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NEUROPHARMACOLOGY

Clozapine Potentiation of N-Methyl-D-aspartate Receptor Currents in the Nucleus Accumbens: Role of NR2B and Protein Kinase A/Src Kinases

Merck Research Laboratories, West Point, Pennsylvania (M.W., M.J.M., D.A.H., G.R.S.); and Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (P.J.C.)

Received November 4, 2004; accepted January 18, 2005.

	Abstract

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Clozapine is an atypical antipsychotic that has a unique clinical profile that distinguishes it from other typical and atypical antipsychotics. At present, the underlying mechanisms of action of clozapine are unclear. Recent studies in the field of schizophrenia suggest that compounds that potentiate N-methyl-D-aspartate (NMDA) receptor function in the appropriate brain regions might be an effective antipsychotic agent. One relevant region in which NMDA receptors play a key role in mediating neurotransmission is the nucleus accumbens. Therefore, we investigated the regulation of NMDA receptor currents and excitatory postsynaptic currents (EPSCs) by clozapine in nucleus accumbens neurons. Whole-cell patch-clamp recordings were performed in rat brain slices. We demonstrate that bath application of clozapine but not haloperidol or the selective 5-hydroxytryptamine 2A antagonist MDL100907 [(R)-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluoro-phenyl)ethyl]-4-piperidine methanol] induces a robust potentiation of NMDA-evoked currents and of glutamatergic EPSCs and that this potentiation is dependent on dopamine release and postsynaptic activation of D1 receptors. Furthermore, the effect of clozapine is selective for NR2B subtype-containing NMDA receptors and is blocked by the selective Src family kinase inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine] and the protein kinase A-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide but not by the protein kinase C-selective inhibitor bisindolylmaleimide I. This effect of clozapine in the nucleus accumbens might underlie the unique clinical profile of this atypical antipsychotic and provides a basis for novel treatment approaches.

Recent findings have led to the development of several complimentary theories on the etiology of schizophrenia. For example, the finding that acute administration of the N-methyl-D-aspartate (NMDA) receptor channel blocker ketamine or phencyclidine to healthy volunteers can elicit a psychosis indistinguishable from acute schizophrenia, including symptoms such as formal thought disorder and auditory hallucinations as well as negative symptoms (Fauman et al., 1976), suggests that NMDA receptor function may play a role in this disorder. Furthermore, when NMDA antagonists are administered to schizophrenic patients, a sustained worsening of symptoms can occur, usually in the form of an acute exacerbation of preexisting symptomatology. These findings have led to the development of the NMDA hypofunction hypothesis of schizophrenia, which proposes a hypoglutamatergic state in the brain leading to a selective hypoactivation of the NMDA subtype of ionotropic glutamate receptors (for review, see Marino and Conn, 2002). According to this model, any treatment that increases NMDA receptor function in the appropriate brain regions would be expected to alleviate the positive, negative, and cognitive symptoms of this disease. However, the anatomical localization of this hypothetical NMDA hypofunction is not known.

A growing body of evidence implicates the importance of the nucleus accumbens in the pathophysiology of schizophrenia. In response to antipsychotic drugs, the nucleus accumbens shows alterations in expression of mRNA (Polese et al., 2002), induction of immediate early genes (Robertson and Fibiger, 1992; Polese et al., 2002; Kinney et al., 2003), and intracellular dye coupling (Onn and Grace, 1995). In addition, the nucleus accumbens represents a site in which NMDA receptors play a critical role in gating cortical information (for review, see Grace, 2000). Nucleus accumbens neurons receive glutamatergic afferent inputs from regions that have been associated with schizophrenia, including the neocortex, hippocampus, and the amygdala (Fuller et al., 1987; Sesack et al., 1989; Finch, 1996). Inhibitory neurons in the nucleus accumbens project to the ventral pallidum (Heimer et al., 1991), which in turn sends inhibitory efferents to the mediodorsal thalamus (Young et al., 1984). The glutamatergic neurons of the mediodorsal thalamus project to (Uylings and van Eden, 1990) and provide a major regulatory control over the prefrontal cortex. Thus, increased activity in the nucleus accumbens would lead to the disinhibition of the mediodorsal thalamus and a consequent increase in glutamate release in the prefrontal cortex. Hypofunction or dysfunction of the prefrontal cortex has been implicated in the severity of negative and cognitive symptoms in schizophrenia patients (for review, see Weinberger and Berman, 1996). An increase in the activity of the nucleus accumbens could therefore ultimately lead to an activation of the prefrontal cortex. Since NMDA receptors are known to play an important role in synaptic transmission and cell excitability, one obvious mechanism whereby nucleus accumbens activity could be increased is the potentiation of NMDA receptor function. Therefore, we tested the hypothesis that clozapine potentiates NMDA receptor function in the nucleus accumbens.

	Materials and Methods

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Electrophysiology
Slice Preparation. All experiments involving rats were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, and the Merck Research Laboratories institutional animal care and use committee approved all studies described in this paper. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility in strict compliance with all of the applicable regulations.

Thirteen- to 20-day-old Sprague-Dawley rats (Taconic Farms, Germantown, NY) were used for all patch-clamp studies. After decapitation, brains were rapidly removed and submerged in an ice-cold choline chloride buffer: 126 mM choline chloride, 2.5 mM KCl, 8 mM MgSO₄, 1.3 mM MgCl₂, 1.2 mM NaH₂PO₄, 10 mM glucose, and 26 mM NaHCO₃, equilibrated with 95% O₂/5% CO₂. Frontal slices (for synaptic stimulation parasagittal slices) (300 µm thick) were made using a Vibraslicer (WPI, Sarasota, FL) or vibratome (Leica, Wetzlar, Germany). Slices were transferred to a holding chamber containing normal artificial cerebrospinal fluid (ACSF): 124 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO₄, 1.0 mM NaH₂PO₄, 2.0 mM CaCl₂, 20 mM glucose, and 26 mM NaHCO₃, equilibrated with 95% O₂/5% CO₂ at room temperature. In all experiments, 5 µM glutathione, 500 µM pyruvate, and 250 µM kynurenic acid were included in the choline chloride buffer and holding chamber to increase slice viability.

Electrophysiological Recordings. Whole-cell patch-clamp recordings were obtained under visual control. During recordings, slices were maintained fully submerged on the stage of a brain slice chamber at 34°C and perfused continuously with equilibrated ACSF (2–3 ml/min). Neurons in the nucleus accumbens were visualized with a 40x water immersion lens mounted on an Axioscope (Carl Zeiss GmbH, Jena, Germany). Patch electrodes were pulled from borosilicate glass on a two-stage vertical patch pipette puller (Heka Elektronik, Lambrecht, Germany) and filled with: 140 mM cesium methane sulfonate, 16 mM HEPES, 10 mM NaCl, 2 mM EGTA, 0.2 mM NaGTP, and 2 mM MgATP, with pH adjusted to 7.5 with 1 M CsOH. Electrode resistance was 5 to 8 M. All recordings were performed using a HEKA EPC10 patch clamp amplifier (Heka Elektronik). For measurement of exogenous NMDA receptor currents, NMDA (100 µM, Tocris Cookson Inc., Ellisville, MO) was applied directly to the postsynaptic cell with a modified fast application system. NMDA-evoked currents were recorded at a holding potential of –60 mV and tetrodotoxin (500 nM–1 µM, Sigma-Aldrich, St. Louis, MO; Calbiochem, San Diego, CA) was present in the bath to block synaptic transmission.

Excitatory postsynaptic currents (EPSCs) were evoked with a bipolar tungsten stimulation electrode placed within the nucleus accumbens 100 to 200 µm rostral to the recording site. EPSCs were recorded from a holding potential of –60 mV unless otherwise stated and low Mg²⁺-ACSF: 124 mM NaCl, 2.5 mM KCl, 1.0 mM NaH₂PO₄, 2.0 mM CaCl₂, 20 mM glucose, and 26 mM NaHCO₃, equilibrated with 95% O₂/5% CO₂ at room temperature), and (–)-bicuculline (20 µM) was bath applied during all EPSC recordings to increase NMDA receptor activation and to block inhibitory synaptic transmission. For exogenous NMDA receptor currents percent potentiation was defined by using the ratio of maximum current during drug application (average of three trials during maximal drug effect) to average current amplitude of three trials immediately preceding drug application. For EPSC recordings, percent potentiation was defined by using the ratio of amplitude or area under the curve (for charge density) during drug application (average of five trials during last 5 min of drug application) to average amplitude or area under the curve of five trials immediately preceding drug application. All data are expressed as mean ± S.E.M.

Catecholamine Depletion
To achieve DA depletion, reserpine (5 mg/kg i.p.) was administered acutely to the rats 1 to 1.5 h before sacrifice, and the brain slices were maintained in the tyrosine hydroxylase inhibitor -methyl-L-P-tyrosine (100 µM) after the dissection to block dopamine synthesis. Reserpine was prepared fresh each day. This acute treatment with reserpine induced a marked catalepsy in all animals (data not shown).

Drugs and Drug Application
Drugs were made fresh into stock solutions of 10 to 100 mM every day and diluted to the desired concentration in ACSF immediately prior to bath application to the slice. Antagonists were applied at least 10 to 15 min prior to application of agonists. Kinase inhibitors were bath applied for at least 15 min before attempting to patch onto a cell. This pretreatment was necessary since prolonged baseline recordings prior to clozapine application lead to a loss of the clozapine effect, likely due to dialysis of intracellular contents. A 10 mM stock of clozapine (in dimethyl sulfoxide) was made fresh every day and slowly diluted 1:10 in water directly before diluting down to its final concentration and bath application. Haloperidol hydrochloride stocks were made in 4 parts 1 M sodium hydroxide and 6 parts 8.5% lactic acid. (S)-(–)-Sulpiride, MDL100907, PP2, H-89, bisindolylmaleimide I (BIS I), and CP101,606 stocks were made in dimethylsulfoxide at 1000 to 10,000x their final concentrations. Tetrodotoxin, SCH23390 SKF 82958, (–)-bicuculline methiodide, ifenprodil hemitartrate, and D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5) were prepared in distilled water. Clozapine, SCH23390 S-(–)-sulpiride, ifenprodil hemitartrate, and D-AP5 were obtained from Tocris Cookson Inc.. H-89 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). BIS I was obtained from EMD Biosciences (San Diego, CA), and PP2 was obtained from Alexis Corporation (Läufelfingen, Switzerland). All other compounds were obtained from Sigma-Aldrich.

