Opioid-Induced Pronociceptive Signaling in the Gastrointestinal Tract Is Mediated by Delta-Opioid Receptor Signaling.

Opioid tolerance (OT) leads to dose escalation and serious side effects, including opioid-induced hyperalgesia (OIH). We sought to better understand the mechanisms underlying this event in the gastrointestinal tract. Chronic in vivo administration of morphine by intraperitoneal injection in male C57BL/6 mice evoked tolerance and evidence of OIH in an assay of colonic afferent nerve mechanosensitivity; this was inhibited by the δ-opioid receptor (DOPr) antagonist naltrindole when intraperitoneally injected in previous morphine administration. Patch-clamp studies of DRG neurons following overnight incubation with high concentrations of morphine, the µ-opioid receptors (MOPr) agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin (DAMGO) or the DOPr agonist [D-Ala2, D-Leu5]-Enkephalin evoked hyperexcitability. The pronociceptive actions of these opioids were blocked by the DOPr antagonist SDM25N but not the MOPr antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 The hyperexcitability induced by DAMGO was reversed after a 1 h washout, but reapplication of low concentrations of DAMGO or [D-Ala2, D-Leu5]-Enkephalin restored the hyperexcitability, an effect mediated by protein kinase C. DOPr-dependent DRG neuron hyperexcitability was blocked by the endocytosis inhibitor Pitstop 2, and the weakly internalizing DOPr agonist ARM390 did not cause hyperexcitability. Bioluminescence resonance energy transfer studies in HEK cells showed no evidence of switching of G-protein signaling from Gi to a Gs pathway in response to either high concentrations or overnight incubation of opioids. Thus, chronic high-dose opioid exposure leads to opioid tolerance and features of OIH in the colon. This action is mediated by DOPr signaling and is dependent on receptor endocytosis and downstream protein kinase C signaling.SIGNIFICANCE STATEMENT Opioids are effective in the treatment of abdominal pain, but escalating doses can lead to opioid tolerance and potentially opioid-induced hyperalgesia. We found that δ-opioid receptor (DOPr) plays a central role in the development of opioid tolerance and opioid-induced hyperalgesia in colonic afferent nociceptors following prolonged exposure to high concentrations of MOPr or DOPr agonists. Furthermore, the role of DOPr was dependent on OPr internalization and activation of a protein kinase C signaling pathway. Thus, targeting DOPr or key components of the downstream signaling pathway could mitigate adverse side effects by opioids.

Mu-Opioids Suppress GABAergic Synaptic Transmission onto Orbitofrontal Cortex Pyramidal Neurons with Subregional Selectivity.

The orbitofrontal cortex (OFC) plays a critical role in evaluating outcomes in a changing environment. Administering opioids to the OFC can alter the hedonic reaction to food rewards and increase their consumption in a subregion-specific manner. However, it is unknown how mu-opioid signaling influences synaptic transmission in the OFC. Thus, we investigated the cellular actions of mu-opioids within distinct subregions of the OFC. Using in vitro patch-clamp electrophysiology in brain slices containing the OFC, we found that the mu-opioid agonist DAMGO produced a concentration-dependent inhibition of GABAergic synaptic transmission onto medial OFC (mOFC), but not lateral OFC (lOFC) neurons. This effect was mediated by presynaptic mu-opioid receptor activation of local parvalbumin (PV+)-expressing interneurons. The DAMGO-induced suppression of inhibition was long lasting and not reversed on washout of DAMGO or by application of the mu-opioid receptor antagonist CTAP, suggesting an inhibitory long-term depression (LTD) induced by an exogenous mu-opioid. We show that LTD at inhibitory synapses is dependent on downstream cAMP/protein kinase A (PKA) signaling, which differs between the mOFC and lOFC. Finally, we demonstrate that endogenous opioid release triggered via moderate physiological stimulation can induce LTD. Together, these results suggest that presynaptic mu-opioid stimulation of local PV+ interneurons induces a long-lasting suppression of GABAergic synaptic transmission, which depends on subregional differences in mu-opioid receptor coupling to the downstream cAMP/PKA intracellular cascade. These findings provide mechanistic insight into the opposing functional effects produced by mu-opioids within the OFC.SIGNIFICANCE STATEMENT Considering that both the orbitofrontal cortex (OFC) and the opioid system regulate reward, motivation, and food intake, understanding the role of opioid signaling within the OFC is fundamental for a mechanistic understanding of the sequelae for several psychiatric disorders. This study makes several novel observations. First, mu-opioids induce a long-lasting suppression of inhibitory synaptic transmission onto OFC pyramidal neurons in a regionally selective manner. Second, mu-opioids recruit parvalbumin inputs to suppress inhibitory synaptic transmission in the mOFC. Third, the regional selectivity of mu-opioid action of endogenous opioids is due to the efficacy of mu-opioid receptor coupling to the downstream cAMP/PKA intracellular cascades. These experiments are the first to reveal a cellular mechanism of opioid action within the OFC.

