Methamphetamine-induced conditioned place preference in LG/J and SM/J mouse strains and an F45/F46 advanced intercross line

The conditioned place preference (CPP) test is frequently used to evaluate the rewarding properties of drugs of abuse in mice. Despite its widespread use in transgenic and knockout experiments, there are few forward genetic studies using CPP to identify novel genes contributing to drug reward. In this study, we tested LG/J and SM/J inbred strains and the parents/offspring of 10 families of an F(45)/F(46) advanced intercross line (AIL) for methamphetamine-induced CPP (MA-CPP) once per week over 2 weeks. Both LG/J and SM/J mice exhibited significant MA-CPP that was not significantly different between the two strains. Furthermore, LG/J mice showed significantly less acute MA-induced locomotor activity as well as locomotor sensitization following subsequent MA injections. AIL mice (N = 105) segregating LG/J and SM/J alleles also demonstrated significant MA-CPP that was equal in magnitude between the first and second week of training. Importantly, MA-CPP in AIL mice did not correlate with drug-free or MA-induced locomotor activity, indicating that MA-CPP was not confounded by test session activity and implying that MA-CPP is genetically distinct from acute psychomotor sensitivity. We estimated the heritability of MA-CPP and locomotor phenotypes using midparent-offspring regression and maximum likelihood estimates derived from the kinship coefficients of the AIL pedigree. Heritability estimates of MA-CPP were low (0-0.21) and variable (SE = 0-0.33) which reflected our poor power to estimate heritability using only 10 midparent-offspring observations. In sum, we established a short-term protocol for MA-CPP in AIL mice that could reveal LG/J and SM/J alleles important for MA reward. The use of highly recombinant genetic populations like AIL should facilitate the identification of these genes and may have implications for understanding psychostimulant abuse in humans.This work was supported by R01DA021336 and K99DA029635

Hnrnph1 Is A Quantitative Trait Gene for Methamphetamine Sensitivity.

Psychostimulant addiction is a heritable substance use disorder; however its genetic basis is almost entirely unknown. Quantitative trait locus (QTL) mapping in mice offers a complementary approach to human genome-wide association studies and can facilitate environment control, statistical power, novel gene discovery, and neurobiological mechanisms. We used interval-specific congenic mouse lines carrying various segments of chromosome 11 from the DBA/2J strain on an isogenic C57BL/6J background to positionally clone a 206 kb QTL (50,185,512-50,391,845 bp) that was causally associated with a reduction in the locomotor stimulant response to methamphetamine (2 mg/kg, i.p.; DBA/2J < C57BL/6J)-a non-contingent, drug-induced behavior that is associated with stimulation of the dopaminergic reward circuitry. This chromosomal region contained only two protein coding genes-heterogeneous nuclear ribonucleoprotein, H1 (Hnrnph1) and RUN and FYVE domain-containing 1 (Rufy1). Transcriptome analysis via mRNA sequencing in the striatum implicated a neurobiological mechanism involving a reduction in mesolimbic innervation and striatal neurotransmission. For instance, Nr4a2 (nuclear receptor subfamily 4, group A, member 2), a transcription factor crucial for midbrain dopaminergic neuron development, exhibited a 2.1-fold decrease in expression (DBA/2J < C57BL/6J; p 4.2 x 10-15). Transcription activator-like effector nucleases (TALENs)-mediated introduction of frameshift deletions in the first coding exon of Hnrnph1, but not Rufy1, recapitulated the reduced methamphetamine behavioral response, thus identifying Hnrnph1 as a quantitative trait gene for methamphetamine sensitivity. These results define a novel contribution of Hnrnph1 to neurobehavioral dysfunction associated with dopaminergic neurotransmission. These findings could have implications for understanding the genetic basis of methamphetamine addiction in humans and the development of novel therapeutics for prevention and treatment of substance abuse and possibly other psychiatric disorders

The heritability of oxycodone reward and concomitant phenotypes in a LG/J x SM/J mouse advanced intercross line

The rewarding property of opioids likely contributes to their abuse potential. Therefore, determining the genetic basis of opioid reward could aid in understanding the neurobiological mechanisms of opioid addiction, provided that it is a heritable trait

Methamphetamine-induced conditioned place preference in LG/J and SM/J mouse strains and an F45/F46 advanced intercross line

The conditioned place preference (CPP) test is frequently used to evaluate the rewarding properties of drugs of abuse in mice. Despite its widespread use in transgenic and knockout experiments, there are few forward genetic studies using CPP to identify novel genes contributing to drug reward. In this study, we tested LG/J and SM/J inbred strains and the parents/offspring of 10 families of an F45/F46 advanced intercross line (AIL) for methamphetamine-induced CPP (MA-CPP) once per week over two weeks. Both LG/J and SM/J mice exhibited significant MA-CPP that was not significantly different between the two strains. Furthermore, LG/J mice showed significantly less acute MA-induced locomotor activity as well as locomotor sensitization following subsequent MA injections. AIL mice (N = 105) segregating LG/J and SM/J alleles also demonstrated significant MA-CPP that was equal in magnitude between the first and second week of training. Importantly, MA-CPP in AIL mice did not correlate with drug-free or MA-induced locomotor activity, indicating that MA-CPP was not confounded by test session activity and implying that MA-CPP is genetically distinct from acute psychomotor sensitivity. We estimated the heritability of MA-CPP and locomotor phenotypes using midparent-offspring regression and maximum likelihood estimates derived from the kinship coefficients of the AIL pedigree. Heritability estimates of MA-CPP were low (0-0.21) and variable (S.E. = 0-0.33) which reflected our poor power to estimate heritability using only 10 midparent-offspring observations. In sum, we established a short-term protocol for MA-CPP in AIL mice that could reveal LG/J and SM/J alleles important for MA reward. The use of highly recombinant genetic populations like AIL should facilitate the identification of these genes and may have implications for understanding psychostimulant abuse in humans

<i>Hnrnph1</i> Is A Quantitative Trait Gene for Methamphetamine Sensitivity

Psychostimulant addiction is a heritable substance use disorder; however its genetic basis is almost entirely unknown. Quantitative trait locus (QTL) mapping in mice offers a complementary approach to human genome-wide association studies and can facilitate environment control, statistical power, novel gene discovery, and neurobiological mechanisms. We used interval-specific congenic mouse lines carrying various segments of chromosome 11 from the DBA/2J strain on an isogenic C57BL/6J background to positionally clone a 206 kb QTL (50,185,512–50,391,845 bp) that was causally associated with a reduction in the locomotor stimulant response to methamphetamine (2 mg/kg, i.p.; DBA/2J −15). Transcription activator-like effector nucleases (TALENs)-mediated introduction of frameshift deletions in the first coding exon of Hnrnph1, but not Rufy1, recapitulated the reduced methamphetamine behavioral response, thus identifying Hnrnph1 as a quantitative trait gene for methamphetamine sensitivity. These results define a novel contribution of Hnrnph1 to neurobehavioral dysfunction associated with dopaminergic neurotransmission. These findings could have implications for understanding the genetic basis of methamphetamine addiction in humans and the development of novel therapeutics for prevention and treatment of substance abuse and possibly other psychiatric disorders

Hnrnph1 Is A Quantitative Trait Gene for Methamphetamine Sensitivity.

Psychostimulant addiction is a heritable substance use disorder; however its genetic basis is almost entirely unknown. Quantitative trait locus (QTL) mapping in mice offers a complementary approach to human genome-wide association studies and can facilitate environment control, statistical power, novel gene discovery, and neurobiological mechanisms. We used interval-specific congenic mouse lines carrying various segments of chromosome 11 from the DBA/2J strain on an isogenic C57BL/6J background to positionally clone a 206 kb QTL (50,185,512-50,391,845 bp) that was causally associated with a reduction in the locomotor stimulant response to methamphetamine (2 mg/kg, i.p.; DBA/2J &lt; C57BL/6J)-a non-contingent, drug-induced behavior that is associated with stimulation of the dopaminergic reward circuitry. This chromosomal region contained only two protein coding genes-heterogeneous nuclear ribonucleoprotein, H1 (Hnrnph1) and RUN and FYVE domain-containing 1 (Rufy1). Transcriptome analysis via mRNA sequencing in the striatum implicated a neurobiological mechanism involving a reduction in mesolimbic innervation and striatal neurotransmission. For instance, Nr4a2 (nuclear receptor subfamily 4, group A, member 2), a transcription factor crucial for midbrain dopaminergic neuron development, exhibited a 2.1-fold decrease in expression (DBA/2J &lt; C57BL/6J; p 4.2 x 10-15). Transcription activator-like effector nucleases (TALENs)-mediated introduction of frameshift deletions in the first coding exon of Hnrnph1, but not Rufy1, recapitulated the reduced methamphetamine behavioral response, thus identifying Hnrnph1 as a quantitative trait gene for methamphetamine sensitivity. These results define a novel contribution of Hnrnph1 to neurobehavioral dysfunction associated with dopaminergic neurotransmission. These findings could have implications for understanding the genetic basis of methamphetamine addiction in humans and the development of novel therapeutics for prevention and treatment of substance abuse and possibly other psychiatric disorders

TALENS-targeted frameshift deletion in <i>Rufy1</i>^+/- mice.

<p><b>(a):</b> A left TALE effector (50,244,600–50,244,616 bp) and a right TALE effector (50,244,569–50,244,585 bp) separated by the <i>Fok</i>I cleavage zone were used to introduce a frameshift deletion that resulted in a premature stop codon in the first coding exon of <i>Rufy1</i> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s019" target="_blank">S8 Fig</a>). <b>(b):</b> A PCR amplicon was generated that captured the <i>Fok</i>I cleavage zone and a single SacII restriction site and was subjected to restriction digest with SacII. <i>Rufy1</i>^+/+ mice contained the SacII restriction site and thus, showed only a single, smaller band. <i>Rufy1</i>^+/- mice showed both the SacII-digested band and a larger, undigested band, indicating the presence of the deletion. <b>(c):</b> There was no effect of genotype or genotype x time interaction in <i>Rufy1</i>^+/- versus <i>Rufy1</i>^+/+ mice from Line #3 on Days 1, 2, or 3 (p > 0.05; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s004" target="_blank">S3 Table</a>). Data are presented as the mean ± S.E.M.</p

QTL for reduced MA sensitivity (D2 < B6) in B6 x D2-F₂ mice.

<p><b>(a):</b> We previously published a genome-wide a significant QTL on chromosome 11 for MA-induced locomotor activity from the same B6 x D2-F₂ dataset (N = 676) that was significant when the data were summed from 15–30 min and 0–30 min but not when the data were summed from 0–15 min [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.ref020" target="_blank">20</a>]. To further dissect the time dependency of this locus, we generated LOD scores from the same mice in six, 5-min time bins over 30 min. The x-axis represents the physical distance (Mb) of the marker on chromosome 11 (mm9). The y-axis represents the LOD score. The dashed, horizontal line represents the genome-wide significance level derived from 1,000 permutations. The dark blue QTL trace (5 min) denotes a distal locus (90 Mb) in which inheritance of the D2 allele caused an <i>increase</i> in locomotor activity relative to the B6 allele that was most likely not associated with MA treatment (see QTL for Days 1 and 2 in response to saline; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s012" target="_blank">S1 Fig</a>; D2 > B6). The remaining red- and pink-shaded QTL traces denote a separate locus (50 Mb) that was specific for MA treatment on Day 3 in which inheritance of the D2 allele caused a <i>decrease</i> in MA-induced locomotor activity. The dashed QTL trace indicates the time bin containing the peak LOD score. (<b>b):</b> The effect plot for the marker nearest the peak LOD score is shown for the six, 5-min time bins. Data are sorted by genotype for each time bin. The time bin with the most significant LOD score is circled. B6 = homozygous for B6 allele (circles); H = heterozygous (triangles); D2 = homozygous for D2 allele (colored squares). Data are presented as the mean ± S.E.M.</p

TALENS-targeted frameshift deletions in <i>Hnrnph</i>1 ^+/- mice reveal <i>Hnrnph1</i> as a quantitative trait gene for MA sensitivity.

<p><b>(a):</b> Left TAL effector (50,191,867–50,191,883 bp) and right TAL effector (50,191,899–50,191,915 bp) separated by the <i>Fok</i>I cleavage zone were used to introduce frameshift deletions in the first coding exon of <i>Hnrnph1</i> (exon 4) that resulted in premature stop codons (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s019" target="_blank">S8 Fig</a>). Founder #28 contained a 16 bp deletion and Founder #22 contained an 11 bp deletion. (<b>b):</b> A PCR amplicon capturing the <i>Fok</i>I cleavage zone was digested with BstNI. <i>Hnrnph1</i>^+/+ mice contained two copies of a functional BstNI restriction site and thus, restriction digest produced a single band containing digested fragments of equal size. <i>Hnrnph1</i>^+/- mice were heterozygous for a deletion of the BstNI site and showed both the digested band and a larger, undigested band. Gel band lanes were cropped and re-ordered to present wild-type first (+/+) followed by B6 control, and heterozygous samples (+/-). (<b>c):</b> There was a significant upregulation of total <i>Hnrnph1</i> transcript levels in <i>Hnrnph1</i>^+/- mice as indicated by cDNA amplification using qPCR primers spanning exons 4–5 that hybridized to both genotypes (t₆ = 5.69; p = 0.0013). (<b>d):</b> An upregulation of total <i>Hnrnph1</i> transcript levels was also indicated by cDNA amplification using qPCR primers spanning untargeted exons 6–7 (t₆ = 8.53; p = 0.00014). <b>(e):</b> A significant downregulation of the <i>Hnrnph1</i>^+/+ transcript levels was observed in <i>Hnrnph1</i>^+/- mice that was indicated by cDNA amplification using primers spanning exons 4–5, one of which hybridized to the deleted <i>Hnrnph1</i>^+/+ sequence (t₆ = 9.45; p = 0.00091; Fig 5e). *p < 0.05. <b>(f):</b> In Line #28, there was no effect of genotype on locomotor activity in response to saline (SAL) on Days 1 or 2 (left, middle panels). On Day 3, <i>Hnrnph1</i>^+/- mice from Line #28 heterozygotes showed a significant reduction in MA-induced locomotor activity compared to <i>Hnrnph1</i>^+/+ littermates (right panel). (<b>g):</b> In Line #22, there was no effect of genotype on locomotor activity in response to SAL on Days 1 or 2 (left, middle panels). On Day 3, <i>Hnrnph1</i>^+/- mice from Line #22 showed significantly reduced MA-induced locomotor activity compared to <i>Hnrnph1</i>^+/+ littermates. Data are presented as the mean ± S.E.M. * = significant genotype x time interaction followed by unpaired t-tests of individual time bins (p < 0.05; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s004" target="_blank">S3 Table</a>; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s001" target="_blank">S1 Text</a>; Supplementary Information).</p

Congenic analysis identifies Line 4 for fine mapping.

<p>Statistical results are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s004" target="_blank">S3 Table</a> and described in the Supplementary Information. <b>(a):</b> Lines 1–6 possessed either one (heterozygous; “H”) or two copies (homozygous, “D2”) of a chromosome 11 interval from the D2 inbred strain (gray region) on an isogenic B6 background (black region; denotes the genotype for the rest of the genome). The white regions represent transitional regions that were not genotyped. The x-axis represents the physical position (Mb) of the SNP marker. The SNP markers that were used to genotype Lines 1–6 are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s002" target="_blank">S1 Table</a>. The y-axis represents the LOD score for the F₂-derived QTL that was causally associated with reduced MA sensitivity on Day 3 (D2 < B6; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.g001" target="_blank">Fig 1</a>; 25 min bin). (+) = congenic line captured the QTL for reduced MA sensitivity on Day 3. (-) = congenic line failed to capture the QTL. <b>(b-e):</b> The three columns represent the phenotypes for Days 1, 2, and 3. The four rows represent Lines 1–4. The negative results for Lines 5 and 6 (-) are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s013" target="_blank">S2 Fig</a> and described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005713#pgen.1005713.s001" target="_blank">S1 Text</a>. <b>“*”</b> indicates a dominant effect of the D2 allele (D2 = H < B6) or H < B6. <b>“$”</b> indicates an additive effect (D2 < H < B6). <b>“#”</b> indicates a recessive effect (D2 < H = B6). “<b>%</b>” indicates that B6 and D2 differ from each other but not from H. “<b>&</b>” indicates that H and D2 differ from each other but not from B6. Data are represented as the mean ± S.E.M. p < 0.05 was considered significant. We estimated the narrow-sense heritability of the QTLs (h²) for Line 3 and Line 4 (25 min) based on the intraclass correlation coefficient using the phenotypic variances from homozygous D2 versus homozygous B6 mice according to the following formula: h² = (between-genotype variance) / (between-genotype variance + within-genotype variance). For Line 3, h² = 0.35; for Line 4, h² = 0.08. Although these h² estimates do not contain confidence intervals, the differences in h² values combined with the different modes of inheritance suggest that Line 3 and Line 4 possess different QTLs.</p