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

Summary

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Abstract

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.

Introduction

The axon initial segment is located at the proximal axon in most vertebrate neurons. Functionally, AIS is where action potentials are initiated due to the high-density of voltage-gated sodium channels in this region. AIS of some excitatory neurons are also targeted by inhibitory interneurons through forming GABAergic synapses^1,2,3. Therefore, AIS is a critical site to integrate cell signaling and modulate the excitability of neurons. AIS is normally 20-60 μm in length and located within 20 μm of the cell body. The length and position of AIS varies in neurons across ....

Protocol

NOTE: This culture method of hippocampal neurons from postnatal 0-day ANK3-E22/23^f/f mice is adapted from Gary Banker’s glia/neuron co-culture system. Therefore, it is critical to perform all steps after dissection in a clean hood using sterilized tools. This protocol takes up to 1 month. The workflow is displayed in Figure 3. The protocol follows the animal guidelines of Duke University.

1. Preparing of coverslips and neuronal plating dish.......

Discussion

The assembly of AIS is organized by 480 kDa ankyrin-G. However, ankyrin-G has shorter isoforms that can target to the AIS of wildtype neurons, which may lead to difficulty in interpretation of structure-function analyses of AIS assembly. Here we present a method using neurons from ANK3-E22/23-flox mice that allows study of de novo assembly of the AIS. By transfecting with Cre-BFP at 3 div, we eliminate the all endogenous isoforms of ankyrin-G. We could also co-transfect 480 kDa ankyrin-G to rescue the f.......

Materials

Name	Company	Catalog Number	Comments
10xHBSS	Thermo Fisher Scientific	14065-056
18mm coverglass (1.5D)	Fisher Scientific	12-545-84-1D
190kDa ankyrin-G-GFP	Addgene	#31059
2.5% Tripsin without phenol red	Thermo Fisher Scientific	14065-056
480kDa ankyrin-G-GFP	lab made	Provide upon request
ANK3-E22/23f/f mice	JAX	Stock No: 029797	B6.129-Ank3tm2.1Bnt/J;
B27 serum-free supplement	Thermo Fisher Scientific	A3582801
Boric acid	Sigma-Aldrich	B6768
Cell strainer with 70-mm mesh	BD Biosciences	352350
Ceramic coverslip-staining rack	Thomas Scientific	8542E40
Cre-BFP	Addgene	#128174
D-Glucose	Sigma-Aldrich	G7021
DMEM	Thermo Fisher Scientific	11995073
GlutaMAX-I supplement	Thermo Fisher Scientific	A1286001
Lipofectamine 2000	Thermo Fisher Scientific	11668030
MEM with Earle’s salts and L-glutamine	Thermo Fisher Scientific	11095-080
Neurobasal Medium	Thermo Fisher Scientific	21103-049
Nitric acid 70%	Sigma-Aldrich	225711
Opti-MEM I Reduced Serum Medium	Thermo Fisher Scientific	31985062
Paraformaldehyde	Sigma-Aldrich	P6148
Penicillin-streptomycin	Thermo Fisher Scientific	15140122
Poly-L-lysine hydrochloride	Sigma-Aldrich	26124-78-7
Potassium hydroxide	Sigma-Aldrich	1310-58-3