Regulation of 5-HT(2A) receptor mRNA levels and binding sites in rat frontal cortex by the agonist DOI and the antagonist mianserin

In the present study we have characterized the time course of effect of administration of the serotonin(2) (5-HT(2)) receptor antagonist mianserin, or the 5-HT(2) receptor agonist (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), on 5-HT(2A) receptor binding sites and mRNA levels in rat frontal cortex. Radioligand binding and ribonuclease protection assays were performed with separate hemispheres of frontal cortex from each animal to examine concomitant changes in 5-HT(2A) receptor sites and mRNA levels. The decrease in cortical 5-HT(2A) receptor sites in response to chronic DOI administration was not accompanied by changes in 5-HT(2A) receptor mRNA. A single injection of DOI produced a transient decrease in 5-HT(2A) receptor mRNA levels detected 1 h post-injection. The density of 5-HT(2A) receptor sites, however, was not significantly reduced following a single injection of DOI. The down-regulation of cortical 5-HT(2A) receptor sites in response to a single injection of mianserin was accompanied by reductions in 5-HT(2A) receptor mRNA levels. Following 4 days of mianserin administration, however, we did not observe a change in 5-HT(2A) receptor mRNA levels, although 5-HT(2A) receptor density was decreased. Thus, changes in receptor mRNA may initially contribute to the down-regulation of 5-HT(2A) receptors in response to acute mianserin administration. Sustained changes in 5-HT(2A) receptor mRNA, however, appear not to be involved in maintaining the down-regulation of 5-HT(2A) receptor number with chronic mianserin administration. Mechanisms other than the regulation of receptor mRNA levels appear to underlie the down-regulation of 5-HT(2A) receptor sites in response to chronic administration of the agonist DOI.     

Substance P and neurokinin levels are decreased in the cortex and hypothalamus of alcohol-preferring (P) rats

OBJECTIVE: Central tachykinin levels (i.e., substance P [SP], neurokinin A [NKA], neurokinin B [NKB] and neurokinin K [NKK]) have been reported to fluctuate in association with stress and anxiety. Ethanol can also modulate stress and anxiety. Further, ethanol intake can change as a result of stress and anxiety. This suggests possible interactions between ethanol and central tachykinins, i.e., changing tachykinin levels could influence ethanol intake and vice versa. However, to date few studies have assessed the potential relationship between tachykinin levels in the brain and ethanol preference. The present study was designed to determine if rodent lines selectively bred for differences in alcohol preference (i.e., alcohol-preferring [P] and non-preferring [NP] rats) have different tachykinin levels in the brain. METHOD: Tissue samples from the brains of ethanol-naive P and NP rats were collected from the frontal cortex, caudate, hippocampus, amygdala and hypothalamus. Using radioimmuno assays, concentrations of SP-like immunoreactivity (SP-LI) and neurokinin-like immunoreactivity (NK-LI, i.e., neurokinin A, B and K) in P and NP rats were determined. RESULTS: In P rats, SP-LI was significantly lower in the frontal cortex (F= 12.80, 1/26 df, p = .001) while NK-LI was significantly lower in the frontal cortex (F = 7.36, 1/26 df, p = .012) and hypothalamus (F = 5.32, 1/26 df, p = .029) compared with NP rats. CONCLUSIONS: These data indicate that endogenous SP and neurokinin levels are reduced in discrete brain regions of P rats compared with NP rats. These decreases may be associated with decreased serotonergic innervation of these brain regions in P rats compared with NP rats.     

Catechol O-methyltransferase (COMT) mRNA expression in the dorsolateral prefrontal cortex of patients with schizophrenia.

Human prefrontal cortical neurons express catechol O-methyltransferase (COMT), an enzyme that inactivates the neurotransmitter dopamine. A functional polymorphism of COMT, Val(108/158) Met, affects prefrontal function, and the high-activity Val allele has been reported to be a genetic risk factor for schizophrenia. We used in situ hybridization histochemistry to measure mRNA levels of COMT in the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia (N=14) and of normal controls (N=15). While the groups did not differ in terms of mean level of COMT mRNA, there was a significantly different laminar pattern of COMT mRNA expression in pyramidal neurons (F=2.68, df=4,108, P <0.04); patients with schizophrenia had relatively lower levels in the superficial (II/III) layers and higher levels in the intermediate/deep (IV/V) layers (P&<0.01), while in controls, the expression was homogeneous across layers. Neither the mean level nor the laminar distribution of COMT mRNA was related to the Val(108/158) Met genotype, suggesting that the feedback regulation of mRNA level is not a compensation for the functional effect of the COMT polymorphism. The disease-related laminar difference of COMT expression may be involved in dysregulation of dopamine signaling circuits in the DLPFC of patients with schizophrenia.     

An investigation of the dihydropyrimidinase-like 2 (DPYSL2) gene in schizophrenia: genetic association study and expression analysis

Several linkage studies support a susceptibility locus for schizophrenia on chromosome 8p21-22. In this study, we investigated a gene mapping to 8p21, dihydropyrimidinase-like 2 (DPYSL2). DPYSL2 plays an important role in axonal formation and dysfunction of DPYSL2 may result in neurodevelopmental abnormalities. In previous studies, the expression of the gene has been shown to display alteration in the brain of schizophrenia patients compared with those of healthy controls. Recently, Nakata and colleagues found polymorphisms in the 3'-end of DPYSL2 to be associated with schizophrenia, especially the paranoid type, in a Japanese population. In this study, we genotyped four SNPs in DPYSL2 in 2552 Chinese Han specimens. Case-control and TDT analyses were performed to detect association of DPYSL2 with schizophrenia. However, no allele, genotype or haplotype association was found. We investigated the expression of DPYSL2 in 29 schizophrenia patients and 54 healthy controls using quantitative real-time PCR and no difference was found between the two groups. In a comparative allele-specific expression test, we used two SNPs as markers. Only a small proportion of heterozygotes revealed a significant difference (>20%) in allele representation. The results indicated the mRNA level did not contribute mainly in the altered expression of the gene in schizophrenia patients. Although our results provided no evidence for DPYSL2 itself as a susceptibility gene for schizophrenia, recent findings have indicated that DPYSL2 may interact with other candidate genes for schizophrenia and be worthy of further studies.     

Differential modulation of extracellular levels of 5-hydroxytryptamine in the rat frontal cortex by (R)- and (S)-zacopride.

1. The ability of various anxiolytic and potential anxiolytic agents to modify 5-hydroxytryptamine (5-HT) release in the frontal cortex of the rat was assessed by the microdialysis technique. 2. The benzodiazepine receptor agonist, diazepam (2.5 mg kg-1, i.p.), the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.32 mg kg-1, s.c.) and the 5-HT1A receptor partial agonist buspirone (4.0 mg kg-1, i.p.) maximally reduced extracellular levels of 5-HT in the rat frontal cortex by approximately 50-60%, 70-80% and 30-40%, respectively. 3. (R)-zacopride (1.0-100 micrograms kg-1, i.p.) dose-dependently reduced extracellular levels of 5-HT in the rat frontal cortex (approximately 80% maximal reduction) whereas the other 5-HT3 receptor antagonists ondansetron (10 micrograms kg-1, i.p.) and (S)-zacopride (10-100 micrograms kg-1, i.p.) were ineffective. 4. In contrast to (S)-zacopride (100 nM; administered via the microdialysis probe), (R)-zacopride (1.0-100 nM; administered via the microdialysis probe) induced a concentration-dependent reduction in extracellular levels of 5-HT in the rat frontal cortex (approximately 70% maximal reduction). 5. In contrast to ondansetron (100 micrograms kg-1, i.p.), (S)-zacopride (10-100 micrograms kg-1, i.p.) dose-dependently reversed the (R)-zacopride (10 micrograms kg-1, i.p.) induced reduction in extracellular levels of 5-HT in the rat frontal cortex. The highest dose of (S)-zacopride (100 micrograms kg-1, i.p.) completely prevented the (R)-zacopride response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylmercury increases glutamate extracellular levels in frontal cortex of awake rats

A current hypothesis about methylmercury (MeHg) neurotoxicity proposes that neuronal damage is due to excitotoxicity following glutamate uptake alterations in the astrocyte. By sampling from a microdialysis probe implanted in the frontal cortex of adult Wistar rats, we measured the effects of acute exposure to either 10 or 100 μM MeHg through the microdialysis probe, on glutamate extracellular levels in 15 awake animals. After baseline measurements, the perfusion of MeHg during 90 min induced immediate and significant elevations in extracellular glutamate at 10 μM (9.8-fold, P<.001) and at 100 μM (2.4-fold, P=.001). This in vivo demonstration of increments of extracellular glutamate supports the hypothesis that dysfunction of glutamate neurotransmission plays a key role in MeHg-induced neural damage.     

Trazodone increases extracellular serotonin levels in the frontal cortex of rats

The effects of the antidepressant drug, trazodone, on the extracellular 5-hydroxytryptamine (5-HT) levels in the frontal cortex of freely moving rats was investigated using microdialysis coupled to a high performance liquid chromatography (HPLC) detection method. Systemic administration of 1.25 and 2.5 mg/kg s.c. of trazodone was followed by a rise in the 5-HT level which reached a 5-fold peak over the basal level 5 h after injection, and a 3-fold peak after 1 h. Higher doses had no effect. The increase was prevented by pretreatment with fluoxetine (10 mg/kg s.c.), a 5-HT uptake inhibitor. Direct administration of trazodone (0.03, 0.1, 1, 2 microg/microl), by reverse dialysis into the frontal cortex, elicited a dose-dependent large increase in 5-HT levels. The increase was not prevented by systemic fluoxetine administration but was reduced by local perfusion of ketanserin (0.1 microg/microl) a 5-HT(2A/C) receptor antagonist. Trazodone s.c. administration for 7 days did not increase 5-HT basal levels but enhanced the effects of challenge doses of 2.5 and 5 mg/kg s.c. The present work demonstrated that trazodone increases the 5-HT extracellular level through a double mechanism which involves the 5-HT transporter and 5-HT(2A/C) receptors. This increase may trigger the chain of events which lead to the therapeutic effects, similar to the case of many other antidepressant drugs.     

Nicotinamide N-methyl transferase (NNMT): An emerging therapeutic target

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (NA) to generate 1-methyl nicotinamide. Since its discovery 70 years ago, the appreciation of the role of NNMT in human health has evolved from serving only metabolic functions to also being a driving force in diseases, including a variety of cancers. Despite the increasing evidence indicating NNMT as a viable therapeutic target, the development of cell-active inhibitors against this enzyme is lacking. In this review, we provide an overview of the current status of NNMT inhibitor development, relevant in vitro and in vivo studies, and a discussion of the challenges faced in the development of NNMT inhibitors.     

Hypothalamic vasopressin mRNA levels in mice are decreased after chronic ethanol ingestion

Vasopressin mRNA levels in the supraoptic and paraventricular nuclei of the hypothalamus, measured by in situ hybridization with a 35S-labeled RNA probe, were decreased by nearly 50% in C57BL/6NCR mice that had ingested an ethanol-containing diet for 7 days, and were tolerant to and physically dependent on ethanol. At 24 h after withdrawal, vasopressin mRNA levels in the supraoptic nucleus were still decreased, while levels in the paraventricular nucleus returned toward control values. Although plasma osmolality was increased in ethanol-fed mice, there was no increase in plasma vasopressin levels, possibly as a result of the effect of chronic ethanol ingestion to decrease vasopressin synthesis. In contrast, in mice that were dehydrated, but not fed ethanol, plasma osmolality, plasma vasopressin levels, and hypothalamic vasopressin mRNA all increased, as expected. The data suggest that chronic ethanol ingestion interferes with the synthesis and secretion of vasopressin, and may result in the reduced ability of an individual to respond to physiological stimuli for vasopressin secretion.     

Decreased expression of mineralocorticoid receptor mRNA in the prefrontal cortex in schizophrenia and bipolar disorder

