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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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) IslMN: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 scarce1,....

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 IslMN:GFP transgenic mice 11.5 days prior to the day of neu.......

Representative Results

The aim of this protocol was to highly purify and culture both primary CN3s/CN4s and SMNs long-term to enable comparative analyses of the mechanisms underlying motor neuron disorders (see Figure 1 and Figure 2 for overview).

Once neurons were successfully isolated and grown in culture, nearly pure primary CN3/CN4 and SMN cultures were obtained (Figu.......

Discussion

Historically, in vitro studies of CN3 and/or CN4 motor neurons have relied on heterogeneous cultures such as dissociated17,18,19,20,21, explant17,22,23,24,25,26, a.......

Acknowledgements

We thank Brigitte Pettmann (Biogen, Cambridge, MA, USA) for instruction in SMN dissection techniques; the Dana Farber Cancer Institute Flow Cytometry Facility, the Immunology Division Flow Cytometry Facility of Harvard Medical School, The Joslin Diabetes Center Flow Cytometry Core, Brigham and Women's Hospital Flow Cytometry Core, and Boston Children's Hospital Flow Cytometry Research Facility for FACS isolation of primary motor neurons; A.A. Nugent, A.P. Tenney, A.S. Lee, E.H. Nguyen, M.F. Rose, additional Engle laboratory members, and Project ALS consortium members for technical assistance and thoughtful discussion. This study was supported by Project ALS. I....

Materials

NameCompanyCatalog NumberComments
Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L)Thermo Fisher ScientificA-110011:400
Alexa Fluor 594-conjugated F(ab')2 goat anti-rabbit IgG (H+L)Thermo Fisher ScientificA-110721:400
B27 Supplement (50X), serum freeThermo Fisher Scientific17504-044
BD FACSAria llu SORP Flow CytometerBD Bioscience-This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers.
BD Falcon 70μm Nylon Cell StrainersCORNING352350For filtering the dissociating cells before FACS.
BD Falcon Round Bottom Test Tubes With Snap CapCORNING352054
BDNF HumanProSpec-Tany TechnoGene, Ltd.CYT-207
Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well)Greiner Bio-One International655090We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC
Circular Cover Glasses for microscopyKarl Hecht & Assistent1001/14We used this coverslip since the area was large (diamater: 14 mm).
CNTF HumanProSpec-Tany TechnoGene, Ltd.CYT-272
Cyclopiazonic acid from Penicillium cyclopiumSigma-AldrichC1530CPA. One of ER stressors.
4′,6-diamidino-2-phenylinodole (DAPI)Thermo Fisher ScientificD1306
Dimethyl sulfoxideSigma-AldrichD2650DMSO
Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie TipsRoboz Surgical InstrumentRS-5015
ForskolinThermo Fisher ScientificBP25205
GDNF HumanProSpec-Tany TechnoGene, Ltd.CYT-305
GlutaMAX supplementThermo Fisher Scientific35050-061
Hanks’ Balanced Salt Solution (HBSS)Thermo Fisher Scientific14175-095
Hibernate EBrainBitsHE
Hibernate E low fluorescenceBrainBitsHELFFluorescence which hinders observation of embryo's GFP expressions should be low.
Horse serum, heat inactivated, New Zealand originThermo Fisher Scientific26050-070
IBMXTocris Cookson2845Isobutylmethylxanthine
LamininThermo Fisher Scientific23017-015
Leibovitz’s L15 mediumThermo Fisher Scientific11415064
2-MercaptoethanolSigma-AldrichM6250
Micro Dissecting ScissorsRoboz Surgical InstrumentRS-5913
Micro Knife 4.75" 1.7 x 27 mm bladeRoboz Surgical InstrumentRS-6272
Moria Mini Perforated SpoonFine Science Tools10370-19
mouse monoclonal antibody to neuronal class III β-tubulin (TUBB3)BioLegend8012021:500, TUJ1
Nikon Perfect Focus Eclipse Ti live cell fluorescence microscope and Elements softwareNikon-Differential interference contrast images and immunocytochemistry images of the cell cultures were captured with these equipments
Nitric Acid 90%, Fuming (Certified ACS)Fisher ScientificA202-212For rinsing coverslips
Olympus 1.7ml Microtubes, ClearGenesee Scientific22-281These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context.
Papain Dissociation SystemWorthington Biochemical CorpLK003150Papain solution and alubumin-ovomucoid inhibitor solution are prepared from this kit.
Penicillin-streptomycin (10,000 U/ml)Thermo Fisher Scientific15140-122
Phosphate buffered saline (PBS)Thermo Fisher Scientific10010-023
Poly D-lysin (PDL)MilliporeSigmaA-003-E
rabbit monoclonal antibody to Islet1Abcamab1095171:200
SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 cameraNikon-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 kitDow Corning1696157We make dissection dishes using this kit.
TC treated Dishes, 100 x 20 mmGenesee Scientific25-202We make dissection dishes using this dish.
Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip WidthRoboz Surgical InstrumentRS-8100
Transducer for LOGOQ e VETGE HealthcareL8-18i-RSFor ultrasound on female mice
Veterinary ultrasound machineGE HealthcareLOGOQ e VETFor ultrasound on female mice
Zeiss LSM 700 series laser scanning confocal microscope and Zen SoftwareCarl Zeiss-Confocal image of the embryo was captured with these equipments

References

  1. Kiernan, M. C., et al. Amyotrophic lateral sclerosis. Lancet. 377 (9769), 942-955 (2011).
  2. Wood-Allum, C., Shaw, P. J. Motor neurone disease: a practical update on diagnosis and management. Clinical Medicine (London, En....

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