Opioid Receptor mRNA Expression Research Articles

Individual Differences in Cue-Induced Motivation and Striatal Systems in Rats Susceptible to Diet-Induced Obesity.

Pavlovian cues associated with junk-foods (caloric, highly sweet, and/or fatty foods), like the smell of brownies, can elicit craving to eat and increase the amount of food consumed. People who are more susceptible to these motivational effects of food cues may have a higher risk for becoming obese. Further, overconsumption of junk-foods leading to the development of obesity may itself heighten attraction to food cues. Here, we used a model of individual susceptibility to junk-foods diet-induced obesity to determine whether there are pre-existing and/or diet-induced increases in attraction to and motivation for sucrose-paired cues (ie, incentive salience or 'wanting'). We also assessed diet- vs obesity-associated alterations in mesolimbic function and receptor expression. We found that rats susceptible to diet-induced obesity displayed heightened conditioned approach prior to the development of obesity. In addition, after junk-food diet exposure, those rats that developed obesity also showed increased willingness to gain access to a sucrose cue. Heightened 'wanting' was not due to individual differences in the hedonic impact ('liking') of sucrose. Neurobiologically, Mu opioid receptor mRNA expression was lower in striatal 'hot-spots' that generate eating or hedonic impact only in those rats that became obese. In contrast, prolonged exposure to junk-food resulted in cross-sensitization to amphetamine-induced locomotion and downregulation of striatal D2R mRNA regardless of the development of obesity. Together these data shed light on individual differences in behavioral and neurobiological consequences of exposure to junk-food diets and the potential contribution of incentive sensitization in susceptible individuals to greater food cue-triggered motivation.

Development of Functionally Selective, Small Molecule Agonists at Kappa Opioid Receptors

The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

Female adolescent exposure to cannabinoids causes transgenerational effects on morphine sensitization in female offspring in the absence of in utero exposure

Female adolescent marijuana use is increasing, yet the effect on future offspring is unknown. Here, adolescent female Sprague Dawley rats (postnatal-day 30; PN30) were given subcutaneous (s.c.) injections with the cannabinoid agonist WIN-55,212 (WIN) or its vehicle (VEH) for three consecutive days using a twice-daily, increasing dosage regimen (1 mg/kg day 1; 2 mg/kg day 2; 4 mg/kg day 3). As adults (PN60), females were mated with drug-naïve males. Their adult female offspring (VEH-F1 or WIN-F1) were tested for behavioral sensitization by administering morphine (0 or 7.5 mg/kg s.c.) every other day for a total of five administrations. Following five days of abstinence, all animals received a morphine challenge (7.5 mg/kg s.c.) and locomotor activity was monitored. At completion of behavioral testing, mu opioid receptor (OPRM1), FosB, cFos, and dopamine receptor mRNA expression was measured in the nucleus accumbens as well as OPRM1 and corticotropin-releasing hormone mRNA in the paraventricular nucleus. In addition, plasma corticosterone levels were examined. On the day of challenge, morphine-pretreated WIN-F1 animals demonstrated a significantly enhanced response to morphine compared to morphine-pretreated VEH-F1 animals. Also following the morphine challenge, significantly higher levels of OPRM1 in the nucleus accumbens were observed in WIN-F1 animals. Together, these findings demonstrate transgenerational effects of adolescent exposure to cannabinoids in the absence of any in utero exposure.

Reduced Supraspinal Nociceptive Responses and Distinct Gene Expression Profile in CXBH Recombinant Inbred Mice

