A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function.

Previous studies have shown that chronic morphine increases levels of the G-protein subunits Gia and Goa, adenylate cyclase, cyclic AMP-dependent protein kinase, and certain phosphoproteins in the rat locus coeruleus, but not in several other brain regions studied, and that chronic morphine decreases levels of Gia and increases levels of adenylate cyclase in dorsal root ganglion/spinal cord (DRG-SC) co-cultures. These findings led us to survey the effects of chronic morphine on the G-protein/cyclic AMP system in a large number of brain regions to determine how widespread such regulation might be. We found that while most regions showed no regulation in response to chronic morphine, nucleus accumbens (NAc) and amygdala did show increases in adenylate cyclase and cyclic AMP-dependent protein kinase activity, and thalamus showed an increase in cyclic AMP-dependent protein kinase activity only. An increase in cyclic AMP-dependent protein kinase activity was also observed in DRG-SC co-cultures. Morphine regulation of G-proteins was variable, with decreased levels of Gia seen in the NAc, increased levels of Gia and Goa in amygdala, and no change in thalamus or the other brain regions studied. Interestingly, chronic treatment of rats with cocaine, but not with several non-abused drugs, produced similar changes compared to morphine in G-proteins, adenylate cyclase, and cyclic AMP-dependent protein kinase in the NAc, but not in the other brain regions studied. These results indicate that regulation of the G-protein/cyclic AMP system represents a mechanism by which a number of opiate-sensitive neurons adapt to chronic morphine and thereby develop aspects of opiate tolerance and/or dependence. The findings that chronic morphine and cocaine produce similar adaptations in the NAc, a brain region important for the reinforcing actions of many types of abused substances, suggest further that common mechanisms may underlie psychological aspects of drug addiction mediated by this brain region.     

Molecular and cellular mechanisms of opiate action: Studies in the rat locus coeruleus

We have studied the molecular and cellular mechanisms underlying the acute and chronic effects of opiates on neurons of the rat locus coeruleus (LC). Acutely, opiates inhibit LC neurons by activating K⁺ channels and inhibiting a novel sodium-dependent inward current. Both of these actions are mediated via pertussis toxin-sensitive G-proteins, and regulation of the sodium current occurs through inhibition of the cyclic AMP pathway. In contrast to the acute effects of opiates, chronic treatment of rats with opiates increases levels of specific G-protein subunits, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the possibility that this upregulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these noradrenergic LC neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms by which opiates regulate the expression of these intracellular messenger proteins in the LC. These studies will lead to an improved understanding of the molecular and cellular basis of opiate addiction.     

Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal

Lewis rats are more likely to self-administer various drugs of abuse than Fischer rats. Here these two strains of rats were compared with regard to basal brain opioid peptide levels and the response to chronic morphine treatment and to naloxone-precipitated withdrawal. Lewis rats had lower basal dynorphin peptides in the substantia nogra, striatum (not Leu-enkephalinArg⁶) and VTA (not dynorphin B) and the pituitary gland. Leu-enkephalinArg⁶ levels were also lower in these structures (with the exception of striatum which had higher levels) and in the nucleus accumbens. There were also strain differences in the response to chronic morphine treatment; in the nucleus accumbens, morphine treatment increased dynorphin A levels in Fischer rats only, in the ventral tegmental area effects were opposite with increased dynorphin levels in Fischer and decreased levels in Lewis rats, in the hippocampus dynorphin levels were markedly reduced in Lewis rats only. In Fischer rats, chronic morphine strongly affected peptide levels in the substantia nigra and striatum, whereas Lewis rats responded less in these areas. Leu-enkephalin, which derives from both prodynorphin and proenkephalin, and Met-enkephalin, which derives from proenkephalin, were effected by chronic morphine mainly in Fischer rats, increasing levels in most of the brain areas examined. The results in this study show (1) strain differences in basal levels of prodynorphin-derived opioid peptides, (2) the prodynorphin system to be differently influenced by morphine in Lewis rats than in Fischer rats and 3) the proenkephalin system to be influenced by chronic morphine in brain areas related to reward processes only in Fischer rats.     

