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EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiology
NOTE: The goal of this protocol is to obtain whole-cell current-clamp recordings from layer II/III pyramidal cell neurons identified visually by GFP expression in GFP-electroporated mouse brains (or any other fluorescent protein previously electroporated). Using this protocol, it is possible to study the effect of a genetic modification introduced by IUE on the electric properties of the neuron. The acquisition of specific firing modes is a gradual process of differentiation that involves the dynamic expression of a wide repertoire of ion channels and that results in the expression of transient firing modes before late postnatal stages. For example, mature electrical responses are not observed in layers II/III of the somatosensory mouse cortex before P16.
<ol>
	<li>Prerequisites for the acute slices
	<ol>
		<li>Prepare 1 L of ACSF using high-purity (double-distilled water) containing 119 mM NaCl, 26 mM NaHCO3, 11 mM glucose, 2.5 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, and 1 mM NaH2PO4. Titrate the pH to 7.3 - 7.4 with HCl or NaOH. Adjust the osmolarity to 290 mOsm.</li>
		<li>Bubble ACSF with carbogen (95% O2/5% CO2) for 15 - 20 min using Teflon tubes (~ 1 mm), to stabilize the pH to 7.3 - 7.4.</li>
	</ol>
	</li>
	<li>Prerequisites for the whole-cell recording
	<ol>
		<li>Make sure that the electrophysiology station is equipped with a recording chamber, a perfusion system, a microscope, electrodes (recording, stimulating, and ground), macro- and micromanipulators, a rigid vibration-resistant table-top and Faraday cage, a stimulator, an amplifier and analog-to-digital (A/D) converter, a computer with acquisition software, and a GFP (or any other fluorochrome) filter for analyzing genetically modified neurons (Figure 1A).</li>
		<li>Prepare the intracellular solution, containing 115 mM potassium gluconate, 2 mM MgCl2, 10 mM HEPES, 20 mM KCl, 4 mM Na2ATP, and 0.3 mM Na3GTP, adjusted to pH 7.2 by KOH and to 290 mOsm by KCl.</li>
		<li>Make patch pipettes by pulling borosilicate glass capillaries. Prepare patch electrodes using a micropipette puller. Use borosilicate capillaries (1.5-mm outer diameter, 0.86-mm inner diameter, 10-cm length). Make patch electrodes showing resistances of 3-10 M&#937; when filled with intracellular solution.</li>
		<li>Perfuse the recording chamber with ACSF at a rate of 2 mL/min. Maintain the chamber&#39;s temperature at approximately 33 &#176;C.</li>
	</ol>
	</li>
	<li>Whole-cell recording
	<ol>
		<li>Transfer the slices into the recording chamber using a Pasteur pipette (cut off the long tip) or a small brush. Hold down the slice with a harp. Perfuse the slices constantly with ACSF at a rate of 2 mL/min.</li>
		<li>Patch a GFP-positive neuron.
		<ol>
			<li>Put the slice into the recording chamber and find the area of interest through the microscope at low magnification (10X). Then, find a GFP-positive cell to patch using the 60x objective.</li>
			<li>Fill the recording electrode with intracellular solution. Use the syringe linked to the filter (4-mm filter) and micro-loader tip to fill the recording electrode with intracellular solution.</li>
			<li>Place the glass pipette in the pipette holder. Place the pipette tip in the bath and focus on the tip. Once the pipette is in the bath, apply positive pressure through the back pressure control system.</li>
			<li>Patch a neuron that is fluorescent (Figure 1B).
			<ol>
				<li>Approach the cell of interest under visual guidance while maintaining back pressure in the pipette. Upon the appearance of a small dimple on the cell surface, release the pressure. At this point, a tight seal (resistance larger than 1 G&#937;) may be formed. Otherwise, apply a light negative pressure (suction) to facilitate it.</li>
				<li>While the seal is being formed, bring the holding voltage clamp to -60 mV. Once the G&#937; seal is formed, apply a pulse of suction to rupture the cell membrane beneath the pipette and go into whole-cell mode.</li>
			</ol>
			</li>
			<li>Record the activity using current-clamp conditions. Once in whole-cell mode, switch from voltage-clamp to current-clamp mode and start recording. For example, to record cell excitability, apply 500 ms-long depolarizing current injections (100 - 400 pA).
			<ol>
				<li>Calculate the firing rates by plotting the number of action potentials along the train for increasing input currents. 
				NOTE: Resting membrane potential, input resistance, and membrane capacitance may also be calculated from the recordings.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Borosilicate glass capillaries (100mm, 1.0/0.58 Outer/Inner diameter)</td>
			<td>Wold Precision Instrument Inc.</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma -Aldrich</td>
			<td>A5177-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma -Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope for Fluorescent Imaging - MZ10F</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 200 Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Acquisition software (MultiClamp 700B Amplifier)</td>
			<td>Molecular Devices</td>
			<td>DD1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized Micromanipulator + Rotating Base</td>
			<td>Sutter Instrument</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Air Table</td>
			<td>Newport</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature Peristaltic Pumps</td>
			<td>WPI</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

studying the excitability of fluorescent neurons using a whole-cell patch clamp

Source: Briz, C. G., et al., <a href="https://app.jove.com/v/55139">In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity.</a> J. Vis. Exp. (2017)
The video demonstrates a method to study the excitability of neuronal cells expressing fluorescent protein using a whole-cell patch current clamp. In the current-clamp mode, action potentials indicative of neuronal excitability are generated.

<img alt="Figure 1" src="/files/ftp_upload/22720/22720_Figure1.jpg" /> 
Figure 1. Electrophysiology Settings and Example of a Firing Response. A. Photograph shows the electrophysiology setup used for patch clamp experiments in acute slices. The setup is included in a Faraday cage to eliminate noise, and the equipment is on top of an anti-vibration table. The controllers of the motorized micromanipulators for the electrodes are observ...

Studying the Excitability of Fluorescent 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. Electrophysiology
NOTE: The ...

Take an immobilized transfected mouse coronal slice in a recording chamber perfused with aCSF.
The slice contains a sparse pyramidal neuron population expressing a target enzyme and a fluorescent protein.
Assemble a recording pipette comprising an intracellular solution and an electrode connected to an amplifier for measuring neuronal signals.
Microscopically identify the target tissue area and locate a fluorescent neuron.
Apply positive pressure to the pipette to prevent clogging and advance it to the target neuron.
The positive pressure causes a slight indentation on the neuronal membrane upon contact.
Release the pressure, forming a tight seal between the membrane and the tip.
Set the holding potential at a constant negative value to stabilize the neuron.
Apply brief negative pressure to rupture the membrane, connecting the cytoplasm to the pipette interior and establishing a whole-cell configuration.
Switch to current-clamp mode and apply positive current pulses, generating action potentials indicative of neuronal excitability.

studying-excitability-fluorescent-neurons-using-whole-cell-patch

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

66 Views. Source: Briz, C. G., et al., In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity. J. Vis. Exp. (2017) The video demonstrates a method to study the excitability of neuronal cells expressing fluorescent protein using a whole-cell patch current clamp. In the current-clamp mode, action potentials indicative of neuronal excitability are generated. 

Video: Studying the Excitability of Fluorescent Neurons using a Whole-Cell Patch Clamp - 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.

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

JoVE Journal

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

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.

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

Studying the Excitability of Fluorescent Neurons using a Whole-Cell Patch Clamp

Transcript

Studying the Excitability of Fluorescent Neurons using a Whole-Cell Patch Clamp

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

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