Name: dorsal root ganglia isolation and primary culture to study neurotransmitter release
Uploaded: 2018-10-06T13:00:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release at JoVE.com

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>
	<li>Anesthetize the rat with a 1:1 mixture of tiletamine and zolazepam (20 mg/kg; intraperitoneal injection (IP)) and wait until the animal shows no foot-withdrawal response in a toe-pinch test. 
	NOTE: Different anesthesia strategies can be used successfully in this protocol.</li>
	<li>Sacrifice the rat by decapitation with a commercial guillotine.</li>
	<li>Use the guillotine to isolate the body trunk of the rat between the forelimb and femur. See Figure 1A for a diagram of the region to be collected. 
	NOTE: The caudal cut line should be just rostral to the femur. The lumbar L6 DRG will be excised if the cut site is too high in the spinal column.</li>
	<li>Cut along the sternum and remove all organs/tissues with dissection scissors (Figure 2A-a).</li>
	<li>Cut along the side of trunk to collect the dorsal part of the rat and remove the skin. See Figure 1B for a photograph of the dissected dorsal trunk.</li>
	<li>Prepare the tissue on ice before collecting DRG. Clean the fur and blood from gloves, and sterilize them with 75% ethanol before proceeding to the next step.</li>
	<li>Remove the muscles covering the lumbar spine. First, make two cuts along the sides of the spinal column (left and right) and one lateral cut to mark the rostral extent of the lumbar spine. Then, remove the dorsal muscles of the spine with bone cutting forceps (Figure 2A-b).</li>
	<li>Remove the dorsal portion of the vertebrae with bone cutting forceps and expose the spinal cord.</li>
	<li>Remove the spinal cord with dissection scissors (Figure 2A-c) and forceps (Figure 2A-d).</li>
	<li>Identify the lumbar DRG by counting vertebrae from the last rib (Thoracic Vertebra 13). See Figure 1C for a diagram of the vertebrae positions.</li>
	<li>Collect the bilateral lumbar DRG (L1-L6) with micro-scissors (Figure 2A-f) into a 35-mm culture dish with 2 mL ice-cold serum-free medium. Remove the neuronal fibers (as indicated in Figure 1C) from connecting DRG, then transfer it into the culture dish to improve the purity of the cultures. 
	NOTE: The collected DRG can be kept in medium on ice for about 1 h. Meanwhile, multiple rats can be euthanized to create a larger pool of DRG.</li>
</ol>
2. Primary Culture of Rat Lumber DRG
NOTE: The following steps should be performed in a laminar flow hood.
<ol>
	<li>Prepare culture medium containing 10% fetal bovine serum, 100 mM sodium pyruvate, and 1x penicillin/streptomycin in 1x DMEM-F12.</li>
	<li>Coat the cell-culture treated 24-well plate with 200 &#181;g/mL poly-L-lysine for 2 h then wash with sterilized water.</li>
	<li>Pre-incubate the culture dish with 1 mL culture medium in a 37 &#176;C CO2 incubator before use for least 30 min.</li>
	<li>Transfer the DRG-containing 35 mm dish into a laminar hood, and wash the DRG with serum-free medium 3 times by pipette. 
	NOTE: The outside of the dish should be cleaned with 75% ethanol before transferring into the hood. The 35 mm dish can contain DRG from a number of rats (this will depend on the demands of the experimental design).</li>
	<li>Move the DRG (from a single rat or combined from multiple rats) to a new 35 mm culture dish, which contains 2 mL of collagenase type IA (1 mg/mL in serum-free medium) with sterile tweezers (Figure 2A-e). 
	NOTE: The collagenase solution should be sterilized by passing it through a 0.22 &#181;m syringe filter.</li>
	<li>Digest the DRG in the collagenase solution in a 37 &#176;C CO2 incubator for 30 min.</li>
	<li>Remove the collagenase solution and wash the DRG 3 times in 2 mL Hank&#39;s balanced salt solution (HBSS). 
	NOTE: There may be residual fibers or tissues that come off the DRG into the solution. Remove them by pipette with the washing solution.</li>
	<li>Add 2 mL pre-warmed 0.05% trypsin-EDTA into the DRG-containing 35 mm dish and digest the DRG in a 37 &#176;C CO2 incubator for 30 min.</li>
	<li>Transfer the 2 mL of DRG-containing solution to a 15 mL centrifuge tube by glass pipette. 
	NOTE: The DRG might stick to the glass pipette so this step should be performed with care. DRG loss can be avoided by keeping the DRG-containing solution in the tapered end of a glass pipette (about 0.5 mL) and transferring the solution into the centrifuge tube slowly but without pause.</li>
	<li>Centrifuge the solution at 290 x g for 5 min at 4 &#176;C. Remove the supernatant and add another 2-mL serum-free medium to resuspend the DRG.</li>
	<li>Repeat step 2.10 2 times but change the serum-free medium to pre-warmed culture medium on the last time.</li>
	<li>Manually triturate the DRG approximately 60 times using a flame-polished Pasteur pipette (length 230 mm and tip head inner diameter 1 mm). See Figure 2B for a photograph comparing the orifice of a flame-polished Pasteur pipette to a non-polished pipette. 
	NOTE: The inside diameter of the flame-polished Pasteur pipette is approximately 10% smaller than the control pipette and the inside of the tapered end should be smoother. Be careful not to create bubbles when triturating the cells.</li>
	<li>Remove the poly-L-lysine-coated dish from the CO2 incubator. Aspirate the incubated culture medium from the dish, and seed the dissociated cells onto the coated dish.</li>
	<li>Seed the DRG cells from one rat (bilateral collection from L1-L6, for 12 total DRG) into four wells of a 24-well plate; there are approximately 5 x 104 cells in one well of a 24-well plate. 
	NOTE: This density is suitable for the detection of the released CGRP or SP and also suitable for immunostaining. For Western blot or RNA extraction, seed the DRG cells from one rat (bilateral L1-L6) into one well of a 6-well plate.</li>
	<li>Replace the culture medium on the following day with the addition of 10 &#181;M cytarabine (Ara-C) and 100 ng/mL NGF, and refresh the medium every two days thereafter. 
	NOTE: The thoracic DRG also can also be cultured by this protocol, if they have been collected from the thoracic spine.</li>
</ol>
3. Transfection of NPFFR2 siRNA in DRG Cells
<ol>
	<li>Perform the transfection of NPFFR2 siRNA and control siRNA according the manufacturer&#39;s protocol. 
	NOTE: The protocol will need to be adapted if the chosen transfection reagent is different from the one we used (see the Table of Materials).</li>
	<li>On Day 3 after cell plating, change the medium to 0.5 mL pre-warm serum-free medium and incubate the DRG in a 37 &#176;C CO2 incubator for 1&#160;h.</li>
	<li>Add 50 nM of siRNA (in 1 &#181;L RNase-free water) into 12.5 &#181;L serum-free medium.</li>
	<li>Add 2.5 &#181;L transfection reagent into 10 &#181;L serum-free medium.</li>
	<li>Mix the solution from steps 3.3 and 3.4 by pipette, and incubate this mixed transfection solution for 10 min at room temperature.</li>
	<li>Add the transfection solution into one DRG-containing 24-well plate and mix the solution with medium by gentle shaking. 
	NOTE: Multiple transfection solutions should be deployed at the same time if multiple wells need to be transfected.</li>
	<li>Incubate the DRG in a 37 &#176;C CO2 incubator for 6 h.</li>
	<li>Add 0.5 mL/well of culture medium containing 20% fetal bovine serum, 100 mM sodium pyruvate, and 1x penicillin/streptomycin in 1x DMEM-F12, with the addition of 10 &#181;M Ara-C and 100 ng/mL NGF, into the 24-well plate.</li>
	<li>Incubate the DRG in a 37 &#176;C CO2 incubator for another 66 h (refresh the medium at 48 h).</li>
</ol>
4. Release of Neurotransmitters from Primary DRG Cells
<ol>
	<li>On Day 6 after cells were plated (72 h after siRNA transfection), change the culture medium to 200 &#181;L serum-free medium, and incubate the cells in a 37 &#176;C CO2 incubator for 30 min.</li>
	<li>Add 1 &#181;L stimulation chemical(s) and gently mix the media by pipetting. Incubate the dish in a 37 &#176;C CO2 incubator for the designated time. 
	NOTE: In this article, the cultured cells were stimulated with the NPFFR2 agonist, dNPA (D.Asn-Pro-(N-Me)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg- Phe-NH2, 5 nmol), for 1 h.</li>
	<li>Collect the culture medium from the culture dish and centrifuge at 5,000 x g for 5 min at 4 &#176;C to remove any suspended impurities.</li>
	<li>Collect the supernatant from the centrifugation and dilute the samples with phosphate-buffered saline (PBS), as needed. Assay the levels of neurotransmitters with enzyme immunoassay (EIA) kits. 
	NOTE: Here, the supernatants were diluted 1:100 before analyzing the level of CGRP. The supernatant was not diluted before analyzing the level of SP.</li>
</ol>
5. CGRP and SP EIA
<ol>
	<li>Analyze the samples immediately according to the CGRP or SP EIA kit manufacturer&#39;s protocol. 
	NOTE: The protocol will vary depending on the kit used.</li>
	<li>Rinse the CGRP EIA wells 5 times with wash buffer supplied within the kit.</li>
	<li>Add 100 &#181;L samples with 100 &#181;L anti-CGRP acetylcholinesterase (AChE) tracer into the CGRP EIA wells, and add 50 &#181;L samples, 50 &#181;L anti-SP AChE tracer and 50 &#181;L anti-SP antiserum into the SP EIA wells.</li>
	<li>Seal the CGRP and SP wells with plastic film which is supplied within the kits.</li>
	<li>Incubate the wells overnight at 4 &#176;C.</li>
	<li>Wash the wells 5 times with CGRP or SP wash buffer and remove all the residual solution from the wells.</li>
	<li>Add 200 &#181;L Ellman&#39;s reagent into the CGRP or SP wells which is supplied within the corresponding EIA kits.</li>
	<li>Incubate the CGRP wells for 30 min at room temperature, and incubate the SP wells for 90 min at room temperature. Protect the wells from light for both assays.</li>
	<li>Read the plates at wavelength 414 nm and calculate the results according to the corresponding EIA instrument. 
	NOTE: Avoid touching the bottom of the wells by hand all the time and clean the water stains from the well bottom by lens cleaning wipes before adding the Ellman&#39;s reagent.</li>
</ol>

