Fig. 5

Ca²⁺ activity measured along the RIS axon in freely moving animals correlates with slowing and reversals: a Strain used for tracking and Ca²⁺ imaging RIS in moving animals, expressing a red fluorescent marker in the pharyngeal terminal bulb (for tracking) and GCaMP6s in RIS. Micrographs of red (II) and green (III) fluorescence and merged color channels (I). Scale bar: 50 µm. b Image analysis after binarization and repositioning the soma involved reorienting the raw image (I), masking unspecific gut fluorescence (II), fitting a parabola (III), and measuring fluorescence intensity in perpendicular rectangular ROIs (IV). Dorsal is up, anterior to the left; CB cell body, NR nerve ring. c, d Upper panels: Representative traces of animal velocity (blue) and fluorescence intensity in the RIS nerve ring portion (green). Lower panels: Corresponding heat maps displaying the normalized fluorescence dynamics along the axon over time. RIS pictograms on the left indicate morphology including nerve ring (NR), branch (Br), and cell body (CB), the distance along the axon as well as the region of the nerve ring (green box) used for calculating the ΔF/F₀ traces in the upper panels, while dashed region shows extent of ROIs analyzed in lower panels (also in e). Distinct Ca²⁺ rise events in the branch region are boxed. e Mean normalized fluorescence heat map of n = 45 acquired Ca²⁺ events along the entire length of RIS, partially excluding the soma, by aligning time windows 6 s prior and post Ca²⁺ peaks (N = 11 animals)