Biochemical adaptations in the mesolimbic dopamine system in response to repeated stress.

We have demonstrated previously that chronic administration of morphine, cocaine, or ethanol produces some common biochemical adaptations in the ventral tegmental area (VTA) and nucleus accumbens (NAc), components of the mesolimbic dopamine system implicated in the reinforcing and locomotor activating properties of these drugs of abuse. Because this neural pathway is also regulated by stress, and because stress has been shown to influence an animal's behavioral responses to drugs of abuse, it was of interest to determine whether repeated exposure to stress results in similar biochemical adaptations. By use of immunoblot analysis, we show here that a course of chronic "unpredictable" stress, like chronic drug exposure, increased levels of immunoreactivity of tyrosine hydroxylase and glial fibrillary acidic protein and decreased levels of immunoreactivity of neurofilament proteins in the VTA. Chronic unpredictable stress also increased levels of cyclic AMP-dependent protein kinase activity and decreased levels of immunoreactivity of the G protein subunit, Gi alpha, in the NAc. These effects required long-term exposure to stress and were in most cases not seen in the substantia nigra and caudate-putamen, components of the nigrostriatal dopamine system studied for comparison. The biochemical effects of chronic stress in the VTA and NAc differed among three strains of rat studied. Fischer 344 rats were the most responsive in that they exhibited all of the aforementioned adaptations, whereas Lewis rats were the least responsive in that they exhibited none of these adaptations; Sprague-Dawley rats exhibited an intermediate number of responses. Taken together, the results of the present study demonstrate that chronic exposure to stress results in biochemical adaptations in the mesolimbic dopamine system that resemble the chronic actions of several drugs of abuse. These adaptations could contribute to the convergent behavioral effects induced by treatments that are mediated via the VTA-NAc pathway.

Messenger RNA editing of the human serotonin 5-HT2C receptor.

RNA encoding the rat serotonin 5-HT2C receptor undergoes editing whereby one to four adenosines are converted to inosines. This conversion can change up to three codons out of a stretch of five in the second intracellular loop of the receptor. RNA editing of the rat 5-HT2C receptor that changes all three codons was shown previously to alter intracellular signaling by 5-HT without changing its receptor-binding affinity. We analyzed 5-HT2C receptor editing in human brain and hypothalamic RNA samples and confirmed that all four adenosine editing sites observed in rat were also present in human samples. Additionally, we identified a novel editing site in the middle edited codon that extends the repertoire of 5-HT2C receptors by six additional protein isoforms. We observed that editing reduces both the binding affinity and functional potency of agonists for recombinant human 5-HT2C receptor isoforms. This effect on binding affinity was proportional to the agonist's intrinsic activity, with full agonists most affected, and antagonists showing no effect. These data suggest that RNA editing may alter coupling energetics within the ternary complex, thereby altering agonist binding affinities, G protein coupling, and functional responses. RNA editing may thus provide a novel mechanism for regulating 5-HT synaptic signaling and plasticity.

4-(1,3-Dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2, 4-dichlorophenyl)pyrazolo[1,5-a]-1,3,5-triazine: a potent, orally bioavailable CRF(1) receptor antagonist.

Structure-activity studies in the pyrazolo[1,5-a]-1,3,5-triazine series led to the discovery that compound 11i (DMP696) is a potent hCRF(1) receptor antagonist (K(i) = 1.7 nM vs 7.5 nM for alpha-hel-CRF(9-41), hCRF(1) adenylate cyclase IC(50) = 82 nM vs 286 nM for alpha-hel-CRF(9-41)). Compound 11i has excellent oral pharmacokinetic profiles in rats and dogs (37% and 50% oral bioavailabilities, respectively). This compound displays good activity in the rat situational anxiety model (MED = 3 mg/kg (po)), whereas a literature standard 1 (CP154526-1) was inactive (MED > 30 mg/kg (po)). Analogue 11i reduced stereotypical mouth movements in rhesus monkeys by 50% at 21 mg/kg (po) using the human intruder paradigm. Overall, the profile of pyrazolotriazine 11i indicates that hCRF(1) receptor antagonists may be anxiolytic agents, which have reduced motor side effect profiles.

Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines.

The synthesis and CRF receptor binding affinities of several new series of N-aryltriazolo- and -imidazopyrimidines and -pyridines are described. These cyclized systems were prepared from appropriately substituted diaminopyrimidines or -pyridines by nitrous acid, orthoester, or acyl halide treatment. Variations of amino (ether) pendants and aromatic substituents have defined the structure-activity relationships of these series and resulted in the identification of a variety of high-affinity agents (Ki's < 10 nM). On the basis of this property and lipophilicity differences, six of these compounds (4d,i,n,x, 8k, 9a) were initially chosen for rat pharmacokinetic (PK) studies. Good oral bioavailability, high plasma levels, and duration of four of these compounds (4d,i,n,x) prompted further PK studies in the dog following both iv and oral routes of administration. Results from this work indicated 4i,x had properties we believe necessary for a potential therapeutic agent, and 4i1 has been selected for further pharmacological studies that will be reported in due course.

Circling behavior exhibited by a transgenic insertional mutant.

We report here of an abnormal circling behavior expressed in the TgX15 transgenic mouse line as a result of insertional mutagenesis. Homozygous transgenic mice expressed the phenotype while heterozygous transgenics were normal. We also found that the dopamine D2 receptor binding sites in the striata of the circling mice were significantly elevated by about 31% compared to normal heterozygous transgenic mice. Other transgenic lines constructed with the same transgene appeared normal suggesting that, in the TgX15 line, a genetic locus significant in mammalian motor behavior has been disrupted.

Ontogeny of hyperactivity and circling behavior in a transgenic insertional mutant mouse.

In a previous study (Ratty et al., 1990), the discovery of a transgenic mutant mouse that displayed abnormal circling behavior was reported. Mice homozygous for the transgene display this phenotype, whereas heterozygotes are phenotypically normal. In this study, circling mutants displayed excessive lateralized circling behavior and locomotor hyperactivity by Postnatal Days (PND) 14 and 16, respectively. These abnormalities persisted unattenuated through adolescence and adulthood. Disturbances in rearing and grooming were also observed in circling mutants. Surface-righting ability in the mutants was normal, and the age of eye opening was only marginally delayed. However, body weights of the mutants were reduced compared with normal mice from PND 15 to adulthood. Possible relationships between the behaviors that are exhibited by circling mutants and previous neurochemical findings are discussed.

Morris water task impairment and hypoactivity following cysteamine-induced reductions of somatostatin-like immunoreactivity.

The effects of cysteamine-induced reductions of somatostatin-like immunoreactivity (SLI) on spatial learning, passive avoidance, and locomotor activity were examined in adult Sprague-Dawley rats. Cysteamine hydrochloride (100 mg/kg, s.c.) produced 54% and 50% reductions in SLI in cortex and hippocampus, respectively, and impaired escape latencies and spatial probe behavior in the Morris water task. Although cysteamine-treated rats displayed hypoactivity in the activity boxes, their swim speed in the Morris water task was unaffected. Cysteamine did not impair passive avoidance retention when administered immediately following training or prior to daily retention testing. These results suggest a role for somatostatin in spatially-mediated behaviors in rats.

Specificity of behavioral and neurochemical dysfunction in the chakragati mouse: a novel genetic model of a movement disorder.

The chakragati (ckr) mouse is a transgenic insertional mutant that displays lateralized circling behavior, locomotor hyperactivity, hyperexcitability as well as body weight deficits. The mutation is autosomal and recessive. We have previously found that ckr mice have bilateral asymmetric elevations in striatal dopamine (DA) D2-like (D2, D3 and/or D4), but not D1-like (D1 and/or D5) receptors. Predictably, these mice increase turning in response to the D2-like agonist quinpirole and spontaneously rotate contralateral to the striatal side with the higher D2-like receptors. The overall objective of the present study was to assess the neurochemical specificity of the mutation in ckr mouse, particularly since motor behaviors can be elicited by a multitude of brain regions and neurotransmitter systems within the basal ganglia. Using quantitative receptor autoradiography, we examined the regional distribution of DA uptake sites and 5-HT1A, 5-HT1B/1D, GABAA and mu opioid receptors. Also, we wanted to determine whether increased behavioral laterality as seen in rotation is evident with another test of laterality, such as lateral paw preference. The ckr mice showed greater paw preferences than normal mice; however, neither the degree nor direction of these preferences correlated with rotational behavior. The ckr mice showed moderate decreases in the density of DA uptake sites in all subregions of the striatum, but not in the nucleus accumbens or olfactory tubercle. Interestingly, these decreases in ckr mice were not accompanied by a reduction in striatal tissue DA content. 5-HT1 and mu opiate receptor populations were normal in ckr mice. However, GABAA sites in the mediodorsal thalamus and superior colliculus were bilaterally and asymmetrically elevated in ckr mice. These data are consistent with the idea that the motor phenotypes of the ckr mouse result from specific disturbances within nigro-striatal, striato-pallido-thalamic and striato-nigro-collicular circuitry. The implications of these and past findings are discussed in relation to current thinking about hyperkinetic motor syndromes in humans involving reduced basal ganglia outflow.

