Activation Of Μ-opioid Receptors Research Articles

β-Casomorphin-7 regulates the secretion and expression of gastrointestinal mucins through a μ-opioid pathway

We have recently shown that beta-casomorphin-7, a milk opioid peptide, strongly stimulates mucin secretion in the rat jejunum through a nervous pathway and opioid receptor activation. In this study, the hypothesis that beta-casomorphin-7 may also act directly on intestinal goblet cells was investigated in vitro in rat and human intestinal mucin-producing cells (DHE and HT29-MTX) using quantitative and semiquantitative RT-PCR and ELISA. The presence of mu-opioid receptors was demonstrated in rat goblet cells in the upper half of the colonic crypt and in the two cell lines by immunohistochemistry and RT-PCR. In rat DHE cells, beta-casomorphin-7 increased the expression of rat mucin (rMuc)2 and rMuc3 but not rMuc1, rMuc4, and rMuc5AC. This effect was time and dose dependent, with the maximum of increase in transcripts being noticed for a concentration of 10(-4) M after 2 h of stimulation for rMuc2 (225% of controls) and 4 h of stimulation for rMuc3 (208% of controls). Mucin secretion was maximally increased after 8 h of stimulation. Interestingly, these effects were prevented by pretreatment of the cells with the mu-opioid antagonist cyprodime. In human HT29-MTX cells, beta-casomorphin-7 (10(-4) M) also increased MUC5AC mRNA levels (219% after 24 h of stimulation) and the secretion of this mucin (169% of controls). In conclusion, beta-casomorphin-7 may contribute significantly to mucin production via a direct effect on intestinal goblet cells and the activation of mu-opioid receptors. Because intestinal mucins have a crucial mucosal protective function, dairy products containing beta-casomorphin-7 may improve intestinal protection and could have dietary and health applications.

A Nuclear Function of β-Arrestin1 in GPCR Signaling: Regulation of Histone Acetylation and Gene Transcription

Chromatin modification is considered to be a fundamental mechanism of regulating gene expression to generate coordinated responses to environmental changes, however, whether it could be directly regulated by signals mediated by G protein-coupled receptors (GPCRs), the largest surface receptor family, is not known. Here, we show that stimulation of delta-opioid receptor, a member of the GPCR family, induces nuclear translocation of beta-arrestin 1 (betaarr1), which was previously known as a cytosolic regulator and scaffold of GPCR signaling. In response to receptor activation, betaarr1 translocates to the nucleus and is selectively enriched at specific promoters such as that of p27 and c-fos, where it facilitates the recruitment of histone acetyltransferase p300, resulting in enhanced local histone H4 acetylation and transcription of these genes. Our results reveal a novel function of betaarr1 as a cytoplasm-nucleus messenger in GPCR signaling and elucidate an epigenetic mechanism for direct GPCR signaling from cell membrane to the nucleus through signal-dependent histone modification.

Antinociception, tolerance and withdrawal symptoms induced by 7-hydroxymitragynine, an alkaloid from the Thai medicinal herb Mitragyna speciosa

7-Hydroxymitragynine is a potent opioid analgesic alkaloid isolated from the Thai medicinal herb Mitragyna speciosa. In the present study, we investigated the opioid receptor subtype responsible for the analgesic effect of this compound. In addition, we tested whether development of tolerance, cross-tolerance to morphine and naloxone-induced withdrawal signs were observed in chronically 7-hydroxymitragynine-treated mice. Subcutaneous (s.c.) administration of 7-hydroxymitragynine produced a potent antinociceptive effect mainly through activation of μ-opioid receptors. Tolerance to the antinociceptive effect of 7-hydroxymitragynine developed as occurs to morphine. Cross-tolerance to morphine was evident in mice rendered tolerant to 7-hydroxymitragynine and vice versa. Naloxone-induced withdrawal signs were elicited equally in mice chronically treated with 7-hydroxymitragynine or morphine. 7-Hydroxymitragynine exhibited a potent antinociceptive effect based on activation of μ-opioid receptors and its morphine-like pharmacological character, but 7-hydroxymitragynine is structurally different from morphine. These interesting characters of 7-hydroxymitragynine promote further investigation of it as a novel lead compound for opioid studies.

μ-Opioid Receptor and CREB Activation Are Required for Nicotine Reward

Environmental cues associated with nicotine delivery are an important part of the stimulus that sustains smoking behavior and is often coupled with craving and relapse; however, the neuronal circuitry and molecular substrates underlying this process are still poorly understood. Exposure to an environment previously associated with rewarding properties of nicotine results in an increase of CREB phosphorylation similar to that seen following nicotine administration, and this response is absent in MOR(-/-) mice. Moreover, a single administration of an opioid receptor antagonist, naloxone, blocks both the conditioned molecular response (CREB phosphorylation) and the conditioned behavioral response (nicotine reward) in a place preference paradigm. Lastly, repeated nicotine administration results in increased expression of MORs. However, this effect, along with rewarding properties of nicotine, is blocked in mice with a targeted disruption in the CREB gene. Together, pharmacologic and genetic manipulations indicate that phosphorylation of CREB and upregulation of functional MORs are required for nicotine-conditioned reward.

Adenylyl cyclase type-VIII activity is regulated by G βγ subunits

The Ca 2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether G i/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by μ-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G βγ subunits inhibit AC-VIII activity, while constitutively active α i/o subunits do not. Different G β isoforms varied in their efficacies, with G β1γ2 or G β2γ2 being more efficient than G β3γ2 and G β4γ2, while G β5 (transfected with γ 2) had no effect. As for the G γ subunits, G β1 inhibited AC-VIII activity in the presence of all γ subunits tested except for γ 5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of G βγ dimers, or to reduce G βγ plasma membrane anchorage, markedly attenuated the μ-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that G βγ (originating from agonist activation of these receptors) and probably not G αi/o subunits are involved in the agonist inhibition of AC-VIII.

In VivoActivation of a Mutant μ-Opioid Receptor by Naltrexone Produces a Potent Analgesic Effect But No Tolerance: Role of μ-Receptor Activation and δ-Receptor Blockade in Morphine Tolerance

Opioid analgesics are the standard therapeutic agents for the treatment of pain, but their prolonged use is limited because of the development of tolerance and dependence. Recently, we reported the development of a mu-opioid receptor knock-in (KI) mouse in which the mu-opioid receptor was replaced by a mutant receptor (S196A) using a homologous recombination gene-targeting strategy. In these animals, the opioid antagonist naltrexone elicited antinociceptive effects similar to those of partial agonists acting in wild-type (WT) mice; however, development of tolerance and physical dependence were greatly reduced. In this study, we test the hypothesis that the failure of naltrexone to produce tolerance in these KI mice is attributable to its simultaneous inhibition of delta-opioid receptors and activation of mu-opioid receptors. Simultaneous implantation of a morphine pellet and continuous infusion of the delta-opioid receptor antagonist naltrindole prevented tolerance development to morphine in both WT and KI animals. Moreover, administration of SNC-80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], a delta agonist, in the naltrexone-pelleted KI animals resulted in a dose-dependent induction in tolerance development to both morphine- and naltrexone-induced analgesia. We conclude that although simultaneous activation of both mu- and delta-opioid receptors results in tolerance development, mu-opioid receptor activation in conjunction with delta-opioid receptor blockade significantly attenuates the development of tolerance.

Interaction of μ-opioid receptor agonists and antagonists with the analgesic effect of buprenorphine in mice

Buprenorphine is a potent opioid analgesic with partial agonistic properties at μ-opioid receptors. This study investigated the interaction potential with several full μ-agonists in the tail-flick test in mice. We further examined the reversibility of buprenorphine antinociception by different μ-opioid receptor antagonists. Combination of buprenorphine with morphine, oxycodone, hydromorphone and fentanyl in the analgesic dose range resulted in additive or synergistic effects. When given after the decline of the acute buprenorphine effect, both morphine and fentanyl also showed full efficacy. A moderate antagonistic effect according to the partial μ-agonistic properties of buprenorphine was only seen when high doses exceeding the therapeutic dose ranges were combined. Under these conditions antinociception of morphine was reduced to the effect of buprenorphine alone. Prophylactic administration of naloxone (10 mg/kg i.v.), naltrexone (1 mg/kg i.v.) and clocinnamox (5 mg/kg s.c.) fully and persistently blocked the antinociception of a high dose of buprenorphine. An established effect of buprenorphine was less sensitive, although repeated administration of naloxone induced complete antagonism, as did the irreversible antagonist clocinnamox under prophylactic and curative treatment conditions. Our results suggest that the antinociceptive effect of buprenorphine is mainly, if not exclusively, mediated by activation of μ-opioid receptors. They confirm clinical experience that in the analgesic dose range a switch between buprenorphine and full μ-agonists is possible without loss of analgesic efficacy and without a refractory period between the termination of buprenorphine analgesia and the onset of action of the new μ-opioid treatment. Antinociception of buprenorphine is sensitive towards μ-opioid receptor antagonists and incomplete inhibition can be improved by increasing the dose or repetitive dosing.

Activation of μ-Opioid Receptors Transfers Control of Gα Subunits to the Regulator of G-protein Signaling RGS9-2: ROLE IN RECEPTOR DESENSITIZATION

In mouse periaqueductal gray matter (PAG) membranes, the mu-opioid receptor (MOR) coprecipitated the alpha-subunits of the Gi/o/z/q/11 proteins, the Gbeta1/2 subunits, and the regulator of G-protein signaling RGS9-2 and its partner protein Gbeta5. RGS7 and RGS11 present in this neural structure showed no association with MOR. In vivo intracerebroventricular injection of morphine did not alter MOR immunoreactivity, but 30 min and 3 h after administration, the coprecipitation of Galpha subunits with MORs was reduced by up to 50%. Furthermore, the association between Galpha subunits and RGS9-2 proteins was increased. Twenty-four hours after receiving intracerebroventricular morphine, the Galpha subunits left the RGS9-2 proteins and re-associated with the MORs. However, doses of the opioid able to induce tolerance promoted the stable transfer of Galpha subunits to the RGS9-2 control. This was accompanied by Ser phosphorylation of RGS9-2 proteins, which increased their co-precipitation with 14-3-3 proteins. In the PAG membranes of morphine-desensitized mice, the capacity of the opioid to stimulate G-protein-related guanosine 5'-O-(3-[35S]thiotriphosphate) binding as well as low Km GTPase activity was attenuated. The in vivo knockdown of RGS9-2 expression prevented morphine from altering the association between MORs and G-proteins, and tolerance did not develop. In PAG membranes from RGS9-2 knockdown mice, morphine showed full capacity to activate G-proteins. Thus, the tolerance that develops following an adequate dose of morphine is caused by the stabilization and retention of MOR-activated Galpha subunits by RGS9-2 proteins. This multistep process is initiated by the morphine-induced transfer of MOR-associated Galpha subunits to the RGS9-2 proteins, followed by Ser phosphorylation of the latter and their binding to 14-3-3 proteins. This regulatory mechanism probably precedes the loss of MORs from the cell membrane, which has been observed with other opioid agonists.

Periodic oscillatory activity in parahippocampal slices maintained in vitro

Brain slices maintained in vitro have been extensively used for studying neuronal synchronization. However, the validity of this approach may be questioned since pharmacological procedures are usually required to elicit spontaneous events similar to the EEG activity recorded in vivo. Here, we report that when superfused with control medium, rat brain slices comprising the entorhinal and perirhinal cortices along with a portion of the basolateral/lateral nuclei of the amygdala can synchronously generate periodic oscillatory activity at 5–11 Hz every 5–30 s. The periodic events: (i) correspond intracellularly to synaptic depolarizations in regularly firing neurons analyzed in the three areas; (ii) have no fixed site of onset; (iii) spread with time lags of 8–20 ms; and (iv) continue to occur asynchronously after their surgical isolation. NMDA receptor antagonism reduced the duration of the oscillatory events, while glutamatergic non-NMDA receptor antagonism abolished them. Activation of μ-opioid receptors, a procedure that hyperpolarizes interneurons thus decreasing GABA release, reversibly decreased the rate of occurrence of periodic oscillatory activity (POA). However, periodic events continued to occur during application of GABA A or GABA B receptor antagonists as well as in the presence of the cholinergic agent carbachol. We also found that POA was abolished by baclofen and irreversibly reduced by the gap junction decoupler carbenoxolone. These findings demonstrate that parahippocampal networks in a brain slice preparation can generate periodic, synchronous activity under quasi-physiological conditions. These network oscillations (i) reflect the activation of ionotropic glutamatergic and GABAergic receptors, (ii) are contributed by gap-junction interactions, and (iii) are controlled by GABA B receptors that are presumably located presynaptically.

Brown adipose tissue thermogenesis contributes to fentanyl-evoked hyperthermia

Mu-opioid receptor activation increases body temperature and affects cardiovascular function. In the present study, fentanyl was administered intravenously [100 mug/kg (300 nmol/kg) iv] and intracerebroventricularly [3.4 mug (10 nmol) in 10 microl icv] in urethane-chloralose-anesthetized, artificially ventilated rats. Increases in brown adipose tissue (BAT) sympathetic nerve activity (SNA) (peak, +326% of control), BAT temperature (peak, +0.8 degrees C), renal SNA (peak, +146% of control), and heart rate (HR; peak, +32 beats/min) produced by intravenous fentanyl were abolished by premamillary transection of the neuraxis but were mimicked by intracerebroventricular administration of fentanyl, which also increased arterial pressure (AP; peak, +12 mmHg). Pretreatment with the opioid antagonist naloxone (100 nmol in 10 microl icv) eliminated the intracerebroventricular fentanyl-evoked responses. Microinjection of glycine (0.5 M, 60 nl) to inhibit local neurons in the rostral raphe pallidus (RPa) selectively reversed the intracerebroventricular fentanyl-evoked increases in BAT SNA and HR, while the fentanyl-evoked excitation in RSNA, the pressor responses, and the tachycardic responses were reversed by inhibition of neurons in the rostral ventrolateral medulla (RVLM). Prior inhibition of neurons in the dorsomedial hypothalamus eliminated the intracerebroventricular fentanyl-evoked increases in BAT SNA, BAT temperature, and HR, but not those in RSNA or AP. These results indicate that activation of central mu-opioid receptors with fentanyl can elicit BAT thermogenesis and cardiovascular stimulation through excitation of the sympathetic outflows to BAT, kidney, and heart. Activation of neurons in the rostral RPa and RVLM are essential for the increases in BAT thermogenesis and renal sympathoexcitation, respectively, induced by activation of central mu-opioid receptors. BAT thermogenesis could contribute to fentanyl-evoked hyperthermia, particularly in infants where BAT plays a significant role in thermoregulation.

Increased gabaergic input to ventral tegmental area dopaminergic neurons associated with decreased cocaine reinforcement in μ-opioid receptor knockout mice

There is general agreement that dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex play a key role in drug reinforcement. The activity of these neurons is strongly modulated by the inhibitory and excitatory input they receive. Activation of μ-opioid receptors, located on GABAergic neurons in the VTA, causes hyperpolarization of these GABAergic neurons, thereby causing a disinhibition of VTA dopaminergic neurons. This effect of μ-opioid receptors upon GABA neurotransmission is a likely mechanism for μ-opioid receptor modulation of drug reinforcement. We studied μ-opioid receptor signaling in relation to cocaine reinforcement in wild-type and μ-opioid receptor knockout mice using a cocaine self-administration paradigm and in vitro electrophysiology. Cocaine self-administration was reduced in μ-opioid receptor knockout mice, suggesting a critical role of μ-opioid receptors in cocaine reinforcement. The frequency of spontaneous inhibitory post-synaptic currents onto dopaminergic neurons in the ventral tegmental area was increased in μ-opioid receptor knockout mice compared with wild-type controls, while the frequency of spontaneous excitatory post-synaptic currents was unaltered. The reduced cocaine self-administration and increased GABAergic input to VTA dopaminergic neurons in μ-opioid receptor knockout mice supports the notion that suppression of GABAergic input onto dopaminergic neurons in the VTA contributes to μ-opioid receptor modulation of cocaine reinforcement.

Neurotransmitters Involved in the Opioid Regulation of Prolactin Secretion at the End of Pregnancy in Rats

Using a pharmacological approach, we explored potential mechanisms for the regulation of prolactin secretion by opioid peptides at the end of pregnancy in rats. On day 19 of pregnancy, intracereboventricular administration of the µ-opioid receptor agonist (D-Ala², NMe-Phe⁴, Gly-ol⁵)-enkephalin (DAMGO) or β-endorphin (β-END) induced a dose-related increase in serum prolactin levels 30 min later. Pretreatment with the opioid antagonist naloxone abolished the increase induced by DAMGO injection. At lower doses, DAMGO and β-END did not modify the 3,4-dihydroxyphenylacetic acid/dopamine ratio, but at higher doses, the µ-agonists evoked a significant increase of the dopaminergic activity as compared with saline control. The time course of the effects of β-END (2.5 µg/rat) showed a higher increase in serum prolactin levels at 15 min than at 30 min after treatment. The 3,4-dihydroxyphenylacetic acid/dopamine ratio increased 15 min after β-END administration and was even higher 30 min later. Neither the selective ĸ-agonist U50,488H nor the selective δ-agonist (D-Pen², D-Pen⁵)- enkephalin were able to modify the serum prolactin levels at the doses studied.To evaluate potential neurotransmitters involved in the regulation of prolactin secretion at the end of pregnancy, we combined the administration of serotoninergic or GABAergic antagonists with the opioid agonist DAMGO. The serotonin 5-HT₂ receptor antagonist ketanserin increased the serum prolactin levels and potentiated the effect of DAMGO. The intracerebroventricular administration of SR-95531 did not modify the serum prolactin concentration under basal conditions, but partially prevented the increase induced by DAMGO injection. The intracerebroventricular administration of the GABA_B receptor antagonist phaclofen had no effect on the serum prolactin levels either in naive or DAMGO-treated rats. The present results support the proposal that activation of µ-opioid receptors stimulates prolactin secretion at the end of pregnancy. Although the exact mechanisms by which the opioid system modulates prolactin secretion at the end of pregnancy are unclear, these results suggest an interaction of the opioidergic system with serotoninergic and GABAergic systems, without ruling out a direct or indirect action on dopaminergic neurons. In conclusion, the opioid system may regulate prolactin secretion at the end of pregnancy through either stimulatory (present results) or inhibitory actions previously described.

Activation of protein kinase C antagonizes the opioid inhibition of calcium current in rat spinal dorsal horn neurons

Spinal dorsal horn (SDH) is one of important regions in both nociceptive transmission and antinociception. Opioid peptides produce analgesia via regulation of neurotransmitter release through modulation of voltage-dependent Ca 2+ channel (VDCC) in neuronal tissues. The modulatory effect of μ-opioid receptor (MOR) activation on VDCC was investigated in acutely isolated rat SDH neurons under the conventional whole-cell patch-clamp recording mode. The Ba 2+ current passing through VDCC was reversibly inhibited by a MOR agonist, [ d-Ala 2, N-MePhe 4,Gly 5-ol]-enkephalin (DAMGO, 1 μM). Among 108 SDH neurons tested, VDCC of 39 neurons (36%) were inhibited by MOR activation, while other 69 neurons (64%) were not affected. The L-, N-, P/Q-, and R-type VDCC components shared 58.4±18.9%, 29.3±12.1%, 8.7±7.2%, and 3.4±4.8% of the total VDCC, respectively. Among VDCC subtypes inhibited by MOR activation, L- and N-types were 61.4±12.8% and 30.7±14.4%, respectively, while both P/Q- and R-types were 7.9±11.8%. A depolarizing pre-pulse increased the amplitude of VDCC and suppressed most of the inhibitory effect of MOR activation. Application of 1 μM phorbol-12-myristate-13-acetate completely antagonized the inhibitory effect of MOR activation without any alteration of basal VDCC amplitude. In contrast, the response of MOR activation was not altered by application of 4-α-phorbol (1 μM), 2-[3-Dimethylaminopropyl]indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X, 1 μM), forskolin (1 μM), N-(2-[ p-Bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 1 μM). These results indicate that activation of MOR coupled to G-proteins inhibits VDCC, and that this G-protein-mediated inhibition is antagonized by PKC-dependent phosphorylation.

Ingested placenta blocks the effect of morphine on gut transit in Long–Evans rats

Opioids produce antinociception, and ingested placenta or amniotic fluid modifies that antinociception. More specifically, ingested placenta enhances the antinociception produced by selective activation of central κ-opioid or δ-opioid receptors but attenuates that produced by activation of central μ-opioid receptors. Opioids also slow gut transit by acting on central or peripheral μ-opioid receptors. Therefore, we hypothesized that ingested placenta would reverse the slowing of gut transit that is produced by morphine, a preferential μ-opioid-receptor agonist. Rats were injected with morphine either centrally or systemically and fed placenta, after which gastrointestinal transit was evaluated. We report here that ingested placenta reversed the slowing of gut transit produced by centrally administered morphine but did not affect the slowing of gut transit produced by systemically administered morphine. These results suggest another likely consequence of placentophagia at parturition in mammals—reversal of opioid-mediated, pregnancy-based disruption of gastrointestinal function—as well as an important consideration in opioid-based treatments for pain in humans—enhancement of desirable effects with attenuation of adverse effects.

Estrogen-induced mu-opioid receptor internalization in the medial preoptic nucleus is mediated via neuropeptide Y-Y1 receptor activation in the arcuate nucleus of female rats.

The endogenous peptides beta-endorphin (beta-END) and neuropeptide Y (NPY) have been implicated in regulating sexual receptivity. Both beta-END and NPY systems are activated by estrogen and inhibit female sexual receptivity. The initial estrogen-induced sexual nonreceptivity is correlated with the activation and internalization of mu-opioid receptors (MORs), in the medial preoptic nucleus (MPN). Progesterone reverses the estrogen-induced activation/internalization of MOR and induces the sexual receptive behavior lordosis. To determine whether NPY and endogenous opioids interact, we tested the hypothesis that estrogen-induced MOR activation is mediated through NPY-Y1 receptor (Y1R) activation. Retrograde tract tracing demonstrated Y1Ron beta-END neurons that projected to the MPN. Sex steroid modulation of MOR in the MPN acts through NPY and the Y1R. Estradiol administration or intracerebroventricular injection of NPY activated/internalized Y1R in the arcuate nucleus and MOR in the MPN of ovariectomized (OVX) rats. Moreover, the selective Y1R agonist [Leu31, Pro34]-Neuropeptide Y (LPNY) internalized MOR in the MPN of OVX rats. The Y1R antagonist (Cys31, Nva34)-Neuropeptide Y (27-36)2 prevented estrogen-induced Y1R and MOR activation/internalization. NPY reversed the progesterone blockade of estradiol-induced Y1R and MOR internalization in the arcuate nucleus and MPN, respectively. Behaviorally, LPNY inhibited estrogen plus progesterone-induced lordosis, and the MOR-selective antagonist D-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide reversed LPNY-induced inhibition of lordosis. These results suggest that a sequential sex steroid activation of NPY and MOR circuits regulates sexual receptivity.

Increased level of neuronal phosphoinositide 3-kinase γ by the activation of μ-opioid receptor in the mouse periaqueductal gray matter: further evidence for the implication in morphine-induced antinociception

It has been proposed that phosphoinositide 3-kinase (PI3K), one of the phosphatidylinositol kinases, can be regulated by G-protein-coupled receptor as well as nerve growth factor-associated receptors. The aim of the present study was to investigate whether in vivo treatment with morphine, a μ-opioid receptor (MOR) agonist, could directly regulate PI3Kγ isoform in the mouse periaqueductal gray matter (PAG). Using the polyclonal antibody recognizing a p110γ catalytic subunit of PI3Kγ, PI3Kγ-like immunoreactivity (IR) was mostly seen in the membrane of the cell labeled by anti-neuron-specific nuclear protein. A single s.c. injection of morphine caused a marked increase in the number of PI3Kγ-IR expressing cells in the PAG. Double immunolabeling assay showed that MOR-IR was mostly overlapped with PI3Kγ-IR on the cell surface in the PAG section. Additionally, phosphorylated-phospholipase Cγ1 (PLCγ1-IR) was highly detected in the membrane compartment of the increased PI3Kγ-IR-positive cells of this region. Further pharmacological evidence for the critical role of PI3Kγ in MOR-mediated antinociceptive response was provided by the warm-plate test. The dose-response lines for antinociceptive effects of morphine were significantly shifted to the right following i.c.v. treatment with PI3K inhibitors. These findings suggested that acute treatment with morphine may directly activate the PI3Kγ/PLCγ1 pathway in the PAG. This effect may, at least in part, result in the expression of morphine-induced pharmacological actions including antinociception in mice.

