Effects of sex and hydration status on kappa opioid receptor-mediated diuresis in rats.

Understanding the function of the kappa opioid receptor (KOP) is crucial for the development of novel therapeutic interventions that target KOP for the treatment of pain, stress-related disorders and other indications. Activation of KOP produces diuretic effects in rodents and man. Sex is a vital factor to consider when assessing drug response in pre-clinical and clinical studies. In this study, the diuretic effect of the KOP agonist, U50488 (1-10 mg/kg), was investigated in both adult female and male Wistar rats that were either normally hydrated or water-loaded. The KOP antagonist norbinaltorphimine (norBNI, 10 mg/kg) was administered 24 h prior to U50488 to confirm the involvement of KOP. U50488 elicited a significant diuretic response at doses ≥ 3 mg/kg in both female and male rats independent of hydration status. U50488 diuretic effects were inhibited by norBNI pre-administration. Water-loading reduced data variability for urine volume in males, but not in females, compared with normally hydrated rats. Sex differences were also evident in U50488 eliciting a significant increase in sodium and potassium ion excretion only in males. This may suggest different mechanisms of U50488 diuretic action in males where renal excretion mechanisms are directly affected more than in females.

Sex Differences in Brain Region-Specific Activation of c-Fos following Kappa Opioid Receptor Stimulation or Acute Stress in Mice.

Kappa opioid receptors (KOPr) are involved in the response to stress. KOPr are also targets for the treatment of stress-related psychiatric disorders including depression, anxiety, and addiction although effects of KOPr are often sex-dependent. Here we investigated c-Fos expression in a range of brain regions in male and female mice following an acute stressor, and a single injection of KOPr agonist. Using adult C57BL/6 c-Fos-GFP transgenic mice and quantitative fluorescence microscopy, we identified brain regions activated in response to a challenge with the KOPr agonist U50,488 (20 mg/kg) or an acute stress (15 min forced swim stress, FSS). In male mice, U50,488 increased expression of c-Fos in the prelimbic area of the prefrontal cortex (PFCx), nucleus accumbens (NAcc), and basolateral nuclei of the amygdala (BLA). In contrast, in female mice U50,488 only activated the BLA but not the PFCx or the NAcc. FSS increased activation of PFCx, NAcc, and BLA in males while there was no activation of the PFCx in female mice. In both sexes, the KOPr antagonist norBNI significantly blocked U50,488-induced, but not stress-induced activation of brain regions. In separate experiments, activated cells were confirmed as non-GABAergic neurons in the PFCx and NAcc. Together these data demonstrate sex differences in activation of brain regions that are key components of the 'reward' circuitry. These differential responses may contribute to sex differences in stress-related psychiatric disorders and in the treatment of depression, anxiety, and addiction.

Simultaneous Transdermal Delivery of Buprenorphine Hydrochloride and Naltrexone Hydrochloride by Iontophoresis.

The opioids buprenorphine hydrochloride (BUP) and naltrexone hydrochloride (NTX) show promise as a combination treatment for addiction, but no means of delivering the two compounds in one medicine currently exist. In this paper, we report sufficient input rates of both these drugs from one iontophoretic transdermal drug delivery system. Experiments were performed using dermatomed pig skin mounted in glass side-bi-side cells. BUP and NTX were iontophoretically delivered together from the anode using direct constant current from Ag/AgCl electrodes. The transdermal drug fluxes and the masses of drugs in both the stratum corneum and the underlying epidermis/dermis were measured. The apparent electroosmotic flow was quantified using a neutral marker (acetaminophen). The effects of donor composition (drug concentration/molar fraction and pH), current density and profile, and the choice of receptor solution were assessed. Iontophoresis dramatically increased the flux of both drugs compared to passive control values. Target fluxes (calculated from literature clearance values and required therapeutic plasma concentrations) were greatly exceeded for NTX and were met for BUP. The latter accumulated in the skin and suppressed electroosmotic flow, inhibiting both its own flux and that of NTX. NTX, in turn, negatively influenced the flux of BUP via co-ion competition. Lowering current density by increasing the delivery area resulted in increased electroosmotic flow but did not significantly affect current-normalized drug fluxes. Delivering the drugs from both electrodes and reversing the polarity for every 2 h did not increase the flux of either compound. In summary, during iontophoresis, BUP and NTX inhibited each other's flux by two distinct mechanisms. While the more complex behavior of BUP complicates the optimization of this drug combination, iontophoresis nevertheless appears to be a feasible approach for the controlled codelivery of NTX and BUP through the skin.

Inhibition of alpha7 nicotinic receptors in the ventral hippocampus selectively attenuates reinstatement of morphine-conditioned place preference and associated changes in AMPA receptor binding.

Recurrent relapse is a major problem in treating opiate addiction. Pavlovian conditioning plays a role in recurrent relapse whereby exposure to cues learned during drug intake can precipitate relapse to drug taking. α7 nicotinic acetylcholine receptors (nAChRs) have been implicated in attentional aspects of cognition and mechanisms of learning and memory. In this study we have investigated the role of α7 nAChRs in morphine-conditioned place preference (morphine-CPP). CPP provides a model of associative learning that is pertinent to associative aspects of drug dependence. The α7 nAChR antagonist methyllycaconitine (MLA; 4 mg/kg s.c.) had no effect on the acquisition, maintenance, reconsolidation or extinction of morphine-CPP but selectively attenuated morphine-primed reinstatement of CPP, in both mice and rats. Reinstatement of morphine-CPP in mice was accompanied by a selective increase in [3 H]-AMPA binding (but not in [3 H]-MK801 binding) in the ventral hippocampus that was prevented by prior treatment with MLA. Administration of MLA (6.7 µg) directly into the ventral hippocampus of rats prior to a systemic priming dose of morphine abolished reinstatement of morphine-CPP, whereas MLA delivered into the dorsal hippocampus or prefrontal cortex was without effect. These results suggest that α7 nAChRs in the ventral hippocampus play a specific role in the retrieval of associative drug memories following a period of extinction, making them potential targets for the prevention of relapse.

Antidepressant-like effects of BU10119, a novel buprenorphine analogue with mixed κ/µ receptor antagonist properties, in mice.

BACKGROUND AND PURPOSE: The κ receptor antagonists have potential for treating neuropsychiatric disorders. We have investigated the in vivo pharmacology of a novel buprenorphine analogue, BU10119, for the first time. EXPERIMENTAL APPROACH: To determine the opioid pharmacology of BU10119 (0.3-3 mg·kg-1 , i.p.) in vivo, the warm-water tail-withdrawal assay was applied in adult male CD1 mice. A range of behavioural paradigms was used to investigate the locomotor effects, rewarding properties and antidepressant or anxiolytic potential of BU10119. Additional groups of mice were exposed to a single (1 × 2 h) or repeated restraint stress (3× daily 2 h) to determine the ability of BU10119 to block stress-induced analgesia. KEY RESULTS: BU10119 alone was without any antinociceptive activity. BU10119 (1 mg·kg-1 ) was able to block U50,488, buprenorphine and morphine-induced antinociception. The κ antagonist effects of BU10119 in the tail-withdrawal assay reversed between 24 and 48 h. BU10119 was without significant locomotor or rewarding effects. BU10119 (1 mg·kg-1 ) significantly reduced the latency to feed in the novelty-induced hypophagia task and reduced immobility time in the forced swim test, compared to saline-treated animals. There were no significant effects of BU10119 in either the elevated plus maze or the light-dark box. Both acute and repeated restraint stress-induced analgesia were blocked by pretreatment with BU10119 (1 mg·kg-1 ). Parallel stress-induced increases in plasma corticosterone were not affected. CONCLUSIONS AND IMPLICATIONS: BU10119 is a mixed κ/µ receptor antagonist with relatively short-duration κ antagonist activity. Based on these preclinical data, BU10119 has therapeutic potential for the treatment of depression and other stress-induced conditions. LINKED ARTICLES: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

Combined administration of buprenorphine and naltrexone produces antidepressant-like effects in mice.

Opiates have been used historically for the treatment of depression. Renewed interest in the use of opiates as antidepressants has focused on the development of kappa opioid receptor (κ-receptor) antagonists. Buprenorphine acts as a partial µ-opioid receptor agonist and a κ-receptor antagonist. By combining buprenorphine with the opioid antagonist naltrexone, the activation of µ-opioid receptors will be reduced and the κ-antagonist properties enhanced. We have established that a combination dose of buprenorphine (1 mg/kg) with naltrexone (1 mg/kg) functions as a short-acting κ-antagonist in the mouse tail withdrawal test. Furthermore, this dose combination is neither rewarding nor aversive in the conditioned place preference paradigm, and is without significant locomotor effects. We have shown for the first time that systemic co-administration of buprenorphine (1 mg/kg) with naltrexone (1 mg/kg) in CD-1 mice produced an antidepressant-like response in behaviours in both the forced swim test and novelty induced hypophagia task. Behaviours in the elevated plus maze and light dark box were not significantly altered by treatment with buprenorphine alone, or in combination with naltrexone. We propose that the combination of buprenorphine with naltrexone represents a novel, and potentially a readily translatable approach, to the treatment of depression.

A non-rewarding, non-aversive buprenorphine/naltrexone combination attenuates drug-primed reinstatement to cocaine and morphine in rats in a conditioned place preference paradigm.

Concurrent use of cocaine and heroin is a major public health issue with no effective relapse prevention treatment currently available. To this purpose, a combination of buprenorphine and naltrexone, a mixed very-low efficacy mu-opioid receptor agonist/kappa-opioid receptor antagonist/nociceptin receptor agonist, was investigated. The tail-withdrawal and the conditioned place preference (CPP) assays in adult Sprague Dawley rats were used to show that naltrexone dose-dependently blocked the mu-opioid receptor agonism of buprenorphine. Furthermore, in the CPP assay, a combination of 0.3 mg/kg buprenorphine and 3.0 mg/kg naltrexone was aversive. A combination of 0.3 mg/kg buprenorphine and 1.0 mg/kg naltrexone was neither rewarding nor aversive, but still possessed mu-opioid receptor antagonist properties. In the CPP extinction and reinstatement method, a combination of 0.3 mg/kg buprenorphine and 1.0 mg/kg naltrexone completely blocked drug-primed reinstatement in cocaine-conditioned rats (conditioned with 3 mg/kg cocaine, drug prime was 3 mg/kg cocaine) and attenuated drug-primed reinstatement in morphine-conditioned rats (conditioned with 5 mg/kg morphine, drug prime was 1.25 mg/kg morphine). These data add to the growing evidence that a buprenorphine/naltrexone combination may be protective against relapse in a polydrug abuse situation.

Endomorphin-2: a biased agonist at the µ-opioid receptor.

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the µ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

Glutamate-induced post-activation inhibition of locus coeruleus neurons is mediated by AMPA/kainate receptors and sodium-dependent potassium currents.

BACKGROUND AND PURPOSE: Locus coeruleus (LC) neurons respond to sensory stimuli with a glutamate-triggered burst of spikes followed by an inhibition. The aim of our work was to characterize the inhibitory effect of glutamate in the LC. EXPERIMENTAL APPROACH: Single-unit extracellular and patch-clamp recordings were performed to examine glutamate responses in rat brain slices containing the LC. KEY RESULTS: Glutamate caused an initial activation followed by a late post-activation inhibition (PAI). Both effects were blocked by an AMPA/kainate receptor antagonist but not by NMDA or metabotropic glutamate receptor antagonists. All glutamate receptor agonists were able to activate neurons, but only AMPA and quisqualate caused inhibition. In neurons clamped at -60 mV, glutamate and AMPA induced inward, followed by outward, currents, with the latter reversing polarity at -110 mV. Glutamate-induced PAI was not modified by alpha(2)-adrenoceptor, micro opioid, A(1) adenosine and GABA(A/B) receptor antagonists or Ca(2+)-dependent release blockade, but it was reduced by raising the extracellular K(+) concentration. Glutamate-induced PAI was not affected by several potassium channel, Na(+)/K(+) pump, PKC and neuronal NO synthase inhibitors or lowering the extracellular Ca(2+) concentration. The Na(+)-activated K channel opener bithionol concentration-dependently potentiated glutamate-induced PAI, whereas partial (80%) Na(+) replacement reduced glutamate- and AMPA-induced PAI. Finally, reverse transcription polymerase chain reaction assays showed the presence of mRNA for the Ca(2+)-impermeable GluR2 subunit in the LC. CONCLUSIONS AND IMPLICATIONS: Glutamate induces a late PAI in the LC, which may be mediated by a novel postsynaptic Na(+)-dependent K(+) current triggered by AMPA/kainate receptors.

