【サテライト国際シンポジウム】13:30-17:00【サテライト国際シンポジウム】

Neurobiology of Opioid Receptor Symposium（オピオイド受容体の神経生物学シンポジウム）［主催］
オピオイド受容体の神経生物学シンポジウム実行委員会
実行委員会委員長：植田弘師（長崎大・院）

13:30

Opening Remarks Hiroshi Ueda

[Chairmen] Shinobu Sakurada / Shiroh Kishioka
13:35 Involvement of the Endogenous Opioid System in Cannabinoid Rewarding
SS1
Effects and Withdrawal Syndrome
R. Maldonado
Univ Pompeu Fabra, Barcelona, Spain
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14:10
SS2

14:30
SS3

15:05
SS4

15:25
SS5

16:00
SS6

16:20

Opioid-Induced Synaptic Plasticity and the Alternation of the Sensitivity
to Opioid Receptor-Mediated Responses under Chronic Pain State
Minoru Narita and Tsutomu Suzuki
Department of Toxicology, Hoshi University School of Pharmacy,Tokyo, Japan

171

Opioid Receptor Genes Inactivated in Mice: The Highlights
Brigitte L. Kieffer
The Faculty of Pharmacy, University Louis Pasteur, Strasbourg, France

168

Neurobiology of Opioid and Plasticity– Toward Drug Discovery for the Pain Resistant to Morphine
Hiroshi Ueda
Division of Molecular Pharmacology and Neuroscience
Nagasaki University Graduate School of Biomedical Sciences,
Nagasaki, Japan170

New Drug Targets for Opioid Receptors.
Christopher J Evans
Neuropsychiatric Institute, UCLA, LA, CA 90024, USA

167

Electrophysiological Characteristic of Corticoaccumbens Synapses in Rat
Mesolimbic System Reconstructed Using Organotypic Slice Cultures
T. Maeda and S. Kishioka
Department of Pharmacology, Wakayama Medical University

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Closing Remarks Masamichi Satoh（国際シンポジウムの進行具合により以下時間変更の可能性あり)
17:10
世話人会報告17:30

次期世話人代表挨拶
次期世話人代表 亀井淳三

17:40

閉会の辞
世話人代表

花岡一雄

ELECTROPHYSIOLOGICAL CHARACTERISTIC OF
CORTICOACCUMBENS SYNAPSES IN RAT MESOLIMBIC SYSTEM
RECONSTRUCTED USING ORGANOTYPIC SLICE CULTURES
T. MAEDA AND S. KISHIOKA
Department of Pharmacology, Wakayama Medical University,
Wakayama 641-0012, JapanThe nucleus accumbens (NAc) is a component of the mesolimbic system involved in
drug dependence. Activity of NAc neurons is increased by glutamatergic afferents fromthe medial prefrontal cortex (mPFC) and is decreased by dopaminergic afferents from theventral tegmental area (VTA). In the present study, we reconstructed the mesolimbic
system using organotypic slice cultures, and examined the effects of dopaminergic agentson synaptic activity in mPFC-NAc synapses. A slice of each of mPFC, NAc and VTA innewborn rat, was arranged on a multi-electrode dish filled with culture medium so thatthey contacted each other, termed 'triple culture'. Neurite outgrowth of mPFC to NAc intriple culture was identified by DiI. Immunohistochemical study with anti-tyrosinehydroxylase (TH) antibody revealed TH-positive cells in VTA and TH-positive fibers inboth mPFC and NAc of triple culture. Extracellular recording using microelectrodes on amulti-electrode dish showed that a single electrical stimulation of mPFC evoked field
excitatory postsynaptic potential (fEPSP), and that spontaneous population spikes (sPS)occurred in NAc of triple culture. Both fEPSP and sPS were suppressed by glutamatergic
antagonists. The D1-like receptor agonist SKF38393, but not the D2-like receptor agonist
quinpirole, reduced both the amplitude of fEPSP and frequency of sPS. Cocaine
depressed fEPSP, and this depression was reversed by D1-like receptor antagonist
SCH23390, but not by D2-like receptor antagonist sulpiride. These results suggest thatglutamatergic synapses were regenerated in triple culture and were subject to exogenousand endogenous dopaminergic modulation that was similar to that shown in in vivo.

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New Drug Targets for Opioid Receptors.

Chris Evans
Neuropsychiatric Institute, UCLA, LA, CA 90024

The variable actions of opioid agonists in vivo are generally attributed to a combination of factors
including receptor selectivity and efficacy for signaling, pharmacokinetics, and ability to reachtarget receptors. Given these parameters, the unique attributes of buprenorphine will be discussedwith regard to opioid receptor trafficking and identifying the pharmacology leading to the bell
shaped dose-response/ceiling effect for buprenorphine analgesia. This study presents new insights
into the clinical possibilities for buprenorphine like drugs. Inverse agonists as potential drug targets
for opioid receptors have not been considered. Inverse agonists would be presumed to have
opposite effects on signaling as agonists. However, with regard adenylate cycalse signaling wehave recently found that chronic treatment with delta receptor inverse agonists mimics the action ofacute treatment with delta agonists. The mechanism we propose is that the constitutive activity ofthe delta receptor induces cyclase supersensitivity in cells containing delta opioid receptors and thissupersensitivity increases the cells response to adenyl cyclase stimulation via Gs coupled receptors
or forskolin. This presents a new signaling rational for the design of inverse agonists of Gi/Go
coupled receptors. Finally, there is accumulating evidence that different ligands may differentiallytrigger various signaling pathways via the same receptor, a phenomenon we term ligand-directedsignaling. In support of this concept, data will be presented that inhibition of adenylate cyclase andactivation of both MAPK and Akt can be regulated independently by different ligands following
opioid receptor activation. Potential models will be presented to explain this observation.

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Opioid receptor genes inactivated in mice:the highlights

Brigitte L. Kieffer
The Faculty of Pharmacy, University Louis Pasteur, Strasbourg, France

The opioid system controls nociception, stress responses and addictive behaviors.
Exogenous alkaloid opiates and endogenous opioid peptides stimulate mu-, delta- andkappa-opioid receptors, whose activities have long been analyzed by pharmacological tools.
Mice lacking opioid receptor and opioid peptide precursor genes have now been produced
by gene targeting and salient phenotypes from these animals will be reviewed. Behavioral
analysis of mutant animals in the absence of drug has highlighted a distinct role of opioid
receptors or peptides in nociception and revealed an important role for delta receptors inemotional behaviors. Recent data on stress-induced analgesia from our laboratory will also
be presented. The examination of responses to drugs has clarified involvement of each
receptor as molecular targets for exogenous opiates in vivo. Those data have also
demonstrated the critical role of mu-receptor in cannabinoid and alcohol reinforcement, andconfirmed the involvement of kappa receptor in several dysphoric responses. On-goingstudies therefore help understanding the molecular basis of opioid-controlled behaviors, andwill contribute to the development of novel therapeutics for pain, anxiety and drug abuse.

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INVOLVEMENT OF THE ENDOGENOUS OPIOID SYSTEMIN CANNABINOID REWARDING EFFECTS
AND WITHDRAWAL SYNDROME
R. Maldonado
Univ Pompeu Fabra, Barcelona, Spain
Several studies have shown functional relationships between the endogenous cannabinoid
and opioid systems. However, acute delta9-tetrahydrocannabinol (THC) pharmacologicalresponses and physical dependence were not modified in knock-out mice with singledeletion of mu, delta or kappa opioid receptors. To further investigate the neurobiologicalbasis of cannabinoid dependence, we have evaluated THC responses in double mu anddelta opioid receptor knock-out mice. Antinociception and hypolocomotion induced byacute THC administration remain unaffected whereas the acute hypothermic effects wereslightly attenuated in these double mutants. During chronic THC treatment, knock-out micedeveloped slower tolerance to the hypothermic effects but the development of tolerance toantinociceptive and hypolocomotor effects was almost unaffected. The rewarding
properties of THC were abolished in knock-out mice. Interestingly, the somatic
manifestations of THC withdrawal were also significantly attenuated in mutant mice,suggesting that a cooperative action of mu and delta opioid receptors is required for theentire expression of THC dependence.

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Neurobiology of opioid and plasticity– toward drug discovery for the pain resistant to morphine
Hiroshi Ueda
Division of Molecular Pharmacology and Neuroscience
Nagasaki University Graduate School of Biomedical Sciences
The study for plasticity in the development of tolerance and dependence following
chronic treatments with morphine or opioids is one of most important issues in theneurobiology. Our approaches are based on two parts, one is the mechanism at the single
cell level, and the other is the one at the synaptic network in the brain. In both approaches
we are in progress of visualizing the molecular mechanisms as one of goals. In cells
expressing µ-opioid receptor (MOP-R), morphine and DAMGO shows differential
MOP-R internalization, and protein kinase C (PKC) inhibition determines this selectivity.
In the peripheral µ-opioid analgesia, similar PKC-regulated acute tolerance is also
observed. On the other hand, chronic tolerance is mostly related to the synaptic plasticity
involving the anti-opioid systems, which is observed in the central nervous system.
NMDA-receptor and nociceptin/OFQ-receptor (NOP-R) are important candidates forsuch mechanisms. In such receptor knock-out mice, morphine tolerance is markedly
reduced, and it is also found that both mechanisms play region-specific roles in theplasticity. Those selective antagonists could be candidates for adjuvants to delay themorphine tolerance. On the other hand, neuropathic pain would be another good target forthe study of plasticity affecting morphine analgesia. Following sciatic nerve injury
peripheral and systemic morphine analgesia are markedly reduced. In such mechanisms
the functional switch of nociceptor fibers involved in pain perception is postulated. Somecandidates as novel analgesics useful for neuropathic pain would be proposed.

170

Opioid-induced synaptic plasticity and the alternation of the sensitivity
to opioid receptor-mediated responses under chronic pain state

Minoru Narita and Tsutomu Suzuki
Department of Toxicology, Hoshi University School of Pharmacy,
Tokyo, JAPANThe stimulation of µ-opioid receptor by morphine can regulate a number of signaling
pathways. These regulated pathways include inhibition of adenylate cyclase activity,activation of inwardly rectifying K+ channels and blockade of Ca2+ entry throughvoltage-dependent Ca2+ channels. A considerable number of evidence indicates that thestimulation of µ-opioid receptors can activate phospholipase C (PLC)-dependent pathway
via Gβγ proteins. We demonstrated that µ-opioid receptor agonists activate PLC,
resulting in the activation of protein kinase C (PKC) and the increase in inositol (1,4,5)
triphosphate (IP3)-dependent Ca2+ signaling. In some cases, phosphoinositide 3-kinase(PI3K)-dependent PLCγ pathway can be involved in this event. We also found that PKC,
especially PKCγ isoform, constitutes an essential pathway through which phosphorylationof µ-opioid receptors occurs. These findings indicate that PKCγ isoform may regulate
the homologous negative feedback pathway for the µ-opioid receptor system.
Furthermore, neuronal PKC and brain-derived neurotrophic factor (BDNF) are essential
for the opioid-induced synaptic plasticity including the rewarding effect and thedevelopment of antinociceptive tolerance. Our recent analysis provides new insightsthat the sensitivity to opioid receptor-mediatedresponses under neuropathic pain state

can be decreased. Sciatic nerve ligation leads to a long-lasting hyperalgesia, which isaccompanied by the sustained increases in PKCγ, BDNF and c-Src family in the dorsal
horn of the spinal cord. As well as the antinociceptive action, the morphine-induced
rewarding effect is dramatically reduced by sciatic nerve ligation. We are currently
investigating the effect of chronic morphine treatment on the nerve and astrocyte growth.
A significant change in nerve growth associated with the activated astrocyte will bediscussed.

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