Selective enhancement of the interneuron network and gamma-band power via GluN2C/GluN2D NMDA receptor potentiation

Abstract

N-Methyl-d-aspartate receptors (NMDARs) are a family of ligand-gated ionotropic glutamate receptors that mediate a slow, calcium-permeable component to excitatory neurotransmission. The GluN2D subunit is enriched in GABAergic inhibitory interneurons in cortical tissue. Diminished levels of GABAergic inhibition contribute to multiple neuropsychiatric conditions, suggesting that enhancing inhibition might have therapeutic utility, thus making GluN2D modulation an attractive drug target. Here, we describe the actions of a GluN2C/GluN2D-selective positive allosteric modulator, (+)-EU1180-453, which has improved drug-like properties, such as increased aqueous solubility, in comparison to the first-in-class GluN2C/GluN2D-selective prototypical positive allosteric modulator, (+)-CIQ. (+)-EU1180-453 doubles the NMDAR response at lower concentrations and produces a greater degree of maximal potentiation at 30 µM compared with (+)-CIQ. Using in vitro electrophysiological recordings, we show that (+)-EU1180-453 potentiates triheteromeric NMDARs containing at least one GluN2C or GluN2D subunit and is active at both exon5-lacking and exon5-containing GluN1 splice variants. (+)-EU1180-453 increases glutamate efficacy for GluN2C/GluN2D-containing NMDARs both by prolonging the deactivation time and by potentiating the peak response amplitude. We show that (+)-EU1180-453 selectively increases synaptic NMDAR-mediated charge transfer onto postnatal day 11-15 CA1 stratum radiatum hippocampal interneurons but is without effect on CA1 pyramidal cells. This increased charge transfer enhances inhibitory output from GABAergic interneurons onto CA1 pyramidal cells in a GluN2D-dependent manner. (+)-EU1180-453 also shifts excitatory-to-inhibitory coupling towards increased inhibition and produces enhanced gamma-band power from carbachol-induced field potential oscillations in hippocampal slices. Thus, (+)-EU1180-453 can enhance overall circuit inhibition, which could prove therapeutically useful for the treatment of anxiety, depression, schizophrenia and other neuropsychiatric disorders. KEY POINTS: (+)EU-1180-453 is a GluN2C/GluN2D positive allosteric modulator and is active at triheteromeric receptors. (+)EU-1180-453 is active at exon5-containing and exon5-lacking GluN1-containing receptors. (+)EU-1180-453 selectively potentiates the interneuron network and can enhance carbachol-induced gamma-band power.

Keywords: N‐methyl‐d‐aspartate receptor; allosteric modulator; drug discovery; excitation–inhibition balance; gamma oscillation; hippocampus; interneuron.

Figure 1.. (+)-EU1180–453 potentiates response of GluN2C- and GluN2D-containing NMDARs expressed in Xenopus oocytes

A and B, representative trace (A) and plotted data (B) for glutamate concentration–response experiments on GluN2C-containing NMDARs in the presence of glycine (30 μM; black) and glycine plus 30 μM (+)-EU1180–453 (purple). C and D, representative trace (C) and plotted data (D) for glutamate concentration–response experiments on GluN2D-containing NMDARs in presence of glycine (30 μM; black) and glycine plus 30 μM (+)-EU1180–453 (red). E–H, representative current responses of GluN2A_C1/GluN2B_C2 (E), GluN2A_C1/GluN2C_C2 (F), GluN2A_C1/GluN2D_C2 (G) and GluN2B_C1/GluN2D_C2 (H) triheteromeric NMDARs to varying concentrations of (+)-EU1180–453 in the presence of saturating concentrations of glutamate (100 μM) and glycine (30 μM). I–L, fitted concentration–response curves of GluN2A_C1/GluN2B_C2 (I), GluN2A_C1/GluN2C_C2 (J), GluN2A_C1/GluN2D_C2 (K) and GluN2BC1/GluN2DC2 (L) triheteromeric NMDARs to varying concentrations of (+)-EU1180–453 in the presence of 30 μM glycine and 100 μM glutamate. Diheteromeric NMDARs with corresponding C1- and C2-tails were included as reference. All data are the mean values ± SD. The dotted line in I–L is 100% response [response without (+)-EU1180–453]. n/n represents the number of oocytes/number of frogs.

Figure 2.. (+)-EU1180–453 strongly potentiates GluN2C- and GluN2D-containing NMDAR current responses recorded from HEK cells

Cells were held at −70 mV and subjected to rapid and brief (10 ms) application of saturating glutamate (1 mM) in the presence of 100 μM glycine. Current responses were recorded in the absence, presence and following removal of (+)-EU1180–453. A–D, representative responses are shown for baseline (glutamate only; black trace), glutamate plus 10 μM (+)-EU1180–453 (coloured trace) and glutamate recovery (grey trace) for GluN1/GluN2A (A), GluN1/GluN2B (B), GluN1/GluN2C (C) and GluN1/GluN2D (D). E, the mean fold-change in charge transfer (response/baseline) is shown. The dotted line is at fold change of one. All data are the mean values ± SD. Statistical significance was determined by Student’s paired t-test of control vs. drug. For full details, see Table 2.

Figure 3.. (+)-EU1180–453 potentiates response of exon5-containing (GluN1–1b) GluN2C and GluN2D NMDARs

A and C, representative current responses from Xenopus oocytes of GluN1–1b/GluN2C (A) and GluN1–1b/GluN2D (C) NMDARs to varying concentrations of (+)-EU1180–453 in the presence of saturating concentrations of glutamate (100 μM) and glycine (30 μM). B and D, fitted concentration–response curves of GluN1–1b/GluN2C (B) and GluN1–1b/GluN2D (D) NMDARs to varying concentrations of (+)-EU1180–453 in presence of 30 μM glycine and 100 μM glutamate. GluN1–1a/GluN2C and GluN1–1a/GluN2D responses are included for reference. E and F, representative responses from HEK293T cells are shown for baseline (glutamate and glycine only; black trace), glutamate and glycine plus 10 μM (+)-EU1180–453 (coloured trace), and glutamate and glycine recovery (grey trace) for GluN1–1b/GluN2C (E) and GluN1–1b/GluN2D (F). Cells were held at −70 mV and subjected to rapid and brief (10 ms) application of saturating glutamate (100 μM) in the constant presence of 30 μM glycine. Current responses were recorded in the absence, presence and following removal of (+)-EU1180–453. G, the mean fold-change in charge transfer (response/baseline) is shown. The dotted line is at fold-change of one. Statistical significance was determined by Student’s paired t-test of control vs. drug. For full details, see Table 2. All data are the mean values ± SD. The dotted line in B and D is 100% response [response without (+)-EU1180–453]. n/n represents the number of oocytes/number of frogs.

Figure 4.. Synaptic NMDAR-mediated EPSCs in CA1 stratum radiatum GABAergic interneurons, but not CA1 pyramidal cells, are potentiated by (+)-EU1180–453 in developing hippocampus

A, and E, diagram of recording arrangement from mouse hippocampal slices. B, representative EPSCs show the effects of 10 μM (+)-EU1180–453 (blue) or vehicle (grey) compared with baseline response (black) at CA1 pyramidal cells. C, average charge transfer vs. time is shown for 10 μM (+)-EU1180–453 (blue) or vehicle (grey). Values are expressed as a percentage of the average of 10 baseline responses. D, application of 10 μM (+)-EU1180–453 (n = 6 cells; blue) or vehicle (n = 6 cells, 0.1% DMSO; grey) does not alter the charge transfer of NMDAR-mediated EPSCs onto CA1 pyramidal cells. F, representative EPSCs show the effects of 10 μM (+)-EU1180–453 (magenta) or vehicle (grey) compared with baseline response (black) at interneurons. G, average charge transfer vs. time is plotted for 10 μM (+)-EU1180–453 (magenta) or vehicle (grey). H, application of 10 μM (+)-EU1180–453 (n = 14 cells; magenta) potentiates the charge transfer of NMDAR-mediated EPSCs onto interneurons [−6.2 ± 4.7 nA ms for baseline vs. −10 ± 7.4 nA ms for (+)-EU1180–453]. Application of vehicle (n = 5 cells, 0.1% DMSO; grey) does not alter the charge transfer. I and J, peak amplitude (I) and weighted τ (J) of NMDAR-mediated EPSCs onto CA1 pyramidal cells were all unchanged following application of 10 μM (+)-EU1180–453 (n = 6 cells; blue) or vehicle (0.1% DMSO, n = 6 cells; grey). Individual components of the weighted τ are as follows, with baseline shown first and (+)-EU1180–453 shown second, (in milliseconds): pyramidal cells (Tau_FAST, 46 vs. 55; Tau_SLOW, 277 vs. 338) and interneurons (Tau_FAST, 64 vs. 53; TauSLOW, 224 vs. 459). K, peak amplitudes of NMDAR-mediated EPSCs onto CA1 interneurons were increased following application 10 μM (+)-EU1180–453 but not with vehicle [−69 ± 42 pA for baseline vs. −93 ± 58 pA for (+)-EU1180–453]. L, weighted τ of NMDAR-mediated EPSCs onto CA1 interneurons was prolonged following application 10 μM (+)-EU1180–453 but not with vehicle [94 ± 55 ms for baseline vs. −120 ± 67ms for (+)-EU1180–453]. All data are mean values ± SD. Student’s paired t-test was used to determine statistical significance. Grey boxes in C and G represent the last 3 min of the baseline and response periods that were used for all analyses. Abbreviations: CA1, cornu Ammonis 1; CA3, cornu Ammonis 3; DG, dentate gyrus; s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum. n/n represents the number of neurons/number of mice.

Figure 5.. Application of (+)-EU1180–453 increases inhibitory tone in developing CA1

Spontaneous IPSCs (sIPSCs) were recorded at +10 mV from mouse CA1 pyramidal cells (postnatal days 11–15). A and B, representative sIPSCs before (black; A₁) and after 10 μM (+)-EU1180–453 (magenta; A₂), and before (black; B₁) and after vehicle (0.1% DMSO, grey; B₂). C and E, average sIPSC frequency is significantly increased in (+)-EU1180–453 [7.7 ± 0.9 Hz for baseline vs. 10.2 ± 1.1 Hz for (+)-EU1180–453; C], whereas there is no change in sIPSC frequency in vehicle (E). D and F, cumulative probability of interevent interval is significantly shifted to the left in (+)-EU1180–453 (Kolmogorov–Smirnov test; D), whereas there is no effect of vehicle (Kolmogorov–Smirnov test; F). G, Plots of average percentage change in frequency from baseline for (+)-EU1180–453 and vehicle. H, representative sIPSCs before (black; H₁) and after 10 μM (+)-EU1180–453 (maroon; H₂) onto CA1 pyramidal cells in Grin2d^−/− mice. I, average sIPSC frequency is significantly decreased in (+)-EU1180–453 [2.1 ± 0.8 Hz for baseline vs. 1.7 ± 0.7 Hz for (+)-EU1180–453] in Grin2d^−/− mice. J, cumulative probability of inter-event interval is significantly shifted to the right in (+)-EU1180–453 in Grin2d^−/− mice (Kolmogorov–Smirnov test). All data are the mean values ± SD. Student’s paired t-test was used to determine statistical significance unless otherwise stated. n/n represents the number of neurons/number of mice.

Figure 6.. (+)-EU-1180–453 has minimal effects on spontaneous inhibitory postsynaptic current amplitude and time course

A, average spontaneous inhibitory postsynaptic current (sIPSC) amplitude is unchanged following 10 μM (+)-EU1180–453. B, cumulative probability of event amplitude is significantly shifted to the right in (+)-EU1180–453 (Kolmogorov–Smirnov test). C and D, average sIPSC amplitude (C) and cumulative probability of event amplitude (D) are both unchanged in response to vehicle (Kolmogorov–Smirnov test). E and F, there is no change in the average event amplitude in Grin2d^−/− mice (E); however, the cumulative probability of event amplitude (F) is significantly shifted to the left (Kolmogorov–Smirnov test). G and I, normalized responses of spontaneous inhibitory postsynaptic currents (sIPSCs) before (black) and after (+)-EU1180–453 (magenta) (G) and before (black) and after vehicle (grey) (I). H and J, average decay time of sIPSCs is unchanged following either (+)-EU1180–453 (H) or vehicle (J). All data are the mean values ± SD. Student’s paired t-test was used to determine statistical significance unless otherwise stated. n/n represents the number of neurons/number of mice.

Figure 7.. (+)-EU1180–453 shifts excitatory-to-inhibitory ratio towards inhibition

A, dual postsynaptic potential where the peak depolarization is used to represent the excitatory postsynaptic (‘EPSP’) and the peak hyperpolarization is used to represent the inhibitory postsynaptic potential (‘IPSP’) before (black) and after 10 μM (+)-EU1180–453 (magenta) or 0.1% DMSO (grey). B, average ‘EPSP/IPSP’ ratio before and after wash-in of (+)-EU1180–453 or vehicle. Dotted line represents no change from baseline. C, ‘EPSP/IPSP’ ratio is significantly reduced after (+)-EU1180–453 application [2.0 ± 0.9 baseline vs. 0.9 ± 0.5 (+)-EU1180–453] but unchanged with vehicle. D and E, (+)-EU1180–453 significantly decreases ‘EPSP’ amplitude [5.5 ± 3.3 mV baseline vs. 4.0 ± 3.3 mV (+)-EU1180–453] (D) and significantly increases ‘IPSP’ amplitude [−2.8 ± 1.2 mV baseline vs. −4.1 ± 1.7 mV (+)-EU1180–453] (E), while ‘EPSP’ and ‘IPSP’ amplitude in vehicle are unchanged. All data are mean values ± SD. Student’s paired t-test was used to determine statistical significance unless otherwise stated. n/n represents the number of neurons or slices/number of mice.

Figure 8.. (+)-EU1180–453 enhances carbachol-induced gamma power in CA1 of hippocampus

A–C, field potential recordings from CA1 at baseline (A), after 20 μM carbachol (B) and after carbachol plus (+)-EU1180–453 (C). D, average power spectral density plot at baseline and in the presence of carbachol and carbachol plus (+)-EU1180–453. E, percentage change in power compared with baseline for carbachol and carbachol (+)-EU1180–453. There are significant interactions at both beta and gamma power bands (two-way ANOVA). F, there are significant increases in fold gamma-band power after carbachol, between carbachol and carbachol plus (+)-EU1180–453, and baseline and carbachol plus (+)-EU1180–453 (one-way ANOVA). G, carbachol-induced average gamma-band power is significantly increased in (+)-EU1180–453 [39 ± 15 μV²/Hz for carbachol alone vs. 100 ± 34 μV²/Hz for carbachol plus (+)-EU1180–453]. All data are mean values ± SD. Student’s paired t-test was used to determine statistical significance unless otherwise stated. n/n represents the number of neurons or slices/number of mice.