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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Cell sealing for recording
<ol>
	<li>Ensure that all the pieces of equipment (microscope, amplifier, digitizer, micromanipulator, and others) are turned on before starting the recording.</li>
	<li>Fill the recording chamber, from a commercial brand (Table of Materials), attach to the microscope with the aCSF solution for recordings. Use a perfusion pump to constantly perfuse the aCSF at a rate of 2 mL/min.</li>
	<li>Transfer a brain slice of interest (one at a time) to the recording chamber. Use an acrylic transfer pipette (inverted Pasteur glass pipette attached to a silicone teat) to transfer the hypothalamic slices to the chamber. Use a slice anchor (Table of Materials) to hold the slice so it does not move during the aCSF perfusion.</li>
	<li>Place a slice at the center of the recording chamber attached to the microscope. The slice position is critical to allow a good view of the desired region under the microscope and for a perfect reach of the recording micropipette.</li>
	<li>Use an immersion microscope&#39;s low-power objective lens (10x or 20x) to assist in positioning the slice and locating the region of interest.</li>
	<li>After locating the region of interest, switch the objective lens to the high-power lens (63x) and focus on the tissue level, observing the endogenous fluorescent protein and shapes of the cells in the target region to locate the kisspeptin cells on the surface of the brain slice.</li>
	<li>When a possible target cell is located, mark it on the computer screen with the mouse cursor or by drawing a format, like a square, over the area of interest. The computer screen mark helps guide the recording micropipette&#39;s position to the cell.</li>
	<li>After determining the exact location of the target cell, lift the objective and introduce the recording micropipette filled with the internal solution. When placing the micropipette in the electrode holder, ensure that the internal solution is in contact with the silver electrode. 
	NOTE: For micropipette preparation, placement, and positioning on the electrode holder.</li>
	<li>Apply positive pressure before submerging the micropipette in the aCSF solution, to prevent debris from entering the micropipette, using a 1-3 mL air-filled syringe connected to the micropipette holder through a polyethylene tubing (&#8776;130 cm longer); apply nearly 100-200 &#956;L of air.</li>
	<li>Using the micromanipulator, guide the micropipette below the center of the objective. Move the buttons on the micromanipulator to guide the micropipette on the X-Y-Z axis toward the cell of interest.</li>
	<li>Adjust the focus to see the tip of the micropipette and bring the focus closer, but not too close, to the slice. Reduce the speed of the micromanipulator and slowly lower the micropipette to the plane of focus. Ensure that the micropipette tip does not abruptly penetrate the slice, but rather slowly descends until it touches the surface of the cell/target region.</li>
	<li>Apply light positive pressure (&#8776;100 &#956;L) with the 1-3 mL air-filled syringe attached to the micropipette holder to clear any debris from the approach path.</li>
	<li>Focus on the target cell and slowly move the micromanipulator on the X-Y-Z axis to bring the micropipette closer to it. When touching the micropipette to the cell, a dimple caused by the pressure applied through the micropipette tip will be observed (Figure 2C).</li>
	<li>After forming the dimple, due to the micropipette&#39;s proximity to the cell, apply weak, brief suction by mouth (1-2 s) through the tube connected to the micropipette holder to generate the seal between the micropipette to the cell (gigaohm seal or gigaseal &#62;1 G&#8486;; Figure 2D). To form the seal, use the voltage-clamp mode on the software.</li>
	<li>If the seal remains stable (the gigaohm seal should be mechanically stable and without noise interference, determined by observation for about 1 min), set the holding voltage at the closest physiological resting potential of the cell of interest. For kisspeptin hypothalamic neurons, -50 mV is recommended.</li>
	<li>Apply brief suction by mouth (negative pressure) with the micropipette sealed to the cell to break the plasma membrane (Figure 2E). Adequate whole-cell configuration is achieved when suction is performed with sufficient force so that the ruptured membrane does not clog the micropipette and does not attract a sizable portion of the membrane or even the cell.</li>
	<li>Check the system settings manual used. Use the software (see Table of Materials) to digitally check and calculate the series resistance (SR) and the whole-cell capacitance (WCC).</li>
