Role of Kappa-Opioid Receptors in Stress-Induced Behaviors

The development of anxiety and mood disorders often coincides with exposure to stress. Accumulating evidence indicates that both corticotropin-releasing factor (CRF) and dynorphin, the endogenous ligand for the kappa-opioid receptor (KOR), can mediate the effects of stress. My dissertation research utilized laboratory animals to investigate the role of KORs in stress-induced increases in the acoustic startle response, a metric often used to study stress effects in humans. Using wild-type mice, I first demonstrated that systemic administration of a KOR antagonist produced an anxiolytic-like effect on acoustic startle following central (intracerebroventricular) infusion of CRF. Immunohistochemical analysis revealed that KOR blockade decreased c-Fos cell counts in the dentate gyrus of the hippocampus in both vehicle- and CRF-treated mice, and reduced CRF-induced increases in the ventral tegmental area (VTA). Within the VTA, reductions were predominantly in dopaminergic neurons. KOR antagonist pretreatment also produced anxiolytic-like effects on footshock-potentiated startle, a model that quantifies context-specific fear conditioning. To complement the antagonist studies, we developed constitutive knockout mice that lack KORs throughout the brain (KOR-/-), and conditional KOs that lack KORs only within dopaminergic neurons (DAT-KORlox/lox). Initial characterization demonstrated that these two mutant lines did not differ from controls in hearing, vision, weight gain, and locomotor activity. KOR-/- mice were similar to controls in unconditioned anxiety-like behavior, but DAT-KORlox/lox mice displayed nominal decreases in anxiety-like behavior in the open field and light/dark box. Unexpectedly, KOR ablation did not affect CRF-induced increases in startle in either mutant line. Importantly, however, KOR antagonist treatment did not alter CRF-induced increases in startle in KOR-/- mice, suggesting that KOR antagonist effects in wild-type mice are due to blockade of KORs. These findings raise the possibility that differences in KOR antagonist and KOR-/- studies may be related to brief KOR blockade during adulthood versus a lack of KORs during the entire lifespan. In the footshock-potentiated startle paradigm, KOR-/- mice were comparable to littermate controls, whereas DAT-KORlox/lox mice showed attenuated effects of footshock. My findings confirm a role for KORs in fear and anxiety-like behavior in rodents, and implicate KORs expressed on dopaminergic neurons in modulating important aspects of stress-related behavior

Long-acting κ opioid antagonists nor-BNI, GNTI and JDTic: pharmacokinetics in mice and lipophilicity

Background: Nor-BNI, GNTI and JDTic induce κ opioid antagonism that is delayed by hours and can persist for months. Other effects are transient. It has been proposed that these drugs may be slowly absorbed or distributed, and may dissolve in cell membranes, thus slowing elimination and prolonging their effects. Recent evidence suggests, instead, that they induce prolonged desensitization of the κ opioid receptor. Methods To evaluate these hypotheses, we measured relevant physicochemical properties of nor-BNI, GNTI and JDTic, and the timecourse of brain and plasma concentrations in mice after intraperitoneal administration (using LC-MS-MS). Results: In each case, plasma levels were maximal within 30 min and declined by >80% within four hours, correlating well with previously reported transient effects. A strong negative correlation was observed between plasma levels and the delayed, prolonged timecourse of κ antagonism. Brain levels of nor-BNI and JDTic peaked within 30 min, but while nor-BNI was largely eliminated within hours, JDTic declined gradually over a week. Brain uptake of GNTI was too low to measure accurately, and higher doses proved lethal. None of the drugs were highly lipophilic, showing high water solubility (> 45 mM) and low distribution into octanol (log D7.4 7% unbound). JDTic showed P-gp-mediated efflux; nor- BNI and GNTI did not, but their low unbound brain uptake suggests efflux by another mechanism. Conclusions: The negative plasma concentration-effect relationship we observed is difficult to reconcile with simple competitive antagonism, but is consistent with desensitization. The very slow elimination of JDTic from brain is surprising given that it undergoes active efflux, has modest affinity for homogenate, and has a shorter duration of action than nor-BNI under these conditions. We propose that this persistence may result from entrapment in cellular compartments such as lysosomes

