Browse
Submit Data
Databases
API
Help

Dataset Information

6 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Protocol for Evaluating Neuronal Polarity in Murine Models.

ABSTRACT: The establishment of polarity is crucial for the physiology and wiring of neurons. Therefore, monitoring the axo-dendritic specification allows the mechanisms and signals associated with development, growth, and disease to be explored. Here, we describe major and minor steps to study polarity acquisition, using primary cultures of hippocampal neurons isolated from embryonic rat hippocampi, for in vitro monitoring. Furthermore, we use in utero electroporated, GFP-expressing embryonic mouse brains for visualizing cortical neuron migration and polarization in situ. Some underreported after-protocol steps are also included. For complete details on the use and execution of this protocol, please refer to Wilson et al. (2020).

SUBMITTER: Wilson C

PROVIDER: S-EPMC7756935 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Protocol for Evaluating Neuronal Polarity in Murine Models.

Wilson Carlos C Rozés-Salvador Victoria V Cáceres Alfredo A

STAR protocols 20200917 3

The establishment of polarity is crucial for the physiology and wiring of neurons. Therefore, monitoring the axo-dendritic specification allows the mechanisms and signals associated with development, growth, and disease to be explored. Here, we describe major and minor steps to study polarity acquisition, using primary cultures of hippocampal neurons isolated from embryonic rat hippocampi, for <i>in vitro</i> monitoring. Furthermore, we use <i>in utero</i> electroporated, GFP-expressing embryoni ...[more]

PMID: 33377010

Similar Datasets

Maintenance of neuronal polarity.

Project description:Neurons are highly polarized cells with distinct domains responsible for receiving, transmitting, and propagating electrical signals. Central to these functions is the axon initial segment (AIS), a short region of the axon adjacent to the cell body that is enriched in voltage-gated ion channels, cell adhesion molecules, and cytoskeletal scaffolding proteins. Traditionally, the function of the AIS has been limited to its role in action potential initiation and modulation. However, recent experiments indicate that it also plays essential roles in neuronal polarity. Here, we review how initial segments are assembled, and discuss proposed mechanisms for how the AIS contributes to maintenance of neuronal polarity.

| S-EPMC3117984 | biostudies-literature

Microtubule polarity is instructive for many aspects of neuronal polarity.

Project description:Many neurons in bilaterian animals are polarized with functionally distinct axons and dendrites. Microtubule polarity, microtubule stability, and the axon initial segment (AIS) have all been shown to influence polarized transport in neurons. Each of these cytoskeletal cues could act independently to control axon and dendrite identity, or there could be a hierarchy in which one acts upstream of the others. Here we test the hypothesis that microtubule polarity acts as a master regulator of neuronal polarity by using a Drosophila genetic background in which some dendrites have normal minus-end-out microtubule polarity and others have the axonal plus-end-out polarity. In these mosaic dendrite arbors, we found that ribosomes, which are more abundant in dendrites than axons, were reduced in plus-end-out dendrites, while an axonal cargo was increased. In addition, we determined that microtubule stability was different in plus-end-out and minus-end-out dendrites, with plus-end-out ones having more stable microtubules like axons. Similarly, we found that ectopic diffusion barriers, like those at the AIS, formed at the base of dendrites with plus-end-out regions. Thus, changes in microtubule polarity were sufficient to rearrange other cytoskeletal features associated with neuronal polarization. However, overall neuron shape was maintained with only subtle changes in branching in mosaic arbors. We conclude that microtubule polarity can act upstream of many aspects of intracellular neuronal polarization, but shape is relatively resilient to changes in microtubule polarity in vivo.

| S-EPMC9058238 | biostudies-literature

Developmental GABA polarity switch and neuronal plasticity in Bioengineered Neuronal Organoids.

Project description:Brain organoids are promising tools for disease modeling and drug development. For proper neuronal network formation excitatory and inhibitory neurons as well as glia need to co-develop. Here, we report the directed self-organization of human induced pluripotent stem cells in a collagen hydrogel towards a highly interconnected neuronal network at a macroscale tissue format. Bioengineered Neuronal Organoids (BENOs) comprise interconnected excitatory and inhibitory neurons with supportive astrocytes and oligodendrocytes. Giant depolarizing potential (GDP)-like events observed in early BENO cultures mimic early network activity of the fetal brain. The observed GABA polarity switch and reduced GDPs in >40 day BENO indicate progressive neuronal network maturation. BENOs demonstrate expedited complex network burst development after two months and evidence for long-term potentiation. The similarity of structural and functional properties to the fetal brain may allow for the application of BENOs in studies of neuronal plasticity and modeling of disease.

| S-EPMC7391775 | biostudies-literature

Algorithms to predict cerebral malaria in murine models using the SHIRPA protocol.

Project description:BackgroundPlasmodium berghei ANKA infection in C57Bl/6 mice induces cerebral malaria (CM), which reproduces, to a large extent, the pathological features of human CM. However, experimental CM incidence is variable (50-100%) and the period of incidence may present a range as wide as 6-12 days post-infection. The poor predictability of which and when infected mice will develop CM can make it difficult to determine the causal relationship of early pathological changes and outcome. With the purpose of contributing to solving these problems, algorithms for CM prediction were built.MethodsSeventy-eight P. berghei-infected mice were daily evaluated using the primary SHIRPA protocol. Mice were classified as CM+ or CM- according to development of neurological signs on days 6-12 post-infection. Logistic regression was used to build predictive models for CM based on the results of SHIRPA tests and parasitaemia.ResultsThe overall CM incidence was 54% occurring on days 6-10. Some algorithms had a very good performance in predicting CM, with the area under the receiver operator characteristic ((au)ROC) curve > or = 80% and positive predictive values (PV+) > or = 95, and correctly predicted time of death due to CM between 24 and 72 hours before development of the neurological syndrome ((au)ROC = 77-93%; PV+ = 100% using high cut off values). Inclusion of parasitaemia data slightly improved algorithm performance.ConclusionThese algorithms work with data from a simple, inexpensive, reproducible and fast protocol. Most importantly, they can predict CM development very early, estimate time of death, and might be a valuable tool for research using CM murine models.

| S-EPMC2850361 | biostudies-literature

Murine models of sleep apnea: functional implications of altered macrophage polarity and epigenetic modifications in adipose and vascular tissues.

Project description:Obstructive sleep apnea (OSA) is a highly prevalent disease across the lifespan, is characterized by chronic intermittent hypoxia and sleep fragmentation, and has been independently associated with substantial cardiometabolic morbidity. However, the reversibility of end-organ morbidity with treatment is not always apparent, suggesting that both tissue remodeling and epigenetic mechanisms may be operationally involved. Here, we review the cumulative evidence focused around murine models of OSA to illustrate the temporal dependencies of cardiometabolic dysfunction and its reversibility, and more particularly to discuss the critical contributions of tissue macrophages to adipose tissue insulin resistance and vascular atherogenesis. In addition, we describe initial findings potentially implicating epigenetic alterations in both the emergence of the cardiometabolic morbidity of OSA, and in its reversibility with treatment. We anticipate that improved understanding of macrophage biology and epigenetics in the context of intermittent hypoxia and sleep fragmentation will lead to discovery of novel therapeutic targets and improved cardiovascular and metabolic outcomes in OSA.

| S-EPMC5955762 | biostudies-literature

Rheb and mTOR regulate neuronal polarity through Rap1B.

Project description:The development of polarized hippocampal neurons with a single axon and multiple dendrites depends on the activity of phosphoinositide 3-kinase (PI3K) and the GTPase Rap1B. Here we show that PI3K regulates axon specification and elongation through the GTPase Rheb and its target mammalian target of rapamycin (mTOR). Overexpression of Rheb induces the formation of multiple axons, whereas its suppression by RNA interference blocks axon specification. mTOR is a central regulator of translation that phosphorylates eIF4E-binding proteins like 4E-BP1. Axon formation was suppressed by inhibition of mTOR and expression of mTOR-insensitive 4E-BP1 mutants. Inhibition of PI3K or mTOR reduced the level of Rap1B, which acts downstream of Rheb and mTOR. The ubiquitin E3 ligase Smurf2 mediates the restriction of Rap1B by initiating its degradation. Suppression of Smruf2 by RNA interference is able to compensate the loss of Rheb. These results indicate that the mTOR pathway is required to counteract the Smurf2-initiated degradation of Rap1B during the establishment of neuronal polarity.

| S-EPMC2662285 | biostudies-literature

Wnt Signaling Directs Neuronal Polarity and Axonal Growth.

