Satoru Ozaki

Suppression of the morphine‐induced rewarding effect in the rat with neuropathic pain: implication of the reduction in µ‐opioid receptor functions in the ventral tegmental area

The present study was designed to investigate the rewarding effect, G‐protein activation and dopamine (DA) release following partial sciatic nerve ligation in the rat. Here we show for the first time that morphine failed to produce a place preference in rats with nerve injury. Various studies provide arguments to support that the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc), is critical of the motivational effects of opioids. In the present study, there were no significant differences between sham‐operated and sciatic nerve‐ligated rats in the increases in guanosine‐5′‐o‐(3‐[35S]thio)triphosphate ([35S]GTPγS) binding to membranes of the N.Acc stimulated by either DA, the D1 receptor agonist SKF81297, the D2 receptor agonist N‐propylnoraporphine or the D3 receptor agonist 7‐hydroxy‐2‐dipropylaminotetralin (7‐OH DPAT). In contrast, the increases in [35S]GTPγS binding to membranes of the VTA induced by either morphine or a selective µ‐opioid receptor agonist [d‐Ala2, NMePhe4, Gly(ol)5]enkephalin were significantly attenuated in nerve‐ligated rats as compared with sham‐ operated rats. Furthermore, the enhancement of DA release in the N.Acc stimulated by morphine was significantly suppressed by sciatic nerve ligation. These findings suggest that attenuation of the morphine‐induced place preference under neuropathic pain may result from a decrease in the morphine‐induced DA release in the N.Acc with reduction in the µ‐opioid receptor‐mediated G‐protein activation in the VTA.

Role of extracellular signal-regulated kinase in the ventral tegmental area in the suppression of the morphine-induced rewarding effect in mice with sciatic nerve ligation

We recently reported that μ‐opioid receptor agonist morphine failed to induce its rewarding effects in rodents with sciatic nerve injury. In the present study, we investigated whether a state of neuropathic pain induced by sciatic nerve ligation could change the activities of the extracellular signal‐regulated kinase (ERK) and p38 in the mouse lower midbrain area including the ventral tegmental area (VTA), and these changes could directly affect the development of the morphine‐induced rewarding effect in mice. The sciatic nerve ligation caused a long‐lasting and profound thermal hyperalgesia. A dose‐dependent place preference induced by s.c. administration of morphine was observed in sham‐operated mice, but not in sciatic nerve‐ligated mice. We found here for the first time that nerve injury produces a sustained and significant reduction in protein levels of phosphorylated‐ERK and ‐p38 in cytosolic preparations of the mouse lower midbrain. The inhibition of ERK activity by i.c.v. pre‐treatment with either PD98059 or U0126 impaired the morphine‐induced place preference. In contrast, i.c.v. treatment with a specific inhibitor of p38, SB203580, did not interfere with the morphine‐induced rewarding effect. Immunohistochemical study showed a drastic reduction in phosphorylated‐ERK immunoreactivity within tyrosine hydroxylase‐positive cells of the VTA. These results suggest that a sustained reduction in the ERK‐dependent signalling pathway in dopamine cells of the VTA may be implicated in the suppression of the morphine‐induced rewarding effect under neuropathic pain.

Involvement of protein kinase Cγ isoform in morphine-induced reinforcing effects

The present study was designed to investigate the role of protein kinase C (PKC) isoform in the morphine-induced reinforcing effect in mice. An intracerebroventricular injection of calphostin C, a specific PKC inhibitor, produced a dose-dependent reduction in the morphine-induced place preference. The protein level of PKCgamma was significantly up-regulated in membrane preparations of the limbic forebrain obtained from the morphine-conditioned mice compared to that from the saline-conditioned mice. However, the protein levels of PKCalpha, betaI, betaII and epsilon were not affected in the same preparation. By contrast, there were no changes in the protein level of all five PKC isoforms in the lower midbrain. Furthermore, we investigated the rewarding properties of morphine in mice lacking PKCgamma gene. A significant place preference was observed following treatment with morphine in wild-type mice, whereas such an effect of morphine was not found in PKCgamma knockout mice. These findings suggest that activated PKCgamma in the limbic forebrain following the treatment with morphine may be critical for the development and/or maintenance of reinforcing effects induced by morphine in mice.

Up-regulation of the TrkB receptor in mice injured by the partial ligation of the sciatic nerve.

Partial nerve injury induced by tying a tight ligature around the sciatic nerve induced a marked hyperalgesia, and this persistent painful state lasted for 14 days in mice. Under these conditions, the nerve injury induced a significant increase in protein level of protein kinase Cgamma isoform in plasma membranes in the spinal cord. We report here for the first time that protein level of TrkB receptor located in plasma membranes was clearly up-regulated in the spinal cord obtained from the nerve-injured mice. These findings suggest that the up-regulation of protein kinase Cgamma associated with activated TrkB receptors following partial sciatic nerve ligation may induce sensitization of synaptic transmission and may in turn cause the persistent pain in mice.

Change in the expression of c-fos in the rat brain following sciatic nerve ligation.

A little or none is known about the direct evidence for the possible change in the expression of c-fos at the supraspinal level after nerve injury. Therefore, the present study was designed to investigate the level of c-fos in some brain regions following sciatic nerve ligation in the rat. Immunoblot analysis clearly showed that the levels of c-fos in the rat frontal cortex, thalamus and periaqueductal gray matter were significantly increased, whereas it was significantly decreased in the nucleus accumbens and ventral tegmental area. Under these conditions, the levels of c-fos in the rat amygdala, hippocampus and hypothalamus were not changed. These results provide direct evidence that the neuropathic pain-like state causes a substantial change in the expression of c-fos in the rat brain.

Different motivational effects induced by the endogenous μ-opioid receptor ligands endomorphin-1 and -2 in the mouse

The present study was designed to investigate the motivational effects of the newly discovered endogenous mu-opioid receptor ligands, endomorphin-1 and endomorphin-2, using the conditioned place preference paradigm in mice. The binding properties of these peptides were first examined using an opioid binding assay. In membranes obtained from the mouse whole brain, the binding of [3H][D-Ala2, NMePhe4, Gly(ol)5]enkephalin (DAMGO; mu), but not of [3H][D-Phe2, D-Phe5]enkephalin (DPDPE; delta) or [3H]U69593 (kappa) selectively and concentration-dependently competed with that of endomorphin-1 and endomorphin-2, indicating that both endomorphin-1 and endomorphin-2 are specific ligands for mu-opioid receptors in the brain. Endomorphin-1 (1-30 nmol/mouse) given i.c.v. produced a dose-related place preference. This effect was abolished by pre-treatment with the mu-opioid receptor antagonist beta-funaltrexamine but not the delta-opioid receptor antagonist naltrindole or the kappa-opioid receptor antagonist nor-binaltorphimine. In contrast, endomorphin-2 (5.6 nmol/mouse) produced place aversion. This aversive effect was inhibited by nor-binaltorphimine as well as beta-funaltrexamine, but not by naltrindole. The place aversion produced by endomorphin-2 was also attenuated by pre-treatment with antiserum against the endogenous kappa-opioid receptor ligand dynorphin A (1-17). These findings indicate that endomorphin-1 may produce its rewarding effect via mu-opioid receptors. On the other hand, the aversive effect induced by endomorphin-2 may be associated with the stimulation of endomorphin-1-insensitive mu-opioid receptors and the activation of dynorphinergic systems in the mouse brain.

Role of the calcium/calmodulin-dependent protein kinase ii (CaMKII) in the morphine-induced pharmacological effects in the mouse

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a family of multifunctional protein kinases that activates signaling pathways. The present study was designed to ascertain whether CaMKII could play a substantial role in the expression of morphine-induced antinociception, hyperlocomotion and rewarding effect in the mouse. An i.c.v. pretreatment with a CaMKII inhibitor KN-93 failed to affect the antinociception and hyperlocomotion induced by s.c. administration of a prototype micro-opioid receptor agonist morphine. In contrast, the morphine-induced place preference was significantly attenuated by i.c.v. pretreatment with KN-93. The levels of phosphorylated-CaMKII (p-CaMKII) in the limbic forebrain, but not in the frontal cortex and the lower midbrain, were significantly increased in morphine-conditioned mice, whereas the levels of CaMKII in three brain regions obtained from morphine-conditioned mice were not changed. This up-regulation of p-CaMKII in the limbic forebrain obtained from morphine-conditioned mice was significantly inhibited by i.c.v. pretreatment with KN-93. These results provide evidence that the increase in CaMKII activity in the mouse limbic forebrain may contribute to the rewarding effect, but not the antinociception and the hyperlocomotion, induced by morphine.

Mechanism of opioid dependence and interaction between opioid receptors

Clinical studies have demonstrated that when opiates are used to control cancer pain, psychological dependence and analgesic tolerance are not a major concern. The present study was, therefore, designed to investigate the modulation of rewarding effects of opiates under inflammatory chronic pain. Formalin or carrageenan was injected into the plantar surface of the rat paw. Formalin and carrageenan reduced the paw pressure threshold. The hyperalgesia lasted for 9–13 days. The rewarding effect of morphine was evaluated by conditioned place preference paradigm. Morphine produced a significant place preference. This effect was significantly attenuated in inflamed groups compared with the respective non‐inflamed groups. Furthermore, the morphine‐induced place preference in the inflamed group gradually recovered to the respective control level as the inflammation healed. We also found that κ‐receptor agonists markedly inhibited the rewarding effect of μ‐receptor agonists. Therefore, to elucidate the mechanism of this attenuation, the effects of pretreatment with κ‐ and δ‐receptor antagonists, nor‐binaltorphimine (nor‐BNI) and naltrindole (NTI), on the development of the morphine‐induced place preference under inflammation were examined. Nor‐BNI, but not NTI, eliminated the suppression of the morphine‐induced place preference in inflamed groups. The morphine‐induced increase in dopamine (DA) turnover in the limbic forebrain was suppressed under inflammation, and the suppression was abolished by the pretreatment with nor‐BNI. These results suggest that endogenous κ‐opioid systems may be activated by chronic inflammatory nociception, and then the activation of κ‐opioid system may inhibit DA release in nucleus accumbens, resulting in the suppression of the development of rewarding effects produced by morphine.

Reduced expression of a novel mu-opioid receptor (MOR) subtype MOR-1B in CXBK mice: implications of MOR-1B in the expression of MOR-mediated responses.

A novel µ‐opioid receptor (MOR) subtype, named MOR‐1B, derived from alternatively spliced variants of MOR gene, has been isolated from the rat brain. Here we found for the first time that CXBK recombinant‐inbred mice display a significant reduction in the expression of MOR‐1B mRNA in the brain as compared to that in their progenitor C57BL/6 mice. In contrast, the expression level of MOR‐1 mRNA in the brain of CXBK mice was similar to that found in C57BL/6 mice. Furthermore, relatively lower levels of MOR‐1B immunoreactivity were detected in the periaqueductal grey matter (PAG) of CXBK mice than that observed in C57BL/6 mice. To investigate further the possible changes in MOR function to activate G‐proteins under the condition of a reduced MOR‐1B expression, the guanosine‐5′‐o‐(3‐[35S]thio)triphosphate ([35S]GTPγS) binding assay was performed. We found that the increased level of [35S]GTPγS bindings to whole brain membranes induced by a selective MOR agonist endomorphin‐1 was significantly decreased in CXBK mice, indicating that CXBK strain can be classified as MOR‐1B‐knockdown mice. We next investigated whether intracerebroventricular (i.c.v.) pretreatment with an antisence oligodeoxynucleotide against exon 5 of MOR gene (MOR‐1B) could affect the endomorphin‐1‐induced supraspinal antinociception. The i.c.v. pretreatment with antisence oligodeoxynucleotide against MOR‐1B produced a significant reduction in the i.c.v.‐administered endomorphin‐1‐induced antinociceptive effect. The present data provide first evidence that a lack of MOR‐1B expression may, at least in part, contribute to the reduced sensitivity to MOR agonists in CXBK mice, and MOR‐1B may play a potential role in the MOR‐mediated supraspinal antinociception.

Heterologous µ‐opioid receptor adaptation by repeated stimulation of κ‐opioid receptor: up‐regulation of G‐protein activation and antinociception

The present study was designed to investigate the effect of repeated administration of a selective κ‐opioid receptor agonist (1S‐trans)‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)cyclohexyl]‐benzeneacetamide hydrochloride [(–)U‐50,488H] on antinociception and G‐protein activation induced by µ‐opioid receptor agonists in mice. A single s.c. injection of (–)U‐50,488H produced a dose‐dependent antinociception, and this effect was reversed by a selective κ‐opioid receptor antagonist nor‐binaltorphimine (nor‐BNI). Furthermore, a single s.c. pre‐treatment with (–)U‐50,488H had no effect on the µ‐opioid receptor agonist‐induced antinociception. In contrast, repeated s.c. administration of (–)U‐50,488H resulted in the development of tolerance to (–)U‐50,488H‐induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (–)U‐50,488H significantly enhanced the antinociceptive effect of selective µ‐opioid receptor agonists endomorphin‐1, endomorphin‐2 and [d‐Ala2,N‐MePhe4,Gly‐ol5] enkephalin (DAMGO). Using the guanosine‐5′‐o‐(3‐[35S]thio) triphosphate ([35S]GTPγS) binding assay, we found that (–)U‐50,488H was able to produce a nor‐BNI‐reversible increase in [35S]GTPγS binding to membranes of the mouse thalamus, which has a high level of κ‐opioid receptors. Repeated administration of (–)U‐50,488H caused a significant reduction in the (–)U‐50,488H‐stimulated [35S]GTPγS binding in this region, whereas chronic treatment with (–)U‐50,488H exhibited the increase in the endomorphin‐1‐, endomorphin‐2‐ and DAMGO‐stimulated [35S]GTPγS bindings in membranes of the thalamus and periaqueductal gray. These results suggest that repeated stimulation of κ‐opioid receptors leads to the heterologous up‐regulation of µ‐opioid receptor functions in the thalamus and periaqueductal gray regions, which may be associated with the supersensitivity of µ‐opioid receptor‐mediated antinociception.