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

Abstract

Source: Lin, Y., et al., Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release. 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.

Protocol

1. 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. Then, use enzyme immunoassay (EIA) kits to assay the levels of neurotransmitters.
   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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM-F12	Invitrogen	12400024	Culture Medium
Hank's balanced salt solution	Invitrogen	14170-112	Culture Medium
Pasteur pipette	Hilgenberg	3150102	Cell trituration
dNPA	Genemed Synthesis	N/A	NPFFR2 agonist