CXBH mice, known as an “opioid receptor-rich” strain, are a recombinant inbred mouse strain established by crossing the C57BL/6By and BALB/cBy strains. In the present study, we investigated nociceptive and antinociceptive sensitivity in CXBH mice and elucidated the underlying molecular mechanisms. CXBH mice exhibited slightly higher morphine-induced antinociception compared with C57BL/6J and BALB/cBy mice in the hot-plate test but not tail-flick test. CXBH mice exhibited a marked reduction of nociceptive sensitivity, regardless of the type of nociceptive stimulus, with the exception of tail stimulation. Changes in gene expression that corresponded to reduced nociceptive sensitivity in the brains of CXBH mice were observed in 62 transcripts, including pain- and analgesia-related transcripts, in a whole-genome expression assay. The total mRNA expression of opioid receptors was higher in CXBH mice than in C57BL/6J and BALB/cBy mice. However, the expression levels of MOR-1 mRNA, a major transcript of the μ opioid receptor gene, were not different among the C57BL/6J, BALB/cBy, and CXBH strains. In conclusion, supraspinal nociceptive responses were reduced in the CXBH mouse strain, and the expression levels of transcripts were altered in the brain of this strain. PerspectiveThis article presents the nociceptive and antinociceptive properties of CXBH recombinant inbred mice and gene expression differences that may underlie nociceptive tolerance in the strain. The CXBH mouse strain may be a useful animal model to investigate the molecular basis of individual differences in supraspinal pain sensitivity.

Absence of μ opioid receptor mRNA expression in astrocytes and microglia of rat spinal cord

Cumulating evidence has demonstrated that μ opioid receptor (MOR) agonists promote spinal glial activation, lead to synthesis and release of proinflammatory cytokines and chemokines, and contribute to opioid-induced hyperalgesia and development of opioid tolerance and dependence. However, whether these MOR agonists directly or indirectly act on spinal cord astrocytes and microglial cells in vivo is unclear. In the present study, by combining the techniques of in-situ hybridization of MOR mRNA with immunohistochemistry of glial fibrillary acidic protein (GFAP; an astrocyte marker) and Iba1 (a microglial marker), we examined expression and distribution of GFAP, Iba1, and MOR mRNA in the spinal cord of rats under chronic morphine tolerance conditions. Intrathecal injections of morphine twice daily for 7 days reduced morphine analgesic effect and increased both GFAP and Iba1 immunostaining densities in the spinal cord. Surprisingly, neither GFAP nor Iba1 colocalized with MOR mRNA in spinal cord cells. Our findings indicate that MOR expression is absent from spinal cord astrocytes and microglia, suggesting that these cell types are indirectly activated by MOR agonists under chronic opioid tolerance conditions.

Dynamic temporal and spatial regulation of mu opioid receptor expression in primary afferent neurons following spinal nerve injury

Despite using prescribed pain medications, patients with neuropathic pain continue to experience moderate to severe pain. There is a growing recognition of a potent peripheral opioid analgesia in models of inflammatory and neuropathic pain. The goal of this study was to characterize the temporal and spatial expression of mu opioid receptor (mOR) mRNA and protein in primary afferent neurons in a rat L5 spinal nerve ligation model of persistent neuropathic pain. Bilateral L4 and L5 dorsal root ganglia (DRGs), L4 and L5 spinal cord segments, and hind paw plantar skins were collected on days 0 (naïve), 3, 7, 14, and 35 post-spinal nerve ligation or post-sham surgery. We found that expression of mOR mRNA and protein in primary afferent neurons changed dynamically and site-specifically following L5 spinal nerve ligation. Real-time RT–PCR, immunohistochemistry, and Western blot analysis demonstrated a down-regulation of mOR mRNA and protein in the injured L5 DRG. In contrast, in the uninjured L4 DRG, mOR mRNA transiently decreased on day 7 and then increased significantly on day 14. Western blot analysis revealed a persistent increase in mOR protein expression, although immunohistochemistry showed no change in number of mOR-positive neurons in the uninjured L4 DRG. Interestingly, mOR protein expression was reduced in the skin on days 14 and 35 post-nerve injury and in the L4 and L5 spinal cord on day 35 post-nerve injury. These temporal and anatomically specific changes in mOR expression following nerve injury are likely to have functional consequences on pain-associated behaviors and opioid analgesia.

Mu opioid receptor mRNA expression, binding, and functional coupling to G‐proteins in human epileptic hippocampus

Mu opioid receptors (MOR) are known to be involved in seizure activity. The main goal of the present study was to characterize the MOR mRNA expression, binding, as well as G protein activation mediated by these receptors in epileptic hippocampus of patients with pharmacoresistant mesial temporal lobe epilepsy (TLE). In contrast with autopsy samples, hippocampus obtained from patients with mesial TLE demonstrated enhanced MOR mRNA expression (116%). Saturation binding experiments revealed significantly higher (60%) B(max) values for the mesial TLE group, whereas the K(d) values were not statistically different. Although mesial TLE group demonstrated high levels of basal binding for the G proteins (136%), DAMGO-stimulated [(35)S]GTPγS binding did not demonstrate significant alterations. In conclusion, our present data provide strong evidence that the epileptic hippocampus of patients with pharmacoresistant mesial TLE presents significant alterations in MOR. Such changes may represent adaptive mechanisms to compensate for other as yet unknown alterations.

A Proteomics Approach for Identification of Single Strand DNA-binding Proteins Involved in Transcriptional Regulation of Mouse μ Opioid Receptor Gene

The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the μ opioid receptor. Previously a single strand DNA element of the mouse μ opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, α-complex proteins (αCP) αCP1, αCP2, αCP2-KL, and αCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, αCP1, αCP2, and αCP2-KL activated the Oprm1 promoter activity, whereas αCP3 acted as a repressor. Ectopic expression of hnRNP K, αCP1, αCP2, and αCP2-KL also led to activation of the endogenous Oprm1 transcripts, and αCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and αCP binding to the single strand DNA element.

Delta opioid receptor mRNA expression is changed in the thalamus and brainstem of monoarthritic rats

Changes in the mRNA expression of neurotransmitters receptors under chronic pain conditions have been described in various areas of the central nervous system (CNS). Delta opioid receptors (DORs) have been implicated in pain mechanisms but, although its mRNA expression has been studied in the rat CNS, there are no reports describing its distribution in specific thalamic and brainstem nuclei during chronic inflammatory pain. Here, in situ hybridization for DOR mRNA was performed in brain sections from control and monoarthritic (MA) rats with 2, 4, 7 and 14 days of inflammation. Grain densities were determined bilaterally in the ventrobasal complex (VB), posterior (Po), centromedial/centrolateral (CM/CL) and reticular (Rt) nuclei of the thalamus, and in the dorsal reticular (DRt), lateral reticular (LRt) and parvocellular reticular (PCRt) nuclei of the brainstem. Control animals exhibited weak mRNA expression in the VB, Po and CM/CL, as well as in PCRt, while moderate grain densities were observed in the Rt, DRt and LRt. During MA, DOR mRNA expression was significantly decreased (22%) in the Rt contralateral to the affected joint at both 7 and 14 days of inflammation, as compared to controls. A bilateral reduction (35%) was also observed in the DRt at 14 days of MA, while a contralateral increase was found in the PCRt at 7 days (+39%). No significant changes were observed in the other regions analyzed. Thus, data show changes in the DOR mRNA expression during the development of chronic inflammatory pain, in thalamic and brainstem nuclei implicated in pain processing mechanisms.

Cell‐specific actions of HIV‐Tat and morphine on opioid receptor expression in glia

HIV-1 patients who abuse opiate-based drugs, including heroin and morphine, are at a higher risk of developing HIV dementia. The effects of opiates are mediated predominantly through opioid receptors, which are expressed on glial cells. As HIV-1 infection in the CNS is restricted to glial cells, experiments were designed to measure the cell-specific effects of HIV Tat and morphine exposure on opioid receptor expression in both astrocytes and microglia. Specifically, the cell-type-specific pattern of mu opioid receptor (MOR), delta opioid receptor (DOR), and kappa opioid receptor (KOR) localization (surface vs. intracellular) and expression of opioid receptor mRNA were determined after exposure to morphine in the presence and the absence of Tat in primary cultured microglia and astrocytes. Data show that morphine treatment caused significantly decreased cell surface expression of opioid receptors in microglia but not in astrocytes. However, morphine treatment in the presence of Tat significantly increased intracellular expression of opioid receptors and prevented morphine-induced cell surface opioid receptor down-regulation in microglia. These findings document that cell surface opioid receptor expression is divergently regulated by morphine in microglia compared with in astrocytes, and further suggest that HIV-Tat could exacerbate opioid receptor signaling in microglia by increasing receptor expression and/or altering ligand-induced trafficking of opioid receptors.

Decreased spinal cord opioid receptor mRNA expression and antinociception in a Theiler's murine encephalomyelitis virus model of multiple sclerosis

Multiple sclerosis patients typically experience increased pain that is relatively insensitive to opiate treatment. The mechanistic basis for this increased nociception is currently poorly understood. In the present study, we utilized the Theiler's murine encephalomyelitis virus (TMEV) model of MS to examine possible changes in spinal cord opioid receptor mRNA over the course of disease progression. TMEV infection led to significantly decreased mu, delta and kappa opioid receptor mRNA expression as analyzed by quantitative real-time PCR in both male and female mice at days 90, 150 and 180 post-infection (PI). Since opioid receptor mRNA expression decreased in TMEV mice, we examined whether opiate analgesia is also altered. TMEV infected female mice had significantly decreased opiate analgesia in thermal nociceptive tests beginning at day 90 PI, while TMEV-infected male mice did not display significantly decreased opiate analgesia until day 120 PI. The novel finding that opioid receptor expression is significantly decreased in the spinal cord of TMEV mice could explain the increased nociception and loss of opiate analgesia observed in both TMEV mice and multiple sclerosis patients.

Subcutaneous, intrathecal and periaqueductal grey administration of asimadoline and ICI-204448 reduces tactile allodynia in the rat

The purpose of this study was to assess the possible antiallodynic effect of asimadoline ([N-methyl-N-[1S)-1-phenyl)-2-(13S))-3-hydroxypyrrolidine-1-yl)-ethyl]-2,2-diphenylacetamide HCl]) and ICI-20448 ([2-[3-(1-(3,4-Dichlorophenyl-N-methylacetamido)-2-pyrrolidinoethyl)-phenoxy]acetic acid HCl]), two peripheral selective kappa opioid receptor agonists, after subcutaneous, spinal and periaqueductal grey administration to neuropathic rats. Twelve days after spinal nerve ligation tactile allodynia was observed, along with an increase in kappa opioid receptor mRNA expression in dorsal root ganglion and dorsal horn spinal cord. A non-significant increase in periaqueductal grey was also seen. Subcutaneous (s.c.) administration of asimadoline and ICI-204448 (1-30 mg/kg) dose-dependently reduced tactile allodynia. This effect was partially blocked by s.c., but not intrathecal, naloxone. Moreover, intrathecal administration of asimadoline or ICI-204448 (1-30 mug) reduced tactile allodynia in a dose-dependent manner and this effect was completely blocked by intrathecal naloxone. Microinjection of both kappa opioid receptor agonists (3-30 mug) into periaqueductal grey also produced a naloxone-sensitive antiallodynic effect in rats. Our results indicate that systemic, intrathecal and periaqueductal grey administration of asimadoline and ICI-204448 reduces tactile allodynia. This effect may be a consequence of an increase in kappa opioid receptor mRNA expression in dorsal root ganglion, dorsal horn spinal cord and, to some extent, in periaqueductal grey. Finally, our data suggest that these drugs could be useful to treat neuropathic pain in human beings.

Site‐specific estrogen and progestin regulation of orphanin FQ/nociceptin and nociceptin opioid receptor mRNA expression in the female rat limbic hypothalamic system

The distributions of orphanin FQ (OFQ/N; also known as nociceptin) and its cognate receptor, opioid receptor-like receptor-1 (NOP), overlap steroid-responsive regions throughout reproductive circuits of the limbic system and hypothalamus. For example, in the ventromedial nucleus of the hypothalamus (VMH), OFQ/N facilitates lordosis in female rats through estrogen and progesterone regulation of nociceptin activity. We studied estrogen and progesterone regulation of OFQ/N and NOP mRNA expression in limbic-hypothalamic reproductive circuits. Ovariectomized rats were treated with 17beta-estradiol-benzoate (2 microg) and 26 hours later with oil or progesterone (500 microg) and were killed 30 hours after initial treatment. Alternate brain sections were processed for OFQ/N or NOP mRNA in situ hybridization. High levels of hybridization for NOP and OFQ/N and overlapping distributions were observed throughout the limbic hypothalamic reproductive circuits; however, in VMH, only NOP expression was observed. Estrogen treatment increased NOP mRNA expression in anteroventral periventricular nucleus (AVPV), median preoptic nucleus, and VMH. Subsequent progesterone treatment did not alter estrogen-induced expression of NOP mRNA in VMH or median preoptic nucleus but reduced expression in the AVPV. OFQ/N mRNA levels were also regulated by steroids. In the caudal part of the posterodorsal medial amygdala, estrogen increased OFQ/N mRNA levels, and progesterone did not alter this increase, whereas, in the medial part of the medial preoptic nucleus, estrogen and progesterone were needed to increase OFQ/N mRNA levels. Steroid regulation of OFQ/N and NOP in the medial preoptic nucleus and VMH is consistent with emerging data indicating that this opioid system regulates female reproduction.

Interleukin-1 beta contributes to the upregulation of kappa opioid receptor mrna in dorsal root ganglia in response to peripheral inflammation

During local painful inflammation, axonal transport of opioid receptors from dorsal root ganglia toward the periphery is increased, associated with a higher receptor density and enhanced efficacy of opioid analgesics at the injured site. To examine whether this increase is related to transcription, mRNA of the kappa opioid receptor in lumbar dorsal root ganglia was quantified by real time light cycler polymerase chain reaction. In dorsal root ganglia of naive rats, kappa opioid receptor mRNA expression was three-fold higher than previously shown for delta opioid receptor and two times lower than mu opioid receptor mRNA, respectively. After induction of unilateral paw inflammation by Freund’s complete adjuvant, kappa opioid receptor mRNA was significantly upregulated with a peak at 12 h in ipsilateral dorsal root ganglia. This effect could be mimicked by intraplantar injection of the proinflammatory cytokine interleukin-1 beta. Kappa opioid receptor mRNA upregulation lasted longer in interleukin-1 beta–treated rats compared with Freund’s complete adjuvant–treated rats. Furthermore, a significant increase in kappa opioid receptor positive neurons was detected by immunohistochemistry 24 h after local injection of Freund’s complete adjuvant or interleukin-1 beta. In Freund’s complete adjuvant–induced inflammation, kappa opioid receptor upregulation was blocked by treatment with interleukin-1 receptor antagonist without changing the leukocyte infiltration in the paw. In conclusion, kappa opioid receptor mRNA and protein in dorsal root ganglia are upregulated in response to peripheral inflammation. This effect can be mimicked by a single local injection of interleukin-1 beta, and Freund’s complete adjuvant–induced upregulation in kappa opioid receptor mRNA and protein can be prevented by treatment with interleukin-1 receptor antagonist. These data suggest that the peripheral production of the proinflammatory cytokine interleukin-1 beta is a specific inducer of kappa opioid receptor expression in the dorsal root ganglia.

Alterations of the preproenkephalin system in cardiac hypertrophy and its role in atrioventricular conduction

The goal of this study was to investigate alterations of the endogenous opioid system in cardiac hypertrophy, to elucidate mechanisms of preproenkephalin (ppENK) gene expression, and to assess effects of endogenous opioids on myocardial contractility and atrioventricular conduction. Hypertrophy was induced by ligation of a renal artery (2K1C) or chronic isoprenaline infusion (ISO). ppENK and opioid receptor (mu-, delta-, kappa-OR) mRNA expression was quantified by Northern blot and quantitative RT-PCR, respectively. Isolated cardiac myocytes and non-myocytes from neonatal rat heart were used for cell culture experiments. Overall expression of OR in the heart was markedly lower than in brain tissue, with kappa-OR being the most abundant isoform in the heart. We did not observe differences in kappa-OR expression in ventricular and atrial myocardium. In contrast, delta-OR expression was markedly higher in atria than in ventricles. Mu-OR expression in the heart was below the detection limit of the developed qRT-PCR assay. In left ventricular myocardium, ppENK mRNA levels were significantly increased in 2K1C rats but decreased in ISO rats. Cell culture experiments from neonatal rat hearts revealed that myocytes and non-myocytes express ppENK mRNA. In these cells, receptor-dependent and -independent stimulation of the beta-adrenergic signalling pathway caused an increase in ppENK mRNA. Furthermore, inactivation of inhibitory G-proteins by pertussis toxin increased basal and noradrenaline-stimulated ppENK mRNA expression. The physiological significance of myocardial opioids was investigated in isolated perfused rat hearts. Opioid receptor antagonists (nor-BNI, naltrindol) and the enkephalinase inhibitor CPL had no effect on contractility but significantly altered atrioventricular conduction. These observations suggest that the cardiac opioid system is activated in cardiac hypertrophy. Pressure overload and stimulation of the beta-adrenergic signalling pathway have been identified as a possible mechanism leading to increased ppENK expression, which may contribute to opioid system activation. Finally, endogenous opioids modulate the dromotropic response to catecholamine stimulation. The latter finding raises the possibility that endogenous opioids may contribute to the pathogenesis of arrhythmias.

Expression of Opioid Receptor Subtypes and Their Ligands in Fibrillating Human Atria

The delta- and kappa-receptor subtypes are both abundantly expressed in the human heart and participate in age- and stress-related alterations of cardiac function. Opioid receptor agonists mediate cardioprotection in response to ischemic preconditioning via increased intracellular Ca(2) (+) levels, opening mitochondrial K(ATP) channels, and PKC activation. We studied the expression of opioid receptor subtypes kappa and delta, and of their ligand precursors, proopiomelanocortin (POMC) and preproenkephalin A (PENKA), in human atrial tissue of patients in sinus rhythm (SR), or persistent atrial fibrillation (AF). The mitochondrial size was also compared between the two groups. The atrial mRNA expression of opioid peptide precursors and receptors was assessed by competitive and real-time RT-PCR in 16 patients in AF and 16 patients in SR. Mitochondria were analyzed in the atrial tissue by electron microscopy in four patients in AF and four patients in SR. Both PENKA (SR: 100 +/- 33% vs AF: 33 +/- 21%; P < 0.05) and kappa-receptor mRNA amounts (AF: 78 +/- 20% vs SR: 100 +/- 11%; P < 0.05) were both decreased in AF in comparison to SR. In addition, POMC mRNA levels were decreased in AF (SR: 100 +/- 54% vs AF: 37 +/- 26%; P < 0.05), whereas the expression of the corresponding delta-opioid receptor was unchanged (AF: 102 +/- 34% vs 100 +/- 44%). Mitochondrial size was increased during persistent AF. Persistent AF is associated with the down-regulation of the opioid receptor/ligand expression. This suggests a loss of protective capacity in the fibrillating atrial tissue, resulting in an ultrastructural remodeling of atrial myocytes.

Alterations in morphine-induced reward, locomotor activity, and thermoregulation in CREB-deficient mice

Previous studies in our lab have shown a robust decrease in the rewarding properties of morphine in CREB αΔ mutant mice. To determine whether the genetic effects of the global CREB αΔ mutation are specific to reward or generalizable, we examined a variety of morphine-induced behaviors regulated by different neural circuitry. At low doses of morphine (5 and 10 mg/kg), CREB αΔ mutant mice show a reduction in reward yet similar locomotor activity in response to morphine compared to wild type littermates. However, at a high dose (20 mg/kg), CREB αΔ mutant mice show an increase in reward and locomotor activity. Morphine-induced thermoregulation is attenuated in CREB αΔ mutant mice at high doses of morphine compared to wild type animals. The behavioral differences in response to morphine seen in CREB αΔ mutant mice are not due to changes in mu opioid receptor (MOR) mRNA expression, as the CREB deletion has no effect on baseline MOR mRNA in three of the brain regions involved in mediating these behaviors: the ventral tegmental area (VTA), nucleus accumbens (NAc), and hypothalamus. These data demonstrate that at low doses, deficits in morphine-induced changes in CREB deficient mice are limited to reward and thermoregulation. However, at higher doses, CREB mutant mice actually find morphine more rewarding and exhibit increased locomotor activity compared to their wild type littermates. Together, these results indicate that the role of CREB in dose-dependent changes in behaviors induced by morphine is different depending on the brain regions involved in mediating the behavior.

Abundant expression of mu and delta opioid receptor mRNA and protein in the cerebellum of the fetal, neonatal, and adult rat

Opioid receptor proteins and mRNAs have been localized to a variety of regions within the rat brain. It is generally accepted that within the lobes of the rat cerebellum, only delta opioid receptor (DOR) is expressed. This is in contrast to that observed in humans and rabbits which express both mu opioid receptor (MOR) and DOR. In this study, we report detection of MOR as well as DOR protein by immunohistochemical localization, and mRNA by fluorescent in situ hybridization (FISH) within Purkinje cells (PK) and the granular layer of neonatal (P6) and adult rat cerebellum. Expression of MOR mRNA was also detected within cells of the molecular layer, but at lower levels than those seen within the PK cells. Abundant expression of MOR and DOR mRNA was detected in the external germinal layer of the immature cerebellum of the fetal (E16) rat, supporting a role for MOR and DOR in regulating neurogenesis of the cerebellum. In addition, using exon-specific cRNA probes, exons 1 and 4, which are both found in the MOR-1 splice variant mRNA, were detected in PK cells in the cerebellum and also within deep cerebellar nuclei in the adult.

Modulation of striatal serotonin and opioid receptor mRNA expression following systemic N-methyl-norsalsolinol administration

The dihydroxylated tetrahydroisoquinoline derivative, 2( N)-methyl-norsalsolinol (NMNorsal), was identified in l-DOPA-treated patients with Parkinson's disease and proposed to be responsible for behavioral changes. In the present study, we investigated the effect of NMNorsal on serotonin and opioid receptors gene expression in caudate nucleus of Wistar rats. Using RT-PCR, serotonin 5-HT2A, μ- and δ-opioid receptor mRNA levels were determined after NMNorsal administration (40 mg/kg, i.p.). There was a marked increase of 5-HT2A and δ-opioid receptor mRNA levels with a maximum after 48 h. In contrast, μ-opioid receptor mRNA levels were significantly decreased to 10% after 24 h and 21% after 48 h, respectively. Our present results demonstrate for the first time that the atypical heterocylic l-DOPA/dopamine metabolite NMNorsal is able to modify long-term regulation of serotonin and opioid receptor expression in striatum. Since the occurrence of hallucinosis or psychosis following l-DOPA treatment is related to the serotonergic system, these results probably reflect a link between NMNorsal and l-DOPA side effects in Parkinson's disease. However, further experiments are needed.

Effects of single and repeated prolonged stress on mu-opioid receptor mRNA expression in rat gross hypothalamic and midbrain homogenates

This study tested the hypothesis that stress-induced opioid peptides may have stimulative and inhibitive influence on mu opioid receptor (MOR) mRNA expression and hypothalamus. Several studies have investigated the effects of stress on MOR mRNA expression in rat brain, but almost none compared the response to single versus repeated stresses. Here, we examined the effects of single and repeated stress on MOR mRNA expression in different rat brain regions using reverse transcriptase-polymerase chain reaction (RT-PCR). Following a single episode of restraint stress for 4 h (1R) or 4 h per day on 2 (2R) or 3 (3R) consecutive days, the hypothalamus and midbrain were removed immediately and MOR mRNA levels in both regions were determined by RT-PCR. Blood samples were also collected for simultaneous measurement of serum adrenocorticotropic hormone (ACTH) and corticosterone (CS). MOR mRNA expression was significantly higher in both regions in the 2R group, whereas expression levels in the 3R group did not differ from controls. In the 1R group, hypothalamic MOR expression was equivalent to that in controls, but expression was significantly higher in the midbrain. Serum ACTH levels were significantly higher only in the 1R group, whereas serum CS was significantly higher in both the 1R and 3R groups. Our findings indicate that the influence of restraint stress on MOR mRNA expression in the hypothalamus is different than in the midbrain region in rats. Endogenous opioid peptides released in response to stress may paradoxically have an effect on the HPA axis.