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. 2018 Mar 5;7:e31659. doi: 10.7554/eLife.31659

Figure 9. The roles of Nlg and Nrx in NAC dependent arbor construction appear decoupled from synapse formation and neurotransmission.

Figure 9.

(A–B) Synapse formation at the light microscope level appears largely unaffected by disruptions to Nrx-Nlg signalling. (A) Active zones at the pleural neuromuscular junctions in adult abdominal fillets from a wild-type control, an Nlg1 null, an Nrx null and an Nlg1 gain-of-function (Mef2-GAL4 > Nlg1 untagged) revealed by antibody staining against BRP. (B) Active zone densities calculated from BRP puncta in synaptic terminals. No significant differences were found between the densities of active zones in controls (0.98 ± 0.23 µm−2, n = 5) and Nlg1 nulls (0.88 ± 0.13 µm−2, n = 5) or controls and Nrx nulls (0.98 ± 0.15 µm−2, n = 5) (Nlg1 vs controls: Mann-Whitney U = 9, p=0.53, two-tailed. Nrx vs controls: Mann-Whitney U = 12, p=0.94, two-tailed). A small, but significant difference was found between active zone densities in Nlg1 gain-of-function terminals (1.29 ± 0.13 µm−2, n = 5) and controls (Mann-Whitney U = 1, p=0.0159, two-tailed). (C–F) Nlg1 overexpression causes changes in PM-Mn axonal arbor morphology, which result in altered connectivity and transmission. (C) GCaMP6m Δfs in a control and in a pupa expressing Nlg1untagged in muscles (Mef2-GAL4) staged within 10 hr of eclosion, (D). In controls, calcium events are generally synchronous across the pleural muscle field whereas in gain-of-function pupae events are often restricted to smaller muscle subsets. This is shown by breaks in the light-coloured bars on the kymograph and in the sequential images (D and F). (G–I) Loss of Nlg1 has a pronounced impact on locomotor ability (G) Automated body tracking performed using FlyLimbTracker (Uhlmann et al., 2017) in Icy (de Chaumont et al., 2012) of a control and an Nlg1 null, 2 days post-eclosion (H) Control flies have a significantly faster climb speed than Nlg1 nulls (controls: 2.25 ± 0.19 cm.s−1, n = 10. Nlg1: 1.60 ± 0.18 cm.s−1, n = 11. t(8.15) = 19, p<0.0001, t-test, two-tailed). (I) Motoneuron axon terminals (VGlut-LexA > myr::GFP) innervating the femur 1 day post-eclosion of an Nlg1 null and of a control. Bars represent SDs. Scale bars: 20 µm (A), 100 µm (D,F,I), 0.2 cm (G).