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.

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.

Contrasting responses by basal ganglia met-enkephalin systems to low and high doses of methamphetamine in a rat model.

The influence of methamphetamine (METH) on basal ganglia met-enkephalin (Menk) was studied by determining levels of this peptide in striatal, pallidal and nigral regions after administering a single low (0.5 mg/kg) or high (10 mg/kg) dose of this stimulant. The Menk levels in the striatal and pallidal areas were reduced and increased after the low- and high-dose METH treatments, respectively, 12 h after drug administration in all striatal and pallidal regions examined. The low-dose effect appeared to be principally influenced by increased activation of the dopamine D2-like receptor, while the high-dose effect seemed to result from dominance of D1-like receptor activation. However, both effects required coactivation of D1- and D2-like receptors. For the most part, both low- and high-dose METH-induced changes in Menk tissue content were fully recovered by 24 h. The Menk levels were not significantly altered in the substantia nigra 3-24 h after either METH treatment. Results reported herein indicated that striatal and pallidal Menk pathways respond differently after acute treatment with low or high doses of METH.

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.

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.

Neuronal and behavioural abnormalities in striatal function in DARPP-32-mutant mice.

We investigated the role of the protein phosphatase inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in the expression of striatal neuropeptides and in biochemical and behavioural responses to repeated cocaine administration, using DARPP-32 knock-out mice. The striatum of DARPP-32-mutant mice showed heightened substance-P-like immunoreactivity, but normal levels of other neuropeptides. Repeated cocaine administration increased levels of DeltaFosB, a Fos family transcription factor, in the striatum of wild-type mice, and this increase was abolished in DARPP-32-mutant mice. Cocaine (20 mg/kg) acutely induced the same level of locomotor activity in the mutant and wild-type mice, but the mutants showed a higher rate of locomotor sensitization to repeated cocaine exposures. These data show that DARPP-32 is involved in regulating substance P expression in the striatonigral pathway, and in biochemical and behavioural plasticity with chronic administration of cocaine.

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.

The involvement of cytochrome P450 3A4 in the N-demethylation of L-alpha-acetylmethadol (LAAM), norLAAM, and methadone.

The N-demethylation of LAAM, norLAAM, and methadone has been investigated in human liver microsomes and microsomes containing cDNA-expressed human P450s. Gas chromatography/mass spectrometry methods allowed detection of norLAAM and dinorLAAM formation from LAAM, dinorLAAM formation from norLAAM, and EDDP and EMDP formation from methadone. The rates of N-demethylation varied 4- to 7-fold in microsomes from four different donors with activities for LAAM and norLAAM consistently greater (5- to 14-fold) than for methadone. The N-demethylation of LAAM, norLAAM, and methadone were significantly inhibited by ketoconazole. IC50s could be determined for ketoconazole inhibition of LAAM and norLAAM N-demethylation of 1.6 and 1.1 microM, respectively. The ability of ketoconazole to reduce methadone N-demethylation below 40% varied in regard to liver donor. No other P450-selective inhibitors reduced the average activities more than 43%. cDNA-expressed P450 3A4 N-demethylated LAAM, norLAAM, and methadone at greater rates than the other cDNA-expressed P450s studied (1A2, 2C9, 2D6, or 2E1). P450 3A N-demethylation of LAAM, norLAAM, and methadone exceeded the next most active P450, respectively, by at least 2.5, 9.6, and 13.4 times when expressed per milligram protein and by 18.2, 6.0, and 6.1 times when expressed per nanomole P450. These results suggest that P450 3A4 is the primary site of N-demethylation of LAAM, norLAAM, and methadone in human liver. Although other enzymes may also be capable of N-demethylating these compounds, identification of specific enzymes, except P450 3A4, has yet to be established. Knowledge of these enzymatic pathways is essential for assessment of the impact of metabolic drug-drug interactions on therapeutic success and/or adverse events.

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.

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.

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.

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.

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.

A sensitive and rapid HPLC-ECD method for the simultaneous analysis of norepinephrine, dopamine, serotonin and their primary metabolites in brain tissue.

This report details a very sensitive, rapid and accurate ion-pair HPLC-ECD method for the analysis of biogenic amines and their metabolites in brain tissue. The method described enables detection of picogram amounts of 3-methoxy-4-hydroxy phenethyleneglycol (MHPG) (37 pg), 3,4-dihydroxy phenylacetic acid (DOPAC) (18 pg), norepinephrine (NE) (12 pg), epinephrine (E) (6 pg), 5-hydroxyindoleacetic acid (5-HIAA) (18 pg), 3,4-dihydroxyphenylethylamine (DA) (6 pg), 3-methoxy-4-hydroxyphenylacetic acid (HVA) (12 pg) and 5-hydroxytryptamine (5-HT) (12 pg). The linearity of the method is from 18.75 ng/mL to 300 ng/mL for MHPG; 9.37 to 150 ng/mL for DOPAC and 5-HIAA; 6.25 to 100 ng/mL for NE, HVA and 5-HT; and from 3.12 to 100 ng/mL for E and DA. The reproducibility, expressed as coefficient of variance (CV%) within-run and between-run groups, was 2.25% and 19.49% for MHPG; 3.84% and 29.84% for DOPAC; 0.89% and 8.97% for NE; 1.26% and 5.61% for E; 1.07% and 28.77% for 5-HIAA; 2.65% and 10.65% for DA; 5.97% and 24.38% for HVA; and 4.44% and 9.45% for 5-HT.

Reduction in striatal D2 dopamine receptor mRNA and binding following AF64A lesions.

Unilateral lesions by a cholinotoxin, receptor autoradiography, and in situ hybridization techniques were employed to determine if dopaminergic receptors are located on cholinergic interneurons in the caudate-putamen (CPu). Lesion of the CPu with small amounts of the cholinotoxin AF64A resulted in a significant decrease in D2 receptor mRNA and D2 receptor binding. The loss was more pronounced in lateral and central portions of the CPu. Results obtained using [3H] SCH23390 binding to D1 receptors indicated that there was no change in this dopamine receptor subtype in the AF64A-lesioned CPu. A decrease in D2 receptor mRNA and receptor binding in AF64A-lesioned animals indicates that a population of postsynaptic D2 receptors is associated with the cholinergic interneurons. Lack of any change in [3H]SCH23390 binding in the AF64A-lesioned animals suggests that D1 receptors are not located on cholinergic neurons. These results provide evidence to support the selectivity of the lesion when used as indicated.

Alterations in the dopaminergic receptor system after chronic administration of cocaine.

Several studies suggest that one of the most important factors contributing to cocaine dependence is an alteration in the actions of the neurotransmitter dopamine in the central nervous system. In order to understand some of the neuroreceptor consequences of cocaine administration, groups of rats were injected with cocaine (2 daily doses of 15 mg/kg) for 1 to 21 days. Binding of [3H]cocaine, [3H]SCH23390, [3H]raclopride, and [3H]BTCP in striatal and cortical tissue from the treated animals was compared to controls. [3H]Cocaine binding was increased by the drug in the striatum and cortex at days 14 and 21, respectively. The binding of [3H]SCH23390 to D1 dopamine receptors was significantly increased at day 3 of cocaine exposure. In striatal membranes, [3H]BTCP binding to dopamine uptake sites was significantly increased after day 7, whereas binding in cortical membranes was increased from day 1. [3H]Raclopride binding to D2 dopamine receptors remained unchanged throughout the study in both cortical and striatal tissues. These results indicate that repeated exposure to cocaine produces an upregulation (possible supersensitivity) in cortical D1, cocaine, and DA-uptake sites which occurs in a time-dependent manner. These increases are coupled with an upregulation in striatal D1, cocaine, and DA-uptake sites, without simultaneous changes in D2 receptors. Thus, cocaine's effects are not uniformly distributed across all brain regions, but rather are focused within areas of the dopamine system.

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.

Unilateral ibotenic acid lesion of the caudate putamen results in D2 receptor alterations on the contralateral side.

Dopaminergic projections to the caudate putamen (CPu) involve fibers in the nigrostriatal pathway from the ipsilateral substantia nigra-pars compacta. Post-synaptic receptor populations on cells receiving this information are composed of both D1 and D2 dopamine receptor subtypes. In the present study, unilateral lesions of the CPu, with ibotenic acid, caused a significant reduction in D2 receptor mRNA on the ipsilateral side, as evidenced by in situ hybridization. Similarly, a reduction of D2 receptor binding (as demonstrated with [3H]raclopride) was observed on the lesioned side. As expected, there was no significant change in the D2 receptor binding on the contralateral side. However, a significant increase of D2 receptor mRNA (> 100%) was found in the CPu on the contralateral side when compared to sham-lesioned animals. These results indicate that compensatory changes may be occurring on the unlesioned side of the brain. These changes may reflect elevated transcription from DNA to mRNA or decreased translation of the D2 mRNA to protein following unilateral damage in the CPu. The observation of bilateral influence in the striatal dopamine receptor system may be of paramount importance in understanding movement disorders. These findings could influence the interpretation of results obtained in animal models of human disease in which the dopamine receptor system of the basal ganglia is compromised.

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.