Lyophilized Kratom Tea as a Therapeutic Option for Opioid Dependence.

BACKGROUND: Made as a tea, the Thai traditional drug "kratom" reportedly possesses pharmacological actions that include both a coca-like stimulant effect and opium-like depressant effect. Kratom has been used as a substitute for opium in physically-dependent subjects. The objective of this study was to evaluate the antinociception, somatic and physical dependence produced by kratom tea, and then assess if the tea ameliorated withdrawal in opioid physically-dependent subjects. METHODS: Lyophilized kratom tea (LKT) was evaluated in C57BL/6J and opioid receptor knockout mice after oral administration. Antinociceptive activity was measured in the 55 °C warm-water tail-withdrawal assay. Potential locomotor impairment, respiratory depression and locomotor hyperlocomotion, and place preference induced by oral LKT were assessed in the rotarod, Comprehensive Lab Animal Monitoring System, and conditioned place preference assays, respectively. Naloxone-precipitated withdrawal was used to determine potential physical dependence in mice repeatedly treated with saline or escalating doses of morphine or LKT, and LKT amelioration of morphine withdrawal. Data were analyzed using one- and two-way ANOVA. RESULTS: Oral administration of LKT resulted in dose-dependent antinociception (≥1 g/kg, p.o.) absent in mice lacking the mu-opioid receptor (MOR) and reduced in mice lacking the kappa-opioid receptor. These doses of LKT did not alter coordinated locomotion or induce conditioned place preference, and only briefly reduced respiration. Repeated administration of LKT did not produce physical dependence, but significantly decreased naloxone-precipitated withdrawal in morphine dependent mice. CONCLUSIONS: The present study confirms the MOR agonist activity and therapeutic effect of LKT for the treatment of pain and opioid physical dependence.

Non-canonical Opioid Signaling Inhibits Itch Transmission in the Spinal Cord of Mice.

Chronic itch or pruritus is a debilitating disorder that is refractory to conventional anti-histamine treatment. Kappa opioid receptor (KOR) agonists have been used to treat chronic itch, but the underlying mechanism remains elusive. Here, we find that KOR and gastrin-releasing peptide receptor (GRPR) overlap in the spinal cord, and KOR activation attenuated GRPR-mediated histamine-independent acute and chronic itch in mice. Notably, canonical KOR-mediated Gαi signaling is not required for desensitizing GRPR function. In vivo and in vitro studies suggest that KOR activation results in the translocation of Ca2+-independent protein kinase C (PKC)δ from the cytosol to the plasma membrane, which in turn phosphorylates and inhibits GRPR activity. A blockade of phospholipase C (PLC) in HEK293 cells prevented KOR-agonist-induced PKCδ translocation and GRPR phosphorylation, suggesting a role of PLC signaling in KOR-mediated GRPR desensitization. These data suggest that a KOR-PLC-PKCδ-GRPR signaling pathway in the spinal cord may underlie KOR-agonists-induced anti-pruritus therapies.

A one-pot multicomponent approach to a new series of morphine derivatives and their biological evaluation.

A novel and facile domino reaction has been developed to synthesize a variety of new derivatives from hydromorphone, amines and paraformaldehyde in good yields in a catalyst-free fashion with high atom efficiency. The products show a mixed MOR/DOR biological characteristic which makes them valuable for further study as opioid analgesics.

A role for the mu opioid receptor in the antidepressant effects of buprenorphine.

Buprenorphine (BPN), a mixed opioid drug with high affinity for mu (MOR) and kappa (KOR) opioid receptors, has been shown to produce behavioral responses in rodents that are similar to those of antidepressant and anxiolytic drugs. Although recent studies have identified KORs as a primary mediator of BPN's effects in rodent models of depressive-like behavior, the role of MORs in BPN's behavioral effects has not been as well explored. The current studies investigated the role of MORs in mediating conditioned approach behavior in the novelty-induced hypophagia (NIH) test, a behavioral measure previously shown to be sensitive to chronic treatment with antidepressant drugs. The effects of BPN were evaluated in the NIH test 24h post-administration in mice with genetic deletion of the MOR (Oprm1-/-) or KOR (Oprk1-/-), or after pharmacological blockade with the non-selective opioid receptor antagonist naltrexone and selective MOR antagonist cyprodime. We found that behavioral responses to BPN in the NIH test were blocked in Oprm1-/- mice, but not in Oprk1-/- mice. Both cyprodime and naltrexone significantly reduced approach latency at doses experimentally proven to antagonize the MOR. In contrast the selective MOR agonist morphine and the selective KOR antagonist nor-BNI were both ineffective. Moreover, antinociceptive studies revealed persistence of the MOR antagonist properties of BPN at 24h post-administration, the period of behavioral reactivity. These data support modulation of MOR activity as a key component of BPN's antidepressant-like effects in the NIH paradigm.

Kappa Opioid Receptor-Induced Aversion Requires p38 MAPK Activation in VTA Dopamine Neurons.

The endogenous dynorphin-κ opioid receptor (KOR) system encodes the dysphoric component of the stress response and controls the risk of depression-like and addiction behaviors; however, the molecular and neural circuit mechanisms are not understood. In this study, we report that KOR activation of p38α MAPK in ventral tegmental (VTA) dopaminergic neurons was required for conditioned place aversion (CPA) in mice. Conditional genetic deletion of floxed KOR or floxed p38α MAPK by Cre recombinase expression in dopaminergic neurons blocked place aversion to the KOR agonist U50,488. Selective viral rescue by wild-type KOR expression in dopaminergic neurons of KOR(-/-) mice restored U50,488-CPA, whereas expression of a mutated form of KOR that could not initiate p38α MAPK activation did not. Surprisingly, while p38α MAPK inactivation blocked U50,488-CPA, p38α MAPK was not required for KOR inhibition of evoked dopamine release measured by fast scan cyclic voltammetry in the nucleus accumbens. In contrast, KOR activation acutely inhibited VTA dopaminergic neuron firing, and repeated exposure attenuated the opioid response. This adaptation to repeated exposure was blocked by conditional deletion of p38α MAPK, which also blocked KOR-induced tyrosine phosphorylation of the inwardly rectifying potassium channel (GIRK) subunit Kir3.1 in VTA dopaminergic neurons. Consistent with the reduced response, GIRK phosphorylation at this amino terminal tyrosine residue (Y12) enhances channel deactivation. Thus, contrary to prevailing expectations, these results suggest that κ opioid-induced aversion requires regulation of VTA dopaminergic neuron somatic excitability through a p38α MAPK effect on GIRK deactivation kinetics rather than by presynaptically inhibiting dopamine release. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) agonists have the potential to be effective, nonaddictive analgesics, but their therapeutic utility is greatly limited by adverse effects on mood. Understanding how KOR activation produces dysphoria is key to the development of better analgesics and to defining how the endogenous dynorphin opioids produce their depression-like effects. Results in this study show that the aversive effects of κ receptor activation required arrestin-dependent p38α MAPK activation in dopamine neurons but did not require inhibition of dopamine release in the nucleus accumbens. Thus, contrary to the prevailing view, inhibition of mesolimbic dopamine release does not mediate the aversive effects of KOR activation and functionally selective κ opioids that do not activate arrestin signaling may be effective analgesics lacking dysphoric effects.

κ-Opioid receptor mediates the antinociceptive effect of nitrous oxide in mice.

BACKGROUND: Our previous reports demonstrated that genetic deletion of µ-opioid receptor has no influence on the anaesthetic and antinociceptive effects of nitrous oxide (N2O) in mice, and that an antagonist selective for κ-opioid receptor (KOP), but not that selective for δ-opioid receptor, suppresses the antinociceptive effect of N2O. However, it is not known whether genetic deletion of KOP affects the N2O actions. METHODS: We measured the minimum alveolar concentration (MAC) of volatile anaesthetics in the absence and presence of N2O. The antinociceptive action of N2O was tested by an acetic acid-writhing test and a hot-plate test. The number of c-Fos-immunopositive cells in sections from the lumbar spinal cord was counted to test whether the descending inhibitory system participates in the pharmacological action of N2O. The hypnotic action of N2O was assessed by measuring the N2O-induced decrease in the EC50 for loss of the righting reflex (EC50-LORR) of sevoflurane. RESULTS: Sevoflurane MAC was not significantly reduced by N2O and its antinociceptive action was almost completely abolished in KOP-knockout (KO) mice. The N2O-induced increase in c-Fos-immunopositive cells in laminae III-IV of the lumbar spinal cord was significant in wild-type (WT), but not in KOP-KO mice. In contrast, sevoflurane EC50-LORR was similarly reduced by N2O in WT and KOP-KO mice. CONCLUSIONS: Our study suggests that N2O demonstrates its antinociceptive action and reduces sevoflurane MAC in mice through KOP activation, whereas its hypnotic potency is not dependent on KOP activation.

Knockout subtraction autoradiography: a novel ex vivo method to detect heteromers finds sparse KOP receptor/DOP receptor heterodimerization in the brain.

Several methodological approaches suggest that receptor heteromers exist in cell systems, but their presence in physiological tissue is widely contentious. We describe a novel method to determine if heterodimers exist in brain tissue sections using autoradiographic binding comparisons from single and double gene knockout mice, where tissues either have a full receptor complement and can form heterodimers, or are incapable of making heterodimers. We have tested this model, which we have named Knockout Subtraction Autoradiography, to determine if heterodimerisation of the kappa (KOP) and delta opioid (DOP) receptors occurs, as evidence from binding studies in cell systems suggest they are present in the brain. Using labeling of putative KOP receptor/DOP receptor heterodimers with either [(3)H]bremazocine or with [(3)H]naltrindole, two ligands which were used to provide evidence suggesting that these opioid receptor subtypes heterodimerize, we have applied a subtraction equation model based on the principle that receptor gene double knockout of either MOP receptor/KOP receptor (DOP receptor expression only) or MOP receptor/DOP receptor (KOP receptor expression only) produces tissue incapable of making the KOP receptor/DOP receptor heterodimer. We have shown in most brain regions that the labeling fits a simple additive model of monomer labeling, but that in a few brain regions opioid receptor heterodimerization does occur. The data does not support the conclusion that KOP receptor/DOP receptor heterodimerisation is widespread in the central nervous system, but does indicate that this novel methodology can detect heterodimerisation, when ligands with distinct binding affinities for monomer and heterodimer forms exist.

Kappa opioid receptors on dopaminergic neurons are necessary for kappa-mediated place aversion.

Kappa-opioid receptor (KOR) agonists have dysphoric properties in humans and are aversive in rodents. This has been attributed to the activation of KORs within the mesolimbic dopamine (DA) system. However, the role of DA in KOR-mediated aversion and stress remains divisive as recent studies have suggested that activation of KORs on serotonergic neurons may be sufficient to mediate aversive behaviors. To address this question, we used conditional knock-out (KO) mice with KORs deleted on DA neurons (DAT(Cre/wt)/KOR(loxp/loxp), or DATCre-KOR KO). In agreement with previous findings, control mice (DAT(Cre/wt)/KOR(wt/wt) or WT) showed conditioned place aversion (CPA) to the systemically administered KOR agonist U69,593. In contrast, DATCre-KOR KO mice did not exhibit CPA with this same agonist. In addition, in vivo microdialysis showed that systemic U69,593 decreased overflow of DA in the nucleus accumbens (NAc) in WT mice, but had no effect in DATCre-KOR KO mice. Intra- ventral tegmental area (VTA) delivery of KORs using an adeno-associated viral gene construct, resulted in phenotypic rescue of the KOR-mediated NAc DA response and aversive behavior in DATCre-KOR KO animals. These results provide evidence that KORs on VTA DA neurons are necessary to mediate KOR-mediated aversive behavior. Therefore, our data, along with recent findings, suggest that the neuronal mechanisms of KOR-mediated aversive behavior may include both dopaminergic and serotonergic components.

Ablation of kappa-opioid receptors from brain dopamine neurons has anxiolytic-like effects and enhances cocaine-induced plasticity.

Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR(-/-)) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KOR(lox/lox)). Autoradiography demonstrated complete ablation of KOR binding in the KOR(-/-) mutants, and reduced binding in the DAT-KOR(lox/lox) mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR(-/-) mice appeared normal in the open field and light/dark box tests, DAT-KOR(lox/lox) mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR(-/-) mutants, but was exaggerated in DAT-KOR(lox/lox) mutants. Increased sensitivity to cocaine in the DAT-KOR(lox/lox) mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR(-/-) mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.

Influence of endogenous opioid systems on T lymphocytes as assessed by the knockout of mu, delta and kappa opioid receptors.

Here, we evaluated the influence of endogenous opioid activation on immune responses by examining consequences of all three opioid receptor gene (mu, delta and kappa) inactivation. In triple-opioid receptor knockout mice, splenocytes and thymocytes numbers, lymphocyte subsets as well as proliferation and cytokines induced by in vitro stimulation of T lymphocytes were measured. Compared with wild-type mice, similar lymphocyte distribution in thymus and spleen as well as comparable T lymphocyte proliferation were observed, while lower levels of IL-2 and IFNγ as well as higher levels of IL-4 and IL-10 were found in triple-opioid receptor knockout mice. Together, our results indicate a shift from TH1 to TH2 cytokines in triple-opioid receptor knockout animals, suggesting that global endogenous opioid tone drives T lymphocytes toward a TH1 profile under non-pathological conditions.

Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia.

Analgesic effects of delta opioid receptor (DOR) -selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) -knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266-273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II-induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR's effects was also independent of beta-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.

Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria.

The molecular mechanisms mediating stress-induced dysphoria in humans and conditioned place aversion in rodents are unknown. Here, we show that repeated swim stress caused activation of both kappa-opioid receptor (KOR) and p38 mitogen-activated protein kinase (MAPK) coexpressed in GABAergic neurons in the nucleus accumbens, cortex, and hippocampus. Sites of activation were visualized using phosphoselective antibodies against activated kappa receptors (KOR-P) and against phospho-p38 MAPK. Surprisingly, the increase in P-p38-IR caused by swim-stress exposure was completely KOR dependent; P-p38-IR did not increase in KOR(-/-) knock-out mice subjected to the same swim-paradigm or in wild-type mice pretreated with the KOR antagonist norbinaltorphimine. To understand the relationship between p38 activation and the behavioral effects after KOR activation, we administered the p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (i.c.v.)] and found that it selectively blocked the conditioned place aversion caused by the kappa agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and the KOR-dependent swim stress-induced immobility while not affecting kappa-opioid analgesia or nonselectively affecting associative learning. We found that the mechanism linking KOR and p38 activation in vivo was consistent with our previous in vitro data suggesting that beta-arrestin recruitment is required; mice lacking G-protein-coupled receptor kinase 3 also failed to increase p-p38-IR after KOR activation in vivo, failed to show swim stress-induced immobility, or develop conditioned place aversion to U50488. Our results indicate that activation of p38 MAPK signaling by the endogenous dynorphin-kappa-opioid system likely constitutes a key component of the molecular mechanisms mediating the aversive properties of stress.

Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase.

Partial sciatic nerve ligation (pSNL) markedly increased glial fibrillary acidic protein immunoreactivity (GFAP-IR) 1 week after lesion in the L4-L5 spinal dorsal horn of wild-type, but not in dynorphin knock-out, mice lacking kappa opioid receptors (KOR-/-) or in wild-type mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). A direct effect of KOR on glial cell proliferation was suggested by the findings that primary cultures of type II GFAP-immunoreactive astrocytes isolated from mouse spinal cord express KOR. Sustained treatment with the kappa agonist U50,488 (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolytinil)-cyclohexyl]-benzeneacetamide methane sulfonate) significantly increased the proliferation rate of GFAP-immunoreactive astrocytes isolated from wild-type mice, and this effect was blocked by norBNI pretreatment. Proliferation of cultured type II astrocytes may have been stimulated by mitogen-activated protein kinase (MAPK) activation by KOR because (1) U50,488 treatment increased phospho-p38 MAPK-immunoreactivity 247 +/- 44% over untreated cells, (2) the increase in phospho-p38 induced by U50,488 was blocked by norBNI and not evident in KOR-/- cultures, and (3) GFAP-immunoreactive astrocyte proliferation induced by U50,488 was blocked by the p38 MAPK inhibitor SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Similar mechanisms of astrocyte activation may also be responsible in vivo because intrathecal injection of SB 203580 blocked the increased GFAP-IR in lumbar spinal cord induced by pSNL. Although the relationship between kappa-stimulated astrocyte proliferation and neuropathic pain mechanisms was not directly established in these studies, the results support the hypothesis that KOR activation induces spinal astrocyte proliferation, which may contribute to cellular reorganization after sciatic nerve damage.

Endogenous kappa opioid receptor systems modulate the responsiveness of mesoaccumbal dopamine neurons to ethanol.

BACKGROUND: Endogenous kappa-opioid receptor (KOPR) systems modulate the actions of several drugs of abuse. Their role in modulating the effects of ethanol is unknown. An increase in nucleus accumbens extracellular dopamine (DA) has been implicated in mediating the rewarding and locomotor-activating effects of ethanol and virtually all drugs of abuse. The present microdialysis studies were conducted to determine whether the lack of KOPR alters ethanol-evoked DA levels in the nucleus accumbens of naïve mice and whether a similar effect is observed in mice repeatedly exposed to ethanol. METHODS: Gene deletion techniques were used in conjunction with in vivo microdialysis to examine the influence of lack of KOPR on ethanol-evoked DA in the nucleus accumbens. To determine whether pharmacological inactivation of KOPR produces similar effects in naïve mice and those repeatedly exposed to ethanol, the KOPR antagonist norbinaltorphimine (n-BNI) was administered in wild-type mice before repeated air or ethanol vapor inhalation. Microdialysis was conducted 24 hours later. RESULTS: Acute ethanol administration increased DA levels in the nucleus accumbens of wild-type mice. In littermates lacking the KOPR gene, ethanol-evoked DA levels were enhanced. Prior ethanol exposure reduced ethanol-evoked DA levels in vehicle-treated and n-BNI-treated mice. Statistical analysis, however, revealed a significant main effect of n-BNI, indicating that KOPR blockade increased ethanol-evoked DA levels in naïve mice and repeated ethanol exposure attenuated, but did not abolish, this effect. CONCLUSIONS: These findings demonstrate that inhibition of KOPR leads to increased sensitivity to the DA-releasing effects of ethanol in the nucleus accumbens.

Prior activation of kappa opioid receptors by U50,488 mimics repeated forced swim stress to potentiate cocaine place preference conditioning.

Repeated forced-swim stress (FSS) produced analgesia, immobility and potentiation of cocaine-conditioned place preference (CPP) in wild-type C57Bl/6 mice, but not in littermates lacking the kappa opioid receptor (KOR) gene. These results were surprising because kappa agonists are known to produce conditioned place aversion and to suppress cocaine-CPP when coadministered with cocaine. The possibility that disruption of the kappa system blocked the stress response by adversely affecting the hypothalamic-pituitary axis was examined by measuring plasma corticosterone levels. However, disruption of the dynorphin/kappa system by gene deletion or receptor antagonism did not reduce the FSS-induced elevation of plasma corticosterone levels. A second explanation for the difference is that kappa receptor activation caused by FSS occurred prior to cocaine conditioning rather than contemporaneously. To test this hypothesis, we measured the effects of the kappa agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) administered to mice at various intervals preceding cocaine conditioning. The results showed that the interaction between the kappa system and cocaine reinforcement depended on the timing of the drug pairing. Mice given U50,488 60 min prior to cocaine showed a robust, nor-BNI-sensitive potentiation of cocaine-CPP, whereas administration 15 min before cocaine significantly suppressed cocaine-CPP. In the absence of cocaine, U50,488 given 60 min prior to saline conditioning produced no place preference, whereas administration 15 min before saline conditioning produced significant place aversion. The results of this study suggest that kappa receptor activation induced by FSS prior to the cocaine-conditioning session may be both necessary and sufficient for potentiation of the reinforcing actions of cocaine.

Endogenous kappa-opioid receptor systems regulate mesoaccumbal dopamine dynamics and vulnerability to cocaine.

Genetic and pharmacological approaches were used to examine kappa-opioid receptor (KOR-1) regulation of dopamine (DA) dynamics in the nucleus accumbens and vulnerability to cocaine. Microdialysis revealed that basal DA release and DA extraction fraction (Ed), an indirect measure of DA uptake, are enhanced in KOR-1 knock-out mice. Analysis of DA uptake revealed a decreased Km but unchanged Vmax in knock-outs. Knock-out mice exhibited an augmented locomotor response to cocaine, which did not differ from that of wild-types administered a behavioral sensitizing cocaine treatment. The ability of cocaine to increase DA was enhanced in knock-outs, whereas c-fos induction was decreased. Although repeated cocaine administration to wild types produced behavioral sensitization, knock-outs exhibited no additional enhancement of behavior. Administration of the long-acting KOR antagonist nor-binaltorphimine to wild-type mice increased DA dynamics. However, the effects varied with the duration of KOR-1 blockade. Basal DA release was increased whereas Ed was unaltered after 1 h blockade. After 24 h, release and Ed were increased. The behavioral and neurochemical effects of cocaine were enhanced at both time points. These data demonstrate the existence of an endogenous KOR-1 system that tonically inhibits mesoaccumbal DA neurotransmission. Its loss induces neuroadaptations characteristic of "cocaine-sensitized" animals, indicating a critical role of KOR-1 in attenuating responsiveness to cocaine. The increased DA uptake after pharmacological inactivation or gene deletion highlights the plasticity of mesoaccumbal DA neurons and suggests that loss of KOR-1 and the resultant disinhibition of DA neurons trigger short- and long-term DA transporter adaptations that maintain normal DA levels, despite enhanced release.

Autoradiography in opioid triple knockout mice reveals opioid and opioid receptor like binding of naloxone benzoylhydrazone.

Naloxone benzoylhydrazone (NalBzoH) is a ligand used to study opioid receptors. It has been suggested to act at a novel kappa3 receptor but also appears to bind to classical opioid receptors, and possibly the ORL1 receptor. We have used opioid receptor triple knockout mice, deficient in genes coding for the mu, delta and kappa-receptor, to characterise the relative contributions of opioid and ORL1 activity to the binding of this ligand, by carrying out receptor autoradiography with [3H]NalBzoH. As competing ligands we have used diprenorphine and nociceptin at 1 microM, alone or in combination, to determine the contribution of opioid and ORL1 receptor binding. At 4 nM [3H]NalBzoH showed labelling in wild-type brains indicative of broad spectrum classical opioid receptor binding. In the triple knockout brains all labelling was completely absent, suggesting that at this concentration there is no binding to ORL1 sites. However at 50 nM [3H]NalBzoH showed labelling in triple knockout brains with a distribution pattern indicative of ORL1 labelling. Quantitative analysis showed that nociceptin displaced typically 30% of the residual labelling in knockout brains whilst diprenorphine had relatively little effect. The data show that at 50 nM NalBzoH no binding was detected other than to classical opioid receptors or to ORL1 in an approximate ratio of 2:1.

Opposite alterations of NPFF1 and NPFF2 neuropeptide FF receptor density in the triple MOR/DOR/KOR-opioid receptor knockout mouse brains.

Mice lacking the mu-delta-kappa-opioid receptor (MOR/DOR/KOR) genes and their corresponding wild-type littermates have been used to quantify NPFF(1) and NPFF(2) (neuropeptide FF) receptors by in vitro autoradiography in the central nervous tissues. Adjacent coronal sections were labelled with [125I]YVP ([125I]YVPNLPQRF-NH(2)) and [125I]EYF ([125I]EYWSLAAPQRF-NH(2)) as specific radioligands for NPFF(1) and NPFF(2) receptors, respectively. NPFF(2) receptors are predominantly expressed in both genotypes, but their density increases significantly in non cortical regions of mutant mice: 64% in the amygdaloid area, 89, 308, 1214 and 49% in the nucleus of the vertical limb of the diagonal band, substantia nigra, the vestibular nucleus and the spinal cord, respectively. In contrast, the density of the NPFF(1) subtype is lower than NPFF(2) in both genotypes and significantly decreased in some brain areas of mutant mice: -99, -90 and -90% in the nucleus of the vertical limb of the diagonal band, substantia nigra and the spinal cord, respectively. This study shows that mice lacking opioid receptors have brain region-dependent increases (NPFF(2)) and decreases (NPFF(1)) in NPFF receptors densities and suggests a different functional participation of each NPFF receptor subtype in the actions of opioids.

The delta agonists DPDPE and deltorphin II recruit predominantly mu receptors to produce thermal analgesia: a parallel study of mu, delta and combinatorial opioid receptor knockout mice.

Delta-selective agonists have been developed to produce potent analgesic compounds with limited side-effects. DPDPE and deltorphin II are considered prototypes, but their delta-selectivity in vivo and the true ability of delta receptors to produce analgesia remain to be demonstrated. Here we have performed a parallel analysis of mu, delta and combinatorial opioid receptor knockout mice, in which we found no obvious alteration of G-protein coupling for remaining opioid receptors. We compared behavioural responses in two models of acute thermal pain following DPDPE and deltorphin II administration by intracerebroventricular route. In the tail-immersion test, both compounds were fully analgesic in delta knockout mice and totally inactive in mu knockout mice. In the hotplate test, the two compounds again produced full analgesia in delta knockout mice. In mu knockout mice, there was significant, although much lower, analgesia. Furthermore, DPDPE analgesia in the delta knockout mice was fully reversed by the mu selective antagonist CTOP in both tests. Together, this suggests that mu rather than delta receptors are recruited by the two agonists for the tail withdrawal and the hotplate responses. Finally, deltorphin II slightly prolonged jump latencies in double mu/kappa knockout mice (delta receptors only) and this response was abolished in the triple knockout mice, demonstrating that the activation of delta receptors alone can produce weak but significant mu-independent thermal antinociception.

Neurofilament proteins and cAMP pathway in brains of mu-, delta- or kappa-opioid receptor gene knock-out mice: effects of chronic morphine administration.

Opiate addiction is associated with abnormalities of neurofilament (NF) proteins and upregulation of cAMP signaling in the brain, which may modulate neuronal plasticity. This study investigated, using gene-targeted mice lacking mu-, delta- or kappa-opioid receptors, the role of these receptors in modulating the basal activity and the chronic effects of morphine on both intracellular targets. In WT mice, chronic treatment (5 days) with morphine (20-100 mg/kg) resulted in decreases in the immunodensity of neurofilament (NF)-L in the cerebral cortex (14-23%). In contrast, chronic morphine did not decrease NF-L in cortices of mu-, delta-, and kappa-KO mice, suggesting the involvement of the three types of opioid receptors in this effect of morphine. Also, the marked increase in phosphorylated NF-H induced by chronic morphine in WT mice (two-fold) was abolished in mu -KO mice. In cortex and/or striatum of mu-, delta- and kappa-KO mice, the basal immunodensities of Galphai1/2 proteins, the catalytic isoform (Calpha) of protein kinase A (PKA) and the total content of cAMP response element-binding protein (CREB, the nuclear target of PKA) were not different from those of WT mice. In contrast, phosphorylated CREB (the active form of this transcription factor) was reduced in cortex and/or striatum (23-26%) of mu- and delta-KO mice, but not in kappa-KO animals. These results suggest that the endogenous opioid tone acting on mu-/delta-receptors tonically stimulate CREB activation in the brain. In cortex and/or striatum of WT mice, chronic morphine did not induce upregulation of the main components of the cAMP signaling pathway. In contrast, chronic morphine treatment in mu-KO mice, but not in delta- or kappa-KO, resulted in a paradoxical upregulation of Galphai1/2 (12-19%), PKA (19-21%,) and phosphorylated CREB (21-73%), but not total CREB, in cortex and/or striatum. The induction of heterologous receptor adaptations in mu-KO mice may explain this paradoxical effect of morphine.