D1 and D2 dopamine receptors differentially mediate the activation of phosphoproteins in the striatum of amphetamine-sensitized rats

Abstract

Rationale: Extracellular signal-regulated kinase (ERK), cAMP response element binding protein (CREB), and protein kinase B (PKB or Akt) in the striatum are differentially activated by acute and repeated amphetamine (AMPH) administration. However, the dopamine receptor subtypes that mediate transient vs. prolonged phosphorylation changes in these proteins induced by AMPH challenge in AMPH-sensitized rats are unknown.

Objectives: The role of the D1 and D2 class of dopamine receptors in the differential phosphorylation of striatal ERK, CREB, Thr308-Akt and Ser473-Akt and the expression of behavioral sensitization induced by AMPH challenge in AMPH-pretreated rats were determined.

Methods: D1 or D2 dopamine receptor antagonists were injected before an AMPH challenge in AMPH-sensitized rats. After behavioral activity was recorded, rats were euthanized either 15 min or 2 h after AMPH challenge and striatal phosphoprotein status was analyzed by Western blotting.

Results: The D1 receptor antagonist (SCH23390) decreased stereotypical behavior whereas the D2 receptor antagonist (eticlopride) decreased all behavioral activity induced by an AMPH challenge in AMPH-sensitized rats. SCH23390, but not eticlopride, significantly decreased ERK, CREB, and Thr308-Akt phosphorylation in the striatum 15 min, and ERK and CREB phosphorylation 2 h, after AMPH challenge in AMPH-sensitized rats. In contrast, eticlopride, but not SCH23390, prevented a decrease in Akt phosphorylation 2 h after AMPH challenge.

Conclusions: These data indicate that the time course of phosphoprotein signaling is differentially regulated by D1 and D2 receptors in the striatum of AMPH-sensitized rats, suggesting that complex regulatory interactions are activated by repeated AMPH exposure.

Fig. 1

The effects of SCH23390 (SCH-a, b) or eticlopride (ETI-c, d) on rat behavioral activity at 15 min (a, c) or 2h (b, d) after amphetamine (A) challenge (1 mg/kg, i.p.) in rats pretreated with AMPH (daily 5 mg/kg, i.p.) once daily for 5 days. (a, b) Pretreatment with SCH (0.01 mg/kg, i.p.) decreased behavioral ratings at 15 min (a) or 2 hr (b) after AMPH challenge in AMPH-sensitized rats. ** p <0.01 vs. S / SCH + S group; *** p <0.001 vs. S / S + S group; # p < 0.001 as compared with A / S + A group. (c), (d): Pretreatment with 0.1 mg/kg ETI attenuated behavioral ratings during both 15 min- and 2 h- period after the AMPH challenge. * p <0.05; ** p <0.01 vs. S / ETI + S group, *** p <0.001 vs. S / S + S group, # p < 0.001 as compared with A+ SAL + A group. *p <0.001 vs. S / S + S or S / SCH + S group. Values are expressed as means ± S.E.M. for 4– 6 rats per group. AUC, area under curve.

Fig. 2

Effects of pre-treatment with SCH23390 on the activation of striatal ERK1/2, CREB, and AktThr308 in AMPH-sensitized rats 15 min (a–d) or 2h (e–h) after challenge with AMPH. Upper panels show representative western blot bands, bottom panels show graphs plotted from data represented as mean ± S.E.M. of p-ERK2/ERK2 (a,e), p-CREB/CREB (b, f), p-Thr308-Akt (c, g), and p-Ser473-Akt (d, h) integrated density ratio (n=4–6 samples/group from three independent determinations). a: * p <0.05 vs. S / S + S group; # p <0.05 vs. A / S + A group. b: *** p <0.001 vs. S / S + S group; ### p <0.01 vs. A / S + A group. c: ** p <0.01 vs. S / S + S group; ## p <0.01 vs. A / S + A group. e: * p <0.05 vs. S / S + S group; # p <0.05 vs. A / S + A group. f: ** p <0.01 vs. S / S + S group; ## p <0.01 vs. A / S + A group. g: ** p <0.01 vs. S / S + S group.

Fig. 3

The blockade of D2 DA receptor with ETI (0.1 mg/kg. i.p.) elevated basal levels of striatal p-ERK (a), p-CREB (b), and p-Thr308-Akt (c) 15 min after SAL challenge injection, and prevented the downregulation of p-Thr308-Akt in AMPH-sensitized rats 2h (g) after challenge with AMPH, without alteration of other phosphoproteins activated by repeated AMPH. Representative immunoblots (upper panel) and quantitative analysis of phosphoproteins (lower panel). Data represented as means ± S.E.M of p-ERK2/ERK2 (a, e), p-CREB/CREB (b, f), p-Thr308-Akt (c, g), and p-Ser473-Akt (d, h) integrated density ratio (n=6 samples/group from three independent determinations). a: *** p <0.001 as compared with S / S + S group; ## p <0.01 as compared with S / S + S. b: * p <0.05 as compared with S / S + S group; # p <0.05 as compared with S /S + S. c: *** p <0.001 as compared with S / S + S group; ## p <0.01 as compared with S / S + S. e: *** p <0.001 as compared with groups challenge with saline. f: *** p <0.001 as compared with groups challenge with saline. g: *** p <0.001 as compared with groups challenge with saline, ##p <0.01 as compared with A / S + A group.

Fig. 4

Correlation of behavioral ratings and phosphoproteins 2 h after AMPH challenge injection in AMPH-sensitize rats. Fig. 4 a–c: Correlation of behavioral ratings and pERK (Fig 4a), pCREB (Fig. 4b), and pAkt (Fig. 4c) 2 h after AMPH challenge injection with (unfilled rectangle) or without SCH 23390 (filled rectangle) in AMPH-sensitize rats. (Fig. 4 d–e): Correlation of behavioral ratings and pERK (Fig. 4a), pCREB (Fig. 4b), and pAkt (Fig. 4c) 2 h after AMPH challenge injection with (unfilled rectangle) or without eticlopride (filled rectangle) in AMPH-sensitize rats. a: r=0.66, p<0.05; b: r=0.67, p<0.05; c: r=0.31, p>0.05; d: r=0.001, p>0.05; e: r=0.08, p>0.05; f: r=−0.84, p<0.01