Motion artifacts remain a barrier to in vivo calcium imaging in Drosophila melanogaster larvae. Here, we evaluate a multimodal immobilization approach that combines a Pluronic F-127 (PF-127) hydrogel with brief diethyl ether vapor exposure (5 minutes, 25 degrees C) and compare it against hydrogel-only immobilization using custom MATLAB-based analysis software that performs NoRMCorre rigid motion correction. In wide-field GFP recordings at 1 Hz over approximately 60 minutes (N = 15 per group), the multimodal condition significantly reduced motion across all three core metrics after FDR correction (all q < 0.001), with large effect sizes for mean speed (Hedges' g = -1.18) and median step size (g = -1.36). In a secondary analysis of the first 30 minutes, uniformly large effect sizes (|g| = 1.10-1.51) were observed, consistent with stronger initial chemical immobilization that partially wanes over the recording period. We implemented a dual-flag quality control system that distinguishes motion data reliability from ROI detection eligibility. Control calcium recordings (33.33 Hz, ~5 minutes; N = 23) yielded 368 ROIs with a mean SNR 30.4 +/- 16.9 and an event rate of 0.228 +/- 0.113 Hz. Experimental recordings (N = 21) yielded 295 ROIs with SNR 18.0 +/- 10.6 and event rate 0.309 +/- 0.188 Hz. SNR was higher in controls (Cliff's delta = 0.50, p < 0.001), while event rate was modestly higher in the experimental group at the ROI level (delta = -0.22, p < 0.001), though this difference did not reach significance at the sample level, suggesting altered but not suppressed calcium dynamics. These results support a practical, accessible immobilization workflow for larval calcium imaging. ### Competing Interest Statement The authors have declared no competing interest.
Homeostatic mechanisms protect synapses from destabilizing challenges throughout an organism's lifespan, ensuring stable yet flexible neural network activity. To delineate the molecular basis of presynaptic homeostatic potentiation (PHP), we conducted a comprehensive, in vivo CRISPR/Cas9-based screen of all 16 glutamate receptor (GluR) genes encoded in the Drosophila genome. We first generated a complete expression atlas across larval and adult stages, identifying nine GluRs expressed in presynaptic motor neurons. We then generated null mutants for all 16 GluRs and screened them at the larval neuromuscular junction. While the loss of any single presynaptic GluR did not affect baseline synaptic growth or neurotransmission, our screen revealed a selective and critical requirement for the kainate receptor subunit ekar in the expression of chronic PHP. Further genetic analysis indicates that Ekar functions coordinately with the kainate receptor subunits KaiRID and Ukar within a shared pathway to promote this plasticity. Mechanistically, Ekar acts downstream of active zone remodeling to drive the homeostatic enhancement of presynaptic Ca2+ influx, which is the defining feature of chronic PHP. Together, this genome-wide analysis establishes a definitive functional atlas for the Drosophila glutamate receptome and highlights a specialized, essential role for Ekar in stabilizing long-term synaptic homeostasis. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Neurological Disorders and Stroke, https://ror.org/01s5ya894, NS091546 U.S. National Science Foundation, https://ror.org/021nxhr62, IOS-2417451 United States-Israel Binational Science Foundation, https://ror.org/00j8z2m73, 2024612
The neuromuscular junction (NMJ) of larval Drosophila is widely used for studying synaptic transmission. Larval body wall muscles express five ionotropic glutamate receptor (iGluR) subunits that assemble into two tetrameric complexes, with subunit composition determining the strength and plasticity of synaptic transmission. Because NMJ function has been extensively characterized in larvae, it is often assumed that adult fly NMJs have similar molecular composition, despite substantial differences between life stages. Here, we systematically compare glutamate receptor expression across larval and adult Drosophila muscles. We find that adult leg and flight muscles exhibit different iGluR expression than larvae, lacking several receptors previously considered essential for viability and NMJ function. Adjacent muscles within the adult femur express distinct iGluRs, suggesting specialization of flexor and extensor muscles. Finally, the glutamate-gated chloride channel (GluClα) is expressed extrasynaptically in adult but not larval muscle fibers. Our results reveal unexpected heterogeneity in glutamate receptor expression across muscles and developmental stages, challenging assumptions about the uniformity of neuromuscular function and demonstrating the need for muscle-specific analyses in flies and other animals. ### Competing Interest Statement The authors have declared no competing interest.
Precancerous oncogenic activation in a target organ often induces senescence, a tumor-suppressive response known as oncogene-induced senescence (OIS). Clinical observations indicate a strong association of metabolic syndrome (MetS) with the precancerous and early-stage cancers. Notably, cells displaying OIS are characterized by a senescence-associated secretory phenotype (SASP), in which they secrete factors, including inflammatory cytokines. Thus, SASP from cells displaying OIS may trigger host MetS, which likely underpins its association with cancers, such as colorectal cancer (CRC). Here, we tested this hypothesis and show that, in Drosophila, the activated RasG12V oncogene, which is frequently implicated in human CRC, induces OIS in imaginal disc epithelium and systemically triggers host larval MetS via the conserved cytokine Upd1/IL6. Thus, the larval host with RasG12V-induced epithelial OIS displays MetS, characterized by obesity, increased lipid and glycogen accumulation in the fat body, and altered insulin signaling, marked by transition from hyperinsulinemia to insulin resistance, all at a precancerous stage. Further, we also noted hyperphagia and increased expression of insulin-like peptides (dILP2/3/5) in the brain of larvae displaying RasG12V-induced OIS. Notably, RasG12V-induced OIS is systemically relayed, leading to activation of a senescence-like program in the distant fat body. Genetic suppression of upd1 or pharmacological intervention with the senomorphic agent, Metformin, attenuated fat body senescence and mitigated MetS-associated phenotypes. Our findings thus identify a causal relationship between OIS and host MetS, suggesting its utility as an early biomarker for detecting cancers such as CRC and its potential as a prophylactic target. ### Competing Interest Statement The authors have declared no competing interest.
Cells with high secretory and metabolic loads such as adipocytes and hepatocytes rely on constitutive activation of stress response pathways for their homeostatic function and to cope with exogenous stressors such as nutrient deprivation or excess lipids. The evolutionarily conserved stress response factor, Activating Transcription Factor 4 (ATF4), is known to be required for both homeostatic function and exogenous burden in these tissues. However, the molecular mechanism by which ATF4 specifies distinct transcriptional targets under homeostasis versus stress conditions remains an open question. Here, we use the Drosophila larval fat tissue as a model to establish that ATF4 interacts with the steroid hormone receptor Ecdysone Receptor (EcR) to transcriptionally activate and repress genes involved in lipid metabolism. Our data show that EcR and its ligand, 20-hydroxyecdysone, are required for transcription activation of the bona fide ATF4 target 4E-BP in the fat body. We also find that ATF4 and EcR co-repress transcription of the triglyceride lipase brummer ( bmm ). In Förster resonance energy transfer (FRET) experiments, we find that ATF4 interacts with EcR and does so by competing with the canonical EcR-binding partner, Ultraspiracle (Usp). Using a genetic model of nutrient deprivation, we find that while EcR is required for homeostatic signaling, it is dispensable for the elevated ATF4 signaling associated with nutrient deprivation as measured by induction of 4E-BP . Together, these data provide a mechanistic starting point for understanding how changes in interaction partner allows ATF4 to engage in context-specific transcriptional programs in metabolic tissues. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences, https://ror.org/04q48ey07, R35GM150516, R00GM149982
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