Opioid analgesics elicit their effects via activation of the mu-opioid receptor (MOR), a G protein-coupled receptor known to interact with Gαi/o-type G proteins. Work in vitro has suggested that MOR couples preferentially to the abundant brain Gαi/o isoform, Gαo. However, studies in vivo evaluating morphine-mediated antinociception have not supported these findings. The aim of the present work was to evaluate the contribution of Gαo to MOR-dependent signaling by measuring both antinociceptive and biochemical endpoints in a Gαo null transgenic mouse strain. Male wild-type and Gαo heterozygous null (Gαo +/−) mice were tested for opioid antinociception in the hot plate test or the warm-water tail withdrawal test as measures of supraspinal or spinal antinociception, respectively. Reduction in Gαo levels attenuated the supraspinal antinociception produced by morphine, methadone, and nalbuphine, with the magnitude of suppression dependent on agonist efficacy. This was explained by a reduction in both high-affinity MOR expression and MOR agonist-stimulated G protein activation in whole brain homogenates from Gαo +/− and Gαo homozygous null (Gαo −/−) mice, compared with wild-type littermates. On the other hand, morphine spinal antinociception was not different between Gαo +/− and wild-type mice and high-affinity MOR expression was unchanged in spinal cord tissue. However, the action of the partial agonist nalbuphine was compromised, showing that reduction in Gαo protein does decrease spinal antinociception, but suggesting a higher Gαo protein reserve. These results provide the first in vivo evidence that Gαo contributes to maximally efficient MOR signaling and antinociception.

Continuing our studies investigating opioids with reduced P-glycoprotein (P-gp) substrate activity, a series of known 3- and 6-hydroxy, -methoxy, and -desoxymorphine analogs was synthesized and analyzed for P-gp substrate activity and opioid binding affinity. 6-Desoxymorphine (7) showed high affinity for opioid receptors and did not induce P-gp-mediated ATP hydrolysis. Additionally, 7 demonstrated morphine-like antinociceptive potency in mice, indicating this compound as an ideal lead to further evaluate the role of P-gp in opioid analgesic tolerance development.

Regulators of G-protein signaling (RGS proteins) comprise a large family of signal transduction molecules that modulate G-protein-coupled-receptor (GPCR) function. Among the RGS proteins expressed in the brain, RGS9-2 is very abundant in the striatum, a brain region involved in movement, motivation, mood and addiction. This protein negatively modulates signal transduction thus playing a key part in striatal function and resultant behavioral responses. In particular, there is evidence of important interactions with μ-opioid- and dopamine D2-receptor signaling pathways. Several studies indicate that manipulations of RGS9-2 levels in the striatum might greatly affect pharmacological responses. These findings indicate that treatment strategies targeting RGS9-2 levels or activity might be used to enhance responses to drugs acting at GPCRs and/or prevent undesired drug actions.

Chronic use of mu-opioid agonists has been shown to cause neurochemical adaptations resulting in tolerance and dependence. While the analgesic effects of these drugs are mediated by mu-opioid receptors (MOR), several studies have shown that antagonism or knockdown of delta-opioid receptors (DOR) can lessen or prevent development of tolerance and dependence. Based on computational modeling of putative active and inactive conformations of MOR and DOR, we have synthesized a series of pentapeptides with the goal of developing a MOR agonist/DOR antagonist peptide with similar affinity at both receptors as a tool to probe functional opioid receptor interaction(s). The eight resulting naphthylalanine-substituted cyclic pentapeptides displayed variable mixed-efficacy profiles. The most promising peptide (9; Tyr-c(S-CH2-S)[D-Cys-Phe-2-Nal-Cys]NH2) displayed a MOR agonist and DOR partial agonist/antagonist profile and bound with equipotent affinity (Ki ~ 0.5 nM) to both receptors, but also showed kappa opioid receptor (KOR) agonist activity.

The development of tolerance to and dependence on opioid analgesics greatly reduces their long-term usefulness. Previous studies have demonstrated that co-administration of a mu opioid receptor (MOR) agonist and delta opioid receptor (DOR) antagonist can decrease MOR agonist induced tolerance and dependence development after chronic exposure. Clinically, a single ligand displaying multiple efficacies (e.g. MOR agonism concurrently with DOR antagonism) would be of increased value over two drugs administered simultaneously. Guided by modeling of receptor-ligand complexes we have developed a series of potent non-selective opioid tetrapeptides that have differing efficacy at MOR and DOR. In particular, our lead peptide (KSK-103) binds with equal affinity to MOR and DOR but acts as a MOR agonist with similar efficacy but greater potency than morphine and a DOR antagonist in cellular assays measuring both G protein stimulation and adenylyl cyclase inhibition.

Three new radiolabeled compounds, [11C]SNC80 ((+)-4-[(αR)- α-{(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl}-3-[11C]methoxybenzyl-N,N-diethylbenzamide), N,N-diethyl-4-[3-methoxyphenyl-1-[11C]methylpiperidin-4-ylidenemethyl)benzamide, and N,N-diethyl-4-[(1-[11C]methylpiperidin-4-ylidene)phenylmethyl]benzamide, were prepared as potential in vivo radiotracers for the δ opioid receptor. Each compound was synthesized by alkylation of the appropriate desmethyl compounds using [11C]methyl triflate. In vivo biodistribution studies in mice showed very low initial brain uptake of all three compounds, and no regional specific binding for [11C]SNC80. A monkey PET study of [11C]SNC80 confirmed low brain permeability and uniform regional distribution of this class of opioid agonists in a higher species. Opioid receptor ligands of this structural class are thus unlikely to succeed as in vivo radiotracers, likely due to efficient exclusion from the brain by the P-glycoprotein efflux transporter.

The highly selective delta opioid agonist, SNC80, elicits dopamine-related behaviors including locomotor stimulation and conditioned place-preference. In contrast, it has been reported that SNC80 fails to promote dopamine efflux from the striatum of freely moving rats. However, SNC80 does enhance behavioral responses to the stimulants, amphetamine and cocaine, suggesting an interaction between delta opioids and psychostimulants. Since the increase in locomotor activity elicited by amphetamine and related stimulants acting at the dopamine transporter is associated with increases in extracellular concentrations of dopamine within the striatum, we hypothesized that SNC80 enhances this activity by potentiating the overflow of dopamine through the transporter. To test this hypothesis, striatal preparations from Sprague-Dawley rats were assayed for dopamine efflux in response to amphetamine challenge. SNC80 was given either in vivo or in vitro directly to rat striatal tissue, prior to in vitro amphetamine challenge. Both in vivo and in vitro administration of SNC80 enhanced amphetamine-mediated dopamine efflux in a concentration- and time-dependent manner. However, SNC80 in either treatment paradigm produced no stimulation of dopamine efflux in the absence of amphetamine. The effect of SNC80 on amphetamine-mediated dopamine overflow, but not the effect of amphetamine alone, was blocked by the delta selective antagonist, naltrindole and was also observed with other delta agonists. The results of this study demonstrate that even though SNC80 does not stimulate dopamine efflux alone, it is able to augment amphetamine-mediated dopamine efflux through a delta opioid receptor mediated action locally in the striatum.

Ligands from the naltrexamine series have consistently demonstrated agonist activity at kappa opioid receptors (KOR), with varying activity at the mu opioid receptor (MOR). Various 6β-cinnamoylamino derivatives were made with the aim of generating ligands with a KOR agonist/MOR partial agonist profile, as ligands with this activity may be of interest as treatment agents for cocaine abuse. The ligands all displayed the desired high affinity, non-selective binding in vitro and in the functional assays were high efficacy KOR agonists with some partial agonist activity at MOR. Two of the new ligands (12a, 12b) have been evaluated in vivo, with 12a acting as a KOR agonist, and therefore somewhat similar to the previously evaluated analogues 3–6, while 12b displayed predominant MOR agonist activity.

Regulators of G protein signaling (RGS) proteins act as GTPase accelerating proteins (GAPs) to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu-opioid signaling, C6 cells stably expressing a mu-opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu-opioid inhibition of forskolin-stimulated adenylyl cyclase (AC) by DAMGO, a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a two-fold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 due to weak physical association with Gαi2 and minimal GAP activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to AC and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.

