Characterization of the binding and comparison of the distribution of benzodiazepine receptors labeled with [3H]diazepam and [3H]alprazolam.

The binding characteristics of [3H]diazepam and [3H]alprazolam were obtained by in vitro analysis of sections of rat brain. Dissociation, association, and saturation analyses were performed to optimize the conditions for obtaining selective labeling of benzodiazepine receptors with the two tritiated compounds. Both drugs approached equilibrium rapidly in vitro. Rosenthal analysis (Scatchard plot) of the saturation data indicated a similar finite number of receptors was being occupied by both ligands. Competition studies, using various ligands to inhibit both [3H]diazepam and [3H]alprazolam indicated that these two compounds bind to the tissue sections as typical benzodiazepine drugs and apparently do not overlap onto other subtypes of receptors. These experiments were performed by both binding assay in tissue sections and by light microscopic autoradiography. The major difference between the labeling of the two compounds is represented by the peripheral benzodiazepine sites, which are recognized by [3H]diazepam, but not occupied by [3H]alprazolam (at nanomolar concentrations). This difference was readily apparent in the autoradiograms. Other pharmacokinetic or pharmacodynamic properties must distinguish these two benzodiazepines.

D1-receptor antagonists: comparison of [3H]SCH39166 to [3H]SCH23390.

A radiolabeled form of the benzonaphthazephine, SCH39166 was used to characterize the binding of this D1 antagonist in cortex, and an autoradiographic comparison of the localization of [3H]SCH39166 to [3H]SCH23390 (D1 antagonist and forerunner of SCH39166) binding was performed. The Kd for [3H]SCH39166, calculated from dissociation and association rate constants (1.09 nM), was comparable to the Kd value derived from Scatchard analyses of saturation data (1.74 nM). [3H]SCH39166 binds to brain tissue in a saturable manner with high affinity and low non-specific binding. Inhibition of [3H]SCH39166 binding by dopaminergic and serotonergic agents supports the hypothesis that this is indeed a D1-specific compound with little overlap onto serotonin (5-HT) receptors. The affinity of [3H]SCH39166 for 5-HT2 and 5-HT1c receptors is at least an order of magnitude lower than the affinity of [3H]SCH23390 for these same receptor sites. Quantitative autoradiographic analysis of [3H]SCH39166 and [3H]SCH23390 binding indicates high D1-receptor density in the caudate-putamen, nucleus accumbens, olfactory tubercle, substantia nigra and entopeduncular nucleus. Low levels of binding (not significantly above background) were detected with [3H]SCH39166 in lamina IV of the cortex and in choroid plexus; areas which had significant [3H]SCH23390 binding and are known to have a high density of 5-HT (5-HT2 and 5-HT1c respectively) receptors.

CNS distribution of D1 receptors: use of a new specific D1 receptor antagonist, [3H]SCH39166.

D1 dopamine receptors have been localized using a radioactive form of a new specific antagonist, [3H]SCH39166. This compound has been shown, in in vitro binding studies, to be highly selective for the D1 receptor subtype; more so than its predecessor, [3H]SCH23390. These ligand binds saturably, reversibly and with high affinity. Use of appropriate conditions produces a high signal to noise binding ratio to D1 receptors in slide-mounted tissue sections. Autoradiographic localization of radiolabeled receptors shows high densities of the D1 receptor subtype in such brain structures as the caudate-putamen, nucleus accumbens, entopeduncular nucleus, and the substantia nigra pars reticulata. A lower density of receptors is found in a few other areas including lamina VI of the cerebral cortex. A distinct paucity of binding was apparent in lamina IV of the cerebral cortex and in the choroid plexus, two areas thought to have D1 receptors. SCH39166 thus represents a superior ligand for obtaining selective labeling of D1 receptors in autoradiographic and binding studies.

Lack of long-term changes in cocaine and monoamine concentrations in rat CNS following chronic administration of cocaine.

