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&#39;s Hospital Flow Cytometry Core, and Boston Children&#39;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. In addition, R.F. was funded by the Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad and NIH Training grant in Genetics T32 GM007748; J.J. was supported by the NIH/NEI training program in the Molecular Bases of Eye Diseases (5T32EY007145-16) through Schepens Eye Research Institute and by the Developmental Neurology Training Program Postdoctoral Fellowship (5T32NS007473-19) through Boston Children&#39;s Hospital; M.C.W was supported by NEI (5K08EY027850) and Children&#39;s Hospital Ophthalmology Foundation (Faculty Discovery Award); and E.C.E. is a Howard Hughes Medical Institute Investigator.

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&#39;s Hospital.
1. Setting Up Timed Matings Prior to the Dissection
<ol>
	<li>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...

<ol>
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The authors declare no conflict of interest.

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...

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 scarce1,3,4,5,6,7,8,9. Therefore, comparative analyses of pure cultures of CN3s/CN4s and SMNs could provide important clues about mechanisms underlying relative vulnerability. Unfortunately, a major barrier to such analyses has been the inability to grow purified cultures of these motor neurons.
Many protocols have been described for the purification of SMNs from animal models. Most of these protocols use density gradient centrifugation10,11,12 and/or p75NTR-antibody-based cell-sorting panning techniques13,14,15,16. Density gradient centrifugation exploits the larger size of SMNs relative to other spinal cells, whereas p75NTR is an extracellular protein expressed exclusively by SMNs in the spinal cord. Nearly 100% pure SMN cultures have been generated by one or both of these protocols11,12,14. However, these protocols have not been successful in generating CN3/CN4 cultures because CN3s/CN4s do not express p75NTR, and other specific CN3/CN4 markers have not been identified. They are also smaller than SMNs and, therefore, more difficult to isolate based on size. Instead, in vitro studies of CN3s or CN4s have relied on dissociated17,18,19,20,21, explant17,22,23,24,25,26, and slice27,28 cultures, which are composed of heterogeneous cell types, and no protocols have existed for the isolation and culture of primary CN3s or CN4s.
Here, a protocol is described for the visualization, isolation, purification, and cultivation of CN3s, CN4s, and SMNs from the same embryonic day 11.5 (E11.5) IslMN:GFP transgenic mice29 (Figure 1, Figure 2A). IslMN:GFP specifically labels motor neurons with a farnesylated GFP that localizes to the cell membrane. This protocol enables species- and age-matched comparison of multiple types of motor neurons in order to elucidate pathological mechanisms in motor neuron disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11001</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594-conjugated F(ab&#39;)2 goat anti-rabbit IgG (H+L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11072</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>B27 Supplement (50X), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria llu SORP Flow Cytometer</td>
			<td>BD Bioscience</td>
			<td>-</td>
			<td>This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers.</td>
		</tr>
		<tr>
			<td>BD Falcon 70&#956;m Nylon Cell Strainers</td>
			<td>CORNING</td>
			<td>352350</td>
			<td>For filtering the dissociating cells before FACS.</td>
		</tr>
		<tr>
			<td>BD Falcon Round Bottom Test Tubes With Snap Cap</td>
			<td>CORNING</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF Human</td>
			<td>ProSpec-Tany TechnoGene, Ltd.</td>
			<td>CYT-207</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well)</td>
			<td>Greiner Bio-One International</td>
			<td>655090</td>
			<td>We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC</td>
		</tr>
		<tr>
			<td>Circular Cover Glasses for microscopy</td>
			<td>Karl Hecht &#38; Assistent</td>
			<td>1001/14</td>
