Effects of TPC2 and LRRK2 expression in Drosophila dopaminergic neurons. (a) Schematic depicting the PER in response to a sucrose solution to assess dopaminergic-mediated movement in vivo. (b) Summary data (mean ± SEM) quantifying PER in transgenic flies (7 days) expressing TPC2-mCherry, LRRK2 G2019S, or both (mean ± SEM) in dopaminergic neurons. Each point represents the proportion of responsive animals from an independent line and trial (n = 7–17). **P < 0.01, ***P < 0.001 (two-way ANOVA, Tukey’s test). (c) Schematic depicting crawling of larvae to assess dopaminergic-mediated movement in vivo. (d) Exemplar crawling tracks recorded over a 60-s period from lines expressing the EV, wild-type LRRK2 (WT), R1441C LRRK2 (RC), G2019S LRRK2 without (GS) and with the K1906M kinase–inactivating mutation (GS KM). Scale bar: 1 mm. (e) Summary data (mean ± SEM) quantifying movement of the indicated single- and double-transgenic larvae (n = 20–42). Each point represents the distance traveled by an individual animal. ***P < 0.001, ****P < 0.0001 (one-way ANOVA, Tukey’s test). (f) Two-photon micrographs of an exemplar third instar larval (L3) Drosophila brain showing the expression of jGCaMP8m in dopaminergic neurons. Images were acquired at increasing zoom levels (I–IV). Scale bar = 10 µm. (g) Spontaneous Ca²⁺ signals recorded from dopaminergic neurons expressing jGCaMP8m from the indicated line. Each trace is a response from an individual neuron. Results are from three independent brain explants for each line. (h) Confocal micrographs of transgenic flies expressing mCherry-tagged TPC constructs. Low magnification image (h) showing the expression of TPC2 in the larval brain. Scale bar: 50 µm. Higher magnification images showing the expression of TPC2, the indicated mutant, and TPC1 in DL2 neurons (i). Scale bar: 5 µm. WT, wild type; PER, proboscis extension reflex; EV, empty vector.