Galpha(i2) inhibition of adenylate cyclase regulates presynaptic activity and unmasks cGMP-dependent long-term depression at Schaffer collateral-CA1 hippocampal synapses.

Cyclic AMP signaling plays a central role in regulating activity at a number of synapses in the brain. We showed previously that pairing activation of receptors that inhibit adenylate cyclase (AC) and reduce the concentration of cyclic AMP, with elevation of the concentration of cyclic GMP is sufficient to elicit a presynaptically expressed form of LTD at Schaffer collateral-CA1 synapses in the hippocampus. To directly test the role of AC inhibition and G-protein signaling in LTD at these synapses, we utilized transgenic mice that express a mutant, constitutively active inhibitory G protein, Galpha(i2), in principal neurons of the forebrain. Transgene expression of Galpha(i2) markedly enhanced LTD and impaired late-phase LTP at Schaffer collateral synapses, with no associated differences in input/output relations, paired-pulse facilitation, or NMDA receptor-gated conductances. When paired with application of a type V phosphodiesterase inhibitor to elevate the concentration of intracellular cyclic GMP, constitutively active Galpha(i2) expression converted the transient depression normally caused by this treatment to an LTD that persisted after the drug was washed out. Moreover, this effect could be mimicked in control slices by pairing type V phosphodiesterase inhibitor application with application of a PKA inhibitor. Electrophysiological recordings of spontaneous excitatory postsynaptic currents and two-photon visualization of vesicular release using FM1-43 revealed that constitutively active Galpha(i2) tonically reduced basal release probability from the rapidly recycling vesicle pool of Schaffer collateral terminals. Our findings support the hypothesis that inhibitory G-protein signaling acts presynaptically to regulate release, and, when paired with elevations in the concentration of cyclic GMP, converts a transient cyclic GMP-induced depression into a long-lasting decrease in release.

mGluR2 acts through inhibitory Galpha subunits to regulate transmission and long-term plasticity at hippocampal mossy fiber-CA3 synapses.

Presynaptic inhibitory G protein-coupled receptors play a critical role in regulating transmission at a number of synapses in the central and peripheral nervous system. We generated transgenic mice that express a constitutively active form of an inhibitory Galpha subunit to examine the molecular mechanisms underlying the actions of one such receptor, metabotropic glutamate receptor (mGluR) 2, at mossy fiber-CA3 synapses in the hippocampus. mGluR2 participates in at least three types of mossy fiber synaptic plasticity, (i) transient suppression of synaptic transmission, (ii) long-term depression (LTD), and (iii) inhibition of long-term potentiation (LTP), and we find that inhibitory Galpha signaling is sufficient to account for the actions of mGluR2 in each. The fact that constitutively active Galphai2 occludes the transient suppression of synaptic transmission by mGluR2, while enhancing LTD, suggests further that these two forms of plasticity are expressed via different mechanisms. In addition, the LTP deficit observed in constitutively active Galphai2-expressing mice suggests that mGluR2 activation may serve as a metaplastic switch to permit the induction of LTD by inhibiting LTP.

Protein kinase C activation enhances morphine-induced rapid desensitization of mu-opioid receptors in mature rat locus ceruleus neurons.

Recent studies have shown that morphine, in contrast to other agonists at the mu-opioid receptor, causes very little rapid mu-opioid receptor desensitization or internalization in adult rat mammalian neurons, raising important questions about how morphine tolerance is induced. Here we show that morphine can indeed cause marked rapid desensitization of mu-opioid receptors in mature rat locus ceruleus neurons when protein kinase C is also activated. Thus, activation of Gq-coupled M3 muscarinic receptors or application of a phorbol ester enhanced the desensitization of the mu-opioid receptor-evoked potassium current in rat locus ceruleus neurons. The enhancement of desensitization was reversible by the protein kinase C inhibitors chelerythrine and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X) and resulted from an effect at the level of the mu-opioid receptor rather than the potassium channel. This is the first finding that morphine can induce rapid mu-opioid receptor desensitization in adult rat neurons, and because reduced protein kinase C activity in vivo attenuates morphine tolerance, we propose that G-protein coupled receptor cross-talk and the level of protein kinase C activity may play critical roles in the desensitization of the mu-opioid receptor and could underlie the development of morphine tolerance.

