Introduction

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The  receptors are membrane-associated binding sites of unknown physiological function that have been implicated in regulating psychotomimetic behaviors in animal models of schizophrenia (Martin et al., 1976; Su, 1982; Tam and Cook, 1984; Taylor and Dekleva, 1987; Snyder and Largent, 1989; Walker et al., 1990). Radioligand binding studies indicate that there are at least three, and possibly more, types of  site; denoted ₁, ₂, ₃ etc. (Hellewell and Bowen, 1990; Quirion et al., 1992; Booth et al., 1993). The ₁ site is modulated by guanosine 5-triphosphate (GTP) and, in rat brain synaptoneurosomes, has effects on phosphatidylinositol turnover stimulated by muscarinic acetylcholine receptor activation (Bowen et al., 1988). A protein with the radioligand binding profile of the ₁ site has recently been cloned (Hanner et al., 1996). The protein is unrelated to any known mammalian receptor. Indeed, the only detected sequence homology is with fungal proteins involved in synthesis of sterols. The ₂ site may mediate the motor effects of many  ligands (Matsumoto et al., 1990), and the regulatory effects of  ligands on K⁺ channels (Wu et al., 1991). The ₃ site is less well characterized, but appears to influence dopamine metabolism in rat and guinea pig forebrain (Booth et al., 1993).

A structurally diverse range of compounds bind to  sites (e.g., Largent et al., 1988; Walker et al., 1990; Rothman et al., 1991; de Costa and He, 1994). Confusion has arisen in the field because many of the early  ligands interact with other types of receptor (Walker et al., 1990). This has made it difficult to positively ascribe biochemical, physiological and behavioral effects of  ligands to actions specifically at  sites. One example of poor specificity is the direct interaction between  site ligands and NMDA receptors (Zukin et al., 1984; Tam and Cook, 1984; Lockhart et al., 1995).

Previous studies have characterized inhibition of NMDA receptor responses by  ligands such as pentazocine and DTG (Church and Lodge, 1990; Fletcher et al., 1993). For these drugs inhibition appeared to be consistent with blockade of the channel pore, probably via the PCP site. Sigma ligands such as ifenprodil, belonging to the N-substituted 4-benzyl-piperidine structural class, similarly antagonize NMDA receptors (Karbon et al., 1990; Legendre and Westbrook, 1991; Carter et al., 1991; Church et al., 1994). For these compounds inhibition is mechanistically distinct from the PCP site and is characterized by pronounced selectivity for receptor subtypes comprised of NR1/2B subunit combinations (Moriyoshi et al., 1991; Monyer et al., 1992; Williams, 1993). The -site ligand haloperidol, belonging to the butyrophenone structural class, is also an NMDA receptor antagonist (Fletcher and MacDonald, 1993; Cougenour and Cordon, 1997; Ilyin et al., 1996). In the initial studies (Fletcher and MacDonald, 1993), inhibition of NMDA responses by haloperidol showed dependence on glycine concentration, leading to the proposal that haloperidol is a partial agonist for the glycine coagonist site. In subsequent studies (Ilyin et al., 1996), inhibition of NMDA responses by haloperidol was found to be insurmountable with respect to glycine and glutamate, and, as with ifenprodil, to have selectivity for NR1/2B receptors.

To complicate matters further, low, systemically administered doses of  site ligands such as DTG and (+)-pentazocine increase the sensitivity of CA3 hippocampal neurons to locally applied NMDA (Monnet et al., 1990, 1992). Other  site ligands, such as haloperidol and R(+)-3-PPP, inhibit this facillitation, suggesting that the drugs are behaving as  receptor agonists and antagonists. Similar types of effect are seen in vitro, for example, in the regulation of NMDA-induced release of [³H]-norepinephrine from rat hippocampal slices (Gonzales-Alvear and Werling, 1995; Monnet, et al., 1996), and [³H]-dopamine release from rat striatal slices (Gonzales-Alvear and Werling, 1994). Collectively, these results suggest that receptors modulate NMDA receptor function, probably via a second messenger system (Debonnel, 1993; Monnet et al., 1996).

Most recently, indirect mechanisms have also been proposed to explain the inhibitory effects of many  ligands on NMDA receptors. For example, Yamamoto et al. (1995) report that there is a close correlation between affinity for ₁ sites and potency for inhibition of TCP binding in cultured rat forebrain neurons (Yamamoto et al., 1995). Similarly, Hayashi et al. (1995) report that in cultured rat cortical neurons inhibition of NMDA-induced Ca⁺⁺ influx by  site ligands is independent of PCP site potency but correlates well with affinity for  sites (Hayashi et al., 1995). In each case, the authors suggest that inhibition of NMDA receptors is due to indirect modulation mediated by  receptors. If correct, this result is important because it means that NMDA receptor inhibition can be used as a simple functional assay of  receptor activation and inhibition (Walker et al., 1990). However, contrary to these findings, Fletcher et al. (1995) assayed some of the same  ligands for inhibition of NMDA-activated membrane currents responses in cultured rat hippocampal neurons and concluded that inhibition is due to direct antagonism of NMDA receptors.

To investigate the issue further we used whole cell electrical recordings to assay 17  site ligands for inhibition of three cloned NMDA receptor subunit combinations expressed in Xenopus oocytes. To confirm that data from the cloned NMDA receptors is relevant to neuronal receptors we tested 10  ligands for inhibition of NMDA responses in cultured rat cortical neurons. We then compared potency, subtype-selectivity and mechanism of NMDA receptor inhibition with previously reported affinities for  sites and PCP sites, as determined by binding assays. A diverse group of  ligands was chosen to lessen the chances of generating false correlations due to a limited sample of compounds. A preliminary report of this work has appeared previously (Ilyin et al., 1995).

	Methods

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Expression of cloned NMDA receptors in Xenopus oocytes. cDNA clones encoding NR1a, NR2A, NR2B and NR2C rat NMDA receptor subunits were generously provided by Dr. P. H. Seeburg (Heidelberg University, Heidelberg, Germany) (Moriyoshi et al., 1991; Monyer et al., 1992). For the NR1 splice variants we adopt the terminology used in Sugihara et al. (1992), denoting the isoform with the lower case letter. Clones were prepared by standard techniques and cRNA was synthesized with T3 RNA polymerase. Xenopus oocytes were prepared using previously described procedures (Woodward et al., 1995), and were stored in Barth's medium with composition (in mM): NaCl, 88; KCl, 1; CaCl₂, 0.41; Ca(NO₃)₂, 0.33; MgSO₄, 0.82; NaHCO₃, 2.4; 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) 5; pH 7.4, with 0.1 mg/ml gentamycin sulfate. Oocytes were injected with between 4 to 40 ng of NMDA receptor-encoding cRNAs. The NR1a + NR2A combination was injected at 1:4 or 1:1 ratios, depending on expressional potency of cRNA preparations. Other subunit combinations were injected 1:1. Drugs were dissolved in Ringer solution with composition (in mM): NaCl, 115; KCl, 2; CaCl₂, 1.8; HEPES, 5; pH 7.4. Solutions were applied to oocytes by bath perfusion or using a linear array system of capillary tubes (Hawkinson et al., 1996). Membrane current responses were recorded with a conventional two electrode voltage-clamp.

Neuronal cultures and electrical recordings. Primary cultures of mixed cortical neurons were prepared using a modification of procedures described previously (Whittemore et al., 1995; Ilyin et al., 1996). Briefly, cortices were obtained from Sprague-Dawley (Charles River, Hollister, CA) rat embryos [gestation day (E) 16-17]. Cells were dissociated by incubation in trypsin followed by light triturization and were plated into poly-D-lysine-coated 35-mm culture dishes at a density of 3 to 5 × 10⁴/cm² in Neurobasal medium (GIBCO, Gaithersburg, MD) supplemented with 5% fetal calf serum, 0.5 mM L-glutamine and 0.25 µM L-glutamate. Cultures were maintained at 37°C in a humidified incubator (5% CO₂/95% air). The first feeding was after 4 days with the same medium minus the glutamate. Subsequent feedings were twice weekly thereafter. Neuronal recordings were made with cultures maintained 5 to 12 days in vitro using methods described previously (Ilyin et al., 1996). The internal pipette solution was either (in mM): 145, CsCl; 10, Cs-HEPES (pH = 7.4); 0.5, CaCl₂; 10, EGTA, with 2, adenosine 5-triphosphate (ATP) and 1, GTP (~290 mOsmol), or the same solution with the CsCl replaced by 134 KF and the ATP and GTP omitted. Whole-cell recordings were made with an Axopatch 200A amplifier (Axon Instruments, Inc., Foster City, CA). Currents were filtered at 2 kHz and analyzed using software provided by the laboratory of Dr. Ricardo Miledi (University of California, Irvine, CA).

