Yasuyuki Nagumo

Direct Involvement of Orexinergic Systems in the Activation of the Mesolimbic Dopamine Pathway and Related Behaviors Induced by Morphine

In this study, we investigated the role of orexinergic systems in dopamine-related behaviors induced by the μ-opioid receptor agonist morphine in rodents. Extensive coexpression of tyrosine hydroxylase with orexin receptors was observed in the mouse ventral tegmental area (VTA). The levels of dopamine and its major metabolites in the nucleus accumbens were markedly increased by the microinjection of orexin A and orexin B into the VTA. The subcutaneous morphine-induced place preference and hyperlocomotion observed in wild-type mice were abolished in mice that lacked the prepro-orexin gene. An intra-VTA injection of a selective orexin receptor antagonist SB334867A [1-(2-methylbenzoxazol-6-yl)-3-[1.5]naphthyridin-4-yl urea] significantly suppressed the morphine-induced place preference in rats. Furthermore, the increased level of dialysate dopamine produced by morphine in the mouse brain was significantly decreased by deletion of the prepro-orexin gene. These findings provide new evidence that orexin-containing neurons in the VTA are directly implicated in the rewarding effect and hyperlocomotion induced by morphine through activation of the mesolimbic dopamine pathway in rodents.

Chronic Pain Induces Anxiety with Concomitant Changes in Opioidergic Function in the Amygdala

Clinically, it has been reported that chronic pain induces depression, anxiety, and reduced quality of life. The endogenous opioid system has been implicated in nociception, anxiety, and stress. The present study was undertaken to investigate whether chronic pain could induce anxiogenic effects and changes in the opioidergic function in the amygdala in mice. We found that either injection of complete Freunds adjuvant (CFA) or neuropathic pain induced by sciatic nerve ligation produced a significant anxiogenic effect at 4 weeks after the injection or surgery. Under these conditions, the selective μ-opioid receptor agonist [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAMGO)- and the selective δ-opioid receptor agonist (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-stimulated [35S]GTPγS binding in membranes of the amygdala was significantly suppressed by CFA injection or nerve ligation. CFA injection was associated with a significant increase in the κ-opioid receptor agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl]acetamide hydrochloride (ICI199,441)-stimulated [35S]GTPγS binding in membranes of the amygdala. The intracerebroventricular administration and microinjection of a selective μ-opioid receptor antagonist, a selective δ-opioid receptor antagonist, and the endogenous κ-opioid receptor ligand dynorphin A caused a significant anxiogenic effect in mice. We also found that thermal hyperalgesia induced by sciatic nerve ligation was reversed at 8 weeks after surgery. In the light–dark test, the time spent in the lit compartment was not changed at 8 weeks after surgery. Collectively, the present data constitute the first evidence that chronic pain has an anxiogenic effect in mice. This phenomenon may be associated with changes in opioidergic function in the amygdala.

Direct Evidence of Astrocytic Modulation in the Development of Rewarding Effects Induced by Drugs of Abuse

Long-term exposure to pyschostimulants and opioids induced neuronal plasticity. Accumulating evidence suggests that astrocytes actively participate in synaptic plasticity. We show here that a glial modulator propentofylline (PPF) dramatically diminished the activation of astrocytes induced by drugs of abuse, such as methamphetamine (METH) and morphine (MRP). In vivo treatment with PPF also suppressed both METH- and MRP-induced rewarding effects. On the other hand, intra-nucleus accumbens (N.Acc.) administration of astrocyte-conditioned medium (ACM) aggravated the development of rewarding effects induced by METH and MRP via the Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway, which modulates astrogliosis and/or astrogliogenesis. Furthermore, ACM, but not METH itself, clearly induced the differentiation of multipotent neuronal stem cells into glial fibrillary acidic protein-positive astrocytes, and this effect was reversed by cotreatment with the Jak/STAT inhibitor AG490. Intra-cingulate cortex (CG) administration of ACM also enhanced the rewarding effect induced by METH and MRP. In contrast to ACM, intra-N.Acc. administration of microglia-conditioned medium failed to affect the rewarding effects of METH and MRP in mice. These findings suggest that astrocyte-, but not microglia-, related soluble factors could amplify the development of rewarding effect of METH and MRP in the N.Acc. and CG. The present study provides direct evidence that astrocytes may, at least in part, contribute to the synaptic plasticity induced by drugs of abuse during the development of rewarding effects induced by psychostimulants and opioids.

Chronic pain-induced emotional dysfunction is associated with astrogliosis due to cortical delta-opioid receptor dysfunction.

It has been widely recognized that chronic pain could cause physiological changes at supraspinal levels. The δ‐opioidergic system is involved in antinociception, emotionality, immune response and neuron‐glia communication. In this study, we show that mice with chronic pain exhibit anxiety‐like behavior and an increase of astrocytes in the cingulate cortex due to the dysfunction of cortical δ‐opioid receptor systems. Using neural stem cells cultured from the mouse embryonic forebrain, astrocyte differentiation was clearly observed following long‐term exposure to the selective δ‐opioid receptor antagonist, naltrindole. We also found that micro‐injection of either activated astrocyte or astrocyte‐conditioned medium into the cingulate cortex of mice aggravated the expression of anxiety‐like behavior. Our results indicate that the chronic pain process promotes astrogliosis in the cingulate cortex through the dysfunction of cortical δ‐opioid receptors. This phenomenon may lead to emotional disorders including aggravated anxiety under chronic pain‐like state.

Protease-Activated Receptor-1 and Platelet-Derived Growth Factor in Spinal Cord Neurons Are Implicated in Neuropathic Pain after Nerve Injury

Recently, it has been reported that both thrombin-sensitive protease-activated receptor 1 (PAR-1) and platelet-derived growth factor (PDGF) are present not only in platelets, but also in the CNS, which indicates that they have various physiological functions. In this study, we evaluated whether PAR-1/PDGF in the spinal cord could contribute to the development of a neuropathic pain-like state in mice. Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were significantly suppressed by repeated intrathecal injection of hirudin, which is characterized as a specific and potent thrombin inhibitor. Furthermore, a single intrathecal injection of thrombin produced long-lasting hyperalgesia and allodynia, and these effects were also inhibited by hirudin in normal mice. In nerveligated mice, the increase in the binding of [35S]GTPγS to membranes of the spinal cord induced by thrombin and PAR-1-like immunoreactivity (IR) in the spinal cord were each greater than those in sham-operated mice. Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were also suppressed by repeated intrathecal injection of either the PDGF α receptor (PDGFRα)/Fc chimera protein or the PDGFR-dependent tyrosine kinase inhibitor AG17 [(3,5-di-tert-butyl-4-hydroxybenzylidene)-malononitrile]. Moreover, thermal hyperalgesia and tactile allodynia induced by thrombin in normal mice were virtually eliminated by intrathecal pretreatment with PDGFRα/Fc. In immunohistochemical studies, PAR-1-like IR-positive cells in the spinal dorsal horn were mostly colocated on PDGF-like IR-positive neuronal cells. These data provide novel evidence that PAR-1 and PDGF-A-mediated signaling pathway within spinal cord neurons may be directly implicated in neuropathic pain after nerve injury in mice.

Implication of protein kinase C in the orexin-induced elevation of extracellular dopamine levels and its rewarding effect

In the present study, we investigated the role of orexinergic systems in the activation of midbrain dopamine neurons. In an in vitro study, exposure to either orexin A or orexin B under superfusion conditions produced a transient increase in the intracellular Ca2+ concentration through the phospholipase C (PLC)/protein kinase C (PKC) pathway via Gq11α or Gβγ subunits in midbrain cultured neurons, which were shown to be tyrosine hydroxylase (TH)‐positive cells, but not in purified midbrain astrocytes. Here we show that in vivo injection with a selective PKC inhibitor chelerythrine chloride or 2‐{8‐[(dimethylamino)methyl]‐6,7,8,9‐tetrahydropyrido[1,2‐a]indol‐3‐yl}‐3‐1‐methyl‐1H‐indol‐3‐ylmaleimide HCl (Ro‐32–0432) into the ventral tegmental area (VTA) significantly suppressed the place preference and increased levels of dopamine in the nucleus accumbens (NAcc) induced by intra‐VTA injection of orexins. These results strongly support the idea that activation of the orexin‐containing neuron in the VTA leads to the direct activation of mesolimbic dopamine neurons through the activation of the PLC/PKC pathway via Gq11α or Gβγ‐subunit activation, which could be associated with the development of its rewarding effect.

Chronic pain-induced astrocyte activation in the cingulate cortex with no change in neural or glial differentiation from neural stem cells in mice

Pain pathways terminate in discrete brain areas that monitor the sensory and affective qualities of the initiating stimulus and show remarkable plasticity. Here, we found that chronic pain by sciatic nerve ligation caused a dramatic increase in glial fibrillary acidic protein (GFAP)-like immunoreactivity (IR), which is located in the dendritic astrocytes, with its expanding distribution in the cingulate cortex (CG) of mice. The branched GFAP-like IR in the CG of nerve-ligated mice was overlapped with S100beta-like IR, which is highly limited to the cell body of astrocytes, whereas there was no difference of S100beta-like IR between sham-operated and nerve-ligated mice. The number of BrdU-positive cells on the CG was not changed by sciatic nerve ligation. Furthermore, subventricular zone (SVZ)-derived neural stem cells marked by pEGFP-C1 did not migrate toward the CG after sciatic nerve ligation. In the behavioral assay, the thermal hyperalgesia observed on the ipsirateral side in nerve-ligated mice was significantly suppressed by a single pre-microinjection of a glial-modulating agent propentofylline into the CG 24 h before nerve ligation. These results suggest that chronic painful stimuli induces astrocyte activation in the CG, whereas they do not affect the cell proliferation/differentiation from neural stem cells in the CG and the migration of neural stem cells from the SVZ area. The astrocyte activation in the CG may, at least in part, contribute to the development of a chronic pain-like state following sciatic nerve ligation in mice.

Implication of cyclin‐dependent kinase 5 in the development of psychological dependence on and behavioral sensitization to morphine

In the present study, we investigated the role of cyclin‐dependent kinase 5 (cdk5) in the brain dynamics changed by repeated in vivo treatment with morphine. The level of phosphorylated‐cdk5 was significantly increased in the cingulate cortex of mice showing the morphine‐induced rewarding effect. Under these conditions, roscovitine, a cdk5 inhibitor, given intracerebroventricularly (i.c.v.) caused a dose‐dependent and significant inhibition of the morphine‐induced rewarding effect. In addition, the dose–response effect of the morphine‐induced rewarding effect was dramatically attenuated in cdk5 heterozygous (+/–) knockout mice. Furthermore, the development of behavioral sensitization by intermittent administration of morphine was virtually abolished in cdk5 (+/–) mice. These findings suggest that the induction and/or activation of cdk5 are implicated in the development of psychological dependence on morphine.

Involvement of mitogen-stimulated p70-S6 kinase in the development of sensitization to the methamphetamine-induced rewarding effect in rats

The neural plasticity associated with behavioral sensitization following repeated administration of a psychostimulant methamphetamine (METH) is thought to require synthesis of new proteins. The aim of the present study was to investigate the role of p70-S6 kinase (p70-S6K) phosphorylation, which contributes to the selective translation of a unique family of mRNA, in mediating both the METH-induced rewarding effect and its sensitization. An intra-nucleus accumbens (N.Acc.) pre-injection with 0.025 pmol/rat of a selective p70-S6K inhibitor rapamycin failed to affect the METH-induced conditioned place preference. However, this treatment clearly abolished the development of sensitization of the METH-induced conditioned place preference. Consistent with the behavioral assay, the level of the immunoreactivity of phosporylated-p70-S6K was not changed in the cytosolic fraction of the N.Acc. obtained from rats that had revealed the METH-induced rewarding effect. In contrast, the immunoreactivities in the cytosolic preparation for Western blotting and immunohistochemical density of phosphorylated-p70-S6K were significantly increased in the N.Acc. obtained from METH-sensitized rats as compared with those with chronic saline treatment. However, the immunoreactivities of phosphorylated-extracellular signal-regulated kinase and phosphorylated-ribosomal S6 protein were not significantly altered in the N.Acc. under the same condition. The present data provide evidence for the change in the translation rate, which can be regulated by S6K phosphorylation, in the N.Acc. during the development of sensitization to METH-induced rewarding effects in rats.

Age-related emotionality is associated with cortical δ-opioid receptor dysfunction-dependent astrogliosis

Multiple changes occur in the aging brain, leading to age-related emotional disorders. A growing body of recent evidence suggests that the cortical delta-opioid receptor system plays a critical role in anxiety- and depressive-like behaviors in the rodent. In this study, we show that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex due to the dysfunction of cortical delta-opioid receptor systems. As well as aging mice, mice with a dysfunction of the delta-opioid receptor system induced by chronic treatment with the selective delta-opioid receptor antagonist naltrindole, revealed astrogliosis in the cingulate cortex, which was associated with anxiety. We also found that the microinjection of cultured astrocytes into the cingulate cortex of young mice enhanced the expression of anxiety-like behavior. Our results indicate that the aging process promotes astrogliosis in the cingulate cortex through the dysfunction of cortical delta-opioid receptors. This phenomenon may lead to emotional disorders including aggravated anxiety during normal aging.