1. 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. 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.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM-F12</td>
			<td>Invitrogen</td>
			<td>12400024</td>
			<td>Culture Medium</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>Pasteur pipette</td>
			<td>Hilgenberg</td>
			<td>3150102</td>
			<td>Cell trituration</td>
		</tr>
		<tr>
			<td>dNPA</td>
			<td>Genemed Synthesis</td>
			<td>N/A</td>
			<td>NPFFR2 agonist</td>
		</tr>
	</tbody>
</table>

stimulation of dorsal root ganglion sensory neurons using a neuropeptide receptor agonist

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

Source: Lin, Y., et al., <a href='https://app.jove.com/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 ...

Take a multi-well plate containing transfected rat dorsal root ganglion sensory neurons.
The test well neurons exhibit reduced neuropeptide receptor expression, while the control well neurons exhibit physiological levels of neuropeptide receptor expression.
Replace the media with serum-free media to provide a controlled environment for accurate stimulation response.
Add a neuropeptide receptor agonist to the wells and mix. Incubate the plate.
In the control well,&#160; the neuropeptide receptor agonist binds to the target receptors, triggering an intracellular signaling cascade that releases neurotransmitters.
However, in the test wells, reduced neuropeptide receptor expression limits agonist binding, leading to decreased neurotransmitter release compared to the control.
After incubation, collect the media from the wells and centrifuge.
Transfer the supernatant into tubes and use a suitable immunoassay to measure neurotransmitter levels. The control sample exhibits higher neurotransmitter release than the test sample following neuroreceptor agonist stimulation.

On day 6, 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 1 microliter of the stimulation chemical, and gently mix by pipetting. After incubating for the required period, collect the culture medium from the culture dish, and centrifuge at 5,000 times g for 5 minutes at 4 degrees Celsius to remove any suspended impurities. Collect the supernatant from the centrifugation, and dilute with phosphate-buffered saline as needed.

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568 Views. Take a multi-well plate containing transfected rat dorsal root ganglion sensory neurons. The test well neurons exhibit reduced neuropeptide receptor expression, while the control well neurons exhibit physiological levels of neuropeptide receptor expression. Replace the media with serum-free media to provide a controlled environment for accurate stimulation response. Add a neuropeptide receptor agonist to the wells and mix. Incubate the plate. In the control well,&#...

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

In This Article

Abstract

Protocol

Disclosures

Materials

Tags

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