We would like to thank Alison Philbrook, Belinda Barbagallo and Michael Francis for insightful comments on this paper. Research reported in this publication was supported by an R15 AREA award 1R15NS070172-01A1 awarded to MLL.

1. Coverslip Preparation: Acid Wash and Bake
<ol>
	<li>At least 2&#160;days prior to dissection (step 2), begin the following steps to prepare coverslips. Perform the acid wash step to remove oils and thoroughly clean coverslips.</li>
	<li>Load coverslips in porcelain holder that vertically positions coverslips and prevents them from touching each other. Submerge holder and coverslips into glass histology container filled with 2 M hydrochloric acid (HCl). Alternatively, if porcelain holder is not available, spread ~20 coverslips horizontally to cover the bottom of 100 mm glass Petri dish and submerge coverslips in 2 M HCl.</li>
	<li>Place glass vessel (with coverslips submerged in acid) on rocker table in fume hood and allow gentle rocking for at least 5 hr at room temperature. Alternatively, acid wash coverslips overnight under the same conditions.</li>
	<li>Wash coverslips with distilled H2O(dH2O) by transferring porcelain holder loaded with coverslips from 2 M HCl to a clean glass histology container filled with dH2O. Alternatively, decant 2 M HCl from glass Petri dish with coverslips and refill with dH2O.</li>
	<li>Wash by decanting dH2O again and refilling glass vessel with fresh dH2O. Repeat for a total of 3&#160;quick washes.</li>
	<li>Begin longer washing regimen by returning glass vessel (with coverslips and dH2O) onto rocker. Allow for gentle agitation for 10-30 min at room temperature. Then, decant dH2O and add fresh dH2O. Repeat for a total of at least ten washes. Alternatively, continue washing overnight.</li>
	<li>Place washed coverslips into furnace preheated to 350 &#176;C. If using porcelain holder, remove holder from dH2O-filled glass container and then place holder with coverslips directly into the furnace. If using a glass Petri dish, decant dH2O and place the glass dish with coverslips into the furnace.</li>
	<li>Bake coverslips for at least five hours or overnight at 350 &#176;C.</li>
	<li>After baking is complete, remove coverslips from porcelain holder with fine forceps and place into sterile 100 mm glass Petri dish. Leave coverslips that were not originally placed in porcelain holder in the glass Petri dish.</li>
</ol>
2. Chick Dissection and Isolation of DRGs
<ol>
	<li>Remove fertile, staged White Leghorn chick egg from incubator (held at 37-38.5 &#176;C with gentle rocking). 
	NOTE: Previous studies have used embryonic day 7-10 for isolation of DRGs2,8,11-13,16.</li>
	<li>Place selected egg in laminar flow hood. Conduct all procedures listed below under aseptic conditions in the laminar flow hood with sterile solutions and instruments. Pre-warm all solutions in a 37 &#176;C water bath.</li>
	<li>Crack egg and place contents into a plastic 100 mm Petri dish. Use standard forceps to transfer embryo to another 100 mm Petri dish.</li>
	<li>Using a Pasteur pipette, add warmed 1x&#160;PBS to Petri dish to keep tissue wet. Decapitate embryo by pinching neck with fine forceps. Transfer body to another clean 100 mm Petri dish.</li>
	<li>Add warm F12HS10 (Ham&#8217;s F12, 10 mM HEPES, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 10% fetal bovine serum) to dish with Pasteur pipette to keep tissue wet. Use forceps to position embryo on its dorsal side (Figure 1A).</li>
	<li>Place 100 mm plastic Petri dish containing embryonic tissue onto a binocular stereovision dissecting microscope in laminar flow hood.&#160;Use fine forceps to make a vertical midline incision from&#160;tail to neck by pinching the developing dermis to expose the heart, lung and other organs. Alternatively, make a series of small tears to expose internal organs.</li>
