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The carrier molecule that transports dopamine (DA) across the synaptic membrane is known as the dopamine transporter (DAT). Depending on the ionic conditions, DAT may function as a mediator of both the inward directed DA transport known as the "reuptake" and the outward directed DA transport known as the "release." The functional significance of DAT is in the regulation of DA neurotransmission by terminating the action of DA in the synapse via reuptake. With use of DAT binding as a presynaptic marker to measure altered DA innervation, abnormalities of the DAT binding have been demonstrated in idiopathic Parkinson's disease, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity, and progressive supranuclear palsy. Moreover, the identification of DAT as the neuronal element that mediates the addictive properties of cocaine highlights its significance in cocaine addiction. Cocaine binding in the brain is heterogeneous, and there is an uneven distribution of the high- and low-affinity binding sites across the anatomical regions. Regional differences in ligand binding are observed by using both [3H]cocaine and the diphenyl-substituted piperazine derivatives known as the "GBR series" of ligands. The identification of compounds that inhibit the binding of medications for cocaine abuse. Furthermore, clarification of the various binding domains that may be relevant to transporter function in human neuropsychiatric disorders may lead to the development of new medications for schizophrenia, Tourette's disease, and drug addiction.

Molecular changes in the neostriatum of human subjects who died with a history of cocaine abuse were revealed in discrete cell populations by means of the techniques of in situ hybridization histochemistry and in vitro receptor binding and autoradiography. Cocaine subjects had a history of repeated cocaine use and had cocaine and/or cocaine metabolites on board at the time of death. These subjects were compared to control subjects that had both a negative history and toxicology of cocaine use. Selective alterations in mRNA levels of striatal neuropeptides were detected in cocaine subjects compared to control subjects, especially for the opioid peptides. Marked reductions in the levels of enkephalin mRNA and mu opiate receptor binding were found in the caudate and putamen, concomitant with elevations in levels of dynorphin mRNA and kappa opiate receptor binding in the putamen and caudate, respectively. Dopamine uptake site binding was reduced in the caudate and putamen of cocaine subjects. The greater magnitude of changes in the dorsolateral striatum (caudate and putamen) as opposed to the ventromedial striatum (nucleus accumbens) suggests that cocaine abuse preferentially alters the biosynthetic activity of striatal systems associated with sensorimotor functioning. Additionally, an imbalance in the activity of the two major striatal output pathways in cocaine users is implicated because peptide mRNA levels were reduced in enkephalinergic striatopallidal neurons and increased in dynorphinergic striatonigral neurons. Another imbalance, that of reductions of transmitter mRNA and receptor expression associated with euphoria (enkephalin and mu opiate receptors), together with elevations in mRNAs of transmitter systems associated with dysphoria (dynorphin and kappa opiate receptors), suggests a model of dysphoria and craving in the human cocaine addict brain.

The acute and long-term effects of a single injection of psychomotor stimulants (amphetamine (1.5 mg/kg i.p.), cocaine (30 mg/kg i.p.) and GBR 12909 (10 mg/kg i.p.)) were studied with in situ hybridization histochemistry to assess alterations in the mRNA expression of enkephalin, dynorphin and substance P in the striatum. The greatest alterations on mRNA levels of enkephalin, dynorphin and substance P were observed 2 h following the first administration of each drug compared to that observed following a second challenge injection 14 days later. Of the drugs tested, the dopamine uptake inhibitory agents cocaine and GBR 12909 acutely elevated mRNA levels of all three neuropeptides, while amphetamine elevated mRNA levels of substance P only. A second challenge administration of the stimulants 14 days subsequent to the initial single injection re-elevated the mRNA level of substance P. An overall tolerance is speculated to account for diminution of the enkephalin and dynorphin responses to a challenge injection while a relative sensitization is suggested for the enkephalin response due to a reduction in the baseline level of expression produced by the first injection. The data also show that there are regional variation within the striatum following systemic administration of psychomotor stimulants, with greater elevations in the sensorimotor dorsolateral striatum than in the ventromedial 'limbic' nucleus accumbens region.

The effects of acute and repeated amphetamine administration on mesolimbic dopamine (DA) neurons was assessed by studying DA and cholecystokinin (CCK) release in the nucleus accumbens (Acc), as well as effects on mRNA genes regulating DA and CCK synthesis in ventral tegmental area (VTA) cells in rats. Amphetamine (1.5 mg/kg) markedly increased extracellular levels of DA in the medial Acc (assessed by in vivo microdialysis) in drug-naive animals, about twice the amount released in animals repeatedly administered the drug for the previous 7 days (twice daily). CCK overflow was found to mirror the DA responses in that the very transient elevation of CCK monitored in drug-naive animals was attenuated in those with prior amphetamine use. The attenuation of both DA and CCK overflow in the medial Acc was found to be associated with a decrease in the number of CCK mRNA-positive VTA neurons (assessed by in situ hybridization histochemistry). Although the number of cells expressing CCK mRNA were decreased, the gene expression in those positive CCK and tyrosine hydroxylase mRNA cells in the VTA was significantly increased. The CCK mRNA neurons in the VTA were positively identified as those projecting to the medial Acc by the local perfusion of Fluoro-gold retrograde tracer via microdialysis probes located in the Acc.

The effect of two consecutive administrations of amphetamine (1.5 mg/kg, s.c.; 2 h apart) on dopamine (DA) and acetylcholine (ACh) efflux in dorsolateral striatum and nucleus accumbens was studied with in vivo microdialysis in freely moving rats. Amphetamine was found to cause a more persistent increase of extracellular DA in the dorsolateral striatum than in the nucleus accumbens. The increase in DA overflow by amphetamine was accompanied by an increase of extracellular ACh in both brain areas. The results are inconsistent with the hypothesis of an amphetamine-induced inhibitory regulation by DA on ACh overflow.

The influence of cocaine self-administration on the expression of messenger RNAs for dynorphin, enkephalin and substance P was analyzed in the rat striatum with in situ hybridization histochemistry. Cocaine, an indirect dopamine agonist, was found to differentially affect the levels of mRNA encoding these neuropeptides in different subregions of the striatum. Following a 7 day period of variable free access to cocaine, dynorphin and substance P mRNA levels were elevated throughout the striatum, but the increases were substantially greater in the dorsal striatum than in the nucleus accumbens. Enkephalin mRNA was not significantly altered in the dorsal striatum but was slightly elevated in the nucleus accumbens. These results suggest that cocaine self-administration has differential effects on striatonigral and striatopallidal projection neurons, and that these effects vary in subregions of the striatum.

In situ hybridization histochemistry and in vivo microdialysis were combined to study the effect of amphetamine on the expression of choline acetyltransferase and glutamate decarboxylase67 mRNA and in vivo release of acetylcholine and GABA in rat medial nucleus accumbens. Differential effects on acetylcholine and GABA neurons by a single challenge injection of amphetamine (1.5 mg/kg, s.c.) were apparent in saline-pretreated and amphetamine-pretreated (same dose, twice daily for the previous seven days) rats. Extracellular acetylcholine levels were increased up to 50% over a prolonged period following both single and repeated amphetamine. In contrast, extracellular concentrations of GABA were gradually decreased to half the control values, but only in rats receiving repeated amphetamine. Although the increase of acetylcholine release was not associated with any change in choline acetyltransferase mRNA levels, the number of neurons expressing high levels of glutamate decarboxylase67 mRNA was decreased (28%) following repeated injections. Thus we suggest that amphetamine decreases extracellular GABA levels by a slow mechanism, associated with the decreased expression of glutamate decarboxylase67 mRNA in a subpopulation of densely labeled neurons in the medial nucleus accumbens. The delayed response by GABA to amphetamine may reflect supersensitivity in the activity of postsynaptic gamma-aminobutyric acid-containing neurons in nucleus accumbens as a consequence of the repeated amphetamine treatment.