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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 by this protocol are suitable for investigations into the cellular mechanisms that regulate sensory neurons. Here, we stimulate the release of neuropeptides, CGRP and SP, from cultured DRG neurons by a selective NPFFR2 agonist, dNPA. NPFFR2 is the cognate receptor for NPFF and has been reported to participate in pain sensation and regulation pathways22,23. The NPFFR2-dependence of CGRP and SP release was further verified by the use of NPFFR2 siRNA.
DRG cultures contain both sensory neurons and satellite glial cells. The satellite glial cells provide metabolic support to neurons and maintain neuronal functions9,10. In the immunostaining pictures, it is easy to identify the neurons and glial cells, since their nuclei are shaped differently (show in Figure 4B). The existence of satellite cells in the culture dish might become problematic if there is an experimental demand to distinguish between the specific function of neurons and glia. For example, it is undeniable that satellite cells are involved in the development and maintenance of pain11,24, and in some studies, the actions of neurons and glial cells would be difficult to distinguish using DRG cultures.
In this protocol, there are a few critical steps that require extra caution. First, since the DRG are collected outside of the laminar hood, extra care should be taken during the tissue collection process to avoid cell contamination. There should be no need to create a sterile space, like in a human surgical room, but instrument sterilization and a clean operating space are essential. Tips to avoid contamination include keeping the sterilized instruments on the sterilization pouch when not in use and avoiding the touching of any unnecessary items. Also, contaminating organisms may be carried on fur that may stick to the rat body trunk or gloves. As such, fur and bloodstains on gloves should be removed by cleaning with 75% ethanol. It is also helpful for the operator to wear a surgical mask to prevent transfer of organisms from the breath or saliva. Furthermore, the 35-mm dish should be kept closed at all the times and only opened when placing dissected DRG inside. It is important to replace the 35 mm dish with a new dish before enzyme digestion. During enzyme digestion, do not extend the incubation time, since over-digestion may damage the neurons. Make sure to pre-warm the trypsin-EDTA to 37 &#176;C in order to achieve appropriate digestion efficiency. The efficiency will be dramatically reduced in lower temperatures, and it will be difficult to achieve a single cell suspension when triturating the DRG by flame-polished Pasteur pipette. Flame polishing the pipette will smooth the orifice and prevent the sharp glass edge from injuring the neurons. However, overheating the pipette with a flame will make the inside diameter too small, and tissue- or cell-containing solution will become difficult to pass through. This reduced diameter may also cause many bubbles to form during the trituration stage, greatly reducing the collectable number of DRG neurons. Finally, DRG cultures should be handled gently at all times, especially when changing medium or performing drug treatment.
The DRG neurons are reported to be the most frequently used primary cultured neuronal cells12. They can be utilized for a variety of different studies, ranging from electrophysiology or cell biology to exploring the physiological or pathological functions of sensory neurons. The major limitation of DRG primary cultures is that they are not well-suited for high-throughput screening. The number of cells that may be collected from the DRG of a single rat are limited, and the neurons are unable to duplicate in culture. Because of the limited cell number, several DRG-hybridoma cell lines or immortalized DRG neuronal cell lines have been developed to replace the primary cultures18,19,20. However, the protein expression profiles of DRG cell lines might not be the same as the original DRG and, thus, each model system needs to be carefully verified. Aside from isolating the cells in the lab, rat embryonic or neonatal DRG neurons have been made commercially available. Therefore, purchase from commercial sources may be a viable alternative to freshly prepared DRG cultures.
DRG primary cultures have been used for many years as a valuable experimental tool that is mostly adopted in pain-related studies. This model system is unlikely to be replaced in the near future. With good quality DRG neurons, scientists can obtain stable and reproducible results that benefit many areas of neuroscience study.

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&#039;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 1–3. The glial cells duplicated and extended processes to surround the cell body of the sensory neuron. In another culture, CGRP protein was stained to reveal the shape of neurons. In Figure 4, 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 glia are more oval in shape (Figure 4B).
The selective NPFFR2 agonist, dNPA, was used to stimulate the release of CGRP and SP. Moreover, the dependence of dNPA-stimulated neurotransmitter release on NPFFR2 was tested by transfecting cells with NPFFR2 siRNA. NPFFR2 siRNA or control siRNA were transfected into the primary DRG cells 72 h prior to agonist treatment. DRG cells were treated with dNPA (5 nmol) for 1 h and the release of CGRP and SP was measured by separate EIA kits. The simulation of DRG with dNPA increased the level of CGRP and SP in the media (Figure 5A, B). However, only the dNPA-induced CGRP release was inhibited by expression of NPFFR2 siRNA in cultured DRG cells. The results shown in Figure 5 were modified from a previous publication and are used here with permission22.
<img alt="Rodent dissection process; anatomy analysis; vertebrae identification; scientific experiment." class="xfigimg" src="/files/ftp_upload/57569/57569fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Tissue processing diagrams. Lumbar DRG are collected from 3 week-old rats. (A) The positions where the guillotine should be used to cut the animal are indicated by dotted lines. (B) The dorsal trunk with skin removed and (C) the locations of lumbar DRG (from L1-L6) are shown. The insert represents the DRG and the connecting fibers (which are indicated by the arrows). <a href="/files/ftp_upload/57569/57569fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical tool comparison, control vs. polished cannula surface for precision and quality evaluation." class="xfigimg" src="/files/ftp_upload/57569/57569fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Special equipment needed for isolating DRG primary cultures. (A) Surgical instruments used in the collection of DRG. From left to right: (a) dissection scissors (large), (b) bone cutting forceps, (c) dissection scissors (small), (d, e) point tweezers, and (f) micro-scissors. (B) A regular Pasteur pipette and a flame-polished Pasteur pipette. "a" denotes the inside diameter of regular Pasteur pipette, and "b" denotes the inside diameter of the flame-polished Pasteur pipette. b/a ≒ 0.9. <a href="/files/ftp_upload/57569/57569fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell differentiation microscope image, Day 1-3, neurite outgrowth tracking experiment." class="xfigimg" src="/files/ftp_upload/57569/57569fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: The morphology of living DRG cells. Live DRG cells were monitored by microscopy. Cells are shown (A) one day after seeding, (B) two days after seeding, and (C) three days after seeding. Arrows indicate neuron and arrowheads indicate glia. Scale bar = 20 µm. <a href="/files/ftp_upload/57569/57569fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="CGRP protein, DAPI staining, and merged fluorescence microscopy images of neuronal cells." class="xfigimg" src="/files/ftp_upload/57569/57569fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Immunostaining of cultured DRG cells. DRG cells were immunostained with anti-CGRP antibody to show neurons, and 4',6-diamidino-2-phenylindole (DAPI) for the nuclei of neurons and glial cells. (A) CGRP protein was expressed in sensory neuron cell bodies and axon fibers. (B) Nuclei of neurons and glia were stained with DAPI. (C) Merged picture from A and B. Arrow indicates a neuron and arrowhead indicates a glial cell. Scale bar = 30 µm. <a href="/files/ftp_upload/57569/57569fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar graph of CGRP and Substance P release; siRNA treatment, comparative analysis, experimental results." class="xfigimg" src="/files/ftp_upload/57569/57569fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: The release of neurotransmitters from cultured DRG cells. The selective NPFFR2 agonist, dNPA, was used to stimulate the release of CGRP and SP from DRG cultures. The dependence of neurotransmitter release on NPFFR2 was verified by transfecting cells with NPFFR2 siRNA. (A and B) After DRG cells were transfected with NPFFR2 siRNA or non-targeting control siRNA (72 h), dNPA (5 nmol) was applied for 1 h to induce the release of CGRP and SP. Data are expressed as mean ± standard error of the mean (SEM) and were analyzed by two-way analysis of variance (ANOVA) with Bonferroni post hoc tests. **p &lt;0.01, ***p &lt;0.001; compared to corresponding vehicle controls (N = 12 per group). Panels A and B have been modified from Lin et al.22 <a href="/files/ftp_upload/57569/57569fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

dorsal-root-ganglia-isolation-primary-culture-to-study

Nanyang Technological University

Research

JoVE Journal

Neuroscience

49.4K Views.  Chang Gung University. Deze methode kan helpen om belangrijke vragen in het gebied van sensorisch onderzoek te beantwoorden, zoals perifere nociception. Het belangrijkste voordeel van deze technische is dat het onderzoeken van het cellulaire mechanisme van sensorische neuronen met het model dat de meeste simuleert tot fysiologische conditie. Verzamel de lendenen rugwortel ganglia uit de stam van een twee tot drie weken oude rat.Begin met het maken van twee sneden langs de zijkanten van de wervelkolom en een ...

Video: Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Biology

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 ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

Author Spotlight: In Vitro and In Vivo Models to Enhance Chronic Pain Treatment Efficacy

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

Bioengineering

Neuronal Enrichment of Dorsal Root Ganglia by Immunopanning

Source: Maguire, A. D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64603">Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning.</a> J. Vis. Exp. (2023)
This video demonstrates a protocol for enriching neuronal cultures from dorsal root ganglia by immunopanning, thereby improving their purity and utility for neurological research.

1. Experimental method

	Day 1: preparation of dishes
	
		In a biosafety cabinet, coat the desired culture surface with enough poly-D-lysine to cover the ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures

Source: de Luca, A. C., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52543">Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration.</a> J. Vis. Exp. (2015).
This video demonstrates a method for harvesting neurons from the dorsal root ganglia (DRG). DRG from a rat&#39;s spinal column is isolated, treated with enzymes, and then mechanically dissociated to obtain a single-cell suspension. The neurons are separated from glial cells using density gradient centrifugation and cultured in a growth medium.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Set-up

	Prior to ...

Primary Neuronal Culture

Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture

Source: Fornaro, M. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56757">Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection.</a> J. Vis. Exp. (2018).
This video demonstrates a method to develop dorsal root ganglia or DRG explant culture and DRG-dissociated cell models using isolated mouse DRG tissues. These models help to study various mechanisms with neuron-glial interactions in a controlled environment.

1. Explant culture of DRG from Adult Mouse

	Place the collected DRG in a 35 mm Petri dish containing 3 mL of ice-cold serum-free media (SFM).
	Transfer ...

Stimulation of Dorsal Root Ganglion Sensory Neurons Using a Neuropeptide Receptor Agonist

Source: Lin, Y., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57569">Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release.</a> J. Vis. Exp. (2018)
This video demonstrates the use of transfected dorsal root ganglion cells to study neurotransmitter release by stimulating them with a neuropeptide receptor agonist.

Source: Lin, Y., et al., Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release. J. Vis. Exp. (2018)
This video demonstrates ...

Neuropathology

Neurodegenerative Diseases

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

Source: Lin, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57569">Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for isolating and culturing dorsal root ganglia (DRG) cells from the rat spinal column. DRGs excised from the spinal column are treated with enzymes and mechanically dissociated to obtain a single-cell suspension. The cells are cultured in a medium containing inhibitors and growth factors that limit glial cell proliferation and promote neuronal growth, respectively.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Collect Lumbar DRG from ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

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