Figure 5. preNMDARs contribute to presynaptic Ca²⁺ rise.

(A) Representative images showing GCs patch-loaded with Alexa 488 (35 µM) to confirm expression of mCherry (bottom). Representative AMPAR-EPSCs recorded from control (top) or Grin1-cKO (middle) GCs. Synaptic responses were elicited by activating medial perforant-path inputs. AMPAR-ESPCs were recorded at V_h = −65 mV in the presence of 100 µM picrotoxin, NMDAR-EPSCs were isolated with 10 µM NBQX and recorded at +40 mV. MK-801 (20 µM) was applied at the end of each experiment. Summary plot (bottom) demonstrating that GluN1 deletion from GCs virtually abolished NMDAR-mediated transmission indicated by a strong reduction of NMDAR/AMPAR in Grin1-cKO granule cells as compared to controls (control 0.90 ± 0.17, n = 7 cells, six animals; Grin1-cKO 0.13 ± 0.05, n = 6 cells, six animals; control vs Grin1-cKO, p=3.81×10⁻⁷, unpaired t-test). (B) Representative control and Grin1-cKO GCs patch-loaded with Fluo-5F (200 µM) and Alexa 594 (35 µM). Arrows indicate the identification of a mf giant bouton, magnified images in white box. (C) Three representative mf boutons (top) and line scan image of calcium transients (CaTs) elicited by five action potentials at 25 Hz (middle, Fluo-5F) and morphological dye (bottom, Alexa 594), in Control and Grin1-cKO animals. Dotted line (yellow) indicates line scan location. Red Channel, Alexa 594; Green Channel, Fluo-5F. (D, E) Peak analysis of the fifth pulse ΔG/R revealed a significant reduction in Ca²⁺ rise of Grin1-cKO animals as compared to Control (control 0.046 ± 0.01, n = 10 boutons, three line scans per bouton, eight animals; Grin1-cKO 0.025 ± 0.004, n = 10 boutons, eight animals; control vs Grin1-cKO, U = 0.017, Mann–Whitney test). Arrows indicate mf activation. Data are presented as mean ± s.e.m. *U < 0.05; ****p<0.001.

Figure 5—figure supplement 1. NMDAR antagonism reveals a reduction in presynaptic Ca⁺² rise in the presence of 1.3 mM Mg⁺² and 2.5 mM Ca⁺².

(A, B) Granule cells were patch-loaded with Fluo-5F (200 µM) and Alexa 594 (35 µM). Line scan analysis of mf giant bouton calcium transients (CaTs) in response to action potential (AP) stimulation (5 APs, 25 Hz). (C) Line scan signals following D-APV application or naïve conditions. (D) D-APV (100 µM) significantly reduced the fifth peak (P5) of CaTs (baseline 0.155 ± 0.04, D-APV 0.138 ± 0.03, n = 13 boutons, three line scans per bouton, 10 animals; baseline vs D-APV, p=0.00642, Wilcoxon signed-rank test). (E) In naïve conditions P5 of CaTs is stable (baseline 0.104 ± 0.026, naïve 0.105 ± 0.026, n = 12 boutons, three line scans per bouton, 10 animals; baseline vs naïve, p=0.255, Wilcoxon signed-rank test). The first peak (P1) of CaTs is not affected by D-APV (baseline 0.05 ± 0.017; D-APV 0.047 ± 0.014; baseline vs D-APV, p=0.485, Wilcoxon signed-rank test) and is stable in naïve conditions (baseline 0.033 ± 0.009; naïve 0.032 ± 0.009, baseline vs naïve, p=0.196, Wilcoxon signed-rank test). Data are presented as mean ± s.e.m. **p<0.01.