	<li>On voltage-clamp mode, after breaking the cell membrane, enable the whole cell option, and click on the Auto command referring to the whole cell tab. The cell&#39;s SR and wcc will be automatically calculated and instantly displayed by the software. These parameters can also be checked by performing the membrane test with the amplifier mentioned in the Table of Materials.</li>
	<li>Make sure to check cell viability parameters. For kisspeptin neurons, check that the electrophysiological measurements are: SR &#60; 25 m&#937;, input resistance &#62; 0.3 G&#937;, and holding current absolute value &#60; 30 pA. The mean value of the WCC of AVPV/PeNKisspeptin or ARHkisspeptin neurons is &#8776; 10-12 pF in gonad-intact mice.</li>
	<li>Monitor the SR and the cell steady-state capacitance during the experiments. Ensure the SR does not change more than 20% during a recording and that the membrane capacitance is stable.</li>
	<li>Check the software settings. Create specific protocols for the recordings according to the type of experiment. To record membrane potential in the current-clamp mode, with equipment mentioned in the Table of Materials, low-pass filter the electrophysiological signals at 2-4 kHz and analyze results offline in software (see the Table of Materials for software information).</li>
	<li>Once the whole-cell configuration is properly achieved, measure synaptic currents in voltage-clamp mode (Figure 2G). Record changes in resting membrane potential (RMP) and induced RMP variations in the current-clamp mode (Figure 2H). Changes in RMP, such as depolarization of the cell membrane, can be induced by administering a known drug/neurotransmitter to the bath (described in step 1.2), as illustrated in Figure 1. 
	NOTE: In current-clamp mode, positive or negative current can be injected to hold the membrane voltage at a desired voltage. For kisspeptin neurons, we usually set zero current injection (I = 0) to record the spontaneous variation of the membrane potential.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compounds for aCSF, internal and slicing solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma Aldrich/various</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich/various</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich/various</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich/various</td>
			<td>O3777</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich/various</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich/various</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma Aldrich/various</td>
			<td>G4500</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma Aldrich/various</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich/various</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich/various</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich/various</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Monosodium phosphate&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma Aldrich/various</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>DIGIDATA 1440 LOW-NOISE DATA ACQUISITION SYSTEM</td>
			<td>Molecular Devices</td>
			<td>DD1440</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital peristaltic pump</td>
			<td>Ismatec</td>
			<td>ISM833C</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>TMC</td>
			<td>81-333-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Camera</td>
			<td>Leica</td>
			<td>DFC 365 FX</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>Roe-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM6000 FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery chamber</td>
			<td>Warner Instruments/Harvard apparatus</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording chamber</td>
			<td>Warner Instruments</td>
			<td>640277</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Fisher Scientific /various</td>
			<td>FISH-14-375-10; FISH-21-401-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Software and systems</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AxoScope 10 software</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Commander Software</td>
		</tr>
		<tr>
			<td>LAS X wide field system</td>
			<td>Leica</td>
			<td>-</td>
			<td>Image acquisition and analysis</td>
		</tr>
		<tr>
			<td>MultiClamp 700B</td>
			<td>Molecular Devices</td>
			<td>MULTICLAMP 700B</td>
			<td>Commander Software</td>
		</tr>
		<tr>
			<td>PCLAMP 10 SOFTWARE FOR WINDOWS</td>
			<td>Molecular Devices</td>
			<td>Pclamp 10 Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ag/AgCl electrode, pellet, 1.0 mm</td>
			<td>Warner Instruments</td>
			<td>64-1309</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice Anchor</td>
			<td>Warner Instruments</td>
			<td>64-0246</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining a test growth hormone effect on kisspeptin neurons using a whole-cell patch clamp

Source: Silva, J. d. N., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64989">Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings.</a> J. Vis. Exp. (2023)
This video demonstrates the use of the whole-cell patch-clamp technique to measure responses in kisspeptin neurons from transfected mouse hypothalamic slices. The process involves achieving whole-cell configuration and recording membrane potential changes induced by the test hormone using current-clamp mode.

<img alt="Figure 1" src="/files/ftp_upload/22745/22745_Figure1.jpg" /> 
Figure 1: Schematic diagram summarizing the whole-cell patch-clamp technique&#39;s contribution to the knowledge of the kisspeptin neurons&#39; activity. Kisspeptin neurons (shown in green) are located in the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeN) and arcuate nucleus of the hypothalamus (ARH). The AVPV/PeNKisspeptin and ARHkisspeptin cells send direct connections to gonadotrophin-releasing hormone (GnRH) neurons&#39; soma located in the preoptic area (POA) and their terminals at the median eminence (ME), culminating in the modulation of the hypothalamus-pituitary-axis (HPG). Different neuromodulators, such as hormones, have been shown to differentially modulate the activity of the AVPV/PeNKisspeptin and ARHkisspeptin neurons. Possible effects on the resting membrane potential are schematically demonstrated by representative tracings obtained using the whole-cell path-clamp technique and current-clamp recordings. The red color indicates that a specific neurotransmitter induces the depolarization of the resting membrane potential (RMP); the blue color indicates no effect on RMP. The dashed line indicates the RMP.
<img alt="Figure 2" src="/files/ftp_upload/22745/22745_Figure2.jpg" /> 
Figure 2: Basic steps to obtain the sealing of the cell of interest by the whole-cell patch-clamp technique. (A,B) Representative photomicrographs of brain slices (250 &#181;m) containing kisspeptin cells at the anteroventral periventricular nucleus (AVPV) and arcuate nucleus of the hypothalamus (ARH). Kisspeptin neurons were identified by green fluorescent protein (GFP) expression. (C) Photomicrograph demonstrating a micropipette (containing electrolyte solution [internal solution]) close enough to the cell to create a dimple in the plasma membrane to perform the seal. (D,E) Mild negative pressure (mouth suction performed on a tube attached to the headstage and micropipette) is required to seal the cell membrane to the micropipette (D). A second application of negative pressure (mild and brief) is necessary to induce the plasma membrane rupture (E). (F) The registration of the cell activity is performed by a mechanical setup used for patch-clamp experiments. After breaking the plasma membrane, currents flowing through the ionic channels in the patched cell can be recorded by an electrode connected to a highly sensitive amplifier. A feedback resistor generates the current needed for voltage-clamp (G) or current-clamp (H) recordings. Abbreviations: 3V = third ventricle; ME = median eminence. Scale bars: A = 130 &#181;m, B = 145 &#181;m, C = 20 &#181;m, D = 15 &#181;m.

Determining a Test Growth Hormone Effect on Kisspeptin Neurons using a Whole-Cell Patch Clamp

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Cell sealing for recording

...

Secure a transgenic mouse hypothalamic slice in a recording chamber perfused with aCSF.
The slice contains fluorescent fusion protein-expressing kisspeptin neurons, crucial for reproductive system regulation.
Assemble a micropipette comprising a recording electrode in an internal solution.
Microscopically locate a fluorescent kisspeptin neuron.
Apply positive pressure to the pipette and advance it toward the target neuron. Upon contact, the pressure creates an indentation on the neuronal membrane.
Apply brief negative pressure, forming a high-resistance seal between the micropipette and the membrane.
Set the holding voltage at the neuron&#39;s physiological resting potential for accurate measurements.
Apply brief negative pressure pulses to break the membrane,&#160; establishing a whole-cell configuration.
Confirm the neuron&#39;s viability by monitoring the parameters.&#160;
Introduce a test growth hormone, triggering signaling pathways and inducing changes in the neuron&#39;s membrane potential.
Measure the changes in the membrane potential to determine the growth hormone&#39;s inhibitory effect on neuronal activity.

determining-test-growth-hormone-effect-on-kisspeptin-neurons-using

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

36 vues. Source : Silva, J. d. N., et al., Neurones hypothalamiques de kisspeptine comme cible pour les enregistrements de patch-clamp de cellules entières. J. Vis. Exp. (2023) Cette vidéo démontre l’utilisation de la technique du patch-clamp de cellules entières pour mesurer les réponses dans les neurones de kisspeptine à partir de tranches hypothalamiques de souris transfectées. Le processus consiste à obtenir une configuration de cellule entière et à enregistrer les changements de potenti...

Vidéo: Détermination d’un test d’effet de l’hormone de croissance sur les neurones de la kisspeptine à l’aide d’un patch clamp à cellules entières - Concept

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Recordings cellules entières Patch-clamp en tranches cérébrales

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

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JoVE Journal

Neurosciences

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Patch Clamp Enregistrement de cellules amacrines Starburst dans un plat de montage Préparation de Deafferentated Souris Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

Évaluation de la multiplicité synaptique à l’aide de Patch-clamp de la cellule entière électrophysiologie

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Determining a Test Growth Hormone Effect on Kisspeptin Neurons using a Whole-Cell Patch Clamp

Transcription

Determining a Test Growth Hormone Effect on Kisspeptin Neurons using a Whole-Cell Patch Clamp