Altered CSMD1 Expression Alters Cocaine-Conditioned Place Preference: Mutual Support for a Complex Locus from Human and Mouse Models

Abstract

The CUB and sushi multiple domains 1 (CSMD1) gene harbors signals provided by clusters of nearby SNPs with 10-2 > p > 10-8 associations in genome wide association (GWAS) studies of addiction-related phenotypes. A CSMD1 intron 3 SNP displays p < 10-8 association with schizophrenia and more modest associations with individual differences in performance on tests of cognitive abilities. CSDM1 encodes a cell adhesion molecule likely to influence development, connections and plasticity of brain circuits in which it is expressed. We tested association between CSMD1 genotypes and expression of its mRNA in postmortem human brains (n = 181). Expression of CSMD1 mRNA in human postmortem cerebral cortical samples differs 15-25%, in individuals with different alleles of simple sequence length and SNP polymorphisms located in the gene's third/fifth introns, providing nominal though not Bonferroni-corrected significance. These data support mice with altered CSMD1 expression as models for common human CSMD1 allelic variation. We tested baseline and/or cocaine-evoked addiction, emotion, motor and memory-related behaviors in +/- and -/- csmd1 knockout mice on mixed and on C57-backcrossed genetic backgrounds. Initial csmd1 knockout mice on mixed genetic backgrounds displayed a variety of coat colors and sizable individual differences in responses during behavioral testing. Backcrossed mice displayed uniform black coat colors. Cocaine conditioned place preference testing revealed significant influences of genotype (p = 0.02). Homozygote knockouts displayed poorer performance on aspects of the Morris water maze task. They displayed increased locomotion in some, though not all, environments. The combined data thus support roles for common level-of-expression CSMD1 variation in a drug reward phenotype relevant to addiction and in cognitive differences that might be relevant to schizophrenia. Mouse model results can complement data from human association findings of modest magnitude that identify likely polygenic influences.

Fig 1. Differential expression of CSMD1 mRNA in cerebral cortical samples from individuals with different rs71534387 SSLP genotypes.

Mean +/- standard error of the mean (SEM) of relative expression of CSMD1 mRNA in cerebral cortical 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.

Fig 2. Cocaine conditioned place preference among mice with different CSMD1 genotypes.

Mean difference ± SEM in time spent on the cocaine-paired 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 = 13–26 mice of each genotype for each dose). Data from male and female mice are combined since gender displayed no significant interaction with genotype.

Fig 3. Locomotion and habituation in a novel 42 x 42 cm chamber: no significant differences between mice with different CSMD1 genotypes.

Values are mean +/- SEM of the number of meters traveled when mice were exposed for the first time to the 42 x 42 cm apparatus (n = 10/genotype, ANOVA p = 0.967).

Fig 4. CSMD1 knockouts display modestly increased locomotion in a brightly lit environment.

Values are mean +/- SEM of the number of cm traveled during the first pretest, in which mice were exposed for the first time to the CPP apparatus and allowed to explore both sides (n = 48/genotype, ANCOVA p = 3.7 x 10⁻¹³).

Fig 5. CSMD1 knockouts display differences in locomotion during the 20 min conditioning sessions when confined to 20 x 20 cm portions of the conditioned place preference boxes after receiving their first saline or cocaine injections.

Saline-injected CSMD1 knockouts displayed significantly increased locomotion (p = 0.002) in comparison to mice of other genotypes. Significant effects of cocaine dose and gender were identified in ANCOVA of data from cocaine-injected mice of all genotypes (p = 5.5 x 10⁻¹⁵ and 0.009, respectively). There was a trend towards genotype * dose interaction (p = 0.054). Values are mean +/- SEM of the number of m traveled.

Fig 6. Morris water maze performance: time to reach platform in mice with different CSMD1 genotypes.

Main Fig: Averaged daily latencies ± SEM to reach the Morris water maze platform for wildtype, heterozygous and homozygous CSMD1 knockout mice (repeated measures ANOVA genotype * day interaction p = 0.004 for learning. There were no significant differences in swimming speed (see text). Inset: More detailed data showing latencies to reach the (visible) platform during the first four trials (day 1). The trend for poorer performance in the homozygous knockouts reached the margin of statistical significance (p = 0.058).