FIGURE LEGENDS
Figure 1. Behavioral tests for phenotype characterization. (A) Timeline of the experimental sequence. (B, C) Short-term memory measured in the V-maze for novel object recognition task, (B) discrimination index and (C) exploration. (D) Locomotor and (E) rearing activity measured in actimetry boxes. (F) Anxiety-like behavior measured by the time spent in the open arms and (G) the percentage of time in the open arms in the elevated plus maze. Motor coordination measured in the rota rod test by the (H) number of r.p.m. that the mice performed in the average trials or the (I) mean time they remain in the apparatus. All data are expressed in median and interquartile range and individual data are shown (WT n=25; Plcb1+/- n=12).
Figure 2. Operant conditioning maintained by cocaine inPlcb1+/-, wild-type (WT) and corresponding control saline mice.(A) Timeline of the experimental sequence. (B) The number of cocaine infusions (0.5 mg/kg/infusion) in both genotypes increased progressively across sessions during FR1 (repeated measures ANOVA, interaction between genotype x drug x sessions, P <0.05) and remained stable during FR3 and higher in mice trained with cocaine (repeated measures ANOVA, main effect of drug, ***P <0.001). The maximum number of infusions was reached on session 5 in both genotypes and was slightly superior in Plcb1+/- (36.35±1.47) than in WT (29.61±2.23) but post hoc analyses demonstrated that this difference was not significant. (C) The motivation for cocaine measured by the breaking point achieved in the progressive ratio schedule of reinforcement (4h) was equivalent in both genotypes trained with cocaine but higher than those trained with saline independently of the genotype (individual data with interquartile range, U Mann-Whitney, ##P <0.01 vs WT cocaine, $$P <0.01 vs Plcb1+/- cocaine). (D) Both genotypes also showed similar levels of extinction that decreased during sessions, but the curve was more pronounced in WT mice than in mutants (repeated measures ANOVA, interaction genotype x sessions, &&&P <0.001). (E) Decreased cue-induced reinstatement of cocaine seeking inPlcb1+/- mice was obtained compared to WT (individual data with interquartile range, U Mann-Whitney, #P <0.05, ##P <0.01 vs WT cocaine). All data are expressed in mean ± SEM when sessions are represented or median and interquartile range when individual data are shown (WT cocaine n=28-36;Plcb1+/- cocaine n=19-26; saline n=6 per genotype). Statistical details are included in Supplementary Table S1.
Figure 3. Gene expression changes in mPFC after cue-induced reinstatement of cocaine seeking in Plcb1+/- versus wild-type (WT) mice. (A) Selection of over-represented KEGG pathways (Kyoto Encyclopedia of Genes and Genomes) and (B) GO (Gene Ontology) identified by DAVID software among the differentially expressed genes. The number of genes with altered expression included in each category is indicated on the right side of the bar. In red, relevant pathways for the addictive process. (C) Gene network involved in cellular development, cellular growth and proliferation, nervous system development and function (score = 62). The green nodes in the pathway indicate genes with downregulated expression in Plcb1 +/- identified in RNAseq.
Figure 4. Alterations in expression in the dopaminergic synapse in mPFC of Plcb1+/- mice after cue-induced reinstatement of cocaine seeking. Adapted from KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways (mmu04728). Enriched pathway in differentially expressed genes in mPFC, 25 out of 131 genes in the pathway were differently expressed (Praw =2e-03;Pajd =0.02). In red, upregulated genes and in green, downregulated genes in mPFC. *Protein complexes including upregulated and downregulated genes. Correspondence between genes and proteins can be found in Supplementary Table S6.