(A) Calcium activity in PM-Mn axon terminals at 32, 42, 48 and 74 hr APF, measured by changes in GCaMP6m fluorescence (Δf) (driven by OK371-GAL4). Sequential images show examples of Δfs displayed as heat registered kymographs (B) GCaMP6m Δfs in PM-Mn axon terminals at 78 hr APF in a pupa injected with a control solution of PBS (top) and a solution of 0.5 mM TTX in PBS (bottom) into the abdomen of a pupa (inset). (C) Arborisations in segment A3 (OK371-GAL4 > myr::GFP) at 79 hr APF following injection at 32 hr APF with a PBS solution and (D) solution containing TTX. (E) Motoneuron axon terminal of a VGlut null MARCM clone (green) alongside a VGlut heterozygous terminal in L3 larva stained with an antibody against VGlut (magenta). (F) The anterior-most arborisation in segment A3 (A3–A) of a heterozygous VGlut control expressing myr::GFP and mCD8::GFP (VGlutNMJX-GAL4) at 85 hr APF (G) an equivalent arborisation in VGlut null MARCM clone at 80 hr APF. (H and I) Arborisations of VGlut null A3-A MARCM clones do not differ significantly from controls in arbor area (controls: 44643 ± 2982 µm2, VGlut: 46518 ± 3083 µm2, n1,2 = 10, t(18) = 1.38, p=0.18, t-test, two-tailed). Marginally greater total arbor lengths (controls: 1812 ± 134 µm, VGlut: 2024 ± 216 µm, n1,2 = 10, t(18) = 2.64, p=0.02, t-test, two-tailed). (J) Muscle GCaMP6m (Mef2-GAL4) Δf in response to motoneuron activation at 49 hr, 54 hr, 68 hr and 71 hr APF, using the warmth-gated ion channel TRPA1 (VGlut-LexA > dTRPA1). Red bars indicate time at the restrictive temperature (22°C), green bars indicate time at the permissive temperature (30°C). (K) Images show muscle GCaMP6m Δfs before and after activation of motoneurons with dTRPA1 at 71 hr APF. (L) Organisation of BRP::RFP puncta through development. Time series of the same arbor segment at 34 hr, 48 hr and 56 hr APF shows a shift from puncta at branch points (white arrowheads) to a distribution along branch lengths (yellow arrowheads). (M) Between 72 hr and 77 hr APF, 12.55 ± 5.79% of total BRP::RFP puncta are found at branch points/bases of filopodia, yet the majority (89.38 ± 6.73%) of branch points/bases of filopodia host puncta (n = 5). (N) Size and distribution of endogenous, GFP labelled BRP. The puncta of BRP (indicated by arrowheads) are larger and more heterogenous in diameter during early arbor growth (32 hr APF) than at later stages (70hAPF), when the major phase of outgrowth has ceased (OK371-GAL4 > mCD8::ChRFP). (O) Diameters of endogenous BRP::GFP puncta measured as the full width at half maximum of peaks in fluorescence are significantly greater at 32 hr APF (435.8 ± 177.8 nm, n = 144) than at 72 hr APF (319.6 ± 87.9 nm, n = 177) (Mann-Whitney U = 6935, p<0.0001, two-tailed). (P) Localisation of GluRIIE at 48 hr APF. This GFP tagged version of GluRIIE (FlyFos; Sarov et al., 2016) driven under the control of the native transcriptional unit cannot be seen in the postsynaptic membrane before or at 48 hr APF. (OK371-GAL4 > mCD8::ChRFP). (Q) At 85 hr APF conspicuous GluRIIE::GFP clusters (arrowheads) are found along the axon terminals. Bars represent SDs. Scale bars: 50 µm (A,C,D,F,G), 10 µm (E), 100 µm (K), 25 µm (L), 5 µm (N,P,Q).