Camron D. Bryant

Behavioral differences among C57BL/6 substrains: implications for transgenic and knockout studies.

Separate breeding colonies of C57BL/6 (“B6”) mice maintained at the Jackson Laboratories (“J”) and NIH (“N”) have led to the emergence of two distinct substrains of C57BL/6 mice: C57BL/6J and C57BL/6N. Molecular genetic studies indicate simple sequence-length polymorphisms, single-nucleotide polymorphisms, and copy-number variants among B6 substrains that may contribute to phenotypic differences. We examined differences in motor coordination, pain sensitivity, and conditional fear in the C57BL/6J strain and three N strains: C57BL/6NCrl (Charles River), C57BL/6NTac (Taconic), and C57BL/6NHsd (Harlan Sprague Dawley). Male C57BL/6J mice demonstrated enhanced motor coordination, as measured by the rotarod assay, markedly enhanced pain sensitivity in two assays of acute thermal nociception (e.g., tail withdrawal and hot plate), and a reduced level of conditional fear. The tail withdrawal result was confirmed in a separate laboratory. We also provide a table reviewing previously reported behavioral differences among various B6 substrains and discuss the significance of environmental differences due to obtaining mice form different vendors. These data may be seen as a potential problem and as a potential opportunity. Great care must be taken when working with mice engineered by using B6 embryonic stem cell lines because control groups, backcrosses, and intercrosses could inadvertently introduce behaviorally significant polymorphic alleles or environmental confounds. On the other hand, deliberate crosses between B6 substrains may provide an opportunity to map polymorphic loci that contribute to variability in a trait on largely homogenous backgrounds, which has the potential to improve mapping resolution and aid in the selection of candidate genes.

Csnk1e Is a Genetic Regulator of Sensitivity to Psychostimulants and Opioids

Csnk1e, the gene encoding casein kinase 1-epsilon, has been implicated in sensitivity to amphetamines. Additionally, a polymorphism in CSNK1E was associated with heroin addiction, suggesting that this gene may also affect opioid sensitivity. In this study, we first conducted genome-wide quantitative trait locus (QTL) mapping of methamphetamine (MA)-induced locomotor activity in C57BL/6J (B6) × DBA/2J (D2)-F2 mice and a more highly recombinant F8 advanced intercross line. We identified a QTL on chromosome 15 that contained Csnk1e (63–86 Mb; Csnk1e=79.25 Mb). We replicated this result and further narrowed the locus using B6.D2Csnk1e and D2.B6Csnk1e reciprocal congenic lines (78–86.8 and 78.7–81.6 Mb, respectively). This locus also affected sensitivity to the μ-opioid receptor agonist fentanyl. Next, we directly tested the hypothesis that Csnk1e is a genetic regulator of sensitivity to psychostimulants and opioids. Mice harboring a null allele of Csnk1e showed an increase in locomotor activity following MA administration. Consistent with this result, coadministration of a selective pharmacological inhibitor of Csnk1e (PF-4800567) increased the locomotor stimulant response to both MA and fentanyl. These results show that a narrow genetic locus that contains Csnk1e is associated with differences in sensitivity to MA and fentanyl. Furthermore, gene knockout and selective pharmacological inhibition of Csnk1e define its role as a negative regulator of sensitivity to psychostimulants and opioids.

Opioids and addiction: Emerging pharmaceutical strategies for reducing reward and opponent processes

Abstract Conventional strategies for treating opioid abuse include cognitive/behavioral therapy, maintenance therapy and managed withdrawal. These strategies deal with the addictive state, but much effort has shifted toward developing preventative measures. One approach is to reduce the rewarding properties of abused drugs, which in the case of prescription pharmaceuticals such as opioid analgesics is clearly a worthwhile endeavor. First, we will review recent studies demonstrating that mice lacking various G-protein coupled receptors and their ligands show diminished opioid reward. These studies point to considerable interdependence among different neuromodulatory systems for establishing drug reward. Thus, we will highlight the potential for combining opioids with other receptor targets to maximize analgesia and minimize reward in order to prevent the establishment of euphoric memories and opponent processes driving addiction. A more complex issue is treating opioid addicts where many physiological shifts have developed to oppose the acute drug effects. These opponent processes occur both at the cellular level (e.g. adenylate cyclase supersensitivity) and behavioral level (e.g. dysphoria and hyperalgesia). Evidence will be presented showing that even weak opioid agonists can effectively induce the intracellular process of adenylate cyclase supersensitivity. Furthermore, opioid-induced hyperalgesia, a behavioral opponent process, may manifest differently depending on the pain stimulus, and may require a priming drug dose in some cases. Environmental stress interacts with these opponent processes and drives relapse and thus, is another variable that needs to be considered in the development of innovative pharmacotherapy options for opioid addicts.

A major QTL on chromosome 11 influences psychostimulant and opioid sensitivity in mice

The identification of genes influencing sensitivity to stimulants and opioids is important for determining their mechanism of action and may provide fundamental insights into the genetics of drug abuse. We used a panel of C57BL/6J (B6; recipient)× A/J (donor) chromosome substitution strains (CSSs) to identify quantitative trait loci (QTL) for both open field activity and sensitivity to the locomotor stimulant response to methamphetamine (MA). Mice were injected with saline (days 1 and 2) and MA (day 3; 2 mg/kg i.p.). We analyzed the total distance traveled in the open field for 30 min following each injection. CSS‐8, ‐11 and ‐16 showed reduced MA‐induced locomotor activity relative to B6, whereas CSS‐10 and ‐12 showed increased MA‐induced locomotor activity. Further analysis focused on CSS‐11 because it was robustly different from B6 following MA injection, but did not differ in activity following saline injection and because it also showed reduced locomotor activity in response to the mu‐opioid receptor agonist fentanyl (0.2 mg/kg i.p.). Thus, CSS‐11 captures QTLs for the response to both psychostimulants and opioids. Using a B6 × CSS‐11 F2 intercross, we identified a dominant QTL for the MA response on chromosome 11. We used haplotype association mapping of cis expression QTLs and bioinformatic resources to parse among genes within the 95% confidence interval of the chromosome 11 QTL. Identification of the genes underlying QTLs for response to psychostimulants and opioids may provide insights about genetic factors that modulate sensitivity to drugs of abuse.

Congenic dissection of a major QTL for methamphetamine sensitivity implicates epistasis

We previously used the C57BL/6J (B6) × A/J mouse chromosome substitution strain (CSS) panel to identify a major quantitative trait locus (QTL) on chromosome 11 influencing methamphetamine (MA)‐induced locomotor activity. We then made an F2 cross between CSS‐11 and B6 and narrowed the locus (Bayes credible interval: 79–109 Mb) which was inherited dominantly and accounted for 14% of the phenotypic variance in the CSS panel. In the present study, we created congenic and subcongenic lines possessing heterozygous portions of this QTL to narrow the interval. We identified one line (84–96 Mb) that recapitulated the QTL, thus narrowing the region to 12 Mb. This interval also produced a small decrease in locomotor activity following prior saline treatment. When we generated subcongenic lines spanning the entire 12‐Mb region, the phenotypic difference in MA sensitivity abruptly disappeared, suggesting an epistatic mechanism. We also evaluated the rewarding properties of MA (2 mg/kg, i.p.) in the 84‐ to 96‐Mb congenic line using the conditioned place preference (CPP) test. We replicated the locomotor difference in the MA‐paired CPP chamber yet observed no effect of genotype on MA‐CPP, supporting the specificity of this QTL for MA‐induced locomotor activity under these conditions. Lastly, to aid in prioritizing candidate genes responsible for this QTL, we used the Affymetrix GeneChip® Mouse Gene 1.0ST Array to identify genes containing expression QTLs (eQTL) in the striatum of drug‐naÏve, congenic mice. These findings highlight the difficulty of using congenic lines to fine map QTLs and illustrate how epistasis may thwart such efforts.

The blessings and curses of C57BL/6 substrains in mouse genetic studies

Phenotypic and genetic differences among C57BL/6 substrains are accumulating. Investigators must address these differences to improve the quality of their studies.

RNA-binding proteins, neural development and the addictions

Transcriptional and post‐transcriptional regulation of gene expression defines the neurobiological mechanisms that bridge genetic and environmental risk factors with neurobehavioral dysfunction underlying the addictions. More than 1000 genes in the eukaryotic genome code for multifunctional RNA‐binding proteins (RBPs) that can regulate all levels of RNA biogenesis. More than 50% of these RBPs are expressed in the brain where they regulate alternative splicing, transport, localization, stability and translation of RNAs during development and adulthood. Dysfunction of RBPs can exert global effects on their targetomes that underlie neurodegenerative disorders such as Alzheimers and Parkinsons diseases as well as neurodevelopmental disorders, including autism and schizophrenia. Here, we consider the evidence that RBPs influence key molecular targets, neurodevelopment, synaptic plasticity and neurobehavioral dysfunction underlying the addictions. Increasingly well‐powered genome‐wide association studies in humans and mammalian model organisms combined with ever more precise transcriptomic and proteomic approaches will continue to uncover novel and possibly selective roles for RBPs in the addictions. Key challenges include identifying the biological functions of the dynamic RBP targetomes from specific cell types throughout subcellular space (e.g. the nuclear spliceome vs. the synaptic translatome) and time and manipulating RBP programs through post‐transcriptional modifications to prevent or reverse aberrant neurodevelopment and plasticity underlying the addictions.

