Protocols in Current Issue
0 Q&A 123 Views Feb 20, 2024

Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest.

Key features

• This protocol requires preexisting experience in hiPSC culturing for a successful outcome.

• The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points.

• The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.

Graphical overview

0 Q&A 86 Views Feb 20, 2024

Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories.

Key features

• Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body.

• Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.

Graphical overview

0 Q&A 110 Views Feb 20, 2024

Structural and functional changes in vascular networks play a vital role during development, causing or contributing to the pathophysiology of injury and disease. Current methods to trace and image the vasculature in laboratory settings have proven inconsistent, inaccurate, and labor intensive, lacking the inherent three-dimensional structure of vasculature. Here, we provide a robust and highly reproducible method to image and quantify changes in vascular networks down to the capillary level. The method combines vasculature tracing, tissue clearing, and three-dimensional imaging techniques with vessel segmentation using AI-based convolutional reconstruction to rapidly process large, unsectioned tissue specimens throughout the body with high fidelity. The practicality and scalability of our protocol offer application across various fields of biomedical sciences. Obviating the need for sectioning of samples, this method will expedite qualitative and quantitative analyses of vascular networks. Preparation of the fluorescent gel perfusate takes < 30 min per study. Transcardiac perfusion and vasculature tracing takes approximately 20 min, while dissection of tissue samples ranges from 5 to 15 min depending on the tissue of interest. The tissue clearing protocol takes approximately 24–48 h per whole-tissue sample. Lastly, three-dimensional imaging and analysis can be completed in one day. The entire procedure can be carried out by a competent graduate student or experienced technician.

Key features

• This robust and highly reproducible method allows users to image and quantify changes in vascular networks down to the capillary level.

• Three-dimensional imaging techniques with vessel segmentation enable rapid processing of large, unsectioned tissue specimens throughout the body.

• It takes approximately 2–3 days for sample preparation, three-dimensional imaging, and analysis.

• The user-friendly pipeline can be completed by experienced and non-experienced users.

Graphical overview

Protocols in Past Issues
0 Q&A 264 Views Feb 5, 2024

Measuring autonomic parameters like heart rate in behaving mice is not only a standard procedure in cardiovascular research but is applied in many other interdisciplinary research fields. With an electrocardiogram (ECG), the heart rate can be measured by deriving the electrical potential between subcutaneously implanted wires across the chest. This is an inexpensive and easy-to-implement technique and particularly suited for repeated recordings of up to eight weeks. This protocol describes a step-by-step guide for manufacturing the needed equipment, performing the surgical procedure of electrode implantation, and processing of acquired data, yielding accurate and reliable detection of heartbeats and calculation of heart rate (HR). We provide MATLAB graphical user interface (GUI)–based tools to extract and start processing the acquired data without a lot of coding knowledge. Finally, based on an example of a data set acquired in the context of defensive reactions, we discuss the potential and pitfalls in analyzing HR data.

Key features

• Next to surgical steps, the protocol provides a detailed description of manufacturing custom-made ECG connectors and a shielded, light-weight patch cable.

• Suitable for recordings in which signal quality is challenged by ambient noise or noise from other recording devices.

• Described for 2-channel differential recording but easily expandable to record from more channels.

• Includes a summary of potential analysis methods and a discussion on the interpretation of HR dynamics in the case study of fear states.

0 Q&A 478 Views Jan 20, 2024

The blood–brain barrier (BBB) is a major obstacle to the diagnostics and treatment of many central nervous system (CNS) diseases. A prime example of this challenge is seen in glioblastoma (GBM), the most aggressive and malignant primary brain tumor. The BBB in brain tumors, or the blood–brain–tumor barrier (BBTB), prevents the efficient delivery of most therapeutics to brain tumors. Current strategies to overcome the BBB for therapeutic delivery, such as using hyperosmotic agents (mannitol), have impeded progress in clinical translation limited by the lack of spatial resolution, high incidences of complications, and potential for toxicity. Focused ultrasound combined with intravenously administered microbubbles enables the transient disruption of the BBB and has progressed to early-phase clinical trials. However, the poor survival with currently approved treatments for GBM highlights the compelling need to develop and validate treatment strategies as well as the screening for more potent anticancer drugs. In this protocol, we introduce an optical method to open the BBTB (OptoBBTB) for therapeutic delivery via ultrashort pulse laser stimulation of vascular targeting plasmonic gold nanoparticles (AuNPs). Specifically, the protocol includes the synthesis and characterization of vascular-targeting AuNPs and a detailed procedure of optoBBTB. We also report the downstream characterization of the drug delivery and tumor treatment efficacy after BBB modulation. Compared with other barrier modulation methods, our optical approach has advantages in high spatial resolution and minimally invasive access to tissues. Overall, optoBBTB allows for the delivery of a variety of therapeutics into the brain and will accelerate drug delivery and screening for CNS disease treatment.

Key features

• Pulsed laser excitation of vascular-targeting gold nanoparticles non-invasively and reversibly modulates the blood–brain barrier permeability.

• OptoBBTB enhances drug delivery in clinically relevant glioblastoma models.

• OptoBBTB has the potential for drug screening and evaluation for superficial brain tumor treatment.

Graphical overview

0 Q&A 529 Views Jan 20, 2024

The central nervous system (CNS) relies on the complex interaction of neuroglial cells to carry out vital physiological functions. To comprehensively understand the structural and functional interplay between these neuroglial cells, it is essential to establish an appropriate in vitro system that can be utilized for thorough investigation. Traditional protocols for establishing primary neuronal and mixed glial cultures from prenatal mice or neural stem cells require sacrificing pregnant mice and have the drawback of yielding only specific types of cells. Our current protocol overcomes these drawbacks by utilizing the brain from day-0 pups to isolate CNS resident neuroglial cells including astrocytes, microglia, oligodendrocytes [oligodendrocyte precursor cells (OPCs) and differentiated oligodendrocytes], and meningeal fibroblasts, as well as hippocampal neurons, avoiding sacrificing pregnant mice, which makes this procedure efficient and cost effective. Furthermore, through this protocol, we aim to provide step-by-step instructions for isolating and establishing different primary neuroglial cells and their characterization using cell-specific markers. This study presents an opportunity to isolate, culture, and establish all major CNS resident cells individually. These cells can be utilized in various cell-based and biochemical assays to comprehensively investigate the cell-specific roles and behaviors of brain resident cells in a reductionist approach.

Key features

• Efficient isolation of major neuroglial cells like meningeal fibroblasts, neurons, astrocytes, oligodendrocytes, and microglia from a single day-0 neonatal mouse pup’s brain.

• Circumvents the sacrifice of pregnant female mice.

• Acts as a bridging experimental method between secondary cell lines and in vivo systems.

• Isolated cells can be used for performing various cell-based and biochemical assays.

Graphical overview

Steps for isolation of meningeal fibroblast and neuroglial cells from day 0 pups of mice (Created using

0 Q&A 237 Views Jan 20, 2024

Capillary density in skeletal muscles is key to estimate exercise capacity in healthy individuals, athletes, and those with muscle-related pathologies. Here, we present a step-by-step, high-throughput semi-automated method for quantifying capillary density from whole human skeletal muscle cross-sections, in areas of the muscle occupied by myofibers. We provide a detailed protocol for immunofluorescence staining, image acquisition, processing, and quantification. Image processing is performed in ImageJ, and data analysis is conducted in R. The provided protocol allows high-throughput quantification of capillary density.

Key features

• This protocol builds upon the method and results described in Abbassi-Daloii et al. (2023b).

• It includes step-by-step details on image acquisition and image processing of the entire muscle section.

• It enables high-throughput and semi-automated image quantification of capillary density.

• It provides a robust analysis for determining capillary density over the entire muscle cross section.

Graphical overview

0 Q&A 518 Views Jan 5, 2024

Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells.

Key features

• Preparation and attachment of PDMS microfluidic chambers to glass coverslips.

• Primary culture of cortical neurons and plating cortical neurons in microfluidic chamber.

• Confirmation of compartmentalization using the retrograde transport of the fluorescently labeled form of cholera toxin subunit B (f-Ctb).

• Immunofluorescence and multilabeling of compartmentalized cortical neurons.

• Retrograde transport of fluorescently labeled BDNF.

0 Q&A 489 Views Jan 5, 2024

In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development. Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes. The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation. We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner. We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line. We validate the use of this tool by silencing MCL-1, an anti-apoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs. In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs). After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression. With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems.

Key features

• Generation of an inducible CRISPRoff Sleeping Beauty transposon system.

• Experiments performed in vitro for generation of inducible CRISPRoff pluripotent stem cell line amenable to oligodendrocyte differentiation.

• Strategy to downregulate an essential gene at different stages of oligodendrocyte development.

Graphical overview

Workflow for generating inducible CRISPRoff stem cell line and assessing knockdown phenotype in stem cell–derived committed oligodendrocyte neural precursor cells

0 Q&A 322 Views Dec 20, 2023

The inferior colliculus (IC) is an important processing center in the auditory system, which also receives non-auditory sensory input. The IC consists of several subnuclei whose functional role in (non-) auditory processing and plastic response properties are best approached by studying awake animals, preferably in a longitudinal fashion. The increasing use of mice in auditory research, the availability of genetic models, and the superficial location of the IC in the mouse have made it an attractive species for studying IC function. Here, we describe a protocol for exposing the mouse IC for up to a few weeks for in vivo imaging or electrophysiology in a stable manner. This method allows for a broader sampling of the IC while maintaining the brain surface in good quality and without reopening the craniotomy. Moreover, as it is adaptable for both electrophysiological recordings of the entire IC and imaging of the dorsal IC surface, it can be applied to answer a multitude of questions.

Key features

• A surgical protocol for long-term physiological recordings from the same or separate neuronal populations in the inferior colliculus.

• Optimized for awake in vivo experiments in the house mouse (Mus musculus).

0 Q&A 427 Views Dec 20, 2023

Satellite glial cells (SGCs) are a type of glial cell population that originates from neural crest cells. They ultimately migrate to surround the cell bodies of neurons in the ganglia of the peripheral nervous system. Under physiological conditions, SGCs perform homeostatic functions by modifying the microenvironment around nearby neurons and provide nutrients, structure, and protection. In recent years, they have gained considerable attention due to their involvement in peripheral nerve regeneration and pain. Although methods for culturing neonatal or rat SGCs have long existed, a well-characterized method for dissociating and culturing adult SGCs from mouse tissues has been lacking until recently. This has impeded further studies of their function and the testing of new therapeutics. This protocol provides a detailed description of how to obtain primary cultures of adult SGCs from mouse dorsal root ganglia in approximately two weeks with over 90% cell purity. We also demonstrate cell purity of these cultures using quantitative real-time RT-PCR and their functional integrity using calcium imaging.

Key features

• Detailed and simplified protocol to dissociate and culture primary satellite glial cells (SGCs) from adult mice.

• Cells are dissociated in approximately 2–3 h and cultured for approximately two weeks.

• These SGC cultures allow both molecular and functional studies.

Graphical overview

Dissociation and culture of mouse satellite glial cells

0 Q&A 257 Views Dec 5, 2023

Habituation, the process by which animals learn to ignore insignificant stimuli, facilitates engagement with salient features of the environment. However, neural mechanisms underlying habituation also allow responses to familiar stimuli to be reinstated when such stimuli become potentially significant. Thus, the habituated state must allow a mechanism for habituation override. The remarkably precise knowledge of cell identity, connectivity, and information coding in Drosophila sensory circuits, as well as the availability of tools to genetically target these cells, makes Drosophila a valuable and important organism for analysis of habituation and habituation-override mechanisms. Studies of olfactory and gustatory habituation in Drosophila suggest that potentiation of GABAergic neurons underlies certain timescales of habituation and have specified some elements of a gustatory habituation-override pathway. More detailed understanding of gustatory habituation and habituation-override mechanisms will benefit from access to robust behavioral assays for (a) the proboscis extension reflex (PER) elicited by a sweet stimulus, (b) exposure paradigms that result in PER habituation, and, most critically, (c) manipulations that result in PER-habituation override. Here, we describe simple protocols for persistent sucrose exposure of tarsal hairs that lead to habituation of proboscis extension and for presentation of a novel appetitive stimuli that reinstate robust PER to habituated flies. This detailed protocol of gustatory habituation provides (a) a simple method to induce habituation by continuous exposure of the flies to sucrose for 10 min without leading to ingestion and (b) a novel method to override habituation by presenting yeast to the proboscis.

Key features

• A protocol for stimulation of Drosophila’s taste (sugar) sensory neurons that induces gustatory habituation without satiation due to ingestion.

• A chemical (yeast) stimulation protocol that rapidly induces habituation override/dishabituation in sugar-habituated Drosophila.

0 Q&A 229 Views Dec 5, 2023

Recent advancements in chemogenetic tools, such as designer receptors exclusively activated by designer drugs (DREADDs), allow the simultaneous manipulation of activity over a specific, broad brain region in nonhuman primates. However, the introduction of DREADDs into large and complexly shaped cortical sulcus regions of macaque monkeys is technically demanding; previously reported methods are time consuming or do not allow the spatial range of expression to be controlled. In the present report, we describe the procedure for an adeno-associated viral vector (AAV2.1) delivery via handheld injections into the dorsolateral prefrontal cortex (Brodmann’s area 9/46) of macaque monkeys, with reference to pre-scanned anatomical magnetic resonance images. This procedure allows the precise delivery of DREADDs to a specific cortical region.

Key features

• This article describes the procedures for injecting viral vectors encoding functional proteins for chemogenetic manipulation into targeted cortical sulcus regions.

• The protocol requires magnetic resonance imaging for the accurate estimation of the injection sites prior to surgery.

• Viral vector solutions are injected using a handheld syringe under microscopic guidance.

• This protocol allows for the precise introduction of designer receptors exclusively activated by designer drugs (DREADDs) to large and complex cortical regions.

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