Contribution of NMDA Receptors to Synaptic Function in Rat Hippocampal Interneurons

Abstract

The ability of neurons to produce behaviorally relevant activity in the absence of pathology relies on the fine balance of synaptic inhibition to excitation. In the hippocampal CA1 microcircuit, this balance is maintained by a diverse population of inhibitory interneurons that receive largely similar glutamatergic afferents as their target pyramidal cells, with EPSCs generated by both AMPA receptors (AMPARs) and NMDA receptors (NMDARs). In this study, we take advantage of a recently generated GluN2A-null rat model to assess the contribution of GluN2A subunits to glutamatergic synaptic currents in three subclasses of interneuron found in the CA1 region of the hippocampus. For both parvalbumin-positive and somatostatin-positive interneurons, the GluN2A subunit is expressed at glutamatergic synapses and contributes to the EPSC. In contrast, in cholecystokinin (CCK)-positive interneurons, the contribution of GluN2A to the EPSC is negligible. Furthermore, synaptic potentiation at glutamatergic synapses on CCK-positive interneurons does not require the activation of GluN2A-containing NMDARs but does rely on the activation of NMDARs containing GluN2B and GluN2D subunits.

Keywords: NMDA receptor; hippocampus; interneuron; long-term potentiation; receptor subunits; whole-cell patch-clamp.

Figure 1.

The absence of GluN2A confers slowed NMDAR kinetics on CA1 PyrCs. A, Reconstruction of a CA1 PyrC showing orientation with respect to the hippocampal layers stratum oriens (Ori.), pyramidale (Pyr.), radiatum (Rad.), or lacunosum-moleculare (L–M). Somatodendritic axis (black) and axonal arborization (red) are indicated. B, Representative EPSCs recorded from CA1 PyrCs from wild-type (black) and GluN2A-null (red) rats. AMPAR-EPSCs (inward currents) and NMDAR-EPSCs (outward currents) are shown. The peak scaled NMDAR traces are shown below to indicate the slowing of the NMDAR-EPSC. C, NMDAR-EPSC rise times, quantified for all recorded CA1 PyrCs. Data from individual cells are shown as filled circles from wild-type rats (black; n = 20 cells; N = 13 rats) and GluN2A-null rats (2A-null, red; n = 21 cells; N = 11 rats). D, NMDAR-EPSC decay time constants (tau) from the weighted tau of a biexponential curve fit. E, NMDAR/AMPAR ratio of EPSCs elicited by Schaffer collateral stimulation. Fewer rats are shown for kinetic values because of the 20 pA cutoff imposed on kinetic data. Data are shown as the mean ± SD, alongside the difference between the means ± CI. *p < 0.05, Student’s t test.

Figure 2.

GluN2A subunits contribute to NMDAR-EPSCs in identified PV INs. A, Reconstruction of a PV IN recorded in CA1 with respect to the hippocampal layers, with the somatodendritic axis indicated in black and axons localized to stratum pyramidale in red. Inset, Immunohistological labeling for PV (green) aligned (asterisk) to the biocytin-filled somata (black and white). Scale bar, 10 μm. B, Representative monosynaptic AMPAR- and NMDAR-mediated EPSCs recorded from PV INs in wild-type (black) and 2A-null (red) rats. The scaled NMDAR-mediated EPSC (bottom) indicates the slowing of response in the GluN2A-null rat line. C, NMDAR-EPSC rise times, quantified for identified PV-INs from wild-type (black; N = 14 rats) and 2A-null (red; N = 13 rats) rats, with individual cells shown overlain (filled circles); fewer rats are shown for kinetic values because of the 20 pA cutoff. D, Quantification of NMDAR-mediated EPSC decay time constants (tau). E, NMDAR/AMPAR ratio of EPSCs elicited by Schaffer collateral stimulation. Data are shown as the mean ± SD. *p < 0.05, Student’s t test.

Figure 3.

GluN2A is a major NMDAR subunit in SSt INs. A, Reconstructed CA1 SSt IN shown with respect to the hippocampal layers, with somatodendritic axis confined to stratum oriens (black) and axons localized to stratum lacunosum-moleculare (L–M) in red. Inset, Immunolabelling for SSt (green) of the biocytin (Bioc)-filled somata (black and white). Scale bar, 10 μm. B, Representative monosynaptic AMPAR- and NMDAR-mediated EPSCs recorded from SSt INs in wild-type (black) and 2A-null (red) rats. The scaled NMDAR-mediated EPSC (bottom) indicates the slower response in the GluN2A-null rats. C, NMDAR-EPSC rise times, quantification in SSt INs, with individual cells from wild-type (black, N = 14 rats) and 2A-null (red, N = 12 rats) rats shown overlain (filled circles); fewer rats are shown for kinetic values because of the 20 pA cutoff. D, Quantification of NMDAR-mediated EPSC decay time constants (tau). E, NMDAR/AMPAR ratio of EPSCs elicited by alveus stimulation. Data are shown as the mean ± SD. *p < 0.05, Student’s t test.

