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. 2021 Jun 1;10:e66612. doi: 10.7554/eLife.66612

Figure 1. Anatomical and functional evidence for preNMDARs at mossy fiber synapses.

(A) Image of a mossy fiber (mf) giant bouton and postsynaptic spines (s). (B, C) Higher magnification of mf synapses. Arrows indicate postsynaptic GluN1, whereas arrowheads indicate presynaptic GluN1. Calibration bars: 500 nm. (D) Mossy fiber (mf) and associational–commissural (ac) synaptic GluN1 immuno-particle radial distribution (30 nm bins), mf: 34 synapses, 100 presynaptic particles; ac: 25 synapses, 24 presynaptic particles; three animals. (E) AMPAR-ESPCs were recorded at Vh = −70 mV in the presence of 0.5 µM LY303070 and 100 µM picrotoxin. Low-frequency facilitation (LFF), induced by stepping stimulation frequency from 0.1 to 1 Hz, was assessed before and after bath application of MK-801 (50 µM). MK-801 significantly reduced LFF (baseline 378 ± 57%, MK-801 270 ± 48%, n = 10 cells, nine animals; baseline vs MK-801, p=3.8×10−5, paired t-test). In all panels of this figure: representative traces (top), representative experiment (middle), and normalized LFF and summary plot (bottom). DCG-IV (1 µM) was applied at the end of all recordings to confirm mf-CA3 transmission. (F) D-APV (100 µM) or R-CPP (50 µM) application also reduced LFF (baseline 546 ± 50%, D-APV/R-CPP 380 ± 38%, n = 7 cells, five animals; baseline vs D-APV/R-CPP, p=0.00743, paired t-test). (G) KAR-EPSCs were recorded at Vh = −70 mV in the presence of 15 µM LY303070 and 100 µM picrotoxin. In addition, NMDAR-mediated transmission was blocked intracellularly by loading MK-801 (2 mM) in the patch-pipette. Bath application of MK-801 (50 µM) significantly reduced LFF (baseline 278 ± 40%, MK-801 195 ± 26% n = 8 cells, six animals; baseline vs MK-801, p=0.00259, paired t-test). Data are presented as mean ± s.e.m. **p<0.01; ***p<0.005; ****p<0.001.

Figure 1.

Figure 1—figure supplement 1. Immunogold-EM reveals negligible presynaptic AMPAR particle distribution.

Figure 1—figure supplement 1.

(A, B) Images of mossy fiber (mf) and associational–commissural (ac) synapses, postsynaptic spines (s). (C) AMPAR immuno-particle distribution (30 nm bins), mf: 102 synapses, eight presynaptic particles; ac: 75 synapses, six presynaptic particles; three animals. Dashed line represents synaptic cleft.
Figure 1—figure supplement 2. Stable low-frequency facilitation of mf-CA3 synaptic transmission in naïve slices.

Figure 1—figure supplement 2.

(A) Stable low-frequency facilitation (LFF) of AMPAR-EPSCs. In naïve slices (interleaved experiments), LFF remained unchanged throughout the recording session (baseline 335 ± 62%, naïve 363 ± 63%, n = 10 cells, nine animals; p=0.185, Wilcoxon signed-rank test, baseline vs naïve). DCG-IV (1 µM) was applied at the end of all recordings to confirm mf-CA3 transmission. (B) LFF of KAR-EPSCs was also stable in interleaved, naïve slices (baseline 274 ± 33%, naïve 278 ± 25%, n = 9 cells, six animals; p=0.236, Wilcoxon signed-rank test, baseline vs naïve). NBQX (10 µM) was applied at the end of all recordings to confirm mf KAR transmission. Data are presented as mean ± s.e.m.
Figure 1—figure supplement 3. Intracellular MK-801 effectively blocked postsynaptic NMDARs.

Figure 1—figure supplement 3.

Representative NMDAR-EPSCs (Vh = +40 mV) from CA3 pyramidal neurons patch-loaded with 2 mM MK-801 (left) or naïve internal solution (right). Mf inputs were stimulated with a bipolar electrode (theta-glass pipette) in stratum lucidum in the presence of picrotoxin (100 µM) and NBQX (10 µM). Bath application of MK-801 (50 µM) blocked NMDAR currents in naïve cells to a similar magnitude as cells patch-loaded with MK-801 (n = 5 cells, four animals in each condition; U = 0.676, Mann–Whitney test). Note that CA3 pyramidal neurons were loaded for at least 3–5 min before recording started at +40 mV.