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. Author manuscript; available in PMC: 2019 Mar 27.
Published in final edited form as: Cell Rep. 2018 May 1;23(5):1259–1274. doi: 10.1016/j.celrep.2018.03.126

Figure 6. An Unc13B Knockdown in Unc13B-Dominated LN:LN Synapses Drastically Reduces Release Amplitude.

Figure 6.

(A) Staining against Unc13A and anti-GFP in the fly AL. The GFP-positive LNs barely show an Unc13A signal.

(B) Scheme of the experimental procedure. Left: recordings were made from LNs postsynaptic to Channelrhodopsin 2 (ChR2+, GFP+) expressing cells. Voltage command (VC, right) measured average postsynaptic currents in response to optogenetical activation of the presynapse (ChR2+) in control (n = 15), unc13A KD (n = 13), and unc13B KD (n = 18) flies. Traces are mean ± SEM.

(C) Whole-cell current-clamp recordings of presynaptic (ChR2+, GFP+) LNs. Left: example of a spike train in presynaptic LNs evoked by a light pulse. Right: light-evoked firing rate of Channelrhodopsin-expressing cells over a 2-s stimulation period. The action potential frequency in presynaptic LNs (ChR2+) is not affected by the expression of unc13A/unc13B-RNAi constructs. (Kruskal-Wallis test, ns = not significant). From left to right: controls (black), unc13A KD (magenta), unc13B (green).

(D) Average peak amplitudes of postsynaptic currents are significantly different between control and unc13B KD flies (Kruskal-Wallis followed by Dunn’s multiple comparison test, Bonferroni-corrected, controls (black): n = 16, unc13A KD (magenta): n = 14, outliers were removed with the fourth spread method [Devore, 2011], p = 0.069, unc13B KD (green): n = 18, p = 0.001).

(E) Average steady-state current (SSC) measured at 1.5 s after light onset, averaged over a period of 0.5 s. SSC was significantly smaller in unc13B KD flies compared to control animals (Kruskal-Wallis followed by Dunn’s multiple comparison test, Bonferroni-corrected, controls (black): n = 16, unc13A KD (magenta): n = 15, p = 0.10, unc13B KD (green): n = 18, p = 0.001).

(F) Time until outward currents reached the maximum after onset of the light stimulus. The time to reach the peak amplitude remained the same in all of the recorded groups (Kruskal-Wallis followed by Dunn’s multiple comparison test, Bonferroni-corrected, controls (black): n = 16, unc13A KD (magenta): n = 14, outliers were removed with the fourth spread method, p = 0.83, unc13B KD (green): n = 18, p = 0.79). Values represent mean ± SD.