In previous studies, we reported time-dependent and dose-dependent changes in the rat dopaminergic receptor system following chronic administration of cocaine. The aim of the present investigation was to monitor the concentration of monoamines (using HPLC-ECD) and cocaine (using GC-PCI/MS) in rat CNS following a dose schedule of 5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d. for 21 days. 12 h after the last cocaine injection, cortical and striatal concentrations of monoamines and their metabolites were not significantly different in saline vs cocaine treated animals. In addition, the cocaine concentration in the brain regions examined did not change with the different doses used. Accumulation of a metabolite of cocaine (ecgonine methyl ester) was the only alteration found. These results indicate that alterations in the dopaminergic receptor system following chronic cocaine administration are not due to changes in neurotransmitter concentration or accumulation of cocaine in the brain.

Ibogaine pretreatment dramatically enhances the dynorphin response to cocaine.

Ibogaine (Endabuse) is a psychoactive indole alkaloid found in the shrub, Tabernanthe iboga, which has been used to treat stimulant addiction. Because ibogaine influences the activity of neurotensin systems, a dopamine-linked neuropeptide, the present study investigated if ibogaine also influences dynorphin (DYN) pathways. Unlike neurotensin responses, ibogaine alone did not alter DYN levels in the striatum, substantia nigra or nucleus accumbens. Interestingly, ibogaine pretreatment dramatically enhanced cocaine-induced increases in DYN content in these structures.

Differential responses by neurotensin systems in extrapyramidal and limbic structures to ibogaine and cocaine.

Ibogaine (Endabuse) is a psychoactive indole alkaloid found in the West African shrub, Tabernanthe iboga. This drug interrupts cocaine and amphetamine abuse and has been proposed for treatment of addiction to these stimulants. However, the mechanism of action that explains its pharmacological properties is unclear. Since previous studies demonstrated differential effects of psychotomimetic drugs (cocaine and methamphetamine) on neuropeptides such as neurotensin (NT), the present study was designed to determine: (1) the effects of ibogaine on striatal, nigral, cortical, and accumbens neurotensin-like immunoreactivity (NTLI); (2) the effects of selective dopamine antagonists on ibogaine-induced changes in NT concentrations in these brain areas; and (3) the effects of ibogaine pretreatment on cocaine-induced changes in striatal, nigral, cortical and accumbens NTLI content. Ibogaine treatments profoundly affected NT systems by increasing striatal, nigral, and accumbens NTLI content 12 h after the last drug administration. In contrast, NTLI concentrations were not significantly increased in the frontal cortex after ibogaine treatment. The ibogaine-induced increases in NTLI in striatum, nucleus accumbens and substantia nigra were blocked by coadministration of the selective D1 receptor antagonist, SCH 23390. The D2 receptor antagonist, eticlopride, blocked the ibogaine-induced increase in nigral NTLI, but not in striatum and nucleus accumbens. Ibogaine pretreatment significantly blocked the striatal and nigral increases of NTLI resulting from a single cocaine administration. Whereas many of the responses by NT systems to ibogaine resembled those which occur after cocaine, there were also some important differences. These data suggest that NT may contribute to an interaction between ibogaine and the DA system and may participate in the pharmacological actions of this drug.

Unique responses of limbic met-enkephalin systems to low and high doses of methamphetamine.

A single administration of a low (0.5 mg/kg) or high (10 mg/kg) dose of methamphetamine (METH) significantly altered the met-enkephalin (M-Enk) systems associated with some, but not all, limbic structures examined. Neither treatment influenced M-Enk levels 3 h after drug exposure in any limbic region studied; however, 12 h after drug administration, 0.5 mg/kg of METH reduced the tissue content of this peptide in both the nucleus accumbens shell (NAs) and the frontal cortex (FrCx). This was similar to the effect of this treatment on the anterior striatal region. In contrast, the high dose of METH increased M-Enk content in the frontal cortex and anterior striatum (AS), but had no effect in the nucleus accumbens shell. By 24 h, the effects of METH in the anterior striatum subsided, but decreases in M-Enk levels were still observed after both the low- and the high-dose METH treatments in the nucleus accumbens shell. The levels of M-Enk were not changed at any of the time points examined in the core of the nucleus accumbens (NAc). In general, treatment with a low or high dose of METH causes distinct and regional selective changes in the tissue levels of M-Enk in the limbic system. These changes appear to be mediated by dopamine (DA) D(2) and D(1) receptor activation.

Responses of the extrapyramidal and limbic substance P systems to ibogaine and cocaine treatments.

Ibogaine is an indolamine found in the West Africa shrub, Tabernanthe iboga, and has been proposed for the treatment of addiction to central nervous system (CNS) stimulants such as cocaine and amphetamine. The mechanism of ibogaine action and its suitability as a treatment for drug addiction still remains unclear. Since previous studies demonstrated differential effects of stimulants of abuse (amphetamines) on neuropeptide systems such as substance P, we examined the impact of ibogaine and cocaine on extrapyramidal (striatum and substantia nigra) and limbic (nucleus accumbens and frontal cortex) substance P-like immunoreactivity. Ibogaine and cocaine treatments altered substance P systems by increasing striatal and nigral substance P-like immunoreactivity concentration 12 h after the last drug treatment. However, substance P-like immunoreactivity content was not significantly increased in nucleus accumbens after treatment with either drug. The ibogaine- and cocaine-induced increases in substance P-like immunoreactivity in striatum and substantia nigra were blocked by coadministration of selective dopamine D(1) receptor antagonist (SCH 23390; R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine hydrochloride) or dopamine D(2) receptor antagonist (eticlopride; S(-)-3-Chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2- methoxy-benzamide hydrochloride). Most of the responses by substance P systems to ibogaine administration resembled those caused by cocaine, except in cortical tissue where multiple administration of cocaine, but not ibogaine increased substance P-like immunoreactivity. These data suggest that substance P systems may contribute to the effects of ibogaine and cocaine treatment.

Evidence for the presence of angiotensin II-type 1 receptors in brain.

Angiotensin II receptor subtypes have been localized in brain by using a novel nonpeptide AII-type 1 receptor antagonist to compete with [125I]AII binding in an autoradiographic study. These data contrast with results obtained using the AII-type 2 antagonist PD123177. Both receptor subtypes appear to exist in the brain, but the type 1 receptor predominates in the small regions of brain where AII binding is highly concentrated. DUP 753 may have a central action involved in its unique antihypertensive effect.

Alpha 2-agonist binding sites in brain: [125I]para-iodoclonidine versus [3H]para-aminoclonidine.

The localization of alpha 2-receptors was determined by quantitative autoradiography using [125I]para-iodoclonidine ([125I]PIC) and [3H]para-aminoclonidine ([3H]PAC). In cortical tissue, [125I]PIC and [3H]PAC were equipotent in their capacity to bind sites recognized by oxymetazoline (preferentially binds to the alpha 2A receptor subtype). The iodinated ligand was about 10 times more potent than [3H]PAC for binding to the heterogenous receptor population labeled by ARC-239 (alpha 2B and alpha 2C). The density of [125I]PIC binding was found to be two-fold higher than that of [3H]PAC in many brain areas and the disparity was even greater in regions such as the dentate gyrus, stria terminalis, and granular layer of the cerebellum. By contrast, other regions of the brain such as the laterodorsal thalamic nucleus, the locus coeruleus, and several amygdaloid areas had equivalent levels of binding. These observations indicate that [3H]PAC has selectivity for the alpha 2A receptor subtype and thus offer a restricted view of alpha 2-adrenergic receptor distribution. The iodinated ligand provides a more complete picture of the overall alpha 2 receptor population.

Differential localization of alpha 2-adrenergic receptor subtypes in brain.

The pharmacological identification and characterization of subtypes of alpha 2-adrenergic receptors have been confirmed by molecular biological investigations. Using receptor autoradiographic techniques, it has been possible to show regions of the brain where alpha 2 agonist binding ([3H]para-aminoclonidine) is preferentially labeling the presumed guaninenucleotide-sensitive, high-affinity conformations of the alpha 2 receptor. Careful examination of autoradiograms generated using the tritiated antagonists yohimbine, idazoxan, and rauwolscine also indicates some disparity in the regions occupied by these radiolabeled ligands. Inhibition of [3H]rauwolscine binding with the subtype selective compounds, ARC-239, or oxymetazoline demonstrates that there are discrete regions of the brain where one receptor subtype predominates over the other. These studies indicate that previous investigations utilizing the agonist para-aminoclonidine as the ligand for obtaining labeling of alpha 2 receptors have overlooked some regions of binding due to the subtype selectivity of this ligand. A more complete localization of alpha 2-adrenergic receptors can be obtained using the tritiated antagonist rauwolscine, and the differential distribution of at least two subtypes of the alpha 2 receptor can be obtained by selective inhibition of this binding.

