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 spat....

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 fo....

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 the c.......

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

List of materials used in this article

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