Antisense oligodeoxynucleotide to δ opioid receptors attenuates morphine dependence in mice

The effect of intracerebroventricular (i.c.v.) treatment with antisense oligodeoxynucleotide (A-oligo) to δ opioid receptor mRNA on the morphine-induced place preference and naloxone-precipitated jumping was examined in morphine-dependent mice. Morphine (5 mg/kg, s.c.) produced a significant place preference. I.c.v. pretreatment with A-oligo (0.01–1 μg/mouse) dose-dependently attenuated this morphine (5 mg/kg, s.c.)-induced place preference, while mismatched oligodeoxynucleotide (M-oligo; 1 μg/mouse, i.c.v.) was ineffective. Naloxone (3 mg/kg, s.c.) precipitated jumping in morphine-dependent mice. I.c.v. pretreatment with A-oligo (1 μg/mouse) attenuated this naloxone (3 mg/kg, s.c.)-precipitated jumping in morphine-dependent mice, while M-oligo (1 μg/mouse, i.c.v.) was ineffective. These data demonstrate that the selective reduction in supraspinal δ opioid receptor function caused by pretreatment with A-oligo attenuated the morphine-induced place preference and naloxone-precipitated jumping in morphine-dependent mice, suggesting that the rewarding effect of and physical dependence on morphine may be modulated by central δ opioid receptors.     

Antibodies raised against the N-terminal sequence of δ opioid receptors blocjed δ-mediated supraspinal antinociception in mice

A polyclonal antiserum directed against the first 16 aminoacids of the N-terminal sequence of the murine δ opioid receptor was raised in rabbits. The intracerebroventricular (i.c.v.) injection to mice of the anti δ receptor IgGs impaired the antinociception produced by DPDPE, [D-Ala²]- Deltorphin II, DADLE and β-endorphin-(1–31) when studied 24 h later in the tail-flick test. Antinociception produced by morphine and DAMGO was fully expressed in mice undergoing this treatment. The selective δ antagonist ICI 174864 (0.8 nmols/mouse, i.c.v.) significantly reduced the antinociceptive activity of opioids to the extent observed after giving the antibodies. ICI 174864 did not decrease further the antinociception that remained after the anti δ receptor serum. The specific binding displayed by 3 nM [³H]-DPDPE was reduced in membranes pre-incubated with the antiserum, whereas no change could be detected for 0.6 nM [³H]-DAMGO labelling μ receptors. This experimental approach revealed the δ component of opioid-evoked supraspinal antinociception in mice.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

Spontaneous light or dark preference in albino mice?

The present experiment examined spontaneous visual choice behaviour and acquisition of a positively reinforced visual discrimination task in Swiss albino mice. In experiment I animals were given 4 consecutive trials in which they could freely enter either a dimly illuminated or a darkened arm of a Y-maze; the position of the light stimulus was randomized across trials. D groups and L groups were tested during the dark and the light period of the day respectively. Results revealed a significant spontaneous preference for the illuminated arm of the maze, independent of the testing period. It is suggested that the dim light has a reinforcing value because it provides additional information about a novel environment. In a second experiment an appetitive visual discrimination task was carried out in the same Y-maze. After a pretraining period, half the animals were reinforced in the illuminated arm and half were reinforced in the darkened one, on five consecutive days. On the first test session all groups of animals chose the illuminated arm significantly more frequently, whereas light/dark choices reached chance level on the last test session. Discrimination learning was not acquired and a behavioural analysis revealed an increasing tendency to a side preference across testing.     

Etonitazene-induced antinociception in μ1 opioid receptor deficient CXBK mice: Evidence for a role for μ2 receptors in supraspinal antinociception

The prevailing view is that supraspinal μ opioid-mediated antinociception in mice is mediated via the μ₁ subtype. The purpose of the present study was to determine if the highly μ-selective compound etonitazene could produce supraspinal (intracerebroventricular; i.c.v.) antinociception in CXBK mice, which are deficient in brain μ₁, but not μ₂, opioid receptors. CXBK or normal Crl:CD-1 ^®(ICR)BR mice were administered graded doses of etonitazene i.c.v. and 15 min later antinociception was assessed by a standard radiant-heat or 55°C water tail-flick test. Etonitazene produced dose-related antinociception that was blocked by naloxone and by β-FNA (demonstrating a μ opioid mechanism), but not by either ICI-174,864 or naltrindole (demonstrating the lack of involvement of δ opioid receptors). These findings suggest that μ₂ opioid receptors are important contributors to opioid-induced supraspinal antinociception in mice.     

L-type voltage-dependent calcium channels facilitate acetylation of histone H3 through PKCγ phosphorylation in mice with methamphetamine-induced place preference

The present study investigated regulation of histone acetylation by L-type voltage-dependent calcium channels (VDCCs), one of the machineries to provide Ca(2+) signals. Acetylation of histone through the phosphorylation of protein kinase Cγ (PKCγ) in the development of methamphetamine (METH)-induced place preference was demonstrated in the limbic forebrain predominantly but also in the nucleus accumbens of α1C subunit knockout mice. Chronic administration of METH produced a significant place preference in mice, which was dose-dependently inhibited by both chelerythrine (a PKC inhibitor) and nifedipine (an L-type VDCC blocker). Protein levels of acetylated histone H3 and p-PKCγ significantly increased in the limbic forebrain of mice showing METH-induced place preference, and it was also significantly attenuated by pre-treatment with chelerythrine or nifedipine. METH-induced place preference was also significantly attenuated by deletion of half the α1C gene, which is one of the subunits forming Ca(2+) channels. Furthermore, increased acetylation of histone H3 was found in specific gene-promoter regions related to synaptic plasticity, such as Nrxn, Syp, Dlg4, Gria1, Grin2a, Grin2b, Camk2a, Creb, and cyclin-dependent kinase 5, in wild-type mice showing METH-induced place preference, while such enhancement of multiple synaptic plasticity genes was significantly attenuated by a deletion of half the α1C gene. These findings suggest that L-type VDCCs play an important role in the development of METH-induced place preference by facilitating acetylation of histone H3 in association with enhanced expression of synaptic plasticity genes via PKCγ phosphorylation following an increase in the intracellular Ca(2+) concentration.     

Role of κ1 opioid receptors and cAMP in regulation of cardiac tolerance to ischemia and reperfusion

Cardioprotective, inotropic, and antiarrhythmic effects of the selective agonist of κ₁ opioid receptors (κ₁-ORs) U-50.488H have been studied after 45-min global ischemia and 30-min reperfusion of isolated perfused rat hearts. The heart κ₁-ORs were stimulated by adding 0.1 or 1 μmol/l U-50.488H to the perfusion solution. The opioid did not affect the frequency of reperfusion arrhythmias. At a concentration of 0.1 μmol/l, it induced a twofold decrease in the reperfusion release of creatine phosphokinase (CPK), which positively correlated with a decrease in the myocardial cAMP level (r = 0.89, p < 0.01). Application of U-50.488H at a final concentration of 1 μmol/l did not change the cAMP level and CPK release. These results suggest that the cardioprotective effect of U-50.488H is due to a decrease in the level of cAMP in cardiomyocytes. Activation of κ₁-ORs decreased the frequency and force of myocardial contractions. It has been shown that the negative inotropic and chronotropic effects of U-50.488H are independent of changes in the myocardial cAMP level. A hypothesis is proposed that the absence of cardioprotective effect of 1μM U-50.488H is a result of activation of nonopioid receptors in cardiomyocytes.     

Induction of conditioned place preference and dopamine release by salsolinol in posterior VTA of rats: involvement of μ-opioid receptors

Salsolinol (Sal), locally administered into the posterior VTA (pVTA) of rats, produces psychomotor responses and reinforcing effects, probably, through the activation of μ-opioid receptors (MORs). The neurochemical correlates of these phenomena are, however, practically unknown. In this paper, we explore the neurochemical events and the mechanisms involved in these behaviors. To do that, we test the ability of Sal, directly microinjected into the pVTA, to induce conditioned place preference (CPP) and to increase dopamine levels in the nucleus accumbens shell. Bilateral injections of 30 pmol of Sal induced a strong CPP (rats spent around 70% of the total test time), a result that could be explained by the fact that Sal microinjected into the pVTA increased DA levels in the ipsilateral accumbens up to 141% of baseline. The local pretreatment with β-FNA, an antagonist of MORs, prevented this increase, supporting our hypothesis on the involvement of MORs in the Sal-derived effects.     

Role of κ1 opioid receptors and cAMP in regulation of cardiac tolerance to ischemia and reperfusion

The cardioprotective, inotropic, and antiarrhythmic effects of U-50.488, a selective agonist of κ₁ opioid receptors (κ₁ ORs), was studied using the model of 45-min total ischemia and 30-min reperfusion of isolated rat heart. Cardiac κ₁ ORs were stimulated by adding U-50.488 to the perfusing solution up to the final concentration of 0.1 or 1 μmol/l. The opioid had no influence on the incidence of reperfusion arrhythmias. The addition of 0.1 μmol/l U-50.488 reduced the reperfusion release of creatine phosphokinase (CPK) by half, which positively correlated with the decrease in the myocardial cAMP content (r = 0.89, p < 0.01). At the same time, the addition of U-50.488 in the higher concentration (1 μmol/l) had no effect on either cAMP level or CPK release. These results indicate that the cardioprotective effect of U-50.488 may be connected with the reduction of myocardial cAMP content. Activation of κ₁ ORs caused a decrease in both frequency and amplitude of myocardial contractions. The negative inotropic and chronotropic effect of U-50.488 was shown to be independent of changes in the myocardial cAMP content. A hypothesis is proposed that the absence of any cardioprotective effect of U-50.488 at the higher concentration (1 μmol/l) is accounted for by its interaction with unknown nonopioid receptors of cardiac myocytes.     

Co-localization of μ and δ opioid receptors on SK-N-SH cells detected by fluorescence microscopy using labeled anti-idiotypic antibodies

Selective fluorescence labeling of opioid receptor subclasses on SK-N-SH cultured cells has been accomplished using labeled polyclonal anti-idiotypic antibodies along with subclass-selective opioid agonists (DPDPE, δ-selective; DAMGO, μ-selective) as blocking reagents. Labeling of the cells was examined using conventional fluorescence microscopy. Co-localization of μ- and δ- opioid receptors on SK-N-SH cells has been studied by double labeling fluorescence experiments. In agreement with our own, and other workers', previous observations on NG108-15 cells, a subpopulation of viable cells in asynchronous cultures are labeled. Amon those SK-N-SH cells that are labeled, both subclasses of receptors are seen. On the basis of sequential blocking experiments we interpret our combined results to be consistent with a model where μ- and δ- binding sites reside on different subunits of a multimeric complex.     

α7-Nicotinic acetylcholine receptor: role in early odor learning preference in mice

Recently, we have shown that mice with decreased expression of α7-nicotinic acetylcholine receptors (α7) in the olfactory bulb were associated with a deficit in odor discrimination compared to wild-type mice. However, it is unknown if mice with decreased α7-receptor expression also show a deficit in early odor learning preference (ELP), an enhanced behavioral response to odors with attractive value observed in rats. In this study, we modified ELP methods performed in rats and implemented similar conditions in mice. From post-natal days 5-18, wild-type mice were stroked simultaneously with an odor presentation (conditioned odor) for 90 s daily. Control mice were only stroked, exposed to odor, or neither. On the day of testing (P21), mice that were stroked in concert with a conditioned odor significantly investigated the conditioned odor compared to a novel odor, as observed similarly in rats. However, mice with a decrease in α7-receptor expression that were stroked during a conditioned odor did not show a behavioral response to that odorant. These results suggest that decreased α7-receptor expression has a role in associative learning, olfactory preference, and/or sensory processing deficits.     

Synthesis and binding affinity of novel mono- and bivalent morphinan ligands for κ, μ, and δ opioid receptors

A novel series of homo- and heterodimeric ligands containing κ/μ agonist and μ agonist/antagonist pharmacophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro binding affinity at κ, μ, and δ opioid receptors, and their functional activities were determined at κ and μ receptors in [(35)S]GTPγS functional assays. Most of these compounds had high binding affinity at μ and κ receptors (K(i) values less than 1nM). Compound 15b, which contains butorphan (1) at one end of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with binding affinity K(i) values of 0.089nM at the μ receptor and 0.073nM at the κ receptor. All of the morphinan-derived ligands were found to be partial κ and μ agonists; ATPM-derived ligands 12 and 11 were found to be full κ agonists and partial μ agonists.     

Are age and sex differences in brain oxytocin receptors related to maternal and infanticidal behavior in naïve mice?

This article is part of a Special Issue “Parental Care”.There is significant variability in the behavioral responses displayed by naïve young and adult mice when first exposed to pups. This variability has been associated with differences in the expression of oxytocin receptors (OXTRs) in the brain in several species. Experiment I investigated the behavioral responses of juvenile, adolescent, and adult CB57BL/6 males and females when first exposed to pups. We found an age increase in maternal females (11% of juveniles, 20% of adolescents, and 50% of young adults), and infanticidal males (0% of juveniles, 30% of adolescents, 44.5% of young adults, and 100% of older adults). Experiment II investigated OXTR density in the brain of juvenile and adult mice. Our results revealed an age decline in the density of OXTR in several brain regions, including the lateral septum, cingulated and posterior paraventricular thalamic nucleus in both males and females. Adult females had higher OXTR density in the ventromedial nucleus/postero-ventral hypothalamus (VMH) and the accessory olfactory bulb (AOB), but lower density in the ventral region of the lateral septum (LSv) than juveniles. Males had lower OXTR density in the anterior olfactory area (AOA) compared to juveniles. No age or sex differences were found in the medial preoptic area, and amygdaloid nuclei, among other brain regions. This study suggests that 1) maturation of parental and infanticidal behavioral responses is not reached until adulthood; 2) the pattern of development of OXTR in the mouse brain is unique, region specific, and differs from that observed in other rodents; 3) either up or down regulation of OXTR in a few brain regions (VMH/AOB/LSv/AOA) might contribute to age or sex differences in parental or infanticidal behavior.     

Spinal β-adrenergic receptors’ activation increases the blood glucose level in mice

We examined the role of spinally located β-adrenergic receptors in the regulation of the blood glucose level. The intrathecal (i.t.) injections with dobutamine (β₁-adrenergic receptor agonist) or terbutaline (β₂-adrenergic receptor agonist) caused an elevation of the blood glucose level, whereas metoprolol (β₁-adrenergic receptor antagonist) or butoxamine (β₂-adrenergic receptor antagonist) did not. In addition, i.t. pretreatment with pertussis toxin (PTX) attenuated the hyperglycemic effect induced by dobutamine or terbutaline. Moreover, plasma insulin level was increased by dobutamine but not by terbutaline, and PTX reduced dobutamine-induced up-regulation of the plasma insulin level. Terbutaline significantly increased plasma corticosterone level, and PTX further enhanced terbutaline-induced corticosterone level. Furthermore, intraperitoneal (i.p.) pretreatment with hexamethonium- (a preganglionic blocker) attenuated dobutamine- and terbutaline-induced hyperglycemic effects. Our results suggest that activation of spinal β₁- and β₂-adrenergic receptors produces hyperglycemic effects in a different manner. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by terbutaline. Furthermore, dobutamine- or terbutaline-induced hyperglycemia appears to be mediated through the spinal nerves.     

Disruption of β-arrestins blocks glucocorticoid receptor and severely retards lung and liver development in mice

In this study, the role of β-arrestin 1 and β-arrestin 2 in fetal lung and liver development was examined using Arrb1(-/-)Arrb2(-/-) mouse embryos. β-Arrestin 1/2 dual-null mice died shortly after birth and morphological examination revealed an obvious pulmonary hypoplasia and severe hepatic impairment. Western blot analysis demonstrated that GR protein levels in Arrb1(-/-)Arrb2(-/-) lung and liver tissues were significantly decreased compared to wild type embryos. Expression of GR proteins was confirmed in the nuclei of type II pneumocytes of 18.5 day embryos (E18.5) by immunofluorescence. The production of hepatic glucose and mRNA level of gluconeogenic enzymes were dramatically reduced in E18.5 Arrb1(-/-)Arrb2(-/-) liver. These results suggest that GR is an important downstream effector of the β-arrestin signaling pathway involved in regulation of lung and liver development. However, no obvious changes in GR expression following in vitro modulation of β-arrestin 1/2 indicated the existence of an indirect regulatory relationship between GR and the β-arrestin signaling pathway.     

Spinal δ2 but not δ1 opioid receptors are involved in intracereboventricular β-endorphin-induced antinociception in the mouse

The antinociception induced by β-endorphin given intracerebroventricularly (i.c.v.) has been previously demonstrated to be mediated by the release of Met-enkephalin and subsequent stimulation of δ receptors in the spinal cord for antinociception. The present study was designed to determine what type of opioid receptor, δ1 or δ2, in the spinal cord is involved in i.c.v. β-endorphin-induced antinociception. Antinociception was assessed by the tail-flick test in male ICR mice. NTB (0.2–20 nmol) and NTI0 (0.22–2.2 nmol),selective δ2 receptor antagonists, given intrathecally (i.t.) dose-dependently attenuated i.c.v. β-endorphin-induced inhibition of the tail-flick response. On the other hand, BNTX (0.02–2.2 nmol), a selective δ1 receptor antagonist, given i.t., did not block i.c.v. β-endorphin-induced antinociception. The tail-flick inhibition induced by DAMGO, a μ receptor agonist, or U50,488H, a к receptor agonist, was not blocked by i.t. BNTX, NTB or NTI. It is concluded that δ2 but not δ1 receptors in the spinal cord are involved in i.c.v. β-endorphin-induced antinociception.     

Replication Stress Leads to Genome Instabilities in Arabidopsis DNA Polymerase δ Mutants

Impeded DNA replication or a deficiency of its control may critically threaten the genetic information of cells, possibly resulting in genome alterations, such as gross chromosomal translocations, microsatellite instabilities, or increased rates of homologous recombination (HR). We examined an Arabidopsis thaliana line derived from a forward genetic screen, which exhibits an elevated frequency of somatic HR. These HR events originate from replication stress in endoreduplicating cells caused by reduced expression of the gene coding for the catalytic subunit of the DNA polymerase δ (POLδ1). The analysis of recombination types induced by diverse alleles of polδ1 and by replication inhibitors allows the conclusion that two not mutually exclusive mechanisms lead to the generation of recombinogenic breaks at replication forks. In plants with weak polδ1 alleles, we observe genome instabilities predominantly at sites with inverted repeats, suggesting the formation and processing of aberrant secondary DNA structures as a result of the accumulation of unreplicated DNA. Stalled and collapsed replication forks account for the more drastic enhancement of HR in plants with strong polδ1 mutant alleles. Our data suggest that efficient progression of DNA replication, foremost on the lagging strand, relies on the physiological level of the polymerase δ complex and that even a minor disturbance of the replication process critically threatens genomic integrity of Arabidopsis cells.