Methylphenidate-induced dendritic spine formation and DeltaFosB expression in nucleus accumbens

Abstract

Methylphenidate is the psychostimulant medication most commonly prescribed to treat attention deficit hyperactivity disorder (ADHD). Recent trends in the high usage of methylphenidate for both therapeutic and nontherapeutic purposes prompted us to investigate the long-term effects of exposure to the drug on neuronal adaptation. We compared the effects of chronic methylphenidate or cocaine (15 mg/kg, 14 days for both) exposure in mice on dendritic spine morphology and DeltaFosB expression in medium-sized spiny neurons (MSN) from ventral and dorsal striatum. Chronic methylphenidate increased the density of dendritic spines in MSN-D1 (MSN-expressing dopamine D1 receptors) from the core and shell of nucleus accumbens (NAcc) as well as MSN-D2 (MSN-expressing dopamine D2 receptors) from the shell of NAcc. In contrast, cocaine increased the density of spines in both populations of MSN from all regions of striatum. In general, the effect of methylphenidate on the increase of shorter spines (class 2) was less than that of cocaine. Interestingly, the methylphenidate-induced increase in the density of relatively longer spines (class 3) in the shell of NAcc was bigger than that induced by cocaine. Furthermore, methylphenidate exposure increased expression of DeltaFosB only in MSN-D1 from all areas of striatum, and surprisingly, the increase was greater than that induced by cocaine. Thus, our results show differential effects of methylphenidate and cocaine on neuronal adaptation in specific types of MSN in reward-related brain regions.

Fig. 1.

Chronic methylphenidate- or cocaine-induced changes in spine density of MSN-D1 in NAcc and dorsal striatum. D1 dopamine receptor promoter driven (Drd1)-EGFP mice were injected daily with saline, cocaine (15 mg/kg) or methylphenidate (15 mg/kg) for 14 days. Two days after the last injection, mouse brains were processed for DiI labeling and immunohistochemistry. Dendritic protrusions of Drd1-EGFP positive MSN in shell (A) or core (B) regions of NAcc, or in dorsal striatum (C) were analyzed. (i) Density of 4 types of protrusions. Data are expressed as number of protrusions per 10-μm dendritic length (mean ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANOVA with Newman-Keuls posttest. (ii and iii) Cumulative frequency plots showing the distribution of the density of class 2 (ii) and class 3 (iii) spines from individual dendrites that were analyzed in i. The total numbers of dendrites analyzed were 37 for saline-shell, 27 cocaine-shell, 40 methylphenidate-shell, 33 for saline-core, 30 cocaine-core, 23 methylphenidate-core, 42 for saline-dorsal striatum, 34 cocaine-dorsal striatum, and 32 methylphenidate-dorsal striatum.

Fig. 2.

Chronic methylphenidate or cocaine-induced changes in spine density of MSN-D2 in NAcc and dorsal striatum. D2 dopamine receptor promoter driven (Drd2)-EGFP mice were treated and mouse brains were processed as described in the legend of Fig. 1. Dendritic protrusions of Drd2-EGFP positive MSN in shell (A) or core (B) regions of NAcc, or in dorsal striatum (C) were analyzed. (i) Density of 4 types of protrusions. Data are expressed as number of protrusions per 10-μm dendritic length (mean ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANOVA with Newman-Keuls posttest. (ii and iii) Cumulative frequency plots showing the distribution of the density of class 2 (ii) and class 3 (iii) spines from individual dendrites that were analyzed in i. The total numbers of dendrites analyzed were 41 for saline-shell, 50 cocaine-shell, 41 methylphenidate-shell, 28 for saline-core, 35 cocaine-core, 26 methylphenidate-core, 63 for saline-dorsal striatum, 50 cocaine-dorsal striatum, and 40 methylphenidate-dorsal striatum.

Fig. 3.

Chronic methylphenidate- or cocaine-induced changes in ΔFosB expression in MSN-D1 from NAcc and dorsal striatum. Drd1-EGFP mice were treated with saline (i), cocaine (ii), or methylphenidate (iii) as described in the legend of Fig. 1. Two days after the last injection, the expression of EGFP (green, for identification of MSN-D1), and ΔFosB (red) were analyzed by immunohistochemistry in the shell (A) and core (B) regions of NAcc, as well as in dorsal striatum (C). The localization of NeuN (blue) was also analyzed to show the position of neuronal nuclei. NeuN, EGFP, and ΔFosB images were merged to examine their co-expression (Merge). Arrows indicate double-positive neurons for ΔFosB and EGFP. (Scale bar, 50 μm.) (iv) The fraction of ΔFosB-positive MSN-D1 was quantified as [the number of pixels containing both a red and a green signal divided by the number of pixels containing a green signal]. S, saline; C, cocaine; M, methylphenidate. Data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Newman-Keuls posttest. A total of 80 images (227 × 227 μm)/group were used for quantification.

Fig. 4.

Chronic methylphenidate- or cocaine-induced changes in ΔFosB expression in MSN-D2 from NAcc and dorsal striatum. Drd2-EGFP mice were treated with saline (i), cocaine (ii), or methylphenidate (iii) as described in the legend of Fig. 1. The localization of NeuN, EGFP and ΔFosB were analyzed in the shell (A) and core (B) regions of NAcc, as well as in dorsal striatum (C) as described in the legend of Fig. 3. Arrows indicate the double-positive neurons for ΔFosB and EGFP. (Scale bar, 50 μm.) (iv) Data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Newman-Keuls posttest. A total of 80 images (227 × 227 μm)/group were used for quantification.