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. Preparation of Standard-artificial Cerebrospinal Fluid (aCSF)
<ol>
	<li>Preparation of Stock Solution A
	<ol>
		<li>Add 700 ml of distilled water to a clean 1 L beaker before adding chemicals.</li>
		<li>While continuously stirring a volume of 500 ml, add 136.75 g of NaCl, 6.99 g of KCl, 2.89 g of MgSO4, and 2.83 g of NaH2PO4.</li>
		<li>Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 117 mM NaCl, 4.69 mM KCl, 1.2 mM MgSO4, and 1.18 mM NaH2PO4.</li>
		<li>Keep refrigerated at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Preparation of Stock Solution B
	<ol>
		<li>Add 700 ml of distilled water to a clean 1L beaker before adding chemicals.</li>
		<li>While continuously stirring a volume of 500 ml, add 41.45 g of D-glucose and 41.84 g of NaHCO3.</li>
		<li>Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 11.5 mM D-glucose and 24.9 mM NaHCO3.</li>
		<li>Keep refrigerated at 4 &#176;C for up to 2 weeks.</li>
		<li>Before each experiment mix 50 ml of stock A with 50 ml stock B and bring to 1 L with distilled water to obtain final concentration aCSF solution and leave at RT while continuously oxygenating it with carbogen (95% O2 - 5% CO2 mix) for at least 30 min. Prepare final concentration aCSF only at the beginning of each experiment and discard at the end of the day.</li>
	</ol>
	</li>
</ol>
2. Preparation of Sucrose-artificial Cerebrospinal Fluid (Sucrose-aCSF)
<ol>
	<li>Preparation of Stock Solution C
	<ol>
		<li>Add 700 ml of distilled water to a clean 1 L beaker before adding chemicals.</li>
		<li>While continuously stirring 500 ml of distilled water, add 240 g of sucrose, 6.55 g of NaHCO3, 0.671 g of KCl, 4.88 g of MgCl2, 0.22 g of CaCl2, and 0.21 g of ascorbic acid.</li>
		<li>Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 701.1 mM sucrose, 78 mM NaHCO3, 9 mM KCl, 24 mM MgCl2, 1.5 mM CaCl2, and 1.2 mM ascorbic acid.</li>
		<li>Keep stock solution C at 4 &#176;C for up to one week.</li>
		<li>Before each experiment, mix 300 ml of 50 ml of stock C with 600 ml distilled water to obtain the sucrose-aCSF solution at the final concentration. Leave at RT while continuously oxygenating it with carbogen (95% O2 - 5% CO2 mix) for at least 30 min.</li>
	</ol>
	</li>
</ol>
3. Slice Preparation
<ol>
	<li>Place a clean beaker with 100 ml sucrose-aCSF solution in ice while oxygenating it with carbogen and fix the pH at 7.4 using a pH meter while adding a few drops of 0.1 M NaOH solution (when pH &#60; 7.4) or 0.1 M HCl solution (when pH &#62; 7.4).</li>
	<li>Fill up a cutting chamber of a vibratome with sucrose-aCSF and oxygenate it. Turn on the glycerol-based refrigerating system coupled to the cutting chamber and wait 15 min to allow it to cool down to 0 - 4 &#176;C.</li>
	<li>Anesthetize rat pups (aged 9 to 12 from adult timed-pregnant Sprague-Dawley rats) with Ketamine (70 mg/kg, i.p.; using &#60; 50 &#181;l final volume). When the pup is calm, double-check that the tail pinch reflex is absent.</li>
	<li>Decapitate pups.
	<ol>
		<li>Cut the head skin longitudinally from the front to back using a carbon steel scalpel blade, and pull the skin to the sides using forceps. Cut the bone covering the brain, move the scissors laterally, and totally remove it to expose the brain.</li>
		<li>Then, rapidly remove the brain using a spatula (initially placed under the olfactory bulb to gently push it out from the most rostral towards the most caudal areas). Gently push the brain into ice-cooled oxygenated sucrose-artificial cerebrospinal fluid (sucrose-aCSF).</li>
	</ol>
	</li>
	<li>Make a parasagittal cut on the right hemisphere (removