The phosphoprotein DARPP-32 mediates cAMP-dependent potentiation of striatal N-methyl-D-aspartate responses

Abstract

The signal transduction pathway underlying the cAMP-dependent modulation of rat striatal N-methyl-D-aspartate (NMDA) responses was investigated by using the two-electrode voltage-clamp technique. In oocytes injected with rat striatal poly(A)+ mRNA, activation of cAMP-dependent protein kinase (PKA) by forskolin potentiated NMDA responses. Inhibition of protein phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A) by the specific inhibitor calyculin A occluded the PKA-mediated potentiation of striatal NMDA responses, suggesting that the PKA effect was mediated by inhibition of a protein phosphatase. Coinjection of oocytes with striatal mRNA and antisense oligodeoxynucleotides directed against the protein phosphatase inhibitor DARPP-32 dramatically reduced the PKA enhancement of NMDA responses. NMDA responses recorded from oocytes injected with rat hippocampal poly(A)+ mRNA were not affected by stimulation of PKA. When oocytes were coinjected with rat hippocampal poly(A)+ mRNA plus complementary RNA coding for DARPP-32, NMDA responses were potentiated after stimulation of PKA. The results provide evidence that DARPP-32, which is enriched in the striatum, may participate in the signaling between the two major afferent striatal pathways, the glutamatergic and the dopaminergic projections, by the cAMP-dependent regulation of striatal NMDA currents.

Figure 1

Striatal NMDA-induced currents are potentiated by forskolin and PMA. The bar indicates drug superfusion. External solution was applied as control (×). (A) Forskolin (50 μM; •), PMA (10 nM; ▪), 1,9-dideoxyforskolin (50 μM; ○) and 4-α-PMA (10 nM; □) were applied for 1 min. (B) After a 10-min incubation with 8-Br-cAMP (10 mM; •), a 1-min incubation with PMA (10 nM; ▪) followed. Each point represents the mean ± SEM of three to seven oocytes.

Figure 4

Forskolin-mediated potentiation of hippocampal NMDA responses is observed in the presence of DARPP-32. Forskolin (50 μM, •), 1,9-dideoxyforskolin (50 μM; ○), and external solution (control, ×) were applied for 1 min as indicated by the bar. NMDA-induced current responses were recorded from oocytes injected with hippocampal mRNA (A) or oocytes injected with hippocampal mRNA and DARPP-32 cRNA (80 ng per oocyte) (B). Each point represents the mean ± SEM of three to nine oocytes.

Figure 2

Calyculin A inhibits forskolin- but not PMA-mediated potentiation of striatal NMDA responses. The NMDA-induced currents of striatal mRNA-injected oocytes were measured before and 8 min after 1-min application of 50 μM forskolin (A) or before and 12 min after 1-min application of 10 nM PMA (C). (B and D) The same protocol was followed after injecting oocytes with calyculin A to a final intracellular concentration of 500 nM 60–90 min before voltage-clamping. The intracellular concentration of calyculin A was calculated by assuming standard oocytes with a volume of 0.5 μl.

Figure 3

Northern blot analysis of poly(A)⁺ mRNA isolated from rat striatum and hippocampus. In each lane 3 μg of RNA was loaded. The full-length cDNA encoding DARPP-32 was radioactively labeled and used as a probe. The lower bands show the hybridization of the RNA probes with mouse β-actin.

Figure 5

Forskolin-mediated potentiation of striatal NMDA responses requires DARPP-32. (A) Representative NMDA responses of oocytes injected with striatal mRNA and ODNs or TE buffer before (controls) and 8 min after 1-min application of forskolin (50 μM). (B) Averaged relative current amplitudes (I/Ic) of the experiments presented in A. Each bar represents the mean ± SEM of four or seven oocytes as indicated.

Figure 6

Schematic diagram illustrating the possible role of DARPP-32 and PP1 in mediating dopamine-induced increase in conductance through NMDA receptors in rat striatum. Glu, glutamate; DA, dopamine; P, phosphate group; PK, protein kinase.