whole-cell patch clamp recordings on intact rat dorsal root ganglia

Whole-Cell Patch Clamp Recordings on Intact Rat Dorsal Root Ganglia

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 in the head stage pipette holder.
Apply gentle positive pressure to the pipette through a 5-milliliter syringe by displacing the plunger about 1 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 reach individual neurons, the recording pipette is maintained at a high positive pressure.
After that, reduce the positive pressure. Achieve a gigaohm seal with gentle suction.
To obtain whole-cell recording configuration, penetrate the neuron cell membrane via 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.

whole-cell-patch-clamp-recordings-on-intact-rat-dorsal-root-ganglia

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

EoE Sub Articles

eoe sub articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. &#160;
1. Patch Clamp Recordings
<ol>
	<li>Place the electrode solution (see Table 1) on ice to prevent degradation. Fill the glass patch pipette with filtered intracellular solution (syringe filter, pore size: 0.2 &#181;m) and place the pipette in the head stage pipette holder. Apply gentle, positive pressure to the pipette through a 5 ml syringe by displacing the plunger about 1 ml before lowering the pipette into the bath solution. 
	Note: This prevents the pipette from becoming blocked.</li>
	<li>Choose &#34;V-clamp&#34; mode by turning the mode knob on the amplifier, then open the &#34;Membrane test&#34; interface in the software. Move the pipette close to the target neuron under microscope inspection. 
	Note: In the intact DRG preparation, a thin layer of satellite glial cells encapsulates each neuron.</li>
	<li>Use 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. Keep moving the pipette towards the neuron until a &#34;dimple&#34; is observed on the neuron. 
	Note: Compared with recordings from disassociated neurons, the positive pressure needs to be slightly larger, which will help penetrate the satellite glial cell layer and increase the chances of successful patching.</li>
	<li>Reduce the positive pressure. Next, achieve a giga ohm seal with gentle suction. 
	Note: With this method, the recording success rate is at least 70%.</li>
	<li>Obtain whole-cell recording configuration as described previously.
	<ol>
		<li>Briefly penetrate the neuron cell membrane via a short but strong suction. Alternatively, use the amplifier&#39;s &#34;zap&#34; function while suction is applied.</li>
		<li>Once a whole-cell mode is established, compensate whole-cell capacitance and series resistance (Rs) by turning the capacitance and resistance compensation knobs on the amplifier. 
		Note: Rs is normally 5-20 M&#937;.</li>
		<li>Abandon the cell if Rs is initially greater than 30 M&#937; or changes by more than 20% during the recording. Also, measure the resting membrane potential; abandon a cell if it is more than -50 mV.</li>
	</ol>
	</li>
	<li>Close the &#34;Membrane test&#34; window. Choose &#34;I-clamp normal&#34; mode by turning the mode knob on the amplifier.</li>
	<li>Click &#34;open protocol&#34; in the software, select and load the protocol for measuring rheobase. Click &#34;record&#34; to start recording. Measure input resistance and rheobase to examine the neuronal excitability by injecting a graded series of depolarizing currents in steps of 100 pA.
	<ol>
		<li>Click &#34;open protocol&#34; in the software, select and load the protocol for measuring rheobase. Click &#34;record&#34; to start recording. Measure input resistance and rheobase to examine the neuronal excitability by injecting a graded series of depolarizing currents in steps of 100 pA.</li>
		<li>Use data acquisition and analysis software (e.g., Clampfit) to analyze the recorded traces according to the manufacturer&#39;s instructions. The software calculates the input resistance (Rin) based on the steady-state I-V relationship during the hyperpolarizing currents delivered. Move the cursor to measure the value for rheobase and membrane threshold. Note: The amplitude of current required to induce the AP is defined as the rheobase, and the lowest voltage for inducing AP is defined as the membrane threshold.</li>
	</ol>
	</li>
	<li>Record the Ligand Induced Currents.
	<ol>
		<li>Fill a pipette with the specific agonists. 
		Note: In the current report, we used 100 &#181;M glutamate and 100 &#181;M AITC to induce the currents mediated by glutamate receptors and TRPA1 receptors respectively.</li>
		<li>Check the pipette to make sure there are no air bubbles inside. Place the pipette in the pipette holder. Connect the pipette holder with a tube connected to a drug dispensing system.</li>
		<li>Use the manipulator to move the pipette within 50 &#181;m of the neuron. Set the drug dispensing system pressure to 1 psi and the duration to 1 sec. Switch the recording mode to voltage clamp, and clamp at -70 mV. Briefly apply the pressure via the drug dispensing system to record a drug-induced current.</li>
	</ol>
	</li>
</ol>
Table 1
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">100 ml of intracellular solution</td>
		</tr>
		<tr>
			<td>Concentration (mM)</td>
			<td>Component</td>
			<td>MW</td>
			<td>g</td>
		</tr>
		<tr>
			<td>130</td>
			<td>KGluconate</td>
			<td>234.24</td>
			<td></td>
		</tr>
		<tr>
			<td>10</td>
			<td>KCl</td>
			<td>74.55</td>
			<td></td>
		</tr>
		<tr>
			<td>10</td>
			<td>HEPES</td>
			<td>238.3</td>
			<td></td>
		</tr>
		<tr>
			<td>10</td>
			<td>EGTA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>MgCl2*6H2O</td>
			<td>203.3</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Notes: Adjust pH to 7.4 with KOH, and osmolarity to 260 - 280 mOsm with sucrose and distilled water. Add 2 mM MgATP, 0.5 mM Na2GTP and filter before immediate use.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass pipette</td>
			<td>Sutter</td>
			<td>BF150-110-7.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Anchor</td>
			<td>Havard apparatus</td>
			<td>64-0250</td>
			<td>stabilize the DRG to avoid drift.</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>WPI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Sutter</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular devices</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Molecular devices</td>
			<td>1440D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON</td>
			<td>FN600</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-manipulator</td>
			<td>Sutter</td>
			<td>MPC200</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen (95% O2, 5% CO2)</td>
			<td>Local Medical Gas supplier</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Gong, K.,et al. <a href="https://app.jove.com/v/54287">Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats.</a> J. Vis. Exp. (2016)
This video demonstrates the examination of sensory neuron excitability in the dorsal root ganglia using the patch clamp technique to measure the rheobase and membrane potential, analyzing the neurons&#39; responses to incremental current injections.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. &#160;
1. Patch Clamp ...

Take immobilized rat dorsal root ganglia, or DRG, in a recording chamber perfused with aCSF.
DRG contains sensory neurons surrounded by satellite glial cells.
Apply positive pressure to a patch pipette filled with intracellular solution and connected to an amplifier, then advance it toward the target neuron.
The positive pressure helps traverse the surrounding satellite glial cell layer and contact the neuron, forming an indentation on its membrane.
Apply negative pressure to form a tight seal with the membrane.
Next, apply negative pressure pulses to establish the whole-cell configuration. Calibrate the amplifier parameters for accurate signal measurements.
To determine the neuron&#39;s excitability,&#160; apply brief incremental currents into the neuron, opening the voltage-gated ion channels and triggering an action potential.
Measure the rheobase, the minimum current needed to trigger an action potential.
Further, apply a ramp current to measure the minimum membrane potential at which the action potential occurs.&#160;

31 ビュー. Intact Rat背根神経節の全細胞パッチクランプ記録

ビデオ: Intact Rat背根神経節の全細胞パッチクランプ記録 - 実験

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

黒質ドーパミンニューロンの細胞体樹状突起のドメインからの亜細胞パッチクランプ記録

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

JoVE Journal

神経科学

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

準備やアフリカツメガエルの視蓋ニューロンの全体セル パッチ ・ クランプ記録のためのプロトコル

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.

Here, we present a protocol to perform a whole-cell patch-clamp on brain slices containing kisspeptin neurons, the primary modulator of gonadotrophin-releasing hormone (GnRH) cells. By adding knowledge about kisspeptin neuron activity, this electrophysiological tool has served as the basis for significant advancements in the neuroendocrinology field over the last 20 years.

全細胞パッチクランプ記録の標的としての視床下部キスペプチンニューロン

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in ...