Fig. 6: Two-photon imaging of ACh activity using a genetically encoded sensor reveals that ACh release is localized to within a micron of its release site.

a A genetically encoded ACh sensor (ACh3.0) was selectively expressed in many starbursts, one of which was stimulated with a patch electrode. A red indicator (SeTau-647) was included in the electrode solution, to reveal the starburst’s morphology. In total, data was collected from 12 starbursts across 8 retinas. b The time course of changes in ACh3.0 sensor fluorescence (ΔF; see Eq. 2) in the boxed region shown in (a), upon depolarizing the starburst (top trace; Starburst stim.). The gray traces show individual trials while the black trace indicates the average over 7 trials. The red dotted line represents an exponential fit to the response decay (decay constant (τ) = 140 ms). c A high-resolution spatial map of the peak ACh3.0 responses in the boxed region shown in (a) (top; average over 7 trials). The white space indicates regions without ACh3.0 expression. The black contours indicate the 1 standard deviation level of a 2D Gaussian fit of the ACh3.0 responses. The bottom image shows the morphology of the stimulated starburst mapped on to the ACh3.0 response sites (dashed white contours). The yellow traces indicate the response timecourse (filtered) at each site. One such window was imaged from each starburst independently. d The average (avg.) spatial profile of the ACh3.0 responses (top) and the starburst morphology (bottom) for 81 sites measured in 12 starbursts (from 8 retinas). This indicated that the ACh3.0 responses were localized to within a micron of the starburst varicosities. Source data are provided as a Source Data file for Fig. 6b.