A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes

Wolfes, Anne C.; Ahmed, Saheeb; Awasthi, Ankit; Stahlberg, Markus A.; Rajput, Ashish; Magruder, Daniel Sumner; Bonn, Stefan; Dean, Camin

doi:10.1085/jgp.201611607

Cited by 33 publications

(58 citation statements)

References 80 publications

(128 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Even though little is known about the in vivo morphology of axonal and dendritic growth cones, some works report evidence that the structure of neuronal growth cones is elaborated with numerous filopodia, especially at early developmental stages 33 . Astrocytes grown in serum -free conditions show a complex morphology that better represents in vivo systems 31,34 comparing to the flat astrocytic cell's bodies observed in FBS (compare astrocytes morphology between ACM and FBS in Fig.3 and Supplementary Fig.3). Indeed, the fibroblast-like morphology of astrocytes is known as an artifact due to the presence of serum in the culturing medium 35,36 .…”

Section: Discussionmentioning

confidence: 99%

An improved method for growing neurons: Comparison with standard protocols

Pozzi

Ban

Iseppon

et al. 2017

Journal of Neuroscience Methods

View full text Add to dashboard Cite

Highlights An improved protocol for primary hippocampal cell cultures is proposed.  The method relies on serum-free astrocytes conditioned medium (ACM).  The ACM method is extensively compared with other two commonly used protocols.  ACM improved morphology and function of both short-and long-term cultures. AbstractBackground: Since different culturing parameters -such as media composition or cell density -lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons -both in vivo and in vitro -is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes.New Method: Our culturing protocol is based on a novel serum-free preparation of astrocyteconditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods: Neurobasal/B27-and FBS-based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1,7,14 and 60 days in vitro (DIV).Results: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone's size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. Comparison with Existing Method(s):ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. Conclusions:We propose our ACM-based culture protocol as an improved and more suitable method for both short-and long-term neuronal cultures.

show abstract

Section: Discussionmentioning

confidence: 99%

An improved method for growing neurons: Comparison with standard protocols

Pozzi

Ban

Iseppon

et al. 2017

Journal of Neuroscience Methods

View full text Add to dashboard Cite

show abstract

“…Moreover, astrocytes also express synaptic vesicle‐associated protein calcitonin‐sensitizing synaptic protein 7 (Wolfes et al, ) and recycling dymanin (Lasic, Stenovec, Kreft, Robinson, & Zorec, ) in addition to VGLUTs, VGAT, and VNUT as mentioned above. Since these proteins are involved in the package, secretion, and exocytosis proteins, their expressions in astrocytes make it possible for GFAP to participate in the membrane protein recycling.…”

Section: Mechanisms Underlying Gfap Guiding Rolementioning

confidence: 99%

Neurochemical regulation of the expression and function of glial fibrillary acidic protein in astrocytes

Liu

et al. 2019

Glia

103

View full text Add to dashboard Cite

Glial fibrillary acidic protein (GFAP), a type III intermediate filament, is a marker of mature astrocytes. The expression of GFAP gene is regulated by many transcription factors (TFs), mainly Janus kinase‐2/signal transducer and activator of transcription 3 cascade and nuclear factor κ‐light‐chain‐enhancer of activated B cell signaling. GFAP expression is also modulated by protein kinase and other signaling molecules that are elicited by neuronal activity and hormones. Abnormal expression of GFAP proteins occurs in neuroinflammation, neurodegeneration, brain edema‐eliciting diseases, traumatic brain injury, psychiatric disorders and others. GFAP, mainly in α‐isoform, is the major component of cytoskeleton and the scaffold of astrocytes, which is essential for the maintenance of astrocytic structure and shape. GFAP also has highly morphological plasticity because of its quick changes in assembling and polymerizing states in response to environmental challenges. This plasticity and its corresponding cellular morphological changes endow astrocytes the functions of physical barrier between adjacent neurons and stabilizer of extracellular environment. Moreover, GFAP colocalizes and even molecularly associates with many functional molecules. This feature allows GFAP to function as a platform for direct interactions between different molecules. Last, GFAP involves transportation and localization of other functional proteins and thus serves as a protein transport guide in astrocytes. This guiding role of GFAP involves an elastic retraction and extension cytoskeletal network that couples with GFAP reassembling, transporting, and membrane protein recycling machinery. This paper reviews our current understanding of the expression and functions of GFAP as well as their regulation.

show abstract

“…For astrocyte cortical cultures, cells were cultured in supplemented NBM plus 10% FBS at a density of 0.21 9 10 6 cells/cm 2 . To remove neurons and obtain an astrocyte monoculture, at day in vitro 4, the dishes were placed on a plate shaker (Grant-bio Orbital Shaking Platform, catalog number: POS-300) in the incubator and shaken at 110 rpm for 6 h. After shaking, the medium was replaced to remove neurons and cellular debris as previously described (Wolfes et al 2017). Cells were plated on poly-D-lysine (Sigma-Aldrich, catalog number: P6407) (coated 24-well culture plates (Thermo Fischer Scientific, catalog number: 142475), except for calcium imaging (12 mm glass bottom dishes, Thermo Fischer Scientific, catalog number: 150680).…”

Section: Cell Culturesmentioning

confidence: 99%

G protein‐coupled estrogen receptor activates cell type‐specific signaling pathways in cortical cultures: relevance to the selective loss of astrocytes

Roque

Mendes-Oliveira

Baltazar

2019

Journal of Neurochemistry

View full text Add to dashboard Cite

Selective activation of the G protein‐coupled estrogen receptor has been proposed to avoid some of the side effects elicited by the activation of classical estrogen receptors α and β. Although its contribution to neuroprotection triggered by estradiol in brain disorders has been explored, the results regarding ischemic stroke are contradictory, and currently, there is no consensus on the role that this receptor may play. The present study aimed to investigate the role of GPER in the ischemic insult. For that, primary cortical cultures exposed to oxygen and glucose deprivation (OGD) were used as a model. Our results demonstrate that neuronal survival was strongly affected by the ischemic insult and concurrent GPER activation with G1 had no further impact. In contrast, OGD had a smaller impact on astrocytes survival but G1, alone or combined with OGD, promoted their apoptosis. This effect was prevented by the GPER antagonist G15. The results also show that ischemia did not change the expression levels of GPER in neurons and astrocytes. In this study, we also demonstrate that selective activation of GPER induced astrocyte apoptosis via the phospholipase C pathway and subsequent intracellular calcium rise, whereas in neurons, this effect was not observed. Taken together, this evidence supports a direct impact of GPER activity on the viability of astrocytes, which seems to be associated with the regulation of different signaling pathways in astrocytes and neurons.

show abstract

A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes

Cited by 33 publications

References 80 publications

An improved method for growing neurons: Comparison with standard protocols

An improved method for growing neurons: Comparison with standard protocols

Neurochemical regulation of the expression and function of glial fibrillary acidic protein in astrocytes

G protein‐coupled estrogen receptor activates cell type‐specific signaling pathways in cortical cultures: relevance to the selective loss of astrocytes

Contact Info

Product

Resources

About