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Mapping Neurotransmitter Identity in the Whole-Mount <i>Drosophila</i> Brain Using Multiplex High-Throughput Fluorescence <i>in Situ</i> Hybridization
摘要： Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence in situ hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount Drosophila melanogaster brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in Drosophila brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter co-expression, reveal neurotransmitter phenotypes of specific cell types and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large scale FISH detection of any mRNA in whole-mount fly brains.
关键词： Neurotransmitter,mRNA,Fluorescence in situ hybridization,Gene expression,Drosophila
更新于2025-11-21 11:08:12
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Behavior Responses to Chemical and Optogenetic Stimuli in Drosophila Larvae
摘要： An animal’s ability to navigate an olfactory environment is critically dependent on the activities of its first-order olfactory receptor neurons (ORNs). While considerable research has focused on ORN responses to odorants, the mechanisms by which olfactory information is encoded in the activities of ORNs and translated into navigational behavior remain poorly understood. We sought to determine the contributions of most Drosophila melanogaster larval ORNs to navigational behavior. Using odorants to activate ORNs and a larval tracking assay to measure the corresponding behavioral response, we observed that larval ORN activators cluster into four groups based on the behavior responses elicited from larvae. This is significant because it provides new insights into the functional relationship between ORN activity and behavioral response. Subsequent optogenetic analyses of a subset of ORNs revealed previously undescribed properties of larval ORNs. Furthermore, our results indicated that different temporal patterns of ORN activation elicit different behavioral outputs: some ORNs respond to stimulus increments while others respond to stimulus decrements. These results suggest that the ability of ORNs to encode temporal patterns of stimulation increases the coding capacity of the olfactory circuit. Moreover, the ability of ORNs to sense stimulus increments and decrements facilitates instantaneous evaluations of concentration changes in the environment. Together, these ORN properties enable larvae to efficiently navigate a complex olfactory environment. Ultimately, knowledge of how ORN activity patterns and their weighted contributions influence odor coding may eventually reveal how peripheral information is organized and transmitted to subsequent layers of a neural circuit.
关键词： olfactory receptor neuron,Drosophila larva,behavior,olfaction,optogenetics
更新于2025-09-23 15:23:52
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Electron microscopic observation of photoreceptor cells in directly inserted anesthetized <i>Drosophila</i> into a high-pressure freezing unit
摘要： The high-pressure freezing (HPF) technique is known to cryofix water-containing materials with little ice-crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light-exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight-exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.
关键词： photoreceptor cell,high-pressure freezing,Drosophila
更新于2025-09-23 15:23:52
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GFP-Forked, a genetic reporter for studying <i>Drosophila</i> oocyte polarity
摘要： The polarized organization of the Drosophila oocyte can be visualized by examining the asymmetric localization of mRNAs, which is supported by networks of polarized microtubules (MTs). In this study, we used the gene forked, the putative Drosophila homologue of espin, to develop a unique genetic reporter for asymmetric oocyte organization. We generated a null allele of the forked gene using the CRISPR-Cas9 system and found that forked is not required for determining the axes of the Drosophila embryo. However, ectopic expression of a truncated form of GFP-Forked generated a distinct network of asymmetric Forked, which first accumulated at the oocyte posterior and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. This localization pattern resembled that reported for the polarized MTs network. Indeed, pharmacological and genetic manipulation of the polarized organization of the oocyte showed that the filamentous Forked network diffused throughout the entire cortical surface of the oocyte, as would be expected upon perturbation of oocyte polarization. Finally, we demonstrated that Forked associated with Short-stop and Patronin foci, which assemble non-centrosomal microtubule-organizing centers. Our results thus show that clear visualization of asymmetric GFP-Forked network localization can be used as a novel tool for studying oocyte polarity.
关键词： ncMTOC,Oocyte,Drosophila,Polarity,Forked,Microtubules,CRISPR
更新于2025-09-23 15:22:29
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Light-induced opening of the TRP channel in isolated membrane patches excised from photosensitive microvilli from Drosophila photoreceptors
摘要： Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin, isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, TRP and TRPL. PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.
关键词： rhabdomere,photoreceptor,phototransduction,Drosophila,diacylglycerol,phospholipase C
更新于2025-09-23 15:21:21
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Probing Cell Mechanics with Bead-Free Optical Tweezers in the <em>Drosophila</em> Embryo
摘要： Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts' rheology.
关键词： Drosophila embryo,Developmental Biology,in vivo imaging,optical tweezers,Light sheet microscopy,force measurements,Issue 141,cell mechanics
更新于2025-09-23 15:21:01
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Maintenance of Rhodopsin levels in <i>Drosophila</i> photoreceptor and phototransduction requires Protein Kinase D
摘要： During Drosophila phototransduction, the G protein coupled receptor (GPCR) Rhodopsin (Rh1) transduces photon absorption into electrical signal via G-protein coupled activation of phospholipase C (PLC). Rh1 levels in the plasma membrane are critical for normal sensitivity to light. In this study, we report that Protein kinase D (dPKD) regulates Rh1 homeostasis in adult photoreceptors. Although eye development and retinal structure are unaffected in the dPKD hypomorph (dPKDH), it exhibited elevated levels of Rh1. Surprisingly, despite having elevated levels of Rh1, no defect was observed in the electrical response to light in these flies. By contrast the levels of another transmembrane protein of the photoreceptor plasma membrane, Transient receptor potential (TRP) was not altered in dPKDH. Our results indicate that dPKD is dispensable for eye development but is required for maintaining Rh1 levels in adult photoreceptors.
关键词： Electroretinogram (ERG),Phototransduction,Retinal degeneration,Rhodopsin,Drosophila,Rhodopsin loaded vesicle (RLVs),Protein Kinase D
更新于2025-09-19 17:15:36
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Distinct metabolic profiles in Drosophila sperm and somatic tissues revealed by two-photon NAD(P)H and FAD autofluorescence lifetime imaging
摘要： Metabolic profiles vary across all levels of biological diversity, from cells to taxa. Two-photon fluorescence lifetime imaging microscopy (FLIM) facilitates metabolic characterisation of biological specimens by assaying the intrinsic autofluorescence of the ubiquitous coenzymes NAD(P)H and FAD. the potential of this method for characterising the diversity of organismal metabolism remains largely untapped. Using FLIM in Drosophila melanogaster, we show tissue-specificity in fluorescence lifetime that reflects variation in redox patterns. In particular, sperm cells exhibited elevated glycolysis relative to other tissues. We also show that sperm metabolism is phenotypically plastic: compared to male-stored sperm, sperm stored in the female’s storage organ showed a substantial reduction in the protein-bound FAD lifetime fraction but no change in the NAD(P)H profile. This study represents the first ex vivo investigation of sperm metabolism using FLIM.
关键词： two-photon fluorescence lifetime imaging microscopy,FAD,Drosophila melanogaster,NAD(P)H,glycolysis,sperm metabolism,OxPHOS
更新于2025-09-12 10:27:22
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Deep-red fluorogenic probe for rapid detection of nitric oxide in Parkinson’s disease models
摘要： Nitric oxide (NO), as one of important gaseous signaling molecule in human body, has been associated with a variety of physiologic processes. However, excessive production of NO has been profoundly implicated in the pathogenesis of neurodegenerative disorders in particular, Parkinson's disease (PD). Therefore, accurate and facile detection of NO is of great significance for investigating its functions in PD, and the subsequent diagnosis and/or treatment. Herein, we developed a deep-red fluorogenic probe (BT-NH), with high sensitivity and good selectivity to detect NO, which was successfully used to visualize exogenous/endogenous NO level in living cells, and further applied to in vitro and in vivo PD models.
关键词： Parkinson’s disease,Drosophila,live cell imaging,nitric oxide,fluorogenic probe
更新于2025-09-04 15:30:14
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Discordant Responses to MAPK Pathway Stimulation Include Axonal Growths in Adult Drosophila Photoreceptors
摘要： Wallenda (WND) is the Drosophila member of a conserved family of dual leucine-zipper kinases (DLK) active in both neuronal regeneration and degeneration. We examined the role of WND over-expression on sensory neuron morphology by driving WND in multiple subtypes of Drosophila photoreceptors. WND overexpression under control of the pan-retinal GAL4 driver GMR causes multiple photoreceptor defects including cell death, rhabdomere degeneration, and axonal sprouting. Individual photoreceptor subtypes were assayed using GAL4 drivers speci?c for each photoreceptor class. Many R7 and R8 cells exhibit axonal sprouting while some show cell degeneration. Delaying the onset of WND overexpression until 20 days of age showed that older adult R7 cells retain the ability to initiate new axon growth. R1–6 photoreceptor cells degenerate in response to WND expression and exhibit rhodopsin loss and rhabdomere degeneration. RNAi knockdown of the MAPK signaling components Kayak (KAY) and Hemipterous (HEP) attenuates the WND-induced loss of Rh1 rhodopsin. UAS-induced HEP expression is similar to WND expression, causing degeneration in R1–6 photoreceptors and axonal sprouting in R7 photoreceptors. These results demonstrate that WND in adult Drosophila photoreceptor cells acts through MAPK signaling activity with both regenerative and degenerative responses. These photoreceptors provide a tractable experimental model to reveal cellular mechanisms driving contradictory WND signaling responses.
关键词： degeneration,dual leucine kinase,Drosophila,axon regeneration,photoreceptors
更新于2025-09-04 15:30:14