Data Analysis
Values are expressed as mean ± S.E.M. Statistical comparisons between two groups were performed by using a Student's t test. Statistical comparisons between several groups were performed by one-way analysis of variance followed by Tukey's post hoc comparisons. Statistical comparisons within groups (EPSCs at different holding potentials) were performed by repeated measures analysis of variance. An effect was considered significant when p < 0.05.

	Results

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Clozapine Potentiates NMDA Receptor Currents in Nucleus Accumbens Neurons. NMDA receptor currents were evoked in nucleus accumbens medium spiny neurons by application of exogenous NMDA (see Materials and Methods). These exogenous NMDA receptor responses were mediated by activation of NMDA receptors because they could be blocked completely by bath application of 20 µM D-AP5 (data not shown). Ten-minute bath application of the atypical antipsychotic clozapine (100 nM) produced a robust increase in NMDA receptor currents in 21 of 25 cells studied in the nucleus accumbens (42.6 ± 6.5% increase, n = 25, p < 0.01; Fig. 1, A and E). This effect of clozapine was not mimicked by the typical antipsychotic haloperidol at a concentration selective for D2 dopamine receptors (100 nM) (Seeman, 1987) (2.5 ± 9.5% increase, n = 6, p > 0.05, paired Student's t test; Fig. 1, B and E). It has been suggested that clozapine might mediate its antipsychotic effects through inhibition of 5-HT2A receptors. Therefore, we tested whether a 5-HT2A-selective antagonist could mimic the potentiating effect of clozapine on NMDA receptor currents. However, bath application of the 5-HT2A-selective antagonist MDL100907 (100 nM) had no significant effect on the amplitude of NMDA-evoked currents in nucleus accumbens neurons (16.3 ± 13.9% increase, n = 4, p > 0.05, paired Student's t test; Fig. 1E). In most experiments, the effect of 100 nM clozapine was not reversible after 10 min (Fig. 1C). The concentration-response relationship for the clozapine-induced potentiation of NMDA receptor currents exhibited a narrow and biphasic shape (Fig. 1D). Lower concentrations (30 and 100 nM) increased NMDA receptor currents, whereas higher concentrations (300 nM) did not.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Clozapine potentiates NMDA receptor currents in nucleus accumbens neurons in rat brain slices. A, example traces showing the effect of 100 nM clozapine on the amplitude of NMDA receptor currents induced by fast application of NMDA (100 µM). B, example traces showing the lack of an effect on the amplitude of NMDA-evoked currents by bath application of the D1/D2 antagonist haloperidol (100 nM). C, average time course of the effect of 10-min bath application of clozapine (100 nM) on NMDA receptor currents (filled circles) and of no drug control (open circles). Each time point represents the normalized mean ± S.E.M. of data from 22 and 7 cells, respectively. D, concentration-response relationship of the clozapine-induced potentiation of NMDA receptor currents. Each point represents the mean ± S.E.M. of the maximal effect during drug application from 3 to 24 experiments. E, bar graph showing the effect of clozapine (100 nM) and the lack of an effect of haloperidol (100 nM) and of the 5-HT2A-selective antagonist MDL100907 (100 nM) on NMDA receptor currents. Each bar represents the mean ± S.E.M. of data from 24, 5, and 4 experiments, respectively. *, p < 0.05, significant effect compared with predrug baseline, Student's paired t test.

Clozapine-Induced Potentiation of NMDA Receptor Currents Is Mediated by Activation of D1 Dopamine Receptors. Previous studies have revealed that clozapine can increase dopamine release in the nucleus accumbens (Yamada et al., 1995). Furthermore, it has been shown that dopamine and D1 agonists can increase NMDA receptor function in the striatum (Cepeda et al., 1998; Flores-Hernandez et al., 2002). Therefore, we performed pharmacological studies using selective dopamine receptor antagonists to test whether clozapine potentiates NMDA receptor currents by a dopaminergic mechanism. Prior bath application (10–15 min) of haloperidol at concentrations sufficient to block both D1 and D2 dopamine receptors (10 µM) (Seeman, 1987) or the D1-selective antagonist SCH23390at a maximal concentration (10 µM) completely blocked the potentiating effect of 100 nM clozapine (haloperidol, –0.4 ± 9.2% increase, n = 6, p > 0.05; SCH23390 –0.07 ± 5.3% increase, n = 3, p > 0.05; Fig. 2, A2, A3, and C). In addition, bath application of the D2-selective antagonist sulpiride at a maximal concentration (10 µM) prior to clozapine partly decreased the effect of clozapine (23.9 ± 14.4, n = 5, p > 0.05; Fig. 2, A4 and C). Bath application of sulpiride (10 µM) alone mimicked the effect of clozapine and significantly increased NMDA receptor currents (43.1 ± 13.2% increase, n = 4, p < 0.05, paired Student's t test; Fig. 2, B and C). Thus, it is possible that the sulpiride-induced blockade was mediated by functional occlusion of the response. In contrast, bath application of the D1 selective antagonist SCH23390(10 µM) alone had no effect on the amplitude of NMDA receptor currents (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 2. The clozapine-induced potentiation of NMDA receptor currents is blocked by D1-selective dopamine antagonists and mimicked by D2-selective antagonists. A1 to A4, example traces showing the effect of clozapine (100 nM) in the absence (A1) and presence (A2–A4) of selective dopaminergic antagonists. B, example traces illustrating the effect of bath application of the D2-selective antagonist sulpiride (10 µM). C, bar graph showing the effect of clozapine (100 nM) in the absence and presence of selective dopaminergic antagonists and the effect of sulpiride (10 µM) on NMDA receptor currents. Each bar represents the mean ± S.E.M. of data from 3 to 12 experiments. *, p < 0.05, significant effect compared with predrug baseline.

Previous work has shown that clozapine, presumably via an inhibitory action on D2 receptors, can release dopamine in the nucleus accumbens of rat brain slices (Yamada et al., 1995). Therefore, one possible mechanism for the NMDA potentiating action of clozapine could be via a similar mechanism. Release of dopamine by inhibition of D2 receptors could then activate postsynaptic D1 receptors leading to an indirect potentiation of NMDA receptor currents.

This hypothesis predicts that it should be possible to mimic the effect of clozapine with a D1-selective agonist. Bath application of 100 nM SKF82958 a highly selective full agonist for D1 receptors, significantly potentiated NMDA receptor currents in 10 of 12 nucleus accumbens neurons recorded (26.1 ± 4.8% increase, n = 12, p < 0.01, paired Student's t test; Fig. 3, A and D). This effect of SKF82958(100 nM), like the clozapine effect, was long-lasting (Fig. 3B) and showed a bell-shaped concentration-response curve (Fig. 3C).

View larger version (26K): [in this window] [in a new window]   Fig. 3. The effect of clozapine is mimicked by the D1-selective dopamine agonist SKF82958 A, example traces showing the effect of bath application of clozapine (100 nM) and SKF82958(100 nM) on the amplitude of NMDA receptor currents. B, average time course of the effect of 10-min bath application of SKF82958(100 nM) on NMDA receptor currents (filled circles) and of no drug control (open circles). Each time point represents the normalized mean ± S.E.M. of data from 11 and 7 cells, respectively. C, concentration response relationship of the SKF82958induced potentiation of NMDA receptor currents. Each point represents the mean ± S.E.M. of the maximal effect during drug application from 4 to 12 experiments. D, bar graph showing the average effects of clozapine (100 nM) and the D1-selective agonist SKF82958(100 nM). Each bar represents the mean ± S.E.M. of the maximal effect during drug application from 24 and 12 experiments, respectively. **, p < 0.01, significant effect compared with predrug baseline, Student's paired t test.

We further tested the hypothesis that the clozapine-induced potentiation of NMDA receptor currents is mediated by clozapine-induced release of dopamine acting on postsynaptic D1 receptors in the nucleus accumbens by repeating the clozapine experiments in slices from acutely dopamine-depleted animals. Consistent with our hypothesis, the effect of clozapine (100 nM) was completely absent in dopamine-depleted slices (3.2 ± 3.4% increase, n = 8, p > 0.05, paired Student's t test; Fig. 4, A2, B, and E). Since the effect of the D1-selective agonist SKF82958should not be influenced by dopamine depletion, we also compared its effects in dopamine-depleted slices. As predicted, the effect of SKF82958(100 nM) on NMDA receptor currents was not abolished by dopamine depletion (27.4 ± 8.6% increase, n = 6, p < 0.05, paired Student's t test; Fig. 4, C2, D, and E). These data suggest that clozapine-induced release of dopamine is an obligatory step in the cascade of events leading to the increase in NMDA receptor currents. The data furthermore show that the potentiation of NMDA receptor currents is not mediated by a direct action of clozapine on D1 receptors.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Acute dopamine depletion induced by in vivo reserpine treatment prevents the potentiating effect of clozapine on NMDA receptor currents in nucleus accumbens neurons. A1 and A2, example traces showing the effect of 100 nM clozapine in slices obtained from control rats (A1) and in slices obtained from reserpine treated animals (A2). B, average time courses showing the effect of clozapine (100 nM) on NMDA receptor currents in slices from control animals (filled circles) and from reserpine-treated animals (open triangles). The potentiating effect of clozapine is absent in dopamine-depleted slices. Each time point represents the normalized mean ± S.E.M. of data from six cells and data from seven cells (three animals), respectively. C1 and C2, example traces showing the effect of SKF82958(100 nM) in slices obtained from control rats (C1) and in slices obtained from reserpine treated animals (C2). D, average time courses showing the effect of SKF82958(100 nM) on NMDA receptor currents in slices obtained from control rats (filled circles) and from reserpine-treated rats (open triangles). Each time point represents the normalized mean ± S.E.M. of data from 11 cells and data from 5 cells (3 reserpinized animals), respectively. E, bar graph showing the average effects of 100 nM clozapine (black bars) and 100 nM SKF82958(gray bars) on NMDA receptor currents in control and reserpine-treated animals. Each bar represents the mean ± S.E.M. of data from 10 and 8 experiments in 3 animals (clozapine) and 11 and 6 experiments in 3 animals (SKF82958, respectively. **, p < 0.01; *, p < 0.05, significant effect compared with predrug baseline, Student's paired t test.

The Potentiating Effect of Clozapine on NMDA Receptor Currents Is Mediated by Activation of Src Family Kinases and Protein Kinase A. It has been hypothesized that protein kinase-mediated phosphorylation of NMDA receptor subunits leads to a potentiation of NMDA receptor function (Blank et al., 1997; Ali and Salter, 2001). Therefore, we investigated the effect of several selective kinase inhibitors on the clozapine-induced potentiation of NMDA receptor currents. Prior bath application (at least 15 min) of maximal concentrations of the selective Src kinase family inhibitor PP2 (10 µM) (Hanke et al., 1996) or the protein kinase A-selective inhibitor H-89 (10 µM) (Chijiwa et al., 1990) blocked the effect of 100 nM clozapine (3.9 ± 8.2% increase, n = 5, p > 0.05, Fig. 5, A2 and B; and 4.5 ± 3.9% increase, n = 6, p > 0.05, Fig. 5, A3 and B). The selective protein kinase C inhibitor BIS I (10 µM), at a concentration that partially blocks protein kinase A (Toullec et al., 1991), also inhibited the effect of 100 nM clozapine on NMDA receptor currents (0.5 ± 8% increase, n = 3, p > 0.05; Fig. 5, A5 and B). When Bis I was applied at a lower concentration that is selective for protein kinase C (1 µM) (Toullec et al., 1991), clozapine potentiation of NMDA receptor currents was not blocked (22.6 ± 5.4% increase, n = 5, p < 0.05; Fig. 5, A4 and B). Together, these data suggest that the mechanism of clozapine-induced potentiation of NMDA receptor function in the nucleus accumbens involves activation of Src family kinases and protein kinase A but not protein kinase C. However, it is possible that one or both (protein kinase A and Src family kinases) may act presynaptically by altering the release of dopamine rather than directly via a postsynaptic mechanism. We addressed this issue by repeating the above experiments and tested the effect of the Src family kinase inhibitor PP2 and the protein kinase A inhibitor H-89 on the effect of the D1 agonist SKF82958(100 nM). Consistent with a direct postsynaptic effect, the potentiating effect of SKF82958(100 nM) on NMDA receptor currents was significantly blocked by prior bath application of either the selective Src family kinase inhibitor PP2 (10 µM) or the protein kinase A inhibitor H-89 (10 µM) (6.0 ± 5.5% increase, n = 9, p > 0.05, and 2.4 ± 4.5% increase, n = 6, p > 0.05; Fig. 6). These data suggest that the potentiating effect of clozapine is mediated by postsynaptic activation of protein kinase A and Src family kinases that provide a mechanistic link between D1 receptor activation and the observed increase in NMDA receptor function.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Acute inhibition of protein kinase A and of Src family kinases, but not protein kinase C, blocks the potentiating effect of clozapine on NMDA receptor currents in nucleus accumbens neurons. A1 to A5, example traces showing the effect of clozapine (100 nM) in the absence (A1) and presence of the Src family kinase inhibitor PP2 (10 µM) (A2), the protein kinase A inhibitor H-89 (10 µM) (A3), and the protein kinase C inhibitor BIS I (1 and 10 µM) (A4 and A5). B, bar graph showing the effect of clozapine in the absence and presence of the selective kinase inhibitors PP2 (10 µM), H-89 (10 µM), and BIS I (1 and 10 µM). Each bar represents the mean ± S.E.M. of data from 3 to 12 experiments. **, p < 0.01, significant effect compared with predrug baseline.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Acute inhibition of protein kinase A and of Src family kinases blocks the potentiating effect of SKF82958on NMDA receptor currents in nucleus accumbens neurons. A1 to A3, example traces showing the effect of SKF82958(100 nM) in the absence (A1) and presence of the Src family kinase inhibitor PP2 (10 µM) (A2) and the protein kinase A inhibitor H-89 (10 µM) (A3). Bar graph showing the average effect of SKF82958(100 nM) in the absence and presence of the selective kinase inhibitors PP2 (10 µM) and H-89 (10 µM). Each bar represents the mean ± S.E.M. of data from 6 to 11 experiments. **, p < 0.01, significant effect compared with predrug baseline.

Potentiation by Clozapine Is Restricted to NMDA Receptors Containing the NR2B Subunit. NMDA receptor subtypes are known to be modulated selectively in other systems. Therefore, we examined clozapine potentiation of NMDA receptor currents in the presence and absence of the NR2B-selective antagonists CP101,606 (Chenard et al., 1995) and ifenprodil (Reynolds and Miller, 1989). Prior bath application of maximal concentrations of CP101,606 (1 µM) or ifenprodil (10 µM) completely blocked the potentiating effect of 100 nM clozapine on NMDA receptor currents (CP101,606, 6.5 ± 5% increase, n = 6, p > 0.05; ifenprodil, – 2.3 ± 2.6% increase, n = 6, p > 0.05; Fig. 7, A2, A3, and B). Next, we examined what proportion of the NMDA-evoked currents in the nucleus accumbens were mediated by activation of NMDA receptors containing the NR2B subunit. Figure 7C shows the time course of the inhibiting effect of 15-min bath application of ifenprodil (10 µM) on the amplitude of evoked NMDA receptor currents in the nucleus accumbens (46.8 ± 5% decrease, n = 5, p < 0.05, paired Student's t test). The remaining NMDA receptor current could be blocked completely by additional application of the nonselective NMDA receptor antagonist D-AP5 (20 µM) (Fig. 7C). Taken together, these data suggest that clozapine selectively potentiates the function of NMDA receptors containing the NR2B subunit and that a significant proportion (46.8%) of the NMDA-evoked current in nucleus accumbens medium spiny neurons is mediated by activation of NR2B subunit-containing NMDA receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Prior bath application of NMDA receptor subunit NR2B-selective antagonists blocks the potentiating effect of clozapine on NMDA receptor currents. A1 to A3, example traces showing the effect of clozapine (100 nM) in the absence (A1) and presence of the NR2B-selective NMDA receptor antagonists CP101,606 (1 µM) (A2) and ifenprodil (10 µM) (A3). B, bar graph showing the effect clozapine in the absence and presence of the NR2B-selective antagonists CP101,606 (1 µM) and ifenprodil (10 µM). Each bar represents the mean ± S.E.M. of data from 6 to 12 experiments. C, average time course showing the effect of 15-min bath application of the NR2B-selective antagonist ifenprodil (10 µM) on NMDA receptor currents and the additional effect of the NMDA receptor antagonist D-AP5 (20 µM). Each point represents the mean ± S.E.M. of data from five experiments. **, p < 0.01, significant effect compared with predrug baseline.

Clozapine Potentiates the Function of Synaptic NMDA Receptors but Not -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors. There is a good possibility that direct application of NMDA (100 µM) to the slices activates mostly extrasynaptic NMDA receptors and very few synaptic NMDA receptors. To test whether the effect of clozapine on NMDA receptor currents is relevant for glutamatergic synaptic transmission, we compared the effects of clozapine on synaptically evoked glutamatergic EPSCs in parasagittal brain slices. Glutamatergic EPSCs were evoked by placing a bipolar stimulation electrode into the nucleus accumbens 100 to 200 µm rostral to the recording site and stimulating synaptic inputs every 15 s. Bath application of clozapine (100 nM) for 15 min produced a small, nonsignificant increase in EPSC amplitude (21.7 ± 12.0% increase, n = 7, p > 0.05, paired Student's t test, data not shown) but did induce a significant increase of charge density (42.6 ± 11.6% increase, n = 7, p < 0.01, paired Student's t test; Fig. 8, A1 and B). This increase in charge density likely is due to the selective potentiation of the kinetically slower NMDA receptor currents (Fig. 8A1). We determined whether this effect of clozapine on charge density is mediated by an increase of NMDA receptor function rather than by effects on AMPA receptors by repeating the experiment in the presence of the NMDA receptor antagonist D-AP5 (20 µM). No significant effect of clozapine (100 nM) on EPSC charge density in the presence of D-AP5 (20 µM) (17.2 ± 13.7, n = 5, p > 0.05, paired Student's t test; Fig. 8, A2 and B) was observed. We also examined whether the effect of clozapine was voltage dependent by repeating the experiment stimulating excitatory inputs every 15 s while holding the cell at different holding potentials, changing from –20 to –80 mV. The results are shown in a charge density voltage plot in Fig. 8C. The charge density voltage plot revealed a profile characteristic of NMDA receptor activation with the peak charge densities observed at a holding potential of –40 mV. There was an overall significant increase of charge density induced by clozapine (100 nM) at the different holding potentials (p < 0.01, n = 5, Fig. 8C). However, there was no significant effect of voltage on the effect of clozapine. When we repeated the experiment in the presence of the NMDA receptor antagonist D-AP5 (20 µM), no significant effect of clozapine was seen (p > 0.05, n = 5, Fig. 8D). Taken together, these data suggest that clozapine potentiates the function of synaptically activated NMDA receptors, but not AMPA receptors, in a voltage-independent manner.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Clozapine increases the charge density but not amplitude of electrically evoked glutamatergic EPSCs in nucleus accumbens. EPSCs were recorded in the presence of 20 µM bicuculline and in low Mg2+ ACSF. A1 and A2, example traces showing the effect of bath application of clozapine (100 nM) on the amplitude and charge density of glutamatergic EPSCs before (predrug) and during clozapine application in the absence (A1) and presence of the NMDA-selective antagonist D-AP5 (20 µM) (A2) at a holding potential of –60 mV. The third traces represent an overlay of the predrug (black trace) and drug condition (gray trace) in the absence (A1) and presence (A2) of D-AP5 showing the effect of clozapine on the slower NMDA component of the EPSCs (A1). B, bar graph showing the average effect of clozapine (100 nM) on EPSC charge density in the absence and presence of D-AP5 (20 µM) at a holding potential of –60 mV. Each bar represents mean ± S.E.M. of data from five experiments each. *, p < 0.05, significant effect compared with predrug baseline. C, normalized average data of charge densities at different holding potentials (–80 to –20 mV) before drug (filled circles) and after 15-min bath application of clozapine (100 nM) (open triangles). Data were obtained from five experiments, and each point represents the mean ± S.E.M. of charge density at a particular holding potential. D, normalized average data of charge densities at different holding potentials in the presence of D-AP5 (20 µM), before (filled circles) and after 15 min of clozapine (100 nM) application (open triangles). Data were obtained from five experiments, and each point represents the mean ± S.E.M. of charge density at a particular holding potential. C and D, each point normalized to maximum control charge density (for C control value at –40 mV, for D control value at –80 mV).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The results of the present study show that acute application of low concentrations of clozapine, but not haloperidol or the 5-HT2A antagonist MDL100907, potentiates NMDA receptor function in nucleus accumbens neurons. This suggests an important mechanism of action for this unique atypical antipsychotic. We also demonstrate that the clozapine effect is mediated by D2-mediated dopamine release, resulting in activation of postsynaptic D1 receptors. Furthermore, we show that the D1-induced potentiation of NMDA receptor currents is limited to receptors containing the NR2B subunit of NMDA receptors and requires activation of the Src and protein kinase A family of kinases.

The current data add to a growing body of evidence demonstrating that clozapine can lead to a potentiation of the function of NMDA receptors in various brain regions. We and others have previously shown that in vivo application of clozapine induces a robust increase in c-fos expression in the nucleus accumbens and the prefrontal cortex with only minimal increase in the medial striatum and no change in the dorsolateral striatum (Robertson and Fibiger, 1992; Kinney et al., 2003). This c-fos expression in the nucleus accumbens is blocked by pretreatment with the NMDA receptor antagonist MK801 (Leveque et al., 2000) or increased by coadministration of D-cycloserine, a partial agonist at the strychnine-insensitive glycine site of the NMDA receptor complex (Leveque et al., 2000; Polese et al., 2002). Furthermore, we reported earlier that an inhibitor of the glycine transporter glycine transporter 1 induces a similar pattern of c-fos expression as clozapine (Kinney et al., 2003), suggesting that increases in NMDA receptor function have a similar effect. Previously, several electrophysiological studies have provided additional functional evidence demonstrating that clozapine potentiates evoked NMDA receptor currents, NMDA EPSCs, and can enhance NMDA-dependent long-term potentiation in the prefrontal cortex (Arvanov et al., 1997; Chen and Yang, 2002; Gemperle et al., 2003).

There is a large body of evidence implicating the importance of the nucleus accumbens in the pathophysiology of schizophrenia. The nucleus accumbens furthermore represents a site in which NMDA receptors may play a critical role in gating the flow of cortical information back to the prefrontal cortex (Grace, 2000). We now show that clozapine can induce a potentiation of NMDA receptor currents resulting in an increase in glutamatergic transmission in the nucleus accumbens. Hypofunction of the prefrontal cortex has been implicated with the severity of negative and cognitive symptoms in schizophrenia patients (Weinberger and Berman, 1996). An increase in activity in the nucleus accumbens would be expected ultimately to lead to increased activation of the prefrontal cortex, representing one possible mechanism by which clozapine might reduce negative and cognitive symptoms in these patients.

Previous studies in the prefrontal cortex have shown a similar effect of low concentrations of clozapine but also haloperidol and the 5-HT2A antagonist MDL100907 on NMDA receptor currents (Arvanov et al., 1997; Arvanov and Wang, 1998). However, in the present study, we demonstrate that only clozapine and neither haloperidol nor MDL100907 mimics this effect in nucleus accumbens neurons, indicating that the mechanism of action of antipsychotics may differ in different brain regions and that this might be one of the factors that produce the unique clinical profile of clozapine. The reason for the lack of effect of haloperidol in the nucleus accumbens remains unknown. However, it has been shown that haloperidol selectively binds to NR2B-containing NMDA receptors (Coughenour and Cordon, 1997) and inhibits NR1/NR2B-NMDA receptor currents at higher concentrations (IC₅₀, 3 µM; Ilyin et al., 1996). Therefore, haloperidol might partially inhibit NR1/NR2B-NMDA receptors in our study and, thus, prevent the D1-induced increase in NR2B-containing NMDA receptor currents.

