Mayumi Miyatake

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.

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.

Direct evidence for the involvement of brain-derived neurotrophic factor in the development of a neuropathic pain-like state in mice

Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were completely suppressed by repeated intrathecal (i.t.) injection of a TrkB/Fc chimera protein, which sequesters endogenous brain‐derived neurotrophic factor (BDNF). In addition, BDNF heterozygous (+/–) knockout mice exhibited a significant suppression of nerve ligation‐induced thermal hyperalgesia and tactile allodynia compared with wild‐type mice. After nerve ligation, BDNF‐like immunoreactivity on the superficial laminae of the ipsilateral side of the spinal dorsal horn was clearly increased compared with that of the contralateral side. It should be noted that a single i.t. injection of BDNF produced a long‐lasting thermal hyperalgesia and tactile allodynia in normal mice, and these responses were abolished by i.t. pre‐treatment with either a Trk‐dependent tyrosine kinase inhibitor K‐252a or a selective protein kinase C (PKC) inhibitor Ro‐32‐0432. Supporting these findings, we demonstrated here for the first time that the increase in intracellular Ca2+ concentration by application of BDNF in cultured mouse spinal neurons was abolished by pre‐treatment with either K‐252a or Ro‐32‐0432. Taken together, these findings suggest that the binding of spinally released BDNF to TrkB by nerve ligation may activate PKC within the spinal cord, resulting in the development of a neuropathic pain‐like state in mice.

Direct evidence for spinal cord microglia in the development of a neuropathic pain-like state in mice

The present study was undertaken to further investigate the role of glial cells in the development of the neuropathic pain‐like state induced by sciatic nerve ligation in mice. At 7 days after sciatic nerve ligation, the immunoreactivities (IRs) of the specific astrocyte marker glial fibrillary acidic protein (GFAP) and the specific microglial marker OX‐42, but not the specific oligodendrocyte marker O4, were increased on the ipsilateral side of the spinal cord dorsal horn in nerve‐ligated mice compared with that on the contralateral side. Furthermore, a single intrathecal injection of activated spinal cord microglia, but not astrocytes, caused thermal hyperalgesia in naive mice. Furthermore, 5‐bromo‐2′‐deoxyuridine (BrdU)‐positive cells on the ipsilateral dorsal horn of the spinal cord were significantly increased at 7 days after nerve ligation and were highly co‐localized with another microglia marker, ionized calcium‐binding adaptor molecule 1 (Iba1), but neither with GFAP nor a specific neural nuclei marker, NeuN, in the spinal dorsal horn of nerve‐ligated mice. The present data strongly support the idea that spinal cord astrocytes and microglia are activated under the neuropathic pain‐like state, and that the proliferated and activated microglia directly contribute to the development of a neuropathic pain‐like state in mice.

Role of δ-opioid receptor function in neurogenesis and neuroprotection

The present study was undertaken to evaluate the implication of δ‐opioid receptor function in neurogenesis and neuroprotection. We found that the stimulation of δ‐opioid receptors by the selective δ‐opioid receptor agonist SNC80 [(+)‐4‐[(αR)‐α‐((2S,5R)‐4‐allyl‐2,5‐dimethyl‐1‐piperazinyl)‐3‐methoxybenzyl]‐N,N‐diethylbenzamide] (10 nm) promoted neural differentiation from multipotent neural stem cells obtained from embryonic C3H mouse forebrains. In contrast, either a selective µ‐opioid receptor agonist, [d‐Ala2, N‐Me‐Phe4, Gly5‐ol]‐enkephalin (DAMGO), or a specific κ‐opioid receptor agonist, (–)‐trans‐(1S,2S)‐U‐50488 hydrochloride (U50,488H), had no such effect. In addition to neural differentiation, the increase in cleaved caspase 3‐like immunoreactivity induced by H2O2 (3 µm) was suppressed by treatment with SNC80 in cortical neuron/glia co‐cultures. These effects of SNC80 were abolished by a Trk‐dependent tyrosine kinase inhibitor: (8R*,9S*,11S*)‐(–)‐9‐hydroxy‐9‐methoxycarbonyl‐8‐methyl‐2,3,9,10‐tetrahydro‐8,11‐epoxy‐1H,8H,11H‐2,7b,11a‐triazadibenzo(a,g)cycloocta(cde)trinden‐1‐one (K‐252a). The SNC80‐induced neural differentiation was also inhibited by treatment with the protein kinase C (PKC) inhibitor, phosphatidylinositol 3‐kinase (PI3K) inhibitor, mitogen‐activated protein kinase kinase (MEK) inhibitor or Ca2+/calmodulin‐dependent protein kinase II (CaMKII) inhibitor. These findings raise the possibility that δ‐opioid receptors play a crucial role in neurogenesis and neuroprotection, mainly through the activation of Trk‐dependent tyrosine kinase, which could be linked to PI3K, PKC, CaMKII and MEK.

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.

Implication of Activated Astrocytes in the Development of Drug Dependence

Astrocytes are a subpopulation of glial cells that directly affect neuronal function. This review focuses on the potential functional roles of astrocytes in the development of behavioral sensitization and rewarding effects induced by chronic treatment with drugs of abuse. In vitro treatment of cortical neuron/glia cocultures with either methamphetamine or morphine caused activation of astrocytes via protein kinase C (PKC). Purified cortical astrocytes were markedly activated by methamphetamine, whereas morphine had no such effect. Methamphetamine, but not morphine, caused a long‐lasting astrocytic activation in cortical neuron/glia cocultures. Morphine‐induced behavioral sensitization, assessed as hyperlocomotion, was reversed by 2 months of withdrawal from intermittent morphine administration, whereas behavioral sensitization to methamphetamine‐induced hyperlocomotion was maintained even after 2 months of withdrawal. In vivo treatment with methamphetamine, which was associated with behavioral sensitization, caused PKC‐dependent astrocytic activation in the mouse cingulate cortex and nucleus accumbens. Furthermore, the glial modulator propentofylline dramatically diminished the activation of astrocytes and the rewarding effect induced by methamphetamine and morphine. On the other hand, intra–nucleus accumbens and intra–cingulate cortex administration of astrocyte‐conditioned medium aggravated the development of rewarding effects induced by methamphetamine and morphine. Furthermore, astrocyte‐conditioned medium, but not methamphetamine itself, clearly induced differentiation of neural stem cells into astrocytes. These findings provide direct evidence that astrocytes may, at least in part, contribute to the development of the rewarding effects induced by drugs of abuse in the nucleus accumbens and cingulate cortex.

Glutamatergic neurotransmission and protein kinase C play a role in neuron–glia communication during the development of methamphetamine-induced psychological dependence

Methamphetamine (METH) is a strongly addictive psychostimulant that dramatically affects the central nervous system (CNS). On the other hand, protein kinase C (PKC) plays a major role in cellular regulatory and signalling processes that involve protein phosphorylation. The purpose of this study was to investigate the role of neuronal and astrocytic PKC in changes in the central glutamatergic system induced by METH. We show here that in vitro treatment with METH caused the phosphorylation of both neuronal and astrocytic PKC and the activation of astrocytes in cortical neuron/glia co‐cultures. Treatment of cortical neuron/glia co‐cultures with either the PKC activator phorbol 12,13‐dibutyrate (PDBu) or glutamate also caused the PKC‐dependent activation of astrocytes. The PKC inhibitor chelerythrine suppressed the Ca2+ responses to glutamate in both cortical neurons and astrocytes. Moreover, a low concentration of PDBu significantly enhanced the Ca2+ responses to glutamate, but not to dopamine, in both cortical neurons and astrocytes. Notably, treatment with METH also enhanced the Ca2+ responses to glutamate in cortical neurons. The activation of astrocytes induced by METH was also reversed by co‐treatment with glutamate receptor antagonists (ifenprodil, DNQX or MPEP) in cortical neuron/glia co‐cultures. In the conditioned place preference paradigm, intracerebroventricular administration of glutamate receptor antagonists (ifenprodil, DNQX or MPEP) attenuated the METH‐induced rewarding effect. These findings provide evidence that the changes in PKC‐dependent neuronal and astrocytic glutamatergic transmission induced by METH may, at least in part, contribute to the development of psychological dependence on METH.

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.

Dynamic Changes in Dopaminergic Neurotransmission Induced by a Low Concentration of Bisphenol-A in Neurones and Astrocytes

One of the most common chemicals that behaves as an endocrine disruptor is the compound 4,4′‐isopronylidenediphenol, called bisphenol‐A (BPA). We previously reported that prenatal and postnatal exposure to BPA potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant‐induced pharmacological actions. Many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. The present study aimed to investigate the role of neurone–astrocyte communication in the enhancement of dopaminergic neurotransmission induced by BPA. We found that treatment of mouse purified astrocytes and neurone/glia cocultures with BPA in vitro caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of glial fibrillary acidic protein. A low concentration of BPA significantly enhanced the Ca2+ responses to dopamine in both neurones and astrocytes. Furthermore, a high concentration of BPA markedly induced the activation of caspase‐3, which is a marker of neuronal apoptotic cell death in mouse midbrain neurone/glia cocultures. By contrast, treatment with 17β‐oestradiol (E2) had no such effects. Prenatal and neonatal exposure to BPA led to an enhancement of the dopamine‐dependent rewarding effect induced by morphine. These findings provide evidence that BPA alters dopamine responsiveness in neurones and astrocytes and that, at least in part, it may contribute to potentiate the development of psychological dependence on drugs of abuse.