EFFECT OF STEROIDS ON NMDA RECEPTORS DEPENDS ON SUBUNIT COMPOSITION

Abstract: Disclosed is a method for identifying a subunit specific modulator of the N-methyl-D-aspartate (NMDA) receptor. The method involves providing a plurality of NMDA receptors which differ in their subunit identity. The receptors are contacted with a neurotransmitter recognition site ligand in the presence and absence of a candidate modulator. Receptor activity is then assayed, with an increase or decrease in activity in at least one, but not all members of the plurality of NMDA receptors, in the presence but not the absence of a candidate modulator, being an indication that the candidate modulator is a subunit specific modulator. The subunit identity of the subset of the NMDA receptors to determine the subunit specificity of the candidate modulator. Various combinations of NMDA receptor subunits are provided.

Description: EFFECT OF STEROIDS ON NMDA RECEPTORS DEPENDS ON SUBUNIT COMPOSITION Background of the Invention The NMDA receptor subtype is known to have a fundamentally important role in CNS function. Ongoing work from many laboratories has established the involvement of NMDA receptors in multiple aspects of brain development, synaptic plasticity associated with long-term potentiation, and pathology related to glutamate-mediated excitotoxicity . In particular, neuropathological mechanisms mediated by NMDA receptors have recently been implicated in neurological disorders including ischemic stroke, kindling epileptogenesis, and schizophrenia . The NMDA receptor is composed of two different types of subunits, the NR1 and the NR2. The NR1 subunit is ubiquitously expressed throughout the CNS, particularly in the cerebral cortex, hippocampus, and olfactory bulb. While homomeric NMDA receptors composed of NR1 subunits are activated by NMDA, heteromeric receptors composed of both NR1 and NR2 subunits exhibit greater responses to NMDA. Alternative splicing of three exons, a , β , and γ (also referred to in the art as Nl, CI , and C2 , respectively) generates eight isoforms of the mRNA encoding the NR1 protein. A schematic of the NR1 splice variants is shown in Fig. 1 in which the presence or absence of any of the three alternatively spliced exons is designated by a subscript within the name. Figure 2 lists the eight splice variants and the alternatively spliced exons present in each variant. Exons, α, β , and γ, code for 21, 37, and 38 amino acid sequences, respectively. The a exon, which corresponds to exon 5, is located in the extracellular amino-terminal portion of the receptor, whereas the β and γ exons, corresponding to exons 21 and 22, respectively, are located at the carboxy-terminal domain. Interestingly, a stop codon is contained within the γ exon. Its removal by alternative splicing leads to the inclusion of a new 22 amino acid sequence . The inclusion of any one or a combination of the three exons imparts differential pharmacological properties to the NMDA receptor. For example, inclusion of the Nl exon leads to a decrease in agonist affinity, but an increase in current amplitude. A number of consensus PKC phosphorylation sites have been identified within the CI exon; however, a functional role for these sites has not been fully elucidated. Other differences imparted by alternative splicing include sensitivity to potentiation by spermine, Zn 2+ , and activators of PKC. An examination of the levels of NRl splice variants in the developing rat brain has revealed regional differences in the pattern of expression. This divergent pattern in the expression of the NRl splice variants may provide a degree of functional diversity in NMDA receptor function that underlies the regional heterogeneity in certain NMDA receptor-dependent processes, including synaptic consolidation, potentiation, and plasticity. The four subtypes of the NR2 subunit: NR2A, NR2B, NR2C, and NR2D were identified through molecular cloning studies. Studies indicate that the inclusion of different NR2 subtypes within the heteromeric NMDA receptor alters the pharmacological properties of NMDA receptor function providing another level of functional diversity to the receptor. For example, differences in Ca 2+ permeability, sensitivity to Mg 2+ block, glycine potentiation, and offset kinetics can be attributed to the presence of specific NR2 subunits in the heteromeric receptor. The NR2 subunits have about 50% sequence homology between members and have approximately 15% sequence homology with the NRl subunits. They are incapable of forming functional NMDA-activiated channels following homomeric expression, but rather serve to potentiate the NMDA response and modify pharmacological properties when coexpressed with NRl subunits. Recent work has shown that the NR2A and NR2B subunits are tyrosine- phosphorylated, whereas the NRl subunits are not. In si tu hybridization experiments have shown that the NR2 subunits exhibit a region-specific and temporal - specific pattern of expression. For example, the NR2B subunit is mainly expressed in the rat forebrain, whereas the NR2C subunit is found predominantly in the cerebellum. Differences in the expression of the NR2 subunits may be responsible for imparting functional diversity to NMDA receptor function from one cell type to the next . Summary of the Invention The present invention relates to a method for identifying a subunit specific modulator of the N-methyl- D-aspartate (NMDA) receptor. The method involves providing a plurality of NMDA receptors which differ in their subunit identity. The receptors are contacted with a neurotransmitter recognition site ligand in the' presence and absence of a candidate modulator. Receptor activity is then assayed, with an increase or decrease in activity in at least one, but not all members of the plurality of NMDA receptors, in the presence but not the absence of a candidate modulator, being an indication that the candidate modulator is a subunit specific modulator. The subunit identity of the subset of the NMDA receptors to determine the subunit specificity of the candidate modulator. Various combinations of NMDA receptor subunits are provided. Brief Description of the Drawings Figure 1 is a schematic of the proposed structure of the alternative splice variants of the NMDARl (NRl) mRNA. Figure 2 is a compilation of graphical representations of data which indicate that PS inhibits AMPA and kainate receptor function. Figures 1(A) through 1(D) are representative traces showing the inhibitory effect of 100 μM PS on kainate- induced currents of oocytes injected with (A) rat brain poly (A) + RNA, (B) GluRl cRNA, (C) GluR3 cRNA, (D) GluR6 cRNA. The kainate concentration used in (A) - (C) was 100 μM, and in (D) was 10 μM. The solid bar represents the period of kainate (KA) application; the open bar indicates the period of PS exposure. Figure 1(E) is a graph of relative current for the indicated Kainate concentration. The administration of PS (open symbols) is seen to decreases maximum kainate responses of GluRl (•, O) , GluR3 (■, D), and GluRβ (A, Δ) receptors. Each data point represents the mean of three experiments. Error bars represent standard error. Smooth curve was determined by nonlinear regression using the logistic equation applied to pooled data. Fitted parameters are (GluRl) J maχ =1.0, EC 50 =27 μM, π H =1.54; (GluRl + PS) I max =0.17, EC 50 =23 μM, n„=0.9; (GluR3) J maχ =1.15, EC 50 =27 μM, n H =1.44; (GluR3 + PS) I maχ =0.33, EC 50 =32 μM, n H =1.93; (GluRδ) I maχ =1.0, EC 50 =550 nM, n H =l.l; (GluR6 + PS) J IΪ13X v =0.69, nM, n„Π=1.2. Figure 1(F) is a graph of data showing the concentration dependence of PS inhibition of recombinant GluRl (O), GluR3 (D), and GluR6 (A) receptors. Results are expressed as percentage change in the peak 100 μM (GluRl and GluR3) or 10 μM (GluR6) kainate- induced current in the presence of PS. Each data point is the mean of three experiments; error bars indicate S.E.M. For GluRl and GluR3 , smooth curves are derived from fits to the Michaelis-Menten equation, as fits to the logistic equation yielded Hill coefficients close to 1, with no significant improvement in sum of squares (F-test, P > 0.05). Fitted parameters are (GluRl) EC 50 =43 μM, E maχ = -99%; (GluR3) EC 50 =12 μM, £ maχ = -90%. For GluR6, the smooth curve is derived from a fit to the logistic equation, as Michaelis-Menten fits were significantly poorer (F-test, P < 0.05). Maximum inhibition was constrained to 100%, as an unconstrained fit yielded an extrapolated maximum inhibition > 100%. Fitted parameters are EC 50 =80 μM, n H =0.29. Figure 3 is a compilation of graphical representations of data which indicate that neuroactive steroids modulate NMDA responses of oocytes injected with specific NMDA receptor subunits. Figure 2(A) indicates the potentiation of the 100 μM NMDA response by PS in oocytes injected with NR1 100 + NR2A cRNA. The solid bar indicates the period of NMDA exposure; the open bar indicates the period of PS exposure. Figure 2(B) indicates inhibition of the 100 μM NMDA response by 3α5/3S in oocytes injected with NR1 100 + NR2A cRNA. The solid bar indicates the period of NMDA exposure; the shaded bar indicates the period of 3α;5j8S exposure. Figure 2(C) indicates modulation of agonist efficacy by PS and 3α5βS in oocytes injected with NR1 100 + NR2A cRNA. PS (100 μM) increases the NMDA T 1U3A v but does not affect the EC—_U. 3α5/3S (100 μM) markedly reduces the NMDA J maχ with little effect on EC 5Q . Peak NMDA responses are normalized to the peak 100 μM NMDA response. Each data point represents the mean of three experiments. Error bars represent standard error. Smooth curves are derived from fits to the logistic equation. Fitted parameters are (control) EC 50 =29 μM, β maχ =1.14, n H =1.43; (+PS) EC 50 =30 μM, E maχ =3.21, n H =1.54; (+3α5/3S) EC 50 =15 μM, £ maχ =0.35, n H =1.6β. Figure 2 (D) is a graph indicating the concentration dependence of steroid modulation of the NMDA response of oocytes injected with NR1 100 + NR2A cRNA. NMDA (100 μM) and the indicated concentration of PS (•), 3/35/3S (Δ) , or 3 5βS (D) were applied simultaneously for 10 s. The peak NMDA- induced current is expressed relative to the average of control NMDA responses determined before application of steroid and after steroid washout. Points indicate mean of 6 (PS and 3o;5/3S) , and 4 (3/35/3S) , experiments. Error jbars indicate S.E.M. Smooth curves are derived from fits to the Michaelis-Menten equation, as fits to the logistic equation yielded Hill coefficients close to 1, with no significant improvement in sum of squares (F-test, P > 0.05). Fitted parameters are (for PS) EC 50 =32 μM, E π , ax = 4 - 43 < for 3α5j8S) EC 50 =41 μM, E maχ =0.1; (for 3/35/3S) EC 50 =79 μM, E maχ =0.26. (E) Concentration dependence for PS enhancement (•) and 3CK5JSS (Δ) and 3050S (D) inhibition of the NMDA response of oocytes injected with NR1 100 cRNA. NMDA (300 μM) and the indicated concentration of steroid were applied simultaneously. The peak NMDA-induced current is expressed relative to the average of control NMDA responses determined before application of steroid and after steroid washout. Points indicate mean of 6 (PS), 3 (3/35/3S) , and 3 (3c*5/3S) experiments. Error bars indicate S.E.M. Smooth curves are derived from fits to the Michaelis-Menten equation, as fits to the logistic equation yielded Hill coefficients close to 1, with no significant improvement in sum of squares (F-test, P > 0.05). Fitted parameters are (for PS) EC 50 =26 μM, E maχ =2.14; (for 3α5/3S) EC 50 =57 μM, E πaχ =0.02; (for 3|35|8S) EC 50 =144 μM, E maχ =0.17. Figure 4 is a graphical representation of data indicating that 3o;5|SS inhibits NMDA receptor function. A, 3θ!5/3S (100 μM) inhibits the 5 μM NMDA-evoked increase in [Ca 2+ ] i . Results are expressed as mean % neuronal death ± S.E.M. of 8 experiments. B, 3α5/3S (100 μM) increases the EC 5o ani reduces the E maχ for neuronal death caused by acute NMDA exposure. 3o;5/3S or DMSO vehicle was present during the 15 min NMDA exposure only. Results are expressed as mean % neuronal death + S.E.M. of 16 (o, DMSO control) and 4 (•, 3α5/3S) experiments. Smooth curves were determined by nonlinear regression using the logistic equation applied to the pooled data (Vehicle treated: E maχ = 80%, EC 50 = 28 μM, n„ = 2.1; 3α:5/?S treated: Emax = 63%,' EC,s n u = 71 r μM, n u π = 1.8) . C, The effect of 3α5/3S on the neuronal death produced by acute 30 μM NMDA exposure is dose dependent. Results are expressed as mean % neuronal death ± S.E.M. of 4 experiments. The smooth curve was determined by nonlinear regression using the logistic equation applied to the pooled data (I = 97%, EC 50 = 45 μM, n H = 2.5) . D, Under chronic (16 hour) exposure conditions, 3o;5|3S (100 μM) reduces the NMDA efficacy but does not alter the affinity. 3o;5j8S or DMSO vehicle was present during the 16 hour NMDA exposure only. Results are expressed as mean % neuronal death ± S.E.M. of 10 (DMSO control; empty circles) and 6 (3o;5(3S; filled circles) experiments. Smooth curves were determined by nonlinear regression using the logistic equation ap * ■ plied to the pooled data (Vehicle treated: Emax = 86%, EC 50 = 12 μM, n H = 1.9; 3o;5j8S treated: E maχ = 70%, EC 50 = 15 μM, n H = 2.0) . EC 50 and E maχ values were determined for each experiment by nonlinear regression using the logistic equation. The break in the x-axis represents a change from linear to logarithmic scale. * Indicates a statistically significant ( P < 0.05) difference from NMDA control. Figure 5 is a bar graph of data indicating inhibition of NMDA- induced neuronal death by 3o;5/3S isomers is stereospecific . The neuronal death caused by acute exposure to 30 μM NMDA is nearly abolished by 5/3 isomers. 3α5αS (100 μM) is only half as effective, whereas 100 μM 3(S5αS potentiates neuronal death. Results are expressed as mean % neuronal death ± S.E.M., with the number of experiments indicated in parentheses. * Indicates a statistically significant (P < 0.05) difference from NMDA control; t indicates a statistically significant difference from 3/35/3S and 3α5/3S . Figure 6 is a graphical representation of data which indicates that modulation of NMDA- induced current by steroid sulfates and hemisuccinates . A: Inhibition of the 100 μM NMDA- induced current by 3ce5/3HS in a Xenopus oocyte expressing NR1 100 and NR2A subunits. B: Inhibition of the NMDA- induced current by 3o;5/5S is shown for comparison. C: Potentiation of the 100 μM NMDA- induced current by PHS in a Xenopus oocyte expressing NR1 100 and NR2A subunits. D: Potentiation of the NMDA- induced current by PS is shown for comparison. Figure 7 indicates inhibition of NMDA-mediated neuronal death by pregnane steroids is dependent upon the C3 ester group. A: Structures of 3α5/?, 3α50S, 3Q;5J8F, 3α5jSHO, 3α50HS, 3α5/3HG. Note that 3α5/3 and 3 x5βF are uncharged, while the other three are negatively charged. B: Negatively charged pregnane steroids are more effective as inhibitors of the NMDA response. Bars show mean percentage decrease in the 100 μM NMDA- induced current in the presence of 100 μM of the indicated steroid in oocytes expressing NR1 100 and NR2A subunits. Error bars indicate S.E.M. Number of oocytes is given in parentheses. * Significant (p < 0.05) decrease in NMDA- induced current. t Significantly (p < 0.05) lower activity than 3α:5|3HO, 3α5j8HS, and 3Q;5J8HG. C: Negatively charged pregnane steroids inhibit the 5 μM NMDA-mediated Ca 2+ influx in rat hippocampal cultures. Bars show mean percentage reduction of the NMDA-induced rise in [Ca 2+ ] . in the presence of the indicated steroid (100 μM, except 3cκ5|8, 50 μM) . Error bars indicate S.E.M.; the number of experiments is given in parentheses. * Significant (p < 0.05) decrease in NMDA- induced elevation of [Ca 2+ ] .. D: 3O5JSHO, 3α5j8HS, and 3α5/3HG (100 μM) inhibit 30 μM NMDA- induced death of rat hippocampal neurons (* p < 0.05) . 30(50 (50 μM) and 3α;5|SF (100 μM) did not protect significantly against NMDA- induced toxicity. Results are expressed as mean % neuronal death ± S.E.M. with the number of experiments indicated in parentheses. t Significantly (p < 0.05) less protection than 3O5]8HO, Figure 8 indicates pregn-5-ene steroid-mediated exacerbation of NMDA receptor function is dependent upon the C3 ester group. A: Structures of pregnenolone (P) , PS, PF, PHO, PHS, and PHG. Note that pregnenolone and PF are uncharged, while the other three are negatively charged. B: PHS and PHG show greatest potentiation of the NMDA response . Bars show mean percentage increase in the 100 μM NMDA-induced current in the presence of 100 μM of the indicated steroid in oocytes expressing NR1 100 and NR2A subunits. Error bars indicate S.E.M. Number of oocytes is given in parentheses. * Significant (p < 0.05) increase in NMDA- induced current. t Significantly (p < 0.05) lower activity than PHO, PHS, and PHG. C: Negatively charged pregn-5-ene steroids potentiate the 5 μM NMDA-mediated Ca 2+ influx in rat hippocampal cultures. Bars show mean percentage potentiation of the NMDA- induced rise in [Ca 2+ ] i in the presence of the indicated steroid (100 μM, except pregnenolone, 20 μM) . Error bars indicate S.E.M.; the number of experiments is given in parentheses. * Significant (p < 0.05) increase in NMDA- induced elevation of [Ca 2+ ] . . t Significantly (p < 0.05) less potentiation than PHG. D: PHO, PHS, and PHG (100 μM) exacerbate NMDA-induced death of rat hippocampal neurons (* p < 0.05) . Results are expressed as mean % neuronal death ± S.E.M. with the number of experiments indicated in parentheses. t Significantly (p < 0. 05) less protection than PHO, PHS, or PHG. Figure 9 is a graphical representation of data indicating steroid modulation of acute NMDA- induced neuronal death is through an interaction with the NMDA receptor. To determine if steroid modulation of acute NMDA- induced cell death is correlated to modulation of NMDA- induced increases in [Ca 2+ ] ■ , the steroid-mediated change (%) in excitotoxicity is plotted against the change (%) in NMDA-induced Ca 2+ influx. Steroid modulation of NMDA-induced cell death is strongly correlated to modulation of NMDA-induced increases in [Ca 2+ ] i (r = 0.87) 1: PS; 2: PHS; 3: PHG; 4: PHO; 5: 3β5o;S; 6: PF; 7: 3Q;5/3F; 8: pregnenolone; 9: 3α5|3; 10: 3θ!5jSHO; 11: 3α5/3HG; 12: 3α5|3HS; 13: 3o;5/3S . Results are expressed as mean ± S.E.M. of at least three experiments. Figure 10 is a schematic of positive modulators and negative modulators of NMDA receptor activity. Steroid activity at the NMDA receptor is stereospecific . 5/3 steroid isomers (with A-B ring junction in the cis conformation) with a negatively charged group at C3 , such as 3α5/3s and 3/35/3S, inhibit NMDA receptor responses. In this orientation, C3 stereochemistry is not a determinant of activity. In contrast, the more planar 5α isomers (with the A-B ring junction in the trans conformation) are active positive modulators only when the negatively charged group at C3 is in the β orientation; 3/35αS and PS potentiate the NMDA response, while 3α5α!S is inactive or possesses much reduced inhibitory activity. Figure 11 is a representation of current recordings obtained from an oocytes bathed in solutions containing either Ba 2+ or Ca + ions. The current responses were induced by co-application of 10 mM glycine and 80 mM NMDA (single bars) . Double bars indicate the co-application of 100 mM PS in addition to NMDA and glycine. Figure 12 contains graphical representations of PS dose-responses for NR1/NR2A receptors. Presented are normalized current responses obtained from oocytes injected with (A) : NR1 000 /NR2A, NR1 100 /NR2A; (B) : NR1 001 /NR2A, NR1 101 /NR2A; (C) : NR1 Q11 /NR2A, NRl ι /NR2A mRNAs . The current was induced by coapplication of 10 mM glycine and 50 mM NMDA (for N-terminal insert lacking NRl isoforms, open symbols) or 80 mM NMDA (for N-terminal insert containing NRl isoforms, closed symbols) and different concentration of PS. Error bars are S.E.M. Solid lines are drawn using equation 1+E max/ (1+ (EC 50 /c) n with parameters from Table 3. Figure 13 contains graphical representations of data which compares 3α5/3S and PS dose-responses for NR1/NR2A receptors. Presented are normalized current responses obtained from oocytes injected with NRl χχχ /NR2A mRNAs . The current was induced by coapplication of 10 mM glycine and 50 mM NMDA (for N-terminal insert lacking NRl isoforms, open symbols) or 80 mM (in B; 100 mM in A) NMDA (for N-terminal insert containing NRl isoforms, closed symbols) and different concentration of 3a5bS (A) or PS (B) . Error bars are S.E.M. Solid lines are drawn using equation 1+E max/ (1+ (EC 50 /c) n with parameters from Table 2 and 3. Figure 14 contains graphical representations of data which compares 3ce5/?S and PS dose-responses for NR1/NR2A receptors. Presented are normalized current responses obtained from oocytes injected with NRl /NR2A mRNAs . The current was induced by coapplication of 10 μM glycine and 50 μM NMDA (for N-terminal insert containingt NRl isoforms, closed symbols) and different concentrations of 3cx5βS (A) or PS (B) . Error bars are S.E.M. Solid lines are drawn using equation 1+E maχ / (1+ (EC 50 /c) n with parameters from Table 3 and 4. Figure 15 contains two graphs of data indicating PS differentially modulates NRl o /2A and NRl ι /2A splice variants at pH 7.5. Data points are averaged normalized peak NMDA-induced current responses obtained from oocytes injected with (A) NR1 011 /2A or (B) NRl ιn /2A mRNAs. Concentration-response data for NMDA (circles) and for NMDA + 100 μM PS (squares) were obtained in the presence of 10 μM glycine. Fitted parameters are (A) (control o, n = 14), EC 50 = 71 μM, Emax = 1.20, n H = 1.47; (+PS open squares, n = 14), EC 50 = 67 μM, Emax = 1.79, n H = 1.24; (B) (control closed circles, n = 14) , EC 50 = 103 μM, Emax = 1.33, n H = 1.67; (+PS closed squares, n = 22), EC 50 = 89 μM, Emax = 2.70, n H = 2.02. The data were normalized relative to the current induced by co-application of 200 μM NMDA and 10 μM glycine to the same oocyte. Error bars represent S.E.M. Figure 16 contains two graphs of data indicating 3α5/3S proportionately modulates NR1 Q11 /2A and NRl ι /2A splice variants at pH 7.5. Data points are averaged normalized peak NMDA-induced current responses obtained from oocytes injected with (A) NR1 Q11 /2A or (B) NRl ι /2A mRNAs. Concentration-response data for NMDA (circles) and for NMDA + 100 μM PS (squares) were obtained in the presence of 10 μM glycine. Fitted parameters are (A) (control O, n = 14), EC 50 = 71 μM, Emax = 1.20, n H = 1.47; (+3α5/3s open squares, n = 6) , EC 50 = 120 μM, Emax = 0.52, n H = 1.35; (B) (control, closed circle, n = 14), EC 50 = 103 μM, Emax = 1.33, n H = 1.67; (+3α5/3S, closed squares, n = 8), EC 50 = 158 μM, Emax = 0.54, n„ = 1.50. The data were normalized relative to the current induced by co- application of 200 μM NMDA and 10 μM glycine to the same oocyte. Error bars represent S.E.M. Figure 17 contains bar graphs of data indicating neuroactive steroid modulation of NR1 Q11 /2A and NRl 1 /2A splice variants at pH 7.5. Bars indicate (A) the percentage potentiation of the maximum NMDA response by 100 μM PS for NR1 Q11 /2A (50%) and on NRl ιn /2A (103%; *p<5xl0 "7 ) receptors and (B) the percentage inhibition of the maximum NMDA response by 100 μM 3cx5βS for NR1 Q11 /2A (56%) and NRl nι /2A (59%; p>0.05) receptors. Dose- response data (from the same oocytes as in Figures 15 and 16) were individually fitted to the logistic equation to determine the maximum NMDA response. Bars depict the average potentiation/inhibition of the maximum NMDA response for the number of oocytes given in parentheses. Error bars represent S.E.M. Figure 18 is a bar graph of data indicating PS induced modulation is NRl χχχ splice variant dependent. A summary chart is shown which illustrates the PS induced increase in the 200 μM NMDA + 10 μM glycine current, expressed relative to the NMDA induced current in the absence of PS (maximum relative current; MRC) ; (for NR1 000 /2A MRC = 1.29, for NR1 100 /2A MRC = 1.73, for NR1 Q11 /2A MRC = 1.41, for NRl ι /2A MRC = 2.28, for NR1 Q01 /2A MRC = 1.72, and for NR1 101 /2A MRC = 1.78). Error bars represent S.E.M. The number of oocytes tested is listed in parentheses. (Significance is indicated by * : p<5xl0 "6 , ** : p<5xl0 "7 ) Figure 19 contains two graphs of data indicating pregnenolone sulfate (PS) differentially modulate NR1 011 /2A and NRl nι /2A splice variants at pH 8.5. Data points are averaged normalized peak NMDA-induced current responses obtained from oocytes injected with (A) NR1 011 /2A or (B) NRl ιn /2A mRNAs. Concentration-response data for NMDA (circles) and for NMDA + 100 μM PS (squares) were obtained in the presence of 10 μM glycine. Fitted parameters are (A) (control o, n = 11) , EC 50 = 94 μM, Emax = 1.30, n H = 1.62; (+PS, open squares, n = 10), EC 50 = 58 μM, Emax = 1.62, n H = 1.44; (B) (control, closed circles, n = 10), EC 50 = HOμM, Emax = 1.35, n H = 1.93; (+PS, closed squares, n = 23), EC 50 = 94 μM, Emax = 1.98, n H = 1.82. The data were normalized relative to the current induced by co-application of 200 μM NMDA and 10 μM glycine to the same oocyte. Error bars represent S.E.M. Figure 20 contains bar graphs of data which indicate PS differentially induced modulation of NR1 011 /2A and NRl ι /2A splice variants at pH 7.5 and 8.5. Bars indicate (A) the percentage potentiation of the maximum NMDA response by 100 μM PS for NR1 011 /2A (50%) and on NRl ι /2A (103%; *p<5xl0 "7 ) receptors at pH = 7.5 and (B) the percentage potentiation of the maximum NMDA response by 100 μM PS for NR1 011 /2A (24%) and NRl nι /2A (47%; *p<0.05) receptors at pH = 8.5. Dose-response data (from the same oocytes as in Figures 15 and 19) were individually fitted to the logistic equation to determine the maximum NMDA response. Bars depict the average potentiation of NMDA response for the number of oocytes given in parantheses . Error bars represent S.E.M Figure 21 is a graphical representation of data indicating effect of pH on PS induced modulation of NR1 011 /2A and NRl ι /2A splice variants. A summary graph is shown which illustrates the 100 μM PS induced increase in the response to 200 μM NMDA + 10 μM glycine for oocytes expressing NR1 011 /2A or NRl ιn /2A receptors. The data were normalized relative to the current response induced by co-application of 200 μM NMDA + 10 μM glycine to the same oocyte. Error bars represent S.E.M of the number of oocytes indicated in parantheses . (Significant difference between NR1 011 /2A and NRl ι /2A is indicated by * : p<0.05, ** : p<5xl0 "6 , *** : p<5xl0 "7 ) . Figure 22 contains bar graphs of data indicating PS induced modulation on NRl subunits is pH dependent. Bars indicate the 100 μM PS induced increase in the response to 200 μM NMDA + 10 μM glycine at different pHs, on oocytes expressing (A.) NR1 Q11 /2A receptors (at pH = 6.5 MRC = 2.24, at pH = 7.5 MRC = 1.41 and at pH = 8.5 MRC = 1.35), and (B.) NRl ι /2A receptors (at pH = 6.5 MRC= 4.06, at pH = 7.5 MRC = 2.28 and at pH = 8.5 MRC = 1.58) . The data were normalized relative to the current response induced by co-application of 200 μM NMDA + 10 μM glycine to the same oocyte. Error bars represent S.E.M of the number of oocytes indicated in parantheses . (Significance is indicated for NR1 011 /2A by * : p<5xl0 "5 and for NR1 011 /2A by * : p<5xl0 "6 and ** : p<5xl0 "7 ) . Figure 23 is a multiple sequence alignment of the ligand binding domains of the human retinoic acid receptor γ (RAR) , the human retinoid X receptor a (RXR- a) , the human progesterone receptor (PR) , the human glucocorticoid receptor (GR) , the human estrogen receptor (ER) and the NMDA receptor NRl subunit (NR1 Q11 ) . Conserved identical residues are bold and underlined and similar residues are bold. The triangles (Δ) under the NR1 011 sequence indicate the five mutation sites in the Penta-mutant . The five mutation sites are R182A, K193A, K202A, R233A, and R252A. Figure 24 is a bar graph of data indicating effect of NRl penta-mutant on NMDA receptor function. Mutation of 5 residues in the NRl subunit (indicated in Figure 23) results in a 47.5 ± 8.80% (mean ± SEM) reduction in pregnenolone sulfate (PS) potentitation of the NMDA response. The bars represent percent increase in maximum inward currents elicited by 300 uM NMDA and 50 uM Glycine in the presence of 100 uM PS. Data is displayed as means plus/minus the standard error fo the mean with the number of individual oocytes used in the experiments indicated as N. Currents were recorded at -70 mV, in Ba 2+ Ringer's solution (in mM) ; 96 NaCl, 2 KC1 , 1.8 BaCl 2 , 5 HEPES, pH = 7.5. Figure 25 is a bar graph of data indicating 3a5β- Pregnanolone Sulfate has similar effect on NMDA receptors consisting of wild-type NRla/NR2A and Penta-Mutant/NR2A subunits. The bars represent percent decrease in maximum inward currents elicited by 300 uM NMDA and 50 urn Glycine in the presence of 100 uM 3α5/3-Pregnanolone Sulfate. 3θf5j8-Pregnanolone Sulfate inhibits wild-type NRla/NR2A responses by 45.49± 5.76% (mean + SEM) and Penta- Mutant/NR2A by 42.79 ± 2.29%. Data is displayed as mean ± the standard error of the mean with the number of individual oocytes used in the experiment indicated as N. The currents were recorded at -70mV, in Ba 2+ -Ringer's solution (in mM) : 96 NaCl, 2KC1, 1.8 BaCl 2 , 5 HEPES, pH = 7.5. Figure 26 contains graphical representations of data indicating inverse modulation of NMDA receptor subtypes by PS. A -D are examples of traces obtained from oocytes previously injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, or NR1/NR2D mRNAs. The bar indicates the period of drug application. Interval between consecutive current traces was 45 s. The receptors were activated by co-application of 10 μM glycine plus 80 μM NMDA (NR1/NR2A, A) , 25 μM NMDA (NR1/NR2B, B and NR1/NR2C, C) , or 10 μM NMDA (NR1/NR2D, D) . Co-application of 100 μM PS to NR1/NR2A or NR1/NR2B receptors resulted in an increase in the agonist response, whereas co-application of 100 μM PS to NR1/NR2C or NR1/NR2D resulted in a decrease in the agonist response. E is dose-response curves for PS effect on NR1/NR2 receptors. Data points are averaged values of normalized peak current responses from oocytes injected with NR1/NR2A (n=8), NR1/NR2B (n=8) , NR1/NR2C (n=4) or NR1/NR2D (n=4) RNAs . Responses were normalized to the control response obtained by application of 10 μM glycine plus 80 μM NMDA (NR2A) , 25 μM NMDA (NR2B, NR2C) or 10 μM NMDA (NR2D) . Error bars indicate SEM. F indicates the effect of holding potential on modulation of the NMDA/glycine response by PS. Points are averaged relative currents obtained in the presence of 100 μM PS, standardized relative to the response induced from the same oocyte by 10 μM glycine plus 80 μM (NR1/NR2A, n=4) , 25 μM (NR1/NR2B, n=7; NR1/NR2C, n=3), or 10 μM NMDA (NR1/NR2D, n=3) . Error bars indicate SEM. Figure 27 contains four dose response curve graphs. The choice of NR2 subunit determines the direction of PS modulation of the glutamate and NMDA concentration- response curves. Data points are averaged normalized peak NMDA-induced current responses obtained from oocytes injected with (A) NR1/NR2A, (B) NR1/NR2B, (C) NR1/NR2C, or (D) NR1/NR2D mRNAs . Concentration-response data for NMDA (squares) and for -glutamate (circles) were obtained in the presence of 10 μM glycine. The data were normalized relative to the current response from the same oocyte induced by co-application of 200 μM NMDA and 10 μM glycine. Open symbols and dashed lines correspond to data obtained in the absence of PS, whereas filled symbols and solid lines correspond to data obtained in the presence of 100 μM PS. Error bars represent SEM. Figure 28 contains graphical representations of data indicating that the choice of NR2 subunit determines the direction of PS modulation of the glycine concentration- response curve. Data points are averaged normalized peak current responses obtained from oocytes injected with (A) NR1/NR2A, (23) NR1/NR2B, (C) NR1/NR2C, or (D) NR1/NR2D mRNAs. Concentration-response data for glycine were obtained in the presence of 10 μM L-glutamate and in the absence (open triangles) and presence (closed triangles) of 100 μM PS. The data for each oocyte were normalized relative to the current response induced by co- application of 200 μM NMDA plus 10 μM glycine. Error bars represent SEM. Figure 29 contains graphical representations of data indicating the choice of NR2 subunit influences 3α5/3S inhibition of the NMDA response. A -D are examples of traces obtained from oocytes previously injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, or NR1/NR2D mRNAs, respectively. The jbar indicates the period of drug application. Interval between consecutive current traces was 45 s. The receptors were activated by co-application of 10 μM glycine plus 80 μM NMDA (NR1/NR2A, A) , 25 μM NMDA (NR1/NR2B, B and NR1/NR2C, C) , or 10 μM NMDA (NR1/NR2D, D) . E is concentration-response curves for 3α5/3S effect on NR1/NR2 receptors. Data points are averaged values of normalized steady-state current responses from oocytes injected with NR1/NR2A (1, n=4), NR1/NR2B (O, n=3), NR1/NR2C (■, n=6) or NR1/NR2D (□, n=4) RNAs . Current responses are expressed relative to the current response in the absence of PS . Error bars represent SEM. F is a graph of data indicating dependence of 3c5/3S effect on membrane potential. Points are averaged relative current obtained in the presence of 100 μM 3α5/3S (1, NR1/NR2A, n=5; O, NR1/NR2B, n=10) or 10 μM 3α5/3S (■, NR1/NR2C, n=4 ; □, NR1/NR2D, n=10) . G is a concentration response curve showing the effect of 3a5βS on glutamate. Data points are averaged normalized peak current responses to glutamate from oocytes injected with NR1/NR2A subunits obtained in the presence of 10 μM glycine and in the absence (1, n = 4) or presence (o, n = 3) of 100 μM 3α;5/3S. The data for each oocyte were normalized to standard current responses induced by co- application of 200 μM NMDA and 10 μM glycine. Concentration response data for glutamate alone is the same as in Fig. 27, and is repeated for comparison. H indicates the effect of 3o;5/3S on glycine concentration- response curve. Data points are averaged normalized peak current responses to glycine from oocytes injected with NR1/NR2A subunits obtained in the presence of 10 μM glutamate and in the absence (s) or presence (Δ) of 100 μM 3o;5/3s. The data for each oocyte were normalized to standard current responses induced by co-application of 200 μM NMDA and 10 μM glycine. Concentration response data for glycine alone is the same as in Fig. 28, and is repeated for comparison. Figure 30 is a graphical representation of data in the form of dose-response curves for Pregnenolone Sulfate (PS) effect on NR1/NR2 receptors. Data points are averaged values of percent changes in peak current response in presence of increasing concentrations of PS, from oocytes injected with NR1/NR2A (n=8) , NR1/NR2B (n=8) , NR1/NR2C (n=4) or NR1/NR2D (n=4) mRNAs. Current response were normalized relative to control response obtained by application of 10 μM glycine and 80 μM NMDA (NR2A) , 25 μM NMDA (NR2B, NR2C) or 10 μM NMDA (NR2D) . Error bars are standard errors of the means . Lines were drawn using equation I + E^/ (1+EC^/ [agonst] )^) . E^, EC^ and h were obtained by averaging maximum responses, EC50 and Hill coefficients of individual experimental dose- response relationships. Figure 31 contains schematic representations of wild-type NMDAR NR2B, NR2D subunits, and the NR2 chimera constructs. Fragments from NR2B are in black and fragments from NR2D are in grey. Vertical bars correspond to the three transmembrane domains and the re- entrance domain. The numbers are the positions of the ligation junctions. Figure 32 is a bar graph of data indicating 100 μM Pregnenolone Sulfate modulates the inward currents elicited by 300 μM NMDA and 50 μM Glycine from Xenopus oocytes expressing recombinant heteromeric NR1 011 /NR2 NMDA receptors. PS potentiates NRl 011 /NR2Bwt by 78.36 ± 7.89% (mean±SEM) , NR1 Q11 /NR2B-D Chimera(I) by 52.69 ± 3.58%, NR1011/NR2B-D chimera (IV) by 66.05±2.76%; inhibits NRl 011 /NR2Dwt by 45.18 ± 2.62%, NR1 Q11 /NR2B-D Chimera (II) by 43.04±5.44%, and NR1 011 /NR2B-D Chimera (III) by 36.74 ± 6.54%. The error bar shows standard error. n is the number of oocytes recorded. The currents were recorded at -70mV, in Ba 2+ -Ringer' s solution (in mM) : 96 NaCl, 2KC1, 1.8 BaCl 2 , 5 HEPES, pH=7.5. Detailed Description of the Invention Aspects of the present invention are based on the identification of steroid molecules which exhibit selectivity with respect to NMDA receptor subunit composition. This observation indicates that it is possible to develop or identify drugs (steroid based as well as non-steroid based) that selectively target specific NMDA receptor subtypes to modulate neurotransmitter recognition site ligands. One aspect of the present invention is a method for identifying a subunit specific modulator of the NMDA receptor. This is accomplished through use of a plurality of NMDA receptors which differ in their subunit identity (NRl and NR2 subunits) . The plurality of NMDA receptors is then contacted with a neurotransmitter site agonist (e.g., L- glutamate or NMDA) in the presence and absence of a candidate modulator. Each NMDA receptor is then assayed for receptor activity produced by the neurotransmitter site agonist, in the presence and absence of the candidate modulator. An increase or decrease in activity of the receptor in the presence of the candidate modulator, as compared to the respective activity in the absence of the candidate modulator, indicates modulatory activity on the receptor. Such an increase or decrease in activity in at least one, but not all members of the plurality of NMDA receptors, is an indication that the candidate modulator is a subunit specific modulator. The subunit specificity of the candidate modulator is identified by comparing the subunit identity of the subset of NMDA receptors which are subject to modulation by the candidate modulator. The candidate modulator is specific for the subunits, or domains of the subunits, which are common to this subset of receptors and absent or non- functional in the remaining receptors of the plurality. One of skill in the art will readily recognize that the potential subunit specificity of modulators identified by this method is vast, and is only limited by the combination of subunits utilized in the method itself. Subunit specificity may be for one or more particular isoforms, chimeras or mutants of either the NRl or NR2 subunit, that is to say it may be independent of one of the subunits. Alternatively, the modulator may be specific for a particular combination of the two subunits . A modulator of the NMDA receptor is defined herein as binding at a modulatory site, and may be either a positive modulator or a negative modulator, or a null modulator. A positive modulator enhances agonist activity. It has been shown that positive and negative modulators of the NMDA receptor act through distinct sites. Pregnenolone sulfate is a known positive modulator of the NMDA receptor. This is often referred to in the art as a modulatory site agonist. A negative modulator inhibits agonist activity. Examples of known negative modulators of the NMDA receptor are 17/3- estradiol and pregnanolone hemisuccinate (3o;5/3HS) , also referred to in the art as inverse agonists. Negative modulators have been shown to be neuroprotective from otherwise lethal exposure to NMDA, whereas positive modulators enhance excitotoxicity . A null modulator has no intrinsic activity in and of itself other than to inhibit the action of a positive and/or negative modulator by competitively binding at the modulatory site . A neurotransmitter recognition site ligand as the term is used herein refers to a ligand (agonist or antagonist) which binds at the neurotransmitter recognition site. Examples of agonists are NMDA, L- glutamate and glycine. An increase or decrease in receptor activity in the presence of the candidate modulator, as compared to activity in the absence of the candidate modulator, is identified by a reproducible, statistically significant change in receptor activity in response to ligand (agonist or antagonist) . Current methodology is sensitive to the detection of approximately a 5% or greater difference in receptor activity, with an 8% difference or greater being a strong indication of modulatory activity. Preferably, a 10% or greater difference in receptor activity in the presence of candidate modulator is used to identify modulator activity. These sensitivity limitations also apply to identifying a subunit specific affect of a modulator, in that down to a 5% difference in receptor activity can be detected and used to identify subunit specific modulation. However, the larger the discrepancy between modulatory activity on one receptor versus another receptor, the more useful the subunit specific modulator will prove in therapeutic applications. Subunit identity, as used herein, refers to the specific subunit components NRl and NR2 used to generate the NMDA receptor. This term encompasses both natural and recombinantly produced subunits and receptors. Preferably, the NMDA receptors used in the method are produced recombinantly as this allows for use of a greater diversity of receptor subunit identities. There are eight known natural isoforms of the NRl subunit (NR1 000 , NR1 001 , NR1 010 , NR1 Q11 , NR1 100 , NR1 101 , NR1 110 , and NRl nι ) , and four known natural isoforms of the NR2 subunit (NR2A, NR2B, NR2C, and NR2D) . Additional isoforms of each subunit will likely be identified in future research, and their use is also encompassed by the present invention. In addition to these natural isoforms, mutant isoforms (e.g., point mutant, deletion mutant, etc.) and also chimeric isoforms are suitable for use in the method. Mutation or other sequence manipulation of an isoform for use in the method should not result in loss of detectable function/activity of the NMDA receptor which is produced. The receptor subunits used in the experiments detailed below in the Exemplification section were of human origin. The present invention is not however limited to the use of human receptors. Homologous subunits or subunit regions of homologous receptors from other species are also suitable for use in the present invention. Experiments detailed in the Exemplification section below have identified several useful sites for point mutation of NRl subunits which contain an a exon encoded protein domain. These sites correspond to residues 182, 193, 202, 233, and 252 of human NR1 Q11 . One or more of these residues, in any combination, may be mutated in the NRl subunit to produce the NMDA receptor for use in the present invention. Preferred point mutations are R182A, K193A, K202A, R233A, and R252A. The nature of the specific substituted amino acid may have an effect on mutant function. As such, substitution of any positively charged amino acid into these positions is expected to produce a mutant having a phenotype similar to the penta- mutant characterized in Example 5. However, the location of the mutated amino acid is also thought to have particular relevance. Therefore, a number of different amino acid substitutions at one of more of these sites is expected to produce a mutant with a phenotype reminiscent of that of the penta-mutant of Example 5. Chimeric NRl or NR2 subunits used in the present invention preferably retain intact protein domains. Such protein domains are usually contained within individual exons, often being delimited by those exons. The a exon of the NRl subunit has been identified as encoding a protein domain which is involved in subunit specific modulation of the receptor. It is likely that domains encoded by other exons will also be useful in identifying additional subunit specific modulators which recognize and/or function through those specific domains. Prime candidates are the other two alternatively spliced exons, β and γ, of the NRl subunit. Some domains of the NR2 subunits identified as useful in the present invention correspond to residues 534-870 of human NR2B, and also residues 548-892 of human NR2D. In addition, the domain which corresponds to residues 703-870 of human NR2B has been identified as necessary and sufficient for stimulation of the receptor by neurosteroids . This domain confers neurosteroid stimulation to the NR2B/NR2D chimera, whereas wild type NR2D is inhibited by the same neurosteroids. The identification of these domains represent the beginnings of a growing understanding of the domains involved in neurosteroid modulation. The use of chimeras made of different combinations of subunit domains in the present invention will identify subunit specific modulators which function through as yet unidentified functional protein domains of the respective subunits. The specific combinations of NRl and NR2 subunits used in the method dictates the types of subunit specific modulator which is identified. For instance, it may be useful to vary the identify of one subunit, either NRl or NR2 , in combination with a fixed identity of the other subunit. Any particular combination of the NRl and NR2 subunits described herein is appropriate for use in the present invention. Assaying for receptor activity can be by any method known in the art. Preferred assay systems allow expression and utilization of recombinant receptors. A preferred assay system is an oocyte expression system. Candidate modulators may be obtained from a variety of sources. They may be naturally occurring molecules, or alternatively synthetic molecules. Although the modulators provided as examples in the Exemplification section below are steroid based molecules, a subunit specific modulator may also be a non-steroid based molecule. Candidate modulators may be known neuromodulators, or may be otherwise derived from known neuromodulators . In one embodiment, candidate modulators are obtained from a library of small molecules, either natural or synthetic (e.g., produced by rational drug design or randomized combination) . Because the different NMDA receptor subunits are differentially distributed throughout the nervous system, therapeutic effects can be achieved through subunit specific intervention, accomplished by targeting a specific receptor, made up of specific subunits, with a subunit specific modulator identified by the above described method. Such targeting has use in therapeutic intervention in neurological disorders which are linked to glutamate receptor function. For example, receptors consisting of specific subunits can be targeted to inhibit the neurodegeneration which results from cerebral ischemia. Specific inhibition of AMPA/kainate receptor function can also be beneficial in the treatment of ischemic stroke. The neuroactive steroid class of glutamate receptor modulatory agents may also prove to be neuroprotective in brain ischemia. Several aspects of the present invention are based upon the identification of specific subunits involved in the modulation of the effect of pregnenolone sulfate on ligand gated ion channel activity. Excitatory neurotransmission in the vertebrate central nervous system is mediated primarily by ionotropic glutamate receptors. A variety of endogenous and synthetic steroids have been found to modulate the function of neuronal glutamate receptors (Farb and Gibbs, Steroids as modulators of amino acid receptor function, in: T.W. Stone (Ed.), CNS Transmitters and Neuromodulators: Neuroactive Steroids, CRC Press, New York, 1996, pp. 23- 36; Irwin et al . , J. Pharmacol . Exp . Ther. 271 : 677-682 (1994); Park-Chung et al . , Mol . Pharmacol . 46 : 146-150 (1994); Park-Chung et al . , Mol . Pharmacol . 52 : 1113-1123 (1997); Wu et al . , Mol . Pharmacol . 40 : 333-336 (1991)). Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS has previously been shown to inhibit Kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition was previously undetermined. To that end, experiments detailed below in Example 1 of the Exemplification section have characterized the effect of PS on recombinant kainate (GluR6) , AMPA (GluRl or GluR3) , and NMDA (NR1 100 + NR2A) receptors electrophysiologically with respect to efficacy and potency of modulation. PS is shown to reduce the efficacy of kainate without affecting its potency, when given to Xenopus oocytes expressing GluRl, GluR3 or GluR6 receptors. This indicates a noncompetitive mechanism of action on these specific receptors. Conversely, PS is shown to enhance the efficacy of NMDA without affecting its potency when given to oocytes expressing NR1 100 + NR2A subunits. The modulatory efficacy, but not the potency, of PS is increased two- fold by co- injection of NR1 100 + NR2A cRNAs as compared with NR1 100 cRNA alone. However, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This indicates that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with previous findings that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. These findings represent a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function. Results presented in Example 1 of the Exemplification section below indicate that selective modulators can be developed which specifically target particular AMPA or kainate receptor subtypes. Only negative steroid modulation of recombinant AMPA and kainate receptors was observed in the experiments described in Example 1 below. At the GluRl, GluR3 and GluR6 receptor subtypes, pregnenolone sulfate reduces the maximum kainate-induced currents without altering the kainate EC50, consistent with a noncompetitive mechanism of inhibition. At GluRl and GluR3 receptors, 100 μM pregnenolone sulfate produces over 70% inhibition of the response to kainate. However, the same concentration of pregnenolone sulfate inhibited GluR6 receptor response to kainate by only 42%. Notably, the concentration response curve for inhibition of GluR6 receptors by pregnenolone sulfate is more shallow than for GluRl or GluR3 receptors, which indicates that pregnenolone sulfate inhibits kainate and AMPA receptors by different mechanisms. Such specific modulators are useful in determining the receptor composition of specific cell populations, as well as probing the functional roles of AMPA and kainate receptors in si tu (Wisden and Seeburg, J " . Neurosci . 13 : 3582-3598 (1993)). In order to investigate if the presence of different exons in the NRl subunit affects NMDA receptor modulation by neurosteroids, an oocyte expression system was employed to electrophysiologically assess the neurosteroid effect on NMDA receptors composed of different NRl subunit isoforms. Experiments detailed in Example 3 indicate NRl subunit dependent effects of neurosteroid modulation on the NMDA receptor. Of note, the presence of the N-terminal 21 amino acid insert in the NRl subunit changes the efficacy of neurosteroid positive modulator PS without altering the effect of neurosteroid negative modulator 3α;5/3S. The fact that uncharged analog of PS, pregnenolone, has little effect on NMDA receptors suggests that some charged amino acids mediate the potentiating effect. The fining of modulation of PS effect by the presence of the n-terminal insert is consistent with this hypothesis because the charged amino acids constitute almost 43% of total of 21 amino acids in the insert. Moreover, empirical analysis of the surface probabilities of amino acids constituting N-terminal insert in the NRl subunit suggests that most of amino acids in the insert are located on the surface of the protein, that position them as good candidates for direct interaction with charged ions, including negatively charged molecules of pregnenalone sulfate. Inhibitory effect of 3α5/3S turned out to be similar for different isoforms of NRl subunit, suggesting that inhibitory steroids do not interact with the N-terminal insert. This observation is consistent with the finding that stimulatory and inhibitory sulfated neurosteroids have distinct sites of action on NMDA receptor. The effect of both potentiating and inhibitory steroids on NMDA receptors was observed at all agonist concentration tested. Furthermore, the potency of agonists L-glutamate, NMDA, and glycine was not changed by neurosteroids indicating that the steroids do not interact with agonist binding sites. The observed dependence of potentiating effect of steroids on the level of NMDA receptor expression in the membrane of oocytes suggests that when too many receptors are expressed in the membrane there are not enough molecules of steroid around the receptor to interact with the receptor. Without being bound by theory, it may be that in order to interact with the receptor the molecules of pregnenolone sulfate must be incorporated into the membrane first, and then, they could approach the receptor's binding site. The molecular structure of pregnenolone sulfate fits this hypothesis quite well. The molecule of pregnenolone sulfate has hydrophobic part that could be incorporated into the membrane, while charged sulfate part remains in aqueous surrounding. According to this hypothesis the molecule of NRl protein folded so that the N-terminal insert is positioned close to the membrane surface . The results detailed in Example 6 are the first to indicate that the modulatory effect of PS is contingent upon the NR2 subunit composition of the NMDA receptor, and that PS inhibits, rather than enhances, the function of NR1/NR2C and NR1/NR2D receptors. The finding that potentiation of the NMDA receptor by PS is dependent upon the presence of the NR2A or NR2B subunit indicates that the steroid binding site responsible for potentiation by PS is partially or entirely located on these subunits. Mutagenesis studies suggest that the glutamate/NMDA binding site resides on the NR2 subunit (Anson et al . , J " . Neurosci . 18 : 581-589 (1998)), while the glycine site resides on NRl (Wafford et al . , Mol . Pharmacol . 41 : 374- 380 (1995) ) , so the NMDA responses observed in oocytes injected only with NRl subunits likely reflect coassembly of NRl with an endogenous NR2A or NR2B-like subunit (Soloviev and Barnard, J " . Mol . Biol . 