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.

<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><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&#039;)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&amp;mu;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 &amp;amp; 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 &lt;em&gt;Penicillium cyclopium&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>C1530</td><td>CPA. One of ER stressors.</td></tr><tr><td>4&amp;prime;,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 0.05 mm x 0.01 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&amp;rsquo; 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&#039;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&amp;rsquo;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&quot; 1.7 mm 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 &amp;beta;-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 1.7 mL Microtubes, Clear</td><td>Genesee Scientific</td><td>22-281</td><td>These are the tubes that we described &quot;1.7 mL microcentrifuge tubes&quot; 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 mm 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&quot; 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...

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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 ...

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10.8K Views.  Boston Children's Hospital. Dit is het eerste protocol om gelijktijdig en efficiënt te zuiveren en cultuur primaire oculaire en spinale motorneuronen van embryonale muizen. Het maakt de studie van mechanismen die ten grondslag liggen aan motorische neuron ziekte. Dit protocol voegt een nieuwe in vitro oculomotorische cultuurcomponent aan bestaande systemen toe en verstrekt zuivere soorten en leeftijdsgelijke motorische neuronculturen voor vergelijking.In amyotrofische laterale sclerose ontaarden spinale motorneu...

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

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

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

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 IslMN-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.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN: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.

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Fujiki, R., et al. <a href="https://app.jove.com/v/60440">Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP transgenic mice.</a> J. Vis. Exp. (2019)
The video demonstrates the isolation of oculomotor, trochlear, and spinal motor neurons from a transgenic mouse embryo. The embryo expresses motor neurons labeled with a green fluorescent protein (GFP) for easy visualization and identification. The motor neurons are surgically isolated under a fluorescence microscope and maintained in a culture medium.

1. Ventral midbrain and Spinal Cord Dissection
NOTE:&#160;Perform all of the following steps except for steps 1.1.1&#8722;1.1.3 and 1.1.5&#8722;1.1.6 ...

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Ex Vivo Culture and Imaging of Oculomotor Slices from Transgenic Mouse Embryos

Source: Whitman, M. C., et al. <a href="https://app.jove.com/v/59911">Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth.</a> J. Vis. Exp. (2019)
This video demonstrates the ex vivo culture and imaging of oculomotor slices from transgenic mouse embryos. The embryos are isolated and embedded in agarose. Using a vibratome, slices containing the oculomotor nuclei and eyes are obtained. The slices are maintained in culture and imaged using a fluorescence microscope.

All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board has reviewed all ...

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

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