Figure 1. Electrically silent neurons do not degenerate while hyperexcitable neurons degenerate faster. A, The Gal4/UAS system was used to express UAS transgenes specifically in two fasciculated motoneurons (using the m12-Gal4 driver; Ritzenthaler et al., 2000; Xiong et al., 2010). UAS-mCD8::GFP was coexpressed with either UAS-Kir2.1 (Baines et al., 2001) to silence the neurons, UAS-SDN (Mosca et al., 2005) to induce hyperexcitability, or no transgene (WT control). Segmental nerves from third instar larvae were coimmunostained for GFP (green) and the neuronal MAP1B homolog Futsch (red). B, The extent of axonal degeneration at different time points after injury was quantified by scoring the axon degeneration index as described in Materials and Methods; n ≥ 20 axons for each condition and genotype. Genotypes used: WT [m12-Gal4, UAS-mCD8::GFP/+ (Canton S background)], Kir2.1 [m12-Gal4, UAS-mCD8::GFP/UAS-Kir2.1], SDN [m12-Gal4, UAS-mCD8::GFP/UAS-SDN]. C, Representative images of muscle 4 NMJs in wild-type (WT) or SDN animals. WT synapses remain predominantly intact at 10 h after injury; however, synapses formed by motoneurons expressing SDN (using the pan-motoneuron driver OK6-Gal4) were significantly degenerated by this time. Genotypes used: WT [OK6-Gal4/+; +/+], SDN [OK6-Gal4/+; UAS-SDN/+]. Scale bars, 100 μm. D, Quantification of the time course (0, 8, 10, 16, or 25 h after injury) of NMJ degeneration. The NMJ degeneration index was calculated from comparing the immunostaining with anti-HRP antibodies (red), and the presynaptic cytoskeletal marker, Futsch (green) as described in Materials and Methods; n ≥ 20 NMJs for each condition and genotype. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, based on one-way ANOVA with Tukey's post hoc test.