Determination of methadone and its N-demethylation metabolites in biological specimens by GC-PICI-MS.

Methadone is often invoked for detoxification and maintenance of the opioid addict. We have developed and validated a sensitive and specific method for the analysis of methadone and its metabolites, 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), in human plasma, urine, and liver microsomes. This assay uses a solid-phase extraction. Separation and analysis of the analytes are performed by capillary gas chromatography-positive ion chemical ionization-mass spectrometry. The protonated molecules (MH+) are monitored at m/z 264 and 267 for EMDP-d0 and -d3, m/z 278 and 281 for EDDP-d0 and -d3, and m/z 310 and 313 for methadone-d0 and -d3. The recovery of methadone and its metabolites exceeded 85% in the different matrices analyzed. Linear standard curves in plasma and in urine were obtained over the concentration range of 10-600 ng/mL (coefficients of determination: methadone, > or = 0.995; EMDP, > or = 0.994; and EDDP, > or = 0.996). With plasma and urine fortified at 25, 100, and 300 ng/mL, the assay was precise (intra-assay coefficients of variation [CVs], 2-12%; interassay CVs, 1-15%) and accurate (intra-assay percent of target, 85-107; interassay percent of target, 88-105) for all three analytes. Stability studies indicated that methadone and its metabolites are stable at room temperature in plasma and in urine for at least 1 week and in liver microsomes for at least 24 h. This method has now been shown to be useful for quantitation of methadone, EDDP, and EMDP in human urine and plasma and is also useful for quantitation of the amount of EDDP produced in human liver microsomes incubated with methadone. It provides an accurate and precise analytical tool for further studies on the metabolism of methadone.

Determination of ibogaine and 12-hydroxy-ibogamine in plasma by gas chromatography-positive ion chemical ionization-mass spectrometry.

Ibogaine, an indolamine derivative, is currently being investigated as a potential agent in the treatment of stimulant and opiate addiction. We developed a rapid, sensitive, and specific method for the analysis of ibogaine and its putative active metabolite, 12-hydroxy-ibogamine (12-OH-ibogamine). This assay employs a one-step basic extraction with n-butyl chloride-acetonitrile (4:1), followed by derivatization of the metabolite using N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide. The derivatized extracts were analyzed by capillary gas chromatography-positive ion chemical ionization-mass spectrometry. The ions monitored were at m/z 311, 314, and 411, which correspond to the protonated molecules (MH+) for ibogaine, ibogaine-d3, and 12-OH-ibogamine.tert-butyldimethylsilyl, respectively. Linear standard curves were obtained over the concentration range of 1 0-1 000 ng/mL (average r2, 0.995 for ibogaine and 0.992 for 12-OH-ibogamine; n = 3). Limits of quantitation were 10 ng/mL. The interrun and intrarun coefficients of variation for the assay of ibogaine at 25, 100, and 300 ng/mL ranged from 2.9 to 8.8%. We also established the extraction and chromatographic conditions to monitor the 12-hydroxylated metabolite. A suitable internal standard was not yet obtained so the method could only provide semiquantitative information for 12-OH-ibogamine. Chemical stability studies of these analytes indicated that ibogaine and 12-OH-ibogamine were stable in a human plasma matrix at room temperature for a period of at least 1 week.

Fentanyl receptor assay. I. Development of a radioreceptor assay for analysis of fentanyl and fentanyl analogs in urine.

Radioreceptor binding assays may be useful methods for determining the plasma and urine concentration of drugs. We have evaluated the possibility of employing CNS receptors in an assay to measure fentanyl and fentanyl-like drugs. This in vitro assay is based on the competition of these drugs with [3H]fentanyl for opioid receptors in membrane preparations of rat forebrain. The binding is stereospecific, reversible, and saturable. Scatchard plots of saturation suggest the presence of high and low affinity binding sites. Naloxonazine, which selectively binds to mu 1-opioid site, competed with [3H]fentanyl for its high affinity binding site. Morphine and hydromorphone competed with [3H]fentanyl for the opioid receptor, but other morphine-like compounds were relatively weak displacers of [3H]fentanyl. Many other commonly abused drugs did not displace [3H]fentanyl from the opiod receptors. Urine samples from animals injected with fentanyl were evaluated by other analytical techniques, including radioimmunoassay (RIA) and gas chromatography/mass spectrometry (GC/MS), and results were compared to those from the radioreceptor assay. Urinary analysis of fentanyl showed a good correlation between all three methods.