Presynaptic dopamine and acetylcholine transmission in the anteroventral (limbic) caudate-putamen were studied by microdialysis in freely moving rats during cocaine self-administration sessions. Acute cocaine exposure elevated dopamine (DA) overflow in drug-naive animals. However, during repeated cocaine administration, the drug-induced elevation of DA was attenuated in animals previously exposed (during the prior 9 days) to cocaine. The diminished dopamine response in repeatedly treated animals while self-administering cocaine was speculated to be due to increased activity of DA transport carrier and/or supersensitive receptors modulating DA release. During repeated cocaine administration, extracellular acetylcholine levels in animals previously exposed to cocaine were found to be significantly decreased compared with controls. The findings of the study may suggest a functional development of supersensitive DA receptors postsynaptic to the DA terminal (located on cholinergic neurons) as a consequence of previous cocaine exposure. Such supersensitivity would allow for a reduced DA signal to be amplified at the postsynaptic level during repeated cocaine self-administration.

The effect of general anaesthesia on extracellular levels of acetylcholine (ACh) in the caudate-putamen of freely moving rats was studied by microdialysis. ACh concentrations were determined in the same perfusate samples by radioenzymatic and HPLC/electrochemical procedures in order to compare the assays. The concentration of ACh in perfusate samples was estimated to be 0.30 microM in conscious unrestrained rats. However, when these rats were administered chloral hydrate (400 mg/kg i.p.), the level of ACh was decreased immediately by 50%, attaining a value of 0.06 microM within 20-40 min following the injection. Upon recovery of the righting reflex, ACh levels were once again re-elevated. The levels of choline (Ch), the precursor of ACh, were unaffected by anaesthesia. It was apparent that the level of consciousness (i.e. awake vs. anaesthetized) is an important factor determining ACh overflow. Radioenzymatic and HPLC assays proved to give identical results for the analysis of ACh and Ch.

The effects of cocaine on dopamine (DA) neurotransmission were evaluated by in vivo microdialysis in the striatum of halothane-anesthetized rats. Intravenous cocaine produced a dose-dependent, transient increase of the extracellular concentration of DA, with a peak response within 10 min and a return to control level by 30 min. The sharp DA response pattern was abolished in a calcium-free environment, indicating that DA release enhanced by cocaine originates from a vesicular storage pool. Continuous administration of cocaine (via the perfusion medium) directly into the nigrostriatal terminal region also produced a dose-dependent increase in DA release. Low concentrations (10(-5) M and 10(-6) M) of cocaine maintained DA at a constant stable level, consistent with the effects observed after potent DA uptake inhibitory agents (e.g., nomifensine and Lu19005). However, continuous exposure to high concentrations (greater than or equal to 10(-4) M) induced a transient elevation of DA within 20 min, following which DA decreased to a stable but high level; this decrease might reflect tolerance to the effect of cocaine. Administration of cocaine (10(-3) M) into the substantia nigra did not change striatal DA release. The local striatal action of cocaine was less potent than amphetamine in elevating DA overflow and in its effect on DA metabolism. These findings suggest that the fast transient enhancement of DA by intravenous cocaine is most likely a consequence of the transient presence of cocaine in the terminal region, correlating with the well-known rapid pharmacokinetic and behavioral aspects of the drug.

The present in vivo microdialysis study examined the role of vesicular- and carrier-mediated mechanisms underlying dopamine (DA) release, uptake and metabolism in halothane-anaesthetized rats. Omission of calcium (Ca2+) from the dialysis perfusing medium, thereby reducing the concentration of Ca2+ in the striatal microenvironment necessary for vesicular DA release, attenuated the elevation of DA normally induced by the potent DA uptake inhibitors, nomifensine and Lu 19-005. Consistent with the results of in vitro studies, amphetamine release DA in a Ca2+-independent manner. The release of DA induced by amphetamine could be effectively blocked by nomifensine and Lu 19-005, demonstrating that the in vivo movement of amines occurred via a transport carried-mediated mechanism. Additionally, the inhibition of DA metabolism produced by amphetamine could be reversed or blocked by prior or delayed treatment with DA uptake inhibitors. The results support a bidirectional in vivo capability of the amine transport carrier.