	<li>Gently use 2&#160;sets of sterile fine forceps to: a) grasp the neck and b) grasp the aorta or other tissue immediately superior to the heart and pull towards the tail, eviscerating the animal.</li>
	<li>Apply warm F12HS10 with Pasteur pipette to tissue. Use forceps to remove any remaining cardiovascular, respiratory or digestive organs to expose the developing ribs, vertebral column and DRGs (Figures 1B-1D).</li>
	<li>Under the dissecting microscope, use fine forceps to further remove organs and collect DRGs along both sides of lumbar vertebral column, inferior to the ribs. Collect DRGs by plucking intact DRGs from the embryo. Pinch and sever the roots that reach from the DRG towards the spinal cord and grasping the developing nerve that reaches from the DRG towards the periphery or vice versa.</li>
	<li>Place intact DRGs into a 35 mm culture dish filled with warm F12HS10. Remove any excess tissue, such as the dorsal or ventral roots that may have adhered to DRGs, from the intact DRGs in 35 mm dish by pinching with fine forceps.</li>
	<li>To obtain DRGs superior to the lumbar region, remove the ventral half of the thoracic and cervical vertebral column. This is done by making a cut in the developing vertebral column perpendicular to the spinal cord axis in the upper cervical region and another cut in the upper lumbar region. Then, make cuts parallel to the spinal cord axis along the right and left sides of the vertebral column. Lift the ventral half of the vertebral column from the embryo, which exposes the exposes the spinal cord (Figures 1E-1F).</li>
	<li>To allow easy access to DRGs, remove the thoracic and cervical spinal cord by grasping the spinal cord with fine forceps. Use the forceps to lift the spinal cord out of the vertebral column. As an approximation, the thoracic spinal cord is at the level of ribs and the cervical area is superior to the ribs.</li>
	<li>Collect intact thoracic and cervical DRGs with fine forceps and place them in warm F12HS10 with other DRGs. Remove excess tissue that may adhere to DRGs.</li>
	<li>Rinse interior of a new glass Pasteur pipette with F12HS10 to help prevent DRGs from sticking to walls of pipette. Use rinsed pipette to transfer all DRGs and F12HS10 from small Petri dish to a sterile 15 ml conical tube and immediately proceed to step 3. 
	NOTE: Additional embryos can be dissected to increase number of&#160;DRGs. DRGs should be kept in F12HS10 at 37 &#176;C until ready for step 3.</li>
</ol>
3. Dissociation, Enriching, and Culturing DRG Neurons &#8211; Part 1
<ol>
	<li>Place conical tube with intact DRGs into centrifuge and gently spin (200 x g) for 2-3 min. Confirm that DRGs have sunk to bottom of tube. If not, spin again.</li>
	<li>Using a pipette, gently remove F12HS10, leaving DRG pellet undisturbed. Add 2 ml of 1x&#160;calcium and magnesium free (CMF) PBS, dislodging the pellet of DRGs. Spin again and remove CMF PBS while leaving the DRG pellet intact. Repeat this wash step for a total of three washes to remove fetal bovine serum in F12HS10, which would interfere with trypsin digestion in next step.</li>
	<li>Add 2 ml of warmed trypsin to 15 ml tube containing 2 ml CMF PBS and DRGs. Place tube in 37 &#176;C water bath for 10-15 min&#160;to digest DRGs into dissociated cells.</li>
	<li>During incubation, make F12H + supplement containing media (Ham&#8217;s F12, 10 mM HEPES 100 units/ml penicillin, 0.1 mg/ml streptomycin, 10 ng/ml NT3, 10 ng/ml NGF, 200 mM l-glutamine and N2) and warm in 37 &#176;C water bath. Typically, 10 ml of total media is sufficient for 4-5 35 mm dishes of primary sensory neurons. Also begin thawing laminin-1 for coating coverslips (step 4).</li>