Lateralized changes in prefrontal cortical dopamine activity induced by controllable and uncontrollable stress in the rat.

Exposure to stressors that are not controlled results in a variety of changes in behavior and in brain chemistry. Among these is the activation of dopamine-containing neuronal systems projecting to the medial prefrontal cortex (PFC), to a lesser extent the nucleus accumbens (NAC) and, in a few studies, the striatum. Previous data have shown that stressor evoked PFC activation is asymmetrical. The present experiments were designed to assess the effects of controlled and uncontrolled stressors on these DA systems using the procedures of the learned helplessness (LH) model. In a first experiment, 80 trials of either a controllable (ESC) or identical uncontrollable footshock stressor (YOK) caused an activation, as indicated by increased metabolite concentrations of DA in the PFC, NAC and striatum. In the PFC, YOK caused a bilateral DA depletion, relative to ESC and control animals, and a right > left increase in DOPAC/DA which was not seen in ESC animals. These findings suggested a preferential effect of YOK in the right PFC. A second experiment used rats that had been grouped according to their turning behavior, YOK right-biased rats showed an increase in DOPAC on the right side of the PFC and YOK left-biased rats displayed a similar increase on the left side in response to a brief (5 min) controllable footshock stressor. Since right-turning rats had been shown to be more sensitive to a LH behavioral phenomenon, the data suggested that right PFC activation is responsible for the greater LH sensitivity. A final experiment evaluated the neurochemical and behavioral responses to a prolonged footshock stressor 24 h after uncontrolled footshock. Right-biased YOK animals displayed depressed footshock escape behavior and a right > left depletion in PFC DA and HVA. Across-groups footshock escape performance was correlated with DA and HVA concentrations on the right but not on the left side of the PFC. Thus a disturbance of right PFC DA utilization was again associated with compromised coping behavior. The data suggest that the inability to control a stressor causes a lateralized alteration of PFC DA and this results in a disruption of the ability to respond to a new stressor. These findings indicate that the two sides of the PFC are differentially specialized for responding to a stressor.

Side and region dependent changes in dopamine activation with various durations of restraint stress.

Exposure to various mild stressors has been shown to result in the activation of dopamine containing neuronal systems projecting to the medial prefrontal cortex (PFC), to a lesser extent the nucleus accumbens septi/olfactory tubercle (NAS) and, in a few studies, the striatum. It has also been shown that dopamine (DA) systems on different sides of the PFC are successively activated as stressors are prolonged. We have therefore examined the effects of variation in the duration of a restraint stressor (15, 30 and 60 min) on region and side dependent alterations in DA utilization in the PFC, NAS and striatum. Increases in the concentrations of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and/or homovanillic acid (HVA) or in their ratios with DA were seen in all regions examined with the largest effects occurring in the PFC and lesser effects in the NAS and striatum. In each region, the magnitude of these effects varied with time of restraint exposure. In the PFC, lateralized alterations in HVA and DA were seen over time with effects progressing from a left greater than right involvement at 15 min to a right greater than left involvement at 60 min. These results are discussed with reference to side and region dependent effects on brain DA systems as stressors are prolonged and the implications they may have for lateralized regional brain activity associated with stressor precipitated psychiatric disease.

Asymmetric elevation of striatal dopamine D2 receptors in the chakragati mouse: neurobehavioral dysfunction in a transgenic insertional mutant.

We have previously reported the discovery of a transgenic insertional mutant, recently named the chakragati (ckr) mouse, which displays lateralized circling, locomotor hyperactivity, hyperreactivity, as well as body weight deficits. Since lateralized dopamine function is associated with circling behavior we sought to determine whether dopamine (DA) D1 and D2 receptors were asymmetrically distributed in the striata of adolescent and adult ckr mice using receptor autoradiography. Stereotypic and rotational responses to quinpirole served as behavioral indices of D2 receptor function. The ckr mice showed hemispherically asymmetric elevations in DA D2 receptors in the lateral subregions of the striatum whereas medial regions of the striatum were symmetrically and bilaterally elevated (overall elevation = 30%). As a group, ckr mice had higher D2 receptor levels on the side which was contralateral to the preferred direction of spontaneous nocturnal rotation. Striatal D1 receptors and mesolimbic D2 and D1 receptors of ckr mice were neither elevated nor differentially asymmetric. Young adult ckr mice showed dose-dependent increases in net rotations in response to quinpirole whereas normal mice showed no change from baseline levels. Both groups showed similar stereotypic responses. Older adult ckr mice, however, showed dose-dependent reductions in rotation after quinpirole whereas normal mice turned at baseline levels. Older ckr mice also displayed significantly greater stereotyped sniffing behavior. This unique mutant provides a novel genetic model of basal ganglia dysfunction, and may be useful in studying aspects of neuropsychiatric disorders associated with dopaminergic abnormalities.

Cholinergic maturation and SCH 23390-induced catalepsy in the male rat pup.

The cataleptogenic effects of the selective dopamine D1 receptor antagonist, SCH 23390, increased between 13 and 17 days of age in male pups. Seventeen- and 21-day-old pups showed equivalent catalepsy. Scopolamine blocked SCH 23390-induced catalepsy in 21-day-old pups but had little effect in 13-day-old pups. The development and cholinergic sensitivity of SCH 23390-induced catalepsy are similar to those seen after D2 or mixed D1/D2 receptor blockade. Cholinergic maturation appears to be an important component in the development of adult-like catalepsy, and the nature of a D1-acetylcholine interaction mediating catalepsy remains to be determined.

Right-sided population bias in male rats: role of stress.

Recent studies have shown that rats develop a pronounced right-sided population bias when tested repeatedly over several days in a shock-escape T-maze paradigm. In the present study we sought to determine if this bias was the result of learning or of the repeated exposure to a stressor (footshock). Rats were tested in the T-maze for 5 trials on each of 6 consecutive days. One group of rats (HS-C) was allowed a free left or right choice on each trial whereas another group of rats (HS-A) was forced to alternate left and right turns on each trial for the first 5 days of testing and then allowed a free choice on Day 6. The population and individual laterality of the HS-A group did not differ from that of the HS-C group, indicating that learning does not account for the biases. A third group of rats (LS-C) was tested using a lower shock level; this decrease in stressor intensity delayed the development of a right-sided population bias without affecting the development of individual laterality. These results indicate that repeated exposure to a stressor, rather than learning, is primarily responsible for the marked right-sided population bias observed in the T-maze.

Absence of differential motoric and thermic responses to clonidine in young rats exposed prenatally to alcohol.

We studied the involvement of altered noradrenergic function in the behavioral consequences of fetal ethanol exposure. Pregnant rats were fed a liquid diet containing 35% Ethanol-Derived Calories (EDC), pair-fed a 0% EDC diet with sucrose substituted isocalorically for ethanol, or fed ad lib lab chow. In Experiment 1, offspring from these prenatal treatment groups were injected with the alpha-adrenergic agonist clonidine (0.5 or 2.0 mg/kg) at 10 or 42 days of age and showed age-characteristic, dose-dependent increases in locomotor activity, exploration, forelimb treading, and catalepsy. In Experiment 2, offspring were injected with clonidine (0.25 or 1.0 mg/kg) at 10, 14, or 18 days of age, and locomotor activity and wall-climbing were observed in a warm chamber (33 degrees C). Catalepsy and rectal temperature were also scored. There were no significant differential effects of clonidine on any prenatal treatment group at any age for any measure of activity or rectal temperature. The results do not support the hypothesis that fetal ethanol exposure alters noradrenergic systems in behaviorally significant ways.

Quantitative autoradiographic analysis of [3H]carfentanil binding to mu opiate receptors in the rat brain.