Fentanyl receptor assay. II. Utilization of a radioreceptor assay for the analysis of fentanyl analogs in urine.

A radioreceptor assay has been developed to measure fentanyl and fentanyl-like drugs in biological specimens. The assay is based on the competition of these drugs with [3H]fentanyl for opioid receptors. Rats were injected intravenously with fentanyl (15 micrograms/kg), alpha-methylfentanyl (15 micrograms/kg), (+/-)-cis-3-methylfentanyl (15 micrograms/kg), butyrylfentanyl (0.48 mg/kg), and benzylfentanyl (3.19 mg/kg). Urine samples were analyzed by radioreceptor assay (RRA), radioimmunoassay (RIA), and gas chromatography/mass spectrometry (GC/MS). The time-course of urinary analysis of fentanyl analogs showed some discrepancies. RRA measurement of urine concentrations of (+/-)-cis-3-methylfentanyl that were undetectable by RIA gave results 5-10 times higher than values obtained by GC/MS. Concentrations of alpha-methylfentanyl obtained by RRA and GC/MS were similar; however, these samples were negative by RIA. Following the administration of benzylfentanyl, urinary concentrations were not detected by RIA and only slightly detectable with RRA; however, high concentrations of benzylfentanyl were found by GC/MS in the same samples. Urine samples from animals injected with butyrylfentanyl showed high cross-reactivity with fentanyl antibody, giving values measured by radioimmunoassay about two times higher than those obtained by the other two methods. These findings suggest that this radioreceptor assay is well-suited as an initial assay for the detection of active analogs of fentanyl in urine and correlates well with other techniques in the analysis of fentanyl; however, there is substantial disagreement between techniques in the quantitation of fentanyl analogs.

Analysis of cocaine and its metabolites from biological specimens using solid-phase extraction and positive ion chemical ionization mass spectrometry.

An accurate and reliable gas chromatographic-mass spectrometric method was developed to analyze tissue, whole blood, plasma, and urine samples for cocaine (COC) and its major metabolites. COC, benzoylecgonine (BZE), and ecgonine methyl ester (EME) were isolated from the biological matrix using solid-phase extraction, and the tert-butyldimethylsilyl derivatives of BZE, EME, and their deuterium-labeled internal standards were formed. Separation of the compounds was performed by capillary chromatography, and analysis was performed by positive ion chemical ionization mass spectrometry using methane and ammonia as the reagent gases. The tert-butyldimethylsilyl derivatives of BZE and EME were stable and produced mass spectral ions with higher mass-to-charge ratios than trimethylsilyl derivatives. Recovery of COC and its metabolites exceeded 80% at all three concentrations tested. Linearity of the method was established from 2.5 to 2000 microg/L. Intra-assay precision had a coefficient of variation (CV) of less than 9% for all analytes when tested at 10, 25, 100, and 200 microg/L. Interassay precision also had a CV of less than 9% for COC, BZE, and EME at 25 and 100 microg/L. At 200 microg/L, %CVs for COC, BZE, and EME were 11.5, 12.0, and 12.7, respectively. In addition to the analysis of COC, BZE, and EME, the method was used to quantitate cocaethylene and to identify norcocaine.

A gas chromatographic-positive ion chemical ionization-mass spectrometric method for the determination of I-alpha-acetylmethadol (LAAM), norLAAM, and dinorLAAM in plasma, urine, and tissue.

l-alpha-Acetylmethadol (LAAM) is approved as a substitute for methadone for the treatment of opiate addiction. Analytical methods are needed to quantitate LAAM and its two psychoactive metabolites, norLAAM and dinorLAAM, to support pharmacokinetic and other studies. We developed a gas chromatographic-positive ion chemical ionization-mass spectrometric method for these analyses. The method uses 0.5 mL urine or 1.0 mL plasma or tissue homogenate, deuterated (d3) isotopomers as internal standards, methanolic denaturation of protein (for plasma and tissue), and extraction of the buffered sample with n-butyl chloride. For tissue homogenates, an acidic back extraction is included. norLAAM and dinorLAAM were derivatized with trifluoroacetic anhydride. Chromatographic separation of LAAM and derivatized norLAAM and dinorLAAM is achieved with a 5% phenyl methylsilicone capillary column. Positive ion chemical ionization detection using methane-ammonia as the reagent gas produces abundant protonated ions (MH+) for LAAM (m/z 354) and LAAM-d3 (m/z 357) and ammonia adduct ions (MNH4+) for the derivatized norLAAM (m/z 453), norLAAM-d3 (m/z 45 6), dinorLAAM (m/z 439), and dinorLAAM-d3 (m/z 442). The linear range of the calibration curves were matrix dependent: 5-300 ng/mL for plasma, 10-1000 ng/mL for urine, and 10-600 ng/g for tissue homogenates. The low calibrator was the validated limit of quantitation for that matrix. The method is precise and accurate with percent coefficients of variation and percent of targets within 13%. The method was applied to the analysis of human urine and plasma samples; rat plasma, liver, and brain samples; and human liver microsomes following incubation with LAAM.

Profiles of delta 9-tetrahydrocannabinol metabolites in urine of marijuana users: preliminary observations by high performance liquid chromatography-radioimmunoassay.

Metabolic profiles of 11-nor-9-carboxylic acid-delta 9-tetrahydrocannabinol (COOH-THC) and other THC metabolites were determined in an infrequent and a frequent marijuana user by high performance liquid chromatography-radioimmunoassay (HPLC-RIA). In the infrequent user, no unconjugated COOH-THC was detected in urine samples for the first 8 h following smoking, whereas this metabolite was detected in the urine samples from a frequent user. A metabolite was also detected in the frequent user, which was not present in the urine sample from the infrequent user.

Effects on monoamine levels in rat CNS after chronic administration of cocaine.

We have previously reported time-dependent and dose-dependent changes in the rat dopaminergic receptor system following chronic administration of cocaine (upregulation of cocaine, D1, and DA-uptake sites). We have now evaluated the effects of chronic cocaine exposure on the central catecholamine/indolamine neurotransmitter systems. Groups of rats were injected with cocaine (15 mg/kg, i.p., b.i.d.) or saline for 1, 3, 7, 14 or 21 days. Cortical and striatal tissues were analyzed for norepinephrine, dopamine, serotonin and their primary metabolites using a HPLC-ECD method. Chronic administration of cocaine did not change the cortical and striatal concentrations of the neurotransmitters under study; except, for a transient increase in the cortical MHPG concentration on day 3. These results suggest that changes in the dopaminergic receptor system following chronic cocaine exposure are not due to changes in the neurotransmitter concentrations.

Response of limbic neurotensin systems to methamphetamine self-administration.

Methamphetamine (METH) abuse is personally and socially devastating. Although effects of METH on dopamine (DA) systems likely contribute to its highly addictive nature, no medications are approved to treat METH dependence. Thus, we and others have studied the METH-induced responses of neurotensin (NT) systems. NT is associated with inhibitory feedback action on DA projections, and NT levels are elevated in both the nucleus accumbens and dorsal striatum after noncontingent treatment with high doses of METH. In the present study, we used a METH self-administration (SA) model (linked to lever pressing) to demonstrate that substitution of an NT agonist for METH, while not significantly affecting motor activity, dramatically reduced lever pressing but was not self-administered per se. We also found that nucleus accumbens NT levels were elevated via a D1 mechanism after five sessions in rats self-administering METH (SAM), with a lesser effect in corresponding yoked rats. Extended (15 daily sessions) exposure to METH SA manifested similar NT responses; however, more detailed analyses revealed (i) 15 days of METH SA significantly elevated NT levels in the nucleus accumbens shell and dorsal striatum, but not the nucleus accumbens core, with a lesser effect in the corresponding yoked METH rats; (ii) the elevation of NT in both the nucleus accumbens shell and dorsal striatum significantly correlated with the total amount of METH received in the self-administering, but not the corresponding yoked METH rats; and (iii) an NT agonist blocked, but an NT antagonist did not alter, lever-pressing behavior on day 15 in SAM rats. After 5 days in SAM animals, NT levels were also elevated in the ventral tegmental area, but not frontal cortex of rats self-administering METH.