Introduction

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The relative ineffectiveness of dopamine antagonists to treat some symptoms of schizophrenia has prompted many investigators to postulate the involvement of other systems in the pathophysiology of this disease. The glutamate hypothesis of schizophrenia proposes a relationship between hypoactive glutamate neurotransmission, particularly NMDA receptor hypofunction, and the pathophysiology of schizophrenia (Carlsson and Carlsson, 1990; Deutsch et al., 1989; Kim et al., 1980; Moghaddam, 1994; Olney and Farber, 1995; Wachtel and Turski, 1990). This hypothesis stems from the observation that in normal human subjects, the NMDA receptor channel blockers ketamine and phencyclidine, can induce psychosis that includes many symptoms and cognitive disturbances commonly observed in patients with schizophrenia (Grotta, 1994; Herrling, 1994; Kristensen et al., 1992). Furthermore, in schizophrenics, NMDA receptor antagonists produce an exacerbation of psychotic symptoms (Lahti et al., 1995). However, evidence for a defect of NMDA receptor function in schizophrenia remains inconclusive. For example, it has been shown that [³H]kainate, D-[³H]aspartate, [³H]tenocyclidine and [³H]glycine binding is increased in the frontal cortex and [³H]dizocilpine (MK-801) binding is increased in the putamen of post-mortem schizophrenics, whereas the [³H]MK-801 binding sites in the temporal lobe areas including the hippocampus are not affected; in contrast, there is a decrease in non-NMDA receptor mRNA in cortex (Deakin et al., 1989; Harrison et al., 1991; Ishimaru et al., 1994; Kerwin et al., 1990; Kornhuber et al., 1989; Nishikawa et al., 1983; Simpson et al., 1991; Ulas and Cottman, 1993). The prefrontal cortex of schizophrenics has recently been shown to exhibit alterations in the expression of NR2 subunit mRNAs, which are potential indicators of deficits in NMDA receptor-mediated neurotransmission accompanying functional hypoactivity of the frontal lobe (Akbarian et al., 1996).

There is evidence suggesting that some APDs might affect the NMDA receptor mediated transmission. For example, clozapine and olanzapine have been shown to reverse noncompetitive NMDA antagonist-induced social withdrawal in rats (Corbett et al., 1995) and prevent MK-801 neurotoxicity (Farber et al., 1996). Moreover, the potency of a series of neuroleptics in blocking phencyclidine-induced hyperlocomotion correlated with the affinity for 5-hydroxytryptamine_2A receptors (Gleason and Shannon, 1997; Maurel-Remy et al., 1995). In agreement with these findings, in vivo microdialysis studies indicate that the acute, systemic administration of clozapine, but not haloperidol, produces an increase in extracellular concentrations of glutamate (Daly and Moghaddam, 1993; Yamamoto et al., 1994) and aspartate (Daly and Moghaddam, 1993) in the mPFC of freely moving rats. Acute clozapine, however, does not alter glutamate levels in the neostriatum (Daly and Moghaddam, 1993). Electrophysiological studies have shown that both haloperidol and clozapine at low and high (>100 nM) concentrations augment and depress, respectively, the amplitude of field potentials in the rat neostriatum slices elicited by electrical stimulation of the overlying white matter (Banerjee et al., 1995). Clozapine has also been shown to potentiate population spikes in the entorhinal-dentate gyrus perforant pathway (Kubota et al., 1996). Currently, the exact explanation for the action of APD on glutamatergic neurotransmission is unknown.

Because a role for glutamate receptors has been suggested in schizophrenia, it is of interest to examine more closely whether APDs affect glutamate receptor subtype-mediated neurotransmission at the cellular level. The present study was designed to investigate and compare effects of haloperidol and clozapine on NMDA- and AMPA-induced responses in pyramidal cells of the mPFC, an area that has been suggested to play a key role in the pathogenesis of schizophrenia and in the working memory and cognitive functions (Berman and Weinberger, 1990), using the techniques of intracellular recording and single-electrode voltage-clamp. The effect of haloperidol and clozapine on NMDA- and non-NMDA receptor-mediated EPSPs/EPSCs evoked by electrical stimulation of the forceps minor was also examined. A portion of these results have appeared in a preliminary form (Wang and Arvanov, 1996).

	Methods

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Preparation of mPFC slices. The procedures for preparation of rat mPFC brain slices have been previously described (Arvanov and Wang, in press; Yang et al., 1996). Briefly, male Sprague-Dawley rats (body weight, 120-200 g; n = 87) were decapitated while under halothane anesthesia, and their brains were removed and cooled in ice-cold aCSF. The coronal (transverse) slices of mPFC (450 µm thick) were cut in ice-cold aCSF containing (in mM) NaCl 117, KCl 4.7, CaCl₂ 2.5, MgCl₂ 1.2, NaHCO₃ 25, NaH₂PO₄ 1.2 and D-glucose 11, aerated with 95% O₂/5% CO₂ (pH 7.4) and kept submerged in room temperature for 1 hr to allow for recovery. A single slice was then transferred to a recording chamber (32°C), in which it was held submerged between two nylon nets. The chamber was continuously superfused with aCSF at a constant rate of 1.5 ml/min.

Identification of pyramidal and nonpyramidal neurons. According to Connors and Gutnick (1990) and McCormick et al. (1985), there are three basic types of neocortical neurons: RS, IB and FS. For each type, classification is based on three general variables: characteristics of individual action potential/afterpotential complexes, response to a just-threshold intracellular current pulse and repetitive response to prolonged, intracellularly applied stimuli. RS and IB are identified as pyramidal neurons, and FS cells are nonpyramidal, presumably GABAergic interneurons (the spike width of FS cells is typically <0.5 msec at half-amplitude; when stimulated with a suprathreshold step of depolarizing current, they generate high frequencies that are sustained for the duration of the stimulus; Connors and Gutnick, 1990; Kawaguchi, 1993; McCormick et al., 1985). Recent studies, however, have revealed that nonpyramidal cells display a great diversity of intrinsic firing properties and can show frequency accommodation (Cauli et al., 1997; Kawaguchi and Kubota, 1996). In addition to RS and IB types pyramidal cells, Yang et al. (1996) added ROB and IM types. In general, the interneurons are characterized by a brief spike duration (<1 msec at half-maximum spike amplitude) and a lack of pronounced spike frequency adaptation in response to constant-current depolarizing pulses, whereas the pyramidal cells have a longer spike duration, particularly the second spike (1-3 msec at half-maximum spike amplitude), and show pronounced spike-frequency adaptation (Cauli et al., 1997; Connors and Gutnick, 1990; Kawaguchi, 1993; Kawaguchi and Kubota, 1996; McCormick et al., 1985).

Intracellular recording and single-electrode voltage-clamp. Standard intracellular and single-electrode voltage-clamp recording techniques were used to record pyramidal cells in layers V and VI of the mPFC in slice preparations as previously described (Arvanov et al., 1996; Arvanov and Wang, in press; Wang and Arvanov, 1996). Sharp electrode recordings were preferable to minimize the washout of intracellular content occurring during the whole-cell recordings (Arvanov et al., 1992; Arvanov and Usherwood, 1991; McDonald et al., 1989). Intracellular recordings were performed using 4 M K-acetate- or 3 M KCl-filled microelectrodes (tip resistances, 60-90 M) with an Axoclamp 2B (Axon Instruments, Burlingame, CA) amplifier. In current-clamp mode, the bridge balance was continually monitored and adjusted as necessary. Single-electrode voltage-clamp was achieved under discontinuous mode at a sampling rate of 5 to 6.2 kHz (30% duty cycle) and a gain of 2.5 to 5 nA/mV. The efficacies of voltage-clamp, electrode "settling time" and input capacitance neutralization at the head stage were continuously monitored on an oscilloscope. Current and voltage records were acquired using digital/analog sampling and acquisition software (pClamp 6; Axon Instruments) and were filtered at 1 kHz and analyzed off-line. Voltage and current signals were also recorded on a Gould (Cleveland, OH) Easy Graph Thermal Recorder (TA 240) and two-channel videotape recorder (Instrutech VR-10B Digital Data Recorder, Elmont, NY). The problems (e.g., space clamping) associated with this method in neurons with extended processes have been previously discussed (Finkel and Redman, 1985). As it has been pointed out, these problems faced during single-electrode voltage-clamp may be less acute when dealing with the relative changes after drug application (Madison et al., 1987; Schweitzer et al., 1993). The voltage-clamp conditions during our experiments were sound because we were able to clamp the fast EPSPs evoked by electrical stimulation (see below). By using a combination of current- and voltage-clamp recordings, the effect produced by APDs on NMDA- and AMPA-induced response could be examined with confidence.