Selective blockade of spinal D2DR by levo-corydalmine attenuates morphine tolerance via suppressing PI3K/Akt-MAPK signaling in a MOR-dependent manner.

Morphine tolerance remains a challenge in the management of chronic pain in the clinic. As shown in our previous study, the dopamine D2 receptor (D2DR) expressed in spinal cord neurons might be involved in morphine tolerance, but the underlying mechanisms remain to be elucidated. In the present study, selective spinal D2DR blockade attenuated morphine tolerance in mice by inhibiting phosphatidylinositol 3 kinase (PI3K)/serine-threonine kinase (Akt)-mitogen activated protein kinase (MAPK) signaling in a µ opioid receptor (MOR)-dependent manner. Levo-corydalmine (l-CDL), which exhibited micromolar affinity for D2DR in D2/CHO-K1 cell lines in this report and effectively alleviated bone cancer pain in our previous study, attenuated morphine tolerance in rats with chronic bone cancer pain at nonanalgesic doses. Furthermore, the intrathecal administration of l-CDL obviously attenuated morphine tolerance, and the effect was reversed by a D2DR agonist in mice. Spinal D2DR inhibition and l-CDL also inhibited tolerance induced by the MOR agonist DAMGO. l-CDL and a D2DR small interfering RNA (siRNA) decreased the increase in levels of phosphorylated Akt and MAPK in the spinal cord; these changes were abolished by a PI3K inhibitor. In addition, the activated Akt and MAPK proteins in mice exhibiting morphine tolerance were inhibited by a MOR antagonist. Intrathecal administration of a PI3K inhibitor also attenuated DAMGO-induced tolerance. Based on these results, l-CDL antagonized spinal D2DR to attenuate morphine tolerance by inhibiting PI3K/Akt-dependent MAPK phosphorylation through MOR. These findings provide insights into a more versatile treatment for morphine tolerance.

Placement of Hydroxy Moiety on Pendant of Peptidomimetic Scaffold Modulates Mu and Kappa Opioid Receptor Efficacy.

In an effort to expand the structure-activity relationship (SAR) studies of a series of mixed-efficacy opioid ligands, peptidomimetics that incorporate methoxy and hydroxy groups around a benzyl or 2-methylindanyl pendant on a tetrahydroquinoline (THQ) core of the peptidomimetics were evaluated. Compounds containing a methoxy or hydroxy moiety in the o- or m-positions increased binding affinity to the kappa opioid receptor (KOR), whereas compounds containing methoxy or hydroxy groups in the p-position decreased KOR affinity and reduced or eliminated efficacy at the mu opioid receptor (MOR). The results from a substituted 2-methylindanyl series aligned with the findings from the substituted benzyl series. Our studies culminated in the development of 8c, a mixed-efficacy MOR agonist/KOR agonist with subnanomolar binding affinity for both MOR and KOR.

µ-Opioid and 5-HT1A receptors in the dorsomedial hypothalamus interact for the regulation of panic-related defensive responses.

The dorsomedial hypothalamus (DMH) and the dorsal periaqueductal gray (DPAG) have been implicated in the genesis and regulation of panic-related defensive behaviors, such as escape. Previous results point to an interaction between serotonergic and opioidergic systems within the DPAG to inhibit escape, involving µ-opioid and 5-HT1A receptors (5-HT1AR). In the present study we explore this interaction in the DMH, using escape elicited by electrical stimulation of this area as a panic attack index. The obtained results show that intra-DMH administration of the non-selective opioid receptor antagonist naloxone (0.5 nmol) prevented the panicolytic-like effect of a local injection of serotonin (20 nmol). Pretreatment with the selective µ-opioid receptor (MOR) antagonist CTOP (1 nmol) blocked the panicolytic-like effect of the 5-HT1AR agonist 8-OHDPAT (8 nmol). Intra-DMH injection of the selective MOR agonist DAMGO (0.3 nmol) also inhibited escape behavior, and a previous injection of the 5-HT1AR antagonist WAY-100635 (0.37 nmol) counteracted this panicolytic-like effect. These results offer the first evidence that serotonergic and opioidergic systems work together within the DMH to inhibit panic-like behavior through an interaction between µ-opioid and 5-HT1A receptors, as previously described in the DPAG.

Mu opioid receptor signaling in the nucleus accumbens shell increases responsiveness of satiety-modulated lateral hypothalamus neurons.

Opioid signaling in the nucleus accumbens shell (sNAcc) has been implicated in hedonic feeding and binge eating behavior. The sNAcc projects to the lateral hypothalamus (LH), and this pathway has been suggested to modulate palatability-driven feeding behavior. In this study, we investigated the effects of sNAcc mu opioid receptor (MOR) stimulation on firing rates of LH neurons in previously sated rats. Neural firing in the LH was recorded while food-deprived rats performed an operant task to obtain sweetened Intralipid (a 4% fat emulsion containing 5% sucrose) before and after bilateral sNAcc infusion of either a MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) or a saline control solution. During sessions in which saline was infused into the sNAcc, the number of trials completed after infusion were significantly lower than the number completed before infusion, likely reflecting animals' increased satiety state. During sessions in which DAMGO was infused into the sNAcc, the decrease in the number of trials completed (comparing post- vs. pre-infusion trials) was significantly attenuated. Electrophysiological recording showed that the percentage of LH neurons showing an excitatory response due to behavioral events (cue presentation, lever press, lever retraction, and consumption) was reduced in post vs. pre-saline infusion period. However, the percentage of LH neurons showing excitatory responses to the same behavioral events was similar in pre- and post-DAMGO infusion periods. These findings suggest that MOR stimulation in sNAcc leads to an increase in stimulus-evoked excitatory signaling in LH neurons which could contribute to preventing satiety-induced decline in palatable food intake.

Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol.

BACKGROUND: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus. METHODS: Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2-6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons. RESULTS: In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol's ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons. CONCLUSIONS: These results suggest that ethanol's neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Activation of mu-opioid receptors in the ventral pallidum decreases the negative hedonic evaluation of a conditioned aversive taste in rats.

Conditioned taste aversion (CTA) causes a shift in the hedonic evaluation of a conditioned stimulus (CS) from positive to negative, and reduces the CS intake. Mu-opioid receptors (MORs) in the ventral pallidum (VP) are known to be involved in the hedonic evaluation of positive rewarding stimuli; however, their involvement in evaluation of a negative aversive stimulus is still unclear. To explore the neural mechanisms of the negative hedonic evaluation of the CS in CTA, we examined the effects of the activation of VP MORs on the behavioral responses of rats to a CS. Rats implanted with guide cannulae into the bilateral VP received a pairing of 5mM saccharin solution as a CS with an intraperitoneal injection of 0.15M lithium chloride as an unconditioned stimulus. On the test day, after microinjections of MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) into the VP, we observed the behavioral responses to the intraorally infused CS solution. The DAMGO injections caused a larger number of ingestive taste reactivity responses to the CS solution. We also measured the consumption of the CS solution in a separate group of rats, using a single-bottle test. The DAMGO injected rats drank a higher volume of the CS solution than the saline injected rats. These results indicate that the activation of MORs in the VP results in the attenuation of aversion to the CS solution, thereby inducing the larger CS intake. Therefore, it is likely that VP MORs are involved in not only positive but also negative hedonic evaluation.

Mu-Opioid (MOP) receptor mediated G-protein signaling is impaired in specific brain regions in a rat model of schizophrenia.

Schizophrenia is a complex mental health disorder. Clinical reports suggest that many patients with schizophrenia are less sensitive to pain than other individuals. Animal models do not interpret schizophrenia completely, but they can model a number of symptoms of the disease, including decreased pain sensitivities and increased pain thresholds of various modalities. Opioid receptors and endogenous opioid peptides have a substantial role in analgesia. In this biochemical study we investigated changes in the signaling properties of the mu-opioid (MOP) receptor in different brain regions, which are involved in the pain transmission, i.e., thalamus, olfactory bulb, prefrontal cortex and hippocampus. Our goal was to compare the transmembrane signaling mediated by MOP receptors in control rats and in a recently developed rat model of schizophrenia. Regulatory G-protein activation via MOP receptors were measured in [(35)S]GTPγS binding assays in the presence of a highly selective MOP receptor peptide agonist, DAMGO. It was found that the MOP receptor mediated activation of G-proteins was substantially lower in membranes prepared from the 'schizophrenic' model rats than in control animals. The potency of DAMGO to activate MOP receptor was also decreased in all brain regions studied. Taken together in our rat model of schizophrenia, MOP receptor mediated G-proteins have a reduced stimulatory activity compared to membrane preparations taken from control animals. The observed distinct changes of opioid receptor functions in different areas of the brain do not explain the augmented nociceptive threshold described in these animals.

Neural activation patterns underlying basolateral amygdala influence on intra-accumbens opioid-driven consummatory versus appetitive high-fat feeding behaviors in the rat.

The present study explored the role of the amygdala in mediating a unique pattern of feeding behavior driven by intra-accumbens (intra-Acb) opioid activation in the rat. Temporary inactivation of the basolateral amygdala (BLA), via GABAA agonist muscimol administration prevents increased consumption following intra-Acb opioid administration of the selective µ-opioid agonist D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO), yet leaves food approach behaviors intact, particularly after consumption has ended. One interpretation is that inactivation of the BLA selectively blocks neural activity underlying DAMGO-driven consummatory (consumption) but not appetitive (approach) behaviors. The present experiments take advantage of this temporal dissociation of consumption and approach behaviors to investigate their associated neural activity. Following either intra-Acb saline or DAMGO administration, with or without BLA muscimol administration, rats were given 2-hr access to a limited amount of high-fat diet. Immediately following the feeding session, rats were sacrificed and brains assayed for neural activity patterns across critical brain regions known to regulate both appetitive and consummatory feeding behaviors. The results show that intra-Acb DAMGO administration increased c-Fos activation in orexin neurons within the perifornical area of the hypothalamus and that this increase in activation is blocked by BLA muscimol inactivation. Intra-Acb DAMGO administration significantly increased c-Fos activation within dopaminergic neurons of the ventral tegmental area, compared to saline controls, and BLA inactivation had no effect on this increase. Overall, these data provide underlying circuitry that may mediate the selective influence of the BLA on driving consummatory, but not appetitive, feeding behaviors in a model of hedonically driven feeding behavior.

GABAB receptors inhibit low-voltage activated and high-voltage activated Ca(2+) channels in sensory neurons via distinct mechanisms.

Growing evidence suggests that mammalian peripheral somatosensory neurons express functional receptors for gamma-aminobutyric acid, GABAA and GABAB. Moreover, local release of GABA by pain-sensing (nociceptive) nerve fibres has also been suggested. Yet, the functional significance of GABA receptor triggering in nociceptive neurons is not fully understood. Here we used patch-clamp recordings from small-diameter cultured DRG neurons to investigate effects of GABAB receptor agonist baclofen on voltage-gated Ca(2+) currents. We found that baclofen inhibited both low-voltage activated (LVA, T-type) and high-voltage activated (HVA) Ca(2+) currents in a proportion of DRG neurons by 22% and 32% respectively; both effects were sensitive to Gi/o inhibitor pertussis toxin. Inhibitory effect of baclofen on both current types was about twice less efficacious as compared to that of the µ-opioid receptor agonist DAMGO. Surprisingly, only HVA but not LVA current modulation by baclofen was partially prevented by G protein inhibitor GDP-ß-S. In contrast, only LVA but not HVA current modulation was reversed by the application of a reducing agent dithiothreitol (DTT). Inhibition of T-type Ca(2+) current by baclofen and the recovery of such inhibition by DTT were successfully reconstituted in the expression system. Our data suggest that inhibition of LVA current in DRG neurons by baclofen is partially mediated by an unconventional signaling pathway that involves a redox mechanism. These findings reinforce the idea of targeting peripheral GABA receptors for pain relief.

Direct inhibition of hypothalamic proopiomelanocortin neurons by dynorphin A is mediated by the µ-opioid receptor.

It has recently been shown that dynorphin A (Dyn A), an endogenous agonist of the κ-opioid receptor (KOR), directly inhibits proopiomelanocortin (POMC) neurons in the hypothalamus through activation of G-protein-coupled inwardly rectifying K(+) channels (GIRKs). This effect has been proposed to be mediated by the putative κ2-opioid receptor (KOR-2), and has been suggested as a possible mechanism for the orexigenic actions of KOR agonists. Using whole-cell voltage clamp recordings in brain slice preparations, the present study demonstrates that Dyn A (1 or 5 µm) induces an outward current in POMC neurons that is reversed by the highly selective µ-opioid receptor (MOR) antagonist CTAP and is absent in mice lacking MORs. Additionally, the KOR-2-selective agonist GR89696 binds MORs on POMC neurons but fails to induce an outward current. Similar to Dyn A, the KOR-selective antagonist nor-binaltorphimine (nor-BNI) lacked specificity when used at sufficiently high concentrations. Maximal concentrations of the MOR-selective agonist DAMGO induced outward currents in POMC neurons that were completely reversed by a relatively high concentration of nor-BNI. Experiments using a half-maximal concentration of DAMGO demonstrate that nor-BNI must be used at concentrations <100 nm to avoid non-specific actions of the antagonist at MORs located on POMC neurons. These data suggest that direct inhibition of POMC neurons by Dyn A is mediated through the MOR, not the KOR-2, which is consistent with previous studies demonstrating that Dyn A can act at the µ-opioid receptor (MOR) when present in high concentrations.

Behavioral and neurochemical characterization of kratom (Mitragyna speciosa) extract.

OBJECTIVE: Mitragyna speciosa and its extracts are named kratom (dried leaves, extract). It contains several alkaloids and is used in traditional medicine to alleviate musculoskeletal pain, hypertension, coughing, diarrhea, and as an opiate substitute for addicts. Abuse and addiction to kratom is described, and kratom has attracted increasing interest in Western countries. Individual effects of kratom on opioidergic, adrenergic, serotonergic, and dopaminergic receptors are known, but not all of the effects have been explained. Pharmacokinetic and pharmacodynamic data are needed. METHODS: The effects of kratom extract on mice behavior were investigated following oral (po), intraperitoneal (ip), and intracerebroventricular (icv) application. Receptor-binding studies were performed. RESULTS: In µ opioid receptor knockout mice (-/-) and wild type (+/+) animals, the extract reduced locomotor activity after ip and low po doses in +/+ animals, but not after icv administration. The ip effect was counteracted by 0.3 mg/kg of apomorphine sc, suggesting dopaminergic presynaptic activity. An analgesic effect was only found in -/- mice after icv application. Norbinaltorphimine abolished the analgesic effect, but not the inhibitory effect, on locomotor activity, indicating that the analgesic effect is mediated via κ opioid receptors. Oral doses, which did not diminish locomotor activity, impaired the acquisition of shuttle box avoidance learning. There was no effect on consolidation. Binding studies showed affinity of kratom to µ, δ, and κ opioid receptors and to dopamine D1 receptors. CONCLUSIONS: The results obtained in drug-naïve mice demonstrate weak behavioral effects mediated via µ and κ opioid receptors.

Mu-opioid stimulation in rat prefrontal cortex engages hypothalamic orexin/hypocretin-containing neurons, and reveals dissociable roles of nucleus accumbens and hypothalamus in cortically driven feeding.

Mu-opioid receptor (µOR) stimulation within ventral medial prefrontal cortex (vmPFC) induces feeding and hyperactivity, resulting possibly from recruitment of glutamate signaling in multiple vmPFC projection targets. We tested this hypothesis by analyzing Fos expression in vmPFC terminal fields after intra-vmPFC µOR stimulation, and by examining of the impact of glutamate receptor blockade in two feeding-related targets of vmPFC, the lateral-perifornical hypothalamic area (LH-PeF) and nucleus accumbens shell (Acb shell), upon behavioral effects elicited by intra-vmPFC µOR stimulation in rats. Intra-vmPFC infusion of the µOR agonist, DAMGO, provoked Fos expression in the dorsomedial sector of tuberal hypothalamus (including the perifornical area) and increased the percentage of Fos-expressing hypocretin/orexin-immunoreactive neurons in these zones. NMDA receptor blockade in the LH-PeF nearly eliminated intra-vmPFC DAMGO-induced food intake without altering DAMGO-induced hyperactivity. In contrast, blocking AMPA-type glutamate receptors within the Acb shell (the feeding-relevant subtype in this structure) antagonized intra-vmPFC DAMGO-induced hyperlocomotion but enhanced food intake. Intra-vmPFC DAMGO also elevated the breakpoint for sucrose-reinforced progressive-ratio responding; this effect was significantly enhanced by concomitant AMPA blockade in the Acb shell. Conversely, intra-Acb shell AMPA stimulation reduced breakpoint and increased nonspecific responding on the inactive lever. These data indicate intra-vmPFC µOR signaling jointly modulates appetitive motivation and generalized motoric activation through functionally dissociable vmPFC projection targets. These findings may shed light on the circuitry underlying disorganized appetitive responses in psychopathology; e.g., binge eating and opiate or alcohol abuse, disorders in which µORs and aberrant cortical activation have been implicated.

Opioids in the perifornical lateral hypothalamus suppress ethanol drinking.

The opioid system is known to enhance motivated behaviors, including ethanol drinking and food ingestion, by acting in various reward-related brain regions, such as the nucleus accumbens, ventral tegmental area and medial hypothalamus. There is indirect evidence, however, suggesting that opioid peptides may act differently in the perifornical lateral hypothalamus (PF/LH), causing a suppression of consummatory behavior. Using brain-cannulated Sprague-Dawley rats trained to voluntarily drink 7% ethanol, the present study tested the hypothesis that opioids in the PF/LH can reduce the consumption of ethanol, with animals receiving PF/LH injections of the δ-opioid receptor agonist D-Ala2-met-enkephalinamide (DALA), the µ-receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), the κ-receptor agonist (±)-trans-U-50,488 methanesulfonate (U-50,488H), or the general opioid antagonist methylated naloxone (m-naloxone). The consumption of ethanol, lab chow, and water was monitored for 4 h after injection. The results showed that the three opioid receptor agonists injected into the PF/LH specifically and significantly reduced ethanol intake, while causing little change in chow or water intake, and the opposite effect, enhanced ethanol intake, was observed with the opioid antagonist. Of the three opioid agonists, the δ-agonist appears to produce the most consistent and long-lasting suppression of consumption. This effect was not observed with injections 2 mm dorsal to this area, focusing attention on the PF/LH as the main site of action. These results suggest that the opioid peptides have a specific role in the PF/LH of reducing ethanol drinking, which is distinct from their more commonly observed appetitive actions in other brain areas. The additional finding, that m-naloxone in the PF/LH stimulates ethanol intake in contrast to its generally suppressive effect in other regions, focuses attention on this hypothalamic area and its distinctive role in contributing to the variable effects sometimes observed with opioid antagonist therapy for alcoholism.

Regulatory mechanism of body temperature in the central nervous system during the maintenance phase of hibernation in Syrian hamsters: involvement of ß-endorphin.

We have shown previously that intracerebroventricular (icv) injection of naloxone (a non-selective opioid receptor antagonist) or naloxonazine (a selective µ1-opioid receptor antagonist) at the maintenance phase of hibernation arouses Syrian hamsters from hibernation. This study was designed to clarify the role of ß-endorphin (an endogenous µ-opioid receptor ligand) on regulation of body temperature (T(b)) during the maintenance phase of hibernation. The number of c-Fos-positive cells and ß-endorphin-like immunoreactivity increased in the arcuate nucleus (ARC) after hibernation onset. In contrast, endomorphin-1 (an endogenous µ-opioid receptor ligand)-like immunoreactivity observed on the anterior hypothalamus decreased after hibernation onset. In addition, hibernation was interrupted by icv injection of anti-ß-endorphin antiserum at the maintenance phase of hibernation. The mRNA expression level of proopiomelanocortin (a precursor of ß-endorphin) on ARC did not change throughout the hibernation phase. However, the mRNA expression level of prohormone convertase-1 increased after hibernation onset. [D-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO, a selective µ-opioid receptor agonist) microinjection into the dorsomedial hypothalamus (DMH) elicited the most marked T(b) decrease than other sites such as the preoptic area (PO), anterior hypothalamus (AH), lateral hypothalamus (LH), ventromedial hypothalamus and posterior hypothalamus (PH). However, microinjected DAMGO into the medial septum indicated negligible changes in T(b). These results suggest that ß-endorphin which synthesizes in ARC neurons regulates T(b) during the maintenance phase of hibernation by activating µ-opioid receptors in PO, AH, VMH, DMH and PH.

Chronic neuropathic pain in mice reduces µ-opioid receptor-mediated G-protein activity in the thalamus.

Neuropathic pain is a debilitating condition that is often difficult to treat using conventional pharmacological interventions and the exact mechanisms involved in the establishment and maintenance of this type of chronic pain have yet to be fully elucidated. The present studies examined the effect of chronic nerve injury on µ-opioid receptors and receptor-mediated G-protein activity within the supraspinal brain regions involved in pain processing of mice. Chronic constriction injury (CCI) reduced paw withdrawal latency, which was maximal at 10 days post-injury. [d-Ala2,(N-Me)Phe4,Gly5-OH] enkephalin (DAMGO)-stimulated [(35)S]GTPγS binding was then conducted at this time point in membranes prepared from the rostral ACC (rACC), thalamus and periaqueductal grey (PAG) of CCI and sham-operated mice. Results showed reduced DAMGO-stimulated [(35)S]GTPγS binding in the thalamus and PAG of CCI mice, with no change in the rACC. In thalamus, this reduction was due to decreased maximal stimulation by DAMGO, with no difference in EC(50) values. In PAG, however, DAMGO E(max) values did not significantly differ between groups, possibly due to the small magnitude of the main effect. [(3)H]Naloxone binding in membranes of the thalamus showed no significant differences in B(max) values between CCI and sham-operated mice, indicating that the difference in G-protein activation did not result from differences in µ-opioid receptor levels. These results suggest that CCI induced a region-specific adaptation of µ-opioid receptor-mediated G-protein activity, with apparent desensitization of the µ-opioid receptor in the thalamus and PAG and could have implications for treatment of neuropathic pain.

Differential expression and sensitivity of presynaptic and postsynaptic opioid receptors regulating hypothalamic proopiomelanocortin neurons.

Hypothalamic proopiomelanocortin (POMC) neurons release the endogenous opioid beta-endorphin and POMC neuron activity is inhibited by opioids, leading to the proposal that beta-endorphin acts to provide feedback inhibition. However, both intrinsic properties and synaptic inputs contribute to the regulation of POMC neurons such that attributing an autoregulatory role to opioids must include consideration of opioid receptor localization and sensitivity at both presynaptic and postsynaptic sites. In the present study, whole-cell recordings were made in POMC cells in mouse brain slices and the presynaptic and postsynaptic regulation of POMC neurons was examined using selective agonists for mu, kappa, and delta opioid receptors. Activation of mu, but not kappa or delta, receptors induced a direct postsynaptic outward current. Agonists for each of the receptors inhibited the frequency of spontaneous IPSCs. Mu and kappa, but not delta, agonists reduced the amplitude of evoked IPSCs and appeared to colocalize in a significant portion of GABAergic terminals onto POMC neurons. The presynaptic inhibition caused by the mu agonist DAMGO had an EC(50) of 80 nM, whereas the EC(50) was 350 nM when measuring the postsynaptic outward current. This differential sensitivity adds an unexpected component of opioid-dependent feedback regulation, where low levels of opioid receptor activation would likely disinhibit POMC neuron activity and higher concentrations would result in an overall inhibition. The results may help explain why it has been difficult to clearly discern the role that opioids play in the regulation of food intake and other processes involving POMC neurons.

