Skip to main content
construction release_alert
Scholars@Duke will be undergoing maintenance April 11-15. Some features may be unavailable during this time.
cancel

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments.

Publication ,  Journal Article
Yang, R; Bennett, V
Published in: J Vis Exp
February 12, 2021

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

J Vis Exp

DOI

EISSN

1940-087X

Publication Date

February 12, 2021

Issue

168

Location

United States

Related Subject Headings

  • Neurons
  • Neuroglia
  • Mice
  • Integrases
  • Hippocampus
  • Gene Editing
  • Cells, Cultured
  • Axon Initial Segment
  • Ankyrins
  • Animals
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Yang, R., & Bennett, V. (2021). Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments. J Vis Exp, (168). https://doi.org/10.3791/61411
Yang, Rui, and Vann Bennett. “Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments.J Vis Exp, no. 168 (February 12, 2021). https://doi.org/10.3791/61411.
Yang, Rui, and Vann Bennett. “Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments.J Vis Exp, no. 168, Feb. 2021. Pubmed, doi:10.3791/61411.

Published In

J Vis Exp

DOI

EISSN

1940-087X

Publication Date

February 12, 2021

Issue

168

Location

United States

Related Subject Headings

  • Neurons
  • Neuroglia
  • Mice
  • Integrases
  • Hippocampus
  • Gene Editing
  • Cells, Cultured
  • Axon Initial Segment
  • Ankyrins
  • Animals