Figure 4.

A decrease in synaptic drive afforded robust and stable muscle activity. A, The firing rate of neurons is positively correlated to the synaptic drive they receive. B, Left, Diagram represents the configuration for recording compound EJPs from the muscle using intracellular electrodes. Middle, Exemplary traces of EJPs from the indicated genotypes. Right, Mean EJP amplitudes in individual experiments in control and RRA>eagSh(DN) larvae. C, mEJP traces (left) and graph of mean mEJP amplitudes in individual experiments (right) for the indicated genotypes. When changes in EJP and mEJP amplitudes in RRA>eagSh(DN) larvae (EJP = 19.37 + 1.12 mV, N = 12; mEJP = 0.68 + 0.02 mV, N = 12) were compared with the UAS/+ control (EJP = 24.76 + 1.0 mV, N = 11; mEJP = 0.60 + 0.02 mV, N = 12), they yielded the same significant results (EJP: p = 0.0018; mEJP: p = 0.0024) as when compared with RRA/+ control (EJP = 27.04 + 0.78 mV, N = 11; mEJP = 0.55 + 0.78 mV, N = 12; comparisons shown in B, C). We also ruled out that synaptic physiology was unaffected by the position of transgene insertion and leaky transgene expression, on finding that there was no significant difference in EJP (p = 0.0883) and mEJP (p = 0.0592) amplitudes between the RRA/+ and UAS/+ controls. Subsequent comparisons of synaptic changes were therefore conducted using only RRA/+ as control. D, Motor nerve terminals double-stained with HRP (red, top panels) and BRP (green, bottom panels) antibodies in the indicated genotypes. Selected BRP-labeled boutons were similarly magnified to highlight the occurrence of the active zones on boutons (1b: *; 1s: #). Scale bar, 10 µm. Active zone numbers per bouton were determined by counting the BRP puncta per bouton for 1s (RRA/+ = 4.50 + 0.26, N = 81; RRA>eagSh(DN) = 6.70 + 0.42, N = 81; p < 0.0001), and 1b (RRA/+ = 10.57 + 0.59, N = 69; RRA>eagSh(DN) = 10.05 + 0.64, N = 94; p = 0.5578) boutons in both genotypes. E, No changes in absolute 1b and 1s nerve terminal bouton numbers (left), or when normalized to muscle surface area (MSA) (right). F, For each motoneuron type within each genotype, individual symbols in each column represent the motoneuron firing rates within individual bursts, pooled from all experiments (whereas in Fig. 3A, right, each symbol represents the mean value across multiple bursts in each experiment). Each column therefore displays the full range of firing rates recorded for each motoneuron type within each genotype. G, For each genotype, individual symbols in each column represent the muscle firing rates within individual bursts, pooled from all experiments (whereas in Fig. 3B, right, each symbol represents the mean value across multiple bursts in each experiment). Each column therefore displays the full range of muscle firing rates recorded for each genotype. The physiological range represents the full range of firing rates observed in control (gray shaded area). H, The CV of muscle firing rates in individual experiments for both genotypes. I, In RRA>eagSh(DN) larvae, the decrease in synaptic drive (black arrow) caused the decrease in mean muscle firing rate from the old set point (black dot) to the new set point (blue dot). The decrease in synaptic drive did not, however, displace firing rates out of physiological range (gray shaded area, as shown in G), or change the relative variation in firing rates around the downshifted set point (CV₂, blue shaded area represents the mean value in H) compared with control (CV₁, charcoal shaded area represents the mean value in H). Muscle activity therefore remained robust and stable. ***p < 0.001, ^#p < 0.0001. NS, Not significant, p > 0.05. Graphs represent individual data points. Error bars indicate average + SEM.