NMDA receptors inhibit synapse unsilencing during brain development

Abstract

How the billions of synapses in the adult mammalian brain are precisely specified remains one of the fundamental questions of neuroscience. Although a genetic program is likely to encode the basic neural blueprint, much evidence suggests that experience-driven activity through NMDA receptors wires up neuronal circuits by inducing a process similar to long-term potentiation. To test this notion directly, we eliminated NMDA receptors before and during synaptogenesis in single cells in vitro and in vivo. Although the prevailing model would predict that NMDA receptor deletion should strongly inhibit the maturation of excitatory circuits, we find that genetic ablation of NMDA receptor function profoundly increases the number of functional synapses between neurons. Conversely, reintroduction of NMDA receptors into NR1-deficient neurons reduces the number of functional inputs, a process requiring network activity and NMDA receptor function. Although NMDA receptor deletion increases the strength of unitary connections, it does not alter neuronal morphology, suggesting that basal NMDA receptor activation blocks the recruitment of AMPA receptors to silent synapses. Based on these results we suggest a new model for the maturation of excitatory synapses in which ongoing activation of NMDA receptors prevents premature synaptic maturation by ensuring that only punctuated bursts of activity lead to the induction of a functional synapse for the activity-dependent wiring of neural circuitry.

Fig. 1.

NMDA receptor deletion results in a strong increase in the number of AMPA receptor-containing synapses. (a) Scatter plot of NMDA EPSCs of pairs of simultaneously recorded neurons in NR1^fl/fl slice culture from CRE-IRES-GFP transfected and neighboring control cells 12–17 days after transfection (n = 41 pairs, P < 10⁻¹⁰). (Inset) Representative traces: black, control cell; green, CRE-IRES-GFP-expressing cell. (b) Scatter plot of AMPA EPSCs from the same cells (n = 41 pairs, P < 10⁻⁵). (c) Bar graph showing the relative change in AMPA and NMDA EPSCs between control and CRE-transfected neurons from the pairs in a. Error bars indicate SEM. (d) Cumulative distribution of mEPSC interintervals from control (n = 16) and CRE-expressing (n = 15) neurons (P < 0.005). Above is an example recording of mEPSCs from a CRE-expressing cell. (e) Cumulative distribution of mEPSC amplitudes from control (n = 16) and CRE-expressing (n = 15) neurons (P = 0.65). Above is an example recording of mEPSCs from a control cell. (f) Bar graph showing average paired-pulse ratio in control and CRE-expressing cells. Above are representative traces.

Fig. 2.

Reintroduction of NMDA receptors to NR1 knockout cells from NEX-CRE;NR1^fl/fl slice cultures reduces AMPA receptor-containing synapses in an activity-dependent manner. (a) Scatter plot of NMDA EPSCs in simultaneous paired recordings from control (NEX-CRE;NR1^fl/fl) and NR1-GFP-transfected neurons 5–10 days after transfection (n = 25 pairs, P < 10⁻⁷). (Inset) Representative traces: black, control cell; green, NR1-GFP-expressing cell. (b) Scatter plot of AMPA EPSCs in the same cells (n = 25 pairs, P < 0.005). (c) Bar graph showing the relative change in AMPA and NMDA EPSCs between control and NR1-GFP-transfected neurons from the pairs in a and in separate experiments when the slices were incubated in a mixture of NMDA receptor antagonists (NMDAR block) (n = 16 pairs, P = 0.46) or TTX (n = 14 pairs, P = 0.48). Error bars indicate SEM. (d) Cumulative distribution of mEPSC interintervals from control and NR1-GFP-expressing neurons (n = 9 each, P < 0.001). Above is an example recording from an NR1-GFP-expressing cell. (e) Cumulative distribution of mEPSC amplitudes from control and NR1-GFP-expressing neurons (n = 9 each, P = 0.72). Above is an example recording from a control cell. (f) Bar graph showing average paired-pulse ratio in control and NR1-GFP-expressing cells. Above are representative traces.

Fig. 3.

NMDA receptor deletion in utero increases the number of AMPA receptor-containing synapses. (a) Scatter plot of NMDA EPSCs of pairs of simultaneously recorded neurons in acute slices from AAV-CRE-GFP-infected and neighboring control cells (n = 32 pairs, P < 10⁻⁹). (Inset) Representative traces: black, control cell; green, CRE-expressing cell. (b) Scatter plot of AMPA EPSCs from the same cells (n = 33 pairs, P < 10⁻⁷). (c) Bar graph showing the relative change in AMPA and NMDA EPSCs between control and CRE-expressing neurons from the pairs in a. Error bars indicate SEM. (d) Cumulative distribution of mEPSC interintervals from control (n = 21) and NR1-deficient (n = 22) neurons (P < 0.001). Above is an example recording of mEPSCs from CRE-expressing cell. (e) Cumulative distribution of mEPSC amplitudes from control (n = 21) and NR1-deficient (n = 22) neurons (P < 0.05). Above is an example recording of mEPSCs from a control cell. (f) Bar graph showing average paired-pulse ratio in control and CRE-expressing cells. Above are representative traces.

Fig. 4.

NR1 deletion in utero results an increase in the strength of unitary connections without any significant changes in cell morphology. (a) Cumulative distribution of sEPSC amplitudes from control (n = 11) and AAV-CRE-GFP-infected (n = 13) neurons (P < 0.05). (b) Cumulative distribution of sEPSC interevent intervals from control (n = 11) and CRE-expressing (n = 13) neurons (P < 0.001). (c) Cumulative distribution of sEPSC and mEPSC amplitudes (after application of TTX) in control neurons (n = 10, P = 0.53). (Inset) Representative traces before (black) and after (gray) application of TTX. (d) Cumulative distribution of sEPSC and mEPSC amplitudes (after application of TTX) in CRE-expressing neurons (n = 10, P < 0.01). (Inset) Representative traces before (thick line) and after (thin line) application of TTX. (e) Representative confocal stacks of 20-μm stretches from secondary apical dendrites of a CRE-expressing cell and a control cell. (f) Representative confocal stacks of CRE-expressing and control cells. (g) Average spine density in control (n = 8) and CRE-expressing (n = 10) neurons (P = 0.88). (h) Average number of dendritic branch points in control (n = 8) and CRE-expressing (n = 9) neurons (P = 0.99). (i) Average dendritic length in control (n = 8) and CRE-expressing (n = 9) neurons (P = 0.87).

Fig. 5.

Postnatal NR1 deletion increases the number of AMPA receptor-containing synapses but does not depend on synaptic competition between neighboring cells. (a) Scatter plot of NMDA EPSCs of pairs of simultaneously recorded neurons in acute slices from postnatally AAV-CRE-GFP-infected and neighboring control cells (n = 32 pairs, P < 10⁻⁸). (Inset) Representative traces. (b) Scatter plot of AMPA EPSCs from the same cells (n = 31 pairs, P < 0.0005). (c) Cumulative distribution of mEPSC interevent interval between control (n = 12) and CRE-expressing cells in slices with sparse (n = 12) or dense (n = 26) infection. (d) Model for the role of NMDA receptors in synaptic maturation. During development, modest activity through NMDA receptors at silent synapses prevents the constitutive trafficking of AMPA receptors to the PSD. This mechanism ensures that synapses become functional only after strong or correlated activity, when enough calcium entry through these NMDA receptors overrides the inhibitory pathway and drives AMPA receptor insertion (like LTP). When NMDA receptors are genetically deleted, the inhibitory signal is absent, and AMPA receptors traffic to the PSD in the absence of any NMDA receptor activity.