Mathieu E. Wimmer

This study demonstrates the utility of a rat model of chemotherapy-induced peripheral neuropathy (CIPN) to assess the ability of the non-psychoactive cannabinoid cannabidiol (CBD) to modulate the development of this syndrome in vivo. The method utilizes the chemotherapeutic agent paclitaxel to generate an allodynic phenotype in the animals. This study describes how to handle and solubilize CBD, administer the chemotherapeutic agent, assess mechanical and cold sensitivity, and apply high-speed videography to measure nocifensive behavior in animals. Using the procedures outlined, the data support that CBD prevents the allodynic phenotype from developing in the treated animals. No difference was observed in the CBD-treated animals from day 0 (pre-paclitaxel baseline) to day 7 (post-sensitization) in mechanical or thermal sensitivity, while the vehicle-treated animals became significantly more sensitive. This response to treatment is durable up to the latest time point where data were collected (7 weeks). The addition of high-speed videography allows for a more granular and unbiased assessment of this behavioral phenotype (e.g., classification of analgesia and anti-allodynia). This demonstrates both the utility of this model for cannabinoid drug characterization and the potential role of CBD in mitigating neuropathic pain.

This study demonstrates the utility of a rat model of chemotherapy-induced peripheral neuropathy (CIPN) to assess the ability of the non-psychoactive cannabinoid cannabidiol (CBD) to modulate the development of this syndrome in vivo. The method utilizes the chemotherapeutic agent paclitaxel to generate an allodynic phenotype in the animals. This study describes how to handle and solubilize CBD, administer the chemotherapeutic agent, assess mechanical and cold sensitivity, and apply high-speed videography to measure nocifensive behavior in animals. Using the procedures outlined, the data support that CBD prevents the allodynic phenotype from developing in the treated animals. No difference was observed in the CBD-treated animals from day 0 (pre-paclitaxel baseline) to day 7 (post-sensitization) in mechanical or thermal sensitivity, while the vehicle-treated animals became significantly more sensitive. This response to treatment is durable up to the latest time point where data were collected (7 weeks). The addition of high-speed videography allows for a more granular and unbiased assessment of this behavioral phenotype (e.g., classification of analgesia and anti-allodynia). This demonstrates both the utility of this model for cannabinoid drug characterization and the potential role of CBD in mitigating neuropathic pain.

Paternal exposure to drugs of abuse can impact addiction-related behaviours in progeny via germline epigenome remodelling. Previously, we found that offspring of morphine-exposed male rats showed increased morphine-taking, diminished adolescent social play, and increased sensitivity to morphine-derived analgesia. Here, we first tested the impact of a 90-day paternal abstinence period following morphine self-administration on the transmission of the aforementioned phenotypes. The previously observed changes in morphine-related behaviours were no longer present in offspring of morphine-abstinent sires. We next compared small RNA (smRNA) content in sperm collected from four sire intravenous self-administration groups: morphine, saline, abstinent morphine, and abstinent saline. Two smRNAs (rno-miR-150-5p and an snoRNA annotated to Snora42/Noc3l) were differentially expressed specifically between morphine- and saline-treated sperm. No differential expression between abstinent morphine and saline sperm was observed. These data begin to delineate the temporal limits of heritable germline modifications associated with morphine exposure, in addition to identifying F0 germline factors coinciding with the manifestation of F1 multigenerational phenotypes. Furthermore, these data suggest that paternal abstinence at conception can prevent inheritance of germline factors that may alter offspring susceptibility to addiction-related endophenotypes.

Background A growing body of preclinical studies report that preconceptional experiences can have a profound and long-lasting impact on adult offspring behavior and physiology. However, less is known about paternal drug exposure and its effects on reward sensitivity in the next generation. Methods Adult male rats self-administered morphine for 65 days; controls received saline. Sires were bred to drug-naïve dams to produce first-generation (F1) offspring. Morphine, cocaine, and nicotine self-administration were measured in adult F1 progeny. Molecular correlates of addiction-like behaviors were measured in reward-related brain regions of drug naïve F1 offspring. Results Male, but not female offspring produced by morphine-exposed sires exhibited dose-dependent increased morphine self-administration and increased motivation to earn morphine infusions under a progressive ratio schedule of reinforcement. This phenotype was drug-specific as self-administration of cocaine, nicotine, and sucrose were not altered by paternal morphine history. The male offspring of morphine-exposed sires also had increased expression of mu-opioid receptors in the ventral tegmental area but not in the nucleus accumbens. Conclusions Paternal morphine exposure increased morphine addiction-like behavioral vulnerability in male but not female progeny. This phenotype is likely driven by long-lasting neural adaptations within the reward neural brain pathways.

