Opioid Peptide Gene Expression Research Articles

Strain and cocaine-induced differential opioid gene expression may predispose Lewis but not Fischer rats to escalate cocaine self-administration

The aim of the present study was to investigate alterations in gene expression of opioid system components induced by extended access (18 h) cocaine self-administration and to determine the impact of genetic background in the vulnerability to escalate cocaine intake. Comparing two inbred rat strains, we previously reported that Lewis rats progressively escalated cocaine consumption compared to Fischer rats, in a new translational model of intravenous cocaine self-administration, which included 14 sessions of 18-h operant sessions in which rats were allowed to select the cocaine unit dose to self-administer. We compare here Fischer and Lewis rats in the gene expression of endogenous opioid peptides (Pomc, Penk, Pdyn) and cognate receptors (Oprm, Oprk and Oprd) in reward-related brain regions, after exposure to either cocaine self-administration or yoked-saline, in the aforementioned translational paradigm. We performed a correlation analysis between the mRNA level, found in the Dorsal Striatum (DS), Nucleus accumbens (NAcc) shell and core respectively, and individual cocaine intake.Our findings show that the gene expression of all the aforementioned opioid genes exhibit strain-dependent differences in the DS, in absence of cocaine exposure. Also, different strain-specific cocaine-induced mRNA expression of Oprm and Oprk was found in DS. Only few differences were found in the ventral parts of the striatum. Moreover, gene expression level of Pdyn, Penk, Oprk, and Oprm in the DS was significantly correlated with cocaine intake only in Fischer rats. Overall, these data shed light on potential genetic differences which may predispose of subjects to initiate and escalate cocaine consumption.

Effect of intravenous general anaesthesia with epidural block on the expression of pre-endogenous opioid peptide genes.

To measure the plasma concentrations of three endogenous opioid peptides and the levels of preproenkephalin (PPE) and preprodynorphin (PPD) mRNA in peripheral blood lymphocytes of patients during scheduled surgery performed under intravenous general anaesthesia combined with an epidural block. Patients were anaesthetized and arterial blood was collected at 0 (baseline), 20, 40, 60, and 80 min during surgery. The plasma concentrations of β-endorphin, leucine-enkephalin and dynorphin A were measured using radioimmunoassay. Reverse transcription-polymerase chain reaction was used to measure the levels of PPD and PPE mRNA in peripheral blood lymphocytes collected during surgery. Fifteen patients participated in this prospective study. The plasma concentrations of β-endorphin were significantly lower at all time-points compared with the baseline value. The plasma concentrations of leucine-enkephalin and dynorphin A were significantly lower at 40, 60, and 80 min compared with baseline. The PPD/β-actin ratio was significantly lower at 80 min compared with baseline, while the PPE/β-actin ratio showed no significant change. The level of mRNA from two pre-endogenous opioid peptide genes either decreased or remained unchanged during surgery under intravenous general anaesthesia with epidural block, suggesting that patients remained pain free during surgery.

Upregulation of Gene Expression in Reward-Modulatory Striatal Opioid Systems by Sleep Loss

Epidemiological studies have shown a link between sleep loss and the obesity 'epidemic,' and several observations indicate that sleep curtailment engenders positive energy balance via increased palatable-food 'snacking.' These effects suggest alterations in reward-modulatory brain systems. We explored the effects of 10 days of sleep deprivation in rats on the expression of striatal opioid peptide (OP) genes that subserve food motivation and hedonic reward, and compared effects with those seen in hypothalamic energy balance-regulatory systems. Sleep-deprived (Sleep-Dep) rats were compared with yoked forced-locomotion apparatus controls (App-Controls), food-restricted rats (Food-Restrict), and unmanipulated controls (Home-Cage). Detection of mRNA levels with in situ hybridization revealed a subregion-specific upregulation of striatal preproenkephalin and prodynorhin gene expression in the Sleep-Dep group relative to all other groups. Neuropeptide Y (NPY) gene expression in the hippocampal dentate gyrus and throughout neocortex was also robustly upregulated selectively in the Sleep-Dep group. In contrast, parallel gene expression changes were observed in the Sleep-Dep and Food-Restrict groups in hypothalamic energy-sensing systems (arcuate nucleus NPY was upregulated, and cocaine- and amphetamine-regulated transcript was downregulated), in alignment with leptin suppression in both groups. Together, these results reveal a novel set of sleep deprivation-induced transcriptional changes in reward-modulatory peptide systems, which are dissociable from the energy-balance perturbations of sleep loss or the potentially stressful effects of the forced-locomotion procedure. The recruitment of telencephalic food-reward systems may provide a feeding drive highly resistant to feedback control, which could engender obesity through the enhancement of palatable feeding.

Electroacupuncture-Induced Dynamic Processes of Gene Expression Levels of Endogenous Opioid Peptide Precursors and Opioid Receptors in the CNS of Goats

In order to investigate the dynamic processes of mRNA levels of proenkephalin, proopiomelanocortin, prodynorphin, and opioid receptors (δ-, μ-, and κ-receptor) induced by electroacupuncture (EA) in the central nerve system, goats were stimulated by EA of 60 Hz for 0.5 h at a set of Baihui, Santai, Ergen, and Sanyangluo points. The pain threshold was measured using the method of potassium iontophoresis. The mRNA levels of the three opioid peptide precursors and three opioid receptors were determined with quantitative real-time PCR and the levels of Met-enkephalin with SABC immunohistochemistry at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA. The results showed that the pain threshold correlated (P < 0.01) with Met-enkephalin immunoactivities in the measured nuclei and areas of goats. The analgesic aftereffect lasted for 12 h at least. The mRNA levels of the three opioid peptide precursors and three opioid receptors began to increase at 0 h, reached the peak during the time from 4 h to 6 h or at 12 h, and remained higher at 24 h after EA was discontinued. These results suggested that the initiation of gene expression of opioid peptides and the three receptors may be associated with EA-induced analgesic aftereffect.

The Role of BDNF/TrkB Signaling in Acute Amphetamine-Induced Locomotor Activity and Opioid Peptide Gene Expression in the Rat Dorsal Striatum

Exposure to psychostimulants increases brain-derived neurotrophic factor (BDNF) mRNA and protein levels in the cerebral cortex and subcortical structures. Because BDNF is co-localized with dopamine and glutamate in afferents to the striatum of rats, it may be co-released with those neurotransmitters upon stimulation. Further, there may be an interaction between the intracellular signaling cascades activated by dopamine, glutamate, and TrkB receptors in medium spiny striatal neurons. In the present study, the effect of acute amphetamine administration on TrkB phosphorylation, as an indirect indicator of activation, and striatal gene expression, was evaluated. In Experiment 1, 15 min or 2 h after a single saline or amphetamine (2.5 mg/kg, i.p.) injection, the caudate–putamen (CPu), nucleus accumbens (NAc), and dorsomedial prefrontal cortex (dmPFC) were extracted and processed for phospho (p)-TrkB immunoreactivity. Immunoprecipitation analyses indicated that neither the tyrosine phosphorylation (p-Tyr) or autophosphorylation sites of TrkB (706) were changed in NAc, CPu, or dmPFC 15 min after amphetamine administration. In contrast, p-Tyr and the PLCγ phosphorylation site of TrkB (816) were increased in the NAc and CPu 2 h after amphetamine. In Experiment 2, intra-striatal infusion of the tyrosine kinase inhibitor, K252a, increased amphetamine-induced vertical activity but not total distance traveled. In addition, K252a inhibited amphetamine-induced preprodynorphin, but not preproenkephalin, mRNA expression in the striatum. These data indicate that acute amphetamine administration induces p-TrkB activation and signaling in a time- and brain region-dependent manner and that TrkB/BDNF signaling plays an important role in amphetamine-induced behavior and striatal gene expression.

Peripheral electrical stimulation-induced suppression of morphine-induced CCP in rats: A role for dopamine in the nucleus accumbens

In the previous study we reported that morphine-induced conditioned place preference (CPP) could be suppressed by peripheral electric stimulation (PES), an effect related to the increased gene expression of opioid peptides in the central nervous system. Considering that opioids were known to elevate dopamine (DA) activity in the mesolimbic brain, the present study was designed to further analyze the possible involvement of the mesolimbic dopaminergic system (MLDS) in the suppressive effect of PES on the rewarding effects of morphine in SD male rats. We found that morphine-induced CPP can be successfully suppressed by PES, an effect accompanied by a reversal of the increased tissue contents of DA and its metabolites in the nucleus accumbens (NAc) of morphine-induced CPP rats. Our results suggest that MLDS seems to play important roles in the mechanisms underlying PES's suppression of the rewarding effect of drug-associated environmental cues in the rat.

Group I Metabotropic Glutamate Receptor-mediated Gene Expression in Striatal Neurons

Group I mGluRs (mGluR1/5) are G-protein-coupled receptors and are abundantly expressed in most of medium spiny projection neurons in the striatum. Recent evidence demonstrates that group I mGluRs are among essential regulators for constitutive and inducible gene expression in host neurons. Upon activation, mGluR1/5 signals activate extracellular signal-regulated kinases (ERKs) which in turn phosphorylate transcription factors such as cAMP response element-binding protein (CREB) and Elk-1, and thereby facilitate immediate early gene and opioid peptide gene expression. The conventional mGluR1/5 signaling cascade (phosphoinositide hydrolysis and intracellular Ca(2+) release) participates in linking mGluR1/5 to ERK. Additionally, the prominent mGluR1/5 adaptor protein Homer contributes to assemble an efficient signaling apparatus connecting mGluR1/5 to gene expression. The mGluR1/5 linkage to transcription also functions in dopamine-stimulated gene expression. Together, the mGluR1/5-mediated gene expression constitutes a transcription-dependent mechanism underlying molecular adaptations and plasticity related to the pathogenesis of various mental illnesses.

Regulation of psychostimulant-induced signaling and gene expression in the striatum.

Amphetamine (AMPH) and cocaine are indirect dopamine agonists that activate multiple signaling cascades in the striatum. Each cascade has a different subcellular location and duration of action that depend on the strength of the drug stimulus. In addition to activating D1 dopamine-Gs-coupled-protein kinase A signaling, acute psychostimulant administration activates extracellular-regulated kinase transiently in striatal cells; conversely, inhibition of extracellular-regulated kinase phosphorylation decreases the ability of psychostimulants to elevate locomotor behavior and opioid peptide gene expression. Moreover, a drug challenge in rats with a drug history augments and prolongs striatal extracellular-regulated kinase phosphorylation, possibly contributing to behavioral sensitization. In contrast, AMPH activates phosphoinositide-3 kinase substrates, like protein kinase B/Akt, only in the nuclei of striatal cells but this transient increase induced by AMPH is followed by a delayed decrease in protein kinase B/Akt phosphorylation whether or not the rats have a drug history, suggesting that the phosphoinositide-3 kinase pathway is not essential for AMPH-induced behavioral sensitization. Chronic AMPH or cocaine also alters the regulation of inhibitory G protein-coupled receptors in the striatum, as evident by a prolonged decrease in the level of regulator of G protein signaling 4 after non-contingent or contingent (self-administered) drug exposure. This decrease is exacerbated in behaviorally sensitized rats and reversed by re-exposure to a cocaine-paired environment. A decrease in regulator of G protein signaling 4 levels may weaken its interactions with metabotropic glutamate receptor 5, Galphaq, and phospholipase C beta that may enhance drug-induced signaling. Alteration of these protein-protein interactions suggests that the striatum responds to psychostimulants with a complex molecular repertoire that both modulates psychomotor effects and leads to long-term neuroadaptations.

Actions of sex steroids on behaviours beyond reproductive reflexes.

The actions of sex steroids in the brain have been shown, from molecular to systems levels, to control reproductive behaviour in a wide range of vertebrates. It has become increasingly clear that gonadal steroid hormones have regulatory functions which extend far beyond the direct coordination of an animal's physiological state and its display of sexual behaviour. While some of these actions may include changes in mood or other behavioural measures, such as exploration or excitability, sex steroid hormones also influence neural plasticity, neuronal activity and, possibly, learning and memory, as reflected by long-term potentiation or age-related deficits. Here we describe two systems that have been used to explore the non-reproductive roles of gonadal steroid hormones. The first of these is to examine the oestrogen-sensitive opioid peptide gene expression in the hypothalamus. Currently, we are attempting to identify the types of behaviour which may be altered consequent to the oestrogenic induction of the preproenkephalin gene. The second approach involves studying the effects of progesterone at the neuronal cell membrane and characterizing the metabolites of progesterone which have benzodiazepine-like actions in the brain. A number of studies suggest that this may provide an alternative mechanism through which progesterone can influence mood or behaviour.

Striatal opioid peptide gene expression differentially tracks short-term satiety but does not vary with negative energy balance in a manner opposite to hypothalamic NPY

It has long been known that central opioid systems play an important role in certain aspects of appetite and food intake, particularly with regard to the hedonic or rewarding impact of calorically dense food, such as fat and sugar. Ventral striatal enkephalin may be a key component of this system, as infusions of mu-opiate agonists into this region strongly increase feeding, whereas infusions of opiate antagonists decrease food intake. While pharmacological analysis has consistently supported such a role, direct measurement of enkephalin gene expression in relation to differing food motivational conditions has not been examined. In this study, the effects of a restricted laboratory chow diet (resulting in negative energy balance) as well has recent consumption of chow (short-term satiety) on striatal preproenkephalin (PPE) and prodynorphin (PD) mRNA expression were measured in rats, using both Northern blot analysis and in situ hybridization methods. As a comparison, hypothalamic (arcuate nucleus) neuropeptide Y (NPY) was also measured in these conditions. PPE expression was broadly downregulated throughout the striatum in animals that had recently consumed a meal, whereas it was unaffected by negative energy balance. Expression of an additional striatal peptide gene, PD, did not follow this pattern, although diet restriction caused a decrease in accumbens core dynorphin mRNA. Conversely, as expected, arcuate nucleus NPY mRNA expression was markedly upregulated by negative energy balance, but was unchanged by recent food consumption. This double dissociation between striatal and hypothalamic peptide systems suggests a specific role for striatal PPE in relatively short-term food motivational states, but not in long-term metabolic responses to diet restriction.

CB 1 knockout mice display significant changes in striatal opioid peptide and D 4 dopamine receptor gene expression

Antagonism of the CB 1 cannabinoid receptor (CB 1 receptor) by rimonabant (SR141716) reduces self-administration of alcohol and other drugs of abuse in animal models. These findings suggest that the CB 1 receptor may be a target for genetic differences that modify the salient features of rewarding drugs. In the present study, wild-type (CB 1 (+/+)) are compared to transgenic mice deficient in CB 1 receptors (CB 1 (−/−)). The goal was to investigate the influences of the cannabinoid receptor system on opioid peptide gene expression and on dopamine receptor gene expression which is commonly influenced by substances of abuse. We demonstrate using reverse transcription and real-time polymerase chain reaction (PCR) that striatal mRNA for preproenkephalin (PPENK) and preprodynorphin (PPDYN) in the CB 1 (−/−) striatum increases when compared to CB 1 (+/+). Real-time PCR analyses to evaluate D 2 and D 4 dopamine receptor gene expression in striatum isolated from CB 1 (+/+) and CB 1 (−/−) revealed a nearly 2-fold increase in D 4 receptor mRNA in the striatum from CB 1 (−/−) mice and no significant change in D 2 expression. In contrast, treatment of C57BL/6 mice with the CB 1 receptor antagonist, rimonabant, produced a reduction of both D 2 and D 4 dopamine receptor expression in the striatum. These data suggest that genetic differences in CB 1 receptor may exert a modulatory effect on D 4 dopamine receptor and opioid peptide gene expression.

Tolerance develops in spinal cord, but not in brain with chronic [Dmt1]DALDA treatment

Previously, we reported that H-2',6'-dimethyltyrosine [Dmt(1)]-d-Arg-Phe-Lys-NH(2) (DALDA), an analogue of the naturally occurring opioid peptide dermorphin, is a highly potent and selective mu receptor agonist with low cross-tolerance to morphine. In the present study, we investigated the effect of treating mice chronically with [Dmt(1)]DALDA. The AD(50) of [Dmt(1)]DALDA (s.c.) increased eight-fold in animals given this drug chronically; in contrast, the AD(50) increased two-fold in mice chronically treated with morphine. The AD(50) of morphine (s.c.) in these [Dmt(1)]DALDA-treated animals was increased more than 120 times, while that of the more selective mu agonist [d-Ala(2)-MePhe(4)-Gly-ol(5)]enkephalin (DAMGO) given intrathecally was increased more than 240 times. However, the AD(50) of DAMGO given intracerebroventricularly was essentially the same in animals treated chronically with [Dmt(1)]DALDA as in naive animals. The dose of naloxone required to precipitate withdrawal in [Dmt(1)]DALDA-treated animals was 20 times lower than that in morphine-tolerant animals. Using real-time quantitative PCR, we found that expression of the mu opioid receptor, delta opioid receptor, preproenkephalin and preprodynorphin genes was upregulated in the brain by [Dmt(1)]DALDA treatment. No significant changes in expression of opioid receptor or opioid peptide genes were detected in the spinal cord of [Dmt(1)]DALDA-treated mice, nor in the brain or spinal cord of morphine-treated mice. We conclude that a high degree of tolerance to [Dmt(1)]DALDA develops in the spinal cord but not brain, and cannot be accounted for by changes in expression of opioid receptors or opioid peptides in these tissues.

Expression of c-fos mRNA is increased and related to dynorphin mRNA expression following excitotoxic spinal cord injury in the rat

Previous studies have demonstrated that excitotoxic spinal cord injury (SCI) created by the intraspinal injection of quisqualic acid (QUIS) is capable of inducing opioid peptide gene expression within the spinal cord and cortex. The opioids are classically involved in the suppression of pain transmission but specifically, dynorphin, has been implicated in the secondary pathophysiologic response to SCI. Activation of the immediate early gene, c-fos, has been implicated in the induction of preprodynorphin (PPD) gene expression and therefore, may be an important intermediate step in the generation of the opioid response to SCI. The purpose of this study was to investigate whether intraspinal QUIS injection induces c-fos expression within the spinal cord. Male, Long–Evans, adult rats ( n=5) received an intraspinal injection of 1.2 μl of 125 mM QUIS directed at spinal segments T12-L2. Four hours post-injection brain and spinal cord tissues were removed and processed for in situ hybridization. Integrated density of c-fos and PPD mRNA expression was increased in the spinal dorsal horn following QUIS injection as compared to sham-injected animals. This indicates that SCI rapidly induces c-fos and PPD expression and suggests that c-fos plays a role in the induction of PPD expression.

Spinal and supraspinal changes in opioid mRNA expression are related to the onset of pain behaviors following excitotoxic spinal cord injury

Excitotoxic spinal cord injury (SCI) causes anatomic, physiologic and molecular changes within the spinal cord and brain. Intraspinal injection of quisqualic acid (QUIS) produces an excitotoxic injury that leads to the onset of behavioral syndromes, believed to be related to the clinical condition of chronic pain. The opioid system, classically involved in the suppression of pain transmission, has been associated with the onset of pain-related behaviors and changes in spinal opioid peptide expression have been demonstrated in various models of SCI and chronic pain. Recently, changes in opioid peptide expression have been demonstrated in both spinal and supraspinal areas following excitotoxic SCI. Therefore, the purpose of this study was to examine changes in opioid peptide gene expression as they relate to the onset of pain behaviors following excitotoxic SCI. Male, Long–Evans rats were given an intraspinal injection of 1.2 μl of 125 mM QUIS and allowed to survive for 10 days, a duration sufficient for the development of pain-related behaviors. Animals were assessed daily for the presence of excessive grooming behavior, i.e. self-directed biting and scratching resulting in damage to superficial and deeper layers of the skin. Animals were also tested for thermal hypersensitivity using a cold plate apparatus on days 5, 7, and 10 following QUIS injection. After sacrifice, quantitative in situ hybridization was performed on regions of the spinal cord surrounding the lesion site as well as whole brain sections through various levels of the thalamus and cortex. Spinal preproenkephalin (PPE) and preprodynorphin (PPD) expression was significantly increased in animals that developed excessive grooming behaviors vs. those that did not. For PPE, this difference was seen bilaterally, in areas of cord caudal to the site of injury. For PPD, this difference was seen only ipsilateral to the site of injection, rostral to the site of injury. In addition, PPE expression in the anterior cingulate cortex and PPD expression in the contralateral parietal cortex were significantly higher in grooming vs. non-grooming animals. These results support previous conclusions that both spinal and supraspinal regulation of endogenous opioid peptide expression plays a role in the response to or onset of post-SCI pain. These results also suggest that the opioid peptides are regulated independently and serve different functions in response to SCI.

Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells.

Magnetic fields have been shown to affect cell proliferation and growth factor expression in cultured cells. Although the activation of endorphin systems is a recurring motif among the biological events elicited by magnetic fields, compelling evidence indicating that magnetic fields may modulate opioid gene expression is still lacking. We therefore investigated whether extremely low frequency (ELF) pulsed magnetic fields (PMF) may affect opioid peptide gene expression and the signaling pathways controlling opioid peptide gene transcription in the adult ventricular myocyte, a cell type behaving both as a target and as a source for opioid peptides. Prodynorphin gene expression was investigated in adult rat myocytes exposed to PMF by the aid of RNase protection and nuclear run-off transcription assays. In PMF-exposed nuclei, nuclear protein kinase C (PKC) activity was followed by measuring the phosphorylation rate of the acrylodan-labeled MARCKS peptide. The effect of PMF on the subcellular distribution of different PKC isozymes was assessed by immunoblotting. A radioimmunoassay procedure coupled to reversed-phase high performance liquid chromatography was used to monitor the expression of dynorphin B. Here, we show that PMF enhanced myocardial opioid gene expression and that a direct exposure of isolated myocyte nuclei to PMF markedly enhanced prodynorphin gene transcription, as in the intact cell. The PMF action was mediated by nuclear PKC activation but occurred independently from changes in PKC isozyme expression and enzyme translocation. PMF also led to a marked increase in the synthesis and secretion of dynorphin B. The present findings demonstrate that an opioid gene is activated by myocyte exposure to PMF and that the cell nucleus and nuclear embedded PKC are a crucial target for the PMF action. Due to the wide ranging importance of opioid peptides in myocardial cell homeostasis, the current data may suggest consideration for potential biological effects of PMF in the cardiovascular system.

STZ-induced diabetes decreases and insulin normalizes POMC mRNA in arcuate nucleus and pituitary in rats.

Effects of streptozotocin (STZ)-induced diabetes and insulin on opioid peptide gene expression were examined in rats. In experiment 1, three groups were administered STZ (75 mg/kg ip single injection). Two groups were killed at either 2 or 4 wk. In the third group, insulin treatment (7.0 IU/kg x 1 day for 3 wk) was initiated 1 wk after STZ injection. STZ induced hyperphagia and reduced weight gain. Insulin decreased food intake and increased body weight relative to diabetes. Proopiomelanocortin (POMC) mRNA in arcuate nucleus (Arc) and pituitary decreased in diabetes and normalized after insulin treatment. Prodynorphin (proDyn) mRNA increased in diabetes and normalized in the pituitary after insulin but not in the Arc. Diabetes did not alter proenkephalin (proEnk) expression in the Arc or pituitary, nor dynorphin A1-17 or beta-endorphin in paraventricular nucleus (PVN). alpha-Melanocyte-stimulating hormone (alpha-MSH) peptide levels were decreased in the PVN and normalized following insulin treatment. Diabetes increased Arc neuropeptide Y mRNA, and insulin suppressed this increase. In experiment 2, insulin (2.5 IU/kg sc) daily for 1 wk in normal rats increased Arc POMC mRNA, but not proDyn and proEnk mRNA. These results suggest that Arc POMC expression and PVN alpha-MSH peptide levels decrease in diabetes. Also, insulin may influence Arc and pituitary POMC activity in neurons that regulate energy metabolism.

Expression of opioid receptors and ligands in pregnant mouse uterus and placenta.

The endogenous opioid system has been implicated in the regulation of hormonal secretion, pain perception, and uterine contractility during pregnancy, but there is only limited information about the cellular location of opioid receptor and opioid peptide gene expression in the pregnant rodent uterus and placenta. In this study, we have used in situ hybridization to identify expression sites of mRNAs encoding the delta (delta), kappa (kappa), and mu ( micro) opioid receptors as well as the endogenous opioid peptide precursors proenkephalin (PENK), prodynorphin (PDYN), and proopiomelanocortin (POMC) in pregnant mouse uterus and placenta. Soon after implantation, all three opioid receptor genes as well as POMC and PENK, but not PDYN, were detected in the uterine environment. Each expressed gene exhibited a distinct expression pattern that was generally retained until late gestation. The delta receptor and POMC were coexpressed in the trophoblast giant cells, which remained the only cells of the placenta/uterus to express these two genes throughout gestation. Cells expressing kappa receptors were absent from the placenta but instead were found in the basal part of the decidualized uterine endometrium. While kappa and micro receptors were transiently expressed in the uterine myometrium (until embryonic day 8.5), substantial levels of PENK were continuously detected in this region until at least embryonic day 18. In addition, complementary expression of the micro receptor and PENK genes in the uterus was detected. Taken together, these results suggest multiple roles for the opioid receptors and opioid peptides in maternal adaptation to pregnancy and in supporting embryo growth.

Distribution of opioid peptide gene expression in the limbic system of Fawn-Hooded (alcohol-preferring) and Wistar-Kyoto (alcohol-non-preferring) rats

Preprodynorphin and preproenkephalin mRNA expression was examined in the CNS of two rat strains, the alcohol-preferring Fawn-Hooded (FH) and the alcohol-non-preferring Wistar-Kyoto (WKY), using in situ hybridisation histochemistry. Relative to the WKY, the FH showed significantly lower levels of preproenkephalin mRNA in the striatum and nucleus accumbens (−24% and −17% respectively), but a higher level of preprodynorphin mRNA in the hippocampus (+33%). The depressed level of preproenkephalin mRNA in the nucleus accumbens may be implicated in alcohol-seeking behaviour.

Alterations in behavior and opioid gene expression induced by the novel tropane analog WF-31

The effects of the acute administration of the serotonin-selective tropane analog, [2β-propanoyl-3β-(4-isopropylphenyl)-tropane, WF-31, on spontaneous locomotor activity were measured and compared to those of the highly selective serotonin uptake inhibitor, fluoxetine and cocaine, a non-selective re-uptake inhibitor of dopamine and serotonin. WF-31 (1, 10 and 30 mg/kg)-elicited increases in locomotor behaviors when compared to vehicle-treated rats. This increased activity was blocked by pre-treatment with the dopaminergic antagonist, flupenthixol, suggesting that these effects may be mediated by dopaminergic mechanisms. Cocaine, but not fluoxetine, also elicited increases in behaviors. In addition, the effects of these three compounds on opioid peptide gene expression were also assessed using in situ hybridization histochemistry in the same animals. The acute administration of both WF-31 and cocaine increased the expression of preprodynorphin mRNA in the dorsal striatum whereas fluoxetine had no effect. Expression of striatal preproenkephalin mRNA was augmented by all three compounds. Within the nucleus accumbens, PPD mRNA levels were affected only by treatment with WF-31, an effect that was blocked by pre-treatment with flupenthixol. In contrast, the acute administration of both WF-31 and fluoxetine, but not cocaine, increased the expression of preproenkephalin mRNA. These increases, however, were not reversed by pre-treatment with flupenthixol. Despite its profile in vitro as a relatively selective serotonin re-uptake inhibitor, some of the in vivo actions of WF-31 appear to be mediated by dopaminergic mechanisms. These data further suggest that the mechanisms underlying expression of the opioid peptides in the nucleus accumbens may vary from those in the dorsal striatum.

Lactation decreases mRNA levels of opioid peptides in the arcuate nucleus of the rat

The state of lactation results in increased food intake to compensate for the increased energy expenditure to produce nutrients supplied to the offspring. In this study, Sprague-Dawley female rats lactating for 10–16 days, and rats 7 days post-lactation were implanted with osmotic minipumps infusing either naltrexone (NTX) (70 μg/h) or saline (0.9%) over a 48 h period. mRNA levels of pro-dynorphin (proDYN), pro-opiomelanocortin (POMC) and pro-enkephalin (proENK) were measured in the arcuate nucleus (ARC) and whole pituitary of both groups. In both saline- and NTX-treated lactating subjects, food intake was higher than in post-lactating subjects ( P<0.01). In post-lactating subjects, NTX decreased food intake by 27% during the infusion period ( P<0.05). There were no significant differences in body weight between the treatment groups; however, naltrexone decreased body weight gain in both lactating and post-lactating subjects. In both saline and NTX-treated lactating subjects, ARC mRNA levels of proDYN, POMC and proENK were significantly decreased compared with the saline or NTX-treated post-lactating subjects ( P<0.01). NTX did not significantly influence gene expression of opioid peptides in the ARC in either the lactating or the post-lactating subjects. Neither the lactation condition nor NTX administration significantly changed mRNA levels of proDYN, POMC or proENK in whole pituitary. Thus, as has been noted in energy-deprived rats, opioid peptide gene expression is decreased in the ARC of lactating rats, a period during which rats have increased energy requirements.