This work was financially supported by project SAF2017-82736-C2-1-R from Ministerio de Ciencia e Innovaci&#243;n to MTM-F and by Fundaci&#243;n Universidad Francisco de Vitoria to JS.

NOTE: Animal experimentation was approved by national and institutional bioethics committees.
1. ihOEG (Ts12 and Ts14) culture
NOTE: This procedure is done under sterile conditions in a tissue culture biosafety cabinet.
<ol>
	<li>Prepare 50 mL ME10 OEG culture medium as provided in Table 1.</li>
	<li>Prepare 5 mL of DMEM/F12-FBS, as provided in Table 1, in a 15 mL conical tube.</li>
	<li>Warm both media at 37 &#176;C in a clean water bath, for 15 min.</li>
	<li>Thaw Ts12 and Ts14 cells vials at 37 &#176;C in a clean water bath.</li>
	<li>Resuspend and add cells to the DMEM/F12-FBS culture medium prepared in step 2.</li>
	<li>Centrifuge for 5 min at 200 x g.</li>
	<li>Aspire the supernatant.</li>
	<li>Add 500 &#181;L of ME10 medium and resuspend the pellet.</li>
	<li>Prepare a p60 cell culture dish with 3 mL of ME10 and add the cellular suspension, dropwise.</li>
	<li>Move to distribute the cells uniformly across the plate.</li>
	<li>Culture cells at 37 &#176;C in 5% CO2. 
	NOTE: After reaching confluence, at least another passage must be done to optimize cells for coculture. 90% confluence is needed before seeding them on the coverslips for coculture. A confluent p-60 has a mean cell number of 7 x 105 for Ts14 and 2.5 x 106 for Ts12 cell lines. Ts12 and Ts14 cell lines should be passaged every 2&#8211;3 days.</li>
</ol>
2. Preparation of ihOEG (Ts12 and Ts14) for the assay
NOTE: This step must be done 24 h before RGN dissection and coculture.
<ol>
	<li>Treat 12 mm &#216; coverslips with 10 &#181;g/mL poly-L-lysine (PLL) for 1 h. 
	NOTE: The coverslips can be left overnight in PLL solution.</li>
	<li>Wash the coverslips with 1x phosphate buffer saline (PBS), three times.</li>
	<li>Detach Ts12 and Ts14 ihOEG cells from p60 cell culture dish.
	<ol>
		<li>Add 4 mL of DMEM/F12-FBS culture medium (Table 1) to a 15 mL conical tube. Warm at 37 &#176;C in a clean water bath.</li>
		<li>Remove the medium from plates and wash cells with 1 mL of 1x PBS-EDTA, once.</li>
		<li>Add 1 mL of trypsin-EDTA to the OEG cells and incubate for 3&#8211;5 min at 37 &#176;C, 5% CO2.</li>
		<li>Collect cells with a p1000 pipette and transfer them to medium prepared in step 3.1.</li>
		<li>Centrifuge for 5 min at 200 x g.</li>
		<li>Aspire the supernatant.</li>
		<li>Add 1 mL of ME10 medium and resuspend the pellet.</li>
		<li>Count the cell number in a hemocytometer.</li>
	</ol>
	</li>
	<li>Seed 80,000 Ts14 cells or 100,000 Ts12 cells/well onto the coverslips in 24-well plates in 500 &#181;L of ME10 medium.</li>
	<li>Culture cells at 37 &#176;C in 5% CO2, for 24 h.</li>
</ol>
3. Retinal tissue dissection
NOTE: 2-month old male Wistar rats are used as RGN source. Two retinas (one rat) for 20 wells of a 24-well cell dish. Autoclave surgical material before use. Papain dissociation kit is commercially purchased (Table of Materials). Follow the provider&#180;s instructions for reconstitution. Reconstitute D,L-2-amino-5-phosphonovaleric acid (APV) in 5 mM stock and prepare the aliquots.
<ol>
	<li>On the day of the assay, prepare the following media.
	<ol>
		<li>Prepare a p60 cell culture dish with 5 mL of cold EBSS (vial 1 of the papain dissociation kit).</li>
		<li>Prepare a p60 cell culture dish with reconstituted vial 2 (papain) of the papain dissociation kit plus 50 &#181;L of APV.&#160; Then, add 250 &#181;L ofreconstituted vial 3 (DNase plus 5 &#181;L of APV).</li>
		<li>In a sterile tube mix 2.7 mL of vial 1 with 300 &#181;L of vial 4 (albumin-ovomucoid protease inhibitor). Add 150 &#181;L of vial 3 (DNase) plus 30 &#181;L of APV.</li>
		<li>Prepare 20 mL of Neurobasal-B27 medium (NB-B27) as provided in Table 1.</li>
	</ol>
	</li>
	<li>Sacrifice a rat by asphyxiation with CO2.</li>
	<li>Remove the head by decapitation with guillotine; place it in a 100 mm Petri dish and spray the head with ethanol 70% before placing it in a laminar flow hood.</li>
	<li>Cut the rat&#180;s whiskers with scissors so they do not interfere with the eye manipulation.</li>
	<li>Grip the optic nerve with forceps to pull out the eyeball enough to be able to make an incision across the eye with a scalpel.</li>
	<li>Remove the lens and vitreous humor and pull out the retina (orange-like tissue), while the remaining layers of the eye stay inside (including the pigment epithelial layer).</li>
	<li>Place the retina in the p60 cell culture dish prepared in step 3.1.1.</li>
	<li>Transfer the retina to the p60 cell culture dish prepared in step 3.1.2 and cut it with the scalpel in small pieces of an approximate size &#60; 1 mm.</li>
	<li>Transfer to a 15 mL plastic tube.</li>
	<li>Incubate the tissue for 30 min, in a humidified incubator at 37 &#176;C under 5% CO2, with agitation every 10 min.</li>
	<li>Dissociate cell clumps by pipetting up and down with a glass Pasteur pipette.</li>
	<li>Centrifuge the cell suspension at 200 x g for 5 min.</li>
	<li>Discard supernatant and to inactivate papain, resuspend the cell pellet in the solution prepared in step 3.1.3. (1.5 mL for 2 eyes).</li>
	<li>Carefully pipet this cell suspension into 5 mL of reconstituted vial 4.</li>
	<li>Centrifuge at 200 x g for 5 min.</li>
	<li>While centrifuging, completely remove the ME-10 medium from the OEG 24 well cell plate (previously prepared in step 2) and replace it with 500 &#181;L of NB-B27 medium per well.</li>
	<li>Discard the supernatant and resuspend the cells in 2 mL of NB-B27 medium.</li>
	<li>Plate 100 &#181;L of retinal cell suspension, per well of the m24 plate, onto PLL-treated or OEG monolayers-coverslips.</li>
	<li>Maintain cultures at 37 &#176;C with 5% CO2 for 96 h in NB-B27 medium.</li>
</ol>
4. Immunostaining
<ol>
	<li>After 96 h, fix the cells for 10 min by adding the same volume of 4% paraformaldehyde (PFA) in 1x PBS to the culture medium (600 &#181;L) (PFA final concentration 2%).</li>
	<li>Remove the media and PFA from the 24-multiwell plate and once again add 500 &#181;L of 4% paraformaldehyde (PFA) in 1x PBS. Incubate for 10 min.</li>
	<li>Discard the fixer and wash 3 times with 1x PBS for 5 min.</li>
	<li>Block with 0.1% Triton X-100/1% FBS in PBS (PBS-TS) for 30&#8211;40 min.</li>
	<li>Prepare the primary antibodies in PBS-TS buffer as follows: SMI31 (against MAP1B and NF-H proteins) monoclonal antibody (1:500). 514 (recognizes MAP2A and B proteins) rabbit polyclonal antiserum (1:400).</li>
	<li>Add primary antibodies to cocultures and incubate overnight at 4 &#176;C.</li>
	<li>Next day, discard the antibodies and wash the coverslips with 1x PBS, 3 times, for 5 min.</li>
	<li>Prepare the secondary antibodies in PBS-TS buffer as follows: For SMI-31, anti-mouse Alexa Fluor 488 (1:500). For 514, anti-rabbit Alexa-594 (1:500).</li>
	<li>Incubate cells with the corresponding fluorescent secondary antibodies for 1 h, at RT, in the dark.</li>
	<li>Wash the coverslips with 1x PBS, 3 times, for 5 min, in the dark.</li>
	<li>Finally, mount coverslips with mounting medium (Table of Materials) and keep at 4 &#176;C. 
	NOTE: Whenever necessary, fluorescent nuclei staining with DAPI (4,6-diamidino-2-phenylindole) may be performed. Before mounting, incubate the cells for 10 min in the dark with DAPI (10 &#181;g/mL in 1x PBS). Wash the coverslips 3 times with 1x PBS and finally, mount the coverslips with the mounting medium.</li>
</ol>
5. Axonal regeneration quantification
NOTE: Samples are quantified under the 40x objective of an epifluorescence microscope. A minimum of 30 pictures should be taken on random fields, with at least 200 neurons, to be quantified for each treatment. Each experiment should be repeated a minimum of three times.
<ol>
	<li>Quantify the percentage of neurons with axon (SMI31 positive neurite) relative to the total population of RGNs (identified with MAP2A/B 514 positive immunostaining of neuronal body and dendrites).</li>
	<li>Quantify the axonal regeneration index or mean axonal length (&#181;m/neuron). This parameter is defined as the sum of the lengths (in &#181;m) of all identified axons, divided by the total number of counted neurons, whether they presented an axon or not. Axonal length is determined using the plugin NeuronJ of the image software ImageJ (NIH-USA).</li>
	<li>Calculate the mean, standard deviation, and statistical significance using the appropriate software.</li>
</ol>

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T., Lim, F., Mart&#237;n-Bermejo, M. J., D&#237;az-Nido, J., Avila, J., Wandosell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+olfactory+ensheathing+glia+promote+axonal+regeneration+of+rat+retinal+ganglion+neurons.">Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons.</a> Journal of Neurochemistry. 85 (4), 861-871 (2003).</li><li>Garc&#237;a-Escudero, V., 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=Patient-derived+olfactory+mucosa+cells+but+not+lung+or+skin+fibroblasts+mediate+axonal+regeneration+of+retinal+ganglion+neurons.">Patient-derived olfactory mucosa cells but not lung or skin fibroblasts mediate axonal regeneration of retinal ganglion neurons.</a> Neuroscience Letters. 509 (1), 27-32 (2012).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proceedings of the National Academy of Sciences of the United States of America. 80 (19), 6126-6130 (1983).</li><li>S&#225;nchez Martin, C., D&#237;az-Nido, J., Avila, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+a+site-specific+phosphorylation+of+the+microtubule-associated+protein+2+during+the+development+of+cultured+neurons.">Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons.</a> Neuroscience. 87 (4), 861-870 (1998).</li><li>Reshamwala, R., Shah, M., Belt, L., Ekberg, J. A. K., St. John, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+cell+purification+and+determination+of+cell+purity:+crucial+aspects+of+olfactory+ensheathing+cell+transplantation+for+spinal+cord+repair.">Reliable cell purification and determination of cell purity: crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair.</a> Neural Regeneration Research. 15 (11), 2016-2026 (2020).</li></ol>

The authors have nothing to disclose.

OEG transplantation at CNS injury sites is considered a promising therapy for CNS injury due to its constitutive pro-neuroregenerative properties7,8,9. However, depending on the tissue source&#8212;olfactory mucosa (OM-OEG) versus olfactory bulb (OB-OEG)&#8212;or the age of the donor, considerable variation exists in such capacity26,31,33</su...

Adult central nervous system (CNS) neurons have limited regenerative capacity after injury or disease. A common strategy to promote CNS regeneration is transplantation, at the injury site, of cell types that induce axonal growth such as stem cells, Schwann cells, astrocytes or olfactory ensheathing glia (OEG) cells1,2,3,4,5.
OEG derives from the neural crest6 and locates in the olfactory mucosa and in the olfactory bulb. In the adult, olfactory sensory neurons die regularly as the result of environmental exposure and they are replaced by newly differentiated neurons. OEG surrounds and guides these new olfactory axons to enter the olfactory bulb and to establish new synapses with their targets in the CNS7. Due to these physiological attributes, OEG has been used in models of CNS injury such as spinal cord or optic nerve injury and its neuroregenerative and neuroprotective properties become proven8,9,10,11. Several factors have been identified as responsible of the pro-regenerative characteristics of these cells, including extracellular matrix proteases production or secretion of neurotrophic and axonal growth factors12,13,14.
Given the technical limitations to expand primary OEG cells, we previously established and characterized reversible immortalized human OEG (ihOEG) clonal lines, which provide an unlimited supply of homogeneous OEG. These ihOEG cells derive from primary cultures, prepared from olfactory bulbs obtained in autopsies. They were immortalized by transduction of the telomerase catalytic subunit (TERT) and the oncogene Bmi-1 and modified with the SV40 virus large T antigen15,16,17,18. Two of these ihOEG cell lines are Ts14, which maintains the regenerative capacity of the original cultures and Ts12, a low regenerative line that is used as a low regeneration control in these experiments18.
To assess OEG capacity to foster axonal regeneration after neural injury, several in vitro models have been implemented. In these models, OEG is applied to cultures of different neuronal origin and neurite formation and elongation&#8212;in response to glial coculture&#8212;are assayed. Examples of such neuronal sources are neonatal rat cortical neurons19, scratch wounds performed on rat embryonic neurons from cortical tissue20, rat retinal explants21, rat hypothalamic or hippocampal postnatal neurons22,23, postnatal rat dorsal root ganglion neurons24, postnatal mouse corticospinal tract neurons25, human NT2 neurons26, or postnatal cerebral cortical neurons on reactive astrocyte scar-like cultures27.
In these models, however, the regeneration assay relies on embryonic or postnatal neurons, which have an intrinsic plasticity that is absent in injured adult neurons. To overcome this drawback, we present a model of adult axonal regeneration in cocultures of OEG lines with adult retinal ganglion neurons (RGNs), based on the one originally developed by Wigley et al.28,29,30,31 and modified and used by our group12,13,14,15,16,17,18,32,33. Briefly, retinal tissue is extracted from adult rats and digested with papain. Retinal cell suspension is then plated on either polylysine-treated coverslips or onto Ts14 and Ts12 monolayers. Cultures are maintained for 96 h before they are fixed and then immunofluorescence for axonal (MAP1B and NF-H proteins)34 and somatodendritic (MAP2A and B)35 markers is performed. Axonal regeneration is quantified as a percentage of neurons with axon, with respect to the total population of RGNs and axonal regeneration index is calculated as the mean axonal length per neuron. This protocol is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

<table>
	<tbody>
		<tr>
			<td>antibody 514</td>
			<td>Reference 34</td>
			<td></td>
			<td>Rabbit polyclonal antiserum, which recognizes MAP2A and B.</td>
		</tr>
		<tr>
			<td>antibody SMI-31</td>
			<td>BioLegend</td>
			<td>801601</td>
			<td>Monoclonal antibody against MAP1B and NF-H proteins</td>
		</tr>
		<tr>
			<td>anti-mouse Alexa Fluor 488 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa Fluor 594 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21207</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-2-amino-5-phosphonovaleric acid</td>
			<td>Sigma</td>
			<td>283967</td>
			<td>NMDA receptor inhibitor</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Nuclei fluorescent stain</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>11573397</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>FBS-Hyclone</td>
			<td>Fisher Scientific</td>
			<td>16291082</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health (NIH-USA)</td>
			<td></td>
			<td>Image software</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605F</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Neuronal cells culture medium</td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>For use in neural cell isolation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-EDTA</td>
			<td>Lonza</td>
			<td>H3BE02-017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Bacteriostatic and bactericidal</td>
		</tr>
		<tr>
			<td>Pituitary extract</td>
			<td>Gibco</td>
			<td>13028014</td>
			<td>Bovine pituitary extract</td>
		</tr>
		<tr>
			<td>Poly -L- lysine (PLL)</td>
			<td>Sigma</td>
			<td>A-003-M</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

In this protocol, we present an in vitro model to assay OEG neuroregenerative capacity after neuronal injury. As shown in Figure 1, the OEG source is a reversible immortalized human OEG clonal cell line -Ts14 and Ts12-, which derives from primary cultures, prepared from olfactory bulbs obtained in autopsies15,17,18. Retinal tissue is extracted from adult rats, digested, and retinal ganglion neurons ...

Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

coculture-axotomized-rat-retinal-ganglion-neurons-with-olfactory

Research

JoVE Journal

Neuroscience

3.8K Views.  Universidad Francisco de Vitoria. We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Video: Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...