eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Surgical Preparation
Note: All incision and pressure points should be repeatedly infiltrated with local anesthetic (lidocaine or bupivacaine). The present procedure is terminal, if an aseptic preparation is required several modifications should be implemented.
<ol>
	<li>Anesthetize a rat with sodium pentobarbital (40 mg/kg) and ketamine (50 mg/kg) in two locally separated intraperitoneal (IP) injections.</li>
	<li>Let the animal go under general anesthesia and repeatedly verify that a surgical plane of anesthesia is attained (no reaction to toe pinching). Place the rat on a feedback heating blanket and insert a rectal probe to maintain a core temperature of around 37 &#176;C.</li>
	<li>Place a catheter in the peritoneal cavity to facilitate subsequent injection of anesthetic agents and to avoid perforating organs by repeated needle punctures.
	<ol>
		<li>Clip the hair over a small (~2 - 3 mm) area above a region located within the stomach&#39;s lower right or left quadrant. 
		Note: Depilatory cream can also be used.</li>
		<li>Make a 2 - 3 mm incision on the skin with sharp scissors or a scalpel. Using blunt dissection, remove fat and muscle layers until the peritoneal cavity is observed.</li>
		<li>Insert about 1 - 2 cm of a small catheter in the cavity and close the wound with surgical glue. 
		Note: The diameter and the total length of the catheter should be minimal (e.g., 2 French - corresponding to 0.043 mm inside diameter) to reduce dead volumes. Polyurethane tubes are most suited.</li>
		<li>Make a loop and suture the catheter to the skin to secure it in place.</li>
	</ol>
	</li>
	<li>Install a tracheal tube to control ventilation during artificial respiration.</li>
	<li>Install the animal in a stereotaxic frame and carefully monitor the following physiological variables: ECoG, SpO2, EtCO2, heart rate (via an electrocardiogram, ECG) and internal temperature. Monitor and adjust these variables to keep a proper depth of anesthesia and physiological state. Specifically, supplement anesthesia with a small dose (10 mg/kg) of sodium pentobarbital if necessary.</li>
	<li>Apply eye ointment on both eyes to avoid desiccation. Clip the hair over the scalp, make a longitudinal incision (~2 cm), and respect the connective tissues overlying the skull using a scalpel or a curette.</li>
	<li>Make a small (~1.5 mm diameter) craniotomy over the region of interest with a dental drill. 
	Note: Here, the barrel field of the primary somatosensory cortex is targeted ( 7 - 8 mm anterior to the interaural line, 4.5 - 5.5 mm lateral to the midline). Rinse repeatedly to dissipate heat.</li>
	<li>Use extra fine forceps to gently make a small hole in the dura. Reserve a ~0.5 mm region within the cranial trepanation to place the ECoG electrode (see next step). Permanently keep the cortex moist with 0.9% Sodium chloride or NaCl solution (or artificial cerebro-spinal fluid).</li>
	<li>Place a low impedance (~60 k&#937;) silver electrode (the ECoG electrode) on the dura, avoiding the cortical region not covered by the meninges, and place the reference electrode on a scalp muscle on the other side of the head.</li>
	<li>At this stage (30 min after the last sodium pentobarbital injection), maintain the anesthesia by repeated injections of sodium pentobarbital (10-15 mg/kg/h) or fentanyl (3-6 &#181;g/kg/h) via the IP catheter. The former will result in a slow oscillatory, sleep-like, ECoG pattern whereas the latter will result in a desynchronized, waking-like, cortical profile.</li>
	<li>Adjust the artificial ventilation system so that the respiratory frequency and volume are similar to those of the rat&#39;s spontaneous breathing (normal range 70 - 115 breaths/min). Then, connect the mechanical ventilation to the tracheal tube and verify the proper thoracic cage inflation (on both sides). If not, adjust the position of the ventilation tubing in the tracheal axis. If necessary, suck up the secretion present in the trachea with a catheter connected to a syringe or a vacuum pump.</li>
	<li>If all physiological variables and ECoG patterns reflect a stable surgical plane of anesthesia, do an intramuscular injection of gallamine triethiodide in each leg to paralyze the rat, 40 mg/kg for the first injection and then 20 mg/kg, every 2hr.</li>
</ol>
2. Intracellular Recordings
<ol>
	<li>Pull a glass micropipette (sharp microelectrode) with a ~0.2 &#181;m tip such that its resistance ranges between 50 and 80 M&#8486; once filled with 2 M potassium acetate (KAc).</li>
	<li>Place the pipette in a specific holder with a silver/silver-chloride (Ag/AgCl) wire to connect the pipette solution to an intracellular amplifier (via a head stage). The holder should be attached to a micromanipulator. Place an Ag/AgCl reference electrode on the rat&#39;s neck muscles.</li>
	<li>Slowly insert the pipette in the brain down to the region of interest and verify its resistance by monitoring the voltage drops in response to current steps. Use the buzz (or zap) button of the amplifier to clear the pipette if needed.</li>
	<li>Use cotton swabs or synthetic absorption triangles to dry the craniotomy {be careful not to touch the ElectroCorticography (ECoG) or intracellular electrodes} before covering it with silicone elastomer or 4% agarose to reduce brain movements.</li>
	<li>Lower the pipette in 1 - 2 &#181;m steps until its resistance increases when approaching a cell. Then use the buzz function of the amplifier to penetrate into the neuron.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ECoG amplifier</td>
			<td>A-M Systems</td>
			<td>AC amplifier, Model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular amplifier</td>
			<td>Molecular Devices</td>
			<td>Axoclamp 900A</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition interface</td>
			<td>Cambridge Electronic Design</td>
			<td>CED power 1401-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Data analysis software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 7</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Scientifica</td>
			<td>IVM-3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Puller</td>
			<td>Narishige</td>
			<td>PE-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC150F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire 0.125mm (intracellular recording)</td>
			<td>WPI</td>
			<td>AGT0525</td>
			<td></td>
		</tr>
		<tr>
			<td>Ag-AgCl reference</td>
			<td>Phymep</td>
			<td>E242</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire 0.25mm (ECoG recording)</td>
			<td>WPI</td>
			<td>AGT1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial respiration system</td>
			<td>Minerve</td>
			<td>Alpha Lab</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological parameters monitoring</td>
			<td>Digicare</td>
			<td>LifeWindow Lite</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Blanket</td>
			<td>Harvard Apparatus</td>
			<td>507215</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon elastomere</td>
			<td>WPI</td>
			<td>KWIK-CAST</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo recording of intracellular signals from a single neuron

