Inactivation of NE in PL cortex blunts amphetamine-induced CPP, thus fostering extinction and showing to be critical for the maintenance of conditioned Amph-seeking behavior. Opposite effects of NE depletion in IL, seemingly in agreement with literature on extinction, are discussed in terms of balance of activity between PL and IL in extinction.

Background and Purpose CR4056 is a first‐in‐class imidazoline‐2 (I2) receptor ligand characterized by potent analgesic activity in different experimental animal models of pain. In a recent phase II clinical trial, CR4056 effectively reduced pain in patients with knee osteoarthritis. In the present study, we investigated the effects of CR4056 on PKCε translocation in vitro and on PKCε activation in vivo in dorsal root ganglia (DRG) neurons. Experimental Approach Effects of CR4056 on bradykinin‐induced PKCε translocation were studied in rat sensory neurons by immunocytochemistry. PKCε activation was investigated by immunohistochemistry analysis of DRG from complete Freund's adjuvant‐treated animals developing local hyperalgesia. The analgesic activity of CR4056 was tested on the same animals. Key Results CR4056 inhibited PKCε translocation with very rapid and long‐lasting activity. CR4056 decreased hyperalgesia and phospho‐PKCε immunoreactivity in the DRG neurons innervating the inflamed paw. The effect of CR4056 on PKCε translocation was blocked by pertussis toxin, implying that the intracellular pathways involved Gi proteins. The inhibition of PKCε translocation by CR4056 was independent of the α2‐adrenoeceptor and, surprisingly, was also independent of idazoxan‐sensitive I2 binding sites. The I2 agonist 2BFI had no effect alone but potentiated the activity of low concentrations of CR4056. Conclusions and Implications Our results demonstrate that CR4056 shares the ability to inhibit PKCε translocation with other analgesics. Whether the inhibition of PKCε involves binding to specific subtype(s) of I2 receptors should be further investigated. If so, this would be a new mode of action of a highly specific I2 receptor ligand.

Prolonged use of opioids causes analgesic tolerance and adverse effects including constipation and dependence. Compounds targeting imidazoline I 2 receptors are known to potentiate opioid analgesia in rodents. We investigated whether combination with the I 2 receptor ligand CR4056 could improve efficacy and safety of morphine and explored the mechanisms of the CR4056-opioid interaction. Experimental Approach: We used the complete Freund's adjuvant (CFA) model in rats to study the effects of treatments on hyperalgesia, morphine tolerance and microglia activation as measured by immunofluorescence. Opioid-induced adverse effects were assessed in rodent models of morphine-induced constipation, sedation (open field, sedation rating scale, and rotarod), physical dependence (naloxoneinduced withdrawal), and abuse (conditioned place preference-associated reward). Chemiluminescence assays tested CR4056 as allosteric modulator of μ-opioid receptors. Key Results: CR4056 (ED 50 = 4.88 mgÁkg −1) and morphine (ED 50 = 2.07 mgÁkg −1) synergized in reducing CFA-induced hyperalgesia (ED 50 = 0.52 mgÁkg −1 ; 1:1 combination). Consistently, low doses of CR4056 (1 mgÁkg −1) spared one third of the cumulative morphine dose administered during 4 days and prevented/reversed the development of tolerance to morphine anti-hyperalgesia. These opioid-sparing effects were associated with decreased activation of microglia, independent of CR4056 interactions on μ-opioid receptors. Importantly, the low doses of CR4056 and morphine that synergize in analgesia did not induce constipation, sedation, physical dependence, or place preference.

Tolerance to opioid administration represents a serious medical alert in different chronic 28conditions. This study compares the effects of the imidazoline compounds 1, 2, and 3 on 29 morphine tolerance in an animal model of inflammatory pain in the rat. 1, 2, and 3 have been 30 selected in that, although bearing a common scaffold, preferentially bind to α 2 -adrenoceptors, 31 imidazoline I 2 receptors, or both systems, respectively. Such compounds have been tested in 32 vivo by measuring the paw withdrawal threshold to mechanical pressure after complete 33Freund's adjuvant injection. To determine the ligand levels in rat plasma, an HPLC-mass 34 spectrometry method has been developed. All the compounds significantly reduced the 35 induction of morphine tolerance, showing different potency and duration of action. Indeed, 36 the selective imidazoline I 2 receptor interaction (2) restored the analgesic response by 37 maintaining the same time-dependent profile observed after a single morphine administration. 38Differently, the selective α 2C -adrenoceptor activation (1) or the combination between α 2C -39 adrenoceptor activation and imidazoline I 2 receptor engagement (3) promoted a change in the 40 temporal profile of morphine analgesia by maintaining a mild but long lasting analgesic 41 effect. Interestingly, the kinetics of compounds in rat plasma supported the pharmacodynamic 42 data. Therefore, this study highlights that both peculiar biological profile and bioavailability 43 of such ligands complement each other to modulate the reduction of morphine tolerance. 44Based on these observations, 1-3 can be considered useful leads in the design of new drugs 45 able to turn off the undesired tolerance induced by opioids. 46 47

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