Opioidergic modulation of excitability of rat trigeminal root ganglion neuron projections to the superficial layer of cervical dorsal horn

The aim of the present study was to investigate the effect of a μ-opioid receptor agonist DAMGO (Tyr- d-Ala-Gly-NMe-Phe-Gly-ol) on the excitability of trigeminal root ganglion (TRG) neurons, projecting onto the superficial layer of the cervical dorsal horn, by using the perforated-patch technique and to determine whether TRG neurons show the expression of mRNA or functional protein for μ-opioid receptors by using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. TRG neurons projecting onto the superficial layer of the cervical dorsal horn were retrogradely labeled with Fluorogold (FG). The cell diameter of FG-labeled TRG neurons was small (<30 μm). Under voltage-clamp (V h=−60 mV), voltage-dependent K + currents were recorded in the TRG neurons and isolated by blocking Na + and Ca 2+ currents with appropriate ion replacement. Separation of the K + current components was achieved by the response to variation in the conditioning voltage. Two distinct K + current components, a transient (I A) and sustained (I K), were identified. DAMGO significantly increased I A by 57% (20 μM) and in a dose-dependent manner (1–50 μM). Similarly, I K was also enhanced by DAMGO administration (42%, 20 μM). The augmentation of both I A and I K was antagonized by a μ-opioid receptor antagonist, CTOP ( d-Phe-Cys-Thr- d-Trp-Orn-Thr-Pen-Thr-NH 2). Hyperpolarization of the membrane potential was elicited by DAMGO (20 μM) and the response was associated with a decrease in the input resistance. DAMGO induced hyperpolarization was blocked by CTOP. DAMGO-sensitive I A and I K currents were antagonized by K + channel blockers, 4-aminopyridine (4-AP) and tetraethylammonium (TEA). In the presence of both 4-AP and TEA, no significant changes in membrane potential induced by DAMGO application were observed. In the presence of BaCl 2, DAMGO evoked hyperpolarization with decreased resistance was observed. The firing rate of action potentials and the first spike duration induced by depolarizing step pulses were decreased in the presence of DAMGO. RT-PCR analysis demonstrated the expression of mRNA for μ-opioid receptors in the trigeminal ganglia. The μ-opioid receptor immunoreactivity was expressed in the small diameter FG-labeled TRG neurons. These results suggest that the activation of μ-opioid receptors inhibits the excitability of rat small diameter TRG neurons projecting on the superficial layer of the cervical dorsal horn and this inhibition is mediated by potentiation of voltage-dependent K + currents. We therefore concluded that modulation of nociceptive transmission in the trigeminal system, resulting in the functional activation of μ-opioid receptors, occurs at the level of small TRG cell bodies and/or their primary afferent terminals, which contribute to opioid analgesia in the trigeminal pain.

Compartment-specific localization of cannabinoid 1 (CB1) and μ-opioid receptors in rat nucleus accumbens

Interactions between cannabinoid and opioid systems have been implicated in reward and drug seeking behaviors involving neuronal circuitry in the nucleus accumbens (Acb) shell and core. To determine the relevant sites, we examined the electron microscopic localization of cannabinoid type-1 (CB1) receptors and μ-opioid receptors in each Acb compartment in rat brain. CB1 receptor immunogold labeling was seen on the plasma membrane and within the cytoplasm of neuronal and glial profiles throughout the Acb. These neuronal profiles included somata and dendrites as well as axon terminals, many of which formed excitatory-type, asymmetric synapses with notable perforations that are often associated with synaptic plasticity. The number of CB1-labeled terminals within the neuropil of the Acb shell was significantly greater than in the core. μ-Opioid receptors were also detected in axonal and dendritic profiles. These dendrites were most prevalent in the Acb shell, where μ-receptors also were located in 21% of the dendritic profiles and 3% of the axon terminals containing CB1 receptors. More of the CB1-labeled terminals contacted dendrites expressing μ-opioid receptors in the shell (19%) compared with the core (13%). Conversely, of the synaptic μ-labeled terminals, 20% in the shell and 10% in the core contacted dendrites containing CB1 receptors. These findings provide ultrastructural evidence that cannabinoid-opioid interactions are mediated by activation of CB1 and μ-opioid receptors within the same or synaptically linked neurons in the Acb shell and core. They also suggest a particularly important role for presynaptic CB1 receptors in the reward circuit of the Acb shell.

Activation of μ-opioid receptors excites a population of locus coeruleus-spinal neurons through presynaptic disinhibition

The nucleus locus coeruleus (LC) plays an important role in analgesia produced by opioids and by modulation of the descending noradrenergic pathway. The functional role of μ-opioid receptors (μOR) in regulation of the excitability of spinally projecting LC neurons has not been investigated. In the present study, we tested the hypothesis that activation of presynaptic μ-opioid receptors excites a population of spinally projecting LC neurons through attenuation of γ-aminobutyric acid (GABA)-ergic synaptic inputs. Spinally projecting LC neurons were retrogradely labeled by a fluorescent dye injected into the spinal dorsal horn of rats. Whole-cell current- and voltage-clamp recordings were performed on labeled LC neurons in brain slices. All labeled LC noradrenergic neurons were demonstrated by dopamine-β-hydroxylase (DβH) immunofluorescence. In 37 labeled LC neurons, ( d-Ala 2, N-Me-Phe 4,Gly-ol 5)-enkephalin (DAMGO) significantly increased the discharge activity of 17 (45.9%) neurons, but significantly inhibited the firing activity of another 15 (40.5%) cells. The excitatory effect of DAMGO on seven labeled LC neurons was diminished in the presence of bicuculline. DAMGO significantly decreased the frequency of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) in all nine labeled LC neurons. However, DAMGO had no effect on glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in 12 of 15 neurons. Furthermore, DAMGO significantly inhibited the peak amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in all 11 labeled neurons, but had no significant effect on the evoked excitatory postsynaptic currents (eEPSCs) in 10 of these 11 neurons. Thus, data from this study suggest that activation of μ-opioid receptors excites a population of spinally projecting LC neurons by preferential inhibition of GABAergic synaptic inputs. These findings provide important new information about the descending noradrenergic modulation and analgesic mechanisms of opioids.

Enhanced Rewarding Properties of Morphine, but not Cocaine, in βarrestin-2 Knock-Out Mice

The reinforcing and psychomotor effects of morphine involve opiate stimulation of the dopaminergic system via activation of mu-opioid receptors (muOR). Both mu-opioid and dopamine receptors are members of the G-protein-coupled receptor (GPCR) family of proteins. GPCRs are known to undergo desensitization involving phosphorylation of the receptor and the subsequent binding of beta(arrestins), which prevents further receptor-G-protein coupling. Mice lacking beta(arrestin)-2 (beta(arr2)) display enhanced sensitivity to morphine in tests of pain perception attributable to impaired desensitization of muOR. However, whether abrogating muOR desensitization affects the reinforcing and psychomotor properties of morphine has remained unexplored. In the present study, we examined this question by assessing the effects of morphine and cocaine on locomotor activity, behavioral sensitization, conditioned place preference, and striatal dopamine release in beta(arr2) knock-out (beta(arr2)-KO) mice and their wild-type (WT) controls. Cocaine treatment resulted in very similar neurochemical and behavioral responses between the genotypes. However, in the beta(arr2)-KO mice, morphine induced more pronounced increases in striatal extracellular dopamine than in WT mice. Moreover, the rewarding properties of morphine in the conditioned place preference test were greater in the beta(arr2)-KO mice when compared with the WT mice. Thus, beta(arr2) appears to play a more important role in the dopaminergic effects mediated by morphine than those induced by cocaine.