Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a behavioral phenomenon termed “incubation of craving.” Mechanistic studies of incubation of craving have focused on specific neurobiological targets within preselected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now allow unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole-brain imaging approaches were developed for mouse brains, while incubation of drug craving has primarily been studied in rats, and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact-brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60 but not 1 or 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we 1) identified brain-wide activity patterns during incubation of food seeking using complementary analytical approaches and 2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying the incubation of food craving.

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Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a phenomenon termed "incubation of craving". Mechanistic studies of incubation of craving have focused on specific neurobiological targets within pre-selected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now enable unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole brain imaging approaches were developed for mouse brains while incubation of drug craving has primarily been studied in rats and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60, but not 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we (1) identified novel brain-wide activity patterns during incubation of food seeking using complementary analytical approaches, and (2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying incubation of food craving.

IBNtxA (3-iodobenzoyl naltrexamine) is a novel μ-opioid receptor (MOR) agonist which is structurally related to the MOR antagonist naltrexone. Recent studies suggest IBNtxA preferentially signals through truncated MOR splice variants, resulting in anti-nociception with reduced side effects, including no conditioned place preference (CPP) when tested at a single dose. IBNtxA represents an intriguing lead compound for preclinical drug development targeting truncated MOR splice variants, but further evaluation of its in vivo pharmacological profile is necessary. The purpose of this study was to independently verify the antinociceptive properties of IBNtxA and to examine more completely the rewarding properties and discriminative stimulus effects of IBNtxA, allowing broader assessment of IBNtxA as a candidate for further medications development. A dose of 3 mg/kg IBNtxA was equipotent to 10 mg/kg morphine in a hot-plate analgesia assay. In drug discrimination testing using mice trained to discriminate between 3 mg/kg IBNtxA and vehicle, the κ-agonist U-50488 fully substituted for IBNtxA. MOR agonist morphine, δ-agonist SNC162, NOP agonist SCH 221510, and MOR/NOP partial agonist buprenorphine each partially substituted for IBNtxA. IBNtxA up to 3 mg/kg did not produce a place preference in CPP. Pretreatment with 3 mg/kg IBNtxA but not 1 mg/kg IBNtxA attenuated acquisition of place preference for 10 mg/kg morphine. A dose of 3 mg/kg IBNtxA attenuated morphine-induced hyperlocomotion but did not alter naloxone-precipitated morphine withdrawal. Overall, IBNtxA has a complicated opioid receptor pharmacology in vivo. These results indicate that IBNtxA produces potent anti-nociception and has low abuse liability, likely driven by substantial κ agonist signaling effects.

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Rationale: IBNtxA (3-iodobenzoyl naltrexamine) is a novel μ opioid receptor (MOR) agonist structurally related to the classical MOR antagonist naltrexone. Recent studies suggest IBNtxA preferentially signals through truncated MOR splice variants, producing a unique pharmacological profile resulting in antinociception with reduced side effects, including no conditioned place preference (CPP) when tested at a single dose. IBNtxA represents an intriguing lead compound for preclinical drug development targeting truncated MOR splice variants but further evaluation of its in vivo pharmacological profile is necessary to evaluate its potential.Objective: The purpose of this study was to independently verify the antinociceptive properties of IBNtxA and to more completely examine the rewarding properties and discriminative stimulus effects of IBNtxA. These results will allow broader assessment of IBNtxA as a translational candidate or lead compound for further development.Results: IBNtxA was synthesized and compared to morphine in a variety of mouse behavioral assays. 3 mg/kg IBNtxA was equipotent to 10 mg/kg morphine in a hot plate analgesia assay. In drug discrimination testing using mice trained to discriminate between 3 mg/kg IBNtxA and DMSO/saline vehicle, the κ agonist U-50488 fully substituted for IBNtxA. Classical μ agonist morphine, δ agonist SNC162, NOP agonist SCH 221510, and μ/NOP partial agonist buprenorphine each partially substituted for IBNtxA. IBNtxA up to 3 mg/kg did not produce a place preference in CPP. Pretreatment with 3 mg/kg IBNtxA but not 1 mg/kg IBNtxA attenuated acquisition of place preference for 10 mg/kg morphine. 3 mg/kg IBNtxA attenuated morphine-induced hyperlocomotion but did not alter naloxone-precipitated morphine withdrawal. Conclusions:Overall IBNtxA has a complicated opioid receptor pharmacology in vivo. These results indicate that IBNtxA produces potent antinociception and has low abuse liability, likely driven by substantial κ agonist signaling effects.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency ('incubate') over time. Animal models of incubation may help in developing treatments for relapse prevention. However, these models have primarily focused on the role of conditioned stimuli (CSs) signaling drug delivery and not on discriminative stimuli (DSs), which signal drug availability and are also known to play a major role in drug relapse. We recently showed that DScontrolled cocaine seeking in rats also incubates during abstinence and persists up to 300 days. We used a trial-based procedure to train male and female rats to discriminate between two light cues: one light cue (DS+) signaled the availability of cocaine reward and the second light cue (DS-) signaled the absence of reward. Rats learned to press a central retractable lever during trials in which the DS+ cue was presented and to suppress responding when the DS-cue was presented. Here, we provide a detailed protocol for the behavioral procedure used in our study. The trial-based design of this behavior lends itself well to time-locked in vivo recording and manipulation approaches that can be used to identify neurobiological mechanisms underlying the contributions of DSs to drug relapse.