Casein kinase 1-epsilon deletion increases mu opioid receptor-dependent behaviors and binge eating1

Abstract

Genetic and pharmacological studies indicate that casein kinase 1 epsilon (Csnk1e) contributes to psychostimulant, opioid, and ethanol motivated behaviors. We previously used pharmacological inhibition to demonstrate that Csnk1e negatively regulates the locomotor stimulant properties of opioids and psychostimulants. Here, we tested the hypothesis that Csnk1e negatively regulates opioid and psychostimulant reward using genetic inhibition and the conditioned place preference assay in Csnk1e knockout mice. Similar to pharmacological inhibition, Csnk1e knockout mice showed enhanced opioid-induced locomotor activity with the mu opioid receptor agonist fentanyl (0.2 mg/kg i.p.) as well as enhanced sensitivity to low-dose fentanyl reward (0.05 mg/kg). Interestingly, female knockout mice also showed a markedly greater escalation in consumption of sweetened palatable food - a behavioral pattern consistent with binge eating that also depends on mu opioid receptor activation. No difference was observed in fentanyl analgesia in the 52.5°C hot plate assay (0-0.4 mg/kg), naloxone conditioned place aversion (4 mg/kg), or methamphetamine conditioned place preference (0-4 mg/kg). To identify molecular adaptations associated with increased drug and food behaviors in knockout mice, we completed transcriptome analysis via mRNA sequencing of the striatum. Enrichment analysis identified terms associated with myelination and axon guidance and pathway analysis identified a differentially expressed gene set predicted to be regulated by the Wnt signaling transcription factor, Tcf7l2. To summarize, Csnk1e deletion increased mu opioid receptor-dependent behaviors, supporting previous studies indicating an endogenous negative regulatory role of Csnk1e in opioid behavior.

Keywords: CK-1; CPP; QTL; Addiction; binge eating; candidate gene; casein kinase; pavlovian conditioning; reward; sex differences; substance use disorder.

Figure 1. Increased opioid and psychostimulant sensitivity in Csnk1e knockout mice

(a,b): Distance traveled in the three day locomotor activity paradigm is shown for the response to saline on Days 1 and 2 (SAL; D1, D2). To increase our power in detecting an effect of Genotype for D1 and D2, we collapsed across eventual Treatment assignment and identified an increase in locomotor activity in KO mice on D1 and D2 (see Fig. S1a,b). (c): On Day 3 (D3), FENT-treated KO mice exhibited increased locomotor activity relative to FENT-treated WT mice. (d, e): Distance traveled in the three day locomotor activity paradigm in response to SAL on D1 and D2. To increase our power in detecting an effect of Genotype for D1 and D2, we collapsed across eventual Treatment assignment and identified an increase in locomotor activity in KO mice on D1 but not on D2 (see Fig. S1c,d). (f): On D3, there was no significant effect of Csnk1e deletion on MA-induced locomotor activity. Data are represented as the mean ± SEM; KO= Csnk1e knockout, WT= wild-type littermates, SAL= saline, FENT= fentanyl, MA= methamphetamine, m= meters, min=minutes.

Figure 2. Increased opioid reward in Csnk1e knockout mice

(a): KO mice exhibited increased FENT-CPP at the 0.05 mg/kg dose relative to WT mice. (b): In contrast, there was no effect of Genotype on MA-CPP. Data are represented as the mean ± SEM, *=p<0.05; Reward was measured via change in time spent on the drug-paired side on D8 versus D1), KO= Csnk1e knockout, WT=wild-type, FENT= fentanyl, MA=methamphetamine, m= meters, s= seconds, i.p.=intraperitoneal.

Figure 3. Female-specific increase in SPF consumption in Csnk1e knockout mice

Because there was an interaction between Genotype and Sex, we present separate analyses for females (a, c, e) and males (b, d, f). (a, b): SPF consumption (% BW) was analyzed across training days and using summed SPF consumption (inset). Female KO mice consumed more SPF relative to female WT. For males, there was no effect of Genotype. (c, d): Slope analysis was completed for SPF consumption across training days. Female KO mice exhibited a significantly greater slope in escalation of SPF consumption that female WT mice (p = 0.005). Top slope value (m=) indicates KO slope, bottom slope value (m=) indicates WT slope. (e, f): Change in time spent of the SPF-paired side (SPF-CPP) was analyzed across assessment days and also using summed SPF-CPP (inset) Female KO mice showed increased SPF-CPP relative to WT mice. Data in a, b, e, and f are represented as the mean ± SEM, *=p<0.05; SPF= sweetened palatable food, % BW= represented as a percentage of body weight, CPP= conditioned place preference (change in time on the SPF-paired side relative to D1), KO= Csnk1e knockout, WT=wild-type, D# - D1 = D8-D1, D15-D1, D22-D1.

Figure 4. “Nervous System Development and Function, Cellular Development, Cellular Growth and Proliferation” IPA network includes top upstream regulator

(a, b): A top IPA network includes TCF7L2 as a hub, with 5 down-regulated genes (green) and 28 up-regulated genes (red) (Score=31). Genes in the network diagram that lack any color were included by the IPA algorithm to facilitate connectivity. IPA=Ingenuity Pathway Analysis, P=p-value, FC=fold-change, FDR= false discovery rate.

Figure 5. Top regulator effect network predicts increased differentiation of neuroglia and decreased hypomyelination

(a, b): Regulator effect networks link upstream regulators with downstream gene expression changes, and hypothesized phenotypic effects. Upstream regulators, FYC and MYOC, affect the expression of 5 genes (CNP, PTEN, PLP1, POU3F1, MBP) which lead to increased differentiation of neuroglia and decreased hypomyelination. P=p-value, FC=fold-change, FDR= false discovery rate.