Behavioral architecture of opioid reward and aversion in C57BL/6 substrains

Drug liking vs. drug disliking is a subjective motivational measure in humans that assesses the addiction liability of drugs. Variation in this trait is hypothesized to influence vulnerability vs. resilience toward substance abuse disorders and likely contains a genetic component. In rodents and humans, conditioned place preference (CPP)/aversion (CPA) is a Pavlovian conditioning paradigm whereby a learned preference for the drug-paired environment is used to infer drug liking whereas a learned avoidance or aversion is used to infer drug disliking. C57BL/6 inbred mouse substrains are nearly genetically identical, yet demonstrate robust differences in addiction-relevant behaviors, including locomotor sensitization to cocaine and consumption of ethanol. Here, we tested the hypothesis that B6 substrains would demonstrate differences in the rewarding properties of the mu opioid receptor agonist oxycodone (5 mg/kg, i.p.) and the aversive properties of the opioid receptor antagonist naloxone (4 mg/kg, i.p.). Both substrains showed similar degrees of oxycodone-induced CPP; however, there was a three-fold enhancement of naloxone-induced CPA in agonist-naïve C57BL/6J relative to C57Bl/6NJ mice. Exploratory factor analysis of CPP and CPA identified unique factors that explain variance in behavioral expression of reward vs. aversion. “Conditioned Opioid-Like Behavior” was a reward-based factor whereby drug-free locomotor variables resembling opioid treatment co-varied with the degree of CPP. “Avoidance and Freezing” was an aversion-based factor, whereby the increase in the number of freezing bouts co-varied with the degree of aversion. These results provide new insight into the behavioral architecture of the motivational properties of opioids. Future studies will use quantitative trait locus mapping in B6 substrains to identify novel genetic factors that contribute to the marked strain difference in NAL-CPA.

Reducing the RNA binding protein TIA1 protects against tau-mediated neurodegeneration in vivo

Emerging studies suggest a role for tau in regulating the biology of RNA binding proteins (RBPs). We now show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo protects against neurodegeneration and prolongs survival in transgenic P301S Tau mice. Biochemical fractionation shows co-enrichment and co-localization of tau oligomers and RBPs in transgenic P301S Tau mice. Reducing TIA1 decreased the number and size of granules co-localizing with stress granule markers. Decreasing TIA1 also inhibited the accumulation of tau oligomers at the expense of increasing neurofibrillary tangles. Despite the increase in neurofibrillary tangles, TIA1 reduction increased neuronal survival and rescued behavioral deficits and lifespan. These data provide in vivo evidence that TIA1 plays a key role in mediating toxicity and further suggest that RBPs direct the pathway of tau aggregation and the resulting neurodegeneration. We propose a model in which dysfunction of the translational stress response leads to tau-mediated pathology.Apicco and colleagues show that reducing TIA1 inhibits tau-mediated neurodegeneration and improves survival in a mouse model of tauopathy. This rescue occurs with a transition in tau aggregation from oligomeric to fibrillar forms of tau. These findings suggest a key role for RNA binding proteins in the pathophysiology of tau.

Extracellular signal-regulated kinase activation in the amygdala mediates elevated plus maze behavior during opioid withdrawal.

This study examined whether activation of extracellular signal-regulated kinase (ERK) contributes to the increased open-arm time observed in the elevated plus maze (EPM) during opioid withdrawal. We applied SL327, a selective ERK kinase (MEK) inhibitor, to specific limbic areas and examined the effect on EPM behaviors of controls and during naloxone-precipitated morphine withdrawal. We next confirmed that ERK activation increased in limbic areas of mice undergoing naloxone-precipitated morphine withdrawal. Direct injection of SL327 into the amygdala blocked the withdrawal-induced increase in open-arm time; however, injecting SL327 into the septum had no effect. Consistent with these results, both 0.2 and 2 mg/kg naloxone increased ERK activation in the central amygdala of morphine-dependent mice. In drug-naive mice, 2 mg/kg naloxone, but not 0.2 mg/kg, increased ERK activation in the central amygdala. During withdrawal, increased ERK activation was also observed in the lateral septum. In the locus coeruleus, a significant increase was observed only in morphine-dependent mice receiving 2 mg/kg, but not 0.2 mg/kg naloxone. In conclusion, ERK activation in limbic areas is likely involved in both the aversive properties of naloxone and in the affective/emotional symptoms of opioid withdrawal, including mediating EPM behaviors.