Selective κ Opioid Antagonists nor-BNI, GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

Background: Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets. Results: In binding assays, the three antagonists showed no detectable affinity (Ki≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α2C-adrenoceptor (Ki = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α1A-adrenoceptor (EC50 = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M1 receptor antagonist (KB = 3.7 µM). JDTic bound to the noradrenaline transporter (Ki = 54 nM), but only weakly inhibited transport (IC50 = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (Ki = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers. Conclusions: Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α1A-adrenoceptors). This may contribute to GNTI's severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists

Tracking Down the Molecular Substrates of Stress: New Roles for p38α MAPK and Kappa-Opioid Receptors

In this issue, Bruchas et al. (2011) uncover a novel stress-induced p38α MAPK signaling cascade within serotonergic neurons of the dorsal raphe nucleus that mediates depressive and drug-seeking behaviors. Their findings have potentially important implications for medication development

Kappa‐opioid receptors differentially regulate low and high levels of ethanol intake in female mice

Abstract Introduction: Studies in laboratory animals and humans indicate that endogenous opioids play an important role in regulating the rewarding value of various drugs, including ethanol (EtOH). Indeed, opioid antagonists are currently a front‐line treatment for alcoholism in humans. Although roles for mu‐ and delta‐opioid receptors have been characterized, the contribution of kappa‐opioid receptors (KORs) is less clear. There is evidence that changes in KOR system function can decrease or increase EtOH drinking, depending on test conditions. For example, female mice lacking preprodynorphin – the precursor to the endogenous KOR ligand dynorphin – have reduced EtOH intake. Considering that KORs can regulate dopamine (DA) transmission, we hypothesized that KORs expressed on DA neurons would play a prominent role in EtOH intake in females. Methods: We used a Cre/loxP recombination strategy to ablate KORs throughout the body or specifically on dopamine uptake transporter (DAT)‐expressing neurons to investigate the role of KORs on preference for and intake of EtOH (2‐bottle choice), the transition from moderate to excessive EtOH drinking (intermittent EtOH access), and binge EtOH drinking (drinking in the dark [DID]). Results: KOR deletion decreased preference for EtOH, although this effect was less pronounced when EtOH intake increased beyond relatively low levels. Discussion Our findings indicate that KOR activation increases EtOH drinking via effects mediated, at least in part, by KORs on DA neurons. While the mechanisms of this regulation remain unknown, previous work suggests that alterations in negative reinforcement processes or sensitivity to the sensory properties of EtOH can affect preference and intake

Mean permeation rates and efflux ratios in Caco-2 cell monolayers.

<p>Data are the mean of three independent determinations (samples in triplicate) each with SEM <10%. A: apical; B: basolateral.</p

Binding affinities of nor-BNI, GNTI and JDTic for 46 neurotransmitter receptors and transporters, determined by radioligand displacement.

<p>Submicromolar affinities are shown in bold; blank cells indicate <i>K</i>_i≥10 µM. For details (uncertainty, radioligand, membrane type, species), see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s007" target="_blank">Table S1</a>. For binding curves, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s006" target="_blank">File S1</a>.</p

Structural similarities between GNTI and α₁-AR ligands.

<p>Structural similarities between GNTI and α₁-AR ligands.</p

GNTI enhances maximal Ca²⁺ mobilization by noradrenaline at α_1A-AR without affecting potency (A); maximal PI hydrolysis is not increased (B).

<p>Some intermediate curves have been omitted for clarity. Error bars represent mean ± S.E.M. For raw data, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s001" target="_blank">Datasets S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070701#pone.0070701.s002" target="_blank">S2</a>.</p