Data analysis. Data were analyzed as reported in Woodward et al. (1995) and Ilyin et al. (1996). Briefly, if antagonists gave, or were presumed to give, full inhibition of NMDA responses the concentration-inhibition data were fit with equation 1: where I is the measured current, I_control is the current in the absence of antagonist, IC₅₀ is the concentration of drug that causes 50% inhibition of the control response and n is the slope factor of the inhibition curve. If antagonists did not inhibit the NMDA responses fully then concentration-inhibition data were fit with equation 2: where min (minimum) is the residual fractional response at concentrations of antagonist that are saturating for the first component of inhibition, and where IC₅₀ is the concentration that causes half this level of inhibition. P values given in the text are results of two-tailed Student's t test or of one-way analysis of variance (Excel, Microsoft).

Drugs. (+)-SKF 10,047, ()-SKF 10,047, carbetapentane, DTG, haloperidol, R(+)-3-PPP, S()-3-(3-hydroxyphenyl)-N-propylpiperidine (S()-3-PPP)ifenprodil, NMDA, (+)-pentazocine, ()-pentazocine, rimcazole and trifluperidol were from Research Biochemicals Inc. (Natick, MA). BD 1008, 4-IBP, IPAG and 4-PPBP were from Tocris/Cookson (St. Louis, MO). Eliprodil was synthesized and generously provided by Dr. C. F. Bigge (Parke-Davis, Pharmaceutical Research, Ann Arbor, MI). Other drugs and reagents were from Sigma Chemical Co. (St. Louis, MO). Drugs was initially made as dimethylsulphoxide (DMSO) or water stocks over the range 0.01 to 100 mM depending on potency. Ringer solutions were made by 300- to 1000-fold dilution of stocks. At these concentrations DMSO caused <5% inhibition of NMDA responses in oocytes. Structures of  ligands are given in figure 1.

View larger version (K): [in this window] [in a new window]   Fig. 1.   Structures of  ligands tested.

	Results

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Inhibition of cloned NMDA receptor responses in Xenopus oocytes. Inhibition of cloned NMDA receptors by  site ligands was measured on responses elicited by saturating, or near saturating, concentrations of agonists. The standard holding potential was 70 mV. As described previously (Williams, 1993; Woodward et al., 1995), the NMDA response followed a multiphasic time course consisting of a transient spike of current, due to secondary activation of Ca⁺⁺-gated Cl channels, and a slowly developing second phase that corresponds more closely to current passing directly through NMDA receptor channels (fig. 2). Oocytes were pretreated for 30 to 60 sec with  site ligands and potency of inhibition was assessed from reductions in amplitude of the second phase of the response. Potency was expressed in terms of the concentration of antagonist that induced 50% inhibition of the control response (IC₅₀ value). A sample experiment assaying()pentazocine on NR1a/2B receptors is shown in figure 2. The 17  site ligands assayed in this study inhibited NMDA responses with a wide range of potencies and varying degrees of subunit selectivity. Sample concentration-inhibition curves for four ligands are given in figure 3. The complete set of IC₅₀ values is given in table 1.

View larger version (K): [in this window] [in a new window]   Fig. 2.   Sample records illustrating inhibition of NMDA responses by the  site ligand ()-pentazocine. The oocyte was expressing NR1a/2B subunits. Drugs were applied as indicated by bars. In the first five records and in the final record the initial spike of current, due to transient activation of Ca++-gated Cl channels, has been cropped during preparation of the figure. Current amplitudes were measured at the second, more slowly developing phase (arrow). Small, transient increases in current are apparent upon wash of 3 and 10 µM pentazocine. Responses were separated by 3- to 6-min wash to minimize response rundown. Inward current is denoted by downward deflection; holding potential was 70 mV.

View larger version (K): [in this window] [in a new window]   Fig. 3.   Concentration-inhibition curves showing subunit-selectivity profiles of four  site ligands: ()-pentazocine and DTG, two weakly selective antagonists, and 4-PPBP and trifluperidol, two NR2B-selective antagonists. In these and following graphs data points are the mean ± S.E.M. Response amplitudes for NR1a/2A are expressed as a fraction of currents elicited by 10 µM glycine and 100 µM glutamate. Amplitudes for NR1a/2B and NR1a/2C are expressed as fractions of currents elicited by 1 µM glycine and 100 µM glutamate. FR, fractional response. Smooth curves are best fits of equation 1 to the data, except for 4-PPBP and trifluperidol inhibition of NR1a/2B that were fit with equation 2. IC50 values (µM) and slopes for the fits to data for NR1a/2A, NR1a/2B and NR1a/2C receptors are, respectively: ()-pentazocine, 0.6, 1.1; 0.82, 0.83; 0.28, 0.88; 4-PPBP, 110, 1.3; 2.2, 0.92; 240, 1.4; DTG, 4.7, 1.1; 4.7, 0.80; 6.0, 0.93; trifluperidol, 69, 0.81; 1.2, 1.0; 390, 1.0 (see table 1). The minimum values (equation 2) for 4-PPBP and trifluperidol on NR1a/2B receptors were 0.05 and 0.06, respectively.

View this table: [in this window] [in a new window]   TABLE 1 Inhibition of NMDA-activated membrane current responses by  site ligands

Mechanisms of inhibition on cloned NMDA receptors in oocytes. Mechanism of antagonism was investigated in three ways: 1) by testing whether inhibition was dependent on agonist concentration, 2) by testing if potency of inhibition was dependent on membrane voltage and 3) for selected compounds, testing if washout of inhibition was dependent on channel activation.

View larger version (K): [in this window] [in a new window]   Fig. 4.   Effect of increasing agonist concentration on inhibition of NMDA responses by  site ligands. Histograms indicate levels of inhibition induced  site ligands (abbreviated) at the fixed concentrations indicated. Inhibition expressed as a fractional response. Solid bars are with control concentrations of glycine and glutamate, open bars are with 10- or 100-fold increases in agonist concentrations (see text). Nonselective antagonists were tested using NR1a/2C receptors (upper and middle panels), selective antagonists were tested using NR1a/2B receptors (lower panel). *indicates P < .05; n = 3 to 5 for each antagonist.

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View larger version (K): [in this window] [in a new window]   Fig. 5.   Sample records illustrating assays to test voltage-dependence of inhibition for ()-SKF 10,047 and 4-PPBP. Records were taken from a single oocyte expressing NR1a/2B receptor subunits. (upper records) Levels of inhibition induced at a holding potential of 20 mV. Currents were measured on the second phase of the response (arrow). (lower records) Inhibition induced by the same concentration of antagonist at a holding potential of 110 mV.

View larger version (K): [in this window] [in a new window]   Fig. 6.   Current-voltage relationships for inhibition of NMDA receptors by  site ligands. (upper panels) Voltage-dependence of inhibition for weakly selective and nonselective antagonists. Experiments were done in oocytes expressing NR1a/2C receptor subunits. (lower panels) Voltage-dependence of inhibition for strongly selective antagonists. (left panel) Low potency inhibition was assayed in oocytes expressing NR1a/2C receptor subunits. (right panel) High potency inhibition was assayed in oocytes expressing NR1a/2B receptor subunits. Inhibition was induced by fixed concentrations of ligands as indicated; n = 3 to 5 for each data point.

View larger version (K): [in this window] [in a new window]   Fig. 7.   Sample records illustrating use-dependence of washout for inhibition induced by (+)-SKF 10,047 on NR1a/2C receptors (upper panel). Inhibition with 5 µM (+)-SKF-10,047 washed out in 5 min with the continuous presence of agonist (lower panel). Washing the oocyte for 5 min in the absence of agonist failed to reverse inhibition. Washout of inhibition occurred only upon reactivation of the channel.

Inhibition of NMDA responses in cultured rat cortical neurons. Neuronal NMDA responses had similar amplitudes and time courses to those described previously (e.g., Fletcher et al., 1993, 1995; Ilyin et al., 1996). IC₅₀ values of  site ligands were measured at a holding potential of 70 mV on the plateau phase of the response. Assays of inhibition were made using cultures that had been maintained for <12 days in vitro, when the neurons are expressing predominantly NR1/NR2B subunits (Zhong et al., 1994; Ilyin et al., 1996). Ten  ligands, covering a wide range of potencies on the cloned NMDA receptors, were assayed for antagonism of neuronal NMDA responses. Results are given in table 1. IC₅₀ values from the neuronal recordings corresponded closely to the values for inhibition of NR1a/2B receptors. We also tested whether low concentrations of DTG had any positive modulatory effects on the neuronal NMDA responses. Neurons were treated with DTG for 0.5 min during established steady-state responses. Under these conditions the current was unaffected by 3 and 10 nM DTG (n = 3) (not illustrated). Thresholds for the inhibitory effects of DTG were approximately 0.3 µM.

	Discussion

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Inhibition of NMDA receptors by  site ligands. Recent reports offer conflicting explanations as to the mechanism by which  site ligands inhibit NMDA receptors. On the one hand, binding and Ca⁺⁺ imaging studies suggest that inhibition is mediated indirectly via  sites (Hayashi et al., 1995; Yamamoto et al., 1995). On the other, electrophysiological studies indicate that inhibition is due to direct effects on NMDA receptors (Fletcher et al., 1993; Fletcher et al., 1995). We reasoned that NMDA receptor subtype-selectivity might reconcile some of the discrepancies in data and differences in interpretation that have arisen between previous studies. Instead of reconciling differences, however, our results strongly support the position that, for the seventeen  ligands tested, inhibition of NMDA responses results from direct actions on the receptor-channel complex. The reasons are as follows: 1) We find no correspondence between patterns of NMDA receptor subtype-selectivity and differences in  subtype-selectivity. 2) We find no correlation between potency of NMDA receptor inhibition and affinity for  sites. 3) Antagonism by the non-selective, or weakly selective, NMDA receptor inhibitors is voltage-dependent and, for the more potent antagonists, is use-dependent. Both effects imply involvement of binding sites located in a transmembrane channel pore. 4) Antagonism by the NR2B-selective inhibitors (ifenprodil, trifluperidol, eliprodil, haloperidol and 4-PPBP) correlates with potency for displacement of radiolabeled ifenprodil, where the ifenprodil binding site is associated with the NMDA receptor.

Patterns of NMDA receptor subtype-selectivity. Plotting the correlation of potency for inhibition of NR1a/2A or NR1a/2C responses vs. potency for NR1a/2B responses illustrates the point that five of the  site ligands are selective for NR1a/2B, whereas the remaining compounds are essentially non-selective (fig. 8, upper panel). The correlation coefficient (r) for the latter group is 0.98 (n = 11, with calculations restricted to pairs of definite values). The slope shows highly significant deviation from zero (P < .0001). Plotting the correlation of potency for NR1a/2A vs. NR1a/2C illustrates that none of the compounds tested has strong selectivity for these receptor subtypes (r = 0.98, P < .0001, n = 14) (fig. 8, lower panel).

View larger version (K): [in this window] [in a new window]   Fig. 8.   Upper panel, correlation between potency of inhibition for NR1a/2A and NR1a/2B receptors. Lower panel, correlation between potency of inhibition for NR1a/2A and NR1a/2C receptors. In this and next graphs, > denotes an indefinite minimum value. The direction of the symbol (vertical, horizontal or diagonal) indicates which value, or whether both values, are indefinite.

View this table: [in this window] [in a new window]   TABLE 2 Binding affinity of  ligands to 1, 2 and PCP sites; comparison with potencies in assays of whole cell [3H]-TCP displacement, inhibition of NMDA-induced Ca++ signals and inhibition of NMDA-activated membrane currents

View larger version (K): [in this window] [in a new window]   Fig. 10.   Upper panel, Correlation between potency of inhibition for NR1a/2B receptors and neuronal NMDA receptors. Solid symbols, inhibition of NMDA responses in rat cortical neurons cultured for <12 days. Open symbols, inhibition of NMDA responses in rat hippocampal neurons 5 to 15 days in vitro (table 2) (lower panel). Correlation between potency of NR1a/2B receptor inhibition and affinity for the NMDA receptor PCP site (table 2).

