This is further supported by our finding that the organization of the ppk11/16 locus is conserved across multiple species of Drosophila ( Figure S2), representing approximately 30 million years of evolutionary divergence, selleck products suggesting that the

tandem placement of these genes is relevant to their regulation. Although the ppk11/16 locus is transcribed as a single RNA, we do not know whether these two could also be transcribed independently. This is important to consider when interpreting the results of transgenic RNAi. Specifically, this concerns whether UAS-ppk11-RNAi targets only the ppk11 gene or whether it will also affect expression of cotranscribed ppk16 and vice versa ( Figures 3 and 4). One further observation is worth considering in the trans-heterozygous data set. When mutations in ppk11 and ppk16 are placed selleck screening library in trans, synaptic homeostasis is blocked but baseline synaptic transmission (in the absence of PhTx) is not statistically different from

wild-type ( Figure 6G). The only significant change observed is a minor decrease in mEPSP amplitude in trans-heterozygous mutants. These data confirm, again, that homeostatic synaptic plasticity can be blocked by disruption of the ppk11/16 locus without a parallel change in baseline synaptic transmission. We hypothesize that PPK11/16-containing DEG/ENaC channels are inserted into the presynaptic plasma membrane to cause a homeostatic increase in presynaptic release and that these channels must be maintained on the plasma membrane in order to sustain the expression of synaptic homeostasis over several days. If this is correct, then pharmacological blockade of the PPK11/16 channel conductance should erase homeostatic potentiation that was previously induced by either PhTx application or by the presence of the GluRIIA mutation. This appears to be the case. DEG/ENaC channels are blocked by amiloride and its however derivatives (Kleyman and Cragoe, 1988). In Drosophila,

Benzamil has been shown to be a potent inhibitor of ENaC channel function ( Liu et al., 2003a). In our first set of experiments, wild-type NMJs were coincubated in 50 μM Benzamil and 10 μM PhTx. After a 10 min incubation, recordings were made in the presence of 50 μM Benzamil. Ten minutes is normally sufficient to induce potent homeostatic compensation ( Figure 7B). However, in the presence of 50 μM Benzamil, we saw a complete block in synaptic homeostasis ( Figures 7A and 7B). Next, we tested whether this effect could be washed out. Benzamil reversibly blocks DEG/ENaC channels, while PhTx irreversibly antagonizes Drosophila glutamate receptors ( Drummond et al., 1998 and Frank et al., 2006). Therefore, we were able to incubate larvae in PhTx and 50 μM Benzamil for 10 min and then wash out only Benzamil, limiting its action to the time when synaptic homeostasis was induced.