Fig. 1: Increased release probability increases the glutamate concentration of synaptic transmission events.

a CA3 pyramidal neuron in organotypic slice culture was patch-clamped to somatically trigger single action potentials while imaging with 2-photon laser scanning microscopy bouton fluorescence in CA1 stratum radiatum. b Response amplitude (green circles) monitored in artificial cerebrospinal fluid (ACSF) containing 2 mM [Ca²⁺]_e at 33 °C was constant over time. Baseline fluorescence before stimulation was analyzed to estimate imaging noise (gray circles). c Monitoring two neighboring boutons located on the same axon. Scale bar, 1 µm. Two trials showing independent release events. Red arrowheads indicate the stimulation onset. Scale bar, 20 ms. iGluSnFR transients between neighboring boutons were not correlated (two-sided Spearman correlation: r = −0.04, p = 0.69). d Glutamate transients in a single bouton, switching from ACSF containing 1 mM [Ca²⁺]_e to 4 mM [Ca²⁺]_e. The light and the dark blue line represent the average amplitude of successes in 1 mM [Ca²⁺]_e and 4 mM [Ca²⁺]_e, respectively. e Summary of all [Ca²⁺]_e switching experiments. The probability of successful glutamate release (left panel) increased from 0.26 ± 0.03 in 1 mM [Ca²⁺]_e to 0.87 ± 0.04 in 4 mM [Ca²⁺]_e (two-sided Wilcoxon-signed rank test, p < 0.0001, n = 27 boutons in 23 slices). The amplitude of fluorescence transients in trials classified as ‘success’ (right panel) increased from 0.84 ± 0.056 ΔF/F₀ in 1 mM [Ca²⁺]_e to 1.76 ± 0.12 ΔF/F₀ in 4 mM [Ca²⁺]_e (paired t test, p < 0.0001, n = 27 boutons in 23 slices). Values are given as mean ± SEM. f The probability of release was correlated with the amplitude of success trials in a non-linear fashion. g In 2 mM [Ca²⁺]_e, the release probability of individual synapses ranged from 0.17 to 0.99. The amplitude of success trials was similar at all boutons with p_syn < 0.5. Source data are provided as a Source Data file.