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Two novel N-substituted-3beta-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically, (-)-N-[4-(3-[(125)I]iodo-4-azidophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ-2-24) and (-)-N-[4-(3-[(125)I]iodo-4-isothiocyanophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ 3-37) were synthesized. Isolation by reversed-phase HPLC and solid-phase extraction gave good average yields of [(125)I]MFZ-2-24 (67%, n = 5) and [(125)I]MFZ-3-37 (45%, n = 3) with high radiochemical purities (96-99%) and specific radioactivities (2000 mCi/micromol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT.

Photoaffinity labeling is a positive function approach that has been used in an effort to identify the cocaine-binding site on the dopamine transporter (DAT). Radioactive and non-radioactive analogs of cocaine and other dopamine uptake blockers are used to irreversibly label the DAT ligand-binding site and the protein is subjected to chemical or enzymatic treatments that cleave at specific amino acid residues. Analysis of cleavage products from radioactively photolabeled DAT using epitope-specific immunoprecipitation, gel electrophoresis, and autoradiography has identified the site of origin in the primary sequence of labeled fragments as small as 4 kDa. More precise localization of the site of labeling is done by subjecting photolabeled DAT to parallel or serial digestion with multiple cleavage methods, followed by analysis of radiolabeled peptides by reverse-phase HPLC. Fragment retention times are compared to calculated retention times of predicted digest peptides and to chemically or photochemically labeled synthetic peptides. The presence of authentic DAT sequence in HPLC fractions of digests from DAT labeled with non-radioactive ligands is further supported by MALDI and nanoelectrospray mass spectrometry. Using these methods we have identified two distinct regions of DAT that interact with multiple structurally related and diverse irreversible ligands, suggesting that these regions may be involved in the formation of ligand binding sites.

Potential irreversible ligands were prepared, based on a series of 3-(1-piperazinyl)propyl-N,N-bis(4-fluorophenyl)amines, as molecular probes for the dopamine transporter (DAT). Both azido- and isothiocyanato-substituted phenylalkyl analogues were synthesized and evaluated for displacement of [(3)H]WIN 35 428 in rat caudate putamen tissue. All of the analogues showed moderate binding potencies at the DAT. The azido analogue, 16b, was radioiodinated and used to photolabel human DAT-transfected HEK 293 cell membranes. [(125)I]16b irreversibly labeled an approximately 80 kDa band corresponding to the DAT detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This radioligand provides a novel addition to the growing arsenal of structurally diverse irreversible ligands that are being used to identify binding domains on the DAT. Characterizing points of attachment of these irreversible probes to the DAT protein will ultimately help elucidate the three-dimensional arrangement of the transmembrane domains, identify individual binding sites of the DAT inhibitors, and direct future drug design.

Although microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady-state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no-net-flux) microdialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast-scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no-net-flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post-implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no-net-flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.

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.

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.

Coadministration of κ-opioid receptor agonists (κ-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 κ-agonist U-69593 (0.32 mg/kg, s.c.), or vehicle, once daily for 5 d. Extracellular DA concentration (DAext) and extraction fraction (Ed), an indirect measure of DA uptake, were determined 3 d later. Repeated cocaine treatment increasedEd, whereas repeated U-69593 treatment decreasedEd, relative to controls. Coadministration of both drugs yielded intermediateEdvalues not different from controls.In vitroDA 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 [125I]RTI-55 binding to the DA transporter (DAT), but did not decrease total DAT protein. These results demonstrate that κ-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. κ-agonist treatment also alters DAT function. The action of κ-agonists on DA uptake or DAT binding, or both, may be the mechanism(s) mediating the previously reported “cocaine-antagonist” effect of κ-opioid receptor agonists.

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.

Rotating disk electrode voltammetry has shown to be a simple, fast, and inexpensive method for studying substrate kinetics at wild-type and mutant catecholamine transporters. The transport of the substrates dopamine (DA) and meta- and para-tyramine (m-TYR, p-TYR) are examined at the human norepinephrine transporter (hNET) expressed stably in LLC cells. Following uptake of DA, monitored at 450 mV (vs. Ag/AgCl), efflux is induced by the uptake of a second substrate. The timecourse of induced efflux of DA is found to be dependent on the structure of the compound used to initiate efflux as well as the concentration of both the preloaded substrate and efflux inducing agent. A double electrode configuration for the electrochemical cell, including one rotating and one stationary working electrode, is described. A potential of 650 mV is applied across the stationary electrode to obtain the timecourse of single-hydroxyl substrates after correction for any catechol substrate oxidation. This dual electrode set-up is useful in that it allows the kinetics of two substrates interacting at the transporter to be monitored simultaneously. The kinetics of the above mentioned substrates are examined at wild-type and mutant hNETs using this two electrode configuration

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.

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 μM) and the GABAB antagonist phaclofen (20–2,000 μM), perfused locally, dose-responsively increased VP extracellular dopamine 2–2.5-fold. Cocaine (10 μM) produced a 6.5-fold increase of VP dopamine. Neither picrotoxin (200 μM), phaclofen (2,000 μM), nor GABA (20–2,000 μM) altered the response of VP dopamine to locally applied cocaine. GBR 12909 (0.5 μM), 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 μM) 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. Synapse 29:406–412, 1998. © 1998 Wiley-Liss, Inc.

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.

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.

1. 1. This study examined the roles of GABAergic and glutamatergic neurotransmission in ventral pallidum (VP) in conditioned place preference and locomotor activity. 2. 2. Picrotoxin (0.1 μg), a GABA antagonist, and (±)α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 0.14μg), a non-NMDA glutamatergic agonist, were injected bilaterally into VP through implanted cannulae. 3. 3. Both drugs produced a robust increase in locomotion, but neither produced conditioned place preference. 4. 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.

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.

Rotating disk electrode (RDE) voltammetry was used to examine the effect of N -methyl- d -aspartate (NMDA) on the initial velocity of dopamine (DA) uptake in suspensions of rat striatal tissue. Transport velocities were determined by measuring the rate of clearance of 2 μM DA added to the tissue suspension. NMDA (200 μM) was found to increase the initial velocity of DA transport from 548 ± 28 to 803 ± 63 pmol/s per g tissue (mean ± SEM; P r = 0.96; P P

The present study was designed to investigate the potential influence of various neuronal processes including uptake, release and metabolism, on the in vivo microdialysis extraction fraction (Ed) of serotonin (5HT) and norepinephrine (NE). Paroxetine administration decreased the Ed of 5HT in the nucleus accumbens from 24 +/- 3 to 18 +/- 0.2% (p < 0.05). Similarly, desipramine infusion reduced the NE Ed from 35 +/- 2 to 26 +/- 1% (p < 0.05). However, perfusion with pargyline or tetrodotoxin had no effect on the Ed of either 5HT or NE. Perfusion with agonists for the 5HT, alpha-adrenergic, D2 and histamine receptors had no effect on the Ed of 5HT. In the same manner, perfusion with the alpha-adrenergic agonists, methoxamine or clonidine, did not affect the Ed of NE. These data are in agreement with experimental results obtained for dopamine in the nucleus accumbens and the theory of quantitative microdialysis which predicts that only changes in the rate of clearance will change Ed of monoamines. These results suggest that, like DA, changes in the Ed for 5HT or NE are indicative of changes in the reuptake of these neurotransmitters. The results also indicate that pharmacological agents which do not affect uptake have no effect on the extraction fraction.

Previous studies have demonstrated that rats showing a strong locomotor response to a novel environment have a greater locomotor response to psycho-stimulant drugs and more rapidly acquire intravenous self-administration of amphetamine. In this report, we examined whether these high-responder (HR) rats would develop place-preference conditioning with cocaine more readily than low-responder (LR) rats. Neither group of rats developed conditioned place preference for cocaine, 2.5 mg/kg, intraperitoneally (IP). Both groups of rats developed conditioned place preference for cocaine, 5.0 and 15 mg/kg, IP. However, we could not find any evidence of enhanced conditioning in the HR rats. HR rats did show a greater locomotor response to cocaine, 15 mg/kg, IP, and the locomotor response of HR and LR rats to cocaine correlated with their response to a novel environment. We conclude that using the place-preference procedure, HR and LR rats do not differ in the rewarding effect of cocaine.

The feasibility of using the difference method of quantitative microdialysis to measure neuropeptide Y (NPY) was evaluated in vitro and in vivo. The accuracy of this method was tested in vitro under steady-state conditions for 3 test solutions containing known concentrations of NPY. The estimated concentrations of NPY were 1.2 +/- 0.6, 3.7 +/- 0.9, and 15.1 +/- 0.7 pg/microliter (mean +/- SEM) in agreement with the actual concentrations of NPY in the test solutions which were 1.1 +/- 0.8, 4.6 +/- 0.6, and 14.6 +/- 0.5 pg/microliter (mean +/- SEM of solution samples), respectively. The responsiveness of the estimated NPYext measure to changes in the external concentration of NPY was also evaluated in vitro. An accurate estimate of NPYext was obtained within the first sampling period (within 15 min) after a 2-3-fold increase in the test solution concentration of NPY and within 2-3 sampling periods (15-45 min) in response to a 2-3-fold decrease in the test solution concentration of NPY. In vivo, the estimated basal concentration of NPY in dialysis samples from probes in the medial basal hypothalamus of anesthetized female rats (n = 4) was 4.0 +/- 1.6 pg/microliters and increased to 9.5 +/- 0.3 pg/microliter during K+ stimulation. Relative recovery was 22% in vivo under steady-state conditions and ranged from 14% to 30% during dynamic conditions. These results demonstrate that the difference method of quantitative microdialysis accurately estimates picomolar concentrations of NPY in vitro, and is sufficiently sensitive to detect basal and increasing concentrations of NPY in vivo.

Rats bearing electrolytic lesions of medial septum and sham-operated controls were trained on cocaine place-preference in a 3-compartment apparatus. Cocaine was paired with a white or a black compartment. An unbiased design was used, in which cocaine was paired with the preferred side in half the animals and with the unpreferred side in the other half. Two low doses of cocaine HCl were used: 2.5 and 5.0 mg/kg. Only two pairings of drug with environment were used to minimize the influence of drug sensitization. Rats with septal lesions, but not controls, showed preference conditioning to the black side at 2.5 mg/kg; lesioned and control animals showed similar conditioning to the black side at 5.0 mg/kg. Lesioned animals could not be conditioned to the white side at either dose. This was attributed to a drug-induced enhancement of a previously described increased reactivity to brightness following septal lesions. Controls conditioned to either side at 5.0 mg/kg. It was concluded that septal lesions lowered the cocaine dose required for preference conditioning, consistent with reports that such damage enhances some behavioral effects of psychostimulants.

