Name: isolation and culture of oculomotor, trochlear, and spinal motor neurons from prenatal islmn:gfp transgenic mice
Uploaded: 2019-11-12T12:00:00
Description: 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

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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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.

isolation-culture-oculomotor-trochlear-spinal-motor-neurons-from

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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 ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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 ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

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.