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
<ol>
	<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 height="20" style="height:20px;width:252px;">Pentobarbital sodium</td>
			<td style="width:172px;">vortech Pharmaceuticals</td>
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			<td style="width:484px;"></td>
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			<td height="20" style="height:20px;">syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>1 ml, 5 ml.</td>
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		<tr height="20">
			<td height="20" style="height:20px;">scalpel</td>
			<td>BD</td>
			<td></td>
			<td>size: 15</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Mayo straight scissor</td>
			<td>Fine Science Tools</td>
			<td>14010-15</td>
			<td></td>
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			<td height="20" style="height:20px;">Mayo curved scissor</td>
			<td>Fine Science Tools</td>
			<td>14011-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16021-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adson toothed forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris Scissor</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Noyes spring scissor</td>
			<td>Fine Science Tools</td>
			<td>15124-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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="21" style="height:21px;">periosteal elevator</td>
			<td>Sklar</td>
			<td>97-0530</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Dissection microscope</td>
			<td>WILD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Fisher brand</td>
			<td>13-711-5AM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish (10 cm)</td>
			<td>Pyrex</td>
			<td></td>
			<td>Glass petri dish can avoid damaging the tips of fine forceps</td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass pipette</td>
			<td>Sutter</td>
			<td>BF150-110-7.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anchor</td>
			<td>Havard apparatus</td>
			<td>64-0250</td>
			<td>stabilize the DRG to avoid drift.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peristaltic pump</td>
			<td>WPI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette puller</td>
			<td>Sutter</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amplifier</td>
			<td>Molecular devices</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digitizer</td>
			<td>Molecular devices</td>
			<td>1440D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>NIKON</td>
			<td>FN600</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Micro-manipulator</td>
			<td>Sutter</td>
			<td>MPC200</td>
			<td></td>
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			<td height="20" style="height:20px;">microinjection dispense system</td>
			<td>General Valve</td>
			<td>Picrospitzer II</td>
			<td>fast drug application system</td>
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			<td height="20" style="height:20px;">Carbogen (95% O2, 5% CO2)</td>
			<td>Local Medical Gas supplier</td>
			<td></td>
			<td></td>
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	</tbody>
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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 ...

The overall goal for this experiment is to introduce methods to prepare the intact dorsal root ganglia and to conduct patch clamp recording using this preparation. This method allows single cell recording from the intact dorsal root ganglia while stimulating the entering spinal nerve or the exiting dorsal root. This approach is especially useful when studying the contribution of primary sensory neurons to pain.The main advantage of this technique is that it maintains both neuron and adjacent satellite glial cells. Therefore, this preparation is closer to in vivo situation. In this procedure, after the rat has been anesthetized, shave its lumbar area.Then, incise the skin and the subcutaneous tissue along the midline. Afterward, detach the perispinal muscles from the spinous processes at the L1 to S2 level using a fine periosteal elevator. Next, cut the spinous processes from L1 to S2.Subsequently, make two transverse cuts in the exposed vertebral column at approximately L1 and S1 and remove this segment of the vertebral column en bloc.Then, quickly submerge it into cold aCSF for two to three minutes. After that, flush off the blood from the removed vertebral column and transfer the vertebral column to a petri dish with cold aCSF, then use a fine rongeur to remove the laminae. Cut the dura mater along the midline with microscissors to expose the spinal cord.Next, transfer the remaining vertebra with DRG to the second petri dish filled with cold aCSF. Identify the L4 and L5 DRGs based on their relative position to transverse processes and the sciatic nerve. Then, free the DRG from the surrounding connective tissues and keep the nerve root and spinal nerve attached to the DRG.After that, transfer the DRG into the third petri dish for further dissection. Cut an opening where the dorsal and ventral roots join the DRG and separate the ventral root from it. Then, use forceps with blunt tips to hold the epineurium and use another fine forceps to roll the DRG from the epineurium.Continue to remove as much epineurium as possible that is attached to the DRG. Now, transfer the DRG to the recording chamber. Make sure the side of the DRG where the nerve roots originate faces down.Next, stabilize the DRG with an anchor. Then, perfuse the DRG with oxygenated aCSF through the plastic tubes connected with a peristaltic pump and allow the cells to recover for 30 minutes. After 30 minutes, assess the DRG quality under IR-DIC optics at 40x magnification through a CCD camera.Individually connected two one milliliter syringes to the pipette holders through two pieces of small diameter rubber tubing. Then, place one glass pipette filled with collagenase into one of the pipette holders and an empty glass pipette into the other one. Subsequently, use the micromanipulator to position the pipettes just above the DRG.Collide the tips of two pipettes against each other gently to enlarge the openings of the pipette tips. After that, move the pipette filled with collagenase close to the DRG surface. Briefly apply positive pressure to the pipette containing collagenase through the tube connecting the holder and the syringe by displacing the plunger about 0.5 milliliters.After 10 to 15 minutes, when the debris of the remaining epineurium is observed, apply gentle negative pressure to the empty pipette to suck away the debris. In this way, the neurons and surrounding satellite glial cells will be clearly exposed and ready for patch recording. In this procedure, place the electrode solution on ice to prevent degradation.Next, fill the glass patch pipette with filtered intracellular solution. Then, place the pipette into the head stage pipette holder. Apply gentle positive pressure to the pipette through a five milliliter syringe by displacing the plunger about one milliliter before lowering the pipette into the bath solution.Subsequently, choose V-Clamp mode on the amplifier and open the membrane test interface in the software. Under the microscope, move the pipette close to the target neuron. Next, apply positive pressure from the pipette to traverse the satellite glial cell layer until a sudden enlargement of space between the neuron and the surrounding layer of satellite glial cells is observed.Then, keep moving the pipette towards the neuron until a dimple is observed on the neuron. In our preparation, the neurons are surrounded by satellite glial cells. Therefore, to penetrate the glial cell layers and to reach individual neurons, the recording pipette is maintained at a high positive pressure.After that, reduce the positive pressure. Achieve a giga-ohm seal with gentle suction. To obtain whole cell recording configuration, penetrate the neuron cell membrane via a short, but strong, suction.Alternatively, use the zap function on the amplifier while suction is applied. Once a whole cell mode is established, compensate the whole cell capacitance and series resistance by turning the capacitance and resistance compensation knobs on the amplifier. Then, close the membrane test window.Choose I-Clamp Normal Mode by turning the mode knob on the amplifier. Afterward, click Open Protocol in the software. Select and load the protocol for measuring rheobase and click record to start recording.To examine the neuronal excitability, measure the input resistance and rheobase by injecting a graded series of depolarizing currents in steps of 100 picoamps. Next, click Open Protocol again and select the protocol for measuring membrane threshold. Subsequently, inject a 500 millisecond depolarizing ramp current to the neuron.To record the ligand induced currents, fill a pipette with specific agonists. Check the pipette to make sure there are no air bubbles inside. Then, place the pipette in the pipette holder which is connected to a drug dispensing system.Use the manipulator to move the pipette to within 15 micrometers of the neuron. Set the drug dispensing system pressure to one PSI and the duration to one second. Then, switch the recording mode to voltage clamp at 70 millivolts.Briefly apply pressure via the drug dispensing system to record a drug induced current. In this figure, rheobase was measured by injecting a series of currents into the DRG neuron. The lowest current intensity which can induce an action potential is defined as rheobase, as indicated by the arrow.The rheobase for this neuron is 300 picoamps. The membrane threshold was measured by injecting a ramp current. The potential is defined as the membrane threshold when an action potential is evoked.The membrane threshold for this neuron is 11.9 millivolts. In this figure, currents were induced by puff application of glutamate or AITC for one second. Both agonists induced inward currents, suggesting the presence of glutamate receptors and TRPA-1 receptors on small DRG neurons.Once mastered, this technique can be finished within one hour if it is performed properly. While attempting this procedure, it is important to remember to remove the epineurium and maintain the high pressure for the recording pipette. Following this procedure, patch clamp recording on satellite glial cells can be performed in order to answer additional questions like receptors and channels in satellite glial cells.This technique has paved the way for researchers in the field of pain to study the contribution of dorsal root ganglia to nociception. After watching this video, you should have a good understanding on how to prepare a whole dorsal root ganglia and then how to patch clamp from single cells on these whole dorsal root ganglia.

patch-clamp-recordings-on-intact-dorsal-root-ganglia-from-adult-rats

Research

JoVE Journal

Biochemistry

14.0K visualizações.  University of California, San Francisco. The overall goal for this experiment is to introduce methods to prepare the intact dorsal root ganglia and to conduct patch clamp recording using this preparation. This method allows single cell recording from the intact dorsal root ganglia while stimulating the entering spinal nerve or the exiting dorsal root. This approach is especially useful when studying the contribution of primary sensory neurons to pain.The main advantage of this technique is that it maintains both neuron and ad...