The marble-burying test is a pharmacologically validated paradigm used to study anxiety-like behaviors in laboratory rodents. Our laboratory has employed this assay as part of a behavioral screen to examine drug-induced negative affective states. Historically, the majority of our prior binge alcohol-drinking studies employed male subjects exclusively and reliably detected adolescent-adult differences in both basal and alcohol withdrawal-induced negative affect. However, age-related differences in marble-burying behavior were either absent or opposite those observed in our prior work when female subjects were included in the experimental design. As chemosensory cues from females are reported to be anxiolytic in males, the present study examined how odors from adult members of the opposite and same sex (obtained from soiled bedding) influence marble-burying behavior in adult, as well as adolescent, mice. Control studies examined the responsiveness of mice in the presence of novel neutral (vanilla) and aversive (tea tree) odors. Adult males exhibited reduced signs of anxiety-like behavior in the presence of female-soiled bedding, while adult females and adolescent mice of both sexes increased marble-burying behavior in the presence of both male- and female-soiled bedding. All mice exhibited increased burying in the presence of an aversive odor, while only adolescents increased marble-burying in response to the novel neutral odor. These data indicate sex by age interactions in the effects of volatile and nonvolatile odors from sexually-naive adult conspecifics on indices of anxiety-like behavior in the marble-burying test of relevance to the experimental design and procedural timing of experiments including sex as a biological variable.

Background

Hnrnph1 is a validated quantitative trait gene for methamphetamine behavioral sensitivity that encodes for heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1). This RNA-binding protein is involved in all stages of RNA metabolism that impacts mesocorticolimbic dopamine neurotransmission to influence addiction-related behavior.

Methods

We characterized the alcohol behavioral phenotypes of mice heterozygous for a deletion in the first coding exon of Hnrnph1 (Hnrnph1+/-). We examined alcohol intake under both continuous- and limited-access procedures, as well as alcohol-induced place-conditioning. Follow-up studies examined genotypic differences in the psychomotor-activating and sedative-hypnotic effects of acute and repeated alcohol, and a behavioral test battery was employed to determine the effects of Hnrnph1 deletion on the manifestation of negative affect during alcohol withdrawal.

Results

Relative to wild-type (WT) controls, Hnrnph1+/- males exhibited blunted intake of high alcohol concentrations under both drinking procedures. Hnrnph1 deletion did not impact the conditioned rewarding properties of low-dose alcohol, but reversed the conditioned place-aversion elicited by higher alcohol doses (2 and 4 g/kg), with more robust effects in male versus female mice. No genotypic differences were observed for alcohol-induced locomotor activity. Hnrnph1+/- mice exhibited a modest increase in sensitivity to alcohol's sedative-hypnotic effects, but did not differ from WT mice with regard to tolerance to alcohol's sedative-hypnotic effects or alcohol metabolism, Inconsistent effects of Hnrnph1 deletion were observed in models for withdrawal-induced negative affect.

Conclusions

These data identify Hnrnph1 as a novel, male-selective, driver of alcohol consumption and high-dose alcohol aversion that is potentially relevant to the neurobiology of alcohol abuse and alcoholism.

Cue-elicited drug-craving is a cardinal feature of addiction that intensifies (incubates) during protracted withdrawal. In a rat model, these addiction-related behavioral pathologies are mediated, respectively, by time-dependent increases in PI3K/Akt1 signaling and reduced Group 1 metabotropic glutamate receptor (mGlu) expression, within the ventromedial prefrontal cortex (vmPFC). Herein, we examined the capacity of single oral dosing with everolimus, an FDA-approved inhibitor of the PI3K/Akt effector mTOR, to reduce incubated cocaine-craving and reverse incubation-associated changes in vmPFC kinase activity and mGlu expression. Rats were trained to lever-press for intravenous infusions of cocaine or delivery of sucrose pellets and then subjected to tests for cue-reinforced responding during early (3 days) or late (30-46 days) withdrawal. Rats were gavage-infused with everolimus (0-1.0 mg/kg), either prior to testing to examine for effects upon reinforcer-seeking behavior, or immediately following testing to probe effects upon the consolidation of extinction learning. Single oral dosing with everolimus dose-dependently blocked cocaine-seeking during late withdrawal and the effect lasted at least 24 h. No everolimus effects were observed for cue-elicited sucrose-seeking or cocaine-seeking in early withdrawal. In addition, everolimus treatment, following initial cue-testing, reduced subsequent cue hyper-responsivity exhibited observed during late withdrawal, arguing a facilitation of extinction memory consolidation. everolimus' "anti-incubation" effect was associated with a reversal of withdrawal-induced changes in indices of PI3K/Akt1/mTOR activity, as well as Homer protein and mGlu1/5 expression, within the prelimbic (PL) subregion of the prefrontal cortex. Our results indicate mTOR inhibition as a viable strategy for interrupting heightened cocaine-craving and facilitating addiction recovery during protracted withdrawal.