0 Q&A 633 Views Sep 5, 2023

The centrosome governs many pan-cellular processes including cell division, migration, and cilium formation. However, very little is known about its cell type-specific protein composition and the sub-organellar domains where these protein interactions take place. Here, we outline a protocol for the spatial interrogation of the centrosome proteome in human cells, such as those differentiated from induced pluripotent stem cells (iPSCs), through co-immunoprecipitation of protein complexes around selected baits that are known to reside at different structural parts of the centrosome, followed by mass spectrometry. The protocol describes expansion and differentiation of human iPSCs to dorsal forebrain neural progenitors and cortical projection neurons, harvesting and lysis of cells for protein isolation, co-immunoprecipitation with antibodies against selected bait proteins, preparation for mass spectrometry, processing the mass spectrometry output files using MaxQuant software, and statistical analysis using Perseus software to identify the enriched proteins by each bait. Given the large number of cells needed for the isolation of centrosome proteins, this protocol can be scaled up or down by modifying the number of bait proteins and can also be carried out in batches. It can potentially be adapted for other cell types, organelles, and species as well.

Graphical overview

An overview of the protocol for analyzing the spatial protein composition of the centrosome in human induced pluripotent stem cell (iPSC)-derived neural cells. ① Human iPSCs are expanded, which serve as the starting cell population for the neural induction (Sections A, B, and C in Procedure). ② Neurons are induced and differentiated for 40 days (Section D in Procedure), in at least four biological replicates. ③ Total protein is isolated either at 15th or 40th day of differentiation, for neural stem cells and neurons, respectively (Sections E and F in Procedure). ④ Selected bait proteins are immunoprecipitated using the respective antibodies (Sections G and H in Procedure). ⑤ Co-immunoprecipitated samples are analyzed with mass spectrometry (Section I in Procedure). ⑥ Mass spectrometry output (.RAW) files are processed using MaxQuant software to calculate intensities (Section A in Data analysis). ⑦ The resulting data are pre-processed, filtered, and statistically analyzed using Perseus and R software (Sections B and C in Data analysis) ⑧ Further analysis is done using software or web tools such as Cytoscape or STRING to gain biological insights (Sections D and E in Data analysis).

0 Q&A 629 Views Feb 20, 2023

The functions of sleep remain largely unclear, and even less is known about its role in development. A general strategy to tackle these questions is to disrupt sleep and measure the outcomes. However, some existing sleep deprivation methods may not be suitable for studying the effects of chronic sleep disruption, due to their lack of effectiveness and/or robustness, substantial stress caused by the deprivation method, or consuming a large quantity of time and manpower. More problems may be encountered when applying these existing protocols to young, developing animals, because of their likely heightened vulnerability to stressors, and difficulties in precisely monitoring sleep at young ages. Here, we report a protocol of automated sleep disruption in mice using a commercially available, shaking platform–based deprivation system. We show that this protocol effectively and robustly deprives both non-rapid-eye-movement (NREM) sleep and rapid-eye-movement (REM) sleep without causing a significant stress response, and does not require human supervision. This protocol uses adolescent mice, but the method also works with adult mice.

Graphical abstract

Automated sleep deprivation system. The platform of the deprivation chamber was programmed to shake in a given frequency and intensity to keep the animal awake while its brain and muscle activities were continuously monitored by electroencephalography and electromyography.

0 Q&A 554 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.

Graphical abstract

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 1200 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.

Graphical abstract:

0 Q&A 940 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.

Graphical abstract:

1 Q&A 3251 Views Mar 5, 2022

Asymmetric cell division (ACD) is fundamental for balancing cell proliferation and differentiation in metazoans. During active neurogenesis in the developing zebrafish forebrain, radial glia progenitors (RGPs) mainly undergo ACD to produce one daughter with high activity of Delta/Notch signaling (proliferative cell fate) and another daughter with low Delta/Notch signaling (differentiative cell fate). The cell polarity protein partitioning-defective 3 (Par-3) is critical for regulating this process. To understand how polarized Par-3 on the cell cortex can lead to differential Notch activity in the nuclei of daughter cells, we combined an anti-Delta D (Dld) -atto 647N antibody uptake assay with label retention expansion microscopy (LR-ExM), to obtain high resolution immunofluorescent images of Par-3, dynein light intermediate chain 1 (Dlic1), and Dld endosomes in mitotic RGPs. We then developed a protocol for analyzing the colocalization of Par-3, Dlic1, and endosomal DeltaD, using JACoP (Just Another Co-localization Plugin) in ImageJ software (Bolte and Cordelières, 2006). Through such analyses, we have shown that cytosolic Par-3 is associated with Dlic1 on Dld endosomes. Our work demonstrates a direct involvement of Par-3 in dynein-mediated polarized transport of Notch signaling endosomes. This bio-protocol may be generalizable for analysis of protein co-localization in any cryosectioned and immunostained tissue samples.

0 Q&A 2364 Views Jan 20, 2022

Larval zebrafish have been established as an excellent model for examining vertebrate biology, with many researchers using the system for neuroscience. Controlling a fast escape response of the fish, the Mauthner cells and their associated network are an attractive model, given their experimental accessibility and fast development, driving ethologically relevant behavior in the first five days of development. Here, we describe methods for immunostaining electrical and chemical synapse proteins at 3-7 days post fertilization (dpf) in zebrafish using tricholoracetic acid fixation. The methods presented are ideally suited to easily visualize neural circuits and synapses within the fish.

0 Q&A 2277 Views Jun 20, 2021

Endothelial cells in the brain interact with other cell types, forming the blood-brain barrier. This barrier controls the movement of solutes into and out of the brain, regulating pathophysiological processes and drug delivery to the brain. Common isolation methods used to study these cells during embryonic development involve enzymatic treatment and cell sorting using specific markers. This process modifies the cell state and produces minute amounts of sample. Here, we describe a protocol for the enrichment of vascular cells from embryonic brains based on dextran separation. In this method, the brain is lightly disrupted with a pestle and then resuspended in a dextran solution. Low-speed centrifugation permits the separation of the parenchymal and vascular fractions. Further centrifugation steps improve fractionation. This method is simple and fast and produces enough sample for biochemical assays.

Graphic abstract:

Purification of vascular fragments from an embryonic brain

0 Q&A 3537 Views May 20, 2021

The size of the neocortex and its morphology are highly divergent across mammalian species. Several approaches have been utilized for the analysis of neocortical development and comparison among different species. In the present protocol (Note: This protocol requires basic knowledge of brain anatomy), we describe three ex vivo neocortical slice/tissue culture methods: (i) organotypic slice culture (mouse, ferret, human); (ii) hemisphere rotation culture (mouse, ferret); and (iii) free-floating tissue culture (mouse, ferret, human). Each of these three culture methods offers distinct features with regard to the analyses to be performed and can be combined with genetic manipulation by electroporation and treatment with specific inhibitors. These three culture methods are therefore powerful techniques to examine the function of genes involved in neocortical development.

0 Q&A 5351 Views Apr 20, 2021

Women are born with an abundant but finite pool of ovarian follicles, which naturally and progressively decreased during their reproductive years until menstrual periods stop permanently (menopause). Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioral changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, around 45-50 years old, women are more likely to express mood disorders, anxiety, irritability and vasomotor symptoms. The current animal models of reproductive aging do not successfully replicate human perimenopause and the gradual changes that occur in this phase. While the traditional rat model of menopause involves ovariectomy or surgical menopause consisting of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones, natural or transitional menopause is achieved by the selective loss of ovarian follicles (perimenopause period). However, the natural aging rodent (around 18-24 months) model fails to reach very low estrogen concentrations and overlaps the processes of somatic and reproductive aging. The chronic exposure of young rodents to 4-vinylcyclohexene diepoxide (VCD) is a well-established experimental model for perimenopause and menopause studies. VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia (apoptosis). The VCD, ovary-intact or accelerated ovarian failure (AOF) model is the experimental model that most closely matches natural human progression to menopause mimicking both hormonal and behavioral changes typically manifested by women in perimenopause.

Graphical abstract:

The female reproductive system is regulated by a series of neuroendocrine events controlled by central and peripheral components. (A). The mechanisms involved in this control are extremely complex and have not yet been fully clarified. In female mammals whose ovulation (the most important event in a reproductive cycle) occurs spontaneously, reproductive success is achieved through the precise functional and temporal integration of the hypothalamus-pituitary-ovary (HPO) axis. (B). In women, loss of fertility appears to be primarily associated with exhaustion of ovarian follicles, and this process occurs progressively until complete follicular exhaustion marked by the final menstrual period (FMP). (C). While in female rodents, reproductive aging seems to begin as a neuroendocrine process, in which changes in hypothalamic/pituitary function appear independently of follicular atresia. The traditional rat model of menopause, ovariectomy or surgical menopause consists of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones. (D). The chronic exposure (15-30 days) to the chemical compound 4-vinylcyclehexene diepoxide (VCD) in young rodents accelerates gradual failure of ovarian function by progressive depletion of primordial and primary follicles, but retains residual ovarian tissue before brain alterations that occurs in women in perimenopause. Low doses of VCD cause the selective destruction of the small preantral follicles of the ovary without affecting other peripheral tissues.