Communication between motor neurons and muscles is established by specialized synaptic connections known as neuromuscular junctions (NMJs). Altered morphology or numbers of NMJs in the developing muscles can indicate a disease phenotype. The distribution and count of NMJs have been studied in the context of several developmental disorders in different model organisms, including zebrafish. While most of these studies involved manual counting of NMJs, a few of them employed image analysis software for automated quantification. However, these studies were primarily restricted to the trunk musculature of zebrafish. These trunk muscles have a simple and reiterated anatomy, but the cranial musculoskeletal system is much more complex. Here, we describe a stepwise protocol for the visualization and quantification of NMJs in the ventral cranial muscles of zebrafish larvae. We have used a combination of existing ImageJ plugins to develop this methodology, aiming for reproducibility and precision. The protocol allows us to analyze a specific set of cranial muscles by choosing an area of interest. Using background subtraction, pixel intensity thresholding, and watershed algorithm, the images are segmented. The binary images are then used for NMJ quantification using the Analyze Particles tool. This protocol is cost-effective because, unlike other licensed image analyzers, ImageJ is open-source and available free of cost.

Alzheimer's disease (AD) poses a global health threat, progressively robbing patients of their memory and cognitive abilities. While it is recognized that meaningful social contact can alleviate the symptoms of dementia in AD patients, the precise mechanisms by which social stimulation mitigates AD symptoms remain poorly understood. We found that social interaction with novel mice, also known as novel social, simulated meaningful socializing. Therefore, we developed the multiple novel social (MNS) stimulation paradigm to train AD model mice and found that MNS effectively alleviated cognitive deficits in AD mice. This discovery not only opens up a new avenue for investigating the relationship between social stimulation and Alzheimer's disease but also lays the groundwork for delving into the underlying mechanisms, thereby providing crucial theoretical support for developing novel strategies to treat Alzheimer's disease.

Sleep is an essential behavior that is still poorly understood. Sleep abnormalities accompany a variety of psychiatric and neurological disorders, and sleep can serve as a modifiable behavior in the treatment of these disorders. Zebrafish (Danio rerio) has proven to be a powerful model organism to study sleep and the interplay between sleep and these disorders due to the high conservation of the neuro-modulatory mechanisms that control sleep and wake states between zebrafish and humans. The zebrafish is a diurnal vertebrate with a relatively simple nervous system compared to mammalian models, exhibiting conservation of sleep ontogeny across different life stages. Zebrafish larvae are an established high-throughput model to assess sleep phenotypes and the biological underpinnings of sleep disturbances. To date, sleep measurement in juvenile and adult zebrafish has not been performed in a standardized and reproducible manner because of the relatively low-throughput nature in relation to their larval counterparts. This has left a gap in understanding sleep across later stages of life that are relevant to many psychiatric and neurodegenerative disorders. Several research groups have used homemade systems to address this gap. Here, we report employing commercially available equipment to track activity and sleep/wake patterns in juvenile and adult zebrafish. The equipment allows researchers to perform automated behavior assays in an isolated environment with light/dark and temperature control for multiple days. We first explain the experimental procedure to track the sleep and activity of adult zebrafish and then validate the protocol by measuring the effects of melatonin and DMSO administration.

Measuring signal propagation through nerves is a classical electrophysiological technique established decades ago to evaluate sensory and motor functions in the nervous system. The whole-nerve preparation provides a valuable model to investigate nerve function ex vivo; however, it requires specific knowledge to ensure successful and stable measurements. Although the methodology for sciatic nerve recordings has long existed, a method for reliable and long-lasting recordings from myelinated and non-myelinated (nociceptive) fibers still needs to be adapted for pharmacological testing. This protocol takes benefits from epineurium sheath removal for pharmacological tests and provides a detailed description of how to make accurate nerve preparations, from the dissection and handling of nerves to epineurium cleaning, fabrication of adaptable suction electrodes for appropriate fiber stimulation and recordings, setting of electrophysiological protocols for compound action potential (CAP) recordings to distinguish between myelinated and non-myelinated (nociceptive) fibers, and finally to the analysis of the datasets of CAP components. We also demonstrate the feasibility of CAP recordings from individual branches in epineurium-free nerve preparations and provide clues to help retain nerve viability and maintain stable recordings over time. Although a sciatic nerve preparation was used here, the methodology can be applied to other nerve-type preparations.

Key features

• Detailed and simplified protocol for peripheral nerve preparation for recording sensory inputs ex vivo.

• Recordings from myelinated and non-myelinated (nociceptive) fibers can be performed hours after nerve preparation.

• The protocol involves the epineurium removal to facilitate drug permeability into nerve tissue for pharmacological tests.

• The protocol allows physiological and pathological studies (pain/chronic pain conditions).

Graphical overview

Preparation and recordings from the sciatic nerve, including myelinated and non-myelinated (nociceptive) fibers

Enzyme immobilization offers a number of advantages that improve biocatalysis; however, finding a proper way to immobilize enzymes is often a challenging task. Implanting enzymes in metal–organic frameworks (MOFs) via co-crystallization, also known as biomineralization, provides enhanced reusability and stability with minimal perturbation and substrate selectivity to the enzyme. Currently, there are limited metal–ligand combinations with a proper protocol guiding the experimental procedures. We have recently explored 10 combinations that allow custom immobilization of enzymes according to enzyme stability and activity in different metals/ligands. Here, as a follow-up of that work, we present a protocol for how to carry out custom immobilization of enzymes using the available combinations of metal ions and ligands. Detailed procedures to prepare metal ions, ligands, and enzymes for their co-crystallization, together with characterization and assessment, are discussed. Precautions for each experimental step and result analysis are highlighted as well. This protocol is important for enzyme immobilization in various research and industrial fields.

Key features

• A wide selection of metal ions and ligands allows for the immobilization of enzymes in metal–organic frameworks (MOFs) via co-crystallization.

• Step-by-step enzyme immobilization procedure via co-crystallization of metal ions, organic linkers, and enzymes.

• Practical considerations and experimental conditions to synthesize the enzyme@MOF biocomposites are discussed.

• The demonstrated method can be generalized to immobilize other enzymes and find other metal ion/ligand combinations to form MOFs in water and host enzymes.

Graphical overview

Circular RNA (circRNA) is an intriguing class of non-coding RNA that exists as a continuous closed loop. With the improvements in high throughput sequencing, biochemical analysis, and bioinformatic algorithms, studies on circRNA expression became abundant in recent years. However, functional studies of circRNA are still limited. Subcellular localization of circRNA may provide some clues in elucidating its biological functions by performing subcellular fractionation assay. Notably, circRNAs that are predominantly found in the cytoplasm are more likely to be involved in post-transcriptional gene regulation, e.g., acting as micoRNA sponge, whereas nuclear-retained circRNAs are predicted to play a role in transcriptional regulation. Subcellular fractionation could help researchers to narrow down and prioritize downstream experiments. The majority of the currently available protocols describe the steps for subcellular fractionation followed by western blot analysis for protein molecules. Here, we present a protocol for the subcellular fractionation of cells to detect circRNA via RT-qPCR with divergent primers. Moreover, detailed steps for the generation of specific circRNAs-enriched cDNA included in this protocol will enhance the amplification and detection of low-abundance circRNAs. This will be useful for researchers studying low-abundance circRNAs.

Key features

• This protocol builds upon the method developed by Gagnon et al. (2014) and extends its application to circRNA study.

• Protocol for amplification of low levels of circRNA expression.

• Analysis takes into consideration the ratio of cytoplasmic RNA concentration to nuclear RNA concentration.

Graphical overview

CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the production of lentiviral supernatants, and the transduction of target cells in a 96-well format. To assess the effect of gene knockouts on cellular fitness, we describe a competition-based cell proliferation assay using flow cytometry, enabling the screening of many genes at the same time in a fast and reproducible manner. This readout can be extended to any parameter that is accessible to flow-based measurements, such as protein expression and stability, differentiation, cell death, and others. In summary, this protocol allows to functionally assess the effect of a set of 50–300 gene knockouts on various cellular parameters within eight weeks.

Graphical abstract

Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a drug screening protocol utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesized that chemicals interrupting zebrafish gastrulation might suppress the metastasis of cancer cells. Thus, we developed a phenotype-based chemical screen that uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours by observing the epiboly progression of chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to the 90% epiboly stage. Finally, positive ‘hit’ chemicals that interrupt epiboly progression are selected by comparing epiboly progression of the chemical-treated and vehicle-treated embryos under a stereoscopic microscope. A previous study subjected 1,280 FDA-approved drugs to the screen and identified adrenosterone and pizotifen as epiboly-interrupting drugs. These were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11β-hydroxysteroid dehydrogenase 1 (HSD11β1) and serotonin receptor 2C (HTR2C), the primary targets of adrenosterone and pizotifen, respectively, promoted metastasis through induction of epithelial-mesenchymal transition (EMT). Therefore, this screen could be converted into a chemical genetic screening platform for identification of metastasis-promoting genes.

Graphical abstract:

The administration of antiretroviral therapy (ART) leads to a rapid reduction in plasma viral load in HIV-1 seropositive subjects. However, when ART is suspended, the virus rebounds due to the presence of a latent viral reservoir. Several techniques have been developed to characterize this latent viral reservoir. Of the various assay formats available presently, the Tat/Rev induced limiting dilution assay (TILDA) offers the most robust and technically simple assay strategy. The TILDA formats reported thus far are limited by being selective to one or a few HIV-1 genetic subtypes, thus, restricting them from a broader level application. The novel TILDA, labelled as U-TILDA ('U' for universal), can detect all the major genetic subtypes of HIV-1 unbiasedly, and with comparable sensitivity of detection. U-TILDA is well suited to characterize the latent reservoirs of HIV-1 and aid in the formulation of cure strategies.

Graphical abstract: