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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