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. Author manuscript; available in PMC: 2020 Mar 6.
Published in final edited form as: Neuron. 2019 Jan 28;101(5):863–875.e6. doi: 10.1016/j.neuron.2019.01.002

Figure 3. Targeting PA-BoNT to Vesicles Improves Its Efficacy.

Figure 3.

(A) Schematic of constructs used to target PA-BoNT to synaptic vesicles.

(B) Left: examples of VAMP2 staining in presynaptic terminals (marked by syph-GFP-BoNT-iLID, pink arrows) from neurons transfected with vPA-BoNT and maintained in the dark (left) or exposed to 15 min blue light (1-s pulse every 2 min) (right). Right: quantification of the VAMP2 signal in terminals from transfected cells relative to neighboring terminals in dissociated hippocampal cultures maintained in the dark (0 min) or exposed to blue light for varying times (p < 0.0001, one-way ANOVA). The kinetics of VAMP2 cleavage by sPA-BoNT (dashed gray line) from Figure 2B is replotted for direct comparison.

(C) Representative traces of mEPSCs from infected cultures kept in the dark (top) or exposed to blue light (bottom; 1-s pulse every 2 min, 4.5 μW/cm2). Scale bars, 20 pA, 5 s.

(D) Left: quantification of AMPA mEPSC frequency (left) and amplitude (right) from uninfected cultures (black) or infected cultures kept in the dark (gray) or exposed to blue light for a minimum of 30 min (blue) (frequency, p < 0.05; amplitude, n.s., one-way ANOVA). Right: cumulative distribution of mEPSC inter-event interval (IEI) for cells kept in the dark (gray) or exposed to blue light (blue, *p < 0.0001, Kolmogorov-Smirnov test).

(E) Time course of VAMP2 recovery following cleavage with vPA-BoNT. After 1 h light exposure, hippocampal neurons were maintained in darkness for varying times to assess the recovery of VAMP2 by immunocytochemistry as in (B). Values represent mean ± SEM from at least 11 cells from 2 independent experiments (***p < 0.0001, *p < 0.05, one-way ANOVA).

(F) Time course of functional recovery. Cultured neurons infected with vPA-BoNT were treated with 1 h of blue light, and mEPSC frequency and amplitude were measured immediately following light exposure or after 8 h or 24 h of dark recovery. Data are normalized to neurons expressing vPA-BoNT but maintained in darkness for the duration. Values represent mean ± SEM from at least 9 cells per condition from two independent experiments (*p < 0.05, Student’s t test).