It has been proposed that on chronic morphine treatment the μ-opioid receptor becomes constitutively active, and as a consequence, the opioid withdrawal response arises from a reduction in the level of this constitutively active receptor. In support of this, the putative μ-opioid receptor inverse agonist naltrexone has been shown to precipitate more severe withdrawal behavior in mice than the putative neutral receptor antagonist 6β-naltrexol. In the present study naltrexone and 6β-naltrexol were compared in NIH Swiss mice to test the hypothesis that their differential ability to precipitate withdrawal is due to differences in their in vivo opioid receptor antagonist potencies caused by differential access to μ-opioid receptors in the central nervous system and not necessarily by intrinsic differences in their opioid receptor activity. In naïve mice both compounds had similar potencies to antagonize morphine-induced antinociception in the hot plate and warm-water tail-withdrawal assays when measured under equilibrium conditions and afforded similar calculated apparent in vivo μ-opioid receptor affinities. In morphine-dependent mice both compounds precipitated withdrawal jumping but naltrexone was between 10- and 100-fold more potent than 6β-naltrexol. A similar potency difference was seen for other withdrawal behaviors. Both naltrexone and 6β-naltrexol at 1 mg/kg reversed antinociception induced by the long-lasting μ-opioid receptor agonist BU72 in the warm-water tail-withdrawal assay, but antagonism by naltrexone was 6-fold more rapid in onset at equal doses. Since the compounds have similar affinity for the μ-opioid receptor in vivo, the results suggest that the differences observed between the ability of naltrexone and 6β-naltrexol to precipitate withdrawal in the mouse may be explained by differential onset of receptor antagonist action.

14β-4’-Chlorocinnamoylaminodihydronormorphinone (2a), and analogues, are selective pseudoirreversible antagonists of the mu opioid receptor (MOR). The preparation of analogues with ethynic bonds, replacing the ethenic bond of 2a, is described. The new ligands, in mouse antinociceptive assays, had pseudoirreversible MOR antagonist activity which, in the case of 8b was of longer duration than that of 2a. The related codeinone (9b) had only antagonist activity in vivo, in contrast to 2a’s codeinone equivalent 3a, which had potent antinociceptive activity.

The collision coupling model describes interactions between receptors and G-proteins as first requiring the molecules to find each other by diffusion. A variety of experimental data on G-protein activation have been interpreted as suggesting (or not) the compartmentalization of receptors and/or G-proteins in addition to a collision coupling mechanism. In this work, we use a mathematical model of G-protein activation via collision coupling but without compartmentalization to demonstrate that these disparate observations do not imply the existence of such compartments. In experiments with GTP analogs (commonly GTPγS), the extent of G-protein activation is predicted to be a function of both receptor number and the rate of GTP analog hydrolysis. The sensitivity of G-protein activation to receptor number is shown to be dependent upon the assay used, with the sensitivity of phosphate production assays (GTPase) > GTPγS binding assays > cAMP inhibition assays. Finally, the amount of competition or crosstalk between receptor species activating the same type of G-proteins is predicted to depend on receptor and G-protein number, but in some (common) experimental regimes this dependence is expected to be minimal. Taken together, these observations suggest that the collision coupling model, without compartments of receptors and/or G-proteins, is sufficient to explain a variety of observations in literature data.

Naltrexone (NTX) exhibited approximately 3-fold higher affinity for sites labeled by [3H]U69,593 (putative κ1-selective ligand) than [3H]bremazocine (non-selective ligand) in the presence of mu and delta receptor blockade in monkey brain membranes. This led us to test an hypothesis that NTX could display in vivo antagonist selectivity for κ1- versus non-k1-mediated effects. Six opioid agonists were characterized by NTX apparent pA2 analysis in a 50°C water tail-withdrawal assay in rhesus monkeys. Constrained NTX pA2 values (95% confidence limits) were: alfentanil, 8.66 (8.47– 8.85); ethylketocyclazocine, 7.97 (7.93– 8.01); U69,593, 7.64 (7.49 –7.79); U50,488, 7.55 (7.42–7.67); bremazocine, 6.92 (6.73–7.12); enadoline, 6.87 (6.69– 7.05). Pretreatment with clocinnamox, an irreversible mu antagonist, confirmed that mu receptors were not involved in the antinociception produced by the kappa agonists, U69,593, U50,488, bremazocine and enadoline; however, both mu and kappa receptors mediated the antinociceptive effects of ethyl-ketocyclazocine. The apparent NTX pA2 profile of opioid agonists correlated highly with the radioligand binding studies, which indicates that U69,593 and U50,488 produced antinociception by acting on kappa-1 receptors, whereas bremazocine and enadoline probably acted via non-kappa-1 receptors. This study provides further functional evidence of kappa opioid receptor multiplicity in primates and suggests that NTX may be a useful tool to study this phenomenon in vivo.

Bombesin along with several closely related neuropeptides elicit scratching behavior when administered centrally. The first part of the study was designed to determine the antagonistic effects of a novel phyllolitorin analogue wdesTrp3,Leu8]phyllolitorin (DTP) on scratching induced by three peptides (bombesin, neuromedin-C, and [Leu8]phyllolitorin). In addition, the binding affinity of each peptide for the bombesin receptor site was determined. DTP (30 μg) inhibited scratching induced by these peptides, but unlike the peptides, DTP had no affinity for the bombesin site, thereby suggesting that DTP is displaying physiological antagonism through an unknown mechanism.

A series of N-substituted-4-cyano-4-phenylpiperidine analogs were synthesized and evaluated for binding affinity at opioid receptors and showed no affinity. The series similarity to previously reported σ ligands prompted analysis at σ receptors to determine the SAR for affinity at σ receptors. Within the N-substituent series the saturated analogs showed increased affinity at both σ receptors. Optimal chain length in the N-arylalkyl series for σ1 and σ2 receptors proved to be N-propylphenyl; extension to a four carbon chain dramatically decreased affinity at both receptors. Substituents in the 4-position affects only σ1 affinity; no change in affinity at σ2 was shown. The N-isobutyl, N-phenylpropyl, and N-benzyl analogs are worth pursuing due to their good affinity and selectivity at the σ1 receptor, whereas the N-benzyl analog exhibits the greatest selectivity for σ1.

To investigate the effects of carboxylic ester and acid moieties as the N-substituent of opioids, a short series of racemic N-substituted normetazocines was prepared. The introduction of both groups as the normetazocine N-substituent produced compounds which displayed low potency in vitro and in vivo, with the esters displaying the greater activity. The pharmacology of the compounds is discussed with implications resulting from potential in vivo metabolic hydrolysis.

BACKGROUND

Endomorphin-1 and endomorphin-2 are endogenous peptides that are highly selective for μ-opioid receptors. However, studies of their functional efficacy and selectivity are controversial. In this study, we systematically compared the effects of intrathecal (i.t.) administration of endomorphin-1 and -2 on nociception assays and G protein activation with those of [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a highly effective peptidic μ-opioid receptor agonist.

METHODS

Male Sprague-Dawley rats were used. Acute and inflammatory pain models were used to compare the duration and magnitude of antinociception. Agonist-stimulated [35S]GTPγS binding was used to observe the functional activity at the level of the receptor-G protein in both spinal cord and thalamic membranes. In addition, antagonists selective for each receptor type were used to verify the functional selectivity of endomorphins in the rat spinal cord.

RESULTS

After i.t. administration, endomorphin-1 and -2 produced less antinociceptive effects than DAMGO in the model of acute pain. Concentration–response curves for DAMGO-, endomorphin-1-, and endomorphin-2-stimulated [35S]GTPγS binding revealed that both endomorphin-1 and -2 produced less G protein activation (i.e., approximately 50%–60%) than DAMGO did in the membranes of spinal cord and thalamus. In addition, i.t. endomorphin-induced antinociception was blocked by μ-opioid receptor selective dose of naltrexone (P < 0.05), but not by δ- and κ-opioid receptor antagonists, naltrindole and nor-binaltorphimine (P > 0.05).

CONCLUSIONS

Endomorphins are partial agonists for G protein activation at spinal and thalamic μ-opioid receptors. Both in vivo and in vitro measurements together suggest that DAMGO is more effective than endomorphins. Spinal endomorphins’ antinociceptive efficacy may range between 53% and 84% depending on the intensity and modality of the nociceptive stimulus.

