Figure 2.
Figure 2. Laser photolysis of caged Ca2+ in MLI axonal varicosities evokes ASCs. (A) MLI loaded with an intracellular solution containing Alexa Fluor 488 and the Ca2+ cage DM-nitrophen. The image corresponds to a Z projection of a stack of epifluorescence images taken at 1-µm steps. (Inset) Detail of the axon centered on the varicosity where the 405-nm laser was focused. (B) The top part of the panel corresponds to a drawing of the cell; the somatodendritic compartment is shown in magenta, and the axonal compartment is in green. The stimulated site is indicated with the arrowhead. Somatic whole-cell recordings of the laser-evoked ASCs recorded when the varicosity shown in A (inset) was stimulated. Black traces are individual sweeps; the gray trace is the average. Black dots in B and G indicate probable multi-vesicular release events. (C) The specific GABAAR antagonist, Gbz, completely blocked the laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during Gbz, and after 5-min wash. (D) Summary plot of the effect of Gbz (control: 27 ± 2 pA; Gbz: 1 pA ± 1 pA; after wash: 18 ± 8 pA; one-way ANOVA; **, P < 0.01; n = 4 sites). (E) Voltage dependence of ASCs is consistent with GABAAR-mediated currents. Representative traces of ASCs evoked at different holding potentials. (F) The extrapolated ASC reversal potential is not different from the EGABA predicted by the Goldman–Hodgkin–Katz equation. Continuous line, linear fit. Circles represent mean ± SEM. The extrapolated reversal potential is 2 ± 2 mV. (G) Block of Nav channels failed to affect laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during TTX, and after 5-min wash. (H) Summary plot of the effect of TTX on laser-evoked ASCs (control: 39 ± 9; in 1 µM TTX: 36 ± 8 pA; after wash: 44 ± 14 pA; one-way ANOVA; P > 0.05, n = 7 sites). In D and H, the black circles represent mean ± SEM for each condition, and gray dots represent the average amplitude of events evoked in individual release sites. In B, C, E, and G, arrows represent timing of the laser pulse.

Laser photolysis of caged Ca2+ in MLI axonal varicosities evokes ASCs. (A) MLI loaded with an intracellular solution containing Alexa Fluor 488 and the Ca2+ cage DM-nitrophen. The image corresponds to a Z projection of a stack of epifluorescence images taken at 1-µm steps. (Inset) Detail of the axon centered on the varicosity where the 405-nm laser was focused. (B) The top part of the panel corresponds to a drawing of the cell; the somatodendritic compartment is shown in magenta, and the axonal compartment is in green. The stimulated site is indicated with the arrowhead. Somatic whole-cell recordings of the laser-evoked ASCs recorded when the varicosity shown in A (inset) was stimulated. Black traces are individual sweeps; the gray trace is the average. Black dots in B and G indicate probable multi-vesicular release events. (C) The specific GABAAR antagonist, Gbz, completely blocked the laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during Gbz, and after 5-min wash. (D) Summary plot of the effect of Gbz (control: 27 ± 2 pA; Gbz: 1 pA ± 1 pA; after wash: 18 ± 8 pA; one-way ANOVA; **, P < 0.01; n = 4 sites). (E) Voltage dependence of ASCs is consistent with GABAAR-mediated currents. Representative traces of ASCs evoked at different holding potentials. (F) The extrapolated ASC reversal potential is not different from the EGABA predicted by the Goldman–Hodgkin–Katz equation. Continuous line, linear fit. Circles represent mean ± SEM. The extrapolated reversal potential is 2 ± 2 mV. (G) Block of Nav channels failed to affect laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during TTX, and after 5-min wash. (H) Summary plot of the effect of TTX on laser-evoked ASCs (control: 39 ± 9; in 1 µM TTX: 36 ± 8 pA; after wash: 44 ± 14 pA; one-way ANOVA; P > 0.05, n = 7 sites). In D and H, the black circles represent mean ± SEM for each condition, and gray dots represent the average amplitude of events evoked in individual release sites. In B, C, E, and G, arrows represent timing of the laser pulse.

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