神经科学


分类

现刊
往期刊物
0 Q&A 418 Views May 20, 2024

Understanding dendritic excitability is essential for a complete and precise characterization of neurons’ input-output relationships. Theoretical and experimental work demonstrates that the electrotonic and nonlinear properties of dendrites can alter the amplitude (e.g., through amplification) and latency of synaptic inputs as viewed in the axosomatic region where spike timing is determined. The gold-standard technique to study dendritic excitability is using dual-patch recordings with a high-resistance electrode used to patch a piece of distal dendrite in addition to a somatic patch electrode. However, this approach is often impractical when distal dendrites are too fine to patch. Therefore, we developed a technique that utilizes the expression of Channelrhodopsin-2 (ChR2) to study dendritic excitability in acute brain slices through the combination of a somatic patch electrode and optogenetic activation. The protocol describes how to prepare acute slices from mice that express ChR2 in specific cell types, and how to use two modes of light stimulation: proximal (which activates the soma and proximal dendrites in a ~100 µm diameter surrounding the soma) with the use of a high-magnification objective and full-field stimulation through a low-magnification objective (which activates the entire somato-dendritic field of the neuron). We use this technique in conjunction with various stimulation protocols to estimate model-based spectral components of dendritic filtering and the impact of dendrites on phase response curves, peri-stimulus time histograms, and entrainment of pacemaking neurons. This technique provides a novel use of optogenetics to study intrinsic dendritic excitability through the use of standard patch-clamp slice physiology.

0 Q&A 967 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 BioRender.com)


0 Q&A 767 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 655 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 607 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 494 Views Oct 5, 2023

Adult neural stem/progenitor cells (NSPCs) in two neurogenic areas of the brain, the dentate gyrus and the subventricular zone, are major players in adult neurogenesis. Addressing specific questions regarding NSPCs outside of their niche entails in vitro studies through isolation and culture of these cells. As there is heterogeneity in their morphology, proliferation, and differentiation capacity between these two neurogenic areas, NSPCs should be isolated from each area through specific procedures and media. Identifying region-specific NPSCs provides an accurate pathway for assessing the effects of extrinsic factors and drugs on these cells and investigating the mechanisms of neurogenesis in both healthy and pathologic conditions. A great number of isolation and expansion techniques for NSPCs have been reported. The growth and expansion of NSPCs obtained from the dentate gyrus of aged rats are generally difficult. There are relatively limited data and protocols about NSPCs isolation and their culture from aged rats. Our approach is an efficient and reliable strategy to isolate and expand NSPCs obtained from young adult and aged rats. NSPCs isolated by this method maintain their self-renewal and multipotency.


Key features

• NSPCs isolated from the hippocampal dentate gyrus of young adult and aged rats, based on Kempermann et al. (2014) and Aligholi et al. (2014).

• Maintenance of NSPCs isolated from the dentate gyrus of aged rats (20–24 months) in our culture condition is feasible.

• According to our protocol, maximum growth of primary neurospheres obtained from isolated NSPCs of young and aged rats took 15 and 35 days, respectively.


Graphical overview



Isolation and expansion of neural stem/progenitor cells

0 Q&A 725 Views Sep 5, 2023

Neurons communicate via synapses—specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit.


Key features

• Confocal imaging and 3D reconstruction of presynaptic terminals.

• Used with a virus-mediated expression of mEGFP to achieve efficient, cell-type specific labeling of the presynaptic compartment.

• Protocol was developed with the calyx of Held but is suitable for pre- and postsynaptic compartments of various neurons across multiple mammalian and invertebrate species.

0 Q&A 1134 Views Sep 20, 2022

The activity of numerous autophagy-related proteins depends on their phosphorylation status, which places importance on understanding the responsible kinases and phosphatases. Great progress has been made in identifying kinases regulating autophagy, but much less is known about the phosphatases counteracting their function. Genetic screens and modern proteomic approaches provide powerful tools to identify candidate phosphatases, but further experiments are required to assign direct roles for candidates. We have devised a novel protocol to test the role of purified phosphatases in dephosphorylating specific targets in situ. This approach has the potential to visualize context-specific differences in target dephosphorylation that are not easily detected by lysate-based approaches such as Western blots.


Graphical abstract:




0 Q&A 1300 Views Sep 5, 2022

Mitochondrial dysfunction is associated with perturbations in the cellular oxidative status, changes in energy production and metabolic rate, and the onset of pathological processes. Classic methods of assessing mitochondrial dysfunction rely on indirect measures, such as evaluating mitochondrial DNA copy numbers, or direct but more costly and skilled techniques, such as electron microscopy. The protocol presented here was recently implemented to evaluate mitochondrial dysfunction in response to insecticide exposure in Drosophila melanogaster larvae, and it relies on the use of a previously established MitoTimer mutant strain. MitoTimer is a genetically engineered mitochondrial protein that shows green fluorescence when newly synthetized, irreversibly turning into red as mitochondria age. The protocol described here allows for the easy and direct assessment of shifts in mitochondrial turnover, with tissue-specific accuracy. This protocol can be adapted to assess changes in mitochondrial turnover in response to drugs, rearing conditions, and/or mutations in larva, pupa, or adult fruit flies.

0 Q&A 2907 Views Jul 5, 2022

The quantification of labeled cells in tissue sections is crucial to the advancement of biological knowledge. Traditionally, this was a tedious process, requiring hours of careful manual counting in small portions of a larger tissue section. To overcome this, many automated methods for cell analysis have been developed. Recent advances in whole slide scanning technologies have provided the means to image cells in entire tissue sections. However, common automated analysis tools do not have the capacity to deal with the large image files produced. Herein, we present a protocol for the quantification of two fluorescently labeled cell populations, namely pericytes and microglia, in whole brain tissue sections. This protocol uses custom-made scripts within the open source software QuPath to provide a framework for the careful optimization and validation of automated cell detection parameters. Images obtained from a whole-slide scanner are first loaded into a QuPath project. Manual counts are performed on small sample regions to optimize cell detection parameters prior to automated quantification of cells across entire brain regions. Even though we have quantified pericytes and microglia, any fluorescently labeled cell with clear labeling in and around the nucleus can be analyzed using these methods. This protocol provides a user-friendly and cost-effective framework for the automated analysis of whole tissue sections.