We thank Dr. M. Calkins for English editing. This work was supported by the Chang Gung Memorial Hospital (CMRPD1F0482), Chang Gung University, Healthy Aging Research Center (EMRPD1G0171) and Ministry of Science and Technology (105-2320-B-182-012-MY2).

All methods described herein that use experimental animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University (CGU 13-014).
1. Collect Lumbar DRG from Experimental Rats
<ol>
	<li>Use 2 to 3 week-old Sprague-Dawley (SD) rats for lumbar DRG collection. 
	NOTE: DRG neurons collected from rats over 4 weeks of age do not grow well under the culture conditions described herein.</li>
	<li>Sterilize all surgical instruments in an autoclave.</li>
	<...

<ol>
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S., Santiago, M. F., Marques, S. A., Allodi, S., Martinez, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+among+satellite+glial+cells+in+dorsal+root+ganglia+of+the+rat.">Diversity among satellite glial cells in dorsal root ganglia of the rat.</a> Braz J Med Biol Res. 41 (11), 1011-1017 (2008).</li><li>Costa, F. A., Moreira Neto, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+glial+cells+in+sensory+ganglia:+its+role+in+pain.">Satellite glial cells in sensory ganglia: its role in pain.</a> Rev Bras Anestesiol. 65 (1), 73-81 (2015).</li><li>Malin, S. A., Davis, B. M., Molliver, D. 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J., Staats, P., Kramer, 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+Dorsal+Root+Ganglion+as+a+Therapeutic+Target+for+Chronic+Pain.">The Dorsal Root Ganglion as a Therapeutic Target for Chronic Pain.</a> Reg Anesth Pain Med. 41 (4), 511-519 (2016).</li><li>Lee, J. S., Han, J. S., Lee, K., Bang, J., Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+peripheral+and+central+mechanisms+underlying+itch.">The peripheral and central mechanisms underlying itch.</a> BMB Rep. 49 (9), 474-487 (2016).</li><li>Valtcheva, M. 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=Surgical+extraction+of+human+dorsal+root+ganglia+from+organ+donors+and+preparation+of+primary+sensory+neuron+cultures.">Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures.</a> Nat Protoc. 11 (10), 1877-1888 (2016).</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 Opin Drug Discov. 4 (10), 1035-1045 (2009).</li><li>Yin, K., Baillie, G. J., Vetter, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cell+lines+as+model+dorsal+root+ganglion+neurons:+A+transcriptomic+comparison.">Neuronal cell lines as model dorsal root ganglion neurons: A transcriptomic comparison.</a> Mol Pain. 12, (2016).</li><li>Chen, W., Mi, R., Haughey, N., Oz, M., 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=Immortalization+and+characterization+of+a+nociceptive+dorsal+root+ganglion+sensory+neuronal+line.">Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line.</a> J Peripher Nerv Syst. 12 (2), 121-130 (2007).</li><li>Doran, C., Chetrit, J., Holley, M. C., Grundy, D., Nassar, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+DRG+Cell+Line+with+Properties+of+Nociceptors.">Mouse DRG Cell Line with Properties of Nociceptors.</a> PLoS One. 10 (6), e0128670 (2015).</li><li>Gouarderes, C., Roumy, M., Advokat, C., Jhamandas, K., Zajac, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+localization+of+neuropeptide+FF+receptors+in+the+rat+dorsal+horn.">Dual localization of neuropeptide FF receptors in the rat dorsal horn.</a> Synapse. 35 (1), 45-52 (2000).</li><li>Lin, Y. 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=Activation+of+NPFFR2+leads+to+hyperalgesia+through+the+spinal+inflammatory+mediator+CGRP+in+mice.">Activation of NPFFR2 leads to hyperalgesia through the spinal inflammatory mediator CGRP in mice.</a> Exp Neurol. 291, 62-73 (2017).</li><li>Yang, H. Y., Tao, T., Iadarola, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulatory+role+of+neuropeptide+FF+system+in+nociception+and+opiate+analgesia.">Modulatory role of neuropeptide FF system in nociception and opiate analgesia.</a> Neuropeptides. 42 (1), 1-18 (2008).</li><li>Takeda, M., Takahashi, M., Matsumoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+activation+of+satellite+glia+in+sensory+ganglia+to+pathological+pain.">Contribution of the activation of satellite glia in sensory ganglia to pathological pain.</a> Neurosci Biobehav Rev. 33 (6), 784-792 (2009).</li></ol>

The authors have nothing to disclose.