273 : 14-18 (1997)). To investigate the structural requirements for steroid inhibition of NMDA-induced neuronal death, 3o;5/3 and stereoisomers of 3α;5/3S were assayed for activity, as were several related synthetic pregnane steroids. The results detailed in Example 2 indicate that stereochemistry at the A-B ring junction is an important determinant of the activity of pregnanes with a negatively-charged group at C3. Since positive and negative modulation by steroids are mediated by distinct sites, the more planar ring structure of the pregn-5-enes and C5α pregnanes is thought to improve the fit of the steroid molecule to a potentiating modulatory site and/or impairs its fit to an inhibitory modulatory site. Such structural information is useful in the identification or rational drug design of additional modulators. Molecules which exhibit the appropriate molecular structure can be synthesized and/or screened for the corresponding activity with the appropriate isoforms of the NRl and NR2 subunits . Results also indicate that the presence of a negative charge adjacent to the C3 position is necessary for the negative modulatory effect of pregnane steroid derivatives on NMDA receptor function. There is limited tolerance for the geometry of the charged group at C3 , since a range of lengths (from the relatively short hemioxalate group to the five-carbon hemiglutarate group) is able to confer inhibitory activity. The effects of the pregnane steroids on NMDA responses of Xenopus oocytes expressing NR1 100 + NR2A subunits are in general agreement with results from hippocampal neurons in culture. The development of a full negative charge may not be absolutely necessary for inhibition of the NMDA receptor by steroids, as 3cn5βF produces a modest but statistically significant inhibition of the NMDA-induced current . This is consistent with the observation that 17/3-estradiol, which has a hydroxyl group at C3 , inhibits the NMDA-induced current and Ca 2+ accumulation, and is neuroprotective against NMDA-induced neuronal death (Weaver, et al . , Brain Res 761 : 338-341 (1997)). A strongly electronegative center attached to C3 is therefore sufficient to support some degree of steroid inhibition of the NMDA receptor. The lack of significant neuroprotection or inhibition of Ca + accumulation with 3α;5/3F is thought to reflect either the difficulty of detecting the effect of such a weak inhibitor, or a difference in the interaction of 3o;5/3F with native NMDA receptors of hippocampal neurons, as compared to the NR1 100 :NR2A subtype used for the electrophysiological studies. These latter results suggest that a negatively charged group at C3 enhances the neuroprotective properties of the pregnane steroid derivatives. These findings, taken in combination with previous findings reported in U.S. Patent 5,888,996, and U.S. Patent 6,083,941, the contents of which are herein incorporated by reference, indicate a wider range of compounds which function in the inhibition of NMDA receptor activation, than was previously appreciated. Another aspect of the present invention is the use of a compound represented by the structural formula : wherein ring A has 0-3 double bonds; R 1 is -OH, =0, or a negatively charged group; R 2 is -H, -CH 3 , or is absent when ring A has three double bonds; R 3 is -H, OH, =0, or - OR' ; R 1 is an aliphatic or aromatic group; and R 4 is -OH, =0 or -COCH 3 , to inhibit NMDA activity resulting from agonist activation. In one embodiment of the invention, R 4 is either hemioxylate, hemisuccinate or hemiglutarate. In preferred embodiments, the compound is either pregnanolone hemioxylate, pregnanolone hemisuccinate, or pregnanolone hemiglutarate . The ability of compounds with the above structural formula to inhibit agonist activation of the NMDA receptor can be therapeutically applied to inhibit NMDA receptor mediated ion-channel activity in an individual in need thereof by administering an effective amount of the compound to the individual. Effective amounts range from about 1 to about 500 μM, with a preferred range being from about 50 to about 250 μM. Such administration can be used to inhibit the toxic effects associated with activation of the NMDA receptor in neurons of the individual. Another useful application of this method of inhibiting agonist activation of the NMDA receptor is in reducing neuronal cell death resulting from L-glutamate activation of the NMDA receptor. This also is achieved by administration of the compound to an individual as described above. Additional diseases which can be treated by said administration of the compound are neuropathic pain, drug withdrawal/dependency, epilepsy, glaucoma, chronic neurodegenerative diseases, amyotrophic lateral sclerosis, anxiety disorders, brain cell death, ischaemia, stroke, and trauma in an individual when said disease results from NMDA induced NMDA receptor activation. Administration of the compound can also be used to inhibit the excitatory or L-glutamate-mediated synaptic activity in an individual in need thereof. Results presented in Example 2 which demonstrate that modulation by steroids of NMDA-induced Ca 2+ uptake is correlated with modulation of NMDA-induced neuronal death, indicate that this rapid functional assay can be usefully employed to identify steroids with neuroprotective activity. Another aspect of the present invention relates to a method for rationally designing steroid-based therapeutics for the treatment of stroke and disorders arising from the overactivation of the NMDA receptor. The finding that a variety of negatively-charged carboxylic acid esters can substitute for the sulfate ester at the C3 position of the above described molecule offers prospects for modifying the steroid nucleus to optimize pharmacological and pharmacokinetic properties. Carboxylic acid derivatives of neuroactive steroids should offer improved penetration into the CNS and reduced susceptibility to hydrolysis by sulfatases. This is supported by the observation that 3o;5/3HS is effective at inhibiting the neuronal death that results from middle cerebral artery occlusion in rats, a model of stroke (Weaver et al . , Proc Natl Acad Sci USA 94 : 10450-10454 (1997)) . Based on the present results, carboxylic acid derivatives of the pregn-5-ene steroids are expected to exhibit memory-enhancing effects such as have been described for pregnenolone sulfate (Isaacson et al . , Behav Neural Biol 61 : 170-176 (1994); Flood et al . Proc Natl Acad Sci USA 92 : 10806-10810 (1995); Vallee et al . , Proc Na tl Acad Sci USA 94 : 14865-14870 (1997)) . Exemplification Example 1 Modulation of recombinant AMPA and kainate receptors by PS and related steroids In addition to altering currents through NMDA receptors, PS inhibits AMPA- and kainate-induced currents recorded from chick spinal cord neurons (Wu et al . , Mol . Pharmacol . 40 : 333-336 (1991)). Similarly, PS produces a 49 ± 3% (n = 4) reduction in kainate-evoked responses from oocytes injected with rat brain poly (A) + RNA (Fig. 2A) . The observation that onset of inhibition is rapid ( < 10 s) , strongly suggests a direct interaction between PS and the AMPA/kainate receptor. However, these results cannot exclude the possibility that PS acts indirectly through some other neuronal receptor or transduction system that is co-expressed from rat brain poly (A) + RNA. Therefore, the ability of steroids to modulate the function of recombinant AMPA and kainate receptors was examined. Injection of GluRl, GluR3 , or GluR6 subunit cRNA into Xenopus oocytes was sufficient to confer kainate sensitivity (Fig. 2B-D) . Oocytes injected with GluR6 cRNA responded to lower concentrations of kainate (EC 50 =550 nM, Fig. 2E) than oocytes injected with GluRl or GluR3 cRNA (EC 50 's 23 μM and 32 μM) , as expected from the classification of GluR6 as a kainate receptor subunit and GluRl and GluR3 as AMPA receptor subunits (Hollmann and Heinemann, Annu . Rev. Neurosci . 17 : 31-108 (1994)). As with the receptors formed from the expression of rat brain poly (A) + RNA, recombinant AMPA and kainate receptor responses were inhibited by PS. In each case, PS functioned as a noncompetitive inhibitor, decreasing the maximum kainate-induced current without affecting the EC 50 (Fig. 2E) . At the GluRl AMPA receptor, PS (100 μM) reduced the kainate I maχ by 83%, while the kainate EC 50 was unchanged (18 μM vs. 23 μM with PS) . GluR3 AMPA receptor function was also markedly reduced by PS, which reduced the kainate I maχ by 71% without altering the EC 50 (27 μM vs. 32 μM in the presence of PS) . Although the extent of inhibition was less, PS also appeared to act as a noncompetitive inhibitor of GluR6 kainate receptor function, reducing the kainate J maχ by 33% with no change in EC 50 (545 nM vs. 567 nM in the absence and presence of PS, respectively) . Inhibition by PS was dose-dependent, with EC 50 's of 43 μM and 12 μM at the GluRl and GluR3 receptors, respectively (Fig. 2F) . PS was also effective at inhibiting kainate responses of GluR6 receptors, but the concentration-response curve was much more shallow (n H = 0.29) than for GluRl and GluR3 receptors. At higher concentrations of PS, the extent of inhibition of GluR6 kainate receptors was considerably less than for the GluRl or GluR3 AMPA receptors. At 200 μM PS, GluRl and GluR3 kainate responses were inhibited by 85 ± 4% and 81 ± 3% {n = 3) , respectively, compared to 64 + 2% (n = 3) at 500 μM PS with the GluR6 receptor. Table 1 tabulates the effects of a series of steroids on kainate responses mediated by GluRl, GluR3 , or GluR6 receptors. As noted above for PS, the GluR6 kainate receptor tended to be less sensitive than GluRl and GluR3 AMPA receptors to inhibition by steroids. Selectively for AMPA over kainate receptors was enhanced by substitution of a hemisuccinate moiety for the C-3 sulfate group of PS, as PS and pregnenolone hemisuccinate were about equally effective in inhibiting GluRl and GluR3 mediated kainate currents, but pregnenolone hemisuccinate was considerably less effective than PS in inhibiting GluR6 mediated responses (15% vs. 42% inhibition) . Not all sulfated steroids were observed to have inhibitory activity; in particular, 17/3-estradiol-3 - sulfate (100 μM) had no effect on GluRl and GluR3 receptors and was only slightly inhibitory at GluR6 receptors (12% inhibition) , demonstrating that inhibition of AMPA receptors is not a universal property of sulfated steroids . Table 1 Effects of selected steroids on recombinant AMPA, kainate, and NMDA receptors expressed in Xenopus oocytes Steroid Percentage change AMPA Kainate NMDA GluRl GluR3 GluR6 NRl ,oo + N Pregnenolone sulfate -85 ± 1(8)* -73 ± 3(8)* -42 ± 3(8)* +281 ± 27ι Pregnenolone hemisuccinate -71 ± 1(4)* -72 ± 3(3)* -15 ± 2(3)* + 118 ± 16ι Pregnanolone sulfate -47 ± 12(3) -63 ± 8(4)* -34 ± 5(7)* 64 ± 3(7)* 17/3-Estradιol-3-sulfate -2 ± 2(3) -8 ± 4(3) -13 ± 4(4)* + 12 ± 3(4) 17α-Estradιol-3-sulfate -22 ± 3(3)* -17 ± 2(3)* -7 ± 1(5)* - Dehydroepiandrosterone sulfate -23 ± 3(3)* -7 ± 1(4)* -17 ± 5(3) + 8 ± 13(3) Dehydroepiandrosterone -16 ± 3(3)* -7 ± 5(3) -5 ± 1(3)* Holding potential during drug application is -1UU mV All steroids were applied at 100 μM Oocytes injected with the indicated subunit cRNA were pre-equilibrated with steroid for 10 s prior to co-application of steroid and NMDA or kainate Values are mean percentage change ± S E in the response to 100 μM NMDA (NR1 I00 + NR2A) or 100 μM (GluRl and GluR3) or 10 μM (GluR6) kainate The number of experiments is given in parentheses * Indicates a statistically (P < 0 05) change in the induced current Modulation of recombinant NMDA receptors by PS and related steroids Xenopus oocytes co- injected with NR1 100 and NR2A cRNAs exhibited NMDA-evoked currents, which were potentiated by PS (Fig. 3A) and inhibited by its reduced metabolite 3cκ5/3S. Steroid modulation was rapid in onset, and the degree of modulation was similar whether NMDA was applied simultaneously with the steroid modulator or after a 10 s preincubation with steroid. Reversal of steroid modulation was also rapid, with NMDA responses returning almost to control levels after a 30 s wash (Fig. 3A and B) . As shown in Figure 3C, the potentiating effect of PS was due primarily to an increase in NMDA efficacy, which was nearly tripled in the presence of 100 μM PS, with no change in the NMDA EC 5Q . The EC 50 for enhancement of the 100 μM NMDA response by PS was 32 μM (Fig. 3D) , with a maximum enhancement of 343%. Pregnenolone hemisuccinate also potentiates the NMDA response (Table 1) , whereas 3/35/3S had an inhibitory effect similar to 3α5/3s, although it was slightly less potent (Fig. 3D) . In contrast, allopregnanolone sulfate (3α5o;S) was without effect on the NMDA induced current (8 ± 8%, n = 7) , while epiallopregnanolone sulfate (3/35αS) had a modest potentiating effect (33 ± 13%, n = 7) . Other steroids tested (Table 1) included 17/3-estradiol-3-sulfate, which had a small but statistically significant potentiating effect, and dehydroepiandrosterone sulfate, which was inactive . When oocytes were injected only with NR1 100 cRNA, the potency of PS in potentiating the NMDA induced current was similar (EC 50 =26 μM) to oocytes injected with both NR1 100 and NR2A cRNA, but the maximum potentiation was decreased to 114% (Fig. 3E) . In contrast, oocytes injected with NR1 100 cRNA were similar to those injected with NR1 100 plus NR2A cRNA with respect to the potency and efficacy of the inhibitory steroids 3O5/3S and 3/35/3S . Methods of the Invention Plasmids were obtained from the following individuals: Dr. Suzanne Zukin (Albert Einstein College of Medicine, Bronx, NY), NR1 100 ; Dr. Shigetada Nakanishi (Kyoto University Faculty of Medicine, Kyoto, Japan) , NR2A; Dr. Stephen Heinemann (Salk Institute, San Diego, CA) , GluRl (flop), GluR3 (flop), and GluR6. Plasmids were linearized with BamHI (NR1 100 ) , Xhol (NR2A, GluRl, GluR3) or Xjbal (GluR6) prior to in vi tro transcription with T3 (GluRl, GluR3 , GluR6) or T7 ( R1 100 , NR2A) RNA polymerase using the Message Machine kit (Ambion, Austin, TX) . Oocytes were harvested and maintained in ND96 (NaCl, 96 mM; MgCl-. 1 mM; KC1, 2 mM; CaCl 2 , 1.8 mM; HEPES, 5mM; pyruvate, 2.5 mM; 50 mg/ml gentamicin) according to published protocols (Stuhmer and Parekh, Electrophysiological recordings from Xenopus oocytes, in: B. Sakmann, E. Neher (Eds.), Single-Channel Recording, 2nd edn., Plenum Press, New York, 1995, pp. 341-356). RNA solutions (50-70 nl) were injected into oocytes for delivery of NR1 100 (5 ng) , NR1 10Q :NR2A (1:10 ng) , GluRl (5 ng) , GluR3 (5 ng) , or GluR6 (20 ng) . Oocytes were incubated in ND96 at 18-20°C for 3-7 days prior to electrophysiological recording. Steroids were obtained commercially from Steraloids, (Wilton, NH) and Sigma (St. Louis, MO), except 3α;5/3S, 335α;S, and 3/35/3S, which were provided by Dr. Robert Purdy (Dept . of Psychiatry, University of California, San Diego, CA) . Steroid solutions were prepared as 70-100 mM frozen stocks in dimethyl sulfoxide (DMSO) . The final DMSO concentration of all recording solutions was between 0.1% to 0.5% and was constant within an experiment. Over this concentration range, DMSO alone does not affect kainate or NMDA responses. Perfusion control and data acquisition were carried out using an automated oocyte electrophysiology workstation that was developed for the rapid collection of concentration-response data and real-time waveform analysis (Yaghoubi et al . , Soc . Neurosci . Abstr. 20 : 1109 (1995)). This system is implemented on a Macintosh Ilci computer (Apple Computer, Cupertino, CA) equipped with MacA-DIOS II data acquisition and control hardware (GW Instruments, Somerville, MA) . Drug application is controlled by the computer through a series of valves, using custom-written software based on the SuperScope II development environment, improving the efficiency and reproducibility of data collection. Experiments were carried out using an Axoclamp-2A voltage clamp amplifier (Axon Instruments, Foster City, CA) in two-electrode voltage clamp mode. Intracellular microelectrodes (1-3 MΩ) were filled with 3 M KCl . Oocytes were perfused continuously with Mg-free Ringer (NaCl, 96 mM; KCl, 2mM; CaCl 2 , 1.8 mM; HEPES, 5mM) . Membrane potential was clamped at -60 to -70 mV and stepped to -100 mV at the start of data acquisition unless otherwise stated. PS concentration-response curves for GluRl and GluR3 experiments were determined at -80 mV due to high levels of receptor expression which resulted in off-scale current responses at -100 mV. Current and voltage recordings were digitized at 100 Hz for analysis. GluR6 injected oocytes were treated for 10 min with 10 μg/ml concanavalin A to prevent fast desensitization. Modulation of NMDA and kainate responses by steroid compounds in expressed as percentage change, [ (T'/J) -1] X 100 %, where J is the average of four control responses, obtained from an oocyte before application and after washout of steroid, and I ' is the average of two responses obtained from the same oocyte in the presence of steroid. Peak NMDA responses were normalized to the maximal (100 μM) NMDA response for analysis and comparison except where otherwise specified. Peak kainate- induced currents are normalized to the maximal (100 μM for GluRl and GluR3 receptors; 10 μM for GluR6 receptors) kainate response. Kainate, rather than AMPA, was used to activate GluRl and GluR3 receptors, because desensitization was much less with kainate than with AMPA. In some experiments, oocytes were pre-equilibrated with steroid modulator for 10 s prior to application of NMDA, but this preincubation was found to have little if any effect on the extent of modulation of the peak NMDA- induced current, and the preincubation period was therefore omitted in subsequent experiments. Results are expressed as mean ± S.E.M. Statistical comparisons were made using Student's t-test. Example 2 The Ca 2+ -sensitive dye Fluo-3, AM and a trypan blue exclusion assay were employed to measure NMDA-induced Ca 2+ influx and cell death, respectively, in primary cultures of rat hippocampal neurons . A number of pregnane and pregn-5-ene steroids were examined for their ability to alter the increase in [Ca 2+ ] i and the neuronal death produced by NMDA exposure . Negative modulators Previously several reduced metabolites of progesterone that modulate currents evoked by NMDA were identified (Farb DH, and Gibbs TT (1996) Steroids as modulators of amino acid receptor function, in CNS neuro transmi tters and neuromodulators: neuroactive steroids (Stone TW, ed.) pp 23-36, CRC Press, New York). 3α5/3S was the first steroid found to inhibit NMDA-induced currents in cultured neurons (Park-Chung et al . , Mol Pharmacol 46: 146-150 (1994)). Consistent with this negative modulation of the NMDA response, 3ce5/3S (100 μM) reduces the 5 μM NMDA-evoked Ca 2+ influx by 32 ± 5% (n = 8) (Fig. 4A) . Further, 3α5/3S protects neurons from the cell death produced by acute (15 min) exposure to NMDA, raising the EC 50 for NMDA-induced neuronal death from 28 μM to 71 μM and lowering the maximal NMDA-induced excitotoxicity from 80% to 63% cell death (Fig. 4B) . This effect is dose-dependent , with an EC 50 of 45 μM and a 97% maximal inhibition (I__,,) of the cell death induced by 30 μM NMDA (Fig. 4C) . The neuronal death caused by chronic (16 h) NMDA treatment is also attenuated by 3α;5/3S, which, under these conditions, reduces the NMDA E m-V from 86% to 70% cell death without affecting the NMDA EC 50 (Fig. 4D) . In addition to its effects on the NMDA response, 3α5/3S inhibits currents elicited by AMPA and kainate (Park-Chung et al . , Mol Pharmacol 46: 146-150 (1994)), raising the possibility that the effect of the steroid on NMDA-induced neuronal death might not be specific to the NMDA receptor. 10 μM DNQX (a selective non-NMDA glutamate receptor antagonist (Honore et al . , Science 84:8215-8219 (1988) ) and 100 μM SR-95531 (a selective GABA A receptor antagonist (Farrant M, and Webster RA (1989) GABA antagonists: their use and mechanisms of action, in Drugs as Tools in Neurotransmi tter Research (Boulton AA, Baker, GB and Juorio, AV, eds.) pp 161-219, Humana Press Inc., Clifton) have no effect on NMDA-induced neuronal death (not shown) , arguing that the GABA A , AMPA, and kainate receptor types do not play a significant role in this process . While 3cx5βS nearly eliminates the toxic effects of an acute exposure to 30 μM NMDA, its stereoisomer 3α- hydroxy-5α-pregnan-20-one sulfate (3Q;5Q;S) is only half as effective, producing a 47 ± 12% (n = 4) inhibition of neuronal death (Fig. 5) . Strikingly, whereas 3/35/3S reduces NMDA-induced currents and neuronal death (86 ± 3% inhibition; n = 6) , its C5α isomer, 3j8-hydroxy-5α- pregnan-20-one sulfate (3/35αS) , both potentiates NMDA- induced currents (Park-Chung et al . , Mol Pharmacol 52 : 1113-1123 (1997)) and exacerbates neuronal death by 40 ± 7% (n = 17) . This shows that, as with their effects on NMDA-evoked currents, the neuroprotective effects of these sulfated steroids are contingent upon the stereochemistry of the A-B ring junction, whereas stereochemistry at C3 appears to be important only for C5α isomers. To elucidate further the structure-activity relationships for modulation of the NMDA receptor by steroids, a series of carboxylic acid derivatives of 3cx5β were synthesized (Fig. 7A) . The three negatively-charged derivatives, pregnanolone hemioxylate (3α;5/3HO) , pregnanolone hemisuccinate (3α5/3HS) , and pregnanolone hemiglutarate (3o;5/3HG) are about equally effective in inhibiting I NMDA/ the NMDA-induced current of Xenopus oocytes expressing NR1 100 + NR2A subunits, but the degree of inhibition is substantially less than is observed with 3α5/3S (Figs. 6A, 6B and 7B) . The uncharged pregnanolone formate (3α:5/3F) significantly inhibits I NHDA , but is significantly less effective than the dicarboxylic acid derivatives, while 3oι5β itself is inactive. Consistent with these results, the negatively- charged 3o;5/3HO, 3α5/3HS, and 3α5jδHG inhibit the NMDA- induced rise in [Ca 2+ ] i by approximately 40% in primary hippocampal cultures, while the uncharged 3α;5/3 and 3o;5/3F have no significant effect on NMDA-induced Ca 2+ accumulation (Fig. 7C) . Similarly, 3α5/3HO, 3α5/3HS, and 3Q;5/3HG are neuroprotective, reducing neuronal death caused by acute exposure to 30 μM NMDA by 35 ± 6% (n = 10), 54 ± 3% (n = 24), and 38 ± 6% (n = 9) , respectively (Fig. 7D) , while 3ot5β and 3o;5/3F do not exhibit significant neuroprotective activity. Positive modulators PS is a potent positive modulator of NMDA receptor function (Wu et al . , Mol Pharmacol 40 : 333-336 (1991); Bowlby MR, Mol Pharmacol 43 : 813-819 (1993)). To evaluate the role of the sulfate ester group at C3 , the effects of pregnenolone, pregnenolone formate (PF) , pregnenolone hemioxylate (PHO) , pregnenolone hemisuccinate (PHS) , and pregnenolone hemiglutarate (PHG) were examined (Fig. 8) . Recordings from Xenopus oocytes injected with NR1 100 + NR2A NMDA receptor subunit cRNA indicate that the negatively-charged PHS and PHG are effective positive modulators of the NMDA receptor, more than doubling the response to 100 μM NMDA (Fig. 8B) . Interestingly, potentiation of I NHDA increased with chain length of the dicarboxylic acid derivatives, with PHS and PHG potentiating the NMDA response to a greater extent than PS (Figs. 6C, 6D, and 8B) , whereas PHO and PF exhibited little activity. Pregnenolone itself is also without effect (5 + 2%, n = 3) . NMDA-induced Ca 2+ accumulation by hippocampal neurons in culture is enhanced by PHS (113 ± 22%, n = 4) and PHG (146 ± 29%, n = 5) , consistent with their potentiation of I NMDA , while pregnenolone and PF are without effect (Fig. 8C) . However, NMDA-induced Ca 2+ accumulation is also significantly increased by PHO (62 ± 17%, n = 7) , even though PHO does not enhance the NMDA-induced current. The effects of these steroids on NMDA-induced excitotoxicity are in agreement with their effects on NMDA-induced Ca 2+ accumulation. The neutral pregnenolone and PF are ineffective, while the negatively charged PHO, PHS and PHG exacerbate NMDA-induced cell death by 48 ± 12% (n = 6) , 80 ± 16% (n = 9) , and 54 ± 9% (n = 6) , respectively (Fig. 8D) . The excitotoxicity produced by excessive NMDA receptor stimulation has been implicated in the neurodegeneration associated with a number of CNS diseases and insults (Rothman et al . , Ann Neurology 19 : 105-111. (1986); Gόmez-Pinilla, et al . , Exp Neurol 104 : 118-124 (1989); Greenamyre, J.T., Neurobiol Aging 12 : 334-336 (1991); Greenamyre et al . , Arch Neurol 48 : 977- 981 (1991); Weaver, et al . , Proc Natl Acad Sci USA 94 : 10450-10454 (1997)). Evidence indicates that neuronal death results from NMDA receptor-mediated activation of a Ca + -dependent enzymatic cascade involving lipid peroxidation and protein and DNA degradation (Choi, D. W., J Neurobio 23 : 1261-1276 (1992); Chan, P. H., Stroke 27 : 1124-1129 (1996)). The present results, demonstrating that modulation by steroids of NMDA-induced Ca 2+ uptake is correlated with modulation of NMDA-induced neuronal death (Fig. 9) , support this view, and indicate that this rapid functional assay can be usefully employed to identify steroids with neuroprotective activity. In the present study, 3α5/3S is also demonstrated to markedly inhibit NMDA-induced changes in [Ca 2+ ] and neuronal death under both acute and chronic exposure conditions, consistent with previous finding that 3o;5/3s inhibits NMDA-induced currents in neurons maintained in primary culture (Park-Chung, et al . , Mol Pharmacol 46 : 146-150 (1994)). It is interesting that, in acute treatments, 3α5βS reduces both the NMDA EC 50 and E maχ for causing cell death, while only reducing the E maχ in chronic treatments. The reason for this difference is unclear, but may indicate metabolic conversion of 30(5/35, such as through the action of a steroid sulfatase, during the course of the chronic treatment, or an adaptive change at the NMDA receptor itself. Stereochemistry To investigate the structural requirements for steroid inhibition of NMDA-induced neuronal death, 3α5/3 and stereoisomers of 3α5/3S were assayed for activity, as were several related synthetic pregnane steroids. 3a5βS is as effective as 3cκ5/3S at protecting against the neuronal death produced by acute exposure to NMDA. This suggests that the stereochemistry at C3 is not critical for inhibition of NMDA-induced neuronal death by the C53 pregnane isomers. Notably, the isomers with C5α stereochemistry exhibit reduced neuroprotective activity as compared to the C5/3 isomers. 3α5o;S is about half as effective as 3α;5/3S and 3/35/3S in protecting against NMDA- induced cell death, whereas 3/35o;s actually exacerbates the toxicity of NMDA and potentiates the NMDA-induced elevation of [Ca 2+ ] i . These results are in agreement with previous electrophysiological studies of voltage-clamped chick spinal cord neurons in primary culture, in which 3α5/3s and 3/35/3S are strong inhibitors of the NMDA-induced current, 3α5o;s is a weaker inhibitor, and 3/35o;s weakly potentiates the NMDA response (Park-Chung, et al . , Mol Pharmacol 52 : 1113-1123 (1997)). The results indicate that stereochemistry at the A-B ring junction is an important determinant of the activity of pregnanes with a negatively-charged group at C3. The effect of C5α stereochemistry on the structure of the steroid molecule is to flatten out the ring system into a more planar configuration, much like the effect of the C5-C6 double bond in the pregn-5-ene series (Fig. 10) . Because competition experiments suggest that positive and negative modulation by steroids are mediated by distinct sites (Park-Chung, et al . , Mol Pharmacol 52 : 1113-1123 (1997) ) , it seems likely that the more planar ring structure of the pregn-5-enes and C5o; pregnanes improves the fit of the steroid molecule to the potentiating site and/or impairs its fit to the inhibitory site. Methods of the Invention Materials. Steroids were used at 100 μM, except where otherwise stated. PS and 17/3-estradiol were obtained commercially from Steraloids (Wilton, NH) . Formate esters were prepared by treating a solution of the steroid (400 mg) in dry dichloromethane (30 ml) with triethylamine (2.4 ml), 4-dimethylpyridine (160 mg) and formic acid (0.32 ml) . The mixture was cooled to -20 °C and acetic anhydride (1.9 ml) added dropwise over a 30 min period with stirring. The reaction mixture was then warmed to 0 °C for 30 min and then the reaction stopped by the addition of methanol (1.0 ml) . After evaporation of the solvents in vacuo, the residue was partitioned between ethyl acetate (10 ml) and aqueous IN HCl . The organic phase was washed twice with IN HCl (10 ml) and water (10 ml), and evaporated to dryness . The product was crystallized twice from a mixture of acetone and hexane . The hemioxalate esters were prepared as described above, using 568 mg oxalic acid instead of formic acid. The hemiglutarate esters were prepared as follows: To a solution of steroid (400 mg) in dry pyridine (6 ml) was added glutaric anhydride (400 mg) and 4-pyrolidinopyridine (40 mg) . The mixture was allowed to stand at room temperature in the dark for 4 days, when thin-layer chromatography showed complete disappearance of steroid starting material. The reaction mixture was then poured into ice water (20 ml) and the product extracted with ethyl acetate (20 ml) , and the extract washed with aqueous IN HCl (5 ml) and water (5 ml) . After drying the ethyl acetate solution over anhydrous sodium sulfate, the product was treated with activated charcoal (200 mg) and crystallized from a mixture of ethyl acetate and hexane . The hemisuccinate esters were prepared as described above, except that succinic anhydride (225 mg) was used in place of glutaric anhydride, and the reaction required 7 days for completion at room temperature. Steroids were initially dissolved in 100% DMSO, then diluted into assay buffer at a final DMSO concentration of 0.5% and sonicated for 20 min. All other solutions also contained 0.5% DMSO. Except where otherwise noted, the final steroid concentration was 100 μM. 3α5/3 and pregnenolone were used at 50 μM and 20 μM, respectively, as higher concentrations tended to precipitate in the assay buffer. Cell Culture. Principally neuronal cultures were prepared from hippocampal tissue of fetal Sprague-Dawley rats on day 18 of embryonic development, as previously described (Brewer et al . , Brain Research 494 : 65- 74(1989)). Briefly, hippocampal cells were dissociated by trituration in Ca 2+ /Mg 2+ -free Hank's basic salt solution (Gibco) supplemented with 4.2 mM bicarbonate, 1 mM sodium pyruvate, 20 mM HEPES, 3 mg/ml bovine serum albumin (BSA) . Dissociated cells were then pelleted by centrifugation (500 x g, 4 min) . The resulting pellet was suspended in Dulbecco's modified eagle medium (DMEM) (Gibco) supplemented with 2.4 mg/ml BSA, 26.5 mM sodium bicarbonate, 1 mM sodium pyruvate, 20 mM HEPES, 10% fetal bovine serum (FBS) (Gibco) , 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco) , and a modification of Brewer's B16 defined components (with 250 nM vitamin B12 and without catalase, glutathione, and superoxide dismutase) (Pike et al . , J Neurosci 13 : 1676-1687 (1993)) . Cells were then plated onto poly-L-lysine- coated 24 -well culture dishes (Nunclon) at a density of 15,000 cell/cm 2 and maintained in a humidified atmosphere containing 5% C0 2 /95% air at 37°C. After 48 h, nonneuronal cell division was inhibited by a 48 h exposure to 1 μM cytosine arabinoside. Cultures were subsequently maintained in serum-free DMEM plus defined components and were used for experiments 16 to 24 days after plating. Intracellular Calcium Concentration Measurements. NMDA- induced increases in [Ca 2+ ] i were measured with the Ca 2+ - sensitive fluorescent dye Fluo-3, AM (Molecular Probes) and a Cytofluor 2350 (Perceptive Biosystems) fluorescence plate reader, using excitation and emission filters of 485 nm and 530 nm, respectively. Hippocampal neurons were loaded with dye by incubating cultures with 10 μM Fluo-3, AM and 0.05% (w/v) Pluronic F-127 (Molecular Probes), a nonionic detergent, for 2 hours at 37°C. Fluo-3, AM and Pluronic F-127 were dissolved in dimethyl sulfoxide (DMSO, final concentration 0.5%). Cultures were then washed 3 times with assay buffer (120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 15 mM glucose, 25 mM Tris HCl, 0.5 μM tetrodotoxin; pH 7.4) to remove excess dye. For the purposes of calibration, other plate wells were rinsed instead with assay buffer in which 1.8 mM MnCl 2 replaced 1.8 mM CaCl ( F r nin buffer). Intracellular free calcium concentrations were calculated using the equation: [Ca 2+ ] ± = K D [F - F m in ] / [F max " F] ' here F is the fluorescence measured, F min is the fluorescence in the absence of calcium (determined in F min buffer after the addition of 10 μM of the Ca 2+ ionophore A-23187) , F maχ is the fluorescence of the Ca 2+ -saturated dye (determined in assay buffer after the addition of 10 μM A-23187) , and K D = 320 nM (the equilibrium dissociation constant for the binding of Ca 2+ to Fluo-3, AM). Fluorescence measurements were made before and 40 s after the addition of NMDA. Steroid or vehicle (0.5% DMSO) was added 10 min prior to the addition of NMDA. NMDA was dissolved in DMEM; steroids and A-23187 were dissolved in DMSO. DMSO was also added to controls to maintain a constant final DMSO concentration of 0.5. Data are expressed as the percentage change in the NMDA-induced increase in [Ca^ ] i in the presence of the indicated steroid. None of the steroids tested significantly altered [Ca 2+ ] j _ in the absence of NMDA. NMDA-induced Cell Death. Primary cultures of rat hippocampal neurons were exposed to NMDA (dissolved in DMEM) for 15 min (acute exposure) or 16 hours (chronic exposure) . In acute exposure experiments, cultures were treated with steroid, MK-801 (Research Biochemicals International; dissolved in DMEM), or vehicle during and/or after NMDA exposure. Steroids were dissolved in DMSO (0.5% final concentration), and all treatment media contained 0.5% DMSO. In chronic exposure experiments, cultures were additionally treated with steroid, vehicle, SR-95531 (Research Biochemicals International; dissolved in DMEM), 6 , 7-dinitroquinoxaline-2 , 3-dione (DNQX; Research Biochemicals International; dissolved in DMEM), or MK-801 (dissolved in DMEM) during NMDA exposure. Following exposure, cultures were washed 3 times with medium from sister cultures (conditioned medium) . After the final wash, steroid or vehicle was reintroduced. Drugs were added to cultures in 25 μl of conditioned medium to yield a final volume of 0.25 ml per well. Except where otherwise noted, final steroid concentration was 100 μM. 3α5/3 and pregnenolone were used at 50 μM and 20 μM, respectively, as higher concentrations tended to precipitate in the culture medium. The ability of neurons to exclude trypan blue was used to quantitate cell viability (Dawson et al . , Proc Natl Acad Sci USA 88 : 6368-6371 (1991)). 24 hours after acute and 16 hours after chronic exposure to NMDA, culture medium was replaced by 0.4% trypan blue in 0.1 M phosphate-buffered saline (PBS; pH 7.4) and placed in a humidified incubator for 10 min. Cultures were then fixed with 4% paraformaldehyde in PBS for 30 min, at which time the fixative was replaced with PBS. The number of stained and unstained neurons were counted in 4 high-power fields per culture well, using an inverted phase contrast microscope under both bright field and phase contrast settings. Experiments were performed in triplicate, and all assessments were made with the experimenter blind to the treatment of each culture well. Percent cell death is expressed as follows: (number of trypan blue stained neurons) / (total number of neurons) x 100%. The basal level of neuronal death (termed background) , measured in controls lacking NMDA and PS, was 0-10% 4 and was subtracted from the raw data in each experiment . Oocyte Electrophysiology. NR1 100 (NR1G) and NR2A clones were kindly provided by Dr. R. S. Zukin (Albert Einstein College of Medicine, New York, NY) and Dr. S. Nakanishi (Faculty of Medicine, Kyoto University, Kyoto, Japan), respectively. mRNA was prepared through in vi tro transcription of NR1 100 and NR2A cDNAs using the mMessage mMachine kit (Ambion, TX) . On the day following isolation, batches of 20-40 selected Xenopus oocytes were injected with 50 nl of prepared RNA solution (0.5 ng NR1 100 and 5 ng of NR2A mRNA/oocyte) . Oocytes were maintained in glass petri dishes containing Barth's solution (84 mM NaCl, 1 mM KCl, 2.4 mM NaHC0 3 , 0.82 mM MgS0 4 , 0.41 mM CaCl 2 , 0.33 mM Ca(N03)2, 2.5 mM pyruvate, 0.1 mg/ml gentamycin, 7.5 mM Tris/HCl, pH 7.4) in an incubator at 18 °C. Oocytes were used for electrophysiological experiments 4-10 days after injection. Recordings from oocytes were obtained using the two electrode voltage-clamp mode with an Axoclamp-2A amplifier (Axon Instruments) . The resistance of microelectrodes, pulled from glass capillaries and filled with 3 M KCl solution, was 2.5-3.5 M. Membrane potential was clamped at -70 mV. Drugs were applied by a gravity- driven external perfusion system. Drug application and data acquisition were carried out with custom-written software implemented in the SuperScopell programming language (GW Instruments, Somerville, MA) . Membrane current was filtered at 500 Hz and sampled at 100 Hz. Statistical Analysis. The degree of modulation of NMDA- induced Ca 2+ influx, cell death, and currents is expressed as the percent change, defined as (I' /I - 1) x 100%, where I and I' are the NMDA-induced responses in the absence and presence of modulator, respectively. All data are expressed as mean ± standard error of the mean. Statistical significance was evaluated using 95% confidence limits unless otherwise noted. Example 3 Figure 11 shows a comparison of traces of NMDA induced responses obtained from oocytes expressing NR1 100 /NR2A receptors bathed in normal (Ca 2+ containing) Ringer solution and Ba-Ringer solution in which Ca 2+ was replaced with Ba 2+ . Current traces obtained in Ba-Ringer solution do not exhibit the rapidly inactivating component that is seen in normal Ringer, and which most likely reflects current through Ca 2+ activated CI " channels (Leonard et al . , Neuron 4 : 53-60 (1990)). All further experiments were performed in Ba-Ringer. The current responses obtained from oocytes injected with NMDA receptors composed of different NRl isoforms differed from each other in agonist EC 5Q s . L-glutamate, glycine and NMDA EC 5Q s for NRl isoforms lacking N-terminal insert were less than ones for isoforms with N-terminal insert (except for NR1 001 , see Table 2) . The absolute current responses induced by saturating concentration of agonists (500 mM NMDA and 10 mM glycine) were in the range from 800 to 1800 nA. Since a steroid' s effect on NMDA receptors was found to depend on level of expression of the receptor in the membrane of oocytes (see below) , oocytes that showed current responses in the range of 0.1 μAmp to 1 μAmp induced by 50 mM (or 80 mM NMDA for isoforms with N-terminal insert) and 10 mM glycine were used to assess the effects of pregnenolone sulfate and pregnanolone sulfate on NMDA receptors comprised of different isoforms of NRl subunit. Co-application of 100 mM PS resulted in potentiation of current through NMDA receptors comprising any NRl isoform. NMDA, L-glutamate and glycine dose-response experiments showed that potentiation by PS was observed even at saturating concentrations of agonists. In addition, the agonists EC 50 obtained in the absence of steroid were similar to ones obtained in the presence of 100 mM PS, suggesting that PS did not compete for the agonists binding sites (see Table 2) . NMDA (PS) Subunits EC o , mM n, Hill —max NR1 011 /NR2A 52±6.5 (43±4.8) 1.39±0.07 (1.11±0.12) 1.14+0.02 (1.68±0.06) n=4 (n=4) NR1 111 /NR2A 88±16 (95±12) 1.49±0.02 (1.33±0.22) 1.34±0.09 (2.27±0.07) n=4 (n=4) NR1 Q01 /NR2A 81±7 (71±18) 1.46±0.07 (1.21±0.15) 1.27+0.04 (2.15±0.17) n=8 (n=4) NR1 Q10 /NR2A 44+8 (22±2) 1.19±0.06 (1.13±0.09) 1.18+0.03 (1.53±0.08) n=7 (n=4) NR1 000 /NR2A 60+7 (33±3) 1.31±0.08 (1.18±0.05) 1.20+0.05 (1.55±0.04) n=7 (n=ll) NR1 1Q1 /NR2A 75±6 (35±5) 1.31±0.05 (1.15±0.12) 1.33+0.07 (1.78±0.06) n=5 (n=4) NR1 100 /NR2A 76±6.2 (40+3.3) 1.13±0.04 (1.20±0.06) 1.36+0.04 (1.60±0.07) n=ll (n=6) L-glutamate (PS) NR1 011 /NR2A 0.56±0.03 (0.39±0.03) 1.17±0.03 (1.13±0.08) 1.59±0.03 (2.08±0.21) n=4 (n=4) R1 111 / R2A 0.71±0.06 (0.51±0.03) 1.39±0.02 (1.58±0.09) 1.87±0.12 (2.62±0.27) n=4 (n=4) NR1 001 /NR2A 1.03 + 0.09 (0.65±0.21) 1.20±0.07 (1.57 + 0.18) 2.10±0.12 (2.85±.26) n=7 (n=4) NR1 010 /NR2A 0.38±0.04 (0.25±0.01) 1.32±0.14 (1.59±0.15) 1.48±0.03 (1.81±0.08) n=9 (n=4) NR1 000 /NR2A 0.65±0.03 (0.59±0.04) 1.05±0.02 (1.17±0.08) 1.75±0.02 (2.92±0.03) n=4 (n=4) NR1 101 /NR2A 0.68±0.06 (0.60±0.04) 1.07+0.06 (1.37±0.06) 1.77+0.09 (2.72±0.07) n=3 (n=4) NR1 1Q0 /NR2A 0.45±0.02 (0.31±0.02) 1.15±0.04 (0.98±0.06) 1.50±0.04 (2.30±0.21) n=4 (n=4) glycine (PS) NR1 011 /NR2A 0.92 + 0.11 (0.85±0.1) 1.30±0.05 (1.34±0.18) 1.67±0.08 (2.24±0.29) n=4 (n=3) NRl 1 /NR2A 1.22±0.09 (1.07±0.09) 1.49+0.06 (1.26±0.10) 1.93±0.07 (2.68±0.16) n=4 (n=8) NR1 001 /NR2A 1.79±0.3 (1.56±0.65) 1.25±0.07 (1.30±0.13) 1.89±0.20 (2.77±0.40) n=6 (n=4) NR1 010 /NR2A 0.90±0.08 (1.07±0.11) 1.32±0.07 (1.38±0.10) 1.51±0.07 (1.92 + 0.19) n=4 (n=4) NR1 000 /NR2A 0.97±0.10 (1.46±0.17) 0.90±0.01 (1.12±0.05) 1.48±0.04 (2.88±0.27) n=3 (n=4) NR1 101 /NR2A 1.74±0.26 (1.07+0.17) 1.37±0.13 (1.32±0.09) 1.63±0.04 (2.46±0.23) n=5 (n=4) NR1 100 /NR2A 0.82±0.11 (0.97±0.07) 1.23±0.07 (1.03±0.05) 1.52±0.07 (2.34±0.09) n=4 (n=4) Table 2. Properties of NMDA receptors comprising different NRl isoforms in the absence and in the presence of 100 mM PS. In order to construct PS dose-response curves different concentrations of PS (in the range from 0.5 to 400 mM) were coapplied with 10 mM glycine and 50 mM (or 80 mM NMDA, which is close to EC 50 values for NRl isoforms without or with N-terminal insert respectively) . The dose-response experiments revealed that PS had similar potency for all combinations of NRl χχχ and NR2A subunits. However, PS was more efficient when applied to NMDA receptors containing NRl isoforms that were lacking N-terminal insert (see Fig. 12) . Maximum potentiation of NMDA-induced current for NRl,,,/, NR1 101 /, NR1 1QQ /NR2A receptors were 1.51±0.04 (n=9) , 1.66+0.10 (n=8), 1.67±0.07 (n=8) fold respectively in comparison to 2.14±0.17 (n=7) , 2.19±0.09 (n=9) , 2.14±0.08 (n=15) fold potentiation for NR1 Q11 /, NR1 Q01 /, NR1 000 /NR2A receptors respectively (see Table 3) . T-test analysis of maximum PS potentiation obtained from receptors comprising NRl isoforms with and without N-terminal insert resulted in p values of 0.024, 0.005, 0.002 for pairs 111 vs. 011, 101 vs. 001 and 100 vs. 000 respectively. NRl ι /NR2A 40±8 (n=9) 1.28±0.19 0.51±0.04 NR1 Q11 /NR2A 32±8 (n=7) 1.18±0.18 1.14±0.17 NR1 101 /NR2A 25±5 (n=8) 1.52±0.24 0.66±0.10 NR1 0Q1 /NR2A 26±2 (n=9) 1.14±0.10 1.19±0.09 NR1 100 /NR2A 24±5 (n=8) 1.33±0.08 0.67±0.07 NR1 0Q0 /NR2A 29±3 (n=15) 1.31±0.06 1.14±0.08 NR1 010 /NR2A 48±12 (n=5) 1.30+0.18 1.03±0.09 NRl nι /NR2B 24±1 (n=3) 1.52±0.10 1.20+0.10 NR1 011 /NR2B 35±10 (n=4) 1.29+0.19 0.78±0.14 NR1 101 /NR2B 32±8 (n=2) 1.63±0.29 1.03±0.02 NR1 0Q1/ NR2B 24±6 (n=4) 1.34±0.17 0.63±0.07 NR1 100 /NR2B 34±4 (n=4) 1.42±0.11 1.02±0.10 NR1 0Q0 /NR2B 22±7 (n=4) 1.52±0.15 0.58±0.04 Table 3. Effect of PS on NMDA receptors comprising different NRl isoforms. In contrast to potentiating effect of PS, 3α5/3S induced current inhibition through receptors comprising any NRl isoform (see Fig. 13) . The inhibitory effect was reversible and concentration dependent . The potency of 3α5/3S obtained for NMDA receptors comprising different NRl isoforms were similar, revealing PS EC 50 s that ranged from 25 to 45 mM. The maximum extent of inhibition obtained for different NRl isoforms ranged from 75±7% (for NRl ι /NR2A) to 99±3% (for NR1 100 /NR2A, see Table 4) . The differences in maximum inhibition were insignificant and no apparent correlation with the presence of N-terminal insert was observed. Splice variant EC 5Q , mM n Hiii E max 111 29±5 (n=4) 1.09±0.9 -0. ,75±0. .07 Oil 38±0. .5 (n= 4) 0.96±0.06 -0. .86+0. .04 101 45±2 (n=3) 1.05±0.25 -0. .90±0. .07 001 25±2 (n=4) 0.95+0.11 -0. ,89±0. .05 100 36±2 (n=4) 0.83±0.02 -0. ,99±0. .03 000 41±0. ,6 (n= 4) 0.98±.03 -0. .89+0. .01 010 34±4 (n=4) 1.04+0.06 -0. .85+0. .02 Table 4. Effect of 3o;5/3s on NMDA receptors comprising different NRl isoforms. Example 4 Differential modulation of NRl /NR2A splice variants by neuroactive steroids Table 5 below shows alternatively spliced exons present in each NRl splice variant. In order to assess the modulatory role or pregnenolone sulfate (PS) on NMDA receptor composed of different NRl χχχ subunits, NMDA dose response of NR1 001 /NR2A and NR1 111 /NR2A splice variants were determined, in the presence or absence of 100 μM PS (Figure 15) . The extrapolated maxima (Emax) obtained from the concentration-response data (fitted by the nonlinear regression logistic equation: relative current = Emax/1+ (EC 50 /c) πH ) , indicate that the relative current change is splice variant dependent. The Emax obtained for NR1 011 /NR2A in the presence of absence of PS was 1.79 and 1.2 respectively, while for NRl nι /NR2A was 2.7 and 1.33. The change in the efficacy was not only significant relative to control but was also between the splice variants (*p<0.0000005) , while the EC 50 s and thus the potency remained unchanged (for NR1 Q11 /NR2A EC 50 = 71 μM, EC 50 (+PS) = 67 μM and for NRl ι /NR2A EC 50 = 103 μM, EC 50 (+PS) = 89 μM) . To further elucidate the role of neuroactive steroids on NRl χχχ /NR2A splice variants, dose response was determined in the presence or absence of 100 μM pregnanolone sulfate (3c5/3S) (Figure 16) . The acquired data indicate that the extrapolated maxima of the relative current change are independent of the splice variant. The Emax obtained for NR1 Q11 /NR2A in the presence or absence of 3α;5/3S was 0.52 and 1.2 respectively, while for NR1 111 /NR2A was 0.54 and 1.33. While the observed change in the efficacy was not significant (p>0.05), the potency relative to control, was decreased by 69% for NR1011/NR2A and 53% for NR1 111 /NR2A, as indicated by the EC 5Q s (for NR1 Q11 /NR2A EC- 0 = 71 μM, EC 50 (+3o;5/3S) = 120 μM and for NR1 111 /NR2A EC 50 = 103 μM, EC 50 (+3α5/3S) = 158 μM) . In summary (Figure 17) , the percentage potentiation of 100 μM PS on NR1 Q11 /NR2A was 50% and on NRl ι /NR2A was 103% suggesting a difference of 106% between them (*p<0.0000005) , while 100 μM of 3α5/3S gave 56% and 59% inhibition for NR1 Q11 /NR2A and NRl ιn /NR2A respective suggesting a difference of 5% (p>0.05). To further test the hypothesis that the presence or absence of the exon (a 21 amino acid insert) controls the modulatory effect of PS on different NRl χχχ /NR2A splice variatns, the data obtained from the max relative currents (mrc) after co-application of 200 μM NMDA + 10 μM glycine ± 100 μM PS were compared (Figure 18) . The data indicate a 1.3 -fold difference between NR1 Q00 /NR2A (mrc = 1.29) and NR1 100 /NR2A (mrc = 1.73), (*p<0.0000005) , a 1.6-fold difference between NR1 Q11 /NR2A (mrc = 1.41) and NR1 111 /NR2A (mrc = 2.28), (p<0.0000005) and significantly no difference between NR1 Q01 /NR2A (mrc = 1.73) and NR1 101 /NR2A (mrc = 1.78), (p>0.05). These results indicate that specificity of PS induced modulation is NR1 XXχ subunit dependent. SPLICE VARIANT EXON PRESENT α β γ NR1, M + - + NR1„, + + + Table 5 Pregnenolone sulfate' s differential modulation of NRl χχχ /NR2A splice variants is pH dependent In order to assess the effect of hydrogen concentration on the modulatory role of pregnenolone sufate (PS) on NMDA receptors composed of different NRl χχχ subunits, NMDA dose response curves of NR1 011 /NR2A and NR1 111 /NR2A splice variants were constructed at pH 8.5 , in the presence or absence of 100 μM PS (Figure 19) . The extrapolated maxima (Emax) obtained from the concentration-response, indicate that the relative current change of each splice variant is pH dependent. Although the potency (EC 50 s) for both NR1 Q11 /NR2A and NR1 111 /NR2A remained the same upon shifting the pH from 7.5 to 8.5, the efficacies of the two splice variants decreased from pH = 7.5 to pH = 8.5. The Emax obtained for NR1 011 /NR2A in the presence or absence of PS was 1.62 and 1.30 (at pH = 8.5) and 1.79 and 1.2 (at pH = 7.5) respectively, while for NR1 111 /NR2A was 1.98 and 1.35 (at pH = 8.5) and 1.79 and 1.20 (at pH = 7.5) respectively. The percentage potentiation of 100 μM PS on NR1 011 /NR2A was 50% and on NRl ιn /NR2A at pH = 7.5 was 103% suggesting a difference of 106% between them (*p<0.0000005) , while 100 μM of PS at pH = 8.5 gave 24% and 47% potentiation for NR1 011 /NR2A and NR1 111 /NR2A respectively suggesting a difference of 91% (*p<0.05) (Figure 20). To further investigate the effect of hydrogen ions and thus the involvement of the proton sensor on PS potentiation, the max relative currents (mrc) after co- application of 200 μM NMDA + 10 μM glycine ± 100 μM PS on NR1 011 /NR2A were compared to that of NRl nι /NR2A, at pH = 6.5, 7.5, 8.5, (Figure 21). These data indicate a 1.8- fold difference between NR1 011 /NR2A (mrc = 2.24) and NR1 111 /NR2A (mrc = 4.06) at pH = 6.5 (***p<0.0000005) , a 1.6-fold difference between NR1 011 /NR2A (mrc = 1.41) and NRl ι /NR2A (mrc = 2.28), (**p<0.0000005) and a 1.2-fold difference between NR1 011 /NR2A (mrc = 1.35) and NR1 111 /NR2A (mrc = 1.59) at pH = 6.5 (*p<0.05). Moreover, the max relative current data for NR1 Q11 /NR2A (Figure 22, panel A) show a 1.7-fold difference between pH = 6.5 and pH = 7.5 (*p<0.00005) , and significantly no difference between pH = 7.5 and pH = 8.5 (*p<0.00005) . The difference for NR1 111 /NR2A (Figure 14, panel B) between pH = 6.5 and pH = 7.5 was 1.8 fold (*p<0.0000005) while between pH = 7.5 and pH = 8.5 was 1.4-fold (*p<0.000005) . These results indicate that protons participate in PS differential modulation of NRl χχχ /NR2A splice variants. Methods of the Invention Materials South African clawed frogs, Xenopus laevis, were purchased from either Xenopus One (Ann Arbor, MI) or Nasco (Fort Atkinson, WI) . Frog brittle is also purchased from the same vendors . Plasmids containing the NR1 100 , NR1 Q00 , NRl ι , NR1 Q11 , NR1 101 , NR1 Q01 inserts (encoding different NMDA receptor splice variants) and NR2A (encoding NMDA receptor subunit) were kindly provided by Dr. Shigetada Nakanishi of Kyoto University Faculty of Medicine, Kyoto, Japan. Competent DH5α cells (used for transformation) were purchased from Gibco (Gaithersburg, MD) . Maxiprep columns were purchased from Qiagen (Chatsworth, CA) . Restriction enzymes were purchased from New England Biolabs (Beverly, MA) and Pharmacia (Piscataway, NJ) . mMessage mMachine in vitro transcription kit (T7 RNA polymerase) was purchased from Ambion (Austin, TX) . NMDA and glycine were purchased from Sigma Chemical Co. (St. Louis, MO). Chemicals used for the preparation of ND96 and Ringer solutions were purchased from Sigma and Gibco. PS and 3α5/3S were purchased commercially from Steraloids (Wilton, NH) and Sigma. Data acquisition and instrument control hardware was purchased from GW Instruments (Somerville, MA) . SuperScope II vl.43 software was also purchased from GW instruments. Miniature teflon-coated valves were purchased from LeeValves (Essx, CT) . Apple Macintosh Ilic was purchased from Apple Computer Inc. (Cupertino, CA) . A microprocessor-controlled fixed or variable volume microinjector was purchased from Drummond Inc. (Broomall, PA) . Micromanipulators (no. M3301) were purchased from World Precision Instruments (Sarasota, FL) , translation stages (single axis, no. 423) , 50mm open loop motorized DC actuators (no. 860-A) , hand-held single axis controller (no. 861) , low profile magnetic base (no. 150), 45° angle bracket (no. 360-45) from Newport Inc. (Irwine, CA) and the headstages for the electrodes from Drummond Inc. The OC-725 Oocyte Clamp amplifier was purchased from Warner Instrument Corp. (Hamden, CT) and the Axoclamp-2A amplifier from Axon Instruments Inc., Foster City, CA) . A programmable horizontal Flaming- Brown micropipette puller was purchased from Sutter Instruments Co., CA. Perfusion manifolds (no. ML-6) and polyethylene (PE) tubing (no. PE-50 and PE-160) were purchased from Warner Instrument Corp. Glass pipettes for RNA microinjection (RNase-free 3.5-in. glass capillaries no. 3-000-203 -Cdx) were purchased from Drummond. Glass electrodes (borosilicate glass 100 μl disposable micropipettes, no. TWI50F-4) were purchased from World Precision Instruments. Preparation of mRNA. For the microinjection into Xenopus oocytes, mRNA was prepared from lyophilized plasmid pellets containing cDNA insets encoding the specific NMDA receptor splice variant. Transformation of competent DH5a (sub-cloning quality) cells resulted in bacterial cultures expressing the desired plasmids. Overnight cultures (500 ml) of transformed bacterial cells were prepared in order to grow sufficient quantities for maxiprep isolation of the plasmid cDNA. Maxipreps were done using a Sorvall RC-5B ultracentrifuge, Qiagen columns and cDNA was quantitated with spectrophotometry (OD 260 and OD 260 / OD 280 ratio) . The resulting plasmid cDNA was linearized by digestion with an appropriate restriction enzyme that cleaves distal to the cDNA insert (Xhol for NR2A or Notl for NR1 100 , NR1 Q00 , NRl ιn , NR1 Q11 , NR1 101 , NR1 001 ) . Following digestion, plasmid cDNA was treated with proteinase K (degradation of RNAse) and extracted with a combination of phenol/chloroform/isoamyl alcohol, after which ethanol precipitation was used to pellet DNA prior to the start of the transcription reaction. cDNA quality was assessed by agarose-gel electrophoresis (1.5% agarose gel). The samples were stored at - 20°C until use. In vitro transcription was performed using the Ambion mMessage mMachine kit with T7 (NR1 100 , NR1 Q00 , NRl ι , NR1 011 , NR1 101 , NR1 Q01 ) RNA polymerase. The reaction was assembled on ice and allowed to incubate at 37°C for 1-2 hr. The resulting mRNA was precipitated (reaction termination) using lithium chloride/50mM EDTA solution and dissolved in RNase-free H 2 O.The concentration (OD 260 ) and OD 260 / OD 280 ratio (should be more than 1.5) was determined by spectrophotometer . The yield of mRNA was generally 15-30 μg . mRNA was stored at - 80°C until use. Electrophysiological recordings of ion currents from oocytes expressing the desired NMDA receptor subtypes (NR1 100 , NR1 000 , NRl 1 , NR1 011 , NR1 101 , NR1 001 / NR2A) were carried out using either the OC-725 Oocyte Clamp amplifier from Warner Instruments or the Axoclamp-2A amplifier from Axon Instruments Inc. in a two-electrode voltage clamp mode. Intracellular recording microelectrodes were fabricated from 100 μl borosilicate glass disposable micropipettes using a 2 -step protocol on a standard horizontal Flaming-Brown micropipette puller. Perfusion control and data acquisition were carried out using an automated oocyte electrophysiology workstation and custom-written software implemented in the SuperScope II environment, which was developed for the rapid collection of concentration-response data and real time waveform analysis (Yaghoubi et al . , Evaluation on neurosteroid modulation of kainate receptors using an automated system for oocyte electrophysiology . In Society for Neuroscience, pp. 1109 (1995)). All experiments were performed at room temperature (22-24°C) . Example 5 Sulfated steroids can act as positive or negative modulators of NMDA receptor function. For instance, pregnenolone sulfate (PS) , an abundant neurosteroid, potentiates NMDA-induced currents, whereas 3α5/3- pregnanolone sulfate (3o;5/3S) inhibits NMDA -induced currents. Moreover, PS does not competitively inhibit the binding of 3α5/3s to the NMDA receptor, indicating the presence of independent binding sites or pathways for negative and positive modulation. The following experiments have identified amino acids on subunit 1 of the NMDA receptor (NRl) that control PS potentiation of NMDA-induced currents in recombinant NRla/NR2A receptors, where NRla corresponds to NR1 011 . These amino acids were identified using multiple sequence alignments and then tested for functional activity in xenopus oocytes using mutated NRla expression constructs. In the first case, the sequence of the NRla subunit was aligned with that corresponding to the ligand binding domain of five nuclear receptors: human retinoid X receptor a (RXR) , the human retinoic acid receptor γ (RAR) , the human progesterone receptor (PR) , the human glucocorticoid receptor (GCR) and the estrogen receptor (ER) . A region was defined in the NRla subunit, residue 1163 to residue N273, which shares 44% sequence similarity to the aforementioned nuclear receptors, spanning helix 3 to helix 6 (Fig. 23) . Using the information from the sequence alignment, the mutant Penta-mutant (PM) was constructed by replacing five positively charged residues (R187, K193, K202, R233, and R252) with alanines (Fig. 23) . PM was co-expressed with NR2A in the xenopus oocyte expression system. The receptors containing PM were functional with an EC50 of 55.0±3.4 uM for the NMDA response, similar to the wild- type receptor. However, the PM containing receptors showed a 40% reduction in sensitivity to PS in the presence of lOOuM PS, 300uM NMDA and 50 uM Glycine (Fig. 24) . In contrast, PM did not effect the response of the NMDA receptor to the negative modulator 3OJ5/3S (Fig. 25) . These results demonstrate for the first time that there is a distinct molecular component for pregnenolone sulfate modulation of NMDA receptor function and are consistent with the hypothesis that PS and 3α5/3s modulate NMDA-induced currents through distinct sites. Methods of the Invention Mutagenesis . Site directed mutagenesis was generated by a method involving PCR. In brief, oligonucleotides were synthesized containing the mutation, and these oligonucleotides were used in combination with other oligonucleotides in PCR amplifications of fragments of the cDNA. The product of the PCR reactions were cut with two different restriction enzymes to generate a cassette containing the mutation. This cassette was then ligated into the cDNA that was cut with the same two restriction enzymes. For all of the mutations, single isolates were selected, and the entire region of the amplified cassette was sequenced to check for the mutation and to insure against second- site mutations. Preparation of RNA. Plasmids containing the NR1 Q11 and NR2A cDNA inserts were kindly provided by Dr. Nakanishi (Kyoto University Faculty of Medicine, Kyoto, Japan) , while plasmids containing the NR2B, NR2C and NR2D cDNA inserts were kindly provided by Dr. Seeburg (Heidelberg University, Heidelberg, Germany) . Plasmids were linearized with appropriate restriction enzyme prior to in vi tro transcription using the Message Machine kit (Ambion, Inc., Austin, TX) . Expression in Xenopus oocytes . Female, oocyte positive Xenopus laevis frogs were purchased from Xenopus I (Dexter, MI) . Following 45 min of 0.15% Tricaine anesthesia, ovarian sections containing the follicular oocytes were removed from the frog through a lateral abdominal incision and were immediately placed in a calcium-free solution (NaCl 96 mM, MgCl 2 1 mM, KCl 2 mM, Hepes 50 mM, pyruvate 2.5 mM, 0.1 mg/ml gentamicin, pH 7.4) . Following 1.5 - 2 hours incubation in 0.2% collagenase (type II, Sigma Chemical Co., St. Louis, MO) at room temperature, individual defolliculated Dumont stage V and VI oocytes were transferred to an incubator and maintained overnight in Barth's solution (NaCl 84 mM, NaHC0 3 2.4 mM, MgS0 4 0.82 mM, KCl 1 mM, Ca (N0 3 ) 2 0.33 mM, CaCl 2 0.41 mM, Tris/HCl 7.5 mM, pyruvate 2.5 mM, 0.1 mg/ml gentamicin, pH 7.4) at 18-20°C. Oocytes were injected with 50 nL of RNA solutions using an electronic microinjector (Drummond Inc., Broomall, PA). The transcripts were injected at a ratio of 0.125/1.25 ng mRNA per oocyte for NRla/NR2A or penta-mutant/NR2A receptors and 0.5/5 ng mRNA per oocyte for NRla/NR2B, NRla/NR2C, and NRla/NR2D receptors. The injected oocytes were used for experiments following 1-5 days of incubation in Barth's solution at 18-20°C. Electrophysiology . Measurements of ion currents from oocytes expressing NMDA receptors were performed using an Axoclamp-2A voltage clamp amplifier (Axon Instruments, Inc., Foster City, CA) in two-electrode voltage clamp mode. The microelectrodes were fabricated with a programmed puller (Sutter Instrument Co., CA) from borosilicate glass capillaries and were filled with 3 M KCl solution. The resistance of filled microelectrodes was in the range of 1-3 MΩ. The oocyte recording chamber was continuously perfused with Mg 2+ -free Ba-Ringer solution (NaCl 96 mM, KCl 2 mM, BaCl 2 1.8 mM, Hepes 5 mM) . In order to minimize activation of Ca -dependent Cl~ conductance the perfusion solutions used did not contain Ca 2+ ions. If not mentioned otherwise, during data acquisition oocytes were clamped at a holding potential of -70 mV. The membrane current was filtered at 500 Hz and sampled at 100 Hz. Drugs were applied using a gravity driven external perfusion system. The working volume of the recording chamber was 30 μl and the rate of the perfusion was 50 μl/sec. The drug application lasted 10 sec and was followed by a 60 sec wash. The data acquisition and external perfusion control were done using the SuperScopell software package (GW Instruments, MA) . All experiments were performed at room temperature (22-24°C). Peak or steady-state current measurements were normalized and expressed as a fraction of the peak or steady-state control current measurements, which were performed before and after application of every single consentration of agonist or steroid. In order to derive the values of NMDA receptor agonists and PS EC 50 s, experimental dose-response data were fit using equations E maχ / (1 + (EC 50 /c) n ) or 1+E maχ / (1 + (EC 50 /c) n ) respectively, where E maχ is a maximum response, n - Hill coefficient, c - concentration of an agonist. The data are presented as mean + S.E.M. with number of experiments in parentheses. Chemicals . Steroids were obtained from Steraloids, Inc. (Wilton, NH) and were dissolved in DMSO prior to use. Other compounds were obtained from Sigma (St. Louis, MO) . Example 6 NMDA receptor expression in Xenopus oocytes To investigate the influence of NMDA receptor subunit composition on the modulatory effects of neuroactive steroids, mRNA coding for the NR1 100 subunit was coinjected into Xenopus laevis oocytes along with mRNA coding for either the NR2A, NR2B, NR2C, or NR2D subunit. All 4 subunit combinations resulted in expression of functional NMDA receptors 1-5 days after injection, as indicated by an inward current in response to application of 80 μM NMDA plus 10 μM glycine. Concentration-response studies showed that the NMDA EC 50 differed substantially across the different subunit combinations. Therefore, to compare modulatory effects of steroids, a concentration of NMDA close to its EC 50 for each subunit combination was used (80 μM for NR1/NR2A, 25 μM for NR1/NR2A and NR1/NR2B, and 10 μM for NR1/NR2D) . Subunit -selective modulation by PS As shown in Figure 26, the choice of NR2 subunit dictated the direction of modulation by PS. Co- application of 100 μM PS with NMDA plus glycine to oocytes expressing NR1/NR2A receptors resulted in an enhancement of 62 ± 8% (n = 8) over the current induced by NMDA plus glycine alone. Similarly, with oocytes expressing NR1/NR2B receptors, the NMDA-induced current was enhanced 78 ± 9% (n = 4) in the presence of 100 μM PS. In contrast, oocytes expressing NR1/NR2C receptors exhibited 35 ± 3% (n = 4) inhibition of the NMDA-induced current, and oocytes expressing NR1/NR2D receptors exhibited 26 ± 1% (n = 9) inhibition of the NMDA response . To examine the influence of subunit composition on the concentration dependence of PS action, PS was applied to oocytes expressing NR1/NR2A, NR1/NR2B, NR1/NR2C, or NR1/NR2D, together with 10 μM glycine and a concentration of NMDA approximately equivalent to its EC 50 for that subunit combination. As shown in Fig. 26E and Table 3, PS was about equally potent in potentiating NR1/NR2A EC 50 = 21 μM) and NR1/NR2B receptors (EC SQ 33 μM) , and 3.4- to 5.6-fold less potent as an inhibitor of NR1/NR2C (EC S0 112 μM) and NR1/NR2D receptors (EC 5Q 118 μM) . Enhancement of NR1/NR2A and NR1/NR2B receptors and inhibition of NR1/NR2C and NR1/NR2D receptors exhibited little if any voltage dependence. (Fig. 26F) . Subunits PS E^ PS EC 50 log PS EC 50 n H (#) (% Change) N 1/NR2A + 167 + 21 μM -4.68 ± 1.33 + ( 8 ) 1% 0.09 0.08 NR1/NR2B +202 ± 33 μM -4.49 ± 1.42 ± (4) 10% 0.05 0.11 NR1/NR2C -63 + 112 μM -3.95 ± 1.32 + (4) 9% 0.29 0.29 NR1/NR2D -70 + 118 μM -3.92 ± 1.16 + (4) 6% 0.11 0.08 Table 3. Concentration dependence of PS modulation of the NMDA response. Results from each oocyte (number given in far right column) were independently fitted to the logistic equation. E max is expressed as percentage change (+ for potentiation, - for inhibition) in the presence of PS, relative to the response induced in the same oocyte by an approximate EC 50 concentration of NMDA in the presence of 10 μM glycine. EC 50 values are averaged as logarithms (De Lean, et al , 1978) ± the SEM of the log EC 50 . Concentration of NMDA was 80, 25, 25, and 10 μM for NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D, respectively (see Methods). To determine how PS enhances or inhibits the response of the NMDA receptor, the glutamate, NMDA, and glycine concentration-response curves were determined in the presence and absence of PS. As shown in Figs. 27 and 28, the nature of the modulatory effect of PS depended not only upon subunit combination, but also upon the specific agonist used. With NR1/NR2A receptors, PS enhanced the efficacy of NMDA, glutamate (Fig. 27A) and glycine (Fig. 28A) , while decreasing the glutamate and glycine EC 50 s (Table 4A) . At NR1/NR2B receptors, however, PS enhanced both potency and efficacy of NMDA, but only enhanced the potency of glutamate (Fig. 27B) , while glycine potency was increased with only a slight increase in efficacy (Fig. 28B and Table 4B) . Glu 1.31 + 0.02 75 nM -7.13 ± 0.03 1.27 ± 0.06 (4 Glu (PS) 0.87 ± 60 nM -7.23 + 0.04 1.18 + 0.06 (6 NMDA 1.05 + 0.01 24 μM -4.62 ± 0.02 1.18 + 0.02 (4 NMDA (PS) 0.69 ± 15 μM** -4.81 + 1.33 + 0.03 (3 Gly 1.31 + 0.01 360 nM -6.45 + 0.02 1.18 + 0.05 (4 Gly (PS) 0.87 + 190 nM** -6.72 + 1.11 + 0.12 (5 D. NR1/NR2D Glu 1.10 + 0.03 25 nM -7.61 + 0.06 1.26 + 0.12 (5 Glu (PS) 0.80 ± 37 nM -7.43 ± 0.11 1.35 + 0.15 (5 NMDA 1.02 + 0.01 8.0 μM -5.09 ± 0.01 1.26 + 0.09 (4 NMDA (PS) 0.71 + 6.2 -5.21 + 1.17 + 0.01 (5 Gly 1.22 ± 0.05 100 nM -7.00 ± 0.08 1.12 + 0.08 (5 Glv (PS) 0.74 + 70 nM -7.17 + 0.02 0.95 + 0.01 (3 Table 4. Effect of PS on agonist concentration-response relationships. Peak agonist-induced currents from each oocyte were separately fitted to the logistic equation to estimate EC 50 , E max , and n H . Values reported are mean parameter values + SEM of the number of oocytes given in the far right column. Responses (and therefore E max estimates) are normalized to the magnitude of the peak 200 μM NMDA plus 10 μM glycine- induced current from the same oocyte. ** Indicates a statistically significant (p < 0.05) change compared to the parameter value in the absence of PS. * Indicates a significant effect of PS on the glycine concentration- response curve but it could not be distinguished whether effect was on EC 50 or E max (see Methods). The inhibitory effect of PS on NR1/NR2C and NR1/NR2D was a consequence of a decrease in the efficacies of glutamate, NMDA (Fig. 27C-D) , and glycine (Figure 28C-D) . Agonist potency was not decreased, and in fact appeared to be somewhat increased, with the result that percentage inhibition by PS tended to be greater at high than at low agonist concentrations (Table 4C and 4D) . Thus, PS did not compete for either the glutamate or glycine recognition site. Inhibitory potency of 3α5/3S depends upon the NR2 subunit As shown in Fig. 29A-D, 100 μM 3o;5/3S reversibly inhibited NMDA-induced currents of Xenopus oocytes expressing NR1/NR2A (Fig. 28A) , NR1/NR2B (Fig. 28B) , NR1/NR2C (Fig. 28C) , or MR1/NR2D (Fig. 28D) receptors. However, the extent of inhibition was significantly greater with NR1/NR2C and NR1/NR2D receptors than with NR1/NR2A and NR1/NR2B receptors. Concentration-response analysis (Fig. 28E, Table 5) indicated that this difference was primarily due to an approximately 4 -fold lower potency of 3α5/3S at NR1/NR2A and NR1/NR2B receptors than at NR1/NR2C and NR1/NR2D receptors (see Table 5 for EC 5 o) • Inhibition of the NMDA induced current by 3α5/3S exhibited little if any voltage dependence from -100 to +20 mV (Fig. 28F) . Subunits 3 5βS E max 3α5/3S EC 50 log 3α5/3S n H (#) (% Change) EC 50 NR1/NR2A -81 + 62 μM -4.20 ± 0.06 1.39 ± (3) 5% 0.07 NR1/NR2B -82 + 38 μM -4.42 ± 0.11 1.06 ± (3) 1 % 0.04 NR1/NR2C -99 ± 12 μM -4.93 ± 0.03 1.34 + (6) 2% 0.04 NR1/NR2D -98 + 14 μM -4.85 ± 0.03 1.14 + (4) 7% 0.16 Table 5. Concentration dependence of 3α5/3S modulation of the NMDA plus glycine response. Results from each oocyte (number given in far right column) were independently fitted to the logistic equation. E max is expressed as percentage change (inhibition) relative to the response induced in the same oocyte by an approximate EC 50 concentration of NMDA in the presence of 10 μM glycine. Concentration of NMDA was 80, 25, 25, and 10 μM for NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D, respectively (see Methods). To determine how 3α5/3S inhibits the glutamate response, concentration-response curves were constructed for glutamate (in the presence of 10 μM glycine) and glycine (in the presence 10 μM glutamate) in the presence and absence of 100 μM 3α5/3S. As shown in Fig. 29, 3α5/3S decreased the efficacy with which glutamate and glycine activate NR1/NR2A (Fig. 29G-H) , NR1/NR2B (Fig. 29I-J) , NR1/NR2C (Fig. 29K-L) , and NRl/NR2D (Fig. 29M-N) receptors . Methods of the Invention Preparation of RNA. Plasmids containing the NR1 100 (NR1G) and NR2A cDNA inserts were kindly provided by Dr. Nakanishi (Kyoto University Faculty of Medicine, Kyoto, Japan) . Plasmids containing the NR2B, NR2C and NR2D cDNA inserts were kindly provided by Dr. P. Seeburg (Heidelberg University, Heidelberg, Germany) . Plasmids were linearized with appropriate restriction enzyme prior to in vi tro transcription using the Message Machine kit (Ambion, Inc., Austin, TX) . NMDA receptor expression in Xenopus oocytes. Female, oocyte-positive Xenopus laevis frogs were purchased from Xenopus I (Dexter, MI). Following 45 min of 0.15% Tricaine anesthesia, ovarian sections containing the follicular oocytes were removed from the frog through a lateral abdominal incision and were immediately placed in a calcium-free solution (NaCl 96 mM, MgCl 2 1 mM, KCl 2 mM, Hepes 50 mM, pyruvate 2.5 mM, 0.1 mg/ml gentamycin, pH 7.4) . Following 1.5 - 2 hours incubation in 0.2% collagenase (type II, Sigma Chemical Co., St. Louis, MO) at room temperature, individual defolliculated Dumont stage V and VI oocytes were transferred to an incubator and maintained overnight in Barth's solution (NaCl 84 mM, NaHC0 3 2.4 mM, MgS0 4 0.82 mM, KCl 1 mM, Ca(N0 3 ) 2 0.33 mM, CaCl 2 0.41 mM, Tris/HCl 7.5 mM, pyruvate 2.5 mM, 0.1 mg/ml gentamycin, pH 7.4) at 18-20°C. Oocytes were injected with 50 nL of RNA solutions using an electronic microinjector (Drummond Inc., Broomall, PA). The transcripts were injected at a ratio of 0.125/1.25 ng mRNA per oocyte for NR1/NR2A receptors and 0.5/5 ng mRNA per oocyte for NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors. The injected oocytes were used for experiments following 1-5 days of incubation in Barth's solution at 18-20°C. Electrophysiolog . Measurements of ion currents from oocytes expressing NMDA receptors were performed using an Axoclamp-2A voltage clamp amplifier (Axon Instruments, Inc., Foster City, CA) in two-electrode voltage clamp mode. The microelectrodes were fabricated from borosilicate glass capillaries with a programmed puller (Sutter Instrument Co., CA) . Microelectrode resistance was 1-3 MΩ_when filled with 3 M KCl. The oocyte recording chamber was continuously perfused with Mg 2+ -free Ba-Ringer solution (NaCl 96 mM, KCl 2 mM, BaCl 2 1.8 mM, Hepes 5 mM) . Ba-Ringer was used to prevent NMDA receptor currents from being complicated by activation of Ca 2+ dependent CI " channels (Leonard and Kelso, Neuron . 4 : 53- 60 (1990) ) . Potentiation of the NMDA-induced current of NR1 100 /NR2A receptors by PS in Ba-Ringer tended to be less than previously observed with using Ca 2+ -containing solution (Yaghoubi et al . , Brain Res . 803 : 153-160 (1998)), possibly reflecting a nonlinear contribution of Ca 2+ dependent CI " channels to the NMDA induced current. Except where otherwise stated, oocytes were clamped at a holding potential of -70 mV during data acquisition. The membrane current was filtered at 500 Hz and sampled at 100 Hz. Drugs were applied using a gravity-driven external perfusion system. The working volume of the recording chamber was 30 μl and the rate of the perfusion was 50 μl/sec. The drug application lasted 10 sec and was followed by 60 s wash. Data acquisition and external perfusion were controlled using custom-written software implemented in the SuperScope II development environment (GW Instruments, MA) . All experiments were performed at room temperature of 22-24°C. Peak or steady-state current measurements were normalized and expressed as a fraction of the peak or steady-state control current measurements, which were performed before and after application of every single concentration of agonists or steroids . Data Analysis. Concentration-response data were analyzed by nonlinear regression using the logistic equation Response = E maχ / (1+ , where c is concentration, ■E ^ is the maximum response, n u is the Hill coefficient. The data are presented as mean ± SEM. EC S0 values are averaged as logarithms (De Lean et al . , Am. J " . Physiol . 235 : E97-E102 (1978)) + the SEM of the log EC 50 . Hence, the reported mean EC^ Q values are geometric means. PS- induced changes in E πaχ and EC 50 for NMDA, glutamate, and glycine were tested for statistical significance by two methods: 1) Concentration-response data from each oocyte were fitted to the logistic equation, and parameter estimates for E maχ and EC S0 in the presence and absence of PS were compared by unpaired, 2 -tailed t-test. 2) The normalized (to the 200 μM NMDA response) responses at each concentration from multiple oocytes were averaged to construct pooled concentration-response curves (points in Fig. 27) in the presence and absence of PS. The pooled data were simultaneously fitted to the logistic equation, either with E m ,„ and EC, n constrained to be identical in the presence and absence of PS, or allowing one or both to vary independently. The improvement in the sum-of- squares due to the introduction of additional free parameters was tested for significance by the "extra sum- of-squares" method (Munson and Rodbard, Anal. Biochem . 107 : 220-239 (1980)). In one case, allowing either E maχ or EC 5Q to vary independently produced a significant improvement in the fit, but allowing both parameters to vary did not produce a significant further improvement. This was regarded as indicating a significant treatment effect of PS, but the change in EC 2,ϋ n and Emax was not considered significant. The PS-induced change in EC 5Q or E maχ was considered significant only if it met the p < 0.05 criterion by both tests. No attempt was made to test the generally small changes in n H for statistical significance . Chemicals . Steroids were obtained from Steraloids, Inc. (Wilton, NH) . Other compounds were obtained from Sigma (St. Louis, MO) . Steroid stocks were prepared in DMSO and diluted into recording medium (final DMSO concentration 0.5%) . Other solutions also contained 0.5% DMSO. Example 7 Differential effects of PS on NMDA receptors Pregnenolone Sulfate (PS) , an endogenous neurosteroid, differentially modulates different subtypes of the NMDA receptor. PS potentiates heteromeric NR1/NR2A and NR1/NR2B, while inhibits NR1/NR2C and NR1/NR2D containing receptors. Following 1 - 5 days after mRNA injection, a 10 sec application of a specific agonist of the NMDA receptor, 80 μM NMDA, plus the co- agonist, 10 μM glycine, gave rise to inward currents in voltage-clamped oocytes that expressed NMDA receptors consisting of NR1/NR2A receptor subunits. Co-application of 100 μM PS resulted in 59 ± 4% (n=33) potentiation of the NMDA responsive membrane current. The observed effect of PS was reversible, as washout of PS for 60 sec led to complete recovery of the initial response. Similar to the effect on the NR1/NR2A receptor, co- application of 100 μM PS to oocytes expressing NR1/NR2B receptors resulted in reversible potentiation (95 ± 6%, (n=20) ) of the membrane current induced by a 10 sec application of 25 μM NMDA and 10 μM glycine. However, in contrast to the potentiating effect of PS on NR1/NR2A and NR1/NR2B receptors, co-application of 100 μM PS to voltage-clamped oocytes injected with NR1/NR2C or NR1/NR2D mRNAs resulted in reversible inhibition (32 ± 3%, n=ll and 29 ± 2%, n=16 respectively) of NMDA-induced membrane currents after a 10 sec application of 10 μM glycine and 25 μM NMDA for NR1/NR2C or 10 μM glycine and 10 μM NMDA for NR1/NR2D receptors. Different concentrations of PS in the range from 0.5 to 200 μM were co-applied with 10 μM glycine and 10, 25 or 80 μM NMDA to oocytes injected with NR1/NR2D, NR1/NR2B (or NR1/NR2C) or NR1/NR2A respectively to study the relationship of PS concentration to potentiation of the NMDA response. The concentration of NMDA chosen to activate the receptor was close to the ECcg for NMDA obtained in the absence of steroids. The PS effect on the NMDA receptors was concentration dependent (Figure 30) with maximum potentiation of 67±7% (n=8) and 119+9% (n=8) for NR1/NR2A and NR1/NR2B respectively and maximum inhibition of 63+9% (n=4) and 70±6% (n=4) for NR1/NR2C and NR1/NR2D respectively. Identification of a Region on the NR2 Subunit Conferring Modulation by PS To identify domains on the NMDA receptor responsible for the differential modulatory effects of PS, four chimeric NR2 subunits were constructed (Fig. 31) . To produce the first subunit, 96% of the cytoplasmic COOH- terminal region of the NR2B subunit (residues 870 to 1482) were replaced with the corresponding region of the NR2D subunit (residues 892 to 1323) . Like the wild-type, NRla/NR2B (NRla is also referred to as NR1 011 ) PS potentiated the NMDA-induced response of receptors containing NRla/NR2 Chimera (I) subunits. IOOUM PS increased NRla/NR2 Chimera (I) responses by 52.69 ± 3.58% in the presence of saturating concentrations of NMDA and Glycine, 300uM and 50uM, respectively (Fig. 32) . In contrast, when residues 524 to 1482 of the NR2B were replaced with those of NR2D, PS inhibited rather than potentiated the NMDA response. IOOUM PS inhibited the NRla/NR2 Chimera (II) by 43.04 ± 5.44%, similar to the wild-type NRla/NR2D (45.18 ± 2.62%) (Fig. 32). These results suggest that the region between residue 524 and 870 on NR2B and residues 548 to 892 on NR2D are sufficient to endow the modulatory effects of PS on the NMDA response that is characteristic to their specific subunits. To confirm this finding, a third chimera was constructed, NR2Chimera (III) , in which the identified region on NR2B was replaced by the one on NR2D. Consistent with the hypothesis, PS inhibited the NMDA- induced currents from the expressed NRla/NR2 Chimera (III) receptors (100 uM PS, 36.74 ± 6.54%) (Fig. 32) . Therefore, the region, reisdues 548 to 892, on the NR2D receptor is necessary and sufficient for the PS inhibitory effect. In the fourth chimera receptor, NR2B-DChimera (IV), the residues 727 to 892 were replaced on NR2D with residues 703 to 870 of NR2B, which contains the second extracellular domain, the fourth hydrophobic transmembrane domain and 31 residues from the C-terminal intracellular tail. 100 μM PS potentiated the NR101l/NR2B-DChimera(IV) by 66.05±2.75%. This demonstrates that the region of residues 703 to 870 on NR2B is necessary and sufficient for the PS potentiating effect . The above results indicate that the determinant of the modulatory effect of Pregnenolone Sulfate resides in the region between residues 703 and 870 on the NR2B subunit and residues 548 and 892 on the NR2D subunit. These findings lay the foundation for further identification of the important amino acid residues underlying the PS modulatory effect and definition of a novel modulatory site on the NR2 subunits of the NMDA receptor. Identification of these sites will lead to the development of pharmacological reagents to regulate NMDA receptor function in neurological and psychiatric diseases . Methods of the Invention Mutagenesis . The chimeric receptors were generated by first introducing a series of silent restriction sites in both the NR2B and NR2D cDNA. The chimeric cDNAs were then constructed by replacing corresponding regions of one cDNA with that of the other. The silence site mutations were generated by a method involving PCR. In brief, oligonucleotides were synthesized containing the mutation, and these oligonucleotides were used in combination with other oligonucleotides in PCR amplifications of fragments of the cDNA. The products of the PCR reactions were cut with two different restriction enzymes to generate A cassette containing the mutation. This cassette was then ligated into the cDNA cut with the same two restriction enzymes. For all of the mutations, single isolates were selected, and the entire region of the amplified cassette was sequenced to check for the mutation and insure against second-site mutations. Preparation of RNA. Same as in Example 5. Expression in Xenopus oocytes. Same as in Example 5. Electrophysiology . Same as in Example 5. Chemicals. Same as in Example 5.

Claims: CLAIMS 1. A method for identifying a subunit specific modulator of the N-methyl-D-aspartate (NMDA) receptor, comprising: a) providing a plurality of NMDA receptors which differ in their subunit identity; b) contacting the NMDA receptors of step a) with a neurotransmitter recognition site ligand in the presence and absence of a candidate modulator; and c) assaying for receptor activity following step b) , wherein an increase or decrease in activity in at least one, but not all members of the plurality of NMDA receptors, in the presence but not the absence of a candidate modulator, is an indication that the candidate modulator is a subunit specific modulator. 2. The method of Claim 1 further comprising comparing the subunit identity of the subset of the NMDA receptors to determine the subunit specificity of the candidate modulator. 3. The method of Claim 1 wherein the plurality of NMDA receptors have identical NR2 subunits, and differ in their NRl subunits. 4. The method of Claim 3 wherein the identical NR2 subunits are selected from the group consisting of NR2A, NR2B, NR2C, and NR2D. 5. The method of Claim 3 wherein at least one of the NRl subunits is a natural isoform selected from the group consisting of NR1 000 , NR1 Q01 , NR1 010 , NR1 Q11 , NR1 100 , NR1 101 , NR1 110 , and NRl ι . 6. The method of Claim 3 wherein at least one of the NRl subunits contain an a exon encoded protein domain. 7. The method of Claim 3 wherein at least one of the NRl subunits is a chimeric isoform. 8. The method of Claim 3 wherein at least one of the NRl subunits is an isoform point mutant. 9. The method of Claim 8 wherein the point mutant contains at least one point mutation at a residue which corresponds to residue 182, 193, 202, 233, or 252 of NR1 011 . 10. The method of Claim 9 wherein the isoform point mutant is a penta-mutant with the amino acid substitution mutations which correspond to mutations R182A, K193A, K202A, R233A, and R252A of NR1 Q11 . 11. The method of Claim 8 wherein the isoform point mutant contains an a exon encoded protein domain and has point mutations within that domain. 12. The method of Claim 1 wherein the plurality of NMDA receptors have identical NRl subunits, and differ in their NR2 subunits. 13. The method of Claim 12 wherein the identical NRl subunits are an isoform selected from the group consisting of NR1 Q00 , NR1 Q01 , NR1 Q10 , NR1 Q11 , NR1 10Q , NR1 101 , NR1 110 , and NRl nι . 14. The method of Claim 12 wherein the identical NRl subunits contain an cϋ-exon encoded protein domain. 15. The method of Claim 12 wherein the identical NRl subunits are a chimeric isoform. 16. The method of Claim 12 wherein the identical NRl subunits are an isoform point mutant which contains an exon encoded protein domain and has point mutations within that domain. 17. The method of Claim 16 wherein the identical NRl subunits contain an α exon encoded protein domain. 18. The method of Claim 17 wherein the identical NRl subunits are point mutants which contain at least one point mutation at a residue which corresponds to residue 182, 193, 202, 233, or 252 of NR1 011 . 19. The method of Claim 18 wherein the identical NRl subunits are a penta-mutant with the amino acid substitution mutations which correspond to mutations R182A, K193A, K202A, R233A, and R252A of NR1 Q11 . 20. The method of Claim 12 wherein at least one of the the NR2 subunits is an isoform selected from the group consisting of NR2A, NR2B, NR2C, and NR2D. 21. The method of Claim 12 wherein at least one of the NR2 subunits is a chimeric isoform. 22. The method of Claim 21 wherein the chimeric isoform contains a. a. 534-870 of NR2B. 23. The method of Claim 22 wherein the chimeric isoform contains amino acid 548-892 of NR2D. 24. The method of Claim 22 wherein the chimeric isoform contains, amino acid 703-870 of NR2B. 25. The method of Claim 12 wherein at least one of the NR2 subunits is an isoform point mutant. 26. The method of Claim 1 wherein assaying step c) is with an oocyte expression system. 27. The method of Claim 1 wherein the neurotrasmitter recongition site ligand is an agonist. 28. The method of Claim 27 wherein the agonist is selected from the group consisting of NMDA, glutamate, and glycine. 29. The method of Claim 1 wherein the neurotrasmitter recongition site ligand is an antagonist. 30. The method of Claim 1 wherein the candidate modulator is a steroid based molecule. 31. The method of Claim 1 wherein the candidate modulator is a non-steroid based molecule. 32. The method of Claim 1 wherein the candidate modulator is obtained from a library of small molecules . 33. The method of Claim 1 wherein the candidate modulator is a known neuromodulator. 34. A method for inhibiting N-methyl-D-aspartate glutamate receptor mediated ion-channel activity in an individual in need thereof comprising administering an effective amount of a compound represented by the following structural formula: wherein : ring A has 0-3 double bonds; R 1 is -OH, =0, or a negatively charged group; R 2 is -H, -CH 3 , or is absent when ring A has three double bonds; R 3 is -H, OH, =0, or -OR' ; R 1 is an aliphatic or aromatic group; and R A is -OH, =0 or -COCH j . 35. The method of Claim 34 wherein R 1 is either hemioxylate, hemisuccinate, or hemiglutarate. 36. The method of Claim 35 wherein the compound is selected from the group consisting of pregnanolone hemioxylate (3ot5/3HO) , pregnanolone hemisuccinate (3QI5/3HS) , and pregnanolone hemiglutarate (3α5/3HG) . 37. The method of Claim 36 wherein the effective amount is a concentration of from about 1 to about 500 μM. 38. The method of Claim 37 wherein the effective amount is from about 50 to about 250 μM. 39. A method for inhibiting the toxic effects associated with activation of the N-methyl-D-aspartate receptor in neurons in an individual in need thereof, comprising administering an effective amount of a compound represented by the following structural formula : wherein : ring A has 0-3 double bonds; R 1 is -OH, =0, or a negatively charged group; R 2 is -H, -CH j , or is absent when ring A has three double bonds; R 3 is -H, OH, =0, or -OR' ; R 1 is an aliphatic or aromatic group; and R 4 is -OH, =0 or -C0CH 3 . 40. The method of Claim 39 wherein R 1 is either hemioxylate, hemisuccinate, or hemiglutarate. 41. The method of Claim 40 wherein the compound is selected from the group consisting of pregnanolone hemioxylate (3α5/3HO) , pregnanolone hemisuccinate (3Q;5J8HS) , and pregnanolone hemiglutarate (3a5βHG) . 42. The method of Claim 39 wherein the effective amount is a concentration of from about 1 to about 500 μM. 43. The method of Claim 42 wherein the effective amount is from about 50 to about 250 μM. 44. The method of Claim 39 wherein the neurons are selected from the group consisting of hippocampal cells and spinal cord cells. 45. A method for reducing neuronal cell death resulting from L-glutamate activation of the N-methyl-D- aspartate receptor in an individual in need thereof, comprising administering an effective amount of a compound represented by the following structural formula: wherein : ring A has 0-3 double bonds; R 1 is -OH, =0, or a negatively charged group; R 2 is -H, -CH 3 , or is absent when ring A has three double bonds; R 3 is -H, OH, =0, or -OR' ; R 1 is an aliphatic or aromatic group; and R 4 is -OH, =0 or -C0CH 3 . 46. The method of Claim 45 wherein R 1 is either hemioxylate, hemisuccinate, or hemiglutarate. 47. The method of Claim 46 wherein the compound is selected from the group consisting of pregnanolone hemioxylate ( 3 x5βKO) , pregnanolone hemisuccinate (3α5/?HS) , and pregnanolone hemiglutarate (3o;5/3HG) . 48. The method of Claim 45 wherein the effective amount is a concentration of from about 1 to about 500 μM. 49. The method of Claim 48 wherein the effective amount is from about 50 to about 250 μM. 50. A method for treating a disease selected from the group consisting of neuropathic pain, drug withdrawal/dependency, epilepsy, glaucoma, chronic neurodegenerative diseases, amyotrophic lateral sclerosis, anxiety disorders, brain cell death, ischaemia, stroke, and trauma in an individual when said disease results from agonist induced NMDA receptor activation comprising administering to the individual an effective amount of a compound represented by the following structural formula: wherein : ring A has 0-3 double bonds; R 1 is -OH, =0, or a negatively charged group; R 2 is -H, -CH 3 , or is absent when ring A has three double bonds; R 3 is -H, OH, =0, or -OR' ; R is an aliphatic or aromatic group; and R is -OH, =0 or -C0CH 3 . 51. The method of Claim 50 wherein R 1 is either hemioxylate, hemisuccinate, or hemiglutarate. 52. The method of Claim 51 wherein the compound is selected from the group consisting of pregnanolone hemioxylate (3α5/3HO) , pregnanolone hemisuccinate (3a5βHS) , and pregnanolone hemiglutarate (3o;5/3HG) . 53. The method of Claim 50 wherein the effective amount is a concentration of from about 1 to about 500 μM. 54. The method of Claim 53 wherein the effective amount is from about 50 to about 250 μM. 55. A method for inhibiting the excitatory L-glutamate- mediated synaptic activity in an individual in need thereof, comprising administering to the individual a compound represented by the following structural formula : wherein : ring A has 0-3 double bonds; R is -OH, =0, or a negatively charged group; R 2 is -H, -CH 3 , or is absent when ring A has three double bonds; R 3 is -H, OH, =0, or -OR' ; R 1 is an aliphatic or aromatic group; and R 4 is -OH, =0 or -C0CH 3 . 56. The method of Claim 55 wherein R 1 is either hemioxylate, hemisuccinate, or hemiglutarate. 57. The method of Claim 56 wherein the compound is selected from the group consisting of pregnanolone hemioxylate (3o;5/3HO) , pregnanolone hemisuccinate (3α5/3HS) , and pregnanolone hemiglutarate (3o;5?HG) .

Inventors: FARB DAVID H, GIBBS TERRELL, JANG MING-KUEI, RUSSEK SHELLEY, FARB, DAVID, H., GIBBS, TERRELL, JANG, MING-KUEI, RUSSEK, SHELLEY
Applicants: UNIV BOSTON, TRUSTEES OF BOSTON UNIVERSITY
Application year: 2000
Main classification: G01N33
IPC codes: C07J1, C07J3, C07K14, G01N33