AChE, the inhibition of this enzyme is expected to result in a substantial increase in ACh levels and a decrease in the E(d) of ACh. The extracellular concentration of ACh increased linearly with increasing concentrations of NEO. The control ACh concentration was determined to be 18.4 +/- 11.8 nM (n = 10; mean +/- S.E.M.) The ACh extracellular concentration for the remaining groups was determined to be 173 +/- 14 nM (n = 5), 329 +/- 52.5 nM (n = 13), and 581 +/- 109 nM (n = 10) for the 100, 200, and 300 nM NEO groups, respectively. Perfusion with 300 nM NEO resulted in a significant reduction in the E(d) of ACh (64.5 +/- 3.5% vs. 43.6 +/- 7.5%, P < 0.05). In contrast to ACh, perfusion with 0, 1, and 10 microM hemicholinium-3, an inhibitor of high-affinity choline uptake, increased choline levels but did not affect the E(d) of choline. The effects on E(d) are consistent with E(d) being influenced by rapid clearance mechanisms.