Mu-opioid receptor desensitization in mature rat neurons: lack of interaction between DAMGO and morphine.

Mu-opioid receptors (MORs) exhibit rapid desensitization and internalization during exposure to various opioid agonists. In some studies, however, morphine has been observed to produce little MOR desensitization or internalization. We examined desensitization in mature rat locus ceruleus (LC) neurons and confirmed that morphine is a very poor desensitizing agent, whereas [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), a high-efficacy agonist, and methadone, an agonist we observed to be of equivalent efficacy to morphine, produced profound rapid desensitization. Similarly, by measuring plasma membrane receptor levels in HEK293 cells stably expressing T7-epitope-tagged rat MOR1 at near physiological levels (HEK293-MOR1 cells), DAMGO and methadone but not morphine caused rapid MOR internalization. It has been reported that a low concentration of DAMGO, coapplied with morphine, caused morphine to induce MOR internalization. We examined whether this interaction occurred in mature mammalian neurons at the level of receptor desensitization. Coapplication of low concentrations of DAMGO did not increase morphine-induced desensitization in LC neurons but caused a lesser degree of desensitization than DAMGO alone. We also failed to observe an enhancement by DAMGO of morphine-induced desensitization in the electrically stimulated guinea pig ileum myenteric plexus-longitudinal muscle preparation. In HEK293-MOR1 cells, low concentrations of DAMGO did not convert morphine into a receptor-internalizing agent. The data presented here fail to support the theory that low concentrations of DAMGO can increase morphine-induced MOR desensitization or internalization.

Long-term depression of presynaptic release from the readily releasable vesicle pool induced by NMDA receptor-dependent retrograde nitric oxide.

Postsynaptic alterations are currently believed to be able to fully account for NMDA-receptor-dependent long-term depression (LTD) and long-term potentiation of synaptic strength, although there is also evidence supporting changes in presynaptic release. Using dualphoton laser scan microscopy of N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1-43) to directly visualize presynaptic vesicular release at Schaffer collateral-CA1 excitatory synapses in hippocampal slices, we demonstrate reduced vesicular release associated with LTD. Selective loading, by hypertonic shock, of the readily releasable vesicle pool (RRP) showed that LTD of release is a selective modification of release from the RRP. Presynaptic LTD of RRP release required activation of NMDA receptors, production and extracellular diffusion of the intercellular messenger NO, and activation of cGMP-dependent protein kinase.

Pairing elevation of [cyclic GMP] with inhibition of PKA produces long-term depression of glutamate release from isolated rat hippocampal presynaptic terminals.

Data suggest both presynaptic and postsynaptic changes contribute to activity-dependent long-term synaptic plasticity. We have shown that pairing elevation of intracellular [cyclic GMP], using the type V phosphodiesterase inhibitor zaprinast, with inhibition of cyclic AMP-dependent protein kinase (PKA), is sufficient to elicit chemical long-term depression (CLTD) of synaptic transmission at Schaffer collateral-CA1 and mossy fibre-CA3 synapses in rat hippocampus. CLTD does not require synaptic activity, and selective postsynaptic drug injections do not affect it, suggesting it is presynaptically induced and expressed. To directly evaluate this hypothesis, we tested whether CLTD of transmitter release can be expressed in isolated presynaptic nerve terminals. Presynaptic nerve terminals (synaptosomes) were isolated from rat hippocampi by Percoll density gradient centrifugation. Synaptosomes were loaded with [3H]glutamate, and basal and depolarisation-induced release of [3H]glutamate measured in control medium versus medium containing zaprinast (20 microm) plus or minus the PKA inhibitor H-89 (10 microm). Zaprinast produced a significant decrease in basal [3H]glutamate release. However, only combining zaprinast with H-89 significantly depressed K+-evoked [3H]glutamate release. After a 20-min drug washout, basal release returned to normal in all conditions, but K+-evoked [3H]glutamate release was persistently reduced only by the combination of zaprinast plus H-89. Long-term reduction of [3H]glutamate release from synaptosomes was completely prevented by the PKG inhibitor KT5823 (5 microm). These data demonstrate the existence of a presynaptic, cyclic GMP-PKG dependent cascade capable of expressing LTD of glutamate release from isolated hippocampal nerve terminals.