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. Surgery
<ol>
	<li>For electrophysiological recordings, use urethane (CAUTION) for anesthesia. 
	Caution: Urethane is toxic and carcinogenic, so always adhere to the safety regulations and data sheet given by the substance&#39;s manufacturer.</li>
	<li>Prepare a solution of 200 mg/mL urethane in 0.9% Sodium Chloride (NaCl) medical saline solution.</li>
	<li>Administer a total of 1.3 g/kg bodyweight urethane intraperitoneally (IP). Depending on the rat strain it might be reasonable to split the dose into two doses with a 15 min interval in between the injections in order to enhance the safety of the anesthesia.</li>
	<li>Check the depth of anesthesia by using pedal-withdrawal reflex and other suitable reflexes. If the anesthesia is not deep enough to perform surgery, inject 0.15 g/kg bodyweight of urethane IP and wait another 15 min.</li>
	<li>Apply an eye ointment to prevent corneal dehydration.</li>
	<li>Constantly monitor respiratory rate and pedal withdrawal reflex during anesthesia. Use a small animal heating pad with temperature control to ensure that a physiological body temperature is maintained throughout the surgery. Before the start electrophysiological recordings, change to a non-electrical alternative (e.g. sodium acetate head pad).</li>
	<li>Shave the fur alongside the dorsal side of the head to achieve a clean surgical field. Disinfect around the incision site with appropriate surgical disinfectant. Fix the animal in the stereotactic frame.</li>
	<li>Perform a 2-cm long incision of the scalp in sagittal direction with a scalpel. Use a scalpel to slightly scrape off the skull aponeurosis and disinfect the skull. Use cotton buds soaked in 3% Hydrogen Peroxide (H2O2) to remove any remaining tissue.</li>
	<li>Use an electrocauter or thermocauter to control bleeding, if necessary. Stop bleedings from the skull bone and hypoderm, if the bleeding does not stop spontaneously after 1-2 min and hinders sight onto the skull.</li>
	<li>Adjust the incisor bar until the head is positioned in flat skull position, which means the bregma and lambda as stereotaxic reference points are in the same plane. This is most important to achieve high surgical precision. Use a standard stereotaxic rat alignment tool, calibrate the designated tip to the bregma under microscopic vision and adjust the incisor bar until the designated points for the bregma and lambda on the tool touch the skull at the same time. 
	NOTE: A view from one side with focused light from the other may help to determine this condition. Alternatively, take a stereotaxic holder with a fine cannula and measure the dorsoventral coordinates of Bregma and lambda under microscopic vision. Adjust the incisor bar until the dorsoventral coordinates of Bregma and lambda are the same.</li>
	<li>Use a stereotaxic holder with a cannula, calibrate to the bregma, and then calculate the position of all drill holes on the skull. Using the stereotaxic holder, mark the positions of the holes to be drilled either by carefully scratching the skull or by using a surgical color marker. The coordinates for this depend on the targets; coordinates with reference to the bregma are given for the hyperdirect pathway in Table 1, including the suggested coordinates for cerebellar reference electrodes.</li>
	<li>Using a micro drill, carefully drill all holes. For the subthalamic nucleus (STN) and the substantia nigra pars reticulate (SNr), drill a common hole (approximately 2 mm x 3 mm in size). All other drill holes should have a diameter of about 1 mm.</li>
	<li>Take two fine cannulas (at least 27 G) and bend their tips to form a hooked shape, using a hard surface or tweezers. Use these to remove any debris from the drill holes, and carefully cut and remove the dura mater in the common STN/SNr hole.</li>
	<li>Flush the drill holes with physiologic saline. Apply a drop of physiologic saline every 15 min to the drill holes to prevent the brain and dura from drying out.</li>
	<li>Take a microdrill and matching stainless steel micro-screw (e.g. a M 1.2 mm x 2 mm screw), drill a hole, and screw in a micro-screw in between the drill holes of the reference epidural electrodes above the cerebellum, do the same for the M1 epidural electrodes.</li>
	<li>Slide the self-built Ag/AgCl epidural electrodes into the drill holes for the reference electrodes and M1 electrodes. Guide the electrode tip with fine tweezers and slide it directly below the skull bone into the drill hole.</li>
	<li>Fix all epidural electrodes with two-component dental acrylic. Make sure not to cover the bregma point nor affect the common STN/SNr hole.</li>
	<li>Insert the prepared holder with the tungsten microwire electrodes into the stereotaxic frame.</li>
	<li>Calibrate the most ventral electrode, which is intended to target the STN, to the bregma. Adjust to the calculated position above the common STN/SNr hole and lower the electrodes down to the brain under microscopic vision. Make sure that the tungsten microwire electrodes go inside the brain smoothly.</li>
</ol>
2. Electrophysiological Mapping and Recordings
NOTE: For this step, a Faraday cage and a multi-channel electrophysiological recording system with recording software capable of online-filtering and online spike-sorting is necessary. Preferably use a system that works with a preamplifier positioned near the head of the animals to keep electrical noise and artifacts to an absolute minimum. Besides the tungsten microwire electrodes, at least one epidural and one reference electrode are necessary to perform recordings of the hyperdirect pathway. It is recommended to insert epidural and reference electrodes pairwise without them touching each other, this helps in case of malfunctioning and allows for different types of referencing in data analysis.
<ol>
	<li>Put a mobile Faraday cage above the stereotaxic frame. If only a stationary Faraday cage is available, carefully move the stereotaxic frame into the Faraday cage while making sure deep brain electrodes are not lowered into the brain until the stereotaxic frame is in its final position.</li>
	<li>Connect the electrodes to the headstage of the electrophysiological setup. Make sure the reference electrodes are connected to appropriate reference channels.</li>
	<li>Set up the recording software: Bandpass filter (0.05-8,000 Hz) and amplify (gain 1,500-2,000x) the raw data signal. Use an online LFP and spike filter with appropriate settings (bandpass filter 0.05-250 Hz for LFPs, bandpass filter 300-8,000 Hz for MUA). For all filters, use a butterworth-type filter.</li>
	<li>Set up a spike threshold, if applicable, for online spike sorting. Most recording software allows for setting up of a spike threshold, which is an amplitude value above which a signal is marked as a spike by the software. This threshold can either be determined mathematically as a factor or standard deviation of the mean amplitude of the filtered spike signal, or can preferably be determined by visual inspection of data segments &#60;500 ms and set up as a line above the signal noise in a graphical user interface. 
	NOTE: The intention in setting up a spike threshold is to count spikes and sort units in order to provide information on how many neurons are presently recorded and how their spikes are shaped.</li>
	<li>Slowly lower the tungsten microwire electrodes to 1 mm dorsal of the target, which is STN for hyperdirect pathway. Wait for the signal to stabilize if necessary.</li>
	<li>For the electrophysiological mapping, advance the electrodes ventrally in steps of 100 &#181;m. At each step, evaluate the firing pattern, firing rate and shape of spikes. Compare those with the typical examples given in Figure 1. Commonly, dense nuclei show fast and continuous spiking over several dorsoventral steps, whereas fiber-rich structures show low firing rates and less homogenous spike activity in subsequent ventral steps.</li>
	<li>For the hyperdirect pathway, make sure that the most ventral electrode is inside STN. 
	NOTE: The STN is reached when a considerable increase in MUA is detected ventral of the Zona incerta. Ventral to the STN, spiking stops almost completely because the electrode has reached the internal capsule. When the most ventral electrode is in STN, the configuration of the tungsten microelectrodes ensures the posterior, second electrode is in SNr. Dorsoventral fine-tuning in small increments might be necessary to record typical MUA in STN and SNr at the same time. Note that the frequency of MUA depends on the number of neurons actually recorded and on the level brain activation.</li>
	<li>Once the electrodes are in the desired structures, set up online filtering and spike-sorting (see Figure 2), and then start the recording of the data. Typical examples for the different cortical synchronization states that can be identified in the LFP recordings are shown in Figure 3.</li>
</ol>
Table 1: Stereotaxic Coordinates for the Recording of the Hyperdirect Cortico-basal Ganglia Pathway. All points are measured from the bregma reference point on the skull in mm; n.a.- not applicable.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Coordinates from Bregma</td>
			<td>STN</td>
			<td>SNr</td>
			<td>M1</td>
			<td>reference 1</td>
			<td>reference 2</td>
		</tr>
		<tr>
			<td>anterior-posterior</td>
			<td>-3.6</td>
			<td>-4.8</td>
			<td>+3.0</td>
			<td>-10.0</td>
			<td>-10.0</td>
		</tr>
		<tr>
			<td>medial-lateral</td>
			<td>+2.5</td>
			<td>+2.5</td>
			<td>+3.0</td>
			<td>+3.0</td>
			<td>-3.0</td>
		</tr>
		<tr>
			<td>dorsal-ventral</td>
			<td>-8.0</td>
			<td>n. a.</td>
			<td>n. a.</td>
			<td>n. a.</td>
			<td>n. a.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ag/AgCl custom epidural electrodes</td>
			<td>Goodfellow GmbH 
			D-61213 Bad Nauheim, Germany 
			info@goodfellow.com</td>
			<td>Product-ID AG005127 for 99.99% silver wire</td>
			<td>Ag/AgCl electrodes will allow for better signal quality, but may only be used in acute experiments. Possible replacement: Stainless steel electrodes</td>
		</tr>
		<tr>
			<td>Stereotaxic holder with acrylic block</td>
			<td>David Kopf Instruments, 
			7324 Elmo Street, Tujunga, CA 91042, USA</td>
			<td>Product ID Model 1770 Standard Electrode Holder</td>
			<td>Make sure the acrylic block has recesses which suit the electrode setup for the desired target. Acrylic blocks can easily be modified with a file to obtain the desired configuration. Possible replacement: Self-constructed electrode holders</td>
		</tr>
		<tr>
			<td>Tungsten microwire electrodes 1.5 M&#937; impedance</td>
			<td>Microprobes.com 
			18247-D Flower Hill Way Gaithersburg, Maryland, 20879 USA</td>
			<td>Product-ID WE3ST31.5A5-250um</td>
			<td>The 1.5 M&#937; is necessary to record MUA and LFP at the same time. Possible replacement: Microelectrodes of different materials can be used. The electrodes have to be straight, robust and as thin as possible.</td>
		</tr>
		<tr>
			<td>Rat alignment tool</td>
			<td>David Kopf Instruments, 
			7324 Elmo Street, Tujunga, CA 91042, USA</td>
			<td>Product ID Model 944 Rat Alignment Tool</td>
			<td>Allows the exact orientation of the brain to match stereotaxic atlases. Possible replacement: Stereotaxic holder with a cannula</td>
		</tr>
		<tr>
			<td>Two-component dental acrylic</td>
			<td>Associated Dental Products Ltd. 
			Kemdent Works, Purton, Swindon 
			Wiltshire, SN5 4HT, United Kingdom</td>
			<td>Simplex Rapid Powder Clear 225g, Product code: ACR803; Simplex Rapid Liquid 150ml, Product code: ACR920</td>
			<td>Depending in the electrodes used, superglue might be an easy alternative, if the electrodes are small and lightweight. Possible replacement: Superglue (Cyanacrylate-based)</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Self-construction</td>
			<td></td>
			<td>A proper Faraday cage will be the best protection from electromagnetic artifacts, but everything which can be formed into a box shape or applied to a frame and is made of conductive material may help. Possible replacement: Aluminum foil or copper mesh</td>
		</tr>
		<tr>
			<td>Electrophysiological setup with recording software and online spike-sorting capabilities</td>
			<td>OmniPlex&#174; Neural Data Acquisition System 
			Plexon Inc 
			6500 Greenville Avenue, Suite 700 
			Dallas, Texas 75206 
			USA</td>
			<td></td>
			<td>Offline sorting software is a potential alternative, multiple scripts and softwares can be found for free in the open source community.</td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological recordings of brain activities from multiple sites in rats

Source: Haumesser, J. K., et al. <a href="https://app.jove.com/v/55940">Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats.</a> J. Vis. Exp.(2017).
This video demonstrates the detailed procedure for preparing an anesthetized rat for electrophysiological recordings of brain activity from multiple sites. The scalp is dissected to access the skull, and holes are drilled at precise locations to install and secure electrodes. Tungsten microwire electrodes are then inserted into the targeted location to record the data.

<img alt="Figure 1" src="/files/ftp_upload/22735/22735_Figure1.jpg" /> 
Figure 1: Characteristic Multi-unit Activity from a Dorsoventral Electrode Trajectory Targeting the STN. (A) Multi-unit recordings of the ventral posteromedial thalamic nucleus (VPM), the zona incerta (ZI), the subthalamic nucleus (STN), and the substantia nigra pars reticularis (SNr). The VPM exhibits sparse and irregularly spaced high amplitude spikes. This pattern of spikes ceases when approac...

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

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

Begin with an anesthetized rat secured in a stereotactic frame with its skull exposed.
Use reference points on the skull to calibrate and mark the desired coordinates.
Drill holes at the marked locations.
Remove debris and cut open the brain&#39;s outer layer.
Frequently flush the holes with saline to prevent drying.
Drill additional holes and anchor screws to support the electrodes.
Install the reference electrodes and electrophysiological recording electrodes. Secure them by applying acrylic resin.
Arrange tungsten microwire electrodes on a frame according to the multi-target locations in the brain.
Multiple electrodes allow simultaneous recording of brain activities from multiple target sites.
Using the reference point on the skull, position the tungsten microwire electrodes.
Gently insert them into the brain.
Configure the recording software to detect brain activity.
Lower the electrodes to the target depth and begin data recording.

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142 Views. Source: Haumesser, J. K., et al. Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats. J. Vis. Exp.(2017). This video demonstrates the detailed procedure for preparing an anesthetized rat for electrophysiological recordings of brain activity from multiple sites. The scalp is dissected to access the skull, and holes are drilled at precise locations to install and secure electrodes. Tungsten microwire ele...

Video: In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats - Concept

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

JoVE Journal

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be user friendly with a graphical user interface, able to read data files in a variety of formats, and provide users with a flexible set of tools giving them the ability to detect and sort extracellular voltage waveforms from different units with some degree of reliability. Previously published spike sorting methods are now available in a software program, SpikeSorter, intended to provide electrophysiologists with a complete set of tools for sorting, starting from raw recorded data file and ending with the export of sorted spikes times. Procedures are automated to the extent this is currently possible. The article explains and illustrates the use of the program. A representative data file is opened, extracellular traces are filtered, events are detected and then clustered. A number of problems that commonly occur during sorting are illustrated, including the artefactual over-splitting of units due to the tendency of some units to fire spikes in pairs where the second spike is significantly smaller than the first, and over-splitting caused by slow variation in spike height over time encountered in some units. The accuracy of SpikeSorter's performance has been tested with surrogate ground truth data and found to be comparable to that of other algorithms in current development.

The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays.

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be ...

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K+ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K+ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood ...

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

Transcript

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress