Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Ya-Tin Lin; Jin-Chung Chen

doi:10.3791/57569

Dorsal kök gangliyon yalıtım ve birincil kültür nörotransmitter yayın çalışmaya

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Neuroscience

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JoVE Journal Neuroscience

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal kök gangliyon yalıtım ve birincil kültür nörotransmitter yayın çalışmaya

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08:15 min

October 6, 2018

DOI: 10.3791/57569-v

Ya-Tin Lin¹, Jin-Chung Chen^1,2,3

¹Graduate Institute of Biomedical Sciences, Department of Physiology and Pharmacology,Chang Gung University, ²Healthy Aging Research Center,Chang Gung University, ³Neuroscience Research Center,Chang Gung Memorial Hospital

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Overview

This study demonstrates the use of lumbar dorsal root ganglia (DRG) primary cultures to explore physiological functions and pathological events in sensory neurons. The research specifically investigates neurotransmitter release after stimulation with a selective neuropeptide FF receptor type 2 agonist.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Physiology

Background

Dorsal root ganglia contain sensory neurons crucial for nociception.
Primary cultures offer insights into neuronal behavior in near-physiological conditions.
Neuropeptide FF receptor type 2 (NPFFR2) plays a role in sensory neuron signaling.
Understanding neurotransmitter release can elucidate sensory processing mechanisms.

Purpose of Study

To develop a methodology for culturing lumbar DRG neurons.
To assess neurotransmitter release in response to NPFFR2 stimulation.
To provide insights into peripheral nociception mechanisms.

Methods Used

Primary cultures of lumbar dorsal root ganglia were prepared from juvenile rats.
The procedure includes careful dissection, enzymatic dissociation, and cell plating.
DRGs were cultured in a medium containing nerve growth factor.
Neurons were transfected with siRNA prior to stimulation with NPFFR2 agonist.
Neurotransmitter levels were assessed using enzyme immunoassays post-stimulation.

Main Results

Successful recovery and culture of sensory neurons from lumbar DRGs were achieved.
Neurotransmitter release was detectable following NPFFR2 activation.
The culture method allows examination of cellular responses under conditions that reflect in vivo physiology.
Cell morphology and viability were maintained throughout the culture period.

Conclusions

The study demonstrates a viable protocol for isolating and culturing lumbar DRG neurons.
It establishes a model for investigating sensory neuronal responses to various stimuli.
The findings contribute to understanding peripheral mechanisms relevant to pain and other sensory disorders.

Frequently Asked Questions

What are the advantages of using lumbar DRG primary cultures?

Lumbar DRG primary cultures provide a model that closely simulates physiological conditions, allowing for direct observation of sensory neuron behavior and neurotransmitter release.

How is the isolation of DRG neurons performed?

DRG neurons are isolated by carefully dissecting lumbar spinal columns, removing unwanted tissues, and using enzymatic solutions to dissociate cells for culture.

What types of data are obtained from this method?

This method allows for the assessment of neurotransmitter release and cellular responses to specific receptor agonists, providing insights into neuronal function.

Can this method be adapted for other types of neurons?

Yes, similar dissection and culture techniques can be adapted for other types of neurons by modifying the dissection protocols and culture conditions.

What considerations should be taken when working with primary cells?

Working with primary cells requires a gentle handling approach, as they are often more sensitive than immortalized cell lines, affecting cell viability and function.

What implications do these findings have for pain research?

The ability to study neurotransmitter release in cultured sensory neurons provides valuable insights into the mechanisms underlying pain perception and could inform therapeutic strategies.

How long does the entire procedure take?

Once mastered, the entire procedure for isolating and culturing DRG neurons can be completed in approximately two and a half hours.

Dorsal kök gangliyon (DRG) birincil kültürler sık fizyolojik fonksiyonları veya patoloji ile ilgili olayların duyusal sinir hücreleri incelemek için kullanılır. Burada, peptit FF reseptör yazdıktan sonra 2 stimülasyon ile seçici bir agonist nörotransmitter sürümü algılamak için bel DRG kültürler kullanımını göstermektedir.

Bu yöntem, periferik nosisepsiyon gibi duyusal araştırma alanında anahtar soruları yanıtlamaya yardımcı olabilir. Bu teknik en büyük avantajı en fizyolojik duruma simüle modeli ile duyusal nöronların hücresel mekanizma incelenmesi olduğunu. 2-3 haftalık bir sıçan ın gövdesinden lomber dorsal kök gangliyonu toplayın.

Omurilik kenarları boyunca iki kesik ve lomber omurgarostral ölçüde işaretlemek için bir lateral kesim yaparak başlayın. Sonra, omurganın sırt kaslarını kaldırmak için kemik kesme forceps kullanın. Sonra, omurların sırt kısmını çıkarın ve omuriliği ortaya çıkarın.

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