Neuronal scaffolding protein spinophilin is integral for cocaine-induced behavioral sensitization and ERK1/2 activation

Abstract

Spinophilin is a scaffolding protein enriched in dendritic spines with integral roles in the regulation of spine density and morphology, and the modulation of synaptic plasticity. The ability of spinophilin to alter synaptic strength appears to involve its scaffolding of key synaptic proteins, including the important structural element F-actin, AMPA/NMDA modulator protein phosphatase 1, and neuromodulatory G-protein coupled receptors, including dopamine receptor D2 and metabotropic glutamate receptor 5. Additionally, spinophilin is highly expressed in the striatum, a brain region that is fundamentally involved in reward-processing and locomotor activity which receives both glutamatergic and dopaminergic inputs. Therefore, we aimed to investigate the role of spinophilin in behavioral responses to cocaine, evaluating wild-type and spinophilin knockout mice followed by the examination of underlying molecular alterations. Although acute locomotor response was not affected, deletion of spinophilin blocked the development and expression of behavioral sensitization to cocaine while maintaining normal conditioned place preference. This behavioral alteration in spinophilin knockout mice was accompanied by attenuated c-Fos and ∆FosB expression following cocaine administration and blunted cocaine-induced phosphorylation of ERK1/2 in the striatum, with no change in other relevant signaling molecules. Therefore, we suggest spinophilin fulfills an essential role in cocaine-induced behavioral sensitization, likely via ERK1/2 phosphorylation and induction of c-Fos and ∆FosB in the striatum, a mechanism that may underlie specific processes in cocaine addiction.

Keywords: Behavioral sensitization; Cocaine; Drug addiction; Spinophilin.

Fig. 1

Experimental design. Wild-type and spinophilin-KO mice were submitted to either the behavioral sensitization or the condition place preference paradigm. After behavioral testing, mice had their brains dissected for biochemical assays or collected for immunostaining

Fig. 2

Spinophilin-KO effects on cocaine-related behaviors. a Locomotor responses to repeated cocaine injections in wild-type and spinophilin-KO mice. b Acute hyperlocomotion induced by cocaine in wild-type and spinophilin-KO mice. c Behavioral sensitization to cocaine treatment in wild-type and spinophilin-KO mice. d Stereotypic rotation behavior during the challenge test calculated by automated analysis. e Stereotypy (including grooming, sniffing, rearing and head bobbing) on the challenge day analyzed by a blinder experimenter. f Conditioned place preference to cocaine in wild-type and spinophilin-KO mice. g Representative western blot confirming the knockout of spinophilin. Data expressed as mean ± SEM. Two-way ANOVA with repeated measures followed by Tukey post-hoc, n = 10–13 per group. **p < 0.01, ***p < 0.001

Fig. 3

CTEP co-treatment affect acute locomotor response but not behavioral sensitization to cocaine in spinophilin-KO mice. a Locomotor responses to repeated co-treatment with cocaine and CTEP in wild-type and spinophilin-KO mice. b Acute hyperlocomotion induced by cocaine and CTEP co-treatment in wild-type and spinophilin-KO mice. c Behavioral sensitization to cocaine and CTEP co-treatment in wild-type and spinophilin-KO mice. d Conditioned place preference to cocaine and CTEP co-treatment in wild-type and spinophilin-KO mice. Data expressed as mean ± SEM. Two-way ANOVA with repeated measures followed by Tukey post-hoc, n = 10–13 per group. **p < 0.01, ***p < 0.001

Fig. 4

Spinophilin deletion affects cocaine induction of Fos family IEGs. a Representative images for immunostaining of c-Fos in the striatum of wild-type and spinophilin-KO mice. Quantification of cocaine-induced expression of b) c-Fos and c) ∆Fosb mRNA levels in wild-type and spinophilin-KO mice. Data expressed as mean ± SEM. Two-way ANOVA followed by Tukey post-hoc. *p < 0.05 for the indicated comparisons, ^###p < 0.001 compared to all other groups and ^&&&p < 0.001 compared to saline-WT and saline-KO

Fig. 5

Cocaine effects on gene expression in the striatum. Cocaine-induced changes in a) D1R, b) D2R, c) mGluR5, d) NR2A and e) NR2B mRNA levels in wild-type and spinophilin-KO mice. Data expressed as mean ± SEM. Two-way ANOVA followed by Tukey post-hoc, n = 6–8 per group. *p < 0.05

Fig. 6

Effects of cocaine on dopamine and glutamate receptors expression in the striatum. a Representative images of immunoblots for D1R, D2R, mGluR5, NR2A, NR2B, PSD95, tyrosine hydroxylase (TH), GAPDH and spinophilin protein expression in the striatum of wild-type and spinophilin-KO mice. Effect of cocaine treatment on b) D1R, c) D2R, d) mGluR5 e) NR2A, f) NR2B, g)PSD95 and h) tyrosine hydroxylase protein expression in wild-type and spinophilin-KO mice. Data expressed as mean ± SEM. Two-way ANOVA followed by Tukey post-hoc, n = 6–8 per group. *p < 0.05

Fig. 7

Cocaine treatment differentially affect signalling pathways in WT and spinophilin-KO mice. a Representative images of immunoblots for pERK, ERK, pAkt, Akt, pGSK3β, GSK3β, pmTOR, mTOR, and pTH in the striatum of wild-type and spinophilin-KO mice. Quantification of cocaine-induced changes in b) pERK, c) pAkt, d) pGSK3β, e) pmTOR, and f) pTH in wild-type and spinophilin-KO mice. Data expressed as mean ± SEM. Two-way ANOVA followed by Tukey post-hoc, n = 6–8 per group. *p < 0.05