Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Summary

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Abstract

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Introduction

Recent estimates suggest that the human brain contains 10¹¹ neurons with 10¹⁴ synaptic connections¹, highlighting the importance of axon branching in vivo. Extracellular axon guidance cues such as netrin-1 guide axons to appropriate synaptic partners and stimulate axonal branching, thereby increasing synaptic capacity^2-5. Netrin-1-dependent axonal arborization involves substantial plasma membrane expansion⁶, which we hypothesized requires delivery of additional membrane components via SNARE complex dependent exocytic vesicle fusion⁷.

Investigating the role of SNARE-....

Protocol

Statement of research ethics: All experiments involving animals detailed herein are subject to the rules and regulations of the UNC Committee on Animal Care and to NIH standards for the care and use of laboratory animals.

1. Preparation and Plating of Dissociated Cortical Neurons

1. Euthanize timed-pregnant females by CO₂ inhalation followed by cervical dislocation. Remove whole brains from the skulls of embryonic day 15.5 (E15.5) mice and microdissect cortices from each hemisphere. For detailed instructions regarding the microdissection of embryonic mouse cortex please see Viesselmann et al

Results

Utilizing in vitro biochemical techniques to assay the amount of SDS-resistant SNARE complexes in a population of neurons. Figure 1 shows the resulting western blot following completion of the SDS-resistant SNARE complex assay probed for SNAP-25, syntaxin1A and VAMP2.

TIRF microscopy at the basal cell membrane provides high resolution images of individual exocytic fusion events in single cells. .......

Discussion

Axon branching is a fundamental neurodevelopmental process and underpins the vast neuroconnectivity of the mammalian nervous system. Understanding the mechanisms involved in localized plasma membrane expansion is integral to our understanding of both normal and pathological neurodevelopment. The use of a multipronged approach incorporating both population level and single cell level methodologies enhances reproducibility and increases spatial and temporal resolution without compromising population level analysis. At the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well tissue culture treated plates	Olympus Plastics	25-105
glass coverslips	Fisher scientific	12-545-81	12CIR-1.5; must be nitric acid treated for 24 hours, rinsed in DI water 2x, and dried prior to use. Must be coated with 1 mg/ml Poly-d-lysine and rinsed prior to plating cells.
Amaxa nucleofection solution	Lonza	VPG-1001	100 ml/transfection
Amaxa Nucleofector/electroporator	Lonza		program O-005
35 mm Glass bottom live cell imaging dishes	Matek Corporation	p356-1.5-14-C	must be coated with 1 mg/ml Poly-d-lysine and rinsed prior to plating cells
Olympus IX81-ZDC2 inverted microscope	Olympus
Lambda LS xenon lamp	Sutter Instruments Company
Environmental Stage top incubator	Tokai Hit
100x 1.49 NA TIRF objective	Olympus
Andor iXon EM-CCD	Andor
Odyssey Licor Infrared Imaging System	LI-COR	Odyssey CL-X	Used for scanning blots
Image studio software suite	LI-COR		Used for scanning on the Odyssey Infrared system; Image studio lite used for offline analysis of blots
Metamorph for Olympus	Molecular devices, LLC	version 7.7.6.0	Software used for all imaging and the analysis of DIC timelapse
CELL TIRF control software	Olympus		Software used to control lasers for TIRF imaging
Fiji (Image J)	NIH	ImageJ Version 1.49t
60x Plan Apochromat 1.4 NA objective	Olympus
40x 1.4 NA Plan Apochromat objective	Olympus
Neurobasal media	GIBCO	21103-049	Base solution for both serum free and trypsin quenching media
Supplement B27	GIBCO	17504-044	500 ml/50 ml Serum free media and Trypsin Quenching media
L-Glutamine		35050-061	1 ml/50 ml Serum free media
Bovine serum albumin	Bio Basic Incorporated	9048-46-8	10% solution in 1x PBS for blocking coverslips; 5% solution in TBS-T for blocking nitrocellulose membranes.
10x  trypsin	Sigma	59427C
HEPES	CELLGRO	25-060-Cl
Dulbecco's Phosphate Buffered Saline (DPBS)+ Ca + Mg	Corning	21-030-cm
Fetal bovine serum	Corning/CELLGRO	35-010-CV
Hank's Balanced Salt Solution (HBSS)	Corning/CELLGRO	20-021-CV
NaCl	Fisher scientific	BP358-10
EGTA	Fisher scientific	CAS67-42-5
MgCl2	Fisher scientific	BP214-500
TRIS HCl	Sigma	T5941-500
TRIS base	Fisher scientific	BP152-5
N-Propyl Gallate	MP Biomedicals	102747
Glycerol Photometric grade	Acros Organics	18469-5000
Glycerol (non optics grade)	Fisher scientific	CAS56-81-5
B-mercaptoethonal	Fisher scientific	BP176-100
SDS	Fisher scientific	BP166-500
Distilled Water	GIBCO	152340-147
Poly-D-Lysine	Sigma	p-7886	Dissolved in sterile water at 1 mg/ml
Botulinum A toxin BoNTA	List Biological Laboratories	128-A
Rabbit polyclonal anti human VAMP2	Cell signaling	11829
Mouse monoclonal anti rat Syntaxin1A	Santa Cruz Biotechnology	sc-12736
Goat polyclonal anti human SNAP-25	Santa Cruz Biotechnology	sc-7538
Mouse monoclonal anti human βIII-tubulin	Covance	MMS-435P
Alexa Fluor 568 and Alexa Fluor 488 phalloidin, or Alexa Fluor 647	Invitrogen
LI-COR IR-dye secondary antibodies	LI-COR	P/N 925-32212,P/N 925-68023, P/N 926-68022	800 donkey anti-mouse, 680 donkey anti rabbit, 680 donkey anti goat
0.2 μm pore size nitrocellulose membrane	Biorad	9004-70-0
Tween-20	Fisher scientific	BP337-500
Methanol	Fisher scientific	S25426A
Bromphenol Blue	Sigma	B5525-5G
Sucrose	Fisher scientific	S6-212
Paraformaldehyde	Fisher scientific	O-4042-500
Triton-X100	Fisher scientific	BP151-500
TEMED	Fisher scientific	BP150-20
40% Bis-Acrylimide	Fisher scientific	BP1408-1
Name	Company	Catalog Number	Comments
Alternative Validated Antibodies
Mouse Monoclonal Anti-Syntaxin HPC-1 clone	Sigma Aldrich	S0664
Mouse Monoclonal Synaptobrevin 2 (VAMP2)	Synaptic Systems	104-211
Mouse Monoclonal SNAP25	Synaptic Systems	111-011