Rationale

Large-scale human candidate gene studies have indicated that a genetic variant (rs2304297) in the alpha(α)6 nicotinic acetylcholine receptor (nAChR) subunit, encoded by the CHRNA6 gene, may play a key role in adolescent nicotine addictive behavior. We hypothesized that the polymorphism selectively enhances nicotine + cue-primed reinstatement, but not nicotine- or cue-reinstatement in α6 ^GG (risk) vs. α6 ^CC (non-risk) allele carriers, without having baseline effects on natural rewards.

Methods

Using CRISPR-Cas9 genomic engineering, we developed a humanized rat line with the human gene variant of the CHRNA6 3'-UTR ^{C 123 G} polymorphism in Sprague-Dawley rats. Genetically modified adolescent male and female rats were food trained under a fixed-ratio (FR)1 schedule of reinforcement and progressively increased to FR5. Animals were implanted with catheters and began nicotine self-administration (15 μg/kg/infusion) at FR5. Upon reaching stable responding, reinforced behavior was extinguished by removal of drug and cues. Reinstatement testing began for cue only, nicotine only, and nicotine + cue in a Latin Square Design. Animals were returned to extinction conditions for 2 days minimum between testing.

Results

For natural food rewards, nicotine self-administration, progressive ratio, and extinction, adolescent male and female (α6 ^GG and α6 ^CC ) rats exhibited equivalent behaviors. Male α6 ^GG rats show enhanced nicotine + cue-primed reinstatement when compared with male α6 ^CC rats. This genotype effect on reinstatement was not seen in female rats.

Conclusion

Our findings support the in vivo functional role of the human CHRNA6 3'-UTR SNP genetic variant in sex-dependently enhancing nicotine seeking behavior in adolescent rats. Overall, the findings support clinical and preclinical data highlighting a role of α6 nAChRs mediating sex heterogeneity in substance use and related phenotypes.

Introduction

The initiation of nicotine and tobacco use peaks during adolescence. How adolescent males and females differ based on the acquisition of nicotine use and nicotine-seeking behavior is less understood. Our current studies develop a preclinical intravenous self-administration and reinstatement paradigm in male and female Sprague Dawley rats to evaluate how sex impacts the acquisition of nicotine self-administration and nicotine-seeking, when behavior is initiated during adolescence.

Aims and methods

Male and female adolescent rats were food trained under a fixed-ratio one (FR1) schedule of reinforcement and progressively increased to FR5. Animals were implanted with catheters and began nicotine self-administration (0.015 mg/kg/infusion) at FR5 during adolescence on postnatal day 34. Upon reaching stable reinforced responding, animals were tested for progressive ratio (PR) schedules of reinforcement followed by extinction via the removal of drug and associated cues for a minimum of 5 days or until responding was reduced to ≤25% of baseline. Reinstatement testing began for cue only, nicotine only, and a combination of nicotine plus cues using a Latin square design. Animals returned to extinction conditions for 2 days minimum between testing.

Results

No sex differences were observed for natural rewards, acquisition, and PR schedules of nicotine self-administration, days to extinction, and reinstatement condition.

Conclusions

Our findings provide evidence that sex does not impact natural rewards, nicotine reinforcement, reward, and reinstatement, when behavior is initiated during adolescence. Our study offers a feasible approach for assessing nicotine-seeking in male and female Sprague Dawley rats during the unique developmental window of adolescence.

Implications

This study demonstrates the impact of nicotine and associated cues in the reinstatement of nicotine-seeking in male and female rats when behavior is initiated during adolescence. Findings support the equivalent impact of nicotine and the nonpharmacological stimuli of cues between male and female adolescent rats that began nicotine self-administration during adolescence.

Introduction

Given the rapid increase in teen vaping over recent years it is critical to understand mechanisms underlying addiction and relapse to tobacco use at this age. To evaluate the role of non-nicotine constituents in cigarette smoke, our lab has previously established a model of intravenous self-administration of aqueous cigarette smoke extract (CSE). We now compare the sensitivity of male adolescent and adult rats who have self-administered CSE or nicotine to reinstatement with the pharmacological stressor, yohimbine, with and without cues.

Methods

Adolescents and adults, aged postnatal day (P) 34 and 84, were tested for the effect of yohimbine (0-2.5 mg/kg) on plasma corticosterone levels to establish a dose that was an effective stressor at both ages. Separate groups of animals were trained to lever press for food before beginning 1-hour drug self-administration sessions for nicotine or CSE (15 μg/kg/infusion nicotine content). Once stable responding was reached, drug was removed, and behavior extinguished. Drug-seeking behavior was reinstated with yohimbine, cues, or a combination of yohimbine and cues.

Results

Although adolescents and adults showed different dose-responses for yohimbine-induced corticosterone release, a dose of 2.5 mg/kg increased stress hormone levels at both ages. Whereas both ages displayed similar responding for CSE and nicotine, adolescents self-administered more CSE and nicotine as compared to adults. Cues and cues + stress reinstated responding to a greater extent in animals that had self-administered CSE, regardless of age.

Discussion

These findings suggest that non-nicotine tobacco smoke constituents influence later but not earlier stages of addiction in both adolescent and adult male rats.

We previously demonstrated that a genetic single-nucleotide polymorphism (SNP, rs2304297) in the 3 untranslated region (UTR) of the human CHRNA6 gene has sex- and genotype-dependent effects on nicotine-induced locomotion, anxiety, and nicotine + cue-induced reinstatement in adolescent rats. This study aims to investigate how the CHRNA6 3-UTR SNP influences dopaminergic and noradrenergic tissue levels in brain reward regions during baseline and after the reinstatement of drug-seeking behavior. Naïve adolescent and adult rats, along with those undergoing nicotine + cue reinstatement and carrying the CHRNA6 3-UTR SNP, were assessed for dopamine (DA), norepinephrine (NE), and metabolites in reward pathway regions. The results reveal age-, sex-, and genotype-dependent baseline DA, NE, and DA turnover levels. Post-reinstatement, male α6GG rats show suppressed DA levels in the Nucleus Accumbens (NAc) Shell compared to the baseline, while nicotine+ cue-induced reinstatement behavior correlates with neurotransmitter levels in specific brain regions. This study emphasizes the role of CHRNA6 3-UTR SNP in the developmental maturation of the dopaminergic and noradrenergic system in the adolescent rat brain, with tissue levels acting as predictors of nicotine + cue-induced reinstatement.

Introduction: The rewarding effects of drugs of abuse are associated with the dopaminergic system in the limbic circuitry. Nicotine exposure during adolescence is linked to increased use of drugs of abuse with nicotine and methamphetamine (METH) commonly used together. Nicotine acts on neuronal nicotinic acetylcholine receptor (nAChR) systems, critical for reward processing and drug reinforcement, while METH leads to a higher dopamine (DA) efflux in brain reward regions. A human single nucleotide polymorphism (SNP) in the 3-untranslated region (UTR) of the α6 nicotinic receptor subunit gene (CHRNA6, rs2304297), has been linked with tobacco/nicotine and general substance use during adolescence. Using CRISPR-Cas9 genomic engineering, our lab recapitulated the CHRNA6 3UTRC123G SNP, generating α6CC and α6GG allele carriers in Sprague Dawley rats. We hypothesized the CHRNA6 3UTRC123G SNP would sex- and genotype-dependently enhance nicotine-induced METH self-administration as well as nicotine-induced DA overflow in the nucleus accumbens shell of adolescent α6GG and α6CC carriers. Methods: Adolescent male and female rats underwent a 4-day sub-chronic, low-dose (0.03 mg/kg/0.1 mL, x2) nicotine pretreatment paradigm to assess intravenous METH (0.02 mg/kg/0.1 mL) self-administration as well as nicotine- and METH (0.02 mg/kg/0.1 mL)-induced DA overflow in the nucleus accumbens shell (NAcS) using in vivo microdialysis coupled with high-performance liquid-chromatography-electrochemical detection (HPLC-ECD). Results: Nicotine pretreatment sex- and genotype-dependently enhanced subsequent METH self-administration in adolescent CHRNA6 3UTRC123G SNP rats. Further nicotine and METH-induced DA overflow is observed in α6CC females as compared to α6GG females, with METH-induced DA overflow enhanced in α6GG males when compared to α6CC males. Conclusion: These findings demonstrate that the CHRNA6 3-UTRC123G SNP can sex- and genotype-dependently impact adolescent nicotine-induced effects on METH self-administration and stimulant-induced DA overflow in reward regions of the brain.