Dynamic changes in synaptic connectivity and strength, which occur during both embryonic development and learning, have the tendency to destabilize neural circuits. To overcome this, neurons have developed a diversity of homeostatic mechanisms to maintain firing within physiologically defined limits. In this study, we show that activity-dependent control of mRNA for a specific voltage-gated Na ϩ channel [encoded by paralytic ( para)] contributes to the regulation of membrane excitability in Drosophila motoneurons. Quantification of para mRNA, by real-time reverse-transcription PCR, shows that levels are significantly decreased in CNSs in which synaptic excitation is elevated, whereas, conversely, they are significantly increased when synaptic vesicle release is blocked. Quantification of mRNA encoding the translational repressor pumilio ( pum) reveals a reciprocal regulation to that seen for para. Pumilio is sufficient to influence para mRNA. Thus, para mRNA is significantly elevated in a loss-of-function allele of pum ( pum bemused ), whereas expression of a fulllength pum transgene is sufficient to reduce para mRNA. In the absence of pum, increased synaptic excitation fails to reduce para mRNA, showing that Pum is also necessary for activity-dependent regulation of para mRNA. Analysis of voltage-gated Na ϩ current (I Na ) mediated by para in two identified motoneurons (termed aCC and RP2) reveals that removal of pum is sufficient to increase one of two separable I Na components (persistent I Na ), whereas overexpression of a pum transgene is sufficient to suppress both components (transient and persistent). We show, through use of anemone toxin (ATX II), that alteration in persistent I Na is sufficient to regulate membrane excitability in these two motoneurons.

An electrophysiology-based forward genetic screen has identified two genes, pickpocket11 (ppk11) and pickpocket16 (ppk16), as being necessary for the homeostatic modulation of presynaptic neurotransmitter release at the Drosophila neuromuscular junction (NMJ). Pickpocket genes encode Degenerin/ Epithelial Sodium channel subunits (DEG/ENaC). We demonstrate that ppk11 and ppk16 are necessary in presynaptic motoneurons for both the acute induction and long-term maintenance of synaptic homeostasis. We show that ppk11 and ppk16 are co-transcribed as a single mRNA that is upregulated during homeostatic plasticity. Acute pharmacological inhibition of a PPK11 and PPK16 containing channel abolishes the expression of short and long-term homeostatic plasticity without altering baseline presynaptic neurotransmitter release, indicating remarkable specificity for homeostatic plasticity rather than NMJ development. Finally, presynaptic calcium imaging experiments support a model in which a PPK11 and PPK16 containing DEG/ENaC channel modulates presynaptic membrane voltage and, thereby, controls calcium channel activity to homeostatically regulate neurotransmitter release.

Summary Two models have been proposed for Endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that Endophilin’s SH3 domain recruits essential endocytic proteins whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, while those disrupting membrane-bending cause significant defects. By anchoring Endophilin to the plasma membrane, we show that Endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of Endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of Endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, Endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This Endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.

Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen we have tested 162 new mutations for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. We show that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. We then provide evidence that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define new roles for Rab3-GAP and Rab3 and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity.