Paternal alcohol exposure reduces acquisition of operant alcohol self-administration and affects Bdnf DNA methylation in male and female offspring.

Familial transmission of alcohol use disorder reflects genetic and environmental factors. Paternal alcohol exposure may affect rodent offspring via epigenetic modifications transmitted through the male germ line. While such exposure alters alcohol sensitivity in mouse offspring, no studies examined if it impacts the development of operant alcohol self-administration in rats. We exposed male (sires) Wistar rats to chronic intermittent ethanol in vapour chambers (16 h/day; 5 days/week) or to air for 6 weeks. Eight weeks later, rats were mated with alcohol-naive females. Adult alcohol- and control-sired F1 offspring were assessed in acquisition of alcohol self-administration in which increasing alcohol concentrations (2.5%, 5% and 10%, v/v) were delivered after one lever press (fixed ratio 1 or FR1). Prior to alcohol sessions, rats were trained to lever press for food delivery under an FR1 schedule of reinforcement. DNA methylation levels of the brain derived neurotrophic factor (Bdnf) gene were measured in sperm, nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) in sires and in offspring. Alcohol-exposed sires had lower Bdnf DNA methylation levels in NAc and greater methylation levels in mPFC. Although this pattern was not recapitulated in offspring, alcohol-sired offspring of both sexes did show aberrant Bdnf DNA methylation patterns compared to control-sired offspring. Alcohol-sired offspring self-administered less alcohol (5% and 10%) with no group differences in food responding. Results indicate that paternal alcohol exposure prior to conception protects against alcohol's initial reinforcing effects but the pattern of dysregulated Bdnf methylation in reward-related circuitry did not mimic changes seen in sires.

Neural signatures of opioid-induced risk-taking behavior in the prelimbic prefrontal cortex.

Opioid use disorder occurs alongside impaired risk-related decision-making, but the underlying neural correlates are unclear. We developed a novel approach-avoidance conflict model using a modified conditioned place preference paradigm to study neural signals of risky opioid seeking in the prefrontal cortex, a region implicated in executive decision making. Upon establishment of morphine conditioned place preference, rats underwent a subsequent conflict test in which fear-inducing cat odor was introduced in the previously drug-paired side of the apparatus. While the saline control group avoided the cat odor side, the morphine group maintained preference for the paired side despite the presence of cat odor. K-means clustering identified two subsets of morphine-treated rats that exhibited either persistent drug seeking (Risk-Takers) or increased avoidance (Risk-Avoiders) during conflict. Single-unit recordings from the prelimbic cortex (PL) revealed decreased neuronal firing rates upon acute morphine exposure in both Risk-Takers and Risk-Avoiders, but this firing rate suppression was absent after repeated administration. Risk-Avoiders also displayed distinct post-morphine excitation in PL which persisted across conditioning. During the preference test, subpopulations of PL neurons in all groups were either excited or inhibited when rats entered the paired side. Interestingly, while this inhibitory signal was lost during the subsequent conflict test in both saline and Risk-Avoider groups, these inhibitory responses persisted in Risk-Takers. Our results suggest that loss of PL inhibition after opioid conditioning is associated with the formation of contextual reward memory. Furthermore, persistent PL inhibitory signaling in the drug-associated context during conflict may underlie increased risk taking following opioid exposure.

Immune receptor toll-like receptor 4 contributes to stress-induced affective responses in a sex-specific manner.

Stress activates innate immune Toll-like receptors (TLRs) and enhances susceptibility to depression, a condition that is more prevalent in females. The TLR4 receptor type is involved in inflammatory responses and its expression levels associate with depressive symptoms and their successful treatment. Yet, little preclinical research has examined the role of TLR4 in stress-induced affective responses to determine if these are sex-specific. One group per genotype of male and female Tlr4 knockout (KO) and wild type (WT) rats were exposed to predator odor in a place conditioning apparatus with others exposed to saline. Affective behaviors evaluated included distance traveled and center time in an open-field apparatus, sucrose preference and fluid intake in a two-bottle test, and conditioned place aversion to the odor-paired compartment. Predator odor exposed rats showed conditioned place aversion to the odor-paired compartment, demonstrating predator odor was aversive. Such exposure led to anhedonia (decreased sucrose preference) across genotypes and sex. Predator odor exposure decreased distance traveled, an effect that was greater in KO rats, especially in females. Tlr4 deletion also resulted in sex-specific effects on anxiety-like behavior. Compared to WTs, female KO rats showed lower center time after predator odor exposure whereas genotype did not affect this response in male rats. Across litters, fewer male KO and heterozygous rats and more WT rats were born whereas female rats showed the typical genotype distribution. Results suggest predator odor alters affective behaviors, consistent with the preclinical literature, and deletion of Tlr4 enhances some stress-induced affective responses, often in a sex-specific manner.