The in vivo neurochemical profile of recently synthesized dopamine (DA) uptake inhibitors (Lu 19-005, Lu 17-133 and GBR 12.921) is described. The antidepressant, nomifensine, as well as another typical DA uptake inhibitor, methylphenidate, was also tested with the microdialysis technique. Most of the new DA uptake inhibitors induced a gradual dose- and time-dependent accumulation of extracellular DA with a weak influence on DA metabolites, similar to that of methylphenidate. Nomifensine, however, caused a DA overflow during the first hour after injection. This was distinguishable from the effect of other uptake inhibitors but comparable to amphetamine. The moderate increase of DOPAC induced by nomifensine compared to the marked decrease produced by amphetamine corroborates reports that the DA 'release' induced by these drugs is mediated by different mechanisms, originating from different intracellular storage pools of DA. The fact that nomifensine can be distinguished from other uptake inhibitors shows clearly that evaluation of dynamic changes in transmitter overflow provides information pertinent to the overall neurochemical characterization of a drug.

A sensitive and rapid HPLC-UV method for in vivo determinations of cocaine levels in extracellular fluid of specific brain regions and plasma is described. Free drug levels resulting from intravenous administration of cocaine were sampled using in vivo microdialysis probes simultaneously located in the jugular vein, nucleus accumbens, and anteromedial caudate-putamen of halothane-anesthetized rats. In a separate group of animals, the influence of cocaine on extracellular dopamine concentrations in the anteromedial caudate-putamen was also assessed. The time dependences of changes in cocaine concentration in each of the above regions were congruent, and peak concentrations were reached 10 min after the drug was administered. The half-lives of cocaine in the blood, nucleus accumbens, and anteromedial caudate-putamen were estimated to be 31.5, 29.1, and 21.4 min, respectively. A repeated injection of cocaine, given 90 min later, produced a maximal cocaine level and pharmacokinetic profile that were indistinguishable from those of the initial infusion. Cocaine was concentrated to a greater extent in brain than in blood, a feature consistent with the action of a lipophilic drug. In addition, extracellular dopamine levels measured in the anteromedial caudate-putamen following cocaine infusions closely mirrored those of cocaine itself. The ability to measure the free concentration of drugs by microdialysis should be applicable to a wide range of in vivo pharmacological studies.

Addictive properties of cocaine have been suggested to be mediated by an interplay of depletion (craving) and re-elevation (reinforcement) of dopamine (DA) levels in limbic brain area. In this study, direct measurement of dopamine in the extracellular fluid of rats freely self-administering cocaine was evaluated using in vivo microdialysis. Acute cocaine administration was associated with enhanced accumulation of DA in the nucleus accumbens, correlated with enhanced locomotor activity. In contrast, the increased DA overflow observed in drug-naive animals was attenuated in animals self-administering cocaine who had previous regular repeated (9-day) exposure to the drug. The results suggest that the absolute amount of DA in the extracellular space is not the critical factor correlated with the self-administration behavior. Additionally, the results indicate that the reduced ability of cocaine to re-elevate DA to first-time drug use is not due to a reduction of DA in the tissue or reduced DA synthesis, but may instead be associated with alterations of release and reuptake processes.

In vivo microdialysis was utilized to evaluate the role of extracellular Na+ in regulating dopamine (DA) neurotransmission in the caudate-putamen of halothane-anaesthetized rats. Reduction of the extracellular Na+ concentration by introduction of a perfusion media containing 50mM Na+ (with choline replacement) produced an excessive release of DA that could be effectively blocked by nomifensine and Lu 19-005, potent inhibitors of an amine transport carrier. These results substantiate reports of a carrier-mediated efflux of DA from presynaptic terminals. Pretreatment with amphetamine, considered both a DA uptake inhibitor and releaser, did not, however, influence the efflux of DA induced by the low extracellular Na+ environment. Although the release of DA from an apparent non-granular cytosolic pool was greatly enhanced by the low extracellular Na+ environment, 3,4-dihydrophenylacetic acid (DOPAC) levels, which supposedly reflect metabolism of non-vesicular DA, were minimally effected. In contrast, homovanillic acid (HVA) was sensitive to extracellular Na+ and not directly related to extracellular levels of either DA or DOPAC, suggesting the possibility of a Na+-sensitive (carrier-mediated?) process involved in the formation of HVA. Overall, the results of this paper cannot be completely reconciled with the traditional concept of intracellular organization of DA pools and suggests the possibility of various non-granular pools being differentially sensitive to efflux and metabolism.