Fentanyl and its derivatives are considered among the most potent opiate analgesic/euphoriants. The pharmacological literature generally supports a mu opiate receptor site of action for the fentanyl derivatives, but some observations suggest that other sites of action may be involved in producing the extremely potent fentanyl effects. In order to investigate the mechanism of action of fentanyl-like drugs further, [3H]carfentanil was used as a radioligand to image high-affinity carfentanil binding sites in slidemounted sections of the rat brain (receptor autoradiography). In parallel studies the prototypical mu opiate agonist radioligand [3H]DAMGO ([D-Ala2-MePhe4-Gly-ol5]enkephalin) was also used. The working hypothesis was that if carfentanil was acting through another high-affinity site besides the mu opiate receptor, the distribution pattern of the autoradiographic image produced by [3H]carfentanil should be significantly different than the autoradiographic pattern displayed by the well-characterized and selective mu opiate [3H]DAMGO. Thirty-five brain regions were examined for specific [3H]carfentanil and [3H]DAMGO binding. The absolute and relative densities of the sites were essentially identical. The highest levels of binding were observed in the "patch" areas of the striatum (131 +/- 5 fmol/mg T.E. for [3H]carfentanil; 162 +/- 13 fmol/mg T.E. for [3H]DAMGO). The lowest levels were observed in the cerebellum where no specific binding of either radioligand was observed. The overall distribution pattern of the two radioligands produced a correlation coefficient of 0.95; the distribution pattern was prototypical for the mu opiate receptor as reported previously by other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular and cellular adaptations in signal transduction pathways following ethanol exposure.

The purpose of this review is to provide an overview of the acute actions of ethanol on signal transduction, as well as a selective consideration of some of the long-term adaptive changes in signal transduction pathways that may underlie clinical manifestations of ethanol dependence, tolerance, withdrawal, and addiction. The acute intoxicating effects of ethanol have been widely attributed to its ability to block voltage-gated Ca2+ and Na+ channels and N-methyl-D-aspartate glutamate receptor cation channels, and to facilitate GABAA receptor Cl- channels. Adaptive changes in these same proteins following chronic ethanol exposure may contribute to physical and psychological signs of ethanol dependence and withdrawal. Ethanol, as with other drugs of abuse, also acutely activates the mesolimbic dopamine pathway, an effect which likely accounts, at least in part, for ethanol's acute reinforcing properties. Studies directed at unraveling the biochemical and molecular basis of ethanol's acute and chronic actions may lead to the development of novel pharmacotherapeutics that mitigate aspects of acute ethanol intoxication and, more importantly, treat the effects of withdrawal and addiction (craving) associated with long-term ethanol abuse.

Serotonin metabolism by monoamine oxidase in rat primary astrocyte cultures.

The oxidative deamination of serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5-HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5-HT concentration over the tested range of 5 x 10(-7) to 10(-4) M. At 5 x 10(-7) M 5-HT, intracellular 5-HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5-HT taken up into astrocytes is not stored for re-release, but is rapidly metabolized to 5-HIAA, which is then extruded from the cell. At 5 x 10(-7) M 5-HT, 5-HIAA formation in intact cells was blocked 63% by the selective high-affinity 5-HT uptake inhibitor fluoxetine. 5-HT oxidation to 5-HIAA is carried out principally by MAO-A, because clorgyline was more effective at inhibiting the production of 5-HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000-fold more effective than pargyline at inhibiting MAO activity toward 14C-labelled 5-HT. However, the relatively selective MAO-B substrate beta-phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO-B activity; the Km values for MAO-A oxidation of 5-HT and MAO-B oxidation of PEA were 135 and 45 microM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (greater than 20 microM) were oxidized by both MAO-A and MAO-B isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cortical and subcortical glutamate receptor subunit expression by antipsychotic drugs.

Because glutamate is an important modulator of subcortical dopamine (DA) function, and abnormal glutamate/DA interactions may be involved in the pathophysiology of schizophrenia, we examined the effect of chronically administered antipsychotic drugs (APDs) on the levels of specific glutamate receptor subunits in the terminal fields of nigrostriatal and mesocorticolimbic DA systems. By immunoblotting procedures using antibodies specific for the NMDAR1, GluR1, and GluR2 subunits, we found that haloperidol (predominantly a D2-like antagonist) increased NMDAR1 subunit immunoreactivity (and mRNA levels) in the striatum, while the D1-like antagonist SCH 23390 had the opposite effect. No effect was seen on GluR1 or GluR2 levels. The result that D1-like and D2-like receptor antagonism can reciprocally regulate NMDAR1 expression is consistent with our observation that complete unilateral destruction of the nigrostriatal DA pathway with 6-hydroxy-dopamine had no effect on striatal NMDAR1 subunit levels. Further examination of these striatal effects revealed that chronic treatment with the D2-like receptor antagonist raclopride significantly increased NMDAR1 levels in the striatum, while the 5-HT2a/2c antagonist mianserin tended to produce an increase that did not achieve statistical significance. These findings indicate that the dopaminergic antagonist properties of haloperidol are likely most responsible for its regulation of this subunit. In contrast, the atypical APD clozapine had no effect on striatal NMDAR1 levels, consistent with the relatively weaker influence of this drug on nigrostriatal DA function. The second major finding of the present study was the ability of haloperidol and clozapine to increase GluR1 levels in the medial prefrontal cortex (PFC), whereas chronic SCH 23390 treatment decreased GluR1 levels.

Pharmacological and biochemical characterization of a recombinant human galanin GALR1 receptor: agonist character of chimeric galanin peptides.

The galanin neuropeptide system is widely distributed throughout the brain and periphery and is thought to play a role in feeding, pain and reproduction. To evaluate the human galanin receptor 1 as a potential therapeutic target, we fully characterized its interaction with several galanin-like peptides. The human galanin receptor 1 receptor was stably expressed using an episomal system in human embryonic kidney 293E cells. Saturation isotherms using 125I-human galanin revealed two distinct populations of receptor affinity states in membranes and whole cells with picomolar and nanomolar affinities at the high- and low affinity states, respectively. A scintillation proximity assay revealed that 125I-human galanin binding in membranes reached steady-state within 2 to 2.5 hr; however, only 50% of galanin radiolabel dissociated from the receptors by excess galanin or guanosine 5'-O-3-thiotriphosphate even after 20 hr. In contrast, galanin binding in whole cells was completely reversible within 1 hr. Competition binding assays showed that galanin-like peptides bound with picomolar affinities in membranes and whole cells. These peptides behaved as full agonists as determined by the inhibition of forskolin-stimulated cyclic 3'5'-adenosine monophosphate production and the stimulation of guanosine 5'-O-(3-[35S]thiotriphosphate binding. The agonist profile of M40, a representative chimeric peptide, was found not to be the result of receptor reserve because receptor inactivation by partial alkylation experiments confirmed its full intrinsic efficacy under conditions of a "zero" reserve state. These observations suggest that the antagonist effects in vivo of M40, and perhaps other chimeric peptides, are not mediated via direct interactions with the galanin receptor 1 receptor.

Drugs of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate receptor subunits in the rat ventral tegmental area: common adaptations among cross-sensitizing agents.

Behavioral and electrophysiological evidence suggests that glutamatergic neurotransmission plays an important role in some of the long-term effects of cocaine and other drugs of abuse on brain function. We therefore examined the effect of repeated cocaine treatment on glutamate receptor subunit expression in central dopamine (DA) pathways implicated in many of cocaine's behavioral actions. By immunoblotting procedures using subunit-specific antibodies, we found that repeated, but not acute, cocaine treatment increased the levels of immunoreactivity of GluR1 (an AMPA receptor subunit) and NMDAR1 (an NMDA receptor subunit) in the ventral tegmental area (VTA), a nucleus containing mesolimbic DA neurons. In contrast, chronic cocaine treatment did not alter levels of GluR2 (an AMPA receptor subunit), NMDA2A/B (NMDA receptor subunits), or GluR6/7 (kainate receptor subunits) in this brain region. Moreover, GluR1 and NMDAR1 levels were not regulated in other regions of the mesolimbic or nigrostriatal DA pathways, including the substantia nigra. Because several drugs of abuse and stress can elicit common and cross-sensitizing effects on mesolimbic DA function, we next examined whether repeated morphine and stress treatments would regulate these proteins similarly in the VTA. Although morphine delivered by subcutaneous pellet implantation had no significant effect on subunit levels, morphine delivered intermittently by subcutaneous injections of escalating doses elevated GluR1 levels in the VTA. Repeated restraint stress also paradigm (2 stressors/d under variable conditions) increased both GluR1 and NMDAR1 levels in this brain region. Unlike cocaine, morphine, and stress, repeated treatment with other psychotropic drugs (haloperidol, raclopride, sertraline, and desipramine) that lack reinforcing or sensitizing properties did not regulate GluR1 or NMDAR1 subunit levels in the VTA. Increased glutamate receptor subunit expression in the VTA may represent an important molecular mechanism by which drugs of abuse and stress exert common, long-term effects on mesolimbic DA function.