			<td>We used this coverslip since the area was large (diamater: 14 mm).</td>
		</tr>
		<tr>
			<td>CNTF Human</td>
			<td>ProSpec-Tany TechnoGene, Ltd.</td>
			<td>CYT-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclopiazonic acid from Penicillium cyclopium</td>
			<td>Sigma-Aldrich</td>
			<td>C1530</td>
			<td>CPA. One of ER stressors.</td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylinodole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie Tips</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP25205</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF Human</td>
			<td>ProSpec-Tany TechnoGene, Ltd.</td>
			<td>CYT-305</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hibernate E</td>
			<td>BrainBits</td>
			<td>HE</td>
			<td></td>
		</tr>
		<tr>
			<td>Hibernate E low fluorescence</td>
			<td>BrainBits</td>
			<td>HELF</td>
			<td>Fluorescence which hinders observation of embryo&#39;s GFP expressions should be low.</td>
		</tr>
		<tr>
			<td>Horse serum, heat inactivated, New Zealand origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>26050-070</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Tocris Cookson</td>
			<td>2845</td>
			<td>Isobutylmethylxanthine</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Fisher Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s L15 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5913</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Knife 4.75&#34; 1.7 x 27 mm blade</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-6272</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria Mini Perforated Spoon</td>
			<td>Fine Science Tools</td>
			<td>10370-19</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse monoclonal antibody to neuronal class III &#946;-tubulin (TUBB3)</td>
			<td>BioLegend</td>
			<td>801202</td>
			<td>1:500, TUJ1</td>
		</tr>
		<tr>
			<td>Nikon Perfect Focus Eclipse Ti live cell fluorescence microscope and Elements software</td>
			<td>Nikon</td>
			<td>-</td>
			<td>Differential interference contrast images and immunocytochemistry images of the cell cultures were captured with these equipments</td>
		</tr>
		<tr>
			<td>Nitric Acid 90%, Fuming (Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>A202-212</td>
			<td>For rinsing coverslips</td>
		</tr>
		<tr>
			<td>Olympus 1.7ml Microtubes, Clear</td>
			<td>Genesee Scientific</td>
			<td>22-281</td>
			<td>These are the tubes that we described &#34;1.7 mL microcentrifuge tubes&#34; in the context.</td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corp</td>
			<td>LK003150</td>
			<td>Papain solution and alubumin-ovomucoid inhibitor solution are prepared from this kit.</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (10,000 U/ml)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly D-lysin (PDL)</td>
			<td>MilliporeSigma</td>
			<td>A-003-E</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit monoclonal antibody to Islet1</td>
			<td>Abcam</td>
			<td>ab109517</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 camera</td>
			<td>Nikon</td>
			<td>-</td>
			<td>Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes.</td>
		</tr>
		<tr>
			<td>Sylgard 170 Black Silicone Encapsulant - A+B 0.9 Kg kit</td>
			<td>Dow Corning</td>
			<td>1696157</td>
			<td>We make dissection dishes using this kit.</td>
		</tr>
		<tr>
			<td>TC treated Dishes, 100 x 20 mm</td>
			<td>Genesee Scientific</td>
			<td>25-202</td>
			<td>We make dissection dishes using this dish.</td>
		</tr>
		<tr>
			<td>Thum Dressing Forceps 4.5&#34; Serrated 2.2 mm Tip Width</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-8100</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer for LOGOQ e VET</td>
			<td>GE Healthcare</td>
			<td>L8-18i-RS</td>
			<td>For ultrasound on female mice</td>
		</tr>
		<tr>
			<td>Veterinary ultrasound machine</td>
			<td>GE Healthcare</td>
			<td>LOGOQ e VET</td>
			<td>For ultrasound on female mice</td>
		</tr>
		<tr>
			<td>Zeiss LSM 700 series laser scanning confocal microscope and Zen Software</td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<td>Confocal image of the embryo was captured with these equipments</td>
		</tr>
	</tbody>
</table>

isolation and culture of oculomotor, trochlear, and spinal motor neurons from prenatal islmn:gfp transgenic mice

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.

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...

Watch this Scientific Journal Video about Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice at JoVE.com

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

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.

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

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

This is the first protocol to simultaneously and efficiently purify and culture primary ocular and spinal motor neurons from embryonic mice. It enables the study of mechanisms underlying motor neuron disease. This protocol adds a novel in vitro oculomotor culture component to existing systems and provides pure species and age matched spinal motor neuron cultures for comparison.In amyotrophic lateral sclerosis, spinal motor neurons degenerate while oculomotor neurons are relatively spared. Comparing these cultures could provide insights into disease mechanism and therapy. This method will facilitate studies of the mechanisms underlying motor neuron development, disease, and selective vulnerability.Our culture system enables characterization of motor neuron morphology, molecular biology, and electrophysiology. For those new to this technique, we recommend lots of practice. Dissection can be technically challenging and multiple dissectors may be required to obtain sufficient neurons for primary culture.Begin by harvesting Islet1 EGF positive embryos from a pregnant mouse approximately 11.5 days post-fertilization. Spray the abdomen thoroughly with ethanol and remove the uterus with sterile microdissection scissors and thumb dressing forceps. Briefly wash the uterus in sterile PBS.Then transfer it to the dissection plate filled with pre-chilled sterile PBS. Under the bright light of the microscope, carefully remove the embryos from the uterus using microdissection scissors, thumb dressing forceps, and Dumont 5 tweezers. Then use a sterile Moria mini perforated spoon to transfer each embryo to a separate well of a 24-well plate filled with pre-chilled hibernate E low fluorescence medium supplemented with 1X V27.While keeping the plate on ice, transfer one embryo to a sterile dissection plate and cover it completely with ice cold sterile Hank's Balanced Salt Solution or HBSS. Use tweezers to remove the face of the embryo and the tail without damaging the midbrain. Then place the embryo prone with limbs straddled and tail pointing toward the front of the microscope.Use tweezers to slit open the roof of the fourth ventricle in order to generate a small opening. Use this opening to hook tweezers into the space created between the fourth ventricle and its roof. Dissect along the dorsal surface of the embryo rostral to the cortex and lateral to the floor plate and motor column.Then open the dissected tissue in an open book manner to reveal the GFP positive CN3 and CN4 nuclei. Carefully separate the ventral midbrain from the embryo and remove meningeal tissue with tweezers and a microdissection knife. Dissect the bilateral GFP positive CN3 and CN4 nuclei away from the floor plate and other GFP positive surrounding tissue taking care to avoid touching or damaging the neurons.If collecting separate CN3 and CN4 nuclei, cut along the midbrain of these two nuclei and use a P1000 pipette to collect the dissected ventral midbrain tissue with minimal HBSS. Place it in a labeled 1.7 milliliter microcentrifuge tube filled with dissection medium and store the tube on ice until dissociation. Continue pulling ventral midbrains from additional embryos in the same tube until the total number meets experimental requirements.To dissect the ventral spinal cord, keep the embryo prone with the head facing the front of the microscope. Hold it with one pair of tweezers and insert the tip of the other pair into the unopened caudal part of the fourth ventricle. Open the rest of the hindbrain and spinal cord dorsally over the whole rostrocaudal extent of the embryo.Cut the dorsal tissue starting from the fourth ventricle and working toward the central canal of the caudal spinal cord using the forceps as scissors. Then hold the embryo with one set of tweezers and pinch off the flap of the dorsal tissue on each side with the other pair. Remove the ventral spinal cord by using the microdissection knife to pierce directly below the GFP positive SMN lifting the ventral spinal cord with saw-like movements on both sides.Cut the spinal cord transversely at the upper boundary of the lower limb and remove the cervical lumbar portion. Cut transversely directly above C1 where the first GFP positive anterior horn projects. Place the ventral spinal cord dorsal side up and hold it by pressing the GFP negative tissue between the GFP positive SMN columns with a pair of tweezers.Remove the remaining attached mesenchyme, DRGs, and dorsal spinal cord by trimming both sides of the GFP positive SMN column with the microdissection knife. Use a P1000 pipette to collect the dissected ventral spinal cord tissue with minimal HBSS and place it in a labeled 1.7 milliliter microcentrifuge tube filled with dissection medium. Store it on ice until dissociation and continue pulling ventral spinal cords from additional embryos in the same tube.Add the appropriate volume of papain solution to each of the microcentrifuge tubes with the dissected tissue samples. Incubate the tubes at 37 degrees Celsius for 30 minutes agitating the tubes every 10 minutes by finger flicking. After the incubation, gently triturate each suspension eight times with a P200 pipette and centrifuge it at 300 times g for five minutes.Resuspend the cell pellets with the appropriate volume of albumin ovomucoid inhibitor solution by gently pipetting up and down. Repeat the centrifugation. Then carefully remove the supernatant with a P1000 pipette.Resuspend the cells in the appropriate volume of dissection medium. Filter the suspensions through a 70 micrometer cell strainer. Next, use FACS sorting to isolate GFP positive cells dissected from CN3/CN4 and SMN.Dilute the isolated cell suspensions with motor neuron culture medium pre-warmed to 37 degrees Celsius to the appropriate densities and add 200 microliters of the suspension to a well of a PDL laminin-coated 96-well plate. Culture the neurons in a 37 degree Celsius and 5%carbon dioxide incubator making sure to refresh the motor neuron culture medium every five days. When successfully isolated neurons were grown in culture, nearly pure primary CN3/CN4 and SMN cultures were obtained and maintained for 14 days in vitro.The purities of the cultures at two days in vitro were 93.5%for CN3/CN4 and 86.7%for SMN when assessed by immunocytochemistry with the motor neuron marker Islet1 and neuronal marker TUJ1. The purities relied heavily on the age of the embryos and on setting the appropriate thresholds for GFP gates during FACS. The purity of neurons isolated from E10.5 embryos were comparable to those of E11.5 embryos.However, the purity decreased significantly when E13.5 embryos were used. To determine if primary CN3/CN4s and SMNs showed differential responses to endoplasmic reticulum stressors, the cells were treated with varying concentrations of Cyclopiazonic Acid or CPA at two days in vitro and fixed three days later for immunocytochemistry to evaluate survival ratios. The number of viable neurons in each sample was counted and survival ratios were calculated.CN3/CN4 monocultures were significantly more resistant to CPA treatment compared to SMN monocultures. Following this procedure, many additional methods can be performed to examine motor neurons. A few examples include studies of electrophysiology, cell biology, transcription, or chromatin accessibility.

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Neuroscience

10.7K Görüntüleme.  Boston Children's Hospital. Bu aynı anda ve verimli bir şekilde arındırmak ve embriyonik farelerden kültür birincil oküler ve spinal motor nöronlar için ilk protokoldür. Motor nöron hastalığının altında yatan mekanizmaların incelenmesini sağlar. Bu protokol mevcut sistemlere bir in vitro okülomotor kültür bileşeni ekler ve karşılaştırma için saf türler ve yaş eşleşen spinal motor nöron kültürleri sağlar.Amiyotrofik lateral sklerozda spinal motor nöronlar dejenere olurken okülo...