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.