DARPP-32 mediates serotonergic neurotransmission in the forebrain

Abstract

Serotonin is implicated in the regulation of complex sensory, motor, affective, and cognitive functions. However, the biochemical mechanisms whereby this neurotransmitter exerts its actions remain largely unknown. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of molecular weight 32,000) is a phosphoprotein that has primarily been characterized in relation to dopaminergic neurotransmission. Here we report that serotonin regulates DARPP-32 phosphorylation both in vitro and in vivo. Stimulation of 5-hydroxy-tryptamine (5-HT4 and 5-HT6 receptors causes an increased phosphorylation state at Thr34-DARPP-32, the protein kinase A site, and a decreased phosphorylation state at Thr75-DARPP-32, the cyclin-dependent kinase 5 site. Furthermore, stimulation of 5-HT2 receptors increases the phosphorylation state of Ser137-DARPP-32, the casein kinase-1 site. Behavioral and gene transcriptional effects induced by compounds that selectively release serotonin were greatly reduced in DARPP-32 knockout mice. Our data indicate that DARPP-32 is essential not only for dopaminergic but also for serotonergic neurotransmission.

Figure 1

Regulation by serotonin of DARPP-32 phosphorylation in slices of neostriatum. (a–c) Time course and (d–f) dose–response studies of regulation by serotonin of phosphorylation of DARPP-32 at (a and d) Thr³⁴, (b and e) Thr⁷⁵, and (c and f) Ser¹³⁷ in neostriatum. (a–c) Serotonin was used at 100 μM. (d–f) Slices were incubated for 2 min for studies of phospho-Thr³⁴–DARPP-32 and phospho-Ser¹³⁷–DARPP-32 and for 10 min for studies of phospho-Thr⁷⁵–DARPP-32. Data represent means (± SE) (n = 6–10). *, P < 0 05; **, P <0.01; ***, P < 0.001 compared with control; one-way ANOVA followed by Dunnett's test.

Figure 2

Regulation of DARPP-32 phosphorylation in vivo by PCA and 5-HTP. Mice were injected i.p. with saline, PCA (4 mg/kg), or 5-HTP (50 mg/kg). Fifteen minutes later mice were killed by focused microwave irradiation. Data represent means ± SE for 5–10 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with saline-treated mice; one-way ANOVA followed by Dunnett's test.

Figure 3

Regulation of c-fos mRNA expression by PCA and 5-HTP in WT and DARPP-32 KO mice. (a) Dark-field autoradiograms showing the expression of c-fos mRNA 20 min after treatment with saline, PCA (4 mg/kg), or 5-HTP (50 mg/kg) in WT and DARPP-32 KO mice. (Magnification: ×5.) (b and c) Histograms show quantification of the expression of c-fos mRNA in (b) periventricular area of striatum and (c) cingulate cortex after each treatment. WT, filled bars; DARPP-32 KO, open bars. Data represent means ± SE for 5–8 mice per group. **, P < 0.01; ***, P < 0.001 compared with saline-treated mice; #, P < 0.05; ##, P < 0.01 compared with WT mice given the same treatment; one-way ANOVA followed by Dunnett's test.

Figure 4

Behavioral responses to treatment with PCA and 5-HTP in WT and DARPP-32 KO mice. Effects of (Left) PCA (4 mg/kg) and (Right) 5-HTP (50 mg/kg) on total locomotion (Upper) and stereotypic movements (Lower) in WT and DARPP-32 KO mice. Data represent means ± SE for 6–10 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with saline-treated mice; ###, P < 0.001; ##, P < 0.01; #, P < 0.05 compared with WT mice given the same treatment; two-way ANOVA followed by Duncan's test.

Figure 5

Effect of clozapine on behavioral responses to treatment with PCA and 5-HTP in WT mice. Effect of pretreatment with clozapine (0.3 mg/kg) on locomotion induced by (Left) PCA (4 mg/kg) or (Right) 5-HTP (50 mg/kg). Data represent means ± SE for 6–10 mice per group. **, P < 0.01 compared with saline-treated mice; ##, P < 0.01 compared with PCA-treated or 5-HTP-treated mice; two-way ANOVA followed by Duncan's test.

Figure 6

Model summarizing pathways by which enhanced serotonergic transmission regulates DARPP-32 phosphorylation at Thr³⁴, Thr⁷⁵, and Ser¹³⁷. Activation of 5-HT₄ or 5-HT₆ receptors sequentially increases cAMP levels, activity of PKA, and phosphorylation of Thr³⁴. Activated PKA also phosphorylates and activates protein phosphatase 2A (PP-2A) (13), causing dephosphorylation of the Cdk5 site, Thr⁷⁵ (14), removing inhibition of PKA (14) and increasing phosphorylation of Thr³⁴. 5-HT₂ receptors activate PLC, increasing casein kinase-1 (CK1) activity (27), phosphorylation of Ser¹³⁷, and inhibition of dephosphorylation by protein phosphatase-2B (PP2B) of Thr³⁴ (15). These various actions of serotonin on DARPP-32 phosphorylation are synergistic, each leading to an increased state of phosphorylation of Thr³⁴ and an increased inhibition of PP-1 (10). Solid lines indicate activation; dashed lines indicate inhibition.