In the present article, we demonstrate the collection, enzyme-dissociation, and culture of rat lumbar DRG. With the neurotrophic support from NGF, the axons of DRG neurons extended within 3 days after cell seeding. The extended axons were clearly observable after cells were stained for CGRP protein, which is synthesized in the cell soma and transported along the axon fibers. The processes of satellite cells also extended, allowing these dividing glial cells to surround the neurons within days. The primary DRG cells grown...

The cell bodies of sensory neurons are contained within DRG. These neurons are pseudo-unipolar and innervate both peripheral tissues and the central nervous system. The peripheral nerve endings of sensory neurons are found in muscle, skin, visceral organs, and bone, among other tissues. They transmit peripheral sensation signals to nerve endings in the spinal dorsal horn and the signals are then transmitted to the brain via different ascending pathways of somatic sensation1,2. Somatic sensation enables the body to feel (i.e., touch, pain, and thermal sensations) and perceive movement and spatial orientation (proprioceptive sensations)1,3. There are four subclasses of primary afferent axons, including group I (A&#945;) fibers that respond to proprioception of skeletal muscles, group II (A&#946;) fibers that respond to mechanoreceptors of the skin, and group III (A&#948;) and group V (C) fibers that respond to pain and temperature. Only the C fibers are unmyelinated, while the rest are myelinated to different degrees.
Nociceptors are primary sensory neurons, which are activated by noxious stimuli (mechanical, thermal, and chemical stimulation) that carry potential for tissue damage. These neurons are composed of myelinated A&#948; fibers and unmyelinated C fibers1,4. The A&#948; fibers express the receptors for nerve growth factor (NGF, trkA receptor), CGRP, and SP. The C fibers are classified as either peptidergic and non-peptidergic C fibers. On the other hand, the non-peptidergic C fibers express the receptors for glial-derived neurotrophic factor (GDNF, RET, and GFR receptors), isolectin IB4, and ATP-gated ion channel subtype (P2X3)5,6,7. Nociceptors can be distinguished by the expression of ion channels and activated by neurotrophic factors, cytokines, neuropeptides, ATP, or other chemical compounds8. Upon stimulation, neurotransmitters, including CGRP, SP, and glutamate may be released from sensory neuron terminals in the spinal dorsal horn to transmit nociceptive signals2. DRG are not only composed of neurons, but also contain satellite glial cells. Satellite cells surround the sensory neurons and provide mechanical and metabolic support9,10. Interestingly, there is a growing body of evidence indicating that satellite glial cells in the DRG may be involved in regulating pain sensation11.
Sensory neurons have been reported to be the most frequently used primary neuronal cells12 and have been utilized for electrophysiology, signal transduction, and neurotransmitter release studies. They are also commonly used to explore the cellular mechanisms of neuronal development, inflammatory pain, neuropathic pain, skin sensation (like itch), and axon outgrowth12,13,14,15. DRG primary cultures can be cultured as dissociated neurons to assess biochemical changes in single or multiple cells, allowing scientists to perform studies that cannot be performed in experimental subjects. Recently, DRG were successfully cultured from human organ donors which might greatly benefit translational research16. On the other hand, sensory neurons can also be cultured as DRG explants. The DRG explants preserve the original tissue architecture of the neurons, including Schwann cells and satellite glial cells, and are especially useful to study interactions between neuronal and non-neuronal cells17. DRG primary cultures can be easily prepared within 2.5 h. The cell composition and properties are highly reflective of the source DRG, and as such, specific DRG (lumbar or thoracic DRG) can be collected according to experimental demands. Cultures of embryonic and neonatal DRG neurons require NGF to survive and induce axon outgrowth, but cultures of adult neurons do not require the addition of neurotrophic factors to the media12,17. There are also commercially available DRG-hybridoma cell lines such as ND7/23 and F11, which do not require the use of experimental animals. However, the lack of the transient receptor potential cation channel subfamily V member 1 (TRPV1) expression (an important marker for small sensory nociceptive neurons) and incongruent gene expression profiles limit their applications18. Recently, immortalized DRG neuronal cell lines have been derived from rat (50B11)19 and mouse (MED17.11)20, which are suitable for use in high-throughput screens for drug targeting studies. However, gene expression profiling for these cell lines has yet to be performed. Thus, the validation experiments comparing these immortalized cells to sensory neurons are still ongoing.
NPFFR2 is synthesized in the DRG and translocated to the sensory nerve terminals in the spinal dorsal horn21. In this article, we provide a protocol for culturing lumbar DRG cells and treating them with an agonist of NPFFR2 to induce the release of neurotransmitters, CGRP and SP. The dependence on NPFFR2 is further tested using NPFFR2 small interfering RNA (siRNA), which may be transfected into the cultured DRG cells.