Project description:The establishment of neuronal polarity is driven by cytoskeletal remodeling that stabilizes and promotes the growth of a single axon from one of the multiple neurites. The importance of the local microtubule stabilization in this process has been revealed however, the external signals initiating the cytoskeletal rearrangements are not completely understood. In this study, we show that local activation of the canonical Wnt pathway regulates neuronal polarity and axonal outgrowth. We found that in the early stages of neuronal polarization, Wnt3a accumulates in one of the neurites of unpolarized cells and thereby could determine axon positioning. Subsequently, Wnt3a localizes to the growing axon, where it activates the canonical Wnt pathway and controls axon positioning and axonal length. We propose a model in which Wnt3a regulates the formation and growth of the axon by activating local intracellular signaling events leading to microtubule remodeling.

| S-EPMC6423405 | biostudies-literature

Establishing neuronal polarity: microtubule regulation during neurite initiation.

Project description:The initiation of nascent projections, or neurites, from the neuronal cell body is the first stage in the formation of axons and dendrites, and thus a critical step in the establishment of neuronal architecture and nervous system development. Neurite formation relies on the polarized remodelling of microtubules, which dynamically direct and reinforce cell shape, and provide tracks for cargo transport and force generation. Within neurons, microtubule behaviour and structure are tightly controlled by an array of regulatory factors. Although microtubule regulation in the later stages of axon development is relatively well understood, how microtubules are regulated during neurite initiation is rarely examined. Here, we discuss how factors that direct microtubule growth, remodelling, stability and positioning influence neurite formation. In addition, we consider microtubule organization by the centrosome and modulation by the actin and intermediate filament networks to provide an up-to-date picture of this vital stage in neuronal development.

| S-EPMC10913830 | biostudies-literature

Developmental GABA polarity switch and neuronal plasticity in Bioengineered Neuronal Organoids

Project description:Brain organoids are promising tools for disease modelling and drug development. For proper neuronal network formation excitatory and inhibitory neurons as well as glia need to co-develop. Here we report the directed differentiation and self-organization of induced pluripotent stem cells in a collagen hydrogel towards a highly interconnected neuronal network in a macroscale tissue format. Bioengineered Neuronal Organoids (BENOs) comprise interconnected excitatory and inhibitory neurons as well as supportive astrocytes and oligodendrocytes. Giant depolarizing potential (GDP)-like events observed within 20-40 days of BENO culture mimic early network activity of the fetal brain. The switch from excitatory to inhibitory GABA activity, and reduced GDPs at >40 day BENO cultures indicate progressive neuronal network maturation. BENOs demonstrate expedited complex network burst development after two months of culture and provide the first evidence for long-term potentiation and plasticity in brain organoids. BENOs exhibit structural and functional properties similar to the fetal brain and thus may be explored as a model to study the development of neuronal plasticity.

2020-07-02 | GSE139101 | GEO

Protocol for the application of single-cell damage in murine intestinal organoid models.

Project description:Spatially defined organoid damage enables the study of cellular repair processes. However, capturing dynamic events in living tissues is technically challenging. Here, we present a protocol for the application of single-cell damage in intestinal organoid models. We describe steps for isolating and cultivating murine colon organoids, lentivirus generation and transduction of organoids, single-cell ablation by a femtosecond laser, and follow-up imaging analysis. We provide examples for the image acquisition pipeline of dynamic processes in organoids using a confocal microscope. For complete details on the use and execution of this protocol, please refer to Donath et al.1,2.

| S-EPMC11342180 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data