Running and cocaine both upregulate dynorphin mRNA in medial caudate putamen

Physical activities such as long-distance running can be habit forming and associated with a sense of well-being to a degree that justifies comparison with drug-induced addictive behaviours. To understand molecular similarities and dissimilarities controlling these behaviours in humans we compared the effects of running in running wheels to the effects of chronic cocaine or morphine administration on mRNA levels in brain reward pathways in the inbred Fischer and Lewis rat strains. These strains are both inbred from the Sprague-Dawley strain; Lewis rats display a higher preference towards addictive drugs and running than do Fischer rats. After chronic cocaine or running a similar increase of dynorphin mRNA in medial caudate putamen was found in the Lewis rat, suggesting common neuronal adaptations in this brain region to both cocaine and running. Fischer and Lewis rats both responded to cocaine with increased dynorphin mRNA levels in medial caudate putamen. However, only Lewis rats increased dynorphin mRNA after running, possibly reflecting the much higher degree of running by the Lewis strain as compared to the Fischer strain. Moreover, the running-induced upregulation of dynorphin mRNA was blocked by the opioid receptor antagonist naloxone. We suggest that running increases dynorphin mRNA by a mechanism that involves endogenous opioids. The voluntary wheel-running model in rats might be used to study natural reward and compulsive behaviours and possibly also to screen candidate drugs for treatment of compulsive disorders.     

Dopaminergic brain reward regions of Lewis and Fischer rats display different levels of tyrosine hydroxylase and other morphine- and cocaine-regulated phosphoproteins

We studied cyclic AMP-dependent protein phosphorylation in the mesolimbic and nigrostriatal dopamine systems of two genetically inbred rat strains, Lewis (LEW) and Fischer (F344) rats. These strains represent genetically divergent populations of rats that have been used to study possible genetic factors involved in a variety of biological processes. We found striking differences in levels of tyrosine hydroxylase, and several other phosphoproteins, in the mesolimbic, but not the nigrostriatal, dopamine system between the two rat strains. Interestingly, in Sprague-Dawley rats, these same phosphoproteins are altered by chronic morphine and chronic cocaine specifically in the mesolimbic dopamine system, generally thought to be a brain reward pathway that mediates some of the reinforcing actions of many drugs of abuse. As LEW and F344 rats have been reported to show different levels of preference for several types of drugs of abuse, the results are consistent with the possibility that these phosphoproteins may mediate aspects of drug reinforcement and contribute to individual differences in vulnerability to drug addiction.     

Identification of MARPP-58, a morphine- and cyclic AMP-regulated phosphoprotein of 58 kDa, as tyrosine hydroxylase: evidence for regulation of its expression by chronic morphine in the rat locus coeruleus

Previously, we have identified a number of morphine- and cyclic AMP-regulated phosphoproteins (MARPPs) in the rat locus coeruleus (LC) and other brain regions. We now show that one of these phosphoproteins, a 58 kDa protein designated MARPP-58, is tyrosine hydroxylase. First, MARPP-58 comigrates with immunolabeled, immunoprecipitated, and purified tyrosine hydroxylase on 1- and 2-dimensional electrophoresis. Second, MARPP-58, immunoprecipitated tyrosine hydroxylase, and purified tyrosine hydroxylase yield identical 1-dimensional phosphopeptide maps. Third, MARPP-58 exhibits a regional and subcellular distribution in brain consistent with tyrosine hydroxylase. Identification of MARPP-58 as tyrosine hydroxylase made it possible to determine whether increases in MARPP-58 phosphorylation induced by chronic morphine in the LC reported previously are associated with alterations in enzyme activity and expression in this brain region. We show that chronic treatment of rats with morphine increases levels of tyrosine hydroxylase activity, immunoreactivity, and mRNA in the LC. Induction of the enzyme by chronic morphine was blocked by concomitant treatment of rats with the opiate receptor antagonist naltrexone, indicating that morphine produces this effect through the activation of opiate receptors. Consistent with previous observations that the chronic morphine-induced change in MARPP-58 phosphorylation is specific to the LC, changes observed in enzyme activity, immunoreactivity, and mRNA were not observed in a number of other brain regions studied. The results indicate that chronic morphine regulates the expression of tyrosine hydroxylase specifically in the LC and suggest that such regulation reflects long-term adaptations of LC neurons to chronic morphine at the level of gene expression.     