	<li>Gently triturate DRGs in trypsin solution with p1000 pipette and visually inspect for absence of intact DRGs. Continue trituration until intact DRGs are no longer seen by eye and/or allow for longer trypsin incubation in 37 &#176;C water bath until intact DRGs are dissociated. To protect DRG neurons from damage, avoid air bubbles during trituration and limit trypsin digestion to &#60;30 min.</li>
	<li>Centrifuge tube containing dissociated DRGs and trypsin at 200 x g for 3-5 min&#160;to form pellet. Spin longer if needed until pellet is formed.</li>
	<li>Carefully aspirate trypsin without disrupting pellet. Add 2 ml of F12HS10 to pellet. Gently triturate DRGs with a p1000 pipette tip.</li>
	<li>Transfer all contents from 15 ml conical tube into 100 mm culture dish. Rinse the tube with 8 ml F12HS10 and transfer it to the culture dish, 2 ml at a time for a total of 10 ml. This rinsing step helps collect cells that may have remained adhered to the walls of the tube.</li>
	<li>Place 100 mm culture dish (with 10 ml F12HS10 and dissociated DRG cells) into a humidified 37 &#176;C incubator for 3 hr. 
	NOTE: CO2 is not required in the incubator because HEPES buffers media pH in the absence of CO2. During incubation, glial cells bind to surface of tissue culture plastic and neurons tend to loosely adhere to glial bed.</li>
</ol>
4. Coating Acid Washed and Baked Coverslips with Laminin-1
<ol>
	<li>Thaw sterile aliquots of frozen laminin-1 on ice.</li>
	<li>Under sterile conditions, add 1 ml of sterile 1x&#160;PBS to 50 &#956;l aliquot of laminin-1.</li>
	<li>Use a spectrophotometer to obtain an absorbance value of laminin-1 at 280 nm. Use this value in the following equation to determine the concentration of laminin-1.</li>
</ol>
ABS 280 = (concentration mg/ml) * (extinction coefficient of protein) 
The extinction coefficient for laminin-1 is 0.86.
<ol start="4">
	<li>Under sterile conditions, dilute laminin-1 with 1X PBS to obtain the concentrations 1 mg/ml and 20 mg/ml. Use these two applied concentrations of laminin-1 to achieve a ten-fold difference of laminin-1 bound to the coverslip (30 ng/cm2 and 300 ng/cm2, respectively) after incubation7,8,10.</li>
	<li>In a laminar flow hood, use fine forceps to place one acid-washed and baked coverslip into the bottom of one 35 mm culture dish. Repeat as necessary to obtain the number of dishes needed.</li>
	<li>Subject the coverslips to UV light for 20-30 min&#160;to further ensure sterilization. Keep dish lid off during this step.</li>
	<li>Add 400 &#956;l of prepared laminin-1 to each glass coverslip. Use pipette tip to spread laminin-1 to ensure full coverage of coverslip. Surface tension will allow laminin-1 to remain on coverslip and not spread onto bottom of culture dish, which is required.</li>
	<li>Place lid onto dish and allow laminin-1 to incubate for 2-3 hr&#160;at room temperature (3 hr&#160;is optimal).</li>
	<li>After laminin-1 incubation, rinse coverslips using aseptic technique with sterile PBS in laminar flow hood. Remove solution on coverslip and add 400 &#956;l of PBS. Wait 5-10 min. Repeat for a total of 3&#160;rinses.</li>
	<li>Leave PBS on coverslip after last rinse. Do not break surface tension during rinsing and do not allow coverslips to dry out. Leave lid on until ready to plate dissociated neurons onto the coverlip.</li>
</ol>
5. Dissociation, Enriching, and Culturing DRG Neurons &#8211; Part 2
<ol>
	<li>After incubation, use a 10 ml sterile pipette to gently rinse the bottom of the 100 mm dish containing dissociated DRG cells. Hold dish at ~45&#176;&#160;angle, pull ~7 ml of F12HS10 with pipette aid, then gently expel 7 ml onto approximately 1/3 of dish. Repeat 5x, gently dislodging neurons.</li>
	<li>Rotate dish and repeat rinsing procedure, for another six rinses, on another 1/3 of the dish. Repeat procedure again for remaining 1/3 of dish. In this way, gently rinse the entire bottom of the dish with F12HS10 to remove neurons.</li>
	<li>Using same pipette, transfer F12HS10 containing neurons from 100 mm dish to 15 conical tube. Centrifuge for 5-10 min&#160;at 200 g to pellet cells.</li>
	<li>Remove F12HS10, leaving pellet undisturbed. Resuspend pellet with 2 ml of warmed F12H + supplements.</li>
	<li>Use a hemocytometer to determine cell density.</li>
	<li>Dilute cells as necessary with F12H + supplements to obtain desired plating concentration (120,000 cells/ml for coverslips coated with 1 &#956;g/ml laminin-1 and 40,000 cells/ml for coverslips coated with 20 &#956;g/ml laminin-18,16).</li>
	<li>Remove PBS from laminin-coated coverslips and immediately place 400 &#956;l of cells onto coverslips. Carefully transfer dish to a humidified, 37 &#176;C cell culture incubator. Allow to incubate for at least 1 hr and up to 3 hr&#160;to allow neurons to adhere to laminin-1.</li>
	<li>Under sterile conditions, gently flood 35 mm dishes containing neurons with 1.6 ml of F12H + supplements. 
	NOTE: At this time, surface tension on coverslip can be broken. Neurons have adequately adhered to laminin-1 on coverslip.</li>
	<li>Return cells to incubator and incubate overnight. Perform experiments such as immunocytochemistry the following day.</li>
</ol>
6. Immunocytochemistry
<ol>
	<li>Prior to immunocytochemistry, warm fixative solution (4% paraformaldehyde, 30% sucrose 2X PBS) in 37 &#176;C water bath. 
	NOTE: Before fixing cells, treat with various reagents, such as netrin-1, Mn2+, sonic hedgehog, semaphorin, and ephrin16,29-31.</li>
	<li>Slowly remove 1 ml of the 2 ml of F12H + supplements from culture dish. Gently add 1 ml of warmed fixative solution. Allow to incubate at room temperature for 10-15 min.</li>
	<li>Carefully remove fixative solution and add 2 ml PBS. Apply solution towards edge of dish, avoiding possible dislodging of fixed cells due to application of PBS. Incubate at room temperature for 5 min.</li>
	<li>Repeat PBS wash for a total of 5 washes. Do not allow cells to dry during any step of immunocytochemistry.</li>
	<li>Remove PBS. Apply 1 ml of blocking solution (0.1% Triton-X, 1X PBS, 5% normal goat serum). Incubate for 1 hr at room temperature. Alternatively, to stain surface molecules, do not use Triton-X in blocking solution8.</li>
	<li>Dilute primary antibodies into blocking solution per manufacture&#8217;s recommendation. Use 1:500 for antibodies against NCAM and activated &#946;1 integrins16. Remove blocking solution, add 1 ml of primary antibody solution and incubate for 1 hr at room temperature or overnight at 4 &#176;C.</li>
	<li>Remove primary antibody solution and add 2 ml of 1X PBS. Incubate for 5-10 min&#160;at room temperature. Repeat for a total of 4 PBS washes. Washes can be extended, if needed.</li>
	<li>Dilute secondary antibody 1:1,000 in blocking solution. Remove PBS, add 1 ml secondary antibody solution and incubate for 1 hr at room temperature. 
	NOTE: Secondary antibodies are light sensitive. Keep solutions and cells in the dark as much as possible from this point forward.</li>
	<li>Remove secondary antibody solution and rinse 5x&#160;with 1x&#160;PBS, as done previously.</li>
	<li>Apply ~60-80 &#956;l of Fluoromount G onto a microscope slide. Using fine forceps, gently lift coverslip out of dish and lower coverslip into Fluoromount G onto microscope slide. Avoid air bubbles.</li>
	<li>Store slides horizontally at 4 &#176;C in the dark. After 12-24 hr, Fluormount G will polymerize and coverslip will be firmly attached to microscope slide. Image cells after this polymerization step is complete.</li>
</ol>