Electrical stimulation-evoked EPSPs/EPSCs. Electrodes for voltage-clamp experiments were filled with 2 M CsCl plus 25 to 50 mM QX314 (lidocaine N-ethyl bromide quaternary salt; tip resistance, 30-50 M) to improve the space clamp and block voltage-activated Na⁺ and K⁺ channels; under these conditions, the membrane resistance was usually increased by 30% to 50% (Wuarin et al., 1992). EPSPs/EPSCs were elicited by passing rectangular current pulses (0.3-0.5-msec duration; 50-250 µA) between the tips of a bipolar stainless steel electrode placed in the medial part of the forceps minor close to the recording electrode (Wuarin et al., 1992). Following the protocol of Tanaka and North (1993), experiments were carried out using a stimulus strength that was 70% of the threshold for evoking an action potential. A train of five electrical pulses was delivered at a rate of 0.05 Hz, three times before and after drug application (i.e., in all experiments), induced synaptic responses before and after drug application were averaged (n = 15). To isolate NMDA receptor-mediated EPSPs, the preparation was superfused with 20 µM CNQX, 5 to 10 µM bicuculline, 0.5 µM CGP 52432 and 1 µM glycine. Under these conditions, the amplitude of EPSPs was reduced to 17%, indicating that under normal conditions EPSPs were mediated primarily through non-NMDA receptors. The stimulus strength was increased 2-fold, and high-intensity electrical stimulation elicited a longer lasting EPSP.

Data analysis. The percent modulation produced by APDs on NMDA- and AMPA-evoked responses was calculated by subtracting the base-line peak amplitude of the responses from that evoked by bath application of APDs. This value was then divided by the base-line response and multiplied by 100. The results were presented as mean ± S.E., except when the data were transformed for analysis of variance (one-way and mixed level; see below). Paired t tests, Student's t tests, analysis of variance and mixed-level analysis of variance models applying SAS Proc Mixed procedure were used; .05 was selected for testing the level of significance after Bonferroni or modified Bonferroni correction. One of the key assumptions of analysis of variance is homoscedasticity of variances in the parameters under study. Due to heteroscedastic variances in the parameters studied, the analysis and significance tests were performed after application of transformations, which included log, square root, reciprocal square root and reciprocal square (Zar, 1996). Note that because transformations were applied, the intervals are not symmetrical around its least-squares mean value; therefore, when transformation was applied, the results were presented as least-squares mean and its corresponding interval (least-squares mean minus S.E., least-squares mean plus S.E.).

Drugs. The compounds AMPA, NMDA, QX314, TTX and bicuculline methochloride were purchased from Research Biochemicals (Natick, MA). Clozapine, haloperidol and CGP 52432 were generous gifts from Sandoz (Hanover, NJ), McNeil Laboratories (Fort Washington, PA) and Ciba-Geigy (Basel, Switzerland), respectively.

	Results

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Presumed pyramidal neurons in the mPFC. All experiments were routinely performed on presumed pyramidal neurons in layers five and six of the prelimbic cortex (anterior cingulate cortex areas 1 and 3; Zilles, 1985), which were located medial to the forceps minor and could be easily identified in the slice. A total of 93 presumed pyramidal cells has been recorded according to established criteria (Connors and Gutnick, 1990; Kawaguchi, 1993; McCormick et al., 1985; Yang et al., 1996). Stable recordings could be maintained for 4 to 5 hr, suggesting a relative lack of injury by the electrode penetration. It is rare to impale FS neurons with a relatively low-resistance unbeveled microelectrode (McCormick et al., 1985), which might account for the fact we have not encountered any FS nonpyramidal cells in our studies. As previously described (for review, see Connors and Gutnick, 1990), most (68%) pyramidal neurons encountered were the RS type. In normal aCSF, the great majority (>90%) of recorded cells were quiescent. They (n = 87) exhibited a mean resting membrane potential of 72.4 ± 1.2 mV, a spike amplitude of 82.9 ± 1.5 mV, a membrane resistance of 51.7 ± 4.1 M (measured from the linear part of current-voltage curve) and a time constant of 10.4 ± 1.2 msec. These results are comparable to those reported by Tanaka and North (1993) and Yang et al. (1996).

Effect of NMDA in presumed pyramidal cells of the mPFC. In all 19 cells studied under the current-clamp mode, NMDA (10 µM) elicited EPSPs (frequency, 0.71 ± 0.03 Hz; amplitude, 7.4 ± 1.2 mV) followed by a membrane depolarization (amplitude, 8.5 ± 3.1 mV, range, 5-17 mV) and bursts of action potentials (fig. 1). In the voltage-clamp mode, at the holding potential (V_h) of 60 mV, NMDA 10 µM produced an inward current of 30-70 pA (46 ± 12 pA, n = 35; fig. 2).

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Potentiation of NMDA-induced membrane depolarization and increase of the number of NMDA-induced EPSPs and action potentials by both clozapine and haloperidol. A, Representative voltage traces illustrating that clozapine, at the concentration of 20 nM, significantly enhanced NMDA-induced responses; NMDA response returned to baseline after 15-min wash. Traces 1 and 2 were expanded from time points of 1 and 2 in A to show clearly that clozapine markedly increased the frequency of NMDA-evoked EPSPs. B, Haloperidol at concentrations of 50 to 100 nM significantly potentiated NMDA-evoked responses. At concentrations of 100 nM, haloperidol tended to hyperpolarize the membrane potential.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The potentiation of NMDA-induced inward current by both clozapine and haloperidol. A, Representative current traces to illustrate that at the concentration of 50 nM, both haloperidol and clozapine, but not raclopride or fluoxetine, strikingly augmented NMDA-induced inward current. B, Comparison of concentration-response curves for clozapine, haloperidol, raclopride and fluoxetine to potentiate NMDA current. Each point represents the least-squares mean of four or five experiments adjusted for cell variability ± S.E. computed from a mixed-level analysis of variance model using SAS Proc Mixed procedure. *, Mean for dose is significantly different than that of control (P < .05, after Bonferroni correction). Due to heteroskedastic variances associated with concentrations, the analysis and significance tests were performed after applying a square root transformation; the adjusted mean ± S.E. was transform back to original scale for this plot.

Comparison of effects of clozapine and haloperidol on NMDA-induced response. Experiments were performed in both current-clamp and voltage-clamp mode to ensure that the effects of clozapine and haloperidol were not the result of altering membrane potentials (see below). To minimize actions of APDs on dopamine inhibition of GABAergic transmission (Kalivas et al., 1993), all voltage-clamp experiments were carried out in the presence of 5 to 10 µM bicuculline (a GABA_A receptor antagonist that has been shown in a separate study to completely block the inhibitory action of GABA).²

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Similarities and differences of effects produced by haloperidol and clozapine on NMDA- and AMPA-induced responses. Each bar represents the least-squares mean of four to six experiments ± S.E. computed from an analysis of variance model (a square root and log transformations were applied). *, Mean is significantly different than that of control (P < .05, after modified Bonferroni correction).

Effects of haloperidol and clozapine on membrane properties of mPFC pyramidal neurons. Facilitation of NMDA-evoked responses by 10 to 50 nM clozapine has not been associated with changes of membrane properties [e.g., neither membrane potential nor input resistance (n = 8) has been altered significantly]. However, at concentrations of 100 nM to 1 µM, clozapine elicited EPSPs (amplitude, 8.6 ± 1.1 mV; frequency, 1.1 ± 0.4 Hz; fig. 4A) and depolarized membrane potential (4.3 ± 1.1 mV) in all seven cells tested. The membrane potential recovered to control values and EPSPs were not observed 30 min after the washout of clozapine.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Voltage-current traces to illustrate clozapine-evoked EPSPs and membrane depolarizations in pyramidal cells of the mPFC. A, Representative voltage traces illustrating that clozapine at 100 nM induced EPSPs and produced a membrane depolarization. B, Comparison of EPSCs evoked by electrical stimulation of the forceps minor and induced by clozapine on the same pyramidal neurons at different levels of Vh. Both EPSCs reversed their polarity at the same Vh of 8 to 3 mV.

Effects of haloperidol and clozapine on AMPA-induced response. Haloperidol inhibited AMPA-induced inward current in a concentration-dependent manner with an EC₅₀ value of 37 nM (fig. 5). In contrast, clozapine did not significantly alter AMPA current. Although at 100 nM, clozapine decreased AMPA-induced inward current to 66 ± 19% (n = 4) of control, the reduction did not reach statistical significance. The latter effect was associated with the appearance of EPSCs. It might be speculated that clozapine-induced reduction of AMPA current is the result of desensitization of AMPA receptors caused by the bombardment of EAAs released by clozapine.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Comparison of effects produced by haloperidol and clozapine on AMPA-activated inward current. A, Representative current traces to illustrate that haloperidol at 20 to 100 nM depressed AMPA current in a concentration-dependent manner, whereas clozapine failed to alter significantly AMPA current. Note that at 100 nM, clozapine produced EPSCs. B, Comparison of the concentration-response curves for haloperidol and clozapine to modulate AMPA-induced inward current. Statistical analyses of the data have been performed as described in the legend of figure 2.

Effects of haloperidol and clozapine on EPSPs/EPSCs evoked by electrical stimulation of the forceps minor. In normal aCSF, haloperidol depressed EPSPs evoked by electrical stimulation of the forceps minor in a concentration-dependent fashion; the depressed EPSPs returned to basal level after 30 min of washout of haloperidol (fig. 6A). At the concentrations of 50 and 100 nM, haloperidol markedly reduced the peak amplitude of EPSPs by 52 ± 7% and 68 ± 4% (P < .05 for both cases, paired t tests; n = 5), respectively. Analyses of the current-voltage relationship of electrical stimulation-evoked EPSCs revealed that the depressant action of haloperidol was voltage independent because 50 nM haloperidol depressed EPSCs to 45 ± 3% and 43 ± 3% of base line at the holding potential of 70 and 20 mV (n = 5; fig. 6, B and D), respectively. The inhibitory effect of haloperidol was blocked by CNQX (fig. 6D3) but undiminished in the presence of D-()-AP-5 (fig. 6D2).These results correspond to the finding that haloperidol markedly depresses AMPA-induced inward current.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Differential effects of clozapine and haloperidol on the amplitude of EPSPs/EPSCs evoked by electrical stimulation of the forceps minor in pyramidal cells. A, Representative voltage traces illustrating that haloperidol depressed EPSPs in a concentration-dependent manner, whereas clozapine enhanced amplitude of EPSPs. B, Representative current traces to show that clozapine potentiated electrical stimulation-evoked EPSCs in a voltage-dependent manner, whereas haloperidol depressed EPSCs in a voltage-independent fashion. C and D, Current-voltage relationship analyses of EPSCs evoked by electrical stimulation of the forceps minor to illustrate the influence of clozapine and haloperidol.  and , Peak amplitude of EPSCs in the presence and absence of APDs, respectively. 1 to 3, Results obtained in normal aCSF, aCSF plus 40 µM D-()-AP-5 and aCSF plus 20 µM CNQX, respectively. The facilitation of EPSCs by clozapine was voltage dependent, and the effect produced by clozapine was abolished by D-()-AP-5. Moreover, CNQX was also effective in blocking the potentiating action of clozapine on electrical stimulation evoked EPSCs. In contrast, the depressant action of haloperidol on EPSCs was voltage independent; CNQX, but not D-()-AP-5, prevented the action of haloperidol.

	Discussion

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The major finding of the present study is that both the classic APD haloperidol and the atypical APD clozapine potently facilitate NMDA-evoked depolarization and membrane current in the rat pyramidal cells of the mPFC in a concentration-dependent manner with an EC₅₀ value of 38 and 14 nM, respectively. Although it is difficult to estimate the precise synaptic concentrations for haloperidol and clozapine to facilitate NMDA response because of the incomplete equilibrium conditions in which the APDs were typically applied for 10 min, the aCSF concentrations in the recording chamber needed for clozapine and haloperidol to exert their action are in a clinically relevant range. For example, serum levels for haloperidol and clozapine of schizophrenic patients are 10 to 70 and 447 to 3387 nM, respectively, after the administration of a range of clinical doses (Baldessarini et al., 1988; Farde et al., 1995; Nordstrom et al., 1995; Verghese et al., 1991). Moreover, it has been estimated that the majority of patients have an approximate CSF or plasma water molarity of 1 to 3 and 11.7 nM for haloperidol and clozapine, respectively (Seeman, 1992). Therefore, based on the estimation of free APDs concentrations in CSF or plasma water of schizophrenic patients, our results suggest that clozapine and, to a much lesser extent, haloperidol may exert some of their antipsychotic effects by altering glutamate neurotransmission in the mPFC if our findings can be generalized to humans. Obviously, further systematic studies of various APDs after both acute and chronic treatment must be performed to have a more thorough evaluation of the effect of APDs on glutamate neurotransmission. At any rate, the facilitating effect of clozapine and haloperidol on the NMDA response may contribute to the compensatory down-regulation of [³H]MK-801 binding in the mPFC observed after subchronic administration of clozapine and haloperidol (Tarazi et al., 1996). On the other hand, it has also been reported that subchronic haloperidol produced an increase in NMDA receptors as quantified by L-[lsqb]³H]glutamate binding (Ulas et al., 1993) and that chronic treatment with either haloperidol or clozapine increases glutamate₁ receptor but not NMDA₁ receptor subunit immunoreactivity in the rat mPFC (Fitzgerald et al., 1995). The alterations of glutamate receptor subtypes produced by repeated treatment with APDs must be verified by functional studies at the cellular level.

It is unlikely that the facilitating effect of clozapine and haloperidol resulted from a direct interaction with NMDA receptors because of the relatively low affinity of these drugs for the [³H]MK-801 binding sites (Kohler et al., 1985; Lidsky et al., 1993; Lynch and Gallagher, 1996; Tarazi et al., 1996). This is supported by our finding that haloperidol and clozapine were not capable of producing the potentiating effect on NMDA-induced inward current in aCSF containing CNQX. Interestingly, we have shown in a separate study (Wang et al., 1997) that MDL 100907, a highly selective 5-hydroxytryptamine_2A receptor antagonist and a purported atypical APD (Kehne et al., 1996), like clozapine, dramatically augments NMDA responses in pyramidal cells of the mPFC; it may enhance NMDA responses by facilitating NMDA-induced release of EAAs, which in turn activate non-NMDA receptors, cause membrane depolarization and remove Mg⁺⁺ block of the NMDA receptor/ionophore complex, thereby facilitating strikingly NMDA responses. Thus, the ability of MDL 100907 to potentiate NMDA-induced inward current is abolished by CNQX and under the conditions [e.g., Ca⁺⁺-free aCSF or low Ca⁺⁺ (0.1 mM) plus Cd⁺⁺ (0.2 mM) aCSF] that prevent Ca⁺⁺-dependent release of neurotransmitters (Wang et al., 1997). It is possible that clozapine may act in the same fashion as MDL 100907 to enhance NMDA responses by facilitating the release of EAAs, although similar experiments as shown with MDL 100907 must be performed to determine the site of action of clozapine. It should be pointed out that TTX does not prevent the potentiating effect of clozapine or haloperidol, possibly because TTX depresses only action potential-dependent release of EAAs, which is ~30%, in rat brain slices (Martin et al., 1991, 1993); the result argues against the involvement of polysynaptic circuitry for APDs to enhance NMDA responses because TTX would have prevented the generation of action potentials.

It might be speculated that the biochemical mechanisms behind clozapine- and haloperidol-induced potentiation of NMDA responses are secondary to their binding to other receptor or effector systems. For example, the blockade of dopamine and serotonin receptors by clozapine and haloperidol could lead to an increase in glutamate release via removal of the inhibitory action of serotonin and dopamine (Kornhuber and Kornhuber, 1986; Maura et al., 1988a, 1988b, 1989; Peris et al., 1988). Our finding that raclopride did not modulate the NMDA response until its concentration was raised to micromolar range suggests that dopamine D₂ receptors may not have a critical role in mediating the action of haloperidol and clozapine. However, it is important to note that raclopride will not occupy D₄ receptors because the raclopride dissociation constant is very high for D₄ receptors (Van Tol et al., 1991). Therefore, the possibility of the involvement of other dopamine receptor subtypes cannot be disregarded.

One of the major differences between haloperidol and clozapine is that clozapine, but not haloperidol, elicited EPSPs/EPSCs at concentrations of 100 nM. The clozapine-evoked EPSPs/EPSCs were similar to those evoked by NMDA and by electrical stimulation of the forceps minor. Both clozapine- and electrical stimulation-evoked EPSCs reversed their polarity at the same V_h, and both were blocked by CNQX plus D-()-AP-5. These findings indicate that EPSPs/EPSCs were the results of increased release of excitatory amino acids. In other words, at concentrations of 100 nM, clozapine, but not haloperidol, produced a dramatic increase of release of EAAs, similar to that produced by electrical stimulation of the forceps minor. Our results are in excellent agreement with those obtained in in vivo microdialysis studies showing that acute clozapine, but not haloperidol, increases extracellular concentrations of glutamate in the mPFC of freely moving rats (Daly and Moghaddam, 1993; Yamamoto et al., 1994), although it is difficult to directly compare the results obtained from in vitro slice vs. in vivo microdialysis. The clozapine-induced marked increase of the release of EAAs in the mPFC may account for, at least in part, the report that clozapine is associated with a higher incidence of seizures than traditional APDs in patients with no previous history of ictal events (Haller and Binder, 1990).

Another major difference between the two APDs is that clozapine markedly potentiated, whereas haloperidol decreased, EPSPs/EPSCs elicited by electrical stimulation of the forceps minor (white matter) in pyramidal neurons of the mPFC. Clozapine-induced facilitation of the evoked EPSPs was voltage dependent (i.e., the potentiating effect of clozapine increased dramatically when the V_h was changed to a more depolarized potential), supporting the finding that clozapine preferentially potentiates NMDA receptor-mediated transmission because membrane depolarization markedly enhances the efficacy of activation of the NMDA receptor/ionophore complex (Nowak et al., 1984). In addition, there is a tendency for clozapine to shift the reversal potential of EPSCs to a more positive level, which presumably could be attributed to the more positive reversal potential of the NMDA component of EPSPs (Arvanov and Wang, in press; Burgard and Hablitz, 1993; Wuarin et al., 1992). Our results indicate that clozapine preferentially potentiated NMDA receptor-mediated transmission, whereas haloperidol produced an overall inhibitory action on glutamate receptor-mediated neurotransmission. The latter is likely due to the fact that the marked depressing action of haloperidol on the non-NMDA component of EPSPs (see below) prevented or obscured the potentiating action on the NMDA component.

Haloperidol, but not clozapine, depressed AMPA-induced response in a concentration-dependent manner. Furthermore, in the presence of D-()-AP-5, haloperidol produced a voltage-independent inhibition of the non-NMDA component of EPSPs evoked by electrical stimulation of the forceps minor. The mechanisms of the inhibitory action of haloperidol on non-NMDA AMPA receptors are not clear at present, although the inhibition of Na⁺ channels by haloperidol (Pencek et al., 1978; Westlind-Danielsson et al., 1992) may in part account for the inhibitory effect. The inhibitory action of haloperidol on AMPA response may contribute to the result that haloperidol, but not clozapine, decreased the amplitude of EPSPs evoked by NMDA administration.

In summary, in the present study, we have shown that both the atypical APD clozapine and the typical APD haloperidol potentiate NMDA-evoked responses in pyramidal cells of the mPFC. In addition, clozapine enhances, whereas haloperidol depresses, electrical stimulation-evoked EPSPs/EPSCs, which might be due to the fact that haloperidol, but not clozapine, depresses AMPA-induced response in a concentration-dependent manner. Further systematic comparison of the similarities and differences in the effect of typical and atypical APDs on NMDA and non-NMDA receptors may help to more fully assess the role of glutamate in schizophrenia and the mechanisms of APD action with regard to therapeutic efficacy and side effects (for review, see Ashby and Wang, 1996). The results generated from these studies may provide a new paradigm to differentiate between typical and atypical APDs.