Subjects that respond more to a novel environment show a greater locomotor response to drugs of abuse such as cocaine and amphetamine. The current study was performed to examine differences between high (HR) and low (LR) responding rats to a novel environment following administration of amphetamine, a selective dopamine uptake blocker (GBR-12909), a nonselective dopamine agonist (apomorphine), and selective dopamine D1 and D2/D3 agonists. A behavioral checklist and a rating scale were used to determine the behavioral arousal caused by administration of amphetamine (0, 0.5, 2.0, and 8.0 mg/kg), GBR-12909 (0, 1.25, 5.0, and 20.0 mg/kg), apomorphine (0, 0.1, 0.3, and 1 mg/kg), SKF 38393 (0, 2.5, 10, and 40 mg/kg), or quinpirole (0, 0.05, 0.5, and 5.0 mg/kg). The five drugs produced behavioral activation profiles distinct from each other. Following amphetamine administration, both HR and LR subjects showed dose dependent increases in behavioral arousal. The behaviors primarily affected were sniffing, locomotor activity, rearing, and oral activity. HR rats showed a greater overall behavioral response to amphetamine administration compared with LR rats and there were differences in specific behaviors between the two groups. Following GBR-12909 administration, all subjects showed dose dependent increases in sniffing, locomotor activity, and rearing. Differences between HR and LR were observed in sniffing, locomotor activity, and rearing behaviors. HR and LR both showed dose dependent increases in behavior following apomorphine administration. HR showed greater behavioral activation after apomorphine than LR. SKF 38393 produced pronounced increases in the amount of sniffing, grooming, and intense grooming, in addition to increasing the overall behavioral rating of all subjects, while quinpirole produced increases in sniffing, locomotor activity, and oral movements. However, the behavioral effects of SKF 38393 and quinpirole did not differ between HR and LR. These results suggest that activation of the dopamine system but probably not only one type of dopamine receptor is sufficient to produce behavioral differences between high and low responding subjects.

The effects of cocaine HCl infusions into either the nucleus accumbens (NACC) or medial prefrontal cortex (PFC) were compared on the performance of schedule-induced polydipsia (SIP) and related behaviours. Food-deprived rats were exposed to a fixed-time 60-s schedule of food delivery in daily 30-min sessions until stable levels of behaviour were obtained (14 days). Rats were then bilaterally infused with cocaine into either the NACC or PFC via chronically indwelling guide cannulae. Each subject received a sequence of five cocaine infusions (0, 12.5, 25, 50, 100 µg) according to a Latin Square design. For comparison, following these intracranial infusions each rat received a sequence of five IP injections of cocaine (0, 2.5, 5, 10, 20 mg/kg) also in a counterbalanced order. NACC and PFC infusions of cocaine and IP cocaine dose-dependently reduced SIP. Cocaine infusions into the NACC, but not the PFC, increased locomotor activity but the characteristic temporal profile of locomotor activity during SIP was retained. IP cocaine also increased locomotor activity in a dose-dependent manner, but the temporal profile of activity was flattened following 20 mg/kg cocaine. NACC and PFC infusions of cocaine had little effect on the total number of panel presses to gain access to the food pellets, but did slightly decrease the high rates of responding immediately prior to the pellet delivery. IP cocaine increased the total number of panel presses at the higher doses, mainly by increasing the low rates of responding. The effects of cocaine infusions into the PFC were behaviourally the most selective, as they reduced SIP without having substantial effects either on locomotor activity or panel pressing. These data therefore implicate a role for the PFC in the performance of SIP.

The temporal and concentration response of microdialysis probes in vivo was characterized. The temporal response was monitored using step changes in dopamine (DA) concentration which were followed with one minute sampling using smallbore HPLC. The response under three conditions was examined: in vitro sampling of DA, in vitro infusion of DA, and local infusion of DA. The dialysate DA concentration stabilized within two minutes after each change. These results suggest that relatively fast changes in DA levels in tissue can be followed by microdialysis probes. The rapid stabilization also suggests that the DA concentration profile does not extend very far from the probe. To characterize the concentration response, the linearity of the gain or loss of DA diffusing between the probe and tissue was determined. The difference in the inflow and outflow concentrations was plotted as a function of the inflow concentration. The correlation coefficient of the linear regression curve was 0.99. The zero point intercept, the extracellular concentration of DA, was 3.8 nM and the slope, or recovery, was 63.4%. There was no break at the zero point which is indicative of symmetrical diffusion into and out of the probe.

The effects of repeated systemic or intra-nucleus accumbens cocaine administration on locomotor activity were examined for environmental dependence. Repeated IP administration of cocaine (15 mg/kg) for 5 days in the context of a given environment increased the locomotor response to a subsequent IP cocaine challenge in that environment. However, there were no differences in the locomotor response to a subsequent IP cocaine challenge in the test chamber in subjects which had received prior repeated IP administration of cocaine in the home-cage. In a second experiment, cocaine (100 micrograms/side) was infused into the nucleus accumbens (NACC) daily for 5 days. This repeated administration produced increases in locomotor activity to subsequent intra-NACC cocaine infusions that were environmentally independent. In contrast to the effects of repeated IP cocaine administration, subjects which received administration of vehicle, acute cocaine, or repeated cocaine in the NACC did not differ following an IP cocaine challenge. The results from these experiments indicate that increases in the response to IP cocaine following repeated IP administration are in part environmentally dependent. Moreover, repeated intra-NACC cocaine infusions increase the responsiveness of the NACC to subsequent intra-NACC cocaine. However, local activation of the NACC alone does not appear to be adequate to produce sensitization to systemically administered cocaine.

The current experiment examined the role of nucleus accumbens (NACC) dopamine in individual differences. Subjects were divided into high responders (HR) and low responders (LR) based on their locomotor response to a novel environment. HR rats were subjects which had a locomotor response to novelty in the upper third of the population sscreened and LR rats in the bottom third of the population. A new method of microdialysis was then used that allowed determination of the extracellular dopamine concentration. This was accomplished by adding various dopamine concentrations (0.0.5.0 and 20.0 nM) to the perfusate. The concentration of dopamine in the dialysate was subsequently determined. The difference in the dialysate and perfusate dopamine was regressed on the perfusate dopamine. The regression yielded the in vivo recovery and the extracellular concentration. HR rats exhibit a 250% higher basal dopamine concentration (6.45 ± 1.01 nM, n = 6) than LR rats (2.58 ± 0.16 nM, n = 7). The in vivo microdialysis recovery was used to estimate the extracellular dopamine followin cocaine challenge (15mg/kg) in the two groups. Following i.p. cocaine administration. HR rats had both a greater locomotor response and increase in absolute dopamine concentration compared to LR rats. The maximum dopamine concentration in the HR group was 2.3 ± 2.9 nM, while that in the LR group was only 8.6 ± 1.1 nM. The maximum on the LR group is comparable to the basal level in the HR group. However, there were no difference in percent change in dopamine following cocaine. These result provide further evidence in support of a role for variation in NACC dopamine in individual differences in vulnerability to psychomotor stimulants.

This study tested the possibility that naloxone (NX), an opioid antagonist, reduces the behavioral effects of amphetamine (AMPH) in rats by attenuating the dopaminergic response to AMPH. In the first experiment, adult, male rats were injected SC with either NX (5.0 mg/kg) or saline and 30 min later received doses of AMPH (0.0, 0.1, 0.4, 1.6, and 6.4 mg/kg) cumulatively at 30-min intervals. Gross locomotor counts following AMPH administration were significantly lower for rats pretreated with NX than for rats pretreated with saline. In the secon experiment, the same drug treatments were given while performing microdialysis in either the striatum (STR) or nucleus accumbens (NACC). STR rats treated with vehicle showed a larger percentage increase in DA levels following AMPH treatment than did NACC rats treated with vehicle. NX pretreatment did not affect dopamine concentrations in either brain region. However, compared to pretreatment with saline pretreatment with NX significantly decreased the dopaminergic response to AMPH in the STR. There was no difference between the two groups in the peak dopaminergic response to AMPH in the NACC, but there was a significant AMPH × treatment × time interaction due to differences between the roups during the later portion of the response to mg/kg AMPH. There was also a difference in locomotor activity following AMPH treatment between NX-and saline-treated subjects during dialysis. These findings suggest that a decrease in the dopaminergic response to AMPH is the mechanism by which NX attenuates behavioral stimulant effects of AMPH. In addition, there is a difference between the STR and NACC in dopaminergic responsiveness to AMPH.

The effect of mazindol on the dopamine (DA) uptake system in the rat striatum was studied by in vivo voltammetry and microdialysis. An increase in the maximal uptake rate was observed by voltammetry 30 min after i.p. administration of 1, 5, 10, 15, and 20 mg/kg doses but not after a dose of 25 mg/kg, while stimulated release was enhanced at all doses. The increased maximal uptake is in contrast to increased levels of extracellular DA observed with microdialysis following mazindol administration. This divergence of action is anomalous in the context of current understanding of the DA uptake system.

Rats were screened for locomotor activity in a novel environment and divided into high (HR) or low (LR) responders based on whether their locomotor score for the first hour was above or below the median. In the first experiment, HR and LR rats were compared for their locomotor response following repeated administration of either 0.0, 0.5, 1.0, or 1.5 mg/kg d-amphetamine sulfate (AMPH). Injections of either 0.5 or 1.0 mg/kg AMPH produced higher locomotor activity in HR rats than in LR rats. Furthermore, there was a correlation between the locomotor response to novelty and the response to either 0.5 or 1.0 mg/kg AMPH. In addition, whereas both groups of rats developed the same degree of sensitization to 0.5 mg/kg AMPH, only the HR rats developed pronounced sensitization to repeated administration of 1.0 mg/kg AMPH. When both HR and LR were considered, there was a significant correlation between response to novelty and the extent of sensitization to the locomotor-stimulating properties of 1.0 mg/kg AMPH. There were no differences in locomotor activity or sensitization between HR and LR rats following the highest dose of AMPH (1.5 mg/kg). In a separate experiment, HR and LR rats were compared for locomotor activity following a series of intracranial infusions of AMPH. There were no overall differences in locomotor activity between the HR and LR groups following AMPH infusions into either the nucleus accumbens (NACC) or the anterior dorsal striatum (ADS). However, the locomotor activity scores in the novel environment significantly correlated with the locomotor response to 3.0 micrograms AMPH infused into either the NACC or ADS.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to test the hypothesis that the active neurotransmitter processes of release and uptake affect the in vivo microdialysis recovery of dopamine (DA) in the nucleus accumbens (N ACC) of the rat. The in vivo recovery for DA was established for rats which had received either unilateral infusions of the neurotoxin 6-hydroxydopamine (6-OHDA, 8 micrograms) or vehicle (0.2 micrograms ascorbate). In the quantitative dialysis method used (point of no net flux method), DA is added to the perfusate at concentrations above and below the expected extracellular concentration (0, 5, 10 and 20 nM) and DA is measured in the dialysate from the brain to generate a series of points. A linear fit is performed, the slope of which is the in vivo recovery of the dialysis probe. The in vivo recovery of the 6-OHDA group was 30 +/- 3% which was significantly lower (P less than 0.002) than the in vivo recovery of the control group which was 60 +/- 3% (mean +/- SEM; n = 6/group). The zero intercept of this regression is the point of no net flux, which is the extracellular concentration of DA independent of the probe sampling characteristics. The extracellular DA concentration for the 6-OHDA group was 7.8 +/- 1.1 nM, which was not significantly different than the control group which was 6.9 +/- 0.7 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

The reinforcing properties of cocaine are thought to be primarily mediated by the release of dopamine (DA) in the nucleus accumbens (N ACC). The extracellular concentration of DA in the N ACC was monitored with in vivo microdialysis procedures during ongoing cocaine self-administration to achieve a more detailed understanding of how DA mediates the reinforcing effects of cocaine. A dose-dependent decrease in lever pressing behavior occurred as the dose of cocaine was increased. The mean number of lever presses (in 20 min intervals) for 0.25, 0.50 and 0.75 mg/infusion doses was 5.6 +/- 0.7, 3.3 +/- 0.3 and 2.4 +/- 0.3, respectively. However, a simple inverse relationship did not occur between lever pressing behavior and the total amount of cocaine injected. Lever pressing behavior significantly increased cocaine intake as the dose of cocaine was increased. The total amount of cocaine intake that occurred during the 3 h self-administration period of the 0.25, 0.50 and 0.75 mg/infusion doses was 12.0 +/- 1.8 mg, 14.6 +/- 0.37 mg and 16.6 +/- 1.2 mg. Correspondingly, the extracellular concentration of DA in the N ACC was increased and maintained at significantly higher levels as the dose of cocaine was increased. The average concentration of DA that occurred during the self-administration of 0.25, 0.50 and 0.75 mg/infusion doses of cocaine was 269 +/- 26%, 381 +/- 21% and 464 +/- 49% of the basal DA concentration. As dose is increased, a corresponding increase occurs in both cocaine intake and in the extracellular concentration of DA in the N ACC.(ABSTRACT TRUNCATED AT 250 WORDS)

Microdialysis in conjunction with thermospray tandem mass spectrometry was employed in following the time course of the experimental drug GBR-12909 in vivo. GBR-12909 is 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-3(phenylpropyl)-piperazine. An important feature of microdialysis exploited in the method is the elimination of sample cleanup procedures. The detection limit was determined to be 100 pg and the relative standard deviation of estimates for standard solution in the range of 50 nmol/L to 1 mumol/L concentrations was found to be 17%. Important factors in obtaining high sensitivity and reproducibility were carrier phase composition and operation in the flow injection mode. The maximum concentration of GBR-12909 in the brain for a dose of 100 mg/kg i.p. was determined to be 250 nmol/L with the maximal concentration occurring approximately 2 h postinjection. This represents a 40-fold lower concentration of GBR-12909 in the brain as compared to cocaine concentration obtained at a dose of 30 mg/kg, which was estimated earlier under similar experimental conditions. This observation could explain the discrepancy between relative in vivo and in vitro potencies of the two drugs.

Background and Purpose— The goal of this study was to compare the predictive ability of S100B, N -methyl- d -aspartate (NMDA) receptor antibodies (NR2Ab) and C-reactive protein (CRP) for neurological deficits after cardiac surgery with cardiopulmonary bypass (CPB). Methods— We investigated 557 high-risk adult patients who underwent coronary artery or valve replacement surgery using CPB as a substudy of a prospective, blinded, multicenter clinical trial. Serum concentrations of S100B (n=513 patients), NR2Ab (n=398) and CRP (n=510) were measured preoperatively, 24 and 48 hours after CPB. Neurological adverse events were assessed at baseline and postoperative days 1 and 2; neurocognitive function (mini-mental status examination) was assessed at baseline and on postoperative days 1, 7 and 28. Results— Fifty-five (9.9%) patients had moderate or severe neurological adverse events (confusion/delirium, transient ischemic attack, or stroke) within 48 hours of CPB. Women had significantly more neurological complications than men (15.5% versus 7.8%; P =0.007). Ninety-six percent (24/25) of patients with NR2Ab concentrations ≥2.0 ng/mL preoperatively had neurological complications within 48 hours post-CPB, versus only 5.4% (20/373) of patients with NR2Ab concentrations Conclusions— Preoperative serum concentrations of NR2Ab, but not S100B or CRP, are predictive of severe neurological adverse events after CPB. Patients with a positive NR2Ab test (≥2.0 ng/mL) preoperatively were nearly 18 times more likely to experience a postoperative neurological event than patients with a negative test (

Voltammetry, a well-characterized electroanalytical technique, has recently been shown to be useful for studying the chemistry of the brain. In particular, the easily oxidizable catecholamine and indoleamine neurotransmitters, metabolites, and related compounds can be studied with this technique. Since the original report from the laboratory of R. N. Adams (Kissinger et al., 1973), more than 150 papers have been published on in vivo voltammetry and its application to the study of monoamines in the various points of view (Adams, 1976; 1978; Adams and Marsden, 1982; Hutson and Curzon, 1983; Marsden et al., 1984; Justice et al., 1985). In this introduction chapter, the theory methods, instrumentation, and interpretation of in vivo voltammetric data are discussed in a tutorial fashion in order to provide the reader with a basis for understanding the chapters that follow. Electrochemists will for obvious reasons not find an extensive treatment of voltammetry, nor will pharmacologists, neuro-chemists, and other neuroscientists find a surfeit of detail about the functioning of the central nervous system. Rather, it is hoped that the chapter will introduce the methods and concepts of voltammetry to those who may find it useful in their research on the central nervous system. Additionally, this chapter and the other chapters in the volume should provide an appreciation of some of the goals of in vivo voltammetry to electrochemists so they may become interested in contributing to the considerable work remaining to be done in developing voltammetry in vivo.

To understand the relationship of dopamine (DA) release to behavior, studies in which DA release, or an index of DA release, is monitored in intact animals are needed to complement studies using lesions of dopaminergic systems. Monitoring DA release directly is relatively difficult, particularly in behaving animals. One approach to the problem of monitoring DA release has been to use metabolite levels, either total tissue levels or extracellular levels, as indices of DA release. It has been known for some time that dopamine turnover in nigrostriatal neurons is dependent on nervous impulse flow (Anden et al., 1971) and electrical stimulation of these neurons produces an elevated turnover (Korf et al., 1976). Tissue measurements are useful for spatial mapping of activity, but require large numbers of animals for temporal information. In order to use extracellular metabolite information effectively, the temporal relationship of the extracellular levels of the metabolite to neurotransmitter release must be known. In this chapter the temporal relationship of extracellular dihydroxyphenylacetic acid (DOPAC) to stimulated dopamine release is described. In vivo voltammetry is used to observe the change in extracellular DOPAC following brief electrical stimulation of the medial forebrain bundle (MFB) (Michael et al., 1985). A similar method has been used to observe homovanillic acid in cerebrospinal fluid (Wightman et al., 1978). Our results indicate that there is considerable delay in the appearance of DOPAC in the extracellular fluid (ECF) following stimulated dopamine release. Because released dopamine is rapidly cleared from ECF following short periods of electrical stimulation (Ewing and Wightman, 1984), the changes in extracellular dopamine and DOPAC following stimulation occur at different times. This has enabled us to monitor directly the appearance and clearance of DOPAC in striatal ECF. The time course of the change in extracellular DOPAC is then used to characterize the kinetics of dopamine metabolism to extra-cellular DOPAC. Using this technique, rate constants for formation of DOPAC and its clearance from the extracellular space have been determined. This approach represents a new, nonpharmacological method for studying neurotransmitter metabolism. When combined with additional information, the compartmentation of dopamine may also be examined. The voltammetric data are validated by pharmacological manipulation of dopamine synthesis and metabolism. This approach should also work for examining serotonin and norepinephrine metabolism and compartmentation. The equivalence of rate constants determined by voltammetry and tissue assays has been demonstrated for clearance of the serotonin metabolite 5-hydroxindole acetic acid (5-HIAA) (Echizen and Freed, 1984).

Although microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady-state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no-net-flux) micro-dialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast-scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no-net-flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post-implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no-net-flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.

Dual-electrode voltammetry is used to characterize induced efflux of dopamine by m-tyramine via the norepinephrine transporter, stably expressed in a LLC-PK(1) cell line. A rotating disk electrode measures solely the dopamine oxidation current, while a stationary electrode held at a higher potential measures both the dopamine and m-tyramine concentrations. The ratio of the rate of dopamine efflux to the rate of m-tyramine uptake exhibits a hyperbolic dependence on initial dopamine concentration (half-maximal initial concentration of 4 microM) and is independent of the concentration of m-tyramine used to induce efflux for the two concentrations of tyramine tested (3 and 10 microM). These results are consistent with the alternating access model of transport, in which the ratio is shown to be independent of the nature of the external substrate used to induce efflux. At a representative initial dopamine concentration of 1 microM, the ratio of efflux to uptake is approximately 0.15 at the time of external substrate addition (zero internal tyramine). The result suggests that at this point, the transporter reorients to the external facing configuration without dopamine approximately 85% of the time, or expressed differently, seven external substrate molecules are taken up, on average, for each one transported outward.

Rats were trained to lever-press for intracranial self-stimulation (ICSS) of the lateral hypothalamus on either a fixed ratio (FR) 1 or 10 schedule. Their brains were removed after a 20 min session and tissue punches taken from the nucleus accumbens, olfactory tubercle, anterior striatum, or central striatum. These punches were assayed for content of dopamine (DA) and the major DA metabolite DOPAC. Compared with implanted controls, only the FR10 group showed significantly elevated DOPAC/DA ratios. These elevations were statistically significant in nucleus accumbens and central striatum and near significance in anterior striatum. They occurred to similar degrees in each hemisphere. In contrast, we found that stimulation of the ventral tegmental area of anesthetized rats asymmetrically increased the DOPAC/DA ratio, being most prominent in the ipsilateral accumbens. Because the FR10 group made only 58% of the responses of the FR1 group and received only 6% of the stimulations of the FR1 group, yet unlike the FR1 group showed a significant increase in the DOPAC/DA ratio, we suggest that the DA release was primarily influenced by the schedule, not the stimulation or the reward of the stimulation. These results were interpreted in terms of a model in which hypothalamic ICSS reward is largely dependent on non-dopaminergic mechanisms, with accumbal DA transmission being strongly dependent on the costs versus benefits of ongoing behavior.

Opioid antagonists attenuate behavioral effects of amphetamine and amphetamine-induced increases in extracellular dopamine levels in nucleus accumbens and striatum of rats but do not alter those effects of cocaine. This study was performed to determine 1) if the effect of opioid antagonists on the dopamine response to amphetamine is mediated in either the terminal or cell body region of the nigrostriatal and mesolimbic pathways, and 2) if the enkephalinase inhibitor thiorphan, which slows degradation of endogenous opioid peptides, increases the dopamine response to amphetamine but not to cocaine. Microdialysis probes were placed either into a dopaminergic terminal region or into both a terminal and cell body region of rats. Naloxone methiodide (1.0 microM), a lipophobic opioid antagonist, was administered into either the terminal or cell body region by reverse dialysis, whereas extracellular dopamine was collected in the terminal region. Increases in extracellular dopamine in nucleus accumbens and striatum caused by amphetamine (0.1-6.4 mg/kg, s.c.) were reduced significantly (28-39%) by naloxone methiodide administered into either substantia nigra or ventral tegmentum but not into terminal regions. Thiorphan (10 microM) administered into substantia nigra increased significantly the dopamine response to amphetamine in the ipsilateral striatum by as much as 42% but did not affect the dopamine response to cocaine (3.0-56 mg/kg, i.p.). These results suggest that amphetamine promotes release of endogenous opioids, which, through actions in the ventral tegmentum and substantia nigra, contribute to amphetamine-induced increases in extracellular dopamine in the nucleus accumbens and striatum.

In vivo voltammetry and microdialysis are two leading techniques for monitoring neurochemistry in the brain. This paper addresses the state of the art of voltammetry and microdialysis as they have evolved from their respective beginnings. Voltammetry is an old and well established electrochemical method. However, the development and application of in vivo voltammetry and microdialysis for measurement of neurotransmitters began about the same time in the 1970’s. In R. N. Adams laboratory at the University of Kansas, carbon paste electrodes were first put in ventricles of the rat brain and then in the brain itself.1,2 The developments that followed have been reviewed.3 About this same time, Urban Ungerstedt, developed the microdialysis probe, based on the idea of exchange of substances across a blood capillary.4 This was an extension of the earlier push-pull device of Gaddum5 and the dialysis devices of Bito et al.6 and Delgado et al.7 for sampling the extracellular environment of the brain. More than a thousand papers a year are now published using microdialysis.

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.

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 externalm-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 them-tyramine-induced DA efflux appeared noncompetitive relative to intracellular DA, but competitive relative to extracellularm-tyramine. Simultaneous measurement ofm-tyramine uptake and accompanying DA efflux at various concentrations of intracellular DA showed that cocaine did not alter the ratio of DA efflux tom-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.

The effects of substrates m-tyramine and β-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.

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.

Publisher Summary Rotating-disk electrode voltammetry (RDEV) has been used to determine kinetic arameters of dopamine (DA) and norepinephrine (NE) at the hNET in LLC-NET cells. As uptake occurs, the decreasing current is recorded for 6 min as a new steady-state is approached. To induce efflux, 6 μ l of unbuffered electrolyte as a control or 1, 10, or 100 μ M of selected substrate is added to the suspension, and the efflux of DA is monitored. At first, there is little l-amphetamine relative to the high concentration of DA on the inside of the cells to compete for outward transport. However, as it accumulates intracellularly, it competes with DA for binding to the empty transporter and DA efflux decreases. At the same time, DA concentration in the medium increases, causing an increase in the inward flux of DA. Amphetamine is known to inhibit the uptake of norepinephrine across the neuronal membrane and participate in the transmitter's release through the transporter. It was hypothesized that amphetamine-induced release was through an exchange diffusion mechanism. Because of the highly lipophilic nature of amphetamine, it is also capable of entering the cell by diffusion, complicating the analysis. The effect of using methylphenidate as an effluxing agent was also examined. Methylphenidate, as with d -amphetamine, has been used to treat attention deficit disorder with hyperactivity. Its mode of action is believed to be similar to that of cocaine in that it inhibits the DA transporter.

