Essential role of postsynaptic NMDA receptors in developmental refinement of excitatory synapses

Abstract

Neurons in the brains of newborns are usually connected with many other neurons through weak synapses. This early pattern of connectivity is refined through pruning of many immature connections and strengthening of the remaining ones. NMDA receptors (NMDARs) are essential for the development of excitatory synapses, but their role in synaptic refinement is controversial. Although chronic application of blockers or global knockdown of NMDARs disrupts developmental refinement in many parts of the brain, the ubiquitous presence of NMDARs makes it difficult to dissociate direct effects from indirect ones. We addressed this question in the thalamus by using genetic mosaic deletion of NMDARs. We demonstrate that pruning and strengthening of immature synapses are blocked in neurons without NMDARs, but occur normally in neighboring neurons with NMDARs. Our data support a model in which activation of NMDARs in postsynaptic neurons initiates synaptic refinement.

Fig. 1.

Mosaic deletion of NMDARs in VPm neurons of SERT-Grin1^−/− mice during early life. (A) Confocal images of the VPm of a SERT-Ai14 mouse at P7. Top: All VPm neurons are shown with an antibody of the neuronal marker NeuN. Middle: Cre-expressing VPm neurons in the same section as revealed by tdTomato signal. Bottom: Overlay of the two images above. (Scale bar: 20 μm.) Approximately 55% of neurons in this section were Cre-positive, and the average was 49 ± 3% for the VPm at P7 (n = 3 mice). (B) EPSCs recorded from a VPm neuron of a SERT-Grin1^−/− mouse at P7. (C) Plot of maximal AMPAR-EPSCs vs. maximal NMDAR-EPSCs for VPm neurons recorded at P7. All VPm neurons recorded at P7 showed NMDAR-EPSCs (14 of 14 neurons from two 2 mice; 1.7 ± 0.2 nA). (D) EPSCs recorded from two VPm neurons of a SERT-Grin1^−/− mouse at P10. Right: Neuron with no NMDAR-EPSCs. Left: Neuron with normal NMDAR-EPSCs. (E) Plot of maximal AMPAR-EPSCs vs. maximal NMDAR-EPSCs for VPm neurons recorded at P10. Fifty percent of cells (12 of 24; box with dashed blue line) had no or small NMDAR-EPSCs (<200 pA) and low NMDAR/AMPAR ratio (<0.3). (F and G) Equivalent results obtained from SERT-Grin1^−/− mice at P13 to P14. Fifty percent of VPm neurons (22 of 44; box with dashed blue line) in SERT-Grin1^−/− mice showed little or no NMDAR-EPSCs (<50 pA) and low NMDAR/AMPAR ratio (<0.1); these neurons were considered to be without NMDARs.

Fig. 2.

Deletion of NMDARs in VPm neurons disrupts pruning of redundant inputs. (A and B) EPSCs recorded from two neighboring VPm neurons in a SERT-Grin1^−/− mouse at P13. Top: EPSCs recorded at +40 mV (in red) and −70 mV (black). Middle: EPSCs at −70 mV in response to a range of stimulus intensity. Bottom: Plots of peak amplitudes of EPSCs at −70 mV vs. stimulus intensities. (C) Equivalent results obtained from a VPm neuron in a SERT-Grin1^+/− mouse at P13. (D–F) Distributions of VPm neurons receiving different numbers of ascending axons for SERT-Grin1^−/− mice at P13 to P14 (D, cells without NMDARs; E, cells with NMDARs) and SERT-Cre control mice at P13 (F). The distribution of cells with NMDARs (E) is significantly different from that of cells without NMDARs (D; P < < 0.00001, χ² test), but not from that of SERT-Grin1^+/− control group (F; P = 0.11, χ² test).

Fig. 3.

Deletion of NMDARs disrupts developmental strengthening of thalamic relay synapses. (A) Maximal EPSCs at −70 mV recorded from three neurons without NMDARs (Left) and three neurons with NMDARs (Right). All six neurons were from SERT-Grin1^−/− mice at P13. (B) Peak amplitudes of AMPAR-EPSCs for neurons without (red) and with NMDARs (gray) of SERT-Grin1^−/− mice at P13 to P14, and for neurons of SERT-Grin1^+/− control mice (green) at P13. Mean amplitudes were 0.45 ± 0.07 nA (n = 22) for neurons without NMDARs, 1.26 ± 0.13 nA (n = 22) for neurons with NMDARs, and 1.49 ± 0.14 nA (n = 21) for neurons of SERT-Grin1^+/− control mice (P < 0.0001, nonparametric ANOVA). (C) Paired pulse responses recorded at −70 mV from a neuron without (Upper) and another with NMDARs (Lower) in a SERT-Grin1^−/− mouse at P13. (D) Paired pulse ratio of EPSCs at −70 mV for the three groups at P13 to P14. The interpulse interval was 100 ms for all neurons. (E) 1, Evoked EPSCs recorded with 3 mM Sr²⁺ from a neuron without (Upper) and another with NMDARs (Lower) in a SERT-Grin1^−/− mouse at P14. 2, Partial views of traces in 1 to illustrate quantal events. (F) Peak amplitude of Sr²⁺-mEPSCs from neurons with and without NMDARs. Mean peak amplitudes were 7.3 ± 0.2 pA (n = 15) for neurons without NMDARs and 8.6 ± 0.3 pA (n = 16) for neurons with NMDARs (P = 0.002, Mann–Whitney test). (G) Averaged Sr²⁺-mEPSCs from 16 neurons with (black) and 15 neurons without (red) NMDARs. Mean decay constants were 1.74 ± 0.03 ms (n = 15) for neurons without NMDARs and 2.01 ± 0.06 ms (n = 16) for neurons with NMDARs (P = 0.0004, Mann–Whitney test).

Fig. 4.

Deletion of NMDARs disrupts pruning of somatic innervation during development. (A) Confocal images of VPm neurons and Pr5 axonal terminals in Ai14;SERT-Cre (SERT-Ai14) mice at P7, P10, and P14. Cre-expressing neurons were visualized with the RFP antibody (green); corticothalamic axons were also labeled with the RFP antibody. Pr5 axonal terminals were visualized with the VGluT2 antibody (red). (B) Confocal images of VPm neurons and Pr5 axonal terminals in SERT-Grin1^+/−;Ai14 and SERT-Grin1^−/−;Ai14 mice at P14. (Scale bar: 10 μm; A and B use the same scale bar.) (C) comparison among the five groups of somatic innervation by VGluT2-positive terminals. Somatic innervation was quantified as the fraction (as a percentage) of the cell body perimeter in contact with VGluT2-positive terminals. In SERT-Ai14 mice, there was a progressive reduction of somatic innervation by VGluT2-positive terminals (71.6 ± 2.8%, n = 12 for P7; 40.2 ± 2.8%, n = 13 for P10; 22.4 ± 1.5%, n = 10 for P14; P < 0.0005, P7 vs. P10, P10 vs. P14). There was no difference between SERT-Grin1^+/− control mice and those in SERT-Ai14 mice (24.7 ± 2.3%, n = 32 for SERT-Grin1^+/− at P14; P = 0.43 vs. SERT-Ai14 at P14); somatic innervation was significantly higher in Cre-positive neurons of SERT-Grin1^−/− mice at P14 than those in SERT-Grin1^+/− or SERT-Ai14 mice at the same age (42.9 ± 2.3%, n = 34 for SERT-Grin1^−/−; P < 0.0001 vs. SERT-Grin1^+/− or SERT-Ai14).