Correlations between potency of NMDA receptor inhibition and  site affinity. Plotting potency for inhibition of any of the three NMDA receptor subtypes against affinity for ₁ sites gives no positive correlation between these two activities. In particular, there is no positive relationship where the correlation curve is displaced to the right, which might have been anticipated considering that we are comparing affinities from binding assays with potencies in a functional assay (Bowen et al., 1988; Walker et al., 1990). Indeed, the only relationship is a negative trend that does not achieve significance (e.g., r = 0.47, P = .092, for NR1a/2A, n = 14; r = 0.14, P = .61, for NR1a/2B, n = 16) (tables 1 and 2) (fig. 9). The negative trend is probably trivial, arising because the second generation of  ligands were specifically designed to have high  potency and weak activity at PCP sites. Some of the more obvious disparities in the data are: 1) The stereoselectivity of benzomorphans for ₁ sites is not evident for the inhibition of NMDA responses. (+)-Pentazocine and (+)-SKF 10,047 have 15- to 30-fold higher affinity for ₁ sites than their ()-enantiomers (Hellewell and Bowen, 1990; Rothman et al., 1991), whereas (+)-pentazocine is 6-fold weaker for inhibition of NMDA responses than the ()-enantiomer. The two SKF 10,047 enantiomers have comparable potency. 2) The stereoselectivity of 3-PPP for ₁ sites is reversed for inhibition of NMDA responses; R(+)-3-PPP has 6-fold higher affinity for ₁ sites than S()-3-PPP but is 5-fold weaker in the NMDA receptor assays. 3) A number of high potency ₁ ligands are either weak or inactive in terms of NMDA receptor inhibition; these include carbetapentane, BD 1008, 4-IBP and R(+)-3-PPP. For these compounds the discrepancy between ₁ affinity and the IC₅₀ for NMDA responses ranges between 9000- to 60,000-fold.

View larger version (K): [in this window] [in a new window]   Fig. 9.   Upper panel, Correlation between potency of NR1a/2B receptor inhibition and affinity for 1 receptors. Lower panel, correlation between potency of NR1a/2A receptor inhibition and affinity for 1 receptors.

Voltage-dependence and use-dependence of NMDA receptor inhibition. All the  ligands tested are noncompetitive NMDA receptors antagonists with respect to glutamate and glycine. Thus the compounds are not ligands for the agonist or coagonist sites. The small changes in apparent potency seen with rimcazole and ifenprodil are presumably due to allosteric interactions between sites (Williams, 1993; Kew et al., 1996). Inhibition by all the nonselective or weakly selective antagonists is voltage dependent. For the high potency antagonists it is also possible to demonstrate use-dependence for washout of the inhibition. The straightforward explanation for the voltage-dependence and use-dependence is that these ligands bind to sites in the NMDA receptor channel pore (Huettner and Bean, 1988; MacDonald et al., 1991). For the inhibition to be indirect, mediated by  receptors, one would have to propose that activation of  receptors causes release of a diffusible messenger molecule that then binds to a site in the pore. No such molecules or mechanisms are known. Moreover, for the nonselective NMDA antagonists, if you plot IC₅₀ for NMDA responses versus affinity for the PCP site there is a good correlation (r = 0.97, P < .0001, n = 8) (fig. 10) (table 2). Therefore, for the majority of the nonselective antagonists, proposing -mediated inhibition of NMDA receptors is simply unnecessary.

Ifenprodil binding studies. Radiolabeled ifenprodil shows multicomponent binding to rat brain membranes (Mercer et al., 1993; Hashimoto and London, 1993, 1995; Coughenour and Cordon, 1997). In the presence of a saturating concentration of a nonselective  site ligand, a high affinity component of ifenprodil binding remains and is modulated by polyamines in a manner consistent with a site located on the NMDA receptor complex (Mercer et al., 1993; Coughenour and Cordon, 1997). Eliprodil, haloperidol and trifluperidol all displace this component of ifenprodil binding, and the potency and rank order of inhibition correspond closely to potency for inhibition of NR1a/2B receptors (Coughenour and Cordon, 1997). These studies provide further evidence that the subtype-selective inhibitors interact directly with NMDA receptors. In addition, the binding studies suggest that either the sites of interaction for butyrophenones such as haloperidol overlap with those for ifenprodil, or that there is strong negative allosteric coupling between the two sites.

Previous studies proposing indirect inhibition of NMDA receptors. Discrepancies between our data and the previously reported [³H]-TCP binding studies are most striking the two enantiomers of SKF 10,047 and ()-pentazocine, which are between 20- to 100-fold weaker in the binding assays, and for R(+)-3-PPP, which is ~15-fold more potent (table 2) (Yamamoto et al., 1995). Reasons for the differences in potency are unclear. In particular, it is difficult to account for SKF 10,047 and ()-pentazocine having such weak activity in the whole cell [³H]-TCP binding studies when our experiments and previous studies indicate that the drugs are potent channel blockers (e.g., Tam and Zhang, 1988; Lockhart et al., 1995; Fletcher et al., 1995; Hayashi et al., 1995). From our perspective, any proposed role for  receptors in the [³H]-TCP binding data for SKF 10,047 and ()-pentazocine would have to be to reduce sensitivity of NMDA receptors to the direct channel blocking effects of the molecules, rather than to mediate indirect inhibition! We find little discrepancy between IC₅₀ values of  ligands tested in our study and steady-state potencies reported previously from NMDA-induced Ca⁺⁺ mobilization assays (Hayashi et al., 1995) (table 2). For these compounds we think that inhibition ascribed to  receptors can be readily explained by direct antagonism of NMDA receptors.

Positive modulation of NMDA receptor function by  site ligands. The only compound that potentiated NMDA receptor responses was haloperidol. This effect was not seen in all oocytes, was specific to the NR1a/2A subunit combination, and only occurred at micromolar concentrations of haloperidol. In the short term, we did not detect potentiation of steady-state NMDA responses with nanomolar concentrations of DTG. Our experiments suggest that the increase in sensitivity of CA3 hippocampal neurons to NMDA induced by  site ligands in vivo is not due to direct facilitation of NMDA receptor currents (Monnet et al., 1990; Debonnel, 1993). The same holds for effects of  ligands on NMDA-induced release of catecholamines from hippocampal and striatal slices (Gonzales-Avear and Werling, 1994, 1995; Monnet et al., 1996). For both types of experiments we would suggest that  receptors affect processes at some stage downstream from the NMDA receptors. In addition, our experiments failed to provide any support for a recent study suggesting that low nanomolar, or subnanomolar, concentrations of haloperidol directly potentiate NMDA receptor function in rat forebrain (Banerjee et al., 1995).

Neuroprotective properties of  ligands. Drugs such as DTG, eliprodil, ifenprodil, haloperidol and 4-PPBP have neuroprotective properties in models of excitotoxicity and acute cerebral ischemia (e.g., MacDonald and Johnston, 1990; Carter et al., 1991; Takahashi et al., 1995; Takahashi et al., 1996). All these drugs antagonize NMDA receptors. In conjunction with previous studies (Fletcher et al., 1993; Kirk et al., 1994), our results raise the issue whether the neuroprotective effects of  ligands are solely due to NMDA receptor antagonism. Recent in vitro studies indicate that the explanation is probably more complex (Lockhart et al., 1995). Specifically, protection against hypoxia-induced neurotoxicity by  ligands does not correlate with NMDA receptor antagonism, suggesting that more than one neuroprotective mechanism is involved. What role  site activation or inhibition actually plays in these additional mechanisms remains uncertain. Still, the surprising potency of drugs such as eliprodil and 4-PPBP in animal models of focal ischemia raises the possibility that a combination of NMDA receptor antagonism and appropriate interactions at  sites may be a favorable profile for a neuroprotectant (Carter et al., 1991; Takahashi et al., 1995; Takahashi et al., 1996).

Conclusion. Antagonism of NMDA receptors by the  site ligands tested in this study is due to direct actions at two, or more, sites on the receptor channel complex. Nonselective antagonism is due to blockade at the PCP-site, or at other sites in the channel pore. Subtype-selective antagonism is mediated by allosteric modulatory sites associated with the NR2B subunit.