Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Summary

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.

Abstract

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)).

Introduction

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 sensation^1,2. 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 (Aα) fibers that respond to proprioception of skeletal muscles, group II (Aβ) fibers that respond to mechanoreceptors of the skin, and group III (Aδ) 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 Aδ fibers and unmyelinated C fibers^1,4. The Aδ 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 support^9,10. 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 outgrowth^12,13,14,15. 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 media^12,17. 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.

Protocol

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

1. 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.
2. Sterilize all surgical instruments in an autoclave.
3. 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.
4. Sacrifice the rat by decapitation with a commercial guillotine.
5. 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.
6. Cut along the sternum and remove all organs/tissues with dissection scissors (Figure 2A-a).
7. 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.
8. 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.
9. 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).
10. Remove the dorsal portion of the vertebrae with bone cutting forceps and expose the spinal cord.
11. Remove the spinal cord with dissection scissors (Figure 2A-c) and forceps (Figure 2A-d).
12. Identify the lumbar DRG by counting vertebrae from the last rib (Thoracic Vertebra 13). See Figure 1C for a diagram of the vertebrae positions.
13. 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.

2. Primary Culture of Rat Lumber DRG

NOTE: The following steps should be performed in a laminar flow hood.

1. Prepare culture medium containing 10% fetal bovine serum, 100 mM sodium pyruvate, and 1x penicillin/streptomycin in 1x DMEM-F12.
2. Coat the cell-culture treated 24-well plate with 200 µg/mL poly-L-lysine for 2 h then wash with sterilized water.
3. Pre-incubate the culture dish with 1 mL culture medium in a 37 °C CO₂ incubator before use for least 30 min.
4. 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).
5. 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 µm syringe filter.
6. Digest the DRG in the collagenase solution in a 37 °C CO₂ incubator for 30 min.
7. Remove the collagenase solution and wash the DRG 3 times in 2 mL Hank'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.
8. Add 2 mL pre-warmed 0.05% trypsin-EDTA into the DRG-containing 35 mm dish and digest the DRG in a 37 °C CO₂ incubator for 30 min.
9. 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.
10. Centrifuge the solution at 290 x g for 5 min at 4 °C. Remove the supernatant and add another 2-mL serum-free medium to resuspend the DRG.
11. Repeat step 2.10 2 times but change the serum-free medium to pre-warmed culture medium on the last time.
12. 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.
13. 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.
14. 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.
15. Replace the culture medium on the following day with the addition of 10 µM 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.

3. Transfection of NPFFR2 siRNA in DRG Cells

1. Perform the transfection of NPFFR2 siRNA and control siRNA according the manufacturer'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).
2. 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 °C CO₂ incubator for 1 h.
3. Add 50 nM of siRNA (in 1 µL RNase-free water) into 12.5 µL serum-free medium.
4. Add 2.5 µL transfection reagent into 10 µL serum-free medium.
5. Mix the solution from steps 3.3 and 3.4 by pipette, and incubate this mixed transfection solution for 10 min at room temperature.
6. 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.
7. Incubate the DRG in a 37 °C CO₂ incubator for 6 h.
8. 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 µM Ara-C and 100 ng/mL NGF, into the 24-well plate.
9. Incubate the DRG in a 37 °C CO₂ incubator for another 66 h (refresh the medium at 48 h).

4. Release of Neurotransmitters from Primary DRG Cells

1. On Day 6 after cells were plated (72 h after siRNA transfection), change the culture medium to 200 µL serum-free medium, and incubate the cells in a 37 °C CO₂ incubator for 30 min.
2. Add 1 µL stimulation chemical(s) and gently mix the media by pipetting. Incubate the dish in a 37 °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.
3. Collect the culture medium from the culture dish and centrifuge at 5,000 x g for 5 min at 4 °C to remove any suspended impurities.
4. 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.

5. CGRP and SP EIA

1. Analyze the samples immediately according to the CGRP or SP EIA kit manufacturer's protocol.
   NOTE: The protocol will vary depending on the kit used.
2. Rinse the CGRP EIA wells 5 times with wash buffer supplied within the kit.
3. Add 100 µL samples with 100 µL anti-CGRP acetylcholinesterase (AChE) tracer into the CGRP EIA wells, and add 50 µL samples, 50 µL anti-SP AChE tracer and 50 µL anti-SP antiserum into the SP EIA wells.
4. Seal the CGRP and SP wells with plastic film which is supplied within the kits.
5. Incubate the wells overnight at 4 °C.
6. Wash the wells 5 times with CGRP or SP wash buffer and remove all the residual solution from the wells.
7. Add 200 µL Ellman's reagent into the CGRP or SP wells which is supplied within the corresponding EIA kits.
8. 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.
9. 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's reagent.

Results

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...

Discussion

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...

Materials

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