A low-density culture method of cerebellar granule neurons with paracrine support applicable for the study of neuronal morphogenesis

Kubota, Kousuke; Seno, Takeshi; Konishi, Yoshiyuki

doi:10.1016/j.brainres.2013.09.046

Cited by 16 publications

(16 citation statements)

References 44 publications

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“…In culture, CGNs extend one or two axons that possess several arbor terminals (Bilimoria et al, 2010;Kubota et al, 2013) (Fig. 1A), thus are thought to be suitable for studying the diversity of arbor terminals.…”

Section: Kinesin-1 Preferentially Accumulates In a Subset Of Axonal Tmentioning

confidence: 99%

“…We also cultured CGNs at a low density (Kubota et al, 2013), and subjected the cells to immunocytochemistry (at 5 DIV) using antibodies specific to kinesin-1 (Fig. 1E) as previously described (Konishi and Setou, 2009).…”

Section: Kinesin-1 Preferentially Accumulates In a Subset Of Axonal Tmentioning

confidence: 99%

“…Neurons were spread on a plate that has been coated with poly-L-ornithine (Sigma-Aldrich). Low-density CGN cultures were prepared by co-cultivating with highdensity CGN culture as described previously (Kubota et al, 2013). Animals were treated according to the institutional ethical guidelines of University of Fukui.…”

Section: Cell Culturementioning

confidence: 99%

“…Finally, the CGN culture was washed twice with DMEM and placed in original medium. In the case of low-density culture, dissociated neurons were placed in DMEM that contained plasmids, and exposed to the square electric pulses (140 V, 5 ms for two times) before plating as described previously (Kubota et al, 2013). To construct a plasmid for KR-KLC, full-length KLC1 cDNA was amplified by PCR, and inserted into pKillerRed-C vector (Evrogen, Moscow, Russia).…”

Section: Transfectionmentioning

confidence: 99%

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Kinesin-1 sorting in axons controls the differential retraction of arbor terminals

Seno

Ikeno

Mennya

et al. 2016

Journal of Cell Science

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The ability of neurons to generate multiple arbor terminals from a single axon is crucial for establishing proper neuronal wiring. Although growth and retraction of arbor terminals are differentially regulated within the axon, the mechanisms by which neurons locally control their structure remain largely unknown. In the present study, we found that the kinesin-1 (Kif5 proteins) head domain (K5H) preferentially marks a subset of arbor terminals. Time-lapse imaging clarified that these arbor terminals were more stable than others, because of a low retraction rate. Local inhibition of kinesin-1 in the arbor terminal by chromophore-assisted light inactivation (CALI) enhanced the retraction rate. The microtubule turnover was locally regulated depending on the length from the branching point to the terminal end, but did not directly correlate with the presence of K5H. By contrast, F-actin signal values in arbor terminals correlated spatiotemporally with K5H, and inhibition of actin turnover prevented retraction. Results from the present study reveal a new system mediated by kinesin-1 sorting in axons that differentially controls stability of arbor terminals.

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Section: Kinesin-1 Preferentially Accumulates In a Subset Of Axonal Tmentioning

confidence: 99%

Section: Kinesin-1 Preferentially Accumulates In a Subset Of Axonal Tmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: Transfectionmentioning

confidence: 99%

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Kinesin-1 sorting in axons controls the differential retraction of arbor terminals

Seno

Ikeno

Mennya

et al. 2016

Journal of Cell Science

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“…[16] To maintain low-density cultures, neurons on glass coverslips (50–130 cells mm –2 ) were maintained as previously described. [17] Nifedipine, tetrodotoxin, and trimethoprim were purchased from Sigma-Aldrich, Abcam (Cambridge, UK), and WAKO (Osaka, Japan), respectively.…”

Section: Methodsmentioning

confidence: 99%

Study of local intracellular signals regulating axonal morphogenesis using a microfluidic device

Uryu

Tamaru

Suzuki

et al. 2016

Science and Technology of Advanced Materials

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The establishment and maintenance of axonal patterning is crucial for neuronal function. To identify the molecular systems that operate locally to control axonal structure, it is important to manipulate molecular functions in restricted subcellular areas for a long period of time. Microfluidic devices can be powerful tools for such purposes. In this study, we demonstrate the application of a microfluidic device to clarify the function of local Ca2+ signals in axons. Membrane depolarization significantly induced axonal branch-extension in cultured cerebellar granule neurons (CGNs). Local application of nifedipine using a polydimethylsiloxane (PDMS)-based microfluidic device demonstrated that Ca2+ entry from the axonal region via L-type voltage-dependent calcium channels (L-VDCC) is required for branch extension. Furthermore, we developed a method for locally controlling protein levels by combining genetic techniques and use of a microfluidic culture system. A vector for enhanced green fluorescent protein (EGFP) fused to a destabilizing domain derived from E. coli dihydrofolate reductase (ecDHFR) is introduced in neurons by electroporation. By local application of the DHFR ligand, trimethoprim (TMP) using a microfluidic device, we were able to manipulate differentially the level of fusion protein between axons and somatodendrites. The present study revealed the effectiveness of microfluidic devices to address fundamental biological issues at subcellular levels, and the possibility of their development in combination with molecular techniques.

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Differentiation of human olfactory bulb‐derived neural stem cells toward oligodendrocyte

Marei

Shouman

Althani

et al. 2017

Journal Cellular Physiology

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In the central nervous system (CNS), oligodendrocytes are the glial element in charge of myelin formation. Obtaining an overall presence of oligodendrocyte precursor cells/oligodendrocytes (OPCs/OLs) in culture from different sources of NSCs is an important research area, because OPCs/OLs may provide a promising therapeutic strategy for diseases affecting myelination of axons. The present study was designed to differentiate human olfactory bulb NSCs (OBNSCs) into OPCs/OLs and using expression profiling (RT-qPCR) gene, immunocytochemistry, and specific protein expression to highlight molecular mechanism(s) underlying differentiation of human OBNSCs into OPCs/OLs. The differentiation of OBNSCs was characterized by a simultaneous appearance of neurons and glial cells. The differentiation medium, containing cAMP, PDGFA, T3, and all-trans-retinoic acid (ATRA), promotes OBNSCs to generate mostly oligodendrocytes (OLs) displaying morphological changes, and appearance of long cytoplasmic processes. OBNSCs showed, after 5 days in OLs differentiation medium, a considerable decrease in the number of nestin positive cells, which was associated with a concomitant increase of NG2 immunoreactive cells and few O4(+)-OPCs. In addition, a significant up regulation in gene and protein expression profile of stage specific cell markers for OPCs/OLs (CNPase, Galc, NG2, MOG, OLIG1, OLIG2, MBP), neurons, and astrocytes (MAP2, β-TubulinIII, GFAP) and concomitant decrease of OBNSCs pluripotency markers (Oct4, Sox2, Nestin), was demonstrated following induction of OBNSCs differentiation. Taken together, the present study demonstrate the marked ability of a cocktail of factors containing PDGFA, T3, cAMP, and ATRA, to induce OBNSCs differentiation into OPCs/OLs and shed light on the key genes and pathological pathways involved in this process.

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A low-density culture method of cerebellar granule neurons with paracrine support applicable for the study of neuronal morphogenesis

Cited by 16 publications

References 44 publications

Kinesin-1 sorting in axons controls the differential retraction of arbor terminals

Kinesin-1 sorting in axons controls the differential retraction of arbor terminals

Study of local intracellular signals regulating axonal morphogenesis using a microfluidic device

Differentiation of human olfactory bulb‐derived neural stem cells toward oligodendrocyte

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