A powerful motivation to seek opioids remains after drug cessation and intensifies during extended periods of abstinence. Unfortunately, biomarkers associated with continued drug seeking have not been described. Moreover, previous studies have focused on the effects of early abstinence with little exploration into the long‐term drug‐induced mechanisms that occur after extended abstinence. Here we demonstrated that 30 days (D) of forced abstinence results in a time‐dependent increase in morphine seeking in a rat model of morphine self‐administration (SA). We measured expression of known drug‐responsive microRNAs (miRNAs) in the nucleus accumbens, an area critical for reward‐related plasticity, during early or late abstinence in animals that underwent either a cue‐induced relapse test or no relapse test. miRNAs are small noncoding RNAs that represent suitable biomarker candidates due to their long‐lasting nature. mir‐32‐5p levels during early abstinence negatively correlated with active lever pressing in both cue‐exposed and cue‐naïve animals. mir‐1298‐5p positively correlated with drug SA history after a relapse test during late abstinence. When animals underwent acute abstinence with no relapse test, mir‐1298‐5p correlated with drug infusions and active lever pressing during SA. In late abstinence with no relapse test, mir‐137‐3p negatively correlated with drug infusions. Regulation of mir‐32‐5p target genes and significant correlation of target gene mRNA with mir‐32‐5p was observed after abstinence. These results indicate that lasting regulation of miRNA expression is associated with drug intake following morphine SA. In addition, we conclude that the miRNA profile undergoes regulation from early to late abstinence and miRNA expression may indicate past drug history.

Incubation of craving refers to the intensification of drug seeking behavior in response to reward-paired cues over the course of abstinence. In rodents, craving and drug-seeking behaviors have been measured by an increase in lever pressing in the absence of reinforcer availability in response to cue presentations. However, craving in rodents is difficult to define and little is known about the behavioral signatures that accompany increased drug-seeking behavior measured by lever pressing. The affective components of relapse are also important, but understudied in rodents. Hormonal fluctuations influence craving for psychostimulants, but little is known about the impact of the estrous cycle on opioid-seeking behavior. This study sought to delineate the behavioral and affective signatures associated with craving, and to examine the influence of the female estrous cycle on craving. Male and female rats underwent 10 days of intravenous opioid self-administration. Separate cohorts of control rats self-administered oral sucrose, a natural non-drug reward. Cue-induced seeking tests were conducted after one or thirty days of forced abstinence. These sessions were recorded and scored for overall locomotion, instances of sniffing, grooming, or hyperactivity. Ultrasonic vocalizations were also recorded to determine affective profiles that accompany opioid seeking. Although active lever presses and overall locomotion increased unanimously over extended abstinence from heroin and sucrose, a sex and reinforcer-specific behavioral and affective signature of craving emerged. Furthermore, though the female estrous cycle did not affect taking or seeking, it appears to influence more granular behaviors. Significance statement Craving was recently added as a diagnostic criterion for substance use disorders. Positive and negative affect may contribute to relapse, though this domain is not fully understood in the context of cue-induced drug seeking in rodents. To enhance the definition of craving and to address potential sex differences, we recorded behaviors and ultrasonic vocalizations during early and late abstinence cue-induced relapse tests in male and female rats. The behavioral signatures associated with craving were sex- and reinforcer-specific. In females, the estrous cycle selectively influenced locomotion and hyperactivity during reinforcer seeking. These findings contribute to a better behavioral definition of craving, which lays the foundation to better understand the neural mechanisms underlying craving and drug seeking behaviors.

Incubation of craving is a well-documented phenomenon referring to the intensification of drug craving over extended abstinence. The neural adaptations that occur during forced abstinence following chronic drug taking have been a topic of intense study. However, little is known about the transcriptomic changes occurring throughout this window of time. To define gene expression changes associated with morphine consumption and extended abstinence, male and female rats underwent 10 days of morphine self-administration. Separate drug-naive rats self-administered sucrose in order to compare opioid-induced changes from those associated with natural, non-drug rewards. After one or 30 days of forced abstinence, rats were tested for craving, or nucleus accumbens shell tissue was dissected for RNA sequencing. Morphine consumption was predictive of drug seeking after extended (30 days) but not brief (1 day) abstinence in both sexes. Extended abstinence was also associated with robust sex- and reinforcer-specific changes in gene expression, suggesting sex differences underlying incubation of morphine and sucrose seeking respectively. Importantly, these changes in gene expression occurred without re-exposure to drug-paired cues, indicating that chronic morphine causes long-lasting changes in gene expression that prime the system for increased craving. These findings lay the groundwork for identifying specific therapeutic targets for curbing opioid craving without impacting the natural reward system in males and females.

Significance: Opioid use disorder is on the rise, as are efforts to understand vulnerability to misuse. However, understanding resilience to drug abuse is also critical to the development of novel treatments. Here we demonstrate that a brief postnatal exposure to a low-resource environment—using the limited bedding and nesting (LBN) manipulation—promoted resilience to addiction-related phenotypes in adult male rats. Specifically, LBN reduced impulsivity, morphine self-administration, and nucleus accumbens (NAc) glutamate transmission in males, effects not seen in females. By comparing the sexes, we found that changes in NAc gene transcription unique to LBN males may contribute to resilience. Importantly, these molecular changes reveal new targets that could guide the development of pharmacotherapies for opioid use disorder.