Figure 4.

GluN2A does not significantly contribute to synaptic NMDAR-mediated EPSCs in CCK INs. A, Reconstructed CCK BC with respect to the hippocampal layers, with the somatodendritic axis covering all layers (black) and axons localized to stratum pyramidale (red). Inset, Immunolabelling for pre-pro-CCK (green) of the IN somata (black and white). Scale bar, 10 μm. B, Monosynaptic AMPAR- and NMDAR-mediated EPSCs recorded from CCK INs in wild-type (black) and 2A-null (red) rats. The scaled NMDAR-mediated EPSC (bottom) indicates no change in decay times in GluN2A-null rats. C, NMDAR-EPSC rise times in CCK INs, with individual cells from wild-type (black, N = 26 rats) and 2A-null (red; N = 16 rats) shown overlain (filled circles); fewer rats are shown for kinetic values because of the 20 pA cutoff. D, Quantification of NMDAR-mediated EPSC decay time constants. E, NMDAR/AMPAR ratio of EPSCs elicited by stratum radiatum stimulation. Data are shown as the mean ± SD.

Figure 5.

GluN2B-containing NMDARs differentially contribute to EPSCs in identified hippocampal neurons. A, Representative NMDAR-mediated EPSCs recorded at +40 mV in the presence of CNQX at (black traces) or following 10 min wash-in of 10 μm ifenprodil (gray traces) from wild-type rats (WT) for the different cell types identified. B, Time course of ifenprodil wash-in (black bar) for CA1 PyrCs (open circles), PV INs (filled black circles), SSt INs (gray circles), and a subset of CCK INs (green circles), measured as a percentage of control EPSCs per minute, compared with 100% at baseline (dashed black line). C, Quantification of ifenprodil block over the last 2 min for the different cell classes identified in wild-type and GluN2A-null neurons (2A-null); number of cells tested is shown in parenthesis. D, Estimation plot showing the difference in ifenprodil block between wild-type and GluN2A-null cells, plotted as the difference between means ± 95% confidence interval. Data are shown as the mean ± SD. *p < 0.05, paired t tests.

Figure 6.

NMDAR-EPSCs in CCK INs are mediated by GluN2D, which is required for LTP induction. A, Representative NMDAR-mediated EPSCs recorded in CCK INs at +40 mV in the presence of CNQX at (black and red traces) or following 10 min wash-in of the GluN2D-negative allosteric modulator NAB-14 (10 μm; gray and pink traces). B, Plot of NMDAR-EPSC amplitudes before and after NAB-14 application in CCK INs. C, Quantification of NMDAR-EPSC amplitudes in CCK INs from GluN2A-null rats before and after NAB-14 application. D, Time course of EPSP amplitude in CCK INs from wild-type rats measured from −70 mV current clamp, following 2× 100 Hz stimulation (double arrow) measured under control conditions (black circles) and in the presence of 10 μm NAB-14 (gray circles). Representative traces are shown above the chart, showing EPSP at baseline (black traces) and 25 min after LTP induction (gray traces). E, Quantification of LTP, reported as the percentage change in EPSP amplitude from baseline (dashed line) for wild-type CCK INs. Control LTP recordings (black circles; n = 9 cells; N = 7 rats) and those performed in the presence of 10 μm NAB-14 (gray circles; n = 5 cells; N = 5 rats) are shown. The number of tested rats is shown in parentheses. F, LTP induction in CCK INs from GluN2A-null rats according to the same scheme as in D. Data are shown for control recordings (red circles) and in the presence of NAB-14 (pink circles). Traces of each treatment are above the chart showing data at baseline (red) and after LTP induction (pink traces). G, Quantification of LTP induction in GluN2A-null rats under control (red; n = 6 cells; N = 5 rats) and in the presence of NAB-14 (pink; n = 5 cells; N = 4 rats). Data are shown as the mean ± SD. *p < 0.05, from paired (B) and unpaired (D, F) Student’s t tests.