<ol>
	<li>Millet, L. J., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over+a+century+of+neuron+culture:+from+the+hanging+drop+to+microfluidic+devices.">Over a century of neuron culture: from the hanging drop to microfluidic devices.</a> The Yale journal of biology and medicine. 85, 501-521 (2012).</li><li>Guan, W., Puthenveedu, M. A., Condic, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+neuron+subtypes+have+unique+substratum+preference+and+receptor+expression+before+target+innervation.">Sensory neuron subtypes have unique substratum preference and receptor expression before target innervation.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23, 1781-1791 (2003).</li><li>Kapitein, L. C., Hoogenraad, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+way+to+go?+Cytoskeletal+organization+and+polarized+transport+in+neurons.">Which way to go? Cytoskeletal organization and polarized transport in neurons.</a> Molecular and cellular neurosciences. 46, 9-20 (2011).</li><li>Kapitein, L. C., Yau, K. W., Hoogenraad, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+in+dendritic+spines.">Microtubule dynamics in dendritic spines.</a> Methods in cell biology. 97, 111-132 (2010).</li><li>Snow, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pioneering+studies+on+the+mechanisms+of+axonal+regeneration.">Pioneering studies on the mechanisms of axonal regeneration.</a> Developmental neurobiology. 71, 785-789 (2011).</li><li>Melli, G., Hoke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+Root+Ganglia+Sensory+Neuronal+Cultures:+a+tool+for+drug+discovery+for+peripheral+neuropathies.">Dorsal Root Ganglia Sensory Neuronal Cultures: a tool for drug discovery for peripheral neuropathies.</a> Expert opinion on drug discovery. 4, 1035-1045 (2009).</li><li>Condic, M. L., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-induced+changes+in+integrin+expression+regulate+neuronal+adhesion+and+neurite+outgrowth.">Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth.</a> Nature. 389, 852-856 (1997).</li><li>Lemons, M. L., Condic, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+integrin+activation+and+intracellular+cAMP+cause+Rho+GTPase+dependent+growth+cone+collapse+on+laminin-1.">Combined integrin activation and intracellular cAMP cause Rho GTPase dependent growth cone collapse on laminin-1.</a> Experimental neurology. 202, 324-335 (2006).</li><li>Beller, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+sensory+neuron+growth+cone+and+filopodial+responses+to+structurally+diverse+aggrecan+variants,+in+vitro.">Comparison of sensory neuron growth cone and filopodial responses to structurally diverse aggrecan variants, in vitro.</a> Experimental neurology. 247, 143-157 (2013).</li><li>Condic, M. L., Snow, D. M., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+neurons+adapt+to+the+inhibitory+proteoglycan+aggrecan+by+increasing+integrin+expression.">Embryonic neurons adapt to the inhibitory proteoglycan aggrecan by increasing integrin expression.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 19, 10036-10043 (1999).</li><li>Lemons, M. L., Barua, S., Abanto, M. L., Halfter, W., Condic, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+sensory+neurons+to+hyalectin+and+decorin+proteoglycans.">Adaptation of sensory neurons to hyalectin and decorin proteoglycans.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 25, 4964-4973 (2005).</li><li>Guan, W., Condic, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Netrin-1,+Neogenin+and+cUNC-5H3+expression+during+chick+dorsal+root+ganglia+development.">Characterization of Netrin-1, Neogenin and cUNC-5H3 expression during chick dorsal root ganglia development.</a> Gene Expr Patterns. 3, 369-373 (2003).</li><li>Guan, W., Wang, G., Scott, S. A., Condic, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shh+influences+cell+number+and+the+distribution+of+neuronal+subtypes+in+dorsal+root+ganglia.">Shh influences cell number and the distribution of neuronal subtypes in dorsal root ganglia.</a> Developmental biology. 314, 317-328 (2008).</li><li>Kolpak, A., Zhang, J., Bao, Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+has+a+dual+effect+on+the+growth+of+retinal+ganglion+axons+depending+on+its+concentration.">Sonic hedgehog has a dual effect on the growth of retinal ganglion axons depending on its concentration.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 25, 3432-3441 (2005).</li><li>Kolpak, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+guidance+factor-induced+macropinocytosis+in+the+growth+cone+plays+a+critical+role+in+repulsive+axon+turning.">Negative guidance factor-induced macropinocytosis in the growth cone plays a critical role in repulsive axon turning.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 29, 10488-10498 (2009).</li><li>Lemons, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+and+cAMP+mediate+netrin-induced+growth+cone+collapse.">Integrins and cAMP mediate netrin-induced growth cone collapse.</a> Brain research. 1537, 46-58 (2013).</li><li>Snow, D. M., Atkinson, P. B., Hassinger, T. D., Letourneau, P. C., Kater, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroitin+sulfate+proteoglycan+elevates+cytoplasmic+calcium+in+DRG+neurons.">Chondroitin sulfate proteoglycan elevates cytoplasmic calcium in DRG neurons.</a> Developmental biology. 166, 87-100 (1994).</li><li>Snow, D. M., Brown, E. M., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone+behavior+in+the+presence+of+soluble+chondroitin+sulfate+proteoglycan+(CSPG),+compared+to+behavior+on+CSPG+bound+to+laminin+or+fibronectin.">Growth cone behavior in the presence of soluble chondroitin sulfate proteoglycan (CSPG), compared to behavior on CSPG bound to laminin or fibronectin.</a> Int J Dev Neurosci. 14, 331-349 (1996).</li><li>Snow, D. M., Lemmon, V., Carrino, D. A., Caplan, A. I., Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+proteoglycans+in+astroglial+barriers+inhibit+neurite+outgrowth+in+vitro.">Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro.</a> Experimental neurology. 109, 111-130 (1990).</li><li>Snow, D. M., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+on+a+step+gradient+of+chondroitin+sulfate+proteoglycan+(CS-PG).">Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG).</a> Journal of neurobiology. 23, 322-336 (1992).</li><li>Snow, D. M., Mullins, N., Hynds, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nervous+system-derived+chondroitin+sulfate+proteoglycans+regulate+growth+cone+morphology+and+inhibit+neurite+outgrowth:+A+light,+epifluorescence,+and+electron+microscopy+study.">Nervous system-derived chondroitin sulfate proteoglycans regulate growth cone morphology and inhibit neurite outgrowth: A light, epifluorescence, and electron microscopy study.</a> Microsc Res Tech. 54, 273-286 (2001).</li><li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1, 791-803 (1988).</li><li>Firnhaber, C., Hammarlund, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-specific+feeding+RNAi+in+C.+elegans+and+its+use+in+a+screen+for+essential+genes+required+for+GABA+neuron+function.">Neuron-specific feeding RNAi in C. elegans and its use in a screen for essential genes required for GABA neuron function.</a> PLoS genetics. 9, e1003921 (2013).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 195, 231-272 (1992).</li><li>Hollyday, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+wing+innervation.+I.+Time+course+of+innervation+and+early+differentiation+of+the+peripheral+nerve+pattern.">Chick wing innervation. I. Time course of innervation and early differentiation of the peripheral nerve pattern.</a> The Journal of comparative neurology. 357, 242-253 (1995).</li><li>Hollyday, M., Morgan-Carr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+wing+innervation.+II.+Morphology+of+motor+and+sensory+axons+and+their+growth+cones+during+early+development.">Chick wing innervation. II. Morphology of motor and sensory axons and their growth cones during early development.</a> The Journal of comparative neurology. 357, 254-271 (1995).</li><li>Honig, M. G., Kueter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+cell+adhesion+molecules+on+the+growth+cones+of+chick+cutaneous+and+muscle+sensory+neurons.">The expression of cell adhesion molecules on the growth cones of chick cutaneous and muscle sensory neurons.</a> Developmental biology. 167, 563-583 (1995).</li><li>Tosney, K. W., Landmesser, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+major+pathways+for+neurite+outgrowth+in+the+chick+hindlimb.">Development of the major pathways for neurite outgrowth in the chick hindlimb.</a> Developmental biology. 109, 193-214 (1985).</li><li>Dontchev, V. D., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+and+semaphorin+3A+signaling+pathways+interact+in+regulating+sensory+neuronal+growth+cone+motility.">Nerve growth factor and semaphorin 3A signaling pathways interact in regulating sensory neuronal growth cone motility.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 22, 6659-6669 (2002).</li><li>Munoz, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin-A5+inhibits+growth+of+embryonic+sensory+neurons.">Ephrin-A5 inhibits growth of embryonic sensory neurons.</a> Developmental biology. 283, 397-408 (2005).</li><li>Roche, F. K., Marsick, B. M., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+in+distal+axons+is+not+required+for+growth+cone+responses+to+guidance+cues.">Protein synthesis in distal axons is not required for growth cone responses to guidance cues.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 29, 638-652 (2009).</li><li>Condic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neuronal+regeneration+induced+by+transgenic+integrin+expression.">Adult neuronal regeneration induced by transgenic integrin expression.</a> Journal of Neuroscience. 21, 4782-4788 (2001).</li><li>Hory-Lee, F., Russell, M., Lindsay, R. M., Frank, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin+3+supports+the+survival+of+developing+muscle+sensory+neurons+in+culture.">Neurotrophin 3 supports the survival of developing muscle sensory neurons in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 90, 2613-2617 (1993).</li><li>Toyofuku, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FARP2+triggers+signals+for+Sema3A-mediated+axonal+repulsion.">FARP2 triggers signals for Sema3A-mediated axonal repulsion.</a> Nature neuroscience. 8, 1712-1719 (2005).</li><li>Steinmetz, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+enhancement+of+the+intrinsic+growth+capacity+of+sensory+neurons+combined+with+the+degradation+of+inhibitory+proteoglycans+allows+functional+regeneration+of+sensory+axons+through+the+dorsal+root+entry+zone+in+the+mammalian+spinal+cord.">Chronic enhancement of the intrinsic growth capacity of sensory neurons combined with the degradation of inhibitory proteoglycans allows functional regeneration of sensory axons through the dorsal root entry zone in the mammalian spinal cord.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 25, 8066-8076 (2005).</li></ol>

The authors have nothing to disclose.

Here we present detailed protocols for isolating and culturing dissociated sensory neurons from a chick embryo. This procedure generates an enriched population of robustly growing neurons in vitro7,8,10,16. Numerous cellular and molecular techniques can be applied to these cultured neurons, including immunocytochemistry, which is described here. This protocol was recently used to quantitatively assess the intensity of immunolabeled activated integrins in sensory growth cones8,16. In these p...

Neurons are complex cells that carry information essential for a variety of functions including sensation, vision, motor movement, learning, and memory. Unique from other cell types, neurons extend arm-like processes, called axons, to form essential neural highways for communication. During development specialized compartments located at the tips of growing axons, called growth cones, navigate through a concert of extracellular cues to lead the axon to its appropriate destination. The intricate molecular mechanisms that underlie growth cone navigation are not fully understood. To better understand these mechanisms, investigators have used cell culture models to study neurons in a defined and simplified in vitro environment. Studying neurons in culture1 has led to significant advances in our understanding of neuronal cell biology including: neuronal differentiation2, cytoskeletal dynamics, endocytosis and trafficking,&#160;dendrite regulation3,4,&#160;axonal regeneration5, and clinical conditions such as neuropathies6. In addition, cultured neurons are highly amenable to a wide range of research techniques including immunocytochemistry, cell surface co-immunoprecipitation, Western blot, transfection, RNAi, and live imaging such as timelapse analysis of growth cone motility. Thus, culturing primary neurons is a powerful approach to elucidate numerous aspects of the cell biology of neurons.
The cell culture model provides investigators with detailed control over environmental conditions and variables. For example, the substrata on which neurons are plated (and grow upon) can be easily manipulated. Here, we provide detailed instructions for generating two distinct substrata, one with a low laminin-1 concentration and the other with saturating concentrations of laminin-1. Surprisingly, different concentrations of the same molecule can have dramatic effects on the internal state of neurons as well as their cell surface composition. For example, intracellular levels of cAMP and surface levels of integrins are significantly different in neurons plated on these two substrata7,8. Additional studies have shown that other molecules, including fibronectin and chondroitin sulfate proteoglycans, impact the expression of cell surface molecules and neuronal motility7-11. In addition, soluble molecules such as neurotrophins and neurotropins also impact cell membrane composition and neuronal motility12-16 and can be easily and accurately manipulated in a cell culture model.
Here, we describe methods to isolate and culture dissociated sensory neurons from chick embryos. This procedure has been used to make significant breakthroughs in neurobiology, including axon outgrowth5,7,8,10,11,16-21 and was modified from a procedure designed to isolate ganglion cells22. There are several advantages to this approach. First, many features of chick dorsal root ganglion (DRG) development are well characterized including the time frame for birth, axon extension, and protein expression profiles2,23-28, thus providing an instructive basis upon which to build informative in vitro experiments. Second, dissociated neuronal cultures allow the investigator to more directly study neurons compared to alternative approaches using intact DRG explants (which contain neurons and non-neuronal cells) and/or mixed cultures containing both dissociated neurons and non-neuronal cells. Third, the procedure described here is straightforward, inexpensive and amenable to undergraduates. Therefore, this technique can be used for research as well as for teaching purposes. Furthermore, minor variations of this protocol should allow fast, high yield purification of neurons from sources other than DRGs. For example, this procedure could be modified to provide neuronally enriched cultures from other tissues such as embryonic forebrain or spinal cord.
Immunocytochemistry protocols have been optimized for these dissociated neuronal cultures and are described in detail here. The procedure for double immunocytochemistry against neural cell adhesion molecule (NCAM) and &#946;1 integrins is provided. Data generated from these immunocytochemical methods have been used to examine the spatial patterning and intensity of several molecules in cultured neurons8,16.

<table border="0" cellpadding="0" cellspacing="0" width="694">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile small culture dishes (35mm)</td>
			<td style="width:176px;">Corning</td>
			<td style="width:135px;">430165</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile large culture dishes (100mm)</td>
			<td style="width:176px;">Falcon</td>
			<td style="width:135px;">353003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile large petri dishes (100mm)</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">89000-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glass petri dish (100mm)</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">D108962&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fine forceps</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:135px;">11251-10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">standard forceps</td>
			<td style="width:176px;">Fine Science Tools</td>
			<td style="width:135px;">1100-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">coverslips&#160; (22x22)</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:135px;">12 518 105K</td>
			<td>0.13-0.17 mm thickness is optimal for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">porcelin coverslip holder</td>
			<td style="width:176px;">Thomas scientific</td>
			<td style="width:135px;">8542E40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine serum</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">17502-048</td>
			<td>use 50ml aliquots per 500 mls of Ham&#39;s F12 media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCL</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">VW3204-1</td>
			<td>use at 2M, can be used twice before discarding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">S9888</td>
			<td>use 5M NaCl solution for laminin-1 stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">laminin-1</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:135px;">23017-015</td>
			<td>Add 72ul of sterile 5M NaCl and aliquot into 50ul</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml conical tube</td>
			<td style="width:176px;">cell treat</td>
			<td style="width:135px;">229411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS CMF 10X</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">14200</td>
			<td>used at 1X, dilute with sterile millipore filtered water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10X</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">14080</td>
			<td>used at 1X, dilute with sterile milliporee filtered water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ham&#39;s F12</td>
			<td style="width:176px;">Lonza</td>
			<td style="width:135px;">12-615F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum Albumin (BSA)</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">10438</td>
			<td>use in F12HS20 at 10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">H3375</td>
			<td>make sterile 1M solution in distilled water, dilute in F12 media to obtain final concentration of 10mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin streptomyocin</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">P0781</td>
			<td>use 5mls in 500mls of F12 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NT3</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">GF031</td>
			<td>Add 1ml of sterile millipore water, aliquot under sterile conditions, store at -20 0C, use at final concentration of 10ng/ml in F12H media, keep in -20 C&#160; non-defrosting freezer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:135px;">17502048</td>
			<td>&#160;aliquot under sterile conditions,stored at -20 0C,&#160; use 100ul per 10ml of F12H media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NGF</td>
			<td style="width:176px;">RnD systems</td>
			<td style="width:135px;">256-GF</td>
			<td>Add 1ml of sterile 1X PBS with 0.1% BSA, aliquot under sterile conditions, store at -20 0C, usd at final concentration of 10 ng/ml in F12H media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">l-glutamine</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">G7513</td>
			<td>aliquot under sterile conditions, store at -20 0C, use at final concentration of 200mM in F12H media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">T4049</td>
			<td>aliquot under sterile conditions, store at -20 0C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:135px;">15260</td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">Other items needed:&#160; general dissection instruments, including&#160; glass pasteur pipettes, fertile white leghorn chicken eggs, check egg incubator (humidified, 37 degrees C), cell incubation chamber (humidified, 37&#160; degrees C), laminar flow hood, binocular stereovision dissecting scope</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immunocytochemistry Reagents Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">S9378</td>
			<td>used in fixative solution (4% paraformaldehye, 30% sucrose, 2X PBS)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton-X 100</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:135px;">P6148</td>
			<td>used in fixative solution (4% paraformaldehye, 30% sucrose, 2X PBS)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluoromount G</td>
			<td style="width:176px;">Southern Biotech</td>
			<td style="width:135px;">0100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">normal goat serum</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">PCN500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microscope slides</td>
			<td style="width:176px;">VWR</td>
			<td style="width:135px;">16004-430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary Antibody Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibody against NCAM</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">AB5032</td>
			<td>polyclonal</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Antibody against activated beta 1 ingegrin</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:135px;">MAB19294</td>
			<td>monoclonal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Seconday Antibody Table</td>
			<td style="width:176px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat anti-mouse IgG Alexa 488</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">A11001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat anti-rabbit IgG Alexa 548</td>
			<td style="width:176px;">Life technologies</td>
			<td style="width:135px;">A11036</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The protocol described here enables investigators to culture an enriched population of dissociated embryonic sensory neurons with very few (e.g., &#60;5%) non-neuronal cells7,8,10,16. Numerous DRGs can be obtained from the lumbosacral, thoracic and cervical regions. Depending upon the needs of the investigator, DRGs from these distinct anatomical regions can be easily isolated. For example, Figure 1 shows images of the chick embryo through various stages of dissection with the lumbar ...

Watch this Scientific Journal Video about Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos at JoVE.com

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

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-dissociated-sensory-neurons-from-chick

Research

JoVE Journal

Neuroscience

14.2K Views.  Assumption College. 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.

Video: Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

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 ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

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 of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

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 ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

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 ...

Methods Article

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos