Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Lopez-Ramirez, Miguel Alejandro; Calvo, Charles‐Félix; Ristori, Emma; Thomas, Jean‐Léon; Nicoli, Stefania

doi:10.3791/53617

Cited by 22 publications

(19 citation statements)

References 14 publications

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“…Larval heads (6-dpf) were dissected on dissection medium (DMEF/F12 with 2% 100X penicillin-streptomycin) and pineal regions were enriched by pipetting the pineal with the whole brain into a tube placed on ice. Then the dissociated cells were obtained according to the protocol from Miguel A. Lopez-Ramirez and colleagues [44]. First, add 300 uL papain solution to the dissected tissue at 37˚C in water heater for 15 minutes, with gentle pipetting during digestion.…”

Section: Scrna-seq and Data Analysismentioning

confidence: 99%

Single-cell in vivo imaging of cellular circadian oscillators in zebrafish

Wang

Yang

et al. 2020

PLoS Biol

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The circadian clock is a cell-autonomous time-keeping mechanism established gradually during embryonic development. Here, we generated a transgenic zebrafish line carrying a destabilized fluorescent protein driven by the promoter of a core clock gene, nr1d1, to report in vivo circadian rhythm at the single-cell level. By time-lapse imaging of this fish line and 3D reconstruction, we observed the sequential initiation of the reporter expression starting at photoreceptors in the pineal gland, then spreading to the cells in other brain regions at the single-cell level. Even within the pineal gland, we found heterogeneous onset of nr1d1 expression, in which each cell undergoes circadian oscillation superimposed over a cell type-specific developmental trajectory. Furthermore, we found that single-cell expression of nr1d1 showed synchronous circadian oscillation under a light-dark (LD) cycle. Remarkably, single-cell oscillations were dramatically dampened rather than desynchronized in animals raised under constant darkness, while the developmental trend still persists. It suggests that light exposure in early zebrafish embryos has significant effect on cellular circadian oscillations.

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Section: Scrna-seq and Data Analysismentioning

confidence: 99%

Single-cell in vivo imaging of cellular circadian oscillators in zebrafish

Wang

Yang

et al. 2020

PLoS Biol

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“…RNA and cDNA were prepared using Qiagen RNeasy mini kit and ThermoFisher High-Capacity cDNA reverse transcription kit respectively. Previously described protocols were used to isolate islet (Emfinger et al 2017) and brain (Lopez-Ramirez et al 2016) tissues. Heart tissue was collected with forceps.…”

Section: Gene Expression Analysesmentioning

confidence: 99%

Beta‐cell excitability and excitability‐driven diabetes in adult Zebrafish islets

et al. 2019

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Islet β ‐cell membrane excitability is a well‐established regulator of mammalian insulin secretion, and defects in β ‐cell excitability are linked to multiple forms of diabetes. Evolutionary conservation of islet excitability in lower organisms is largely unexplored. Here we show that adult zebrafish islet calcium levels rise in response to elevated extracellular [glucose], with similar concentration–response relationship to mammalian β ‐cells. However, zebrafish islet calcium transients are nor well coupled, with a shallower glucose‐dependence of cytoplasmic calcium concentration. We have also generated transgenic zebrafish that conditionally express gain‐of‐function mutations in ATP‐sensitive K + channels (K ATP ‐GOF) in β ‐cells. Following induction, these fish become profoundly diabetic, paralleling features of mammalian diabetes resulting from equivalent mutations. K ATP ‐GOF fish become severely hyperglycemic, with slowed growth, and their islets lose glucose‐induced calcium responses. These results indicate that, although lacking tight cell‐cell coupling of intracellular Ca 2+ , adult zebrafish islets recapitulate similar excitability‐driven β ‐cell glucose responsiveness to those in mammals, and exhibit profound susceptibility to diabetes as a result of inexcitability. While illustrating evolutionary conservation of islet excitability in lower vertebrates, these results also provide important validation of zebrafish as a suitable animal model in which to identify modulators of islet excitability and diabetes.

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“…These results were in agreement with those reported by Salamat, Erfani Majd, Hashemitabar, Mesbah, and Ahangarpoor () on the ovarian cell culture from Cyprinus carpio . Based on the results of other studies on Oreochromis mossambicus (Gardell, Qin, Rice, Li, & Kültz, ) and Danio rerio (Lopez‐Ramirez, Calvo, Ristori, Thomas, & Nicoli, ), in optimal conditions, cultivated brain cells began to grow between 5 and 8 days after incubation, and most of the cells were epithelioid.…”

Section: Discussionmentioning

confidence: 99%

Using ovarian and brain cell culture from the Mullet, Liza klunzingeri, to assess the inhibitory effects of benzo[a]pyrene on aromatase activity

Sheibani

Salamat

Movahedinia

et al. 2020

J of Applied Toxicology

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We assessed the toxic effects of benzo[a]pyrene (BaP) on cell viability, aromatase (Aro) activity and steroid production using ovarian and brain cell cultures obtained from Mullet, Liza klunzingeri. The brain and ovary were minced and digested, and the cells were suspended in Leibovitz's L-15 medium supplemented with 15% and 20% fetal bovine serum. The cell suspensions were seeded on 25-cm 2 cell-culture flasks at 1 × 10 6 cells/mL and incubated at 25 C for 2 weeks. A BaP concentration of 10 −5 mol/L was accepted as the half-maximal inhibitory concentration. Ovarian and brain cells were exposed to different concentrations of BaP [0 (control), 10 −6 , 2 × 10 −6 , 3 × 10 −6 mol/L] and incubated at 30 C. At different sampling times (0, 12, 24 and 48 h) 40 ng/10 5 cells of 1,4,6-androstatriene-3,17-dione (ATD) was added to each well. Aro activity, 17β-estradiol (E2) and ATD production were determined. The sensitivity of the cultivated ovarian and brain cells to BaP increased dose dependently. BaP was a potent inhibitor of Aro activity at 2 × 10 −6 and 3 × 10 −6 mol/L, both in the cultivated brain and ovarian cells at different sampling times, with 10 −6 mol/L BaP found to be the least potent Aro inhibitor. E2 production decreased from cultivated ovarian and brain cells treated by different concentrations of BaP. In conclusion, BaP is able to change the activity of Aro and disrupt the biosynthesis of estrogens, and thus affects reproduction in fish. K E Y W O R D Saromatase, benzo (a) pyrene, brain cell culture, Liza klunzingeri, ovarian cell culture, steroid

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Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Cited by 22 publications

References 14 publications

Single-cell in vivo imaging of cellular circadian oscillators in zebrafish

Single-cell in vivo imaging of cellular circadian oscillators in zebrafish

Beta‐cell excitability and excitability‐driven diabetes in adult Zebrafish islets

Using ovarian and brain cell culture from the Mullet, Liza klunzingeri, to assess the inhibitory effects of benzo[a]pyrene on aromatase activity

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