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

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

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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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>

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.

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비디오: Stimulation of Dorsal Root Ganglion Sensory Neurons Using a Neuropeptide Receptor Agonist - 실험

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

니코틴 유도 칼슘 신호 전달 및 복부 해마 축삭을 따라 신경 전달 물질 방출의 라이브 영상

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

급성 뇌 슬라이스에서 내인성 모아민 방출 측정을 위한 판 기반 분석

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

생체 외 설치류의 삼차 혈관 시스템에서 칼시토닌 유전자 관련 펩타이드의 방출

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...

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

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