Figure 3. Evoked glutamate release from Schaffer collaterals shows lower paired-pulse ratios at greater distances from pyramidal cell bodies.

(A) Experimental design: area of the hippocampal slice with iGluSnFR expressed in neuronal membranes (green channel); arrow, measured distance d between CA1 pyramidal cell body layer and the axonal bouton of interest; stim, stimulating electrode. (B) Examples of recorded axonal boutons (image panels, dotted circles; position of spiral 'Tornado' linescans is illustrated) showing characteristic glutamate signals in response to two afferent stimuli 50 ms apart (green traces, individual trials; black, average), at two distances from the s. pyramidale, as indicated. The lack of release failures reflects detection of glutamate escaping from multiple neighbouring synapses. (C) Paired-pulse ratio for optical glutamate signals: ΔF/F₀(1) / ΔF/F₀(2) averaged over 18–36 trials at individual boutons, plotted against distance to the soma. Other notations as in Figure 1C (n = 33). (D) Amplitude of the first glutamate response, ΔF/F₀(1) averaged over 18–36 trials at individual boutons, plotted against distance to the soma. The amplitude values reflect the average amount of glutamate released from the bouton of interest, and glutamate escaping from its neighbours; other notations as in (C) (n = 33).

Figure 3—figure supplement 1. Optical (multi-synaptic) glutamate signal evoked by short bursts of Schaffer collateral stimulation at varied distances from s.pyramidale.

(A) Examples of recorded ROIs (iGluSnFR green fluorescence channel, dotted ovals) in s. radiatum, with groups of stimulation-responding tentative axonal boutons expressing iGluSnFR, at different distances to the s. pyramidale border (as illustrated in Figure 3A; distance reference shown is measured to the horizontal midline of the area shown; distance to each ROI was measured to the ROI centroid). (B) ROI-average iGluSnFR responses (green line, temporal resolution ~25 ms) to five stimuli 50 ms apart. The underlying sensor signal kinetics (light grey line) was reconstructed using the fitting algorithm $\Delta F/F_0={\displaystyle {\sum }_{}{A}_i\exp \left(-\left(t-\Delta t\cdot (i-1)\right)\cdot {\tau }^{-1}\right)}$ (i = 1,..., 5) where A_i is the ith signal amplitude (fitted directly to the recoded amplitude), Δt = 50 ms, and the decay constant τ obtained directly from fitting the signal tail after the fifth pulse; dash-dotted lines, the linear regression line for the five ΔF/F₀ response peaks (at the five pulse onsets). (C) Average slope of linear regression for the five ΔF/F₀ response peaks as shown in (B), plotted against the distance from the s. pyramidale. (n = 61 boutons from N = 7 animals / slices).