eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Animals
NOTE: Mice were obtained from the Australian Rodent Centre (ARC; Perth, Australia) and held at the Medical Foundation Building Animal Facility at the University of Sydney.
<ol>
	<li>Maintain the mice on a standard 12 h light/dark cycle with environmental enrichment.</li>
	<li>Use male and female C57BL/6 mice (3&#8211;5 weeks old) for all experiments.</li>
</ol>
2. Preparation of Solutions
<ol>
	<li>Prepare 1 L of artificial cerebrospinal fluid (ACSF) composed of 29 mM NaHCO3, 11 mM glucose, 120 mM NaCl, 3.3 mM KCl, 1.4 mM NaH2PO4, 2.2 mM MgCl2, 2.77 mM CaCl2.</li>
	<li>Prepare 200 mL of sucrose-ACSF (sACSF) containing 29 mM NaHCO3, 11 mM glucose, 241.5 mM sucrose, 3.3 mM KCl, 1.4 mM NaH2PO4, 2.2 mM MgCl2, 2.77 mM CaCl2. Prior to adding CaCl2 to the ACSF and sACSF, gas the solutions with carbogen (95 % O2 and 5 % CO2) to establish a pH of 7.4 and avoid calcium precipitation (cloudiness).</li>
	<li>Prepare K+-based intracellular solution composed of 70 mM potassium gluconate, 70 mM KCl, 2 mM NaCl, 10 mM HEPES, 4 mM EGTA, 4 mM Mg2-ATP, 0.3 mM Na3-GTP, with a final pH of 7.3 (adjusted using KOH). 
	NOTE: It is recommended that intracellular solutions be filtered with 0.22 &#956;m filters and stored in 0.5 mL aliquots at -20 &#176;C.</li>
</ol>
3. Preparation of the Brainstem
<ol>
	<li>Before brainstem extraction, equilibrate the sACSF with carbogen and cool at -80 &#176;C for 25 min to form an ice slurry.</li>
	<li>Anaesthetize the mouse with isoflurane (3&#8211;5 %) saturated in oxygen (3 mL/min). Once the hind paw reflexes are absent, decapitate the mouse with sharp stainless-steel scissors.</li>
	<li>Expose the skull by making a sagittal incision in the skin using a razor blade (#22 rounded).</li>
	<li>Using the pointed end of a pair of standard pattern scissors, make a small incision at the lambda and cut along the longitudinal fissure.</li>
	<li>Carefully reflect away the paired parietal and occipital bones using a pair of shallow-bend Pearson rongeurs. 
	NOTE: The brain is continuously bathed in situ using the previously prepared ice-cold sACSF slurry during this procedure.</li>
	<li>Isolate the brainstem from the forebrain and its bony encasing using a razor blade (#11 straight) to cut down the parieto-occipital sulcus and at the caudal medulla.</li>
	<li>Mount the isolated brainstem ventral end on a previously cut trapezoidal polystyrene block. Use a wick of tissue paper to remove excess fluid around the dissected tissue and ensure good tissue adhesion to the cutting stage. 
	NOTE: The polystyrene block is cut in a trapezoidal shape to ensure the rostral end of the midbrain fits and tapers into the spinal cord.</li>
	<li>Use cyanoacrylate glue to fix the polystyrene block with the attached brainstem rostral end down to the cutting stage.</li>
	<li>Using an advance speed of 0.16 mm/s and vibration amplitude of 3.00 mm, prepare 200 &#956;m transverse slices of the MVN. 
	NOTE: The location of the MVN is determined using the Paxinos and Franklin mouse brain atlas. The MVN (listed as MVe in the atlas) lies immediately ventrolateral to the 4th ventricle and is largest right before the attachment of the cerebellum (between the inferior colliculi and the obex).</li>
	<li>Use a plastic-trimmed pipette to transfer slices onto a filter paper disc sitting in carbogenated ACSF at 25 &#176;C for at least 30 min before recording.</li>
</ol>
4. Instruments
<ol>
	<li>Use a standard electrophysiological setup to perform the whole-cell patch clamp technique.</li>
	<li>Prepare micropipettes using a two-step protocol (heat step 1: 70; heat step 2: 45) on a micropipette puller (see the Table of Materials).</li>
	<li>When placed in the bath, micropipettes should have a final resistance ranging from 3 to 5 M&#937; with an internal solution. 
	NOTE: Settings used may vary depending on the temperature within the room and can change quite frequently.</li>
</ol>
5. Whole-cell Patch Clamp Electrophysiology
<ol>
	<li>To obtain whole-cell patch clamp recordings from individual neurons in the MVN, a K+-based internal solution is used within the recording pipette.</li>
	<li>Transfer a single tissue slice from the incubation chamber to the recording chamber and secure it using nylon thread on a U-shaped weight. Continuously perfuse the recording chamber with carbogenated-ACSF at 25 &#176;C at a 3 mL/min flow rate.</li>
	<li>After filling a micropipette with internal solution, locate the MVN using a low-power (10x) objective lens. Using a high-power (40x) objective, individual neurons within the MVN can be located. 
	NOTE: Cell quality is essential in ensuring quality recordings and durability of the cell when attempting to achieve the whole-cell configuration. A suitable cell will demonstrate a spherical shape, a reflective cell membrane, and an invisible nucleus. An unsuitable cell will have a large visible nucleus (egg-like) and a swollen/shrunken appearance.</li>
	<li>Before breaching the tissue with the pipette, apply a small amount of positive pressure to push debris away from the pipette tip.</li>
	<li>Move the pipette using the micromanipulator towards the chosen neuron, and a small dimple should form on the neuronal membrane. 
	Release positive pressure and apply a small amount of negative pressure.</li>
	<li>Once a 1 G&#937; seal is achieved, apply gentle, short, and sharp negative pressure to the pipette holder through the suction port to rupture the membrane and create a whole-cell configuration.</li>
	<li>Make whole-cell current clamp recordings using standard techniques.</li>
</ol>
6. Applying Sinusoidal and Stochastic Noise to Individual Medial Vestibular Nucleus Neurons
<ol>
	<li>Apply the stochastic and sinusoidal noise at a range of amplitudes from 3 to 24 pA to determine neuronal threshold and firing rate.</li>
	<li>Determine the sensory threshold by grouping lower and higher stimulus intensities and perform an ANOVA to observe any differences (as shown in Figure 1).</li>
	<li>Calculate the average firing rate over the 10 s period where the depolarizing current step was/will be injected for each individual current level (i.e., 7 total episodes; Figure 2).</li>
	<li>Use the average firing rate values to generate a firing rate versus the current plot and perform a linear regression analysis to determine the gradient of the line of best fit. The gradient of the best-fit line indicates neuronal gain.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl2</td>
			<td>Scharlau</td>
			<td>CA01951000</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E0396-25G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-25G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Chem-supply</td>
			<td>PA054-500G</td>
			<td>Used for ACSF, sACSF and intracellular solution</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>P1847-100G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187-500MG</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>MgCl</td>
			<td>Chem-supply</td>
			<td>MA00360500</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>Na3-GTP</td>
			<td>Sigma</td>
			<td>G8877-100MG</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chem-supply</td>
			<td>SO02270500</td>
			<td>Use for ACSF and intracellular solution</td>
		</tr>
		<tr>
			<td>NaH2PO4.2H2O</td>
			<td>Ajax</td>
			<td>AJA471-500G</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761-1KG</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Chem-supply</td>
			<td>SA030-500G</td>
			<td>Used for sACSF</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>1169567762</td>
			<td>Used for anaesthetising mice</td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Warner instruments</td>
			<td>GC150T-7.5</td>
			<td>1.5mm OD, 1.16mm ID, 7.5cm 
			&#160;length</td>
		</tr>
		<tr>
			<td>Data acquisition software</td>
			<td>Axograph</td>
			<td></td>
			<td>Used for electrophysiology and analysis</td>
		</tr>
		<tr>
			<td>Friedmen-Pearson Rongeurs</td>
			<td>World precision instruments</td>
			<td>14089</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige</td>
			<td>PP-830</td>
			<td>Used for micropipette</td>
		</tr>
		<tr>
			<td>Multiclamp amplifier</td>
			<td>Axon instruments</td>
			<td>700B</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Sper scientific</td>
			<td>860033</td>
			<td>Used for internal solution</td>
		</tr>
		<tr>
			<td>Standard pattern scissors</td>
			<td>FST</td>
			<td>14028-10</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>Sutter micromanipulator</td>
			<td>Sutter</td>
			<td>MP-225/M</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>Used for slicing brain tissue</td>
		</tr>
	</tbody>
</table>

applying noise currents to medial vestibular nucleus neurons using whole-cell patch-clamp technique

Source: Stefani, S. P., et al. <a href="https://app.jove.com/v/60044">Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro.</a> J. Vis. Exp. (2019).
This video demonstrates a protocol involving the application of subthreshold noise currents to medial vestibular nucleus neurons using the whole-cell patch-clamp technique to study the impact of the noise currents on neuronal sensitivity to electrical stimuli.

<img alt="Figure 1" src="/files/ftp_upload/22708/22708_Figure1.jpg" /> 
Figure 1: Objective determination of 12 pA threshold. Firing rates for less than 12 pA (3 and 6 pA) and more than 12 pA (18 and 24 pA) were pooled and averaged. These averages were then analyzed using an ANOVA and statistical significance between sham and &#62;12 pA and between &#60;12 pA and &#62;12 pA. *p &#60; 0.05.
<img alt="Figure ...

Applying Noise Currents to Medial Vestibular Nucleus Neurons Using Whole-Cell Patch-Clamp Technique

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Animals
NOTE: Mice were obtained ...

Take a mouse brainstem slice containing the medial vestibular nucleus, or MVN.
Secure the slice in a recording chamber filled with artificial cerebrospinal fluid to maintain neuronal viability.
Visualize the slice under a microscope to locate MVN neurons.
Apply positive pressure to advance a micropipette filled with an intracellular environment-mimicking solution through the tissue.
As the pipette contacts a neuron, the neuronal membrane will dimple.
Apply negative pressure to aspirate a small membrane portion, creating a tight seal.
Next, apply negative pressure pulses to rupture the membrane, creating a continuous connection between the neuron and the pipette.
Apply random noise amplitudes to the neuron and identify a subthreshold amplitude beyond which the noise induces neuronal firing.
Finally, apply a range of electrical stimuli to the neuron combined with the subthreshold random noise amplitude.
Measure neuronal gain &#8212; the neuron&#39;s ability to modulate its firing response to the stimuli in the presence of noise.

applying-noise-currents-to-medial-vestibular-nucleus-neurons-using

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

57 visualizações. Source: Stefani, S. P., et al. Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro. J. Vis. Exp. (2019). This video demonstrates a protocol involving the application of subthreshold noise currents to medial vestibular nucleus neurons using the whole-cell patch-clamp technique to study the impact of the noise currents on neuronal sensitivity to electrical stimuli. 

Vídeo: Applying Noise Currents to Medial Vestibular Nucleus Neurons Using Whole-Cell Patch-Clamp Technique - Conceito

Using Unidirectional Rotations to Improve Vestibular System Asymmetry in Patients with Vestibular Dysfunction

The vestibular system provides information about head movement and mediates reflexes that contribute to balance control and gaze stabilization during daily activities. Vestibular sensors are located in the inner ear on both sides of the head and project to the vestibular nuclei in the brainstem. Vestibular dysfunction is often due to an asymmetry between input from the two sides. This results in asymmetrical neural inputs from the two ears, which can produce an illusion of rotation, manifested as vertigo. The vestibular system has an impressive capacity for compensation, which serves to rebalance how asymmetrical information from the sensory end organs on both sides is processed at the central level. To promote compensation, various rehabilitation programs are used in the clinic; however, they primarily use exercises that improve multisensory integration. Recently, visual-vestibular training has also been used to improve the vestibulo-ocular reflex (VOR) in animals with compensated unilateral lesions. Here, a new method is introduced for rebalancing the vestibular activity on both sides in human subjects. This method consists of five unidirectional rotations in the dark (peak velocity of 320&#176;/s) toward the weaker side. The efficacy of this method was shown in a sequential, double-blinded clinical trial in 16 patients with VOR asymmetry (measured by the directional preponderance in response to sinusoidal rotations). In most cases, VOR asymmetry decreased after a single session, reached normal values within the first two sessions in one week, and the effects lasted up to 6 weeks. The rebalancing effect is due to both an increase in VOR response from the weaker side and a decrease in response from the stronger side. The findings suggest that unidirectional rotation can be used as a supervised rehabilitation method to reduce VOR asymmetry in patients with longstanding vestibular dysfunction.

A new rehabilitation method is presented for rebalancing the vestibular system in patients with asymmetric responses, which consists of unidirectional rotations toward the weaker side. By directly modifying the vestibular pathway rather than enhancing the multisensory aspects of compensation, asymmetry can be normalized within 1-2 sessions and show lasting effects.

Uso de rotações unidirecionais para melhorar a assimetria do sistema vestibular em pacientes com disfunção vestibular

The vestibular system provides information about head movement and mediates reflexes that contribute to balance control and gaze stabilization during ...

JoVE Journal

Comportamento

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.

Gravações de células inteiras de patch-clamp em 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 ...

Neurociência

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

Método multifuncional, baseado em Micropipeta de Incorporação e estimulação do bacteriana Mechanosensitive canais iônicos em gotículas de interface Bicamadas

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

Applying Noise Currents to Medial Vestibular Nucleus Neurons Using Whole-Cell Patch-Clamp Technique

Transcrição

Applying Noise Currents to Medial Vestibular Nucleus Neurons Using Whole-Cell Patch-Clamp Technique