The 14-aminodihydromorphinone and codeinone series of opioid ligands have produced a number of ligands of substantial interest. In order to investigate the importance of the 14-substituent a series of analogues in which the side chain length is varied and the amide and alkene functions are reduced have been prepared. Binding affinity, particularly at the mu opioid receptor (MOR), was largely determined by the aromatic group of the side chain. In the [35S]GTPγS functional assay, the ligands having a three carbon side chain were more potent antagonists than their longer chain counterparts, whilst shorter, 2 carbon chain analogues were of higher MOR efficacy, an effect that was confirmed in vivo. Wash-resistant binding was observed within this series and appeared to be unrelated to side chain length.

In recent years there has been substantial interest in the 14-aminodihydromorphinone derivatives methoclocinnamox (MC-CAM) and clocinnamox (C-CAM). In order to investigate the importance of the cinnamoyl ring substituent a series of analogues have been prepared with chloro, methyl and nitro-substituents in the 2’- and 4’-positions. Despite some discrepancies between the in vitro and in vivo data, a clear SAR could be observed where the 2’-chloro and 2’-methyl ligands consistently displayed higher efficacy than their 4’-substituted analogues. The new series also followed the well established SAR that 17-methyl ligands have greater efficacy at the mu opioid receptor than their 17-cyclopropylmethyl counterparts.

Previous studies have demonstrated that peptidic and nonpeptidic δ-opioid receptor agonists have different effects depending on the measure. For example, nonpeptidic δ-opioid agonists, but not peptidic agonists, produce convulsions in rats, and in vitro studies suggested that peptidic and nonpeptidic δ-opioid agonists might have differential mechanisms of receptor down-regulation. The present study evaluated potential differences between peptidic and nonpeptidic δ-opioid agonists in their ability to activate G proteins using guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) autoradiography experiments in rat brain slices. The peptidic agonist [D-Pen2,D-Pen5]-enkephalin and the nonpeptidic agonist (+)BW373U86 [(+)-4-[α(R)-α-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide] demonstrated concentration-dependent increases in [35S]GTPγS binding that were attenuated by the δ-opioid antagonist naltrindole. (+)BW373U86 was more potent and efficacious than the peptidic agonist, and this difference remained consistent across brain regions where significant stimulation was observed. In addition, multiple δ-opioid compounds were evaluated for their agonist activity in this assay. These data suggested that differences between peptidic and nonpeptidic δ-opioid agonists in behavioral studies were most likely caused by differences in agonist efficacy. Finally, these data also revealed that [35S]GTPγS autoradiography could be used to compare efficacy differences among agonists across various brain regions in rat brain slices.

6β-Naltrexol is the major metabolite of the opioid receptor antagonist, naltrexone, in humans. However, there are no functional studies of 6β-naltrexol in primates. The aim of this study was to compare the in vitro and in vivo potencies of naltrexone and 6β-naltrexol in rhesus monkeys. Affinity and potency were determined using radioligand displacement and stimulation of 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding in monkey brain membranes. In vivo apparent pA2 analysis was applied to compare the μ-opioid receptor (MOR) antagonist potency of both compounds in nondependent monkeys. In addition, the potencies of both compounds were determined in precipitating withdrawal manifested by increased respiratory parameters in acute morphine-dependent monkeys. In vitro assays revealed that naltrexone displayed 2-fold higher affinity and potency than 6β-naltrexol for the MOR binding site and for MOR agonist-stimulated [35S]GTPγS binding, respectively. 6β-Naltrexol (0.32–3.2 mg/kg) dose-dependently produced parallel rightward shifts of the dose-response curve of alfentanil-induced antinociception. Nevertheless, the apparent pA2 value of 6β-naltrexol (6.5) was 100-fold less potent than that of naltrexone (8.5) determined previously. 6β-Naltrexol was also less potent than naltrexone in antagonizing other MOR-mediated effects including respiratory depression and itch/scratching. Naltrexone (0.0032–0.032 mg/kg) and 6β-naltrexol (0.32–3.2 mg/kg) retained the same potency difference in precipitating withdrawal to a similar degree. Furthermore, 6β-naltrexol failed to block naltrexone-precipitated withdrawal in morphine-dependent monkeys. These results indicate that naltrexone and 6β-naltrexol display similar pharmacological actions with a large in vivo potency difference in monkeys such that 6β-naltrexol may play a minimal role in the therapeutic or antagonist effects of naltrexone in primates.

