Protocols in Current Issue
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0 Q&A 350 Views Jan 20, 2023

Anorexia nervosa (AN) is a devastating neuropsychiatric disease with a prevalence rate of approximately 0.3%–1% among women and morbidity and mortality rates among the highest of all neuropsychiatric disorders. The disease etiology is complex but primarily characterized by reduced food intake and body weight, and intense anxiety and fear associated with gaining weight. Existing rodent models of AN are useful and capture features of the disease, but either require specialized genetic mouse models or are difficult to implement in mice. Here, we describe two simple mouse models of stress-induced anorexia that are easy to implement in basic research labs, and capture core features associated with AN, such as reduced food intake in the context of social/physical stress and increased anxiety-related behavior. These protocols provide reproducible and robust assays for stress-induced anorexia and may be implemented with additional assays to probe the neural circuitry mediating the effects of psychological stress on feeding in mice.

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0 Q&A 333 Views Jan 20, 2023

Targeted protein degradation (TPD) facilitates the selective elimination of unwanted and pathological cellular cargoes via the proteasome or the lysosome, ranging from proteins to organelles and pathogens, both within and outside the cell. Currently, there are several in vitro and in vivo protocols that assess the degradative potency of a given degrader towards a myriad of targets, most notably soluble, monomeric oncoproteins. However, there is a clear deficiency of methodologies to assess the degradative potency of heterobifunctional chimeric degraders, especially those in the autophagy space, against pathological, mutant tau species, such as detergent-insoluble oligomers and high-molecular aggregates. The protocol below describes both in vitro and in vivo biochemical assays to induce tau aggregation, as well as to qualitatively and quantitatively measure the degradative potency of a given degrader towards said aggregates, with specific applications of the AUTOTAC (AUTOphagy-TArgeting Chimera) platform provided as an example. A well-defined set of methodologies to assess TPD-mediated degradation of pathological tau species will help expand the scope of the TPD technology to neurodegeneration and other proteinopathies, in both the lab and the clinic.

Graphical abstract

Overview of assays observing elimination of tauP301L aggregates with AUTOTAC. (A) Description of the biological working mechanism of heterobifunctional chimeric AUTOTAC degraders. (B) Schematic illustration of assays described in this paper.

0 Q&A 215 Views Jan 20, 2023

Lysosomes play a central role in signaling, nutrient sensing, response to stress, and the degradation and recycling of cellular content. Defects in lysosomal digestive enzymes or structural components can impair lysosomal function with dire consequences to the cell, such as neurodegeneration. A number of methods exist to assess lysosomal stress in the model Drosophila, such as specific driver and reporter strains, transmission electron microscopy, and the investigation of gene expression. These methods, however, can be time consuming and, in some cases, costly. The procedure described here provides a quick, reliable, and low-cost approach to measure lysosomal stress in the Drosophila brain. Using fluorescence confocal microscopy and the LysoTracker staining, this protocol allows for the direct measurement of lysosome size and number. This method can be used to assess lysosomal stress under a number of different genetic and environmental scenarios in the Drosophila brain.

0 Q&A 377 Views Jan 5, 2023

Molecular characterization of different cell types in rodent brains is a widely used and important approach in neuroscience. Fluorescent detection of transcripts using RNAscope (ACDBio) has quickly became a standard in situ hybridization (ISH) approach. Its sensitivity and specificity allow for the simultaneous detection of between three and forty-eight low abundance mRNAs in single cells (i.e., multiplexing or hiplexing), and, in contrast to other ISH techniques, it is performed in a shorter amount of time. Manual quantification of transcripts is a laborious and time-consuming task even for small portions of a larger tissue section. Herein, we present a protocol for creating high-quality images for quantification of RNAscope-labeled neurons in the rat brain. This protocol uses custom-made scripts within the open-source software QuPath to create an automated workflow for the careful optimization and validation of cell detection parameters. Moreover, we describe a method to derive mRNA signal thresholds using negative controls. This protocol and automated workflow may help scientists to reliably and reproducibly prepare and analyze rodent brain tissue for cell type characterization using RNAscope.

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0 Q&A 167 Views Jan 5, 2023

In nature, parasitoid wasp infections are a major cause of insect mortality. Parasitoid wasps attack a vast range of insect species to lay their eggs. As a defense, insects evolved survival strategies to protect themselves from parasitoid infection. While a growing number of studies reported both host defensive tactics and parasitoid counter-offensives, we emphasize that this parasite–host relationship presents a unique ecological and evolutionary relevant model that is often challenging to replicate in a laboratory. Although maintaining parasitoid wasp cultures in the laboratory requires meticulous planning and can be labor intensive, a diverse set of wasp species that target many different insect types can be maintained in similar culture conditions. Here, we describe the protocol for culturing parasitoid wasp species on Drosophila larvae and pupae in laboratory conditions. We also detail an egg-laying assay to assess the reproductive modification of Drosophila females in response to parasitoid wasps. This behavioral study is relatively simple and easily adaptable to study environmental or genetic influences on egg-laying, a readout for female germline development. Neither the parasitoid culture conditions or the behavioral assay require special supplies or equipment, making them a powerful and versatile approach in research or teaching laboratory settings.

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0 Q&A 287 Views Dec 5, 2022

Pathogen invasion of the central nervous system (CNS) is an important cause of infection-related mortality worldwide and can lead to severe neurological sequelae. To gain access to the CNS cells, pathogens have to overcome the blood–brain barrier (BBB), a protective fence from blood-borne factors. To study host–pathogen interactions, a number of cell culture and animal models were developed. However, in vitro models do not recapitulate the 3D architecture of the BBB and CNS tissue, and in vivo mammalian models present cellular and technical complexities as well as ethical issues, rendering systematic and genetic approaches difficult. Here, we present a two-pronged methodology allowing and validating the use of Drosophila larvae as a model system to decipher the mechanisms of infection in a developing CNS. First, an ex vivo protocol based on whole CNS explants serves as a fast and versatile screening platform, permitting the investigation of molecular and cellular mechanisms contributing to the crossing of the BBB and consequences of infection on the CNS. Then, an in vivo CNS infection protocol through direct pathogen microinjection into the fly circulatory system evaluates the impact of systemic parameters, including the contribution of circulating immune cells to CNS infection, and assesses infection pathogenicity at the whole host level. These combined complementary approaches identify mechanisms of BBB crossing and responses of a diversity of CNS cells contributing to infection, as well as novel virulence factors of the pathogen.

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Procedures flowchart.
Mammalian neurotropic pathogens could be tested in two Drosophila central nervous system (CNS) infection setups (ex vivo and in vivo) for their ability to: (1) invade the CNS (pathogen quantifications), (2) disturb blood–brain barrier permeability, (3) affect CNS host cell behaviour (gene expression), and (4) alter host viability.

0 Q&A 241 Views Dec 5, 2022

Pavlovian fear conditioning is a widely used procedure to assess learning and memory processes that has also been extensively used as a model of post-traumatic stress disorder (PTSD). Freezing, the absence of movement except for respiratory-related movements, is commonly used as a measure of fear response in non-human animals. However, this measure of fear responses can be affected by a different baseline of locomotor activity between groups and/or conditions. Moreover, fear conditioning procedures are usually restricted to a single conditioned stimulus (e.g., a tone cue, the context, etc.) and thus do not depict the complexity of real-life situations where traumatic memories are composed of a complex set of stimuli associated with the same aversive event. To overcome this issue, we use a conditioned lick suppression paradigm where water-deprived mice are presented with a single conditioned stimulus (CS, a tone cue or the context) previously paired with an unconditioned stimulus (US, a foot shock) while consuming water. We use the ratio of number of licks before and during the CS presentation as a fear measure, thereby neutralizing the potential effect of locomotor activity in fear responses. We further implemented the conditioned lick suppression ratio to assess the effect of cue competition using a compound of contextual and tone cue conditioned stimuli that were extinguished separately. This paradigm should prove useful in assessing potential therapeutics and/or behavioral therapies in PTSD, while neutralizing potential confounding effects between locomotor activity and fear responses on one side, and by considering potential cue-competition effects on the other side.

Graphical abstract

Schematic representation of the compound context-cue condition lick suppression procedure. Illustration reproduced from Bouchekioua et al. (2022).

0 Q&A 806 Views Nov 20, 2022

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)–acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13–18 candidate biosensors and 44–84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner.

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0 Q&A 417 Views Nov 20, 2022

Actin filaments are essential for various biological activities in eukaryotic cellular processes. Available in vitro experimental data on these systems often lack details and information on sample preparation protocols and experimental techniques, leading to unreproducible results. Additionally, different experimental techniques and polymerization buffers provide different, sometimes contradictory results on the properties of these systems, making it substantially difficult to gather meaningful data and conclusive information from them. This article presents a robust, accurate, detailed polymerization protocol to prepare high-quality actin filament samples for light scattering experiments. It has been shown to provide unicity and consistency in preparing stable, dispersed, aggregates-free, homogenous actin filament samples that could benefit many other scientific research groups currently working in the field. To develop the protocol, we used conventional actin buffers in physiological conditions. However, it can easily be adapted to prepare samples using other buffers and biological fluids. This protocol yielded reproducible results on essential actin filament parameters such as the translational diffusion coefficient and electrophoretic mobility. Overall, suitable modifications of the proposed experimental method could generate accurate, reproducible light scattering results on other highly charged anionic filaments commonly found in biological cells (e.g., microtubules, DNAs, RNAs, or filamentous viruses).

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0 Q&A 798 Views Oct 20, 2022

When understanding the neuronal function of a specific neural circuit, single-cell level photoablation of a targeted cell is one of the useful experimental approaches. This protocol describes a method to photoablate specific motor neurons via the mini singlet oxygen generator (miniSOG2), a light–oxygen–voltage (LOV)-based optogenetic tool used for ablating targeted cells in arbitrary areas. MiniSOG2 could induce the cell death pathway by generating reactive oxygen species (ROS) upon blue light illumination. Photoablation of a specific cell using the miniSOG2 was performed to show that, in Ciona intestinalis type A (Ciona robusta), a single pair of motor neurons, MN2/A10.64, is necessary to drive their tail muscle contraction. The membrane targeted miniSOG2 combined with neuron-specific promoter (pSP-Neurog::miniSOG2-CAAX) was electroplated into the Ciona egg and transiently expressed at specific neurons of the embryo. MN2 labeled with pSP-Neurog:mCherry-CAAX was irradiated using a 440-nm laser from the lateral side for 10 min to ablate its neural function. The behavior of the embryo before and after the irradiation was recorded with a high-speed camera.

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