Citation:Brusich DJ, Spring AM and Frank CA (2015) A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity. Front. Cell. Neurosci. 9:107. doi: 10.3389/fncel.2015.00107 A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity Homeostatic synaptic plasticity (HSP) helps neurons and synapses maintain physiologically appropriate levels of output. The fruit fly Drosophila melanogaster larval neuromuscular junction (NMJ) is a valuable model for studying HSP. Here we introduce a genetic tool that allows fruit fly researchers to examine the lifelong maintenance of HSP with a single cross. The tool is a fruit fly stock that combines the GAL4/UAS expression system with RNA interference (RNAi)-based knock down of a glutamate receptor subunit gene. With this stock, we uncover important new information about the maintenance of HSP. We address an open question about the role that presynaptic Ca V 2-type Ca 2+ channels play in NMJ homeostasis. Published experiments have demonstrated that hypomorphic missense mutations in the Ca V 2 α1a subunit gene cacophony (cac) can impair homeostatic plasticity at the NMJ. Here we report that reducing cac expression levels by RNAi is not sufficient to impair homeostatic plasticity. The presence of wild-type channels appears to support HSP-even when total Ca V 2 function is severely reduced. We also conduct an RNAi-and electrophysiology-based screen to identify new factors required for sustained homeostatic signaling throughout development. We uncover novel roles in HSP for Drosophila homologs of Cysteine string protein (CSP) and Phospholipase Cβ (Plc21C). We characterize those roles through follow-up genetic tests. We discuss how CSP, Plc21C, and associated factors could modulate presynaptic Ca V 2 function, presynaptic Ca 2+ handling, or other signaling processes crucial for sustained homeostatic regulation of NMJ function throughout development. Our findings expand the scope of signaling pathways and processes that contribute to the durable strength of the NMJ.

Forms of homeostatic plasticity stabilize neuronal outputs and promote physiologically favorable synapse function. A well-studied homeostatic system operates at the Drosophila melanogaster larval neuromuscular junction (NMJ). At the NMJ, impairment of postsynaptic glutamate receptor activity is offset by a compensatory increase in presynaptic neurotransmitter release. We aim to elucidate how this process operates on a molecular level and is preserved throughout development. In this study, we identified a tyrosine kinase-driven signaling system that sustains homeostatic control of NMJ function. We identified C-terminal Src Kinase (Csk) as a potential regulator of synaptic homeostasis through an RNAi- and electrophysiology-based genetic screen. We found that Csk loss-of-function mutations impaired the sustained expression of homeostatic plasticity at the NMJ, without drastically altering synapse growth or baseline neurotransmission. Muscle-specific overexpression of Src Family Kinase (SFK) substrates that are negatively regulated by Csk also impaired NMJ homeostasis. Surprisingly, we found that transgenic Csk-YFP can support homeostatic plasticity at the NMJ when expressed either in the muscle or in the nerve. However, only muscle-expressed Csk-YFP was able to localize to NMJ structures. By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII). By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals–but markedly increased in Csk mutant animals. Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis. Based on these data, we propose that Csk and its SFK substrates impinge upon homeostatic control of NMJ function by regulating downstream expression or localization of FasII.

Several million traumatic brain injury (TBI) events are reported in the United States annually. However, mild TBI events often go unreported, and mild and repetitive mild TBI conditions are challenging to model. Fruit flies (Drosophila melanogaster) have gained traction for the study of TBI. The best-characterized fly TBI model is the high-impact trauma (HIT) method. We replicated the HIT method and confirmed several previous findings at the standard level of injury severity. We then expanded upon the HIT model by characterizing mortality across three reduced levels of injury severity. Importantly, we found reduced mortality with reduced injury severity and synergistic effects on mortality in response to repetitive TBI by our moderate injury conditions. Last, we compared moderate, repetitive TBI to a single severe TBI via assessment of the pattern of mortality and geotaxis performance in the 24 h following TBI. We found the number and severity of injuries could result in different patterns of death, while all TBI conditions led to impaired geotaxis compared to uninjured flies at 0.5 h and 6 h post-TBI. Thus, we have extended a wellcharacterized model of TBI in flies, and shown the utility of this model for making unique insights into TBI across various severities, injury numbers, and time-points post-injury.

Gain-of-function mutations in the human CaV2.1 gene CACNA1A cause familial hemiplegic migraine type 1 (FHM1). To characterize cellular problems potentially triggered by CaV2.1 gains of function, we engineered mutations encoding FHM1 amino-acid substitutions S218L (SL) and R192Q (RQ) into transgenes of Drosophila melanogaster CaV2/cacophony. We expressed the transgenes pan-neuronally. Phenotypes were mild for RQ-expressing animals. By contrast, single mutant SL- and complex allele RQ,SL-expressing animals showed overt phenotypes, including sharply decreased viability. By electrophysiology, SL- and RQ,SL-expressing neuromuscular junctions (NMJs) exhibited enhanced evoked discharges, supernumerary discharges, and an increase in the amplitudes and frequencies of spontaneous events. Some spontaneous events were gigantic (10–40 mV), multi-quantal events. Gigantic spontaneous events were eliminated by application of TTX–or by lowered or chelated Ca2+–suggesting that gigantic events were elicited by spontaneous nerve firing. A follow-up genetic approach revealed that some neuronal hyperexcitability phenotypes were reversed after knockdown or mutation of Drosophila homologs of phospholipase Cβ (PLCβ), IP3 receptor, or ryanodine receptor (RyR)–all factors known to mediate Ca2+ release from intracellular stores. Pharmacological inhibitors of intracellular Ca2+ store release produced similar effects. Interestingly, however, the decreased viability phenotype was not reversed by genetic impairment of intracellular Ca2+ release factors. On a cellular level, our data suggest inhibition of signaling that triggers intracellular Ca2+ release could counteract hyperexcitability induced by gains of CaV2.1 function.

2Gain-of-function mutations in the human Ca V 2.1 gene CACNA1A cause familial hemiplegic migraine type 2 3 1 (FHM1). To characterize cellular problems potentially triggered by Ca V 2.1 gains of function, we 2 4 engineered mutations encoding FHM1 amino-acid substitutions S218L (SL) and R192Q (RQ) into 2 5 transgenes of Drosophila melanogaster Ca V 2/cacophony. We expressed the transgenes pan-neuronally. 2 6Phenotypes were mild for RQ-expressing animals. By contrast, single mutant SL-and complex allele 2 7 RQ,SL-expressing animals showed overt phenotypes, including sharply decreased viability. By 2 8 electrophysiology, SL-and RQ,SL-expressing neuromuscular junctions (NMJs) exhibited enhanced 2 9 evoked discharges, supernumerary discharges, and an increase in the amplitudes and frequencies of 3 0 spontaneous events. Some spontaneous events were gigantic (10-40 mV), multi-quantal events. 1Gigantic spontaneous events were eliminated by application of TTX -or by lowered or chelated Ca 2+ -3 2 suggesting that gigantic events were elicited by spontaneous nerve firing. A follow-up genetic approach 3 3 revealed that some neuronal hyperexcitability phenotypes were reversed after knockdown or mutation of 3 4 Drosophila homologs of phospholipase Cβ (PLCβ), IP 3 receptor, or ryanodine receptor (RyR) -all factors 3 5 known to mediate Ca 2+ release from intracellular stores. Pharmacological inhibitors of intracellular Ca 2+ 3 6 store release produced similar effects. Interestingly, however, the decreased viability phenotype was not 3 7 reversed by genetic impairment of intracellular Ca 2+ release factors. On a cellular level, our data suggest 3 8 inhibition of signaling that triggers intracellular Ca 2+ release could counteract hyperexcitability induced by 3 9 gains of Ca V 2.1 function. 4 0 4 1 AUTHOR SUMMARY 4 2Prior research has demonstrated that gain-of-function mutations in a gene important for 4 3 neurotransmission (CACNA1A) are known to cause migraine in humans. We attempted to mimic some of 4 4 those gain-of-function mutations in a simple genetic model organism and to examine neurotransmission 4 5 by electrophysiology. Our findings yield potential clues as to how particular migraine-causing mutations 4 6 may impact neurophysiology on a cellular level. We used the fruit fly Drosophila melanogaster and its 4 7 3 model synapse, the neuromuscular junction (NMJ) to perform our studies. We document three main 4 8 advances: 1) characterization of fruit fly models harboring gain-of-function calcium channel alterations 4 9 known to cause human familial hemiplegic migraine type 1 (FHM1); 2) characterization of hyperactive 5 0 neurotransmission caused by one of these alterations; and 3) an ability to quell hyperactive 5 1 neurotransmission by impairing intracellular Ca 2+ store release, through both genetic and 5 2 pharmacological means. Our work contributes to a broader understanding of how pathological mutations 5 3 could impact cellular physiology. More generally, the utilization of genetic model organisms promises to 5 4uncover...