Journal: bioRxiv
Article Title: Synaptotagmin-7 is required for synchronous but not asynchronous facilitation of glutamate release at cortical boutons
doi: 10.64898/2025.12.19.695358
Figure Lengend Snippet: (a) Experimental design. Schematic of the imaging configuration. Whole-cell voltage-clamp recordings were used to evoke action potentials (APs) in SF-iGluSnFR-transfected pyramidal neurons while monitoring glutamate release from their axonal boutons. (b) Identification of active boutons. Image analysis from an axonal fragment of a representative wild type (WT) neuron. Top: stimulation paradigm showing the consecutive paired-pulse (20 Hz) and train (50 APs at 20 Hz) protocols. Example raw fluorescence images acquired after AP stimulation during paired-pulse trials and the train. Bottom: corresponding images after application of a spatiotemporal filter, illustrating vesicular release successes and failures in active boutons within the selected region of interest. Bottom right: maximal projection of the filtered stack, visualising all boutons that released at least one vesicle during the stimulation protocol (five active boutons in this example). (c) Automatic event detection. Top: raw fluorescence trace from an example bouton (b1, WT) indicated by an arrowhead in (b). Bottom: corresponding deconvolved trace showing detected quantal release events. Vertical dotted lines mark AP timings; gaps between traces correspond to 10 s intersweep intervals. Events were identified as local maxima exceeding the detection threshold θ (horizontal dashed line), defined as 4 standard deviations (σ) of baseline fluorescence noise (see Methods). Blue and red circles denote synchronous and asynchronous events (<10 ms and >10 ms after the preceding AP, respectively). (d) Quantal analysis. Amplitudes of all detected events (top raster) were used to generate a quasi-continuous amplitude distribution by bootstrap resampling with bouton specific noise, which improved convergence and stability of the Gaussian mixture fit (see Methods). The resulting distribution was fitted with a sum of Gaussian functions to estimate the quantal amplitude (q, green line on the deconvolved traces in (c)), corresponding to the mean single vesicle SF iGluSnFR response. Peaks at q and 2q indicate one and two vesicle release events, respectively. See additional examples in and . (e) Functional bouton parameters for the example bouton b1 (WT). n T , n S , and n A represent total, synchronous, and asynchronous release efficacies (average number of quanta released per action potential); n A / n T denotes the asynchronous release fraction, and FDI is Facilitation-Depression Index. Parameters were calculated for the 1 st (AP1) and 2 nd (AP2) stimuli of the paired-pulse protocol and for the steady-state phase of the train (Train; APs 11– 50). See Methods for exact definitions.
Article Snippet: At 4-6 days in vitro (DIV 4-6), neurons were transfected with the plasmid pAAV.hSynap.SF-iGluSnFR.A184V (Addgene #106174; ( )) using Neuromag reagent (OZ Biosciences).
Techniques: Imaging, Transfection, Fluorescence, Functional Assay
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