PMID- 23469201
OWN - NLM
STAT- MEDLINE
DCOM- 20130827
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 8
IP  - 3
DP  - 2013
TI  - Primary neuron culture for nerve growth and axon guidance studies in zebrafish 
      (Danio rerio).
PG  - e57539
LID - 10.1371/journal.pone.0057539 [doi]
LID - e57539
AB  - Zebrafish (Danio rerio) is a widely used model organism in genetics and 
      developmental biology research. Genetic screens have proven useful for studying 
      embryonic development of the nervous system in vivo, but in vitro studies 
      utilizing zebrafish have been limited. Here, we introduce a robust zebrafish 
      primary neuron culture system for functional nerve growth and guidance assays. 
      Distinct classes of central nervous system neurons from the spinal cord, 
      hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor 
      neurons from transgenic reporter zebrafish lines, were dissociated and plated 
      onto various biological and synthetic substrates to optimize conditions for axon 
      outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the 
      tips of extending axons. The mean rate of axon extension in vitro was 21.4+/-1.2 microm 
      hr(-1) s.e.m. for spinal cord neurons, which corresponds to the typical  approximately 0.5 mm 
      day(-1) growth rate of nerves in vivo. Fluorescence labeling and confocal 
      microscopy demonstrated that bundled microtubules project along axons to the 
      growth cone central domain, with filamentous actin enriched in the growth cone 
      peripheral domain. Importantly, the growth cone surface membrane expresses 
      receptors for chemotropic factors, as detected by immunofluorescence microscopy. 
      Live-cell functional assays of axon extension and directional guidance 
      demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent 
      stimulation of outgrowth and growth cone chemoattraction, whereas mammalian 
      myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell 
      Ca(2+)-imaging revealed local elevation of cytoplasmic Ca(2+) concentration in 
      the growth cone induced by BDNF application. Moreover, BDNF-induced axon 
      outgrowth, but not basal outgrowth, was blocked by treatments to suppress 
      cytoplasmic Ca(2+) signals. Thus, this primary neuron culture model system may be 
      useful for studies of neuronal development, chemotropic axon guidance, and 
      mechanisms underlying inhibition of neural regeneration in vitro, and complement 
      observations made in vivo.
FAU - Chen, Zheyan
AU  - Chen Z
AD  - Mayo Graduate School, College of Medicine, Mayo Clinic, Rochester, Minnesota, 
      United States of America.
FAU - Lee, Han
AU  - Lee H
FAU - Henle, Steven J
AU  - Henle SJ
FAU - Cheever, Thomas R
AU  - Cheever TR
FAU - Ekker, Stephen C
AU  - Ekker SC
FAU - Henley, John R
AU  - Henley JR
LA  - eng
GR  - T32 AR007612/AR/NIAMS NIH HHS/United States
GR  - R01 NS067311/NS/NINDS NIH HHS/United States
GR  - DA14546/DA/NIDA NIH HHS/United States
GR  - R01 DA014546/DA/NIDA NIH HHS/United States
GR  - NS067311/NS/NINDS NIH HHS/United States
GR  - F32 NS080322/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20130304
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Brain-Derived Neurotrophic Factor)
RN  - 0 (Myelin-Associated Glycoprotein)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Animals, Genetically Modified
MH  - Axons/drug effects/*ultrastructure
MH  - Brain-Derived Neurotrophic Factor/pharmacology
MH  - Calcium/*metabolism
MH  - Calcium Signaling/drug effects
MH  - Gene Expression Regulation, Developmental/drug effects
MH  - Microscopy, Fluorescence
MH  - Motor Neurons/*cytology/drug effects
MH  - Myelin-Associated Glycoprotein/pharmacology
MH  - Primary Cell Culture/*methods
MH  - Prosencephalon/cytology/drug effects/growth & development
MH  - Retina/cytology/drug effects/growth & development
MH  - Rhombencephalon/cytology/drug effects/growth & development
MH  - Spinal Cord/cytology/drug effects/growth & development
MH  - Time-Lapse Imaging
MH  - Zebrafish/anatomy & histology/genetics/*growth & development
PMC - PMC3587632
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2013/03/08 06:00
MHDA- 2013/08/28 06:00
PMCR- 2013/03/04
CRDT- 2013/03/08 06:00
PHST- 2012/10/19 00:00 [received]
PHST- 2013/01/25 00:00 [accepted]
PHST- 2013/03/08 06:00 [entrez]
PHST- 2013/03/08 06:00 [pubmed]
PHST- 2013/08/28 06:00 [medline]
PHST- 2013/03/04 00:00 [pmc-release]
AID - PONE-D-12-32522 [pii]
AID - 10.1371/journal.pone.0057539 [doi]
PST - ppublish
SO  - PLoS One. 2013;8(3):e57539. doi: 10.1371/journal.pone.0057539. Epub 2013 Mar 4.