Name: patch clamp recordings on intact dorsal root ganglia from adult rats
Uploaded: 2016-09-29T13:00:00
Description: Watch this Scientific Journal Video about Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats at JoVE.com

The authors would like to acknowledge the Painless Research Foundation for support of the work. This work was also supported by the NIH grants R01 NS080921-01 and R21 NS079897-01A1.

Ethics Statement: All procedures for the maintenance and use of the experimental animals conformed to the regulations of UCSF Committees on Animal Research and were carried out in accordance with the guidelines of the NIH regulations on animal use and care (Publication 85 - 23, Revised 1996). The UCSF Institutional Animal Care and Use Committee approved the protocols used in this study.
1. Preparation of Instruments, Solutions and Dishes
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	<li>Prepare Artificial Cerebrospinal Fluid (aCSF...

<ol>
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We report a method to prepare whole DRGs for patch clamp studies. There are several key elements for preparing an ideal specimen. Firstly, it is important to dissect the DRGs with dorsal roots attached. After that, the epineurium need to be carefully removed while avoiding damage to the neurons. Finally, to expose the neurons and their surrounding satellite glial cells, it is necessary to digest the remaining connective tissue. Intact DRGs from adult rats prepared with the method described here will maintain good viabili...

Sensation is essential to an organism&#39;s survival and wellbeing. The transmission of stimuli is dependent on the sensory pathways starting at peripheral endings of axons from primary sensory neurons. Primary sensory neurons, with the exception of the mesencephalic nucleus of the trigeminal nerve, are located in the trigeminal ganglia and dorsal root ganglia (DRGs). They serve as gatekeepers of the sensory information 1. At the perikarial membrane, just as at the central and peripheral terminals, DRG neurons express receptors and ion channels, such as glutamate receptors, TNF alpha receptors, transient receptor potential cation channel subfamily V member 1 (TRPV1), sodium channels, etc. 2-7. Patch clamp recordings of the perikarial membrane allow understanding functional changes of many of these receptors and channels throughout the neuron.
The patch clamp recording technique is a powerful tool for studying the activities of channels or receptors and a great number of studies have been conducted by applying this technique on DRG neurons 8-10. In most studies the DRG is removed by cutting the dorsal rootlets and spinal nerve close to the ganglion. After mincing, the ganglion is then placed in digestive enzymes that result in dissociation of the DRG neurons, which may then be recorded immediately or cultured for several days prior to recording. Unfortunately, dissociation of DRG neurons involves a necessary axotomy close to the perikarya. Once dissociated and axotomized, DRG neurons undergo phenotypic changes as well as changes in membrane excitability 11,12. The loss of contact between the perikarya of individual neurons and the satellite glial cells that normally surround them is likely to contribute to these changes 13. The crosstalk between neurons and satellite glial cells is both essential in physiological conditions and in adaptation to pathological conditions such as those leading to intractable pain 14,15. It would be challenging to study the interaction between neurons and satellite glial cells using a dissociated DRG preparation.
Intact DRGs, on the other hand, provide closer to in vivo conditions. In the past several years, our laboratory, as well as some other groups, has been using intact DRGs from adult rats to investigate changes of primary sensory neurons in different conditions associated with chronic pain 3-5,11,15-17. Although the techniques used in these studies are somewhat established, a step-by-step description has not yet been published. In the present manuscript, we describe a convenient and fast way to prepare intact DRGs and their use for patch clamp recordings.

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			<td>1 ml, 5 ml.</td>
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			<td>size: 15</td>
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			<td height="20" style="height:20px;">Adson toothed forceps</td>
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			<td height="20" style="height:20px;">Iris Scissor</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
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			<td height="20" style="height:20px;">Noyes spring scissor</td>
			<td>Fine Science Tools</td>
			<td>15124-12</td>
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			<td height="20" style="height:20px;">Bone scissors</td>
			<td>Fine Science Tools</td>
			<td>16044-10</td>
			<td>Special for cutting the bones.&#160;</td>
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			<td height="20" style="height:20px;">Forceps: Dumont, Dumoxel Biologie #5</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td>These have the fine tips that do not need sharpening when first purchased.</td>
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			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Fisher brand</td>
			<td>13-711-5AM</td>
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			<td height="20" style="height:20px;">Petri dish (10 cm)</td>
			<td>Pyrex</td>
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			<td>Glass petri dish can avoid damaging the tips of fine forceps</td>
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			<td height="20" style="height:20px;">Collagenase (Liberase TM)</td>
			<td>Roche</td>
			<td>05-401-119-001</td>
			<td>dissolve at the concentration of 13 u/ml, aliquot into glass pipette. Avoid repeated freeze and thaw.</td>
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			<td height="20" style="height:20px;">filter</td>
			<td>Thermo scientific</td>
			<td>7232520</td>
			<td>Filter the internal solutions for patch clamp recording to avoid clog.</td>
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			<td height="20" style="height:20px;">Glass pipette</td>
			<td>Sutter</td>
			<td>BF150-110-7.5</td>
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			<td height="20" style="height:20px;">Anchor</td>
			<td>Havard apparatus</td>
			<td>64-0250</td>
			<td>stabilize the DRG to avoid drift.</td>
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			<td height="20" style="height:20px;">Peristaltic pump</td>
			<td>WPI</td>
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			<td>1440D</td>
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patch clamp recordings on intact dorsal root ganglia from adult rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

Figure 1 shows the process of preparing intact DRG for patch recording. Figure 1A shows the exposure and location of the ganglia after laminectomy. Figure1B shows L3, L4 and L5 DRGs with the nerve roots attached after removing the spinal cord. Then L4 and 5 DRGs are carefully dissected and freed from the vertebrae. Next, the epineurium, a transparent membrane surrounding the DRG, is removed (yellow arrow, Figure 1D). The ...

Watch this Scientific Journal Video about Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats at JoVE.com

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

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Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...