Plasticity-associated gene Krox24/Zif268 is required for long-lasting behavioral effects of cocaine

Abstract

The extracellular signal-regulated kinases (ERKs) 1/2 pathway is stimulated by drugs of abuse in striatal neurons through coincident activation of dopamine D1 and glutamate NMDA receptors and is critical for long-lasting behavioral effects of these drugs. Although regulation of transcription is a major target of ERK, the precise mechanisms by which it contributes to behavioral alterations is not known. We examined the role of Zif268, an immediate-early gene induced by drugs of abuse under the control of ERK, in behavioral responses to cocaine using knock-in mutant mice in which Zif268 was replaced by LacZ. No biochemical or behavioral differences between mutant and wild-type mice were observed in basal conditions or in acute responses to cocaine injection. In contrast, locomotor sensitization to single or repeated cocaine injections was dramatically diminished in both heterozygous and homozygous Zif268 mutant mice. Conditioned place preference in response to cocaine was prevented in Zif268-deficient mice. This effect was not attributable to a general learning deficit because the mutant mice displayed normal conditioned place preference when food was used as reward. Our results provide direct genetic evidence for the requirement of Zif268 for long-lasting association of environmental context with specific behavioral responses after short exposures to cocaine. They also underline the common molecular machinery involved in long-lasting drug-induced behavioral alterations and the formation of other types of memory.

Figure 1.

Normal gene induction by cocaine in Zif268^−/− mice. a, β-Galactosidase immunostaining in knock-in mutant mice (Zif268^−/−) in which LacZ replaces Zif268 90 min after acute administration of saline or cocaine (20 mg/kg). No immunostaining was detected in wild-type littermates (Zif268^+/+). Scale bars, 40 μm. b, c-fos induction was quantified in the DStr and in the shell of the NAcc in the same conditions in Zif268^−/− and Zif268^+/+ mice. Data (means ± SEM; n = 3 per group) were analyzed using two-way ANOVA: effect of treatment, DStr, F_(1,8) = 38.55, p < 0.01; shell, F_(1,8) = 82.38, p < 0.01; effect of genotype, DStr, F_(1,8) = 0.171, not significant (NS); shell, F_(1,8) = 2.054, NS. Post hoc comparison (Bonferroni's test), ∗∗p < 0.01 saline versus cocaine.

Figure 2.

Effects of chronic cocaine treatment on the induction of β-galactosidase in the dorsal striatum and nucleus accumbens shell of Zif268^+/− mice. The induction of β-galactosidase was tested in Zif268^+/− mice in which an allele of Zif268 was replaced by LacZ. Naive mice (Naive) received an acute injection of saline (Sal) or cocaine (20 mg/kg; Coc), as indicated. Pretreated mice (Chronic Coc) were injected daily for five consecutive days with cocaine (20 mg/kg) and, after a 7 d withdrawal, received an additional injection of saline or cocaine (20 mg/kg). β-Galactosidase-immunoreactive cells (as in Fig. 1a) were quantified in DStr and nucleus accumbens shell using an image analyzer (Image Pro Plus). Data are means ± SEM (n = 3 mice per group) and were analyzed using one-way ANOVA: effect of treatment in the DStr, F_(3,11) = 11.20, p < 0.001; NAcc, F_(3,11) = 10.76, p < 0.001. Post hoc comparison (Newman–Keuls test), ∗p < 0.01 saline versus cocaine; °p < 0.05 cocaine versus chronic cocaine.

Figure 3.

Alteration of cocaine-induced locomotor sensitization in Zif268 mutant mice. Locomotor sensitization after a single injection of cocaine (a, b). a, Time course of locomotor activity in Zif268^+/+, Zif268^+/−, and Zif268^−/− mice after the first injection of cocaine (20 mg/kg; 1st inj) and a second injection 7 d later (Test inj). b, Total activity during 60 min. Data (means ± SEM, n = 6–8 per group) were analyzed using two-way ANOVA: effect of treatment, F_(1,34) = 118.4, p < 0.01; effect of genotype, F_(2,34) = 27.26, p < 0.001. Post hoc comparison (Bonferroni's test), ∗p < 0.05, ∗∗p < 0.001 test versus first injection; °p < 0.01 mutant versus wild type. c, Locomotor sensitization to repeated cocaine injections. After 3 d of habituation, Zif268^+/+ and Zif268^+/− mice were injected daily for 5 consecutive days with cocaine (10 and 20 mg/kg; n = 10–11 per group) and, after a 7 d withdrawal, received an additional injection of cocaine on day 15. Sal, Saline. Data were analyzed with a mixed-factor ANOVA (repeated measure over time): 10 mg/kg cocaine: effect of time, F_(8,171) = 17.23, p < 0.01; effect of genotype, F_(1,171) = 42.35, p < 0.01; 20 mg/kg cocaine: effect of time, F_(8,177) = 25.80, p < 0.01; effect of genotype, F_(1,177) = 66.15, p < 0.01. Post hoc comparison (Bonferroni's test), ∗p < 0.01 later days versus day4; °p < 0.01 Zif268^+/+ versus Zif268^+/−.

Figure 4.

Absence of cocaine-induced, but not food-induced, conditioned place preference in Zif268^−/− mice. a, Cocaine CPP. Before conditioning (Preconditioning), mice showed no initial preference for either side in any of the experimental groups (effect of treatment, F_(1,34) = 0.02, NS; effect of genotype, F_(2,34) = 0.12, NS). After conditioning (Postconditioning), Zif268^+/+ and Zif268^+/−, but not Zif268^−/−, mice developed a significant place preference for the cocaine-paired side (effect of treatment, F_(1,34) = 10.02, p < 0.01; effect of genotype, F_(2,34) = 2.46, NS). Data are means ± SEM (n = 6–8 per group). Bonferroni's test, ∗p < 0.05 cocaine versus saline; °p < 0.01 Zif268^−/− versus Zif268^+/+. b, Food-induced CPP. Before conditioning, no group of mice showed any initial preference for either side (Preconditioning) (effect of treatment, F_(1,24) = 0.53, NS; effect of genotype, F_(1,24) = 0.07, NS). After conditioning (Postconditioning), Zif268^+/+ and Zif268^−/− mice showed a significant place preference for the food-paired side (effect of treatment, F_(1,24) = 25.39, p < 0.001; effect of genotype, F_(1,24) = 0.62, NS). Data are means ± SEM (n = 6–8 per group). ∗p < 0.01 food versus no food.