Preference for a high fat diet, but not hyperphagia following activation of mu opioid receptors is blocked in AgRP knockout mice.

Activation of mu opioid receptors (MOR) makes animals hyperphagic and selectively increases their preference for a high fat diet independent of their dietary preference. The orexigenic peptide Agouti Related Peptide (AgRP) also produces hyperphagia and increased the preference for a high fat diet. In this paper, we tested the hypothesis that the effect of MOR on feeding behavior will be attenuated in the absence of the orexigenic peptide AgRP. Immunohistochemical studies demonstrated that MOR are co-localized on AgRP neurons located in the arcuate nucleus. This finding is consistent with a role of MOR in mediating the release of AgRP. Our data also demonstrated that the wild-type (FVB) animals preferred a diet high in fat whereas the AgRP knockout (AgRP KO) mice did not. mRNA expression of MOR in the hypothalamus was not significantly different between AgRP KO mice and their wild-type control. In a dose-response experiment, the low dose (0.025 microg) of a MOR agonist, DAMGO, increased cumulative food intake in wild-type and AgRP KO mice. The low and middle (0.25 microg) dose of DAMGO significantly increased the amount of high fat diet eaten by the wild-type animals, but did not significantly change the amount of high fat diet eaten by the AgRP KO mice. The highest dose of DAMGO (2.5 microg) reduced food intake in the control and AgRP KO mice, probably due to somnolence. These data demonstrate that the increased preference for a high fat diet after stimulation of MOR is attenuated in the absence of AgRP, but the increase in food intake (i.e. hyperphagia) is not.

The effects of daikenchuto (DKT) on propulsive motility in the colon.

BACKGROUND: The purpose of this study is to examine the use of daikenchuto (DKT), a traditional Japanese medicine, as a potential treatment for opiate-induced slowing of intestinal transit in an isolated guinea pig colon model of motility. METHODS: Isolated segments of distal guinea pig colon were mounted in a perfusion chamber and imaged with a digital video camera interfaced with a computer. Fecal pellets were inserted into the oral end of the colonic segment and the rates of propulsive motility over a 3 to 4 cm segment of colon were determined in the presence and absence of test compounds. In addition, intracellular recordings were obtained from intact circular muscle, and the responsiveness of inhibitory and excitatory junction potentials to DKT was evaluated. RESULTS: The addition of D-Ala2, N-Me-Phe4, Gly-ol5 (DAMGO), a selective µ-receptor agonist, caused a concentration dependent decrease in colon motility. Naloxone did not affect basal activity, but partially restored motility in the DAMGO treated preparations. DKT (1 × 10(-4)-3 × 10(-4)g/mL) also reversed the inhibitory effect of DAMGO treated colon in a concentration dependent manner. At higher concentrations (1 × 10(-3)-3 × 10(-3)g/mL), however, this effect was lost. Motility slowed even further when naloxone and DKT were combined with noticeable disruptions in spatiotemporal patterns. Interestingly, when added alone, DKT resulted in reverse peristalsis of the pellet. In electrophysiologic studies DKT inhibited both excitatory and inhibitory junction potentials. CONCLUSIONS: DKT appears to be as effective as naloxone in restoring motility in DAMGO treated colon. These two agents, however, do not appear to have an additive effect. When used on untreated colon segments, DKT appears to cause disruptions in the intrinsic reflex circuit of the gut resulting in a disruption of neuromuscular communication.