Source: Altwegg-Boussac, T., et al. <a href="https://app.jove.com/v/53576">Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo.</a> J. Vis. Exp. (2016).
This video demonstrates the recording of electrical signals from single brain neurons using a fine-tipped glass micropipette and a specialized setup. The micropipette is carefully inserted into the rat&#39;s brain and is buzzed to penetrate the neuron to record internal neuronal electrical signals.

In Vivo Recording of Intracellular Signals from a Single Neuron

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a glass micropipette and fill it with an ionic solution.
Attach the prepared micropipette to a holder with a connector wire. The holder is secured in a micromanipulator.
The setup is connected to an intracellular amplifier, which converts ionic signals within the neuron into readable electrical outputs.
Place a reference electrode on the neck muscles of an anesthetized rat with its skull exposed.
Insert the micropipette into the target brain region. This results in a voltage change. Rectify this change using the amplifier.
Remove any moisture at the site and seal the opening with a sealing agent to minimize brain movements.
Gradually lower the micropipette until a significant increase in resistance is observed, indicating contact of the micropipette with the membrane of a neuron.
Use a transient electrical pulse to disrupt the membrane, creating a temporary opening for micropipette penetration.
Record the electrical signals from within the neuron.

in-vivo-recording-of-intracellular-signals-from-a-single-neuron

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

268 Views. Source: Altwegg-Boussac, T., et al. Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo. J. Vis. Exp. (2016). This video demonstrates the recording of electrical signals from single brain neurons using a fine-tipped glass micropipette and a specialized setup. The micropipette is carefully inserted into the rat's brain and is buzzed to penetrate the neuron to record internal neuronal electrical signals. 

Video: In Vivo Recording of Intracellular Signals from a Single Neuron - 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

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

In Vivo Recording of Intracellular Signals from a Single Neuron

Transcript

In Vivo Recording of Intracellular Signals from a Single Neuron

Whole-cell Patch-clamp Recordings in Brain Slices

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity