Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Summary

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Abstract

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) Isl^MN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Introduction

The culture of primary motor neurons is a powerful tool which enables the study of neuronal development, function, and susceptibility to exogenous stressors. Motor neuron cultures are particularly useful for the study of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS)^1,2, whose disease mechanisms are incompletely understood. Interestingly, despite the significant cell death of spinal motor neurons (SMNs) in both ALS patients and ALS model mice, cell death in oculomotor neurons (CN3s) and trochlear neurons (CN4s) are relatively scarce^1,....

Protocol

All experiments utilizing laboratory animals were performed in accordance with NIH guidelines for the care and use of laboratory animals and with the approval of the Animal Care and Use Committee of Boston Children's Hospital.

1. Setting Up Timed Matings Prior to the Dissection

1. To generate prenatal embryonic mice for motor neuron harvest, weigh each female mouse and set up timed mating between adult Isl^MN:GFP transgenic mice 11.5 days prior to the day of neu.......

Discussion

Historically, in vitro studies of CN3 and/or CN4 motor neurons have relied on heterogeneous cultures such as dissociated^{17,18,19,20,21}, explant^{17,22,23,24,25,26}, a.......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L)	Thermo Fisher Scientific	A-11001	1:400
Alexa Fluor 594-conjugated F(ab')2 goat anti-rabbit IgG (H+L)	Thermo Fisher Scientific	A-11072	1:400
B27 Supplement (50X), serum free	Thermo Fisher Scientific	17504-044
BD FACSAria llu SORP Flow Cytometer	BD Bioscience	-	This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers.
BD Falcon 70μm Nylon Cell Strainers	CORNING	352350	For filtering the dissociating cells before FACS.
BD Falcon Round Bottom Test Tubes With Snap Cap	CORNING	352054
BDNF Human	ProSpec-Tany TechnoGene, Ltd.	CYT-207
Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well)	Greiner Bio-One International	655090	We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC
Circular Cover Glasses for microscopy	Karl Hecht & Assistent	1001/14	We used this coverslip since the area was large (diamater: 14 mm).
CNTF Human	ProSpec-Tany TechnoGene, Ltd.	CYT-272
Cyclopiazonic acid from Penicillium cyclopium	Sigma-Aldrich	C1530	CPA. One of ER stressors.
4′,6-diamidino-2-phenylinodole (DAPI)	Thermo Fisher Scientific	D1306
Dimethyl sulfoxide	Sigma-Aldrich	D2650	DMSO
Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie Tips	Roboz Surgical Instrument	RS-5015
Forskolin	Thermo Fisher Scientific	BP25205
GDNF Human	ProSpec-Tany TechnoGene, Ltd.	CYT-305
GlutaMAX supplement	Thermo Fisher Scientific	35050-061
Hanks’ Balanced Salt Solution (HBSS)	Thermo Fisher Scientific	14175-095
Hibernate E	BrainBits	HE
Hibernate E low fluorescence	BrainBits	HELF	Fluorescence which hinders observation of embryo's GFP expressions should be low.
Horse serum, heat inactivated, New Zealand origin	Thermo Fisher Scientific	26050-070
IBMX	Tocris Cookson	2845	Isobutylmethylxanthine
Laminin	Thermo Fisher Scientific	23017-015
Leibovitz’s L15 medium	Thermo Fisher Scientific	11415064
2-Mercaptoethanol	Sigma-Aldrich	M6250
Micro Dissecting Scissors	Roboz Surgical Instrument	RS-5913
Micro Knife 4.75" 1.7 x 27 mm blade	Roboz Surgical Instrument	RS-6272
Moria Mini Perforated Spoon	Fine Science Tools	10370-19
mouse monoclonal antibody to neuronal class III β-tubulin (TUBB3)	BioLegend	801202	1:500, TUJ1
Nikon Perfect Focus Eclipse Ti live cell fluorescence microscope and Elements software	Nikon	-	Differential interference contrast images and immunocytochemistry images of the cell cultures were captured with these equipments
Nitric Acid 90%, Fuming (Certified ACS)	Fisher Scientific	A202-212	For rinsing coverslips
Olympus 1.7ml Microtubes, Clear	Genesee Scientific	22-281	These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context.
Papain Dissociation System	Worthington Biochemical Corp	LK003150	Papain solution and alubumin-ovomucoid inhibitor solution are prepared from this kit.
Penicillin-streptomycin (10,000 U/ml)	Thermo Fisher Scientific	15140-122
Phosphate buffered saline (PBS)	Thermo Fisher Scientific	10010-023
Poly D-lysin (PDL)	MilliporeSigma	A-003-E
rabbit monoclonal antibody to Islet1	Abcam	ab109517	1:200
SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 camera	Nikon	-	Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes.
Sylgard 170 Black Silicone Encapsulant - A+B 0.9 Kg kit	Dow Corning	1696157	We make dissection dishes using this kit.
TC treated Dishes, 100 x 20 mm	Genesee Scientific	25-202	We make dissection dishes using this dish.
Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip Width	Roboz Surgical Instrument	RS-8100
Transducer for LOGOQ e VET	GE Healthcare	L8-18i-RS	For ultrasound on female mice
Veterinary ultrasound machine	GE Healthcare	LOGOQ e VET	For ultrasound on female mice
Zeiss LSM 700 series laser scanning confocal microscope and Zen Software	Carl Zeiss	-	Confocal image of the embryo was captured with these equipments