We also generated a precise excision of the ppk11Mi transposon that restores the ppk11 gene locus, assessed by sequence analysis. After the addition PD0325901 cell line of PhTx to the precise excision background (ppk11Precise),

the EPSP amplitude is returned to the size it was in the absence of PhTx (ppk11Precise; Figure 1D), and the homeostatic enhancement of presynaptic neurotransmitter release is restored to wild-type levels ( Figure 1E). Taken together, these data support the conclusion that disruption of the ppk11 gene blocks the rapid induction of synaptic homeostasis. Despite observing a complete block of synaptic homeostasis, there is no consistent alteration in baseline synaptic transmission caused by the loss of ppk11. First, we compare wild-type Screening Library chemical structure ( Figure 2B, black bars) with the ppk11PBac mutation ( Figure 2B, dark blue bars) and find no significant change in presynaptic release and only a minor change in mEPSP amplitude. Second, we compare the ppk11Mi mutation ( Figure 2B, light blue bars) with its appropriate genetic control, the precise excision of the ppk11Mi transposon (ppk11Precise; Figure 2B, open bars). There is no change in baseline release when this comparison is made. We note that there is a significant (p < 0.01) difference

in baseline release when we compare wild-type with either ppk11Mi or the ppk11Precise control line, and we attribute this to differences in genetic background. Third, we analyzed a trans-heterozygous combination of independently derived ppk11 mutations

(ppk11Mi/ ppk11PBac) and find no change in quantal content compared to wild-type ( Figure S1). In this trans-heterozygous combination, there is a decrease in mEPSP amplitude that correlates with a decrease in postsynaptic muscle input resistance (WT = 8.1 MΩ compared to ppk11Mi/ ppk11PBac = 3.9 MΩ; p < 0.01). Terminal deoxynucleotidyl transferase From these data, we conclude that disruption of ppk11 blocks synaptic homeostasis without altering baseline release, specifically when mutations are compared to their appropriate genetic control. This conclusion is supported by several additional experiments, presented below. We next examined synaptic homeostasis and baseline transmission at elevated external calcium (1 mM) that is within the range of what is thought to be physiological calcium (Figures 2C–2E). We first quantified mEPSP amplitudes in current-clamp mode, in which the signal-to-noise ratio is excellent, and then switched to two-electrode voltage-clamp mode to measure evoked synaptic currents. We observe a decrease in mEPSP amplitude when PhTx is applied to the wild-type NMJ at 1 mM calcium and we find that EPSC amplitudes are unchanged in the presence of PhTx, as expected for precise homeostatic compensation.