Toll-like receptor 4 mediates the development of fatigue in the murine Lewis Lung Carcinoma model independently of activation of macrophages and microglia.

Cancer-related fatigue at the time of tumor diagnosis is commonly attributed to inflammation associated with the disease process. However, we have previously demonstrated that running wheel deficits occur well before increased expression of proinflammatory cytokines in the liver and brain in a murine model of human papilloma virus-related head and neck cancer (mEER). Further, we have demonstrated that genetic deletion of type I interleukin-1 receptor and MyD88 has no effect. In the current investigation we sought to test the generality of this finding by assessing whether there is a role for toll-like receptor (TLR) 4-dependent inflammation in the fatigue-like behavior observed in mice with Lewis Lung Carcinoma (LLC) or mEER tumors. Genetic deletion of TLR4 attenuated tumor-induced elevations in liver pro-inflammatory cytokine expression in both models. However, it only abrogated wheel running deficits in LLC tumor bearing mice. To determine whether TLR4 signaling in the LLC model involves innate immune cells, mice were treated with the colony stimulating factor (CSF)-1 receptor antagonist PLX-5622 before and throughout tumor development to deplete microglia and peripheral macrophages. Administration of PLX-5622 had no protective effect on wheel running deficits in either mEER or LLC tumor models despite effective depletion of microglia and a down regulation of peripheral proinflammatory cytokine expression. These results indicate that the TLR4 signaling that mediates fatigue-like behavior in LLC mice is not dependent upon microglial or peripheral macrophage activation. Based on the literature and our data demonstrating attenuation of ubiquitin proteasome pathway activation in the gastrocnemius muscle of Tlr4-/- mice implanted with LLC cells, we interpret our current findings as indication that skeletal muscle TLR4 signaling may be involved. These results are important in that they add to the evidence that tumor-induced fatigue develops independently from classical neuroinflammation.

Naltrexone alters alcohol self-administration behaviors and hypothalamic-pituitary-adrenal axis activity in a sex-dependent manner in rats.

BACKGROUND: The mu-opioid antagonist, naltrexone (NTX), is a FDA-approved treatment for alcohol use disorder (AUD); however, the data on whether it differentially affects males vs. females are mixed. NTX increases hypothalamic-pituitary-adrenal (HPA) axis activity that associates with subjective responses to alcohol and craving in individuals with AUD. The present study tested for sex differences in the ability of NTX to decrease appetitive and consummatory behaviors in rats in operant alcohol self-administration. Because the opioid system and HPA axis are sexually dimorphic, we examined NTX's effect on adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels. METHODS: Male and female Sprague-Dawley rats (n's = 6-8) were trained to lever press for alcohol (10% v/v) under a fixed-ratio 2 schedule of reinforcement. NTX doses (0, 0.1-10 mg/kg) were assessed in tests conducted under a progressive ratio schedule of reinforcement. Separate groups of alcohol and water drinking rats (n's = 8) were used to assess NTX's (10 mg/kg) effects on HPA axis hormones. RESULTS: NTX decreased consummatory behaviors for alcohol in a dose-related manner, but not appetitive behaviors in males. In females, NTX decreased appetitive behaviors for alcohol in a dose-dependent manner, but only decreased consummatory behaviors at the highest (10 mg/kg) NTX dose. NTX increased ACTH levels in alcohol drinking females in diestrus, but not in other groups. However, NTX increased CORT levels for longer durations in alcohol drinking males relative to alcohol drinking females in diestrus. CONCLUSIONS: Our findings suggest that NTX selectively reduces consummatory behaviors for alcohol in males and appetitive behaviors in females, while also showing differential sex effects on HPA hormones.