Regulation of G proteins by chronic morphine in the rat locus coeruleus

A possible role for G proteins in contributing to the chronic actions of opiates was investigated in the rat locus coeruleus (LC). The LC is a relatively homogeneous brain region that appears to play an important role in mediating acute and chronic opiate action in animals, as well as in humans. It was found that chronic, but not acute, treatment of rats with morphine, under conditions known to induce states of opiate tolerance and dependence, produced an increase in the level of pertussis toxin-mediated ADP-ribosylation of G proteins in the LC. The morphine-induced increase in ADP-ribosylation occurred in both Gi and Go, and was observed over a 30-fold range of NAD concentrations used. Concomitant treatment of rats with the opiate receptor antagonist naltrexone blocked the ability of morphine to produce this effect. In contrast, chronic morphine had no effect on pertussis toxin-mediated ADP-ribosylation of Gi and Go in the other brain regions studied, including the neostriatum, frontal cortex, and dorsal raphe. Chronic morphine also had no effect on cholera toxin-mediated ADP-ribosylation of Gs in the LC and these other brain regions. Preliminary immunoblot analysis revealed that increased ADP-ribosylation levels of the alpha subunit of Go in the LC were associated with equivalent increases in the immunoreactivity of this protein in this brain region. It is possible that the observed regulation of G-proteins by morphine in the LC represents part of the changes that underlie opiate addiction in these neurons.     

Biochemical adaptations in the mesolimbic dopamine system in response to heroin self-administration

Previous studies have shown that chronic, forced exposure to opiates produces specific biochemical adaptations in the ventral tegmental area (VTA) and nucleus accumbens (NAc). The functional consequences of these adaptations have been hypothesized to contribute to certain motivational aspects of drug addiction. In this study, the possibility that similar adaptations could occur in response to intermittent heroin self-administration was tested by comparing homogenates of VTA and NAc from rats self-administering heroin, rats receiving yoked injections of heroin, and rats receiving yoked injections of saline (controls). Tyrosine hydroxylase (TH) immunoreactivity was increased (31–38%) in the VTA and decreased (11%) in the NAc of heroin-exposed rats relative to controls. Heroin exposure also increased cAMP-dependent protein kinase (PKA) activity in both particulate (19–27%) and soluble (17–20%) fractions of the NAc, and decreased (16–17%) the level of G_iα, immunoreactivity in this brain region. In contrast, no significant biochemical changes were found in the substantia nigra or caudate-puta-men, indicating a selective effect on the mesolimbic dopamine system. Overall, adapta-tions in the VTA and NAc of heroin-exposed rats were similar to, but generally smaller in magnitude than, adaptations produced by chronic morphine administration. However, in contrast to morphine-treated animals, heroin-exposed animals failed to display overt signs of opiate physical dependence, suggesting that adaptations in motivational systems may occur more readily than adaptations in brain regions associated with physical dependence. © 1995 Wiley-Liss, Inc.     

Opiate withdrawal and the rat locus coeruleus: behavioral, electrophysiological, and biochemical correlates

We have compared the time course of the behavioral manifestations of opiate withdrawal to the in vivo activity of locus coeruleus (LC) neurons and to increases in the levels of G-proteins, adenylate cyclase, and cAMP-dependent protein kinase known to occur in the LC in opiate-dependent animals. Rats were given morphine by daily subcutaneous implantation of morphine pellets for 5 d. On the sixth day, morphine withdrawal was induced by subcutaneous administration of naltrexone, an opiate receptor antagonist, with additional doses given 6 and 24 hr later, conditions that resulted in sustained, maximal levels of withdrawal over the duration of the experiment. We found a striking parallel between the time courses of the behavioral signs and the increased activity of LC neurons during withdrawal, both of which appeared to follow 2 phases. There was an early, rapid phase, during which withdrawal signs and increased LC activity became most pronounced within 15-30 min after naltrexone administration, and then recovered rapidly by over 50% within 4 hr of withdrawal. Subsequently, there was a slower phase, during which the persisting withdrawal signs and elevated LC activity remained roughly constant from 4 to 24 hr and did not recover completely until after 72 hr of continuous withdrawal. Adenylate cyclase and cAMP-dependent protein kinase activities in isolated LC subcellular fractions, both elevated in dependent animals, recovered to control levels after 6 hr of withdrawal, in parallel with the rapid phase of withdrawal. Levels of G1 and Go, also elevated in dependent animals, remained only slightly elevated at 6 hr and returned to normal by 24 hr. Taken together, these data suggest that increased neuronal activity in the LC is associated temporally with the behavioral morphine withdrawal syndrome and that increased levels of G-proteins and an up-regulated cAMP system may contribute to the early withdrawal activation of these neurons.     

Downregulation of cAMP response element-binding protein by lentiviral vector-mediated RNAi attenuates morphine withdrawal syndromes in rats

cAMP response element-binding protein(CREB) and the cAMP cascade play a pivotal role in the opiate-dependence. The blockade of this cascade is believed to attenuate signs of physical opiate withdrawal. A lentiviral vector (LV) expressing a small hairpin RNA (shRNA) to silence CREB in vitro and in vivo was used in this study. The effect of the shRNA on the regulation of the relevant protein expression and the signs of opiate withdrawal were subsequently evaluated in rats undergoing chronic morphine treatment. In cultured primary locus coeruleus (LC) neurons, the designed lentiviral vectors were successfully infected into the cells and led to 70% knockdown in CREB expression. In cells treated with chronic morphine, the expression of CREB, adenylyl cyclase (AC) and protein kinase A (PKA) were increased, while in cells infected with LV-CREB3, treated with chronic morphine treatment failed to increase the expressions of CREB and AC. Consistently, in the rat model for chronic morphine treatment, morphine increased the expression of CREB, AC and PKA in LC neurons. However, in rats received bilateral microinjections of LV-CREB3 into the LC, morphine did not alter the levels of these proteins. Moreover, microinjection of LV-CREB3 significantly attenuated the appearance of certain withdrawal behaviors. In conclusion, the lentiviral vectors expressing CREB shRNA inhibited the increase of CREB and AC expression induced by chronic morphine treatment both in vivo and in vitro. This inhibition was associated with the alleviation of some withdrawal behaviors. These findings suggested that lentivirus-mediated RNA interference could be useful for opiate-dependence therapy.     

Biochemical actions of chronic ethanol exposure in the mesolimbic dopamine system

In previous studies, we have demonstrated that chronic administration of morphine or cocaine 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 actions of these and other drugs of abuse. Since this neural pathway is also implicated in the reinforcing actions of ethanol, it was of interest to determine whether chronic ethanol exposure results in similar biochemical adaptations. Indeed, as seen for chronic morphine and cocaine treatments, we show here that chronic ethanol treatment increased levels of tyrosine hydroxylase and glial fibrillary acidic protein immunoreactivity, and decreases levels of neurofilament protein immunoreactivity, in the VTA. Also like morphine and cocaine, ethanol increases levels of cyclic AMP-dependent protein kinase activity in the NAc. These actions of ethanol required long-term exposure to the drug, and were in most cases not seen in the substantia nigra or caudate-putamen, components of the nigrostriatal dopamine system studied for comparison. Altered levels of tyrosine hydroxylase in catecholaminergic cells frequently reflect altered states of activation of the cells. Moreover, increasing evidence indicates that ethanol produces many of its acute effects on the brain by regulating NMDA glutamate and GAB_A receptors. We therefore examined the influence of chronic ethanol treatment on levels of expression of specific glutamate and GABA receptor subunits in the VTA. It was found that long-term, but not short-term, ethanol exposure increased levels of immunoreactivity of the NMDARl subunit, an obligatory component of NMDA glutamate receptors, and of the Glu Rl subunit, a component of many AMPA glutamate receptors; but at the same time, long-term ethanol exposure decreased immunoreactivity levels of the α1 subunit of the GABA_A receptor complex. These changes are consistent with an increased state of activation of VTA neurons inferred from the observed increase intyrosine hydroxylase (TH) expression. These results demonstrate that chronic ethanol exposure results in several biochemical adaptations in the mesolimbic dopamine system, which may underlie prominent changes in the structural and functional properties of this neural pathway related to alcohol abuse and alcoholism. © 1995 Wiley-Liss, Inc.     

Differential basal proenkephalin gene expression in dorsal striatum and nucleus accumbens, and vulnerability to morphine self-administration in Fischer 344 and Lewis rats

We have previously shown that the acquisition rate of intravenous morphine self-administration under a fixed ratio one (FR1) schedule of reinforcement was greater in Lewis (LEW) than Fischer 344 (F344) rats. The purpose of the present experiment was to examine the relative motivational properties of morphine (1 mg/kg) or food under progressive ratio (PR) schedules of reinforcement in LEW and F344 rats. In addition, by using in situ hybridization histochemistry we have measured in both strains of rats the basal level of proenkephalin (PENK) gene expression in dorsal striatum and nucleus accumbens (NAcc). The results show that LEW rats responded to significantly higher breaking points (BPs) than F344 rats for intravenous morphine self-administration. In contrast, no differences were found in BPs for food pellets. Basal PENK mRNA levels were significantly higher in the dorsal striatum and nucleus accumbens of F344 than in LEW rats. Taken together, these results reveal a strain difference in the reinforcing efficacy of morphine and in the basal PENK gene expression in brain regions involved in the reinforcing actions of opiates. These data also suggest that the strain differences in opiate self-administration behavior found in this and other studies may be related, at least in part, to differences in basal opioid activity between LEW and F344 rats.     

Chronic morphine treatment inhibits oxytocin synthesis in rats

The changes of oxytocin content and mRNA expression in some nuclei were investigated in morphine-dependent rats using radioimmunoassay (RIA) and in situ hybridization (ISH). After chronic administration of morphine, the oxytocin content in supraoptic nucleus (SON) and nucleus accumbens (NAc) decreased, and increased in the ventral tegment area (VTA) and locus coeruleus (LC), but did not change in other nuclei including the paraventricular nucleus (PVN), lateral septum (SEPTUM), raphe magnus nucleus (NRM) and periaquaductal gray (PAG). In morphine-L dependent rats, naloxone increased the levels of oxytocin in SON and PVN, but decreased that in LC. ISH first showed that chronic morphine treatment inhibited the oxytocin synthesis in SON but not in PVN. The present study demonstrates that chronic morphine treatment alters the brain oxytocin system, suggesting that oxytocin might contribute to the behavioral and neuroendocrine responses to morphine.     

Differential behavioral responses to cocaine are associated with dynamics of mesolimbic dopamine proteins in Lewis and Fischer 344 rats

Differential behavioral and biochemical responses to drugs of abuse may reflect genetic makeup as suggested by studies of inbred Lewis (LEW) and Fischer 344 (F344) rats. We investigated locomotor activity, stereotypy signs, and levels of specific proteins in the nucleus accumbens (NAc) and ventral tegmental area (VTA) in these strains at baseline and following chronic administration of cocaine (30 mg/kg/day for 14 days). Using Western blot analysis, we replicated our previous findings of baseline strain differences and found lower levels of DeltaFosB immunoreactivity in NAc of F344 vs. LEW rats. F344 rats showed greater baseline locomotor activity, sniffing, and grooming compared to LEW rats. Chronic cocaine increased DeltaFosB levels in NAc in both strains, whereas adaptations in other proteins were induced in F344 rats only. These included reduced levels of tyrosine hydroxylase (TH) in NAc and increased TH and glial fibrillary acidic protein (GFAP) immunoreactivity in VTA. Chronic cocaine led to greater increases in overall stereotypy in F344 vs. LEW rats and decreased exploratory behaviors in LEW rats. Opposing effects by strain were seen in locomotor activity. Whereas F344 rats showed higher initial activity levels that decreased with cocaine exposure (tolerance), LEW rats showed increased activity over days (sensitization) with no strain differences seen at 14 days. Further, conditioned locomotor activation to vehicle injections was greater in F344 vs. LEW rats. These results suggest that behavioral responsiveness to chronic cocaine exposure may reflect dynamics of mesolimbic dopamine protein levels and demonstrate the role of genetic background in responsiveness to cocaine.     

Altered subcellular signaling in murine peritoneal macrophages upon chronic morphine exposure

Alterations in opioid signaling that take place in murine peritoneal macrophages in vitro are variably dependent on opiate exposure conditions. Acute exposure to morphine inhibits Fc-mediated phagocytosis by a pertussis toxin (PT)-sensitive mechanism, but has no effect on cAMP levels. In contrast, chronic exposure to morphine results in a "tolerant" state, wherein test and control values for both phagocytosis and cAMP are equivalent. However, drug withdrawal after chronic exposure to morphine results in inhibition of phagocytosis and a concomitant 4-fold increase in cAMP by a PT-insensitive mechanism. This increase is causally related to inhibition of phagocytosis since an artificial increase in cAMP inhibits phagocytosis in non-withdrawn cells exposed chronically to morphine. We suggest that macrophage opioid receptors signaling switches from a Gi/o-mediated mechanism that does not involve adenylate cyclase in acute exposure to a non-Gi/o-mediated adenylate cyclase superactivation during chronic exposure.     

N-phenylisopropyladenosine stimulates in normal and inhibits in adrenalectomized rats the low KM cyclic AMP phosphodiesterase in the brain

In crude rat brain membranes, apparent adenylate cyclase activity tested in the absence of phosphodiesterase inhibitors was dose-dependently inhibited by the adenosine ‘R-site’ agonist N⁶-phenylisopropyladenosine (N⁶-PIA). In membranes from adrenalectomized rats, however, N⁶-PIA induced, under the same conditions, an activation. However, in the presence of the phosphodiesterase inhibitor Ro-20-1724, brain adenylate cyclase responsiveness to N⁶-PIA resulted in a dose-dependent inhibition in both sham-operated and adrenalectomized rats. Thus, the low K_M cyclic AMP phosphodiesterase activity present in these brain membranes was investigated. Although this activity (tested in the presence of GTP) was unaltered by adrenalectomy, the dose-response curve of this enzyme to low concentrations of N⁶-PIA showed an activation in sham-operated and an inhibition in adrenalectomized rats, two effects which were suppressed by sodium (80 mM). These results showing that N⁶-PIA modulates both adenylate cyclase and cyclic AMP phosphodiesterase in the brain, provide an additional argument for a potential role of adenosine in the regulation of cyclic AMP metabolism in normal as well as in pathological brain.     

Induction of the c-fos proto-oncogene during opiate withdrawal in the locus coeruleus and other regions of rat brain.

Opiate regulation of the nuclear proto-oncogene c-fos was studied in the locus coeruleus (LC) and other regions of rat brain by immunoblotting, northern blotting, and in situ hybridization procedures. Precipitation of opiate withdrawal in rats, which is known to increase LC firing rates 4-fold, led to a two- to three-fold increase in levels of mRNA and protein for c-fos in the LC 1-2 h after initiation of withdrawal. In contrast, levels of c-fos expression were decreased in LC from rats treated acutely or chronically with morphine but not experiencing withdrawal, conditions under which LC firing rates are depressed. Similar regulation of c-fos expression during opiate withdrawal was found in the amygdala, ventral tegmentum, nucleus accumbens, neostriatum, and cerebral cortex, but not in a number of other brain regions studied, which included the hippocampus, dorsal raphe, periaqueductal gray, and paragigantocellularis. In the LC and some other brain regions, induction of c-fos during opiate withdrawal was associated with a parallel induction of c-jun, another nuclear proto-oncogene, which, like c-fos, is expressed rapidly in brain in response to certain extracellular stimuli. The results demonstrate a novel use of c-fos in neuropharmacology, namely to map neuronal pathways and neuronal cell types activated in response to acute and chronic opiate administration and during opiate withdrawal, as well as in response to other psychotropic drug treatments.     

A comparative study of the morphine effect on the level of cyclic AMP in lymphocytes of smokers and nonsmokers

A study of the effect of morphine on the level of cAMP in the lymphocytes of peripheral blood in smokers and nonsmokers was carried out. It was shown that morphine enhances the activity of adenylate cyclase, and that this effect can be abolished by naloxone. The dependence of the enzyme activity on the concentration of morphine is complex. The maximal effect of the investigated opiate on the level of cAMP in lymphocytes occurs among smokers at a tenfold concentration compared with nonsmokers. On the basis of our data two suggestions are made: (i) there are opiate receptors on lymphocytes with a mechanism of action connected with the regulation of adenylate cyclase activity; (ii) smoking develops tolerance to morphine at the level of the opiate-dependent adenylate cyclase of lymphocytes.     

Morphine differentially regulates hsp90beta expression in the nucleus accumbens of Lewis and Fischer 344 rats

We have comparatively studied hsp90beta gene and protein expression in the nucleus accumbens of Lewis and Fischer 344 (F344) rats, two inbred strains that exhibit prominent behavioural differences in drug-seeking behaviours. Phenotypical studies confirmed that Lewis rats developed a higher preference for morphine-paired environments after conditioning. RT-PCR assays did not reveal strain-related differences in hsp90beta gene expression in basal conditions; however, acute morphine treatment provoked an increase of hsp90beta mRNA 2h after injection only in the case of Lewis rats. We also found a significant upregulation of the Hsp90beta protein in both strains 8h after morphine injection, this increase being significantly higher in Lewis rats. Taking into account the suggested roles for Hsp90 in the brain, the data suggest that Lewis and F344 strain differences concerning opioid-seeking behaviours could be related to differential sensitivity to opioid-induced neuronal plasticity within the brain reward system, an effect that could be mediated (at least partially) by stress proteins.