We have shown that inhibition of both protein kinase A and Src family kinases blocks the potentiating effect of clozapine and D1 receptor activation on NMDA receptor currents, indicating that both kinases are necessary for this effect. Both Src family tyrosine kinases and protein kinase A have been shown to phosphorylate NMDA receptor subunits. In particular, the Src family kinase fyn has been identified as the main enzyme phosphorylating the NR2B subunit (Nakazawa et al., 2001). Protein kinase A also has been shown to phosphorylate the NR1 subunit of NMDA receptors, leading to an increase in function (Blank et al., 1997; Tingley et al., 1997). Furthermore, biochemical studies have demonstrated that D1-induced activation of protein kinase A leads to a protein kinase A-mediated synergistic mechanism of increased phosphorylation and decreased dephosphorylation of the NR1 subunit in nucleus accumbens slices and isolated striatal cells (Snyder et al., 1998; Flores-Hernandez et al., 2002). Our data show that only NR2B-containing NMDA receptors are potentiated by clozapine; however, activation of both Src family kinases and protein kinase A are necessary to induce the clozapine-mediated changes. The regulatory steps between the activation of D1 receptors and the increase of NMDA receptor currents are unclear; however, recent studies suggest three possible mechanisms. First, protein kinase A phosphorylation of NR1 subunits in addition to NR2B phosphorylation by Src family kinases might lead to an increase in NMDA receptor function. Second, RACK1 has recently been identified as a scaffolding protein for fyn as well as the NR2B subunit of the NMDA receptor, presumably providing a novel mechanism that localizes fyn in the proximity of NR2B subunits and at the same time prevents fyn phosphorylation of the NR2B subunit (Yaka et al., 2002). Interestingly, activation of the cAMP/protein kinase A pathway has been identified as an important modulator of the fyn/RACK1/NR2B complex leading to protein kinase A-induced release of RACK1 from the NR2B subunit and its subsequent fyn-mediated tyrosine phosphorylation (Yaka et al., 2003). Finally, a mechanism underlying increased NMDA receptor currents may be insertion of new receptors into the membrane. There is evidence that in the striatum D1-induced tyrosine kinase, phosphorylation of NR2A and NR2B subunits, possibly by Src family tyrosine kinases, leads to a rapid trafficking of NMDA receptors containing these subunits from intracellular membranous structures to the postsynaptic membrane (Dunah and Standaert, 2001). In addition, phosphorylation of NR1 subunits at serine⁸⁹⁰ has been shown to produce a redistribution of NR1 subunits from intracellular sites to the cell surface (Tingley et al., 1997). Therefore, phosphorylation of NR1 and NR2B subunits might lead to the trafficking of the NR1/NR2B NMDA receptor complex to the cell surface. Recent data give further support to the idea that receptor trafficking may underlie the clozapine-induced increase in NMDA receptor currents. In this study, chronic treatment of rats with clozapine induced increased MK801 binding only in the nucleus accumbens but not in other brain regions (Schmitt et al., 2003), without any changes in gene expression of NMDA receptor subunits (Hanaoka et al., 2003; Schmitt et al., 2003).

In summary, the present results demonstrate that acute clozapine application potentiates NR2B-containing NMDA receptor currents in the nucleus accumbens by a distinct mechanism involving Src kinases and protein kinase A, possibly leading to an increase of cortical information flow through the nucleus accumbens. This effect of clozapine could be an important possible mechanism underlying the significant clinical benefit of clozapine on cognitive and negative symptoms in schizophrenic patients.

	Footnotes

 
This work was supported by Merck and Co., Inc.

doi:10.1124/jpet.104.080200.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; ACSF, artificial cerebrospinal fluid; EPSC, excitatory postsynaptic current; MDL100907, (R)-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluoro-phenyl)ethyl]-4-piperidine methanol; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; BIS I, bisindolylmaleimide I; CP101,606, (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-proponal; SCH23390 (R)-(+)-7-chloro-8-hyrdoxy-3-methyl-1-phenyl-2,3,4,5-tetrahyrdo-1H-3-benzazepine hydrochloride; SKF 82958, 6-chloro-N-allyl-SKF-38393 hydrobromide; D-AP5, D-(–)-2-amino-5-phosphonopentanoic acid; 5-HT2A, 5-hydroxytryptamine 2A; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; MK801, 5H-dibenzo[a,d]cyclohepten-5,10-imine.

Address correspondence to: Dr. Marion Wittmann, Alzheimer's Research, Merck Research Laboratories, 770 Sumneytown Pike, WP44E-200, West Point, PA 19486. E-mail: marion_wittmann{at}merck.com

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