Abnormal prefrontal cortical activity, activation of the hypothalamic-pituitary-adrenal (HPA) axis, and deficits in slow-wave sleep (SWS) have been extensively reported in patients with affective disorders and schizophrenia, yet the underlying pathophysiological mechanisms have not been completely elucidated. Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are two nuclear hormone receptors of primary importance in the control of stress-related and circadian HPA activity. A recent study showed that blocking brain MR activity not only enhances CRF-induced ACTH and cortisol release, but also significantly reduces SWS in humans. We hypothesized that the expression of MR would be deficient in the prefrontal cortex of patients with schizophrenia and affective disorders. The MR mRNA expression in the post-mortem prefrontal cortex of patients with major depression (MD), bipolar (BP), and schizophrenic (SZ) disorders and non-psychiatric controls (n=15 for each patient group, and n=14 for controls) was determined by in-situ hybridization. In the dorsolateral prefrontal cortex Brodmann's area 9 (BA 9), MR mRNA was significantly lower (p<0.05) in all laminae (I-VI) in BP, and in laminae I, III, IV and VI in SZ than in the controls. MR mRNA in BA 9 was negatively correlated with the duration of psychiatric illnesses. In BA 46, MR mRNA was not significantly different among groups, but was positively correlated with brain pH. These results provide the first evidence of deficient prefrontal MR mRNA expression in BP and SZ. Whether these findings may be linked to the abnormal prefrontal function, HPA axis activation, or the deficits in SWS found in these major psychiatric illnesses remains to be further explored.     

Effect of chronic ritanserin or clorgyline on amine and metabolite levels in rat frontal cortex

As part of an investigation of ritanserin-induced receptor down-regulation, monoamine and metabolite levels in rat frontal cortex were measured following chronic ritanserin (2 mg/kg per day) or clorgyline (10 mg/kg per day) administration. Clorgyline increased 5-hydroxytryptamine (5-HT) by 83%, noradrenaline (NA) by 54%, and dopamine (DA) by 16% and decreased 5-hydroxyindoleacetic acid (5-HIAA) by 28%, homovanillic acid (HVA) by 57% and 3,4-dihydroxyphenylacetic acid (DOPAC) by 67%. All these changes were statistically significant (P less than 0.001) except for the increase in DA. Ritanserin increased 5-HT by 30%, NA by 33% and DA by 26% and decreased 5-HIAA by 22%, HVA by 23% and DOPAC by 40%; however, only the increases in 5-HT and NA reached statistical significance (P less than 0.05). Monoamine oxidase (MAO) activity in cortical homogenates was also measured following the chronic ritanserin and clorgyline regimens and also following ritanserin administration in vitro. Chronic clorgyline and ritanserin inhibited MAO activity by 60 and 39%, respectively. In vitro, ritanserin administration at concentrations of less than 10(-6) M had no effect on MAO activity but at doses higher than 10(-6) M, MAO activity was inhibited in a dose-dependent manner from 18 +/- 0.5% at 3 x 10(-6) M to 63 +/- 9% at 10(-4) M. Thus, ritanserin appears to act as an MAO inhibitor in addition to being a 5-HT2 antagonist and this may be related to its ability to induce 5-HT2 receptor down-regulation.     

Expression of muscarinic and dopaminergic receptors and monoamine levels frontal cortex of epileptic rats

Apart from stroke, epilepsy is the most common neurological disorder with 0.5% of prevalence. The present study was performed in order to determine the monoamine levels, (M(1)-like) muscarinic and (D(1)- and D(2)-like) dopaminergic receptor changes in frontal cortex of adult rats after pilocarpine-induced status epilepticus (SE). Male Wistar rats were treated with a single dose of pilocarpine (400 mg/kg, s.c.) and the control group received 0.9% saline (s.c.). Both groups were sacrificed 1 h after treatment. The frontal cortex was dissected for neurochemical assays. The results show a downregulation of 27% in M(1) muscarinic receptor density, but in the dissociation constant (K(d)) value remained unaltered. D(1) and D(2) dopaminergic receptor densities and their K(d) values remained unaltered. Monoamine and metabolites levels presented decreases of 44%, 27%, 30% and 42% in dopamine (DA), homovanilic acid (HVA), norepinephrine (NE) and 5-hydroxyindoleacetic acid (5-HIAA) contents, respectively. Moreover, in serotonin (5-HT) level remained unaltered and the 4-hydroxy-3-methoxy-phenylacetic acid (DOPAC) concentration was augmented by 34%. The results suggest that dopaminergic system in this area studied may not be directly involved in the seizures and status epilepticus, but different monoamines and metabolites can be modified in this cerebral area during seizure process. In conclusion, the neurochemical alterations that occur in frontal cortex of adult rats observed during the establishment of the status epilepticus induced by pilocarpine are decrease in M(1) receptor density concentration and a reduction in DA and NE levels.     

Reduced pyramidal cell somal volume in auditory association cortex of subjects with schizophrenia.

Subjects with schizophrenia have decreased gray matter volume of auditory association cortex in structural imaging studies, and exhibit deficits in auditory sensory memory processes subserved by this region. In dorsal prefrontal cortex (dPFC), similar in vivo observations of reduced regional volume and working memory deficits in subjects with schizophrenia have been related to reduced somal volume of deep layer 3 pyramidal cells. We hypothesized that deep layer 3 pyramidal cell somal volume would also be reduced in auditory association cortex (BA42) in schizophrenia. We used the nucleator to estimate the somal volume of pyramidal neurons in deep layer 3 of BA42 in 18 subjects with schizophrenia, each of whom was matched to one normal comparison subject for gender, age, and post-mortem interval. For all subject pairs, somal volume of pyramidal neurons in deep layer 3 of dPFC (BA9) had previously been determined. In BA42, somal volume was reduced by 13.1% in schizophrenic subjects (p=0.03). Reductions in somal volume were not associated with the history of antipsychotic use, alcohol dependence, schizoaffective disorder, or death by suicide. The percent change in somal volume within-subject pairs was highly correlated between BA42 and BA9 (r=0.67, p=0.002). Deep layer 3 pyramidal cell somal volume is reduced in BA42 of subjects with schizophrenia. This reduction may contribute to impairment in auditory function. The correlated reductions of somal volume in BA42 and BA9 suggest that a common factor may affect deep layer 3 pyramidal cells in both regions.     

Antinociceptive activity of (-)-carvone: evidence of association with decreased peripheral nerve excitability

(-)-Carvone is a monoterpene ketone that is the main active component of Mentha plant species like Mentha spicata. This study aimed to investigate the antinociceptive activity of (-)-carvone using different experimental models of pain and to investigate whether such effects might be involved in the nervous excitability elicited by others monoterpenes. In the acetic acid-induced writhing test, we observed that (-)-carvone-treated mice exhibited a significant decrease in the number of writhes when 100 and 200 mg/kg was administered. It was also demonstrated that (-)-carvone inhibited the licking response of the injected paw when 100 and 200 mg/kg was administered (i.p.) to mice in the first and second phases of the formalin test. Since naloxone (5 mg/kg, s.c.), an opioid antagonist, showed no influence on the antinociceptive action of (-)-carvone (100 mg/kg), this suggested nonparticipation of the opioid system in the modulation of pain induced by (-)-carvone. Such results were unlikely to be provoked by motor abnormality, since (-)-carvone-treated mice did not exhibit any performance alteration on the Rota-rod apparatus. Because the antinociceptive effects could be associated with neuronal excitability inhibition, we performed the single sucrose gap technique and observed that (-)-carvone (10 mM) was able to reduce the excitability of the isolated sciatic nerve through a diminution of the compound action potential amplitude by about 50% from control recordings. We conclude that (-)-carvone has antinociceptive activity associated with decreased peripheral nerve excitability.     

Psychotropic drugs affect Ser9-phosphorylated GSK-3 beta protein levels in rodent frontal cortex

Glycogen synthase kinase (GSK)-3beta, a serine/threonine kinase highly abundant in brain is a negative regulator of signal transduction cascades including the phosphatidylinositol-3-kinase (PI3-K)/Akt and the Wnt. GSK-3beta has recently been suggested to be an intracellular target of the mood stabilizers lithium and valproate and of the typical and atypical antipsychotic agents haloperidol and clozapine. We have previously shown that these agents do not alter frontal cortex GSK-3beta protein levels or activity. The current study was conducted to assess the effect of psychotropic drugs on phospho-Ser9-GSK-3beta levels in rodents. Chronic administration of haloperidol to rats resulted in a significant reduction in frontal cortex phospho-Ser9-GSK-3beta protein levels and no change in those of GSK-3alpha, while chronic administration of clozapine or subchronic administration of valproate caused significant elevation of GSK-3beta protein levels. Mice treated chronically with lithium exhibited the most prominent elevation in phospho-Ser9-GSK-3beta. The results support the notion that GSK-3beta may be a common target for mood stabilizers and neuroleptics.     

Resistance to antidepressant treatment is associated with polymorphisms in the leptin gene,decreased leptin mRNA expression,and decreased leptin serum levels

Leptin, a peptide hormone from adipose tissue and key player in weight regulation, has been suggested to be involved in sleep and cognition and to exert antidepressant-like effects, presumably via its action on the HPA-axis and hippocampal function. This led us to investigate whether genetic variants in the leptin gene, the level of leptin mRNA-expression and leptin serum concentrations are associated with response to antidepressant treatment. Our sample consisted of inpatients from the Munich Antidepressant Response Signature (MARS) project with weekly Hamilton Depression ratings, divided into two subsamples. In the exploratory sample (n=251) 17 single nucleotide polymorphisms (SNPs) covering the leptin gene region were genotyped. We found significant associations of several SNPs with impaired antidepressant treatment outcome and impaired cognitive performance after correction for multiple testing. The SNP (rs10487506) showing the highest association with treatment response (p=3.9×10^?5) was analyzed in the replication sample (n=358) and the association could be verified (p=0.021) with response to tricyclic antidepressants. In an additional meta-analysis combining results from the MARS study with data from the Genome-based Therapeutic Drugs for Depression (GENDEP) and the Sequenced Treatment Alternatives to Relieve Depression (STAR^?D) studies, nominal associations of several polymorphisms in the upstream vicinity of rs10487506 with treatment outcome were detected (p=0.001). In addition, we determined leptin mRNA expression in lymphocytes and leptin serum levels in subsamples of the MARS study. Unfavorable treatment outcome was accompanied with decreased leptin mRNA and leptin serum levels. Our results suggest an involvement of leptin in antidepressant action and cognitive function in depression with genetic polymorphisms in the leptin gene, decreased leptin gene expression and leptin deficiency in serum being risk factors for resistance to antidepressant therapy in depressed patients.     

Prefrontal cortex glutathione S-transferase levels in patients with bipolar disorder, major depression and schizophrenia

Antioxidant defence systems have received increasing attention in the pathophysiology of psychiatric disorders, including: bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ). Recently, we reported decreased glutathione (GSH) levels in post-mortem prefrontal cortex from patients with BD, MDD, and SCZ. To explore this further, we evaluated the levels of two glutathione S-transferase (GST) isoforms via immunoblotting: GST Pi and GST Mu. GST Pi levels were not affected in any of the patients groups vs. controls. GST Mu levels were significantly decreased in patients with MDD and SCZ but not BD. Compared to controls, GST Mu levels were not different in BD patients who had been treated with mood stabilizers at the time of death but were significantly lower in those not taking mood stabilizers at the time of death. These data suggest that GST Mu may be a target for mood stabilizers.     

Conditioned opioid withdrawal decreases nociceptin/orphanin FQ levels in the frontal cortex and olfactory tubercle.

Clinical evidence suggests that individuals experiencing drug withdrawal can become conditioned to environmental situations, whereby previously neutral stimuli can produce symptoms of withdrawal. It is believed that this "conditioned withdrawal" can have motivational significance, but the neurobiological basis for conditioned withdrawal is unknown. The goal of this study was to determine adaptations in endogenous opioid systems that may be responsible for expression of conditioned withdrawal. Opioid-dependent rats trained to lever press for food were exposed to tone and scent cues in the presence of naloxone or saline. Naloxone but not saline predictably suppressed responding for food. One month later and in a post-dependent state, all rats again were exposed to the cues but not naloxone. The conditioned cues alone suppressed responding for food in the rats previously paired with naloxone, but no suppression was seen in rats previously paired with saline. Radioimmunoassay (RIA) analysis for nociceptin/orphanin FQ (nociceptin), met-enkephalin-Arg-Phe (MEAP), and dynorphin A (dyn A) was performed from dissections of various brain regions of the rats undergoing conditioned withdrawal. Significant reductions in nociceptin peptide levels were seen in the frontal cortex and olfactory tubercle of these rats. Unconditioned opioid withdrawal and unconditioned footshock stress produced different patterns of opioid peptide regulation in separate groups of rats. These results shed light on adaptations of endogenous opioid systems to conditioned cues, stress, and withdrawal, all factors that play a role in motivating drug intake.