Glutamate Imaging Reveals Multiple Sites of Stochastic Release in the CA3 Giant Mossy Fiber Boutons

Abstract

One of the most studied central synapses which have provided fundamental insights into cellular mechanisms of neural connectivity is the "giant" excitatory connection between hippocampal mossy fibers (MFs) and CA3 pyramidal cells. Its large presynaptic bouton features multiple release sites and is densely packed with thousands of synaptic vesicles, to sustain a highly facilitating "detonator" transmission. However, whether glutamate release sites at this synapse act independently, in a stochastic manner, or rather synchronously, remains poorly understood. This knowledge is critical for a better understanding of mechanisms underpinning presynaptic plasticity and postsynaptic signal integration rules. Here, we use the optical glutamate sensor SF-iGluSnFR and the intracellular Ca²⁺ indicator Cal-590 to monitor spike-evoked glutamate release and presynaptic calcium entry in MF boutons. Multiplexed imaging reveals that distinct sites in individual MF giant boutons release glutamate in a probabilistic fashion, also showing use-dependent short-term facilitation. The present approach provides novel insights into the basic mechanisms of neurotransmitter release at excitatory synapses.

Keywords: CA3 pyramidal cell; action potential; dentate gyrus; giant mossy fiber bouton; glutamate release; short-term plasticity.

FIGURE 1

Experimental Protocol. (A) Dentate gyrus granule cell expressing SF-iGluSnFR A184V, maintained in current-clamp. Collage of 20 μm deep image stacks, axon was followed to the bouton of interest (white square). (B) Area shown by the white square in panel (A). A small varicosity (<2μm) reveals a typical en-passant bouton. The fast “Tornado” (spiral) line-scan was set-up to cover most of the bouton visible area. (C) Typical imaging protocol. Traces: Five APs initiated by brief (5 ms) current pulses at 20 Hz (holding voltage −80 mV); Image panels: Tornado line-scans recorded in the Cal-590 (upper) and SF-iGluSnFR (lower) emission channels. (D) Same recordings as in panel (C), but pixel values were averaged over the length of the tornado line-scan and converted as ΔF/F. Note that for the Cal-590 signal, each AP induced a calcium entry in the presynaptic bouton (middle) whereas APs 2 and 5 failed to induce a glutamate release (bottom, black arrows). (E) Average release probability (mean ± SEM) for the AP train (n = 11 boutons). Note the increasing probability with the AP number, reflecting presynaptic facilitation.

FIGURE 2

Monitoring distinct glutamate release sites in MFBs. (A) Two examples of large (>2μm) MF boutons showing multiple un-synchronized release sites. Left panels: Z-projection of a 3D stack, with Tornado line scan superimposition (red); arrows 1 and 2, locations of glutamate-releasing sites (white transparent areas). Middle panels: tornado line-scan of the iGluSnFR signal, showing un-synchronized releases of glutamate. Note the repeating patterns, as the tornado line-scan enters and exits the area periodically, at each turn of the spiral. Right: ΔF/F traces for areas 1 and 2, as indicated. Note unsynchronized peaks of glutamate release during APs (black arrows), and some spontaneous releases (asterisks). Scale Bars: 0.2 ΔF/F and 200 ms. (B) Example of line-scan acquisition on a giant (>5μm) MB bouton. Left: Z-projection of a 3D stack, with line-scan superimposition (red). Other notations as in panel (A). Scale Bars: 0.2 ΔF/F and 200 ms.