Figure 2. Ca²⁺ uncaging dose-response curve measured with presynaptic capacitance measurements.

(A) Illustration of the experimental setup showing the light path of the two-photon laser illumination (red line), the UV laser illumination (blue line), the electrophysiology amplifier (‘ephys.’), the red and green gate-able photomultiplier tubes (PMTs), and infrared LED illumination with oblique illumination via the condenser for visualization of the cells at the specimen plane by the camera (gray line) when the upper mirror is moved out of the light path (gray arrow). (B) Top: Two-photon microscopic image of a cMFB in the whole-cell configuration loaded with OGB-5N, Atto594, and DMn/ Ca²⁺. Positions of the patch pipette and line scan are indicated. Bottom: Two-photon line scan showing the fluorescence signal as measured through the green PMT, red PMT, and an overlay of the green and red channels. Arrow indicates the onset of the UV flash and dashed lines represent the flash-induced luminescence artefact as detected outside the cMFB. The lookup tables for the green and red channel were arbitrarily adjusted independent of the absolute values in C. (C) Top: change in fluorescence intensity within the cMFB for the green channel along with the corresponding flash-induced green artefact measured in the background. Middle: change in fluorescence intensity within the cMFB for the red channel along with the corresponding flash-induced red artefact. Bottom: green and red fluorescence signal after subtracting the flash-induced artefacts. (D) Top: Ca²⁺ signals of different concentrations elicited through Ca²⁺ uncaging in three different cells, the flash was blanked. Bottom: corresponding traces of capacitance recordings measured using a 5 kHz (left and middle) or 10 kHz sinusoidal stimulation (right). $\tau$ represents the time constant from a mono-exponential fit, ${\tau }_1$ represents the time constant of the fast component of a bi-exponential fit. (E) Traces of capacitance recordings showing the resolution limit in detecting fast release rates of >5 ms⁻¹ using 5 kHz sinusoidal stimulation or >10 ms⁻¹ using 10 kHz sinusoidal stimulation. (F) Plot of release rate versus post-flash Ca²⁺ concentration (n = 65 from 5-kHz- and from 15 10-kHz-recordings obtained from 80 cMFBs). The line represents a fit with a Hill equation (Equation 2) with best-fit values V_max = 1.7*10⁷ ms⁻¹, K_D = 7.2*10⁶ µM, and n = 1.2. Color coded symbols correspond to traces in (D – E). Gray symbols represent values above the resolution limit. (G) Plot of synaptic delay versus post-flash Ca²⁺ concentration (n = 64 from 5-kHz- and 15 from 10-kHz-recordings obtained from 79 cells). Note that one recording was removed from the analysis because the exponential fit led to a negative value of the delay. Color coded symbols correspond to traces in (D – E).

Figure 2—figure supplement 1. Measurement of the UV energy profile with caged fluorescein.

(A) 3D plot of the fluorescence profile in response to UV uncaging of caged-fluorescein at different z-positions. (B) Magnification of the middle part in panel (A) over a range of 10 μm.