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Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2022)

Adolescence is an important period in development where brain maturation is uniquely vulnerable to nicotine. Approximately one in three high school-aged students currently use tobacco/nicotine-containing products in the United States. Epidemiological and preclinical studies suggest that adolescent nicotine exposure results in long-lasting effects including nicotine dependence and progression to illegal substance use. Nicotine is the exogenous ligand for nicotinic acetylcholine receptors (nAChRs). Alpha(α)6 nAChR subunits reach peak expression in the dopaminergic neurons of the midbrain during adolescence, when initiation and establishment of tobacco/nicotine-containing product use usually occurs. Furthermore, a C to G single nucleotide polymorphism (SNP), rs2304297, in the 3’-untranslated region (UTR) of the α6 nicotinic receptor subunit gene, CHRNA6, has been associated with nicotine dependence and substance use. With the popularity of e-cigarette use among adolescents, it is essential to identify mechanisms underlying the health consequences of developmental nicotine exposure.I explore the effects of nicotine on subsequent opioid and stimulant self-administration using a low-dose, 4-day nicotine pretreatment paradigm in adolescent and adult male and female rats. To explore the role of α6-containing (α6*, denotes nicotinic receptors with the α6 subunit) nAChRs in nicotine-induced behaviors, I characterize a novel humanized CHRNA6 3’-UTR mutant rat line in which the 3’-UTR of the CHRNA6 gene in the rat was replaced with the human 3’-UTR containing the SNP alleles (C or G) associated with nicotine behaviors. To evaluate the role of the CHRNA6 3’-UTR SNP in adolescent substance use, I assessed the role of the CHRNA6 3’-UTR SNP in nicotine-induced mRNA expression, locomotor activity, anxiety-like behavior, and methamphetamine (METH) self-administration. I additionally investigated the role of α6* nAChRs in nicotine-induced METH self-administration via a targeted knock down approach. Taken together, findings show that adolescent, but not adult, nicotine exposure enhances subsequent cocaine, fentanyl, and METH self-administration. In the humanized CHRNA6 3’-UTR mutant line, I show that the human SNP is functional in vivo and that there are no alterations in behaviors that have been shown not to be impacted by α6* nAChRs; however, there are bidirectional sex-dependent nicotine-induced behavioral effects. Finally, I show that α6 nAChR subunit knockdown impacts adolescent nicotine-induced methamphetamine self-administration behavior. Overall, the dissertation emphasizes the complexity of risk factors that impact the behavioral effects of nicotine, including timing of exposure, sex, and genetics, and highlights potential molecular therapeutic targets.

    Cover page: Age, Genetic, and Sex Differences in Alpha(α)6-Containing Nicotinic Receptors and Nicotine-Induced Behaviors
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2022)

    Introduction

    Initiation of tobacco products typically occurs in adolescence. Adolescence is a critical period in development where the maturation of brain neurocircuitry is vulnerable to nicotine. Nicotine-containing products and psychostimulants, such as methamphetamine (METH), are often coabused. Rodent studies have shown that nicotine exposure in early adolescence increases subsequent drug intake and reward. Given the exponential increase in e-cigarette use among adolescents, there is a pressing need to understand whether adolescent nicotine exposure impacts concurrent increased METH use. The objective of this study is to evaluate age, sex, and longitudinal effects of nicotine pretreatment on METH reinforcement.

    Aims and methods

    Male and female Sprague-Dawley rats were pretreated with a subchronic, low-dose nicotine (2×, 30 µg/kg/0.1 mL, intravenous) or saline during early adolescence (postnatal days [PN] 28-31) or adulthood (PN 86-89). Following nicotine pretreatment, on PN 32 or PN 90, animals underwent operant intravenous self-administration for METH (20 µg/kg/inf) over a 2-hour period for five consecutive days.

    Results

    Early adolescent nicotine exposure enhances intravenous METH self-administration in male, but not female adolescents. Male adult rats self-administer METH over the 5-day testing period, independent of nicotine exposure. In contrast, nicotine exposure increases METH self-administration in female adults during the later sessions of the 5-day testing period.

    Conclusions

    Taken together, our data highlight age- and sex-dependent effects of low dose, subchronic nicotine pretreatment on subsequent intravenous METH self-administration.

    Implications

    A majority of polysubstance users begin smoking before the age of 18. Mounting evidence highlights adolescent susceptibility to nicotine exposure on brain and behavior. With the escalation in nicotine-containing products and stimulant use among adolescents, it is important to identify the consequences from adolescent nicotine use, including polysubstance use. Our study provides evidence that adolescent nicotine exposure enhances subsequent METH use, with important sex- and age-dependent effects.
      Cover page: Age- and Sex-Dependent Nicotine Pretreatment Effects on the Enhancement of Methamphetamine Self-administration in Sprague-Dawley Rats
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2020)
      Alpha(α)6-containing nicotinic acetylcholine receptors (nAChRs) have been implicated in nicotine reward and reinforcement. To date, a commercially available, validated α6 nAChR subunit antibody has not been reported. To evaluate a commercially available neuronal α6 nAChR subunit antibody we performed quantitative western blots on protein from the ventral tegmental area of wild type Sprague Dawley rats. As a first approach to determine the specificity of the antibody, we used a control antigen to block the α6 antibody from binding. Next, we tested the antibody in brain tissue of wild type and α6 knockout (KO) C57BL/6J mice. The α6 antibody was present at a higher than expected molecular weight (63 versus 57 kDa) and the control antigen blocked the α6 antibody, suggesting specificity. However, when we genetically validated the antibody, bands were present in both α6 KO mice and C57BL/6J samples. Taken together, our study highlights the necessity to genetically validate antibodies when possible and we report that a commercially available α6 nAChR subunit antibody is non-specific.
        Cover page: Specificity of a rodent alpha(α)6 nicotinic acetylcholine receptor subunit antibody
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2021)
        An exponential rise in nicotine-containing electronic-cigarette use has been observed during the period of adolescence. Preclinical studies have shown that nicotine exposure during early adolescence, but not adulthood, increases subsequent drug intake and reward. Although growing clinical trends highlight that stimulant use disorders are associated with the opioid epidemic, very few studies have assessed the effects of adolescent nicotine exposure on opioid intake. The objective of our current study is to develop a new animal model to assess the causal relationship of adolescent nicotine exposure on subsequent opioid intake. In this effort, we first replicate previous studies using a well-established 4-day nicotine paradigm. Rats are pretreated with a low dose of nicotine (2 × , 30 μg/kg/0.1 mL, intravenous) or saline during early adolescence (postnatal days 28-31) or adulthood (postnatal days 86-89). Following nicotine pretreatment on postnatal day 32 or postnatal day 90, animals underwent operant intravenous self-administration for the psychostimulant, cocaine [500 μg/kg/infusion (inf)] or the opioid, fentanyl (2.5 μg/kg/inf). We successfully show that adolescent but not adult, nicotine exposure enhances cocaine self-administration in male rats. Furthermore, we illustrate early adolescent but not adult nicotine exposure enhances fentanyl self-administration, independent of sex. Overall, our findings highlight that adolescence is a unique period of development that is vulnerable to nicotine-induced enhancement for cocaine and fentanyl self-administration in rats.
          Cover page: Early adolescent subchronic low-dose nicotine exposure increases subsequent cocaine and fentanyl self-administration in Sprague-Dawley rats.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2024)
          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.
            Cover page: Sub-chronic nicotine exposure influences methamphetamine self-administration and dopamine overflow in a sex-and genotype-dependent manner in humanized CHRNA6 3-UTR SNP (rs2304297) adolescent rats.
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2022)
            In human adolescents, a single nucleotide polymorphism (SNP), rs2304297, in the 3'-UTR of the nicotinic receptor subunit gene, CHRNA6, has been associated with increased smoking. To study the effects of the human CHRNA6 3'-UTR SNP, our lab generated knock-in rodent lines with either C or G SNP alleles. The objective of this study was to determine if the CHRNA6 3'-UTR SNP is functional in the knock-in rat lines. We hypothesized that the human CHRNA6 3'-UTR SNP knock-in does not impact baseline but enhances nicotine-induced behaviors. For baseline behaviors, rats underwent food self-administration at escalating schedules of reinforcement followed by a locomotor assay and a series of anxiety tests (postnatal day (PN) 25-39). In separate cohorts, adolescent rats underwent 1- or 4-day nicotine pretreatment (2×, 30 μg/kg/0.1 mL, i.v.). After the last nicotine injection (PN 31), animals were assessed behaviorally in an open-field chamber, and brain tissue was collected. We show the human CHRNA6 3'-UTR SNP knock-in does not affect food reinforcement, locomotor activity, or anxiety. Further, 4-day, but not 1-day, nicotine exposure enhances locomotion and anxiolytic behavior in a genotype- and sex-specific manner. These findings demonstrate that the human CHRNA6 3'-UTR SNP is functional in our in vivo model.
              Cover page: Sex- and Genotype-Dependent Nicotine-Induced Behaviors in Adolescent Rats with a Human Polymorphism (rs2304297) in the 3′-UTR of the CHRNA6 Gene
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2019)
              Despite persistent public health initiatives, many women continue to smoke during pregnancy. Since maternal smoking has been linked to persisting sex-dependent neurobehavioral deficits in offspring, some consider nicotine to be a safer alternative to tobacco during pregnancy, and the use of electronic nicotine delivery systems is on the rise. We presently show, however, that sustained exposure to low doses of nicotine during fetal development, approximating plasma levels seen clinically with the nicotine patch, produces substantial changes in developing corticostriatal dopamine systems in adolescence. Briefly, pregnant dams were implanted on gestational day 4 with an osmotic minipump that delivered either saline (GS) or nicotine (3 mg/kg/day) (GN) for two weeks. At birth, pups were cross-fostered with treatment naïve dams and were handled daily. Biochemical analyses, signaling assays, and behavioral responses to cocaine were assessed on postnatal day 32, representative of adolescence in the rodent. GN treatment had both sex-dependent and sex-independent effects on prefrontal dopamine systems, altering Catechol-O-methyl transferase (COMT)-dependent dopamine turnover in males and norepinephrine transporter (NET) binding expression in both sexes. GN enhanced cocaine-induced locomotor activity in females, concomitant with GN-induced reductions in striatal dopamine transporter (DAT) binding. GN enhanced ventral striatal D2-like receptor expression and G-protein coupling, while altering the roles of D2 and D3 receptors in cocaine-induced behaviors. These data show that low-dose prenatal nicotine treatment sex-dependently alters corticostriatal dopamine system development, which may underlie clinical deficits seen in adolescents exposed to tobacco or nicotine in utero.
                Cover page: Prenatal nicotine sex-dependently alters adolescent dopamine system development
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2018)

                Background

                Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy.

                Methods

                To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures.

                Results

                An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization.

                Conclusions

                These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.
                  Cover page: A molecular cascade modulates MAP1B and confers resistance to mTOR inhibition in human glioblastoma
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