Al samples of 72 EuAm individuals with no rs71534387 allele > 203 bp vs 96 individuals with at least one rs71534387 allele > 203 bp. Triplicate RT-PCR assays. Relative expression was determined by the mean of two CSMD1 amplimers in relation to the geometric mean of three control mRNAs from the same sample. doi:10.1371/journal.pone.0120908.gMiceInitial characterization of mice on mixed genetic backgrounds. Heterozygote x heterozygote crosses of mice on mixed genetic backgrounds supplied by Taconic produced offspring with genotypes in expected Mendelian ratios that displayed differences in coat colors; different mice had agouti, black or white coat colors. Initial tests of mice of these mixed genetic backgrounds revealed no significant influences of genotype on locomotion after injections of saline or 10 mg/kg cocaine doses (Table A in S2 Table; ANCOVA p = 0.729 and 0.650, respectively) or performance on the rotorod test of motor coordination/motor learning (Table B in S2 Table; ANCOVA Quisinostat site effect of genotype p = 0.771). Characterization of mice on C57Bl genetic backgrounds. We thus backcrossed csmd1 knockout mice of the original mixed genetic background for 5 generations to mice with C57Bl/ 6J genetic backgrounds as noted above, producing backcrossed csmd1 knockouts that we termPLOS ONE | DOI:10.1371/journal.pone.0120908 July 14,6 /CSMD1 Variants and AddictionFig 2. Cocaine conditioned place preference among mice with different CSMD1 genotypes. Mean difference ?SEM in time spent on the cocainepaired side (Y axis) before and after conditioning with different doses of cocaine for wildtype heterozygous and homozygote knockout mice (ANCOVA effect of genotype p = 0.024; n = 13?6 mice of each genotype for each dose). Data from male and female mice are combined since gender displayed no significant interaction with genotype. doi:10.1371/journal.pone.0120908.gcsmd1 knockouts in the rest of this paper. There was significance for the modest differences in weights for the knockout males but not for the females. Weights for male/female were wildtype: 28.6/21.3 g; heterozygotes 28.6/21.5 g and homozygotes 27.1/20.7 g, n = 39?5/genotype, (Table C in S2 Table; ANCOVA with age as covariate p = 0.019 for males and 0.237 for females). Since there were no significant sex ?genotype interactions, the small, sex-specific differences did not provide effects on behavioral SP600125 cost results of the knockout. These backcrossed mice were subjected to a number of physiologic, pharmacological and behavioral tests. The csmd1 +/- and-/- knockouts displayed no evidence for gross alterations in motor function. They were similar to wildtype littermates in screen hang time and rotarod testing (Tables D and E in S2 Table; ANCOVA p values for effects of genotype 0.346 and 0.402, respectively). csmd1 knockout mice failed to display significant differences from their wild type siblings in the amounts of time spent in the center of an open field or their latencies before emerging from a dark box (Table F in S2 Table; ANCOVA p = 0.216 and 0.263, respectively).Cocaine conditioned place preferenceIn wildtype mice, cocaine-conditioned place preference (CPP) was maximal at 5?0 mg/kg doses, as we and others have observed in studies of many other knockout mouse strains (Fig 2; Table G in S2 Table) [33]. There was a significant main effect of csmd1 genotype (p = 0.024).PLOS ONE | DOI:10.1371/journal.pone.0120908 July 14,7 /CSMD1 Variants and AddictionFig 3. Locomotion and habituation in a novel 42 x.Al samples of 72 EuAm individuals with no rs71534387 allele > 203 bp vs 96 individuals with at least one rs71534387 allele > 203 bp. Triplicate RT-PCR assays. Relative expression was determined by the mean of two CSMD1 amplimers in relation to the geometric mean of three control mRNAs from the same sample. doi:10.1371/journal.pone.0120908.gMiceInitial characterization of mice on mixed genetic backgrounds. Heterozygote x heterozygote crosses of mice on mixed genetic backgrounds supplied by Taconic produced offspring with genotypes in expected Mendelian ratios that displayed differences in coat colors; different mice had agouti, black or white coat colors. Initial tests of mice of these mixed genetic backgrounds revealed no significant influences of genotype on locomotion after injections of saline or 10 mg/kg cocaine doses (Table A in S2 Table; ANCOVA p = 0.729 and 0.650, respectively) or performance on the rotorod test of motor coordination/motor learning (Table B in S2 Table; ANCOVA effect of genotype p = 0.771). Characterization of mice on C57Bl genetic backgrounds. We thus backcrossed csmd1 knockout mice of the original mixed genetic background for 5 generations to mice with C57Bl/ 6J genetic backgrounds as noted above, producing backcrossed csmd1 knockouts that we termPLOS ONE | DOI:10.1371/journal.pone.0120908 July 14,6 /CSMD1 Variants and AddictionFig 2. Cocaine conditioned place preference among mice with different CSMD1 genotypes. Mean difference ?SEM in time spent on the cocainepaired side (Y axis) before and after conditioning with different doses of cocaine for wildtype heterozygous and homozygote knockout mice (ANCOVA effect of genotype p = 0.024; n = 13?6 mice of each genotype for each dose). Data from male and female mice are combined since gender displayed no significant interaction with genotype. doi:10.1371/journal.pone.0120908.gcsmd1 knockouts in the rest of this paper. There was significance for the modest differences in weights for the knockout males but not for the females. Weights for male/female were wildtype: 28.6/21.3 g; heterozygotes 28.6/21.5 g and homozygotes 27.1/20.7 g, n = 39?5/genotype, (Table C in S2 Table; ANCOVA with age as covariate p = 0.019 for males and 0.237 for females). Since there were no significant sex ?genotype interactions, the small, sex-specific differences did not provide effects on behavioral results of the knockout. These backcrossed mice were subjected to a number of physiologic, pharmacological and behavioral tests. The csmd1 +/- and-/- knockouts displayed no evidence for gross alterations in motor function. They were similar to wildtype littermates in screen hang time and rotarod testing (Tables D and E in S2 Table; ANCOVA p values for effects of genotype 0.346 and 0.402, respectively). csmd1 knockout mice failed to display significant differences from their wild type siblings in the amounts of time spent in the center of an open field or their latencies before emerging from a dark box (Table F in S2 Table; ANCOVA p = 0.216 and 0.263, respectively).Cocaine conditioned place preferenceIn wildtype mice, cocaine-conditioned place preference (CPP) was maximal at 5?0 mg/kg doses, as we and others have observed in studies of many other knockout mouse strains (Fig 2; Table G in S2 Table) [33]. There was a significant main effect of csmd1 genotype (p = 0.024).PLOS ONE | DOI:10.1371/journal.pone.0120908 July 14,7 /CSMD1 Variants and AddictionFig 3. Locomotion and habituation in a novel 42 x.