Cold Spring Harbor Protocols

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Drosophila dendritic arborization (da) neurons are a powerful model for studying neuronal differentiation and sensory functions. A general experimental strength of this model is the examination of the neurons in situ in the body wall. However, for some analyses, restricted access to the neurons in situ causes difficulty; da neuron cultures circumvent this. Here, we outline isolation and culture techniques for larval and pupal da neurons. Investigators can use these cultures to perform high-resolution imaging, quantitative immunohistochemistry, and electrophysiology...

Sleep is likely a whole-brain phenomenon, with most of the brain probably benefiting from this state of decreased arousal. Recent advances in our understanding of some potential sleep functions, such as metabolite clearance and synaptic homeostasis, make it evident why the whole brain is likely impacted by sleep: All neurons have synapses, and all neurons produce waste metabolites. Sleep experiments in the fly Drosophila melanogaster suggest that diverse sleep functions appear to be conserved across all animals...

Nervous system formation involves the specification of neuron identity, followed by precise circuit construction; this includes controlling the pattern and connectivity of the dendrite arbor. Drosophila dendritic arborization (da) neurons are a powerful experimental model for studying dendrite arbor differentiation mechanisms. da neuron dendrite arbors elaborate in two dimensions in the body wall, making it easy to visualize them with high resolution. Immunostaining is a conventional method to examine arbor pattern and the subcellular distribution of proteins...

Mosaic analysis with a repressible cell marker (MARCM) is used in Drosophila research to create labeled homozygous mutant clones of cells in an otherwise heterozygous fly. It allows the study of the effect of embryonically lethal genes and the determination of cell autonomy for a mutant phenotype. When used in dendritic arborization (da) neurons with a fluorescent protein targeted to the plasma membrane, MARCM allows the identification of homozygous mutant neurons and clear imaging of the dendrite arbor in both live and fixed preparations...

Neurons have a complex dendritic architecture that governs information flow through a circuit. Manual quantification of dendritic arbor morphometrics is time-consuming and can be inaccurate. Automated quantification systems such as DeTerm help to overcome these limitations. DeTerm is a software tool that automatically recognizes dendrite branch terminals with high precision. It uses an artificial neural network to label the terminals, count them, and provide each terminal's positional data. DeTerm can recognize the dendritic terminals of Drosophila dendritic arborization (da) neurons, and it can also examine other types of neurons, including mouse Purkinje cells...

Genetically encoded calcium indicators (GECIs) allow for the noninvasive evaluation of neuronal activity in vivo, and imaging GECIs in Drosophila has become commonplace for understanding neural functions and connectivity in this system. GECIs can also be used as read-outs for studying sleep in this model organism. Here, we describe a methodology for tracking the activity of neurons in the fly brain using a two-photon (2p) microscopy system. This method can be adapted to perform functional studies of neural activity in Drosophila under both spontaneous and evoked conditions, as well as during spontaneous or induced sleep...

Live imaging approaches are essential for monitoring how neurons go through a coordinated series of differentiation steps in their native mechanical and chemical environment. These imaging approaches also allow the study of dynamic subcellular processes such as cytoskeleton remodeling and the movement of organelles. Drosophila dendritic arborization (da) neurons are a powerful experimental system for studying the dendrite arbor in live animals. da neurons are located on the internal surface of the body wall and, therefore, are easily accessible for imaging...

Sleep studies in Drosophila melanogaster rely mostly on behavioral read-outs to support molecular or circuit-level investigations in this model. Electrophysiology can provide an additional level of understanding in these studies to, for example, investigate changes in brain activity associated with sleep manipulations. In this protocol, we describe a procedure for performing multichannel local field potential (LFP) recordings in the fruit fly, with a flexible system that can be adapted to different experimental paradigms and situations...

Neurons receive, process, and integrate inputs. These operations are organized by dendrite arbor morphology, and the dendritic arborization (da) neurons of the Drosophila peripheral sensory nervous system are an excellent experimental model for examining the differentiation processes that build and shape the dendrite arbor. Studies in da neurons are enabled by a wealth of fly genetic tools that allow targeted neuron manipulation and labeling of the neuron's cytoskeletal or organellar components. Moreover, as da neuron dendrite arbors cover the body wall, they are highly accessible for live imaging analysis of arbor patterning...

Anautogenous female mosquitoes, which ingest a blood meal from warm-blooded vertebrates to produce eggs, have become a valuable model organism for investigating signaling pathways and physiological processes that occur during egg development. Different molecular pathways tightly regulate the initiation of egg development and are governed by a balance among different insect hormones. Gravid (mature egg-carrying) females deposit fully developed eggs at the end of each gonotrophic cycle, which is defined as the time interval between the ingestion of a blood meal to oviposition...

The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos...

Understanding how female mosquitoes find a prospective host is crucial to developing means that can interfere with this process. Many methods are available to researchers studying cues and orientation mechanisms that modulate female mosquito attraction to hosts. Behaviors that can be monitored with these assays include activation, taking flight, upwind flight along an odor plume (optomotor anemotaxis), close approach to the stimulus (including hovering), and landing. Video recording can three-dimensionally document flight tracks and can correlate overall distribution patterns and moment-to-moment movements with odor contact and the presence of nearby cues such as a visual target...

In insects, oocyte resorption (oosorption) or follicular atresia is one of the key physiological processes and evolutionary strategies used to optimize reproductive fitness. Mosquitoes are ideal model organisms for studying egg maturation in arthropods, as their follicle development is initiated only following the ingestion of a blood meal, followed by a carefully orchestrated series of hormonally regulated events leading to egg maturation. A cohort of approximately 100 follicles per mosquito ovary begin developing synchronously...

Western blot analysis is a well-known and dependable technique used to quantify protein abundance in a wide variety of samples. A major consideration for running a successful western blot is ensuring that the protein to be analyzed is purified appropriately. For work with membrane-bound proteins, traditional methods of protein processing such as the use of high-frequency sonication and ultracentrifugation to separate proteins from the membrane are being replaced with less time-consuming approaches. The use of a membrane fractionation kit, which involves the separation of membrane proteins from soluble (cytosolic) proteins, is effective in allowing for the quantification and analysis of membrane-bound proteins...

Immunohistochemistry (IHC) is an important technique that permits visualization of cellular components and for determining the presence and/or distribution of proteins or other macromolecules in tissue samples. Normally, IHC involves the detection of epitopes using an antigen-specific primary antibody and a secondary antibody coupled with a reporter molecule or fluorophore that can bind to the primary antibody, allowing for the spatial distribution of a protein of interest to be detected. Although normally IHC does not provide quantitative results compared to techniques such as enzyme-linked immunoassay or western blotting, it permits the localization, expression mapping, and distribution of target proteins in intact tissues...

Immunohistochemistry (IHC) is a powerful technique used for visualizing cellular components and determining the presence and/or location of proteins or other macromolecules in tissue samples. The classical IHC process involves the detection of epitopes using a highly specific primary antibody. This is followed by a secondary antibody that is coupled to a reporter molecule or fluorophore and capable of binding to the primary antibody and allowing for protein immunodetection. Although IHC does not routinely provide quantitative results compared to an enzyme-linked immunoassay or western blotting, it permits the localization of the proteins in intact tissues...

Fluorescence in situ hybridization (FISH) is a macromolecular recognition tool that uses RNA or DNA fragments combined with fluorophore- or digoxigenin-coupled nucleotides as probes to examine transcript localization through the presence or absence of complementary sequences in fixed tissues or samples under a fluorescent microscope. FISH technology has been highly effective for mapping genes and constructing a visual map of animal genomes. Here, we describe the application of FISH technology in the Aedes aegypti mosquito, where it is specifically used to localize receptor transcripts in gut tissues/organs...

The female mosquito is endowed with the responsibility of gene transfer from one generation to another through careful delivery of her eggs to an appropriate environment. To guarantee a successful reproduction process, female mosquitoes use an evaluative approach to select an oviposition site that will ensure that their progeny successfully hatch and develop from larvae to pupa to adult. Specific conditions must be met during oviposition site selection before gravid females deposit eggs, including but not limited to appropriate temperature, salinity, and pH; the presence of nutrition; and a low risk of predation...

This introduction reviews techniques used to examine the distribution and expression of gene transcripts and proteins in a variety of tissues/organs in the medically important global disease vector mosquito, Aedes aegypti Specifically, these methods allow the detection of cell-specific transcript expression by fluorescent in situ hybridization; facilitate immunohistochemical mapping of a protein of interest in whole-mount small tissue/organ samples; examine the subcellular localization of proteins, such as membrane transporters, through sectioning of paraffin-embedded tissue/organ samples; and finally, enable the efficient separation of cytosolic and membrane proteins for western blot analysis without the need for specialized equipment (e...