<table>
	<tbody>
		<tr>
			<td>Mixture of tiletamine and zolazepam (Zoletil)</td>
			<td>Virbac</td>
			<td>Zoletil 50</td>
			<td>anaesthetic</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biological Industries</td>
			<td>04-001-1</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Biological Industries</td>
			<td>03-033-1</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Invitrogen</td>
			<td>12400024</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>Poly-l-lysine</td>
			<td>Sigma</td>
			<td>P9011</td>
			<td>Coating dish</td>
		</tr>
		<tr>
			<td>Collagenase IA</td>
			<td>Sigma</td>
			<td>9001-12-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Biological Industries</td>
			<td>03-051-5</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Hilgenberg</td>
			<td>3150102</td>
			<td>Cell trituration</td>
		</tr>
		<tr>
			<td>Cytarabine (Ara-C)</td>
			<td>Sigma</td>
			<td>C6645</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Millipore</td>
			<td>NC011</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>NPFFR2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-099691-02-0005</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>Non-targeting siRNA</td>
			<td>Dharmacon</td>
			<td>L-001810-10-05</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>NeuroPORTER Reagent</td>
			<td>Genlantis</td>
			<td>T400150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>dNPA</td>
			<td>Genemed Synthesis</td>
			<td>N/A</td>
			<td>NPFFR2 agonist</td>
		</tr>
		<tr>
			<td>CGRP ELISA</td>
			<td>Cayman</td>
			<td>589001</td>
			<td>EIA</td>
		</tr>
		<tr>
			<td>SP ELISA</td>
			<td>Cayman</td>
			<td>583751</td>
			<td>EIA</td>
		</tr>
		<tr>
			<td>CGRP antibody</td>
			<td>Calbiochem</td>
			<td>PC205L</td>
			<td>IHC</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td>IHC</td>
		</tr>
	</tbody>
</table>

dorsal root ganglia isolation and primary culture to study neurotransmitter release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Rat lumbar DRG neurons, cultured in a 24-well plate, were grown in culture medium with additional Ara-C to inhibit glial cell proliferation and NGF to support neuronal growth. The morphology of living DRG cells was observed. As shown in Figure 3, the cell body of a single neuron was attached on the bottom of a dish at Day 1 and selected for observation. Axon growth was monitored from Day 1&#8211;3. The glial cells duplicated and extended processes to surround...

Watch this Scientific Journal Video about Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release at JoVE.com

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

This method can help to answer key questions in the sensory research field, such as peripheral nociception. The main advantage of this technical is that investigating the cellular mechanism of sensory neurons with the model that most simulates to physiological condition. Collect the lumbar dorsal root ganglia from the trunk of a two to three week old rat.Begin by making two cuts along the sides of the spinal column and one lateral cut to mark the rostral extent of the lumbar spine. Then, use bone-cutting forceps to remove the dorsal muscles of the spine. Next, remove the dorsal portion of the vertebrae and expose the spinal cord.Then use dissection scissors and forceps to remove the spinal cord. Identify the lumbar DRG by counting vertebrae from the last rib. This diagram shows the vertebrae positions.Use micro scissors to collect the 12 bilateral lumbar DRG from L1 to L6.Remove any attached neuronal fibers to improve the purity of the culture. Transfer each lumbar DRG to a 35 millimeter culture dish containing two milliliters of ice cold serum-free medium. Transfer the DRG-containing 35 millimeter dish into a laminar hood, and wash the DRG with serum-free medium three times by pipette.Use sterile tweezers to move the DRGs to a new 35 millimeter culture dish containing two milliliters of sterile collagenase type 1A. Place the tissue in collagenase solution in the 37 degree Celsius tissue culture incubator for 30 minutes to begin dissociation of the cells. Following the incubation, remove the collagenase solution and wash the DRG three times in two milliliters of Hank's balanced salt solution.Next, add two milliliters of prewarmed 0.05%trypsin EDTA to the dish, and digest the DRG in the incubator for 30 minutes as before. After digestion, use a glass pipette to transfer the two milliliters of DRG-containing solution to a 15 milliliter centrifuge tube. The DRG might stick to the glass pipette so this step should be performed with care.The actual loss can be avoid by keeping the DRG-containing solution in the taper end of the glass pipette, and transferring the solution into the centrifuge tube slowly but without pause. Next, centrifuge the solution at 290 times G for five minutes at four degrees Celsius. After centrifuging, remove the supernatant and add another two milliliters of serum-free medium to resuspend the DRG.Repeat the wash twice, using two milliliters of prewarmed culture medium to resuspend the tissue after the final centrifugation. Use the previously prepared sterile flame polished pipette to manually triturate the DRG approximately 60 times. Next, remove a poly-L-lysine coated 24 well plate containing one milliliter of culture medium per well from the CO2 incubator.Aspirate the incubated culture medium from the dish. Seed the DRG cells from one rat into four of the wells. This gives a seeding density of approximately 500, 000 cells per well.The following day, replace the culture medium with medium supplemented with 10 micromolar AraC, and 100 nanograms per milliliter NGF. On day three after cell plating, change the medium to 0.5 milliliters of prewarmed serum-free medium, and incubate for one hour. During the incubation, add 50 millimolar siRNA in one microliter of RNase-free water to 12.5 microliters of serum-free medium per transfection.Then pipette 2.5 microliters of transfection reagent into 10 microliters of serum-free medium for each transfection. Mix both solutions by pipette, and incubate this mixed transfection solution for 10 minutes at room temperature. After 10 minutes, add the transfection solution into the wells of the DRG-containing 24 well plate and mix by shaking gently.Following a six hour incubation, add 0.5 milliliters of culture medium with 20%FBS, 10 micromolar AraC, and 100 nanograms per milliliter NGF into each well of cells. Finally, incubate the DRG in a 37 degree Celsius CO2 incubator for another 66 hours. On day six after plating, and 72 hours after siRNA transfection, change the culture medium to 200 microliters of serum-free medium.Following a 30 minute incubation, add one microliter of the stimulation chemical and gently mix by pipetting. The NPFFR2 agonist, dNPA, and corresponding vehicle are used here. After incubating for the required period, collect the culture medium from the culture dish, and centrifuge at 5000 times G for five minutes at four degrees Celsius to remove any suspended impurities.Collect the supernatant from the centrifugation and dilute with phosphate-buffered saline as needed. Assay the levels of neurotransmitters with commercially available enzyme immunoassay kits. On day one, the cell body of a single neuron indicated by the arrow was attached on the bottom of a dish.A glial cell indicated by an arrowhead is also present. The glial cells duplicated and extended processes to surround the cell body of the sensory neuron on day two, a process that was even more prominent on day three. In another culture, CGRP protein was stained to reveal the shape of neurons.CGRP protein staining appears in the cytoplasm and axons of sensory neurons. The nuclear morphologies of neurons and glial cells are distinct when stained with DAPI. The neurons have a larger and more rounded nucleus than glial cells.By comparison, the nuclei of glial are more oval in shape. Once mastered, this technique can be done in two and half hours if it is performed properly. After watching this video, you should have a good understanding of how to dissect and culture the dorsal root ganglion, and use it as a tool to investigate underlying physiological functions.Don't forget that working with primary cells can be much more difficult than working with regular cell lines, and being gentle is always the best way when performing these procedure.

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JoVE Journal

Neuroscience

47.8K 조회수.  Chang Gung University. 이 방법은 주변 고치와 같은 감각 연구 분야의 주요 질문에 대답하는 데 도움이 될 수 있습니다. 이 기술의 주요 장점은 대부분의 생리적 상태를 시뮬레이션 하는 모델감각 뉴런의 세포 메커니즘을 조사 하는 것입니다. 2~3주 된 쥐의 트렁크에서 요추 등 뿌리 간리아를 수집합니다.척추의 측면을 따라 두 개의 컷을 만들고 요추 척추의 장밋빛 정도를 표시하기 위해 측면 ?...