Intracellular modulation of NMDA receptor function by antipsychotic drugs

Abstract

The present study deals with the functional interaction of antipsychotic drugs and NMDA receptors. We show that both the conventional antipsychotic drug haloperidol and the atypical antipsychotic drug clozapine mediate gene expression via intracellular regulation of NMDA receptors, albeit to different extents. Data obtained in primary striatal culture demonstrate that the intraneuronal signal transduction pathway activated by haloperidol, the cAMP pathway, leads to phosphorylation of the NR1 subtype of the NMDA receptor at (897)Ser. Haloperidol treatment is likewise shown to increase (897)Ser-NR1 phosphorylation in rats in vivo. Mutation of (896)Ser and (897)Ser to alanine, which prevents phosphorylation at both sites, inhibits cAMP-mediated gene expression. We conclude that antipsychotic drugs have the ability to modulate NMDA receptor function by an intraneuronal signal transduction mechanism. This facilitation of NMDA activity is necessary for antipsychotic drug-mediated gene expression and may contribute to the therapeutic benefits as well as side effects of antipsychotic drug treatment.

Fig. 1.

MK 801 inhibits haloperidol-mediatedc-fos gene expression. A, Haloperidol (0.3 and 1 mg/kg) induced c-fos gene expression in the striatum in a dose-dependent manner. B,c-fos induction after treatment with 0.3 mg/kg haloperidol (i.p.) was completely inhibited by MK 801 (1 mg/kg).C, c-fos induction after treatment with 1 mg/kg haloperidol was partially inhibited by MK 801. RNA blots with duplicate samples are shown. For a statistical analysis of multiple experiments, see Figure 4, A and B.

Fig. 2.

MK 801 attenuates Fos protein induction after haloperidol administration. A, An immunoblot of rat striata with the M-peptide antiserum shows an attenuation of haloperidol (1 mg/kg)-induced Fos expression by MK 801 (1 mg/kg). At least two regulated bands are observed between the 53 and 78 kDa size markers. The uppermost band (arrow onleft) is the same size as a band observed with a Fos antiserum. B, mRNA induction in the contralateral striata of the animals shown in A. All treatments are shown in triplicate.

Fig. 3.

d-cycloserine promotes haloperidol-mediated c-fos expression. A, In multiple experiments, a trend toward increased c-fosexpression after cotreatment with haloperidol (0.3 mg/kg) and DCS (0.5 and 1 mg/kg) was observed. This trend was significant for 1 mg/kg DCS (see Fig. 4C). B, The trend toward increased c-fos expression after cotreatment with haloperidol (1 mg/kg) and DCS was variable and not significant in multiple experiments (see Fig. 4D).

Fig. 4.

Statistical analysis demonstrates an involvement of NMDA receptors in haloperidol-mediated c-fos mRNA expression in the rat striatum. A, c-fosinduction by 0.3 mg/kg haloperidol (H 0.3) was completely blocked by the NMDA antagonist MK 801 (1 mg/kg). The average fold induction ± SEM of 12 experiments is shown.B, c-fos induction by 1 mg/kg haloperidol (H1) was significantly blocked by MK 801 (MK/H 1). The average fold induction ± SEM of three experiments is shown. C, d-cycloserine (DCS), a partial agonist at the glycine site of the NMDA receptor enhanced at 1 mg/kg haloperidol (0.3 mg/kg)-mediatedc-fos induction (compare H 0.3 withDCS 1 H 0.3) but had no significant effect at 5 mg/kg (compare H 0.3 with DCS 5 H 0.3).c-fos levels after DCS alone were equivalent to control levels at all concentrations used. The average fold induction ± SEM of nine (DCS 1 mg/kg) and six (DCS 5 mg/kg) striata is shown.D, The increase of haloperidol (1 mg/kg)-mediatedc-fos expression after treatment with DCS (1 mg/kg) did not reach significance. The average fold induction ± SEM of four striata is shown. All data are compared with c-foslevels in saline-treated rats, which were arbitrarily set to onefold induction. Asterisks indicate statistically significant differences with haloperidol-treated rats.

Fig. 5.

NMDA receptors play a role in clozapine-mediatedc-fos expression. A, c-fosexpression after treatment with the atypical antipsychotic drug clozapine (20 mg/kg) was inhibited by MK 801 (1 mg/kg).B, Average fold induction ± SEM ofc-fos after treatment with clozapine (20 mg/kg), and the inhibition of c-fos induction by MK 801, of 10 striata.C, No significant increase of clozapine-mediatedc-fos expression by DCS (1 mg/kg) was observed.D, The average fold induction ± SEM ofc-fos after treatment with clozapine (20 mg/kg) was not significantly changed by 0.5 mg/kg DCS (n = 12) or 1 mg/kg DCS (n = 8). Data in B andD are compared with c-fos levels in saline-treated rats, which were arbitrarily set to onefold induction.Asterisks indicate statistically significant differences with clozapine-treated rats.

Fig. 6.

Immunocytochemical analysis of Fos-positive nuclei in the striatum after treatment with haloperidol (0.3 mg/kg) and clozapine (20 mg/kg). DCS (1 mg/kg) did not change the number of Fos-positive nuclei, whereas MK 801 caused a significant reduction (see also Fig. 8). No Fos-positive nuclei were observed after treatment with saline. Scale bar, 0.25 mm.

Fig. 7.

All treatments (haloperidol 0.3 and 1 mg/kg, clozapine 20 mg/kg) induce Fos levels in the striatum with variations in anatomical distribution. A, Fos protein staining in the lateral striatum after treatment with haloperidol (0.3 and 1 mg/kg) or with clozapine (20 mg/kg). No Fos-positive nuclei were observed after treatment with saline (data not shown). Scale bar, 0.25 mm.B, Comparison of the number of Fos-positive nuclei in the nucleus accumbens, the medial striatum, and the lateral striatum after treatment with haloperidol (0.3 and 1 mg/kg, H 0.3, H 1) and with clozapine (20 mg/kg, CLZ).n = 3 for H 1 and CLZ, n = 5 for H 0.3 (see also Fig. 8). Bars in graphs present the average fold induction ± SEM of six striatal areas of three rats treated with haloperidol (1 mg/kg) or clozapine (20 mg/kg), or five rats treated with haloperidol (0.3 mg/kg). C, The ratio of Fos-positive nuclei in the medial over the lateral striatum after treatment with haloperidol (0.3 and 1 mg/kg) and clozapine (20 mg/kg). The medial striatum was particularly sensitive to clozapine and 0.3 mg/kg haloperidol, whereas the lateral striatum responded strongly to 1 mg/kg haloperidol.

Fig. 8.

Distribution of Fos-positive nuclei in the nucleus accumbens, the medial striatum, and the lateral striatum, after treatment with haloperidol (0.3 and 1 mg/kg) and clozapine (20 mg/kg), and the involvement of NMDA receptors. MK 801 reduced the number of Fos-positive nuclei in the nucleus accumbens and the medial striatum in all treatment paradigms. In the lateral striatum, MK 801 affected only haloperidol at 1 mg/kg. DCS (1 mg/kg) did not significantly affect the number of Fos-positive nuclei in any brain area examined (DCS was not used with 1 mg/kg haloperidol treatment). Bars in graphs represent the average fold induction ± SEM of six striatal areas of each rat. Number of rats per experiment is shown in the top left corner of each individual graph.Asterisks indicate statistically significant differences between agonists (haloperidol or clozapine) and agonists pretreated with MK 801. No Fos-positive nuclei were observed after treatment with saline or DCS alone.

Fig. 9.

The NMDA antagonist MK 801 affects the chronic regulation of the c-fos and proenkephalingenes by haloperidol. A, The attenuated induction ofc-fos after chronic treatment with haloperidol was prevented by the NMDA antagonist MK 801. Rats were treated for 11 d with MK 801, haloperidol, or combined MK 801 and haloperidol. Rats that were chronically treated with MK 801 and haloperidol responded to a singular haloperidol injection similar to acute haloperidol-treated rats (C). B, Theproenkephalin gene was upregulated after chronic treatment with haloperidol. This upregulation was prevented by pretreatment with MK 801. C, Chronic treatment with haloperidol attenuated c-fos induction after the final haloperidol injection [compare HAL (d 1–12) withHAL (d 12)]). This attenuation was prevented by pretreatment with MK 801 during the chronic haloperidol administration [compare HAL-MK (d1-d11)/HAL (d 12) with HAL (d 12)]). Average fold induction ± SEM of six experiments. For treatment paradigms see Table 1.

Fig. 10.

Upregulation by forskolin of theproenkephalin and c-fos genes is prevented by MK 801 and enhanced by glutamate in primary striatal culture. A, Primary striatal cultures were treated with the adenylate cyclase-inducing agent forskolin (10 μm forproenkephalin, 5 μm forc-fos), which increased the levels of both genes. This induction was prevented by pretreatment with the NMDA antagonist MK 801 (1 μm). B, Induction ofproenkephalin and c-fos by forskolin (5 μm for proenkephalin, 2.5 μmfor c-fos) is enhanced by simultaneous treatment with glutamate (50 μm for proenkephalin, 10 μm for c-fos). All treatments are shown in duplicate and are representative of n = 4.

Fig. 11.

Transfected pENKAT12 and3xCRE-luciferase constructs are induced by forskolin in primary striatal cultures. MK 801 blocks this induction.A, Transfection with pENKAT12. B, Transfection with 3xCRE-luciferase. Transfected cells were treated with MK 801 (1 μm), forskolin (10 μm; FOR), or both, and CAT activity (pENKAT12) or luciferase activity (3xCRE-luc) were measured. Bars present the average fold induction ± SEM over baseline activity.Asterisks indicate statistically significant difference with forskolin treatment. Five experiments performed in triplicate were averaged.

Fig. 12.

Forskolin induces phosphorylation of⁸⁹⁷Ser of the NR1 receptor in a PKA-dependent manner.A, An immunoblot with an antiserum specific for⁸⁹⁷Ser–NR1 shows that forskolin (2.5 μm)-induced phosphorylation of ⁸⁹⁷Ser–NR1 is blocked by pretreatment with the PKA antagonist H-89 (20 μm) (top panel). Levels of NR1 protein were not changed (second panel).¹³³Ser-CREB was induced by forskolin and blocked by H-89 (third panel). CREB protein levels were unchanged (bottom panel). Blot was cut at 80 kDa, and the upper part was exposed to ⁸⁹⁷Ser–NR1 antiserum, stripped, and reprobed with NR1 antiserum, whereas the lower part was exposed to¹³³Ser-CREB antiserum, stripped, and reprobed with CREB antiserum. B, The CaM kinase antagonist KN-62 (30 μm) does not block forskolin-induced phosphorylation of⁸⁹⁷Ser-NR1.

Fig. 13.

In vivo treatment with haloperidol causes ⁸⁹⁷Ser–NR1 phosphorylation. Rats were treated with 1 mg/kg haloperidol for 15 min and killed, and their striata were quickly frozen. The top panel is an immunoblot with the⁸⁹⁷Ser-NR1 antiserum; the bottom panel is the same immunoblot stripped and exposed to an NR1 antiserum.n = 3 in each group.

Fig. 14.

Mutation of ^896/897Ser–NR1 to alanine blocks forskolin-mediated gene expression. Primary striatal cultures were cotransfected with 3xCRE-luciferase and control expression vector, NR1 wild-type DNA (NR1wt), or mutated ^896/897S/A DNA. Forskolin (2.5 μm) activated the 3xCRE-luciferaseconstruct. This activation was enhanced by cotransfection with wild-type NR1 and completely blocked by the mutated construct.Asterisks indicate statistically significant induction.n = 6.

Fig. 15.

Hypothesized interaction of D₂receptors and NMDA receptors in the striatum. Inhibition of D₂ receptors or stimulation with forskolin increases cAMP levels and activates the PKA–second messenger pathway. The second messenger pathway increases NMDA receptor function, e.g., via⁸⁹⁷Ser-NR1 phosphorylation, such that the NMDA receptor activates a signal transduction pathway that translocates to the nucleus and causes phosphorylation of the transcription factor CREB. Phosphorylation of CREB leads to an increase in mRNA synthesis of thec-fos and proenkephalin genes. Although CREB may be necessary for the transactivation of c-fosand the proenkephalin gene in the striatum, additional transcription factors may be involved and activated by the same signal transduction pathway. Inset, D₂ receptor activity depresses PKA activity. Inhibition of D₂ receptors with drugs such as haloperidol disinhibits PKA activity and leads to⁸⁹⁷Ser–NR1 phosphorylation. Activation of PKA with low levels of forskolin has a similar effect, whereas high levels of forskolin can mediate CREB phosphorylation in addition to NMDA receptor phosphorylation. Thus, high levels of forskolin can mediate gene expression independent of NMDA receptors (Rajadhyaksha et al., 1998).