The specific opioid receptor antagonist naloxone attenuates the behavioral and neurochemical effects of amphetamine. Furthermore, the amphetamine-induced increase in locomotor activity is attenuated by intracisternally administered naltrindole, a selective delta-opioid receptor antagonist, but not by the irreversible mu-opioid receptor antagonist beta-funaltrexamine. Therefore, this research was designed to determine if naltrindole would attenuate the neurochemical response to amphetamine as it did the behavioral response. In vivo microdialysis was used to monitor the change in extracellular concentrations of dopamine in awake rats. Naltrindole (3.0, 10, or 30 micrograms) or vehicle was given 15 min before and beta-funaltrexamine (10 micrograms) or vehicle 24 h before the start of cumulative dosing, intracisternally in a 10-microliters volume, while the rats were lightly anesthetized with methoxyflurane. Cumulative doses of subcutaneous d-amphetamine (0.0, 0.1, 0.4, 1.6, and 6.4 mg/kg) followed pretreatment injections at 30-min intervals. Dialysate samples were collected every 10 min from either the striatum or nucleus accumbens and analyzed for dopamine content by HPLC. Amphetamine dose-dependently increased dopamine content in both the striatum and nucleus accumbens, as reported previously. Naltrindole (3.0, 10, and 30 micrograms) significantly reduced the dopamine response to amphetamine in the striatum. In contrast, 30 micrograms of naltrindole did not modify the dopamine response to amphetamine in the nucleus accumbens. On the other hand, beta-funaltrexamine (10 micrograms) had no effect in the striatum but significantly attenuated the amphetamine-induced increase in extracellular dopamine content in the nucleus accumbens. These data suggest that delta-opioid receptors play a relatively larger role than mu-opioid receptors in mediating the amphetamine-induced increase in extracellular dopamine content in the striatum, whereas mu-opioid receptors play a larger role in mediating these effects in the nucleus accumbens.

The ventral pallidum (VP) is often viewed as an output structure of the nucleus accumbens septi (NAS). However, VP, like NAS, receives a dopaminergic input from the ventral tegmental area. These experiments investigated some behavioral effects of microinjection into VP of drugs which enhance dopaminergic transmission. Injection of 25 micrograms dopamine or 5-10 micrograms amphetamine into VP produced hypermotility. In contrast, injection of 12.5-50 micrograms cocaine initially suppressed, then increased, activity. Injection of 100 micrograms cocaine only produced hypomotility in the 1-h period examined. The hypomotility following cocaine seemed to be a local anesthetic effect, because it was mimicked by 50-200 micrograms procaine. Procaine did not, however, produce subsequent hypermotility. Conditioned place preference (CPP) was produced by 10 micrograms amphetamine and 50 micrograms cocaine but not 100 micrograms procaine. We conclude that injection of cocaine into VP unlike similar injections into NAS, produces CPP. These results support the idea of an involvement of dopamine in VP in reward and locomotor activation, independent of dopamine in NAS. The use of intracerebral injections of cocaine is complicated, however, by an apparent local anesthetic effect of the drug.

Fluctuations in extracellular dopamine and DOPAC levels in nucleus accumbens septi (NAS) were monitored in 1-min microdialysis samples taken from rats engaged in intravenous cocaine self-administration. For four rats the dose per injection was fixed at 2.0 mg/kg; for four others the dose per injection was varied irregularly, from one response to the next, between three levels (0.5, 1.0 and 2.0 mg/kg). Regardless of the dosing regimen, extracellular dopamine levels were tonically elevated by 200-800% within the cocaine self-administration periods, fluctuating phasically within this range between responses. In the fixed dose condition, the phasic increases following each injection (and the phasic decreases preceding them) averaged approximately 50% of the mean tonic elevation. Phasic fluctuations in dopamine levels remained time-locked to lever-presses even when response rate was irregular, because of the variable dose condition. In the variable dose condition greater increases in dopamine and longer inter-response times followed injections of the higher doses; dopamine fluctuations were consistent with the multiple-infusion pharmacokinetics of cocaine. DOPAC levels showed a slow tonic depression during cocaine self-administration, but individual injections were not associated with discernible phasic fluctuations of DOPAC. These data are consistent with the hypothesis that falling dopamine levels trigger successive responses in the intravenous cocaine self-administration paradigm, but inconsistent with the notion that extracellular dopamine levels are depleted at the times within sessions when the animal initiates drug-seeking responses.

The specific opioid receptor antagonist naloxone modifies the effects of amphetamine in a wide variety of behavioral paradigms. Naloxone also attenuates the amphetamine-induced increase in extracellular dopamine in the brain of rats. Therefore, these experiments were designed to replicate the neurochemical and behavioral interactions between naloxone and amphetamine, and to extend these observations to interactions between naloxone and cocaine. Microdialysis was performed on adult male rats of Sprague-Dawley descent. Rats were pretreated with a subcutaneous injection of 5.0 mg/kg naloxone or vehicle, which was followed 30 min later by cumulative doses of subcutaneous d-amphetamine (0.0, 0.1, 0.4, 1.6, 6.4 mg/kg) or intraperitoneal cocaine (0, 3, 10, 30, 56 mg/kg) at 30 min intervals. The microdialysis probes were perfused at a flow rate of 0.6 microliter/min with artificial cerebrospinal fluid. Dialysate samples were collected every 10 min from either the nucleus accumbens or striatum and analyzed for dopamine content by high-performance liquid chromatography (HPLC). Locomotor activity (photobeam breaks) was monitored simultaneously with the collection of dialysate samples. Amphetamine and cocaine dose-dependently increased extracellular dopamine in both the nucleus accumbens and striatum. Naloxone pretreatment significantly reduced the amphetamine-induced increase in extracellular dopamine in both brain regions and also attenuated the increase in locomotor activity elicited by amphetamine. Naloxone pretreatment had no effect, however, on the cocaine-induced increase in extracellular dopamine or locomotor activity. These findings suggest that endogenous opioid systems play a role in mediating the neurochemical and behavioral effects of amphetamine, but not those of cocaine.