title: Long-term primary culture of neurons taken from chick embryo brain: A model to study neural cell biology, synaptogenesis and its dynamic properties.
abstract: BACKGROUND
  Studying neuronal growth, development and synaptogenesis are among the hot research topics. However, it is faced with various challenges and technical limitations that include but not limited to donor's species and health, threat to life, age of embryo, glial contamination, real-time tracking, and follow-up.
  
  
  METHODS
  We have successfully standardized a method for long-term primary culture of neurons collected from post-fertilized 9 day incubated chicken embryo brain overcoming the limitations mentioned above. Fertilized eggs were incubated in the laboratory and neurons from the embryonic brain were collected and low-density culture, apparently without glial contamination, was studied at least for 35 days in vitro (DIV).
  
  
  RESULTS
  Neurons were characterized by double immunostaining using stringent neuronal and glial markers. Neuronal differentiation, cytomorphology, neurite and axon formation, development and maturation, spine formation and synaptogenesis were tracked in real-time in a stage and time dependent manner. The neurons were transfected with Synaptophysin-RFP to label synaptic vesicles, which were followed in real-time under live-cell imaging.
  
  
  METHODS
  Every step was carried out under controlled laboratory conditions. Eggs are easily available, easy to handle, neurons from desired day of incubation could be conveniently studied for long period in apparently glia-free condition. In addition to common factors affecting primary culture, selection of culture media and cover glass coating are other key factors affecting neuronal cultures.
  
  
  CONCLUSIONS
  We describe an inexpensive, simpler pure primary neuronal culture method for studying neuronal cell-biology, synaptogenesis, vesicular dynamics and it has potential to grow 3D-multilayered brain in vitro.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Awanish
authors.initials: A
authors.lastname: Kumar
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. Electronic address: remsbnm@yahoo.com.
authors.forename: Birendra Nath
authors.initials: BN
authors.lastname: Mallick
mesh_terms: D000818:Animals; D001369:Axons / Q000502:physiology; D001921:Brain / Q000166:cytology*; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D019394:Ki-67 Antigen / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D055495:Neurogenesis / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D017711:Nonlinear Dynamics; D013569:Synapses / Q000502:physiology*; D013572:Synaptic Vesicles / Q000502:physiology
chemical_list: D019394:Ki-67 Antigen; D009419:Nerve Tissue Proteins; D020932:Nerve Growth Factor
keywords: Growth cone; Neurite growth; Neuronal differentiation and growth; Primary culture of neurons; Spines; Synaptic vesicles; Synaptogenesis