The Cre-lox recombination approach is commonly used to generate cell-specific gene inactivation (or activation). We have noticed that the breeding and genotyping sections of papers utilizing Cre-lox techniques are frequently incomplete. While seemingly straightforward, there are important considerations that need to be implemented in the breeding and genotyping methods to prevent the introduction of experimental confounds. Germline recombination and transient expression of Cre recombinase during development are some examples of the complications that can occur, and conventional genotyping methods may fail to identify these events. In this opinion article, we highlight the importance of testing for unexpected recombination events, suggest strategies to isolate and minimize adverse recombination events, and encourage editors and reviewers to expect more definitive statements regarding the validation of genotyping.

Nalfurafine is a moderately selective kappa opioid receptor (KOR) analgesic with low incidence of dysphoric side effects in clinical development for the treatment of uremic pruritis. The basis for its reduced dysphoric effect compared to other KOR agonists is not clear, but prior studies suggest that the aversive properties of KOR agonists require p38α MAPK activation through an arrestin-dependent mechanism. To determine whether nalfurafine is a functionally selective KOR agonist, we measured its potency to activate the G protein-dependent early phase of Extracellular Signal-Regulated Kinase (ERK1/2) phosphorylation and the arrestin-dependent late phase of p38 MAPK signaling. Nalfurafine was approximately 250 fold more potent for ERK1/2 activation as compared to p38 MAPK activation in human KOR (hKOR) expressing HEK293 cells, and approximately 20 fold more potent for ERK1/2 activation than p38 activation in rodent KOR (rKOR) expressing HEK293 cells. The 10-fold greater G-bias at the hKOR than rKOR was unexpected, however the G protein biased effect of nalfurafine is consistent with its reduced dysphoric effects in human and rodent models. Although nalfurafine is reported to have low receptor selectivity in radio ligand binding assays, its antinociceptive effect was blocked by the selective KOR antagonist norbinaltorphimine. Nalfurafine pretreatment also resulted in a KOR-dependent and mu opioid receptor-independent reduction in scratching induced by 5′-GNTI. These findings suggest that nalfurafine is a functionally selective KOR agonist and that KOR agonists able to selectively activate G protein signaling without activating p38α MAPK may have therapeutic potential as non-dysphoric antipruritic analgesics.

Activation of κ opioid receptors (KORs) produces analgesia and aversion via distinct intracellular signaling pathways, but whether G protein-biased KOR agonists can be designed to have clinical utility will depend on a better understanding of the signaling mechanisms involved. We found that KOR activation produced conditioned place aversion and potentiated CPP for cocaine in male and female C57BL/6N mice. Consistent with this, males and females both showed arrestin-mediated increases in phospho-p38 MAPK following KOR activation. Unlike in males, however, KOR activation had inconsistent analgesic effects in females and KOR increased Gβγ-mediated ERK phosphorylation in males, but not females. KOR desensitization was not responsible for the lack of response in females because neither nor gene knock-out enhanced analgesia. Instead, responsiveness was estrous cycle dependent because KOR analgesia was evident during low estrogen phases of the cycle and in ovariectomized (OVX) females. Estradiol treatment of OVX females suppressed KOR-mediated analgesia, demonstrating that estradiol was sufficient to blunt Gβγ-mediated KOR signals. G protein-coupled receptor kinase 2 (GRK2) is known to regulate ERK activation, and we found that the inhibitory, phosphorylated form of GRK2 was significantly higher in intact females. GRK2/3 inhibition by CMPD101 increased KOR stimulation of phospho-ERK in females, decreased sex differences in KOR-mediated inhibition of dopamine release, and enhanced mu opioid receptor and KOR-mediated analgesia in females. In OVX females, estradiol increased the association between GRK2 and Gβγ. These studies suggest that estradiol, through increased phosphorylation of GRK2 and possible sequestration of Gβγ by GRK2, blunts G protein-mediated signals. Chronic pain disorders are more prevalent in females than males, but opioid receptor agonists show inconsistent analgesic efficacy in females. κ opioid receptor (KOR) agonists have been tested in clinical trials for treating pain disorders based on their analgesic properties and low addictive potential. However, the molecular mechanisms underlying sex differences in KOR actions were previously unknown. Our studies identify an intracellular mechanism involving estradiol regulation of G protein-coupled receptor kinase 2 that is responsible for sexually dimorphic analgesic responses following opioid receptor activation. Understanding this mechanism will be critical for developing effective nonaddictive opioid analgesics for use in women and characterizing sexually dimorphic effects in other inhibitory G protein-coupled receptor signaling responses.

The dynorphin/κ-opioid receptor (KOR) system has been previously implicated in the regulation of cognition, but the neural circuitry and molecular mechanisms underlying KOR-mediated cognitive disruption are unknown. Here, we used an operational test of cognition involving timing and behavioral inhibition and found that systemic KOR activation impairs performance of male and female C57BL/6 mice in the differential reinforcement of low response rate (DRL) task. Systemic KOR antagonism also blocked stress-induced disruptions of DRL performance. KOR activation increased 'bursts' of incorrect responses in the DRL task and increased marble burying, suggesting that the observed disruptions in DRL performance may be attributed to KOR-induced increases in compulsive behavior. Local inactivation of KOR by injection of the long-acting antagonist nor-BNI in the ventral tegmental area (VTA), but not the infralimbic prefrontal cortex (PFC) or dorsal raphe nucleus (DRN), prevented disruption of DRL performance caused by systemic KOR activation. Cre-dependent genetic excision of KOR from dopaminergic, but not serotonergic neurons, also blocked KOR-mediated disruption of DRL performance. At the molecular level, we found that these disruptive effects did not require arrestin-dependent signaling, because neither global deletion of G-protein receptor kinase 3 (GRK3) nor cell-specific deletion of GRK3/arrestin-dependent p38α MAPK from dopamine neurons blocked KOR-mediated DRL disruptions. We then showed that nalfurafine, a clinically available G-biased KOR agonist, could also produce DRL disruptions. Together, these studies demonstrate that KOR activation in VTA dopamine neurons disrupts behavioral inhibition in a GRK3/arrestin-independent manner and suggests that KOR antagonists could be beneficial for decreasing stress-induced compulsive behaviors.