Nonpeptidic δ-opioid agonists produce a number of behaviors, such as antidepressant-like effects, locomotor stimulation, antinociception, and convulsions. To consider this class of compounds as potential therapeutics for humans, the effects of δ-opioid agonists after repeated administration must be evaluated. Therefore, the present study investigated the effects of repeated δ-opioid agonist, SNC80 ([(+)-4-[(αR)-α-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)-methyl]-N,N-diethylbenzamide), administration on its antidepressant-like effects in the forced swim test, locomotor activity, and convulsions in male Sprague-Dawley rats. Tolerance developed rapidly to the convulsive and locomotor-stimulating effects of SNC80 but not to the antidepressant-like effects. In addition, tolerance was evaluated at the level of the receptor-G protein interaction by measuring 5′-O-(3-[35S]thio)triphosphate binding in brains from rats that were pretreated with SNC80. With various exposure durations to SNC80, some brain regions demonstrated tolerance at different times, suggesting that adaptations in the δ-opioid system may occur during agonist exposure. Overall, the lack of observable tolerance to the antidepressant-like effects of SNC80 indicates that this class of compounds has potential as a novel antidepressant therapy.

We have previously shown that cinnamoyl derivatives of 14β-amino-17-cyclopropylmethyl-7,8-dihydronormorphinone and 7α-aminomethyl-6,14-endoethanonororipavine have pronounced pseudoirreversible μ opioid receptor (MOR) antagonism. The present communication describes the synthesis and evaluation of fumaroylamino analogues of these cinnamoylamino derivatives together with some related fumaroyl derivatives. The predominant activity of the new ligands was MOR antagonism. The fumaroylamino analogues (2a, 5a) of the pseudoirreversible antagonist cinnamoylamino morphinones and oripavines (2b, 5b) were themselves irreversible antagonists in vivo. However the fumaroylamino derivatives had significantly higher MOR efficacy than the cinnamoylamino derivatives in mouse antinociceptive tests. Comparison of 2a and 5a with the prototypic fumaroylamino opioid β-FNA (1a) shows that they have similar MOR irreversible antagonist actions but differ in the nature of their opioid receptor agonist effects; 2a is a predominant MOR agonist and 5a shows no opioid receptor selectivity, whereas the agonist effect of β-FNA is clearly κ opioid receptor (KOR) mediated.

3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH) are amphetamine analogues with similar persistent neurochemical effects in the mouse which some have described as neurotoxicity. We attempted to identify dose regimens of MDMA and METH with similar effects on behavioral and physiological variables in the mouse, then quantified the effects of these dose regimens on neurochemistry and microglial markers. Four discrete injections of saline, MDMA (10, 20, or 30 mg/kg), or METH (5 or 10 mg/kg) were administered to mice at 2 h intervals. Body weight was quantified immediately before each injection, and 2 h after the last injection, while core temperature and locomotor activity were continuously monitored via radiotelemetry. Mice were sacrificed 72 h after the final injection and brains were rapidly dissected on ice. Dopamine content in various brain regions was quantified via high pressure liquid chromatography (HPLC), and microglial activation was assessed by saturation binding of the peripheral benzodiazepine receptor (PBR) ligand [3H]PK11195. Specific dose regimens of MDMA and METH induced similar reductions in body weight, depletions of dopamine and its metabolites, and similar hyperthermic and locomotor stimulant effects, but only METH activated microglia in striatum. These results suggest that repeated high doses of MDMA and METH that produce hyperthermia, locomotor stereotypy, weight loss and neurochemical depletion are not consistently accompanied by microglial activation. The finding that METH, but not MDMA, induces microglial effects in the striatum consistent with neurotoxicity might imply different mechanisms of toxic action for these two psychostimulants.