Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Summary

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 Isl^MN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Abstract

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 Isl^MN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

Introduction

The ocular motor system provides an elegant system for investigating axon guidance mechanisms. It is relatively uncomplicated, consisting of three cranial nerves innervating six extraocular muscles (EOMs) which move the eye, and the levator palpebrae superioris (LPS) which lifts the eyelid. The oculomotor nerve innervates the LPS and four EOMs - the inferior oblique and the medial, inferior, and superior rectus muscles. The other two nerves, the trochlear and abducens, each only innervate one muscle, the superior oblique and lateral rectus muscle, respectively. Eye movements provide an easy readout, showing if innervation was appropriate, missing, or aberrant. Additio....

Protocol

All animal work described here was approved and performed in compliance with the Boston Children's Hospital Institutional Animal Care and Use Committee (IACUC) protocols.

1. Timed matings

1. Place ISL^MN:GFP (Islet Motor Neuron Green Fluorescent Protein; MGI: J:132726; Jax Tg(Isl-EGFP*)1Slp/J Stock No: 017952) male and female mice together overnight. Weigh the females and record weights prior to mating.
   NOTE: ISL.......

Discussion

This ex vivo slice culture protocol provides significant advantages over traditional axon guidance assays²³. The size of each cranial motor nucleus is not a limiting factor, and no difficult dissection is necessary. The endogenous microenvironment through which the axons travel is maintained, allowing modification of one signaling pathway while maintaining other signaling pathways. Additionally, effects can be assessed at different points along the axon trajectory. Since axon guidance requires mul.......

Materials

Name	Company	Catalog Number	Comments
24-Well Tissue Culture Plate	Genesee Scientific	25-107
6-Well Tissue Culture Plate	Genesee Scientific	25-105
Disposable Pasteur Pipet (Flint Glass)	VWR	14672-200
Fine Forceps	Fine Science Tools	11412-11
Fluorobrite DMEM	Thermo Fisher Scientific	A1896701
Glucose (200 g/L)	Thermo Fisher Scientific	A2494001
Hank's Balanced Salt Solution (1X)	Thermo Fisher Scientific	14175-095
Heat Inactivated Fetal Bovine Serum	Atlanta Biologicals	S11550H
HEPES Buffer Solution (1M)	Thermo Fisher Scientific	15630106
L-Glutamine (250 nM)	Thermo Fisher Scientific	25030081
Loctite Superglue	Loctite
Low Melting Point Agarose	Thermo Fisher Scientific	16520050
Millicell Cell Culture Insert (30mm, hydrophilic PTFE, 0.4 um)	Millipore Sigma	PICM03050
Moria Mini Perforated Spoon	Fine Science Tools	10370-19
Penicillin/Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122
Petri Dish (100 x 15mm)	Genesee Scientific	32-107G
Phosphate Buffered Saline (1X, pH 7.4)	Thermo Fisher Scientific	10010049
Razor Blades	VWR	55411-050
Surgical Scissors - Blunt	Fine Science Tools	14000-12
Ti Eclipse Perfect Focus with TIRF	Nikon
Vibratome (VT 1200S)	Leica	1491200S001
Vibratome Blades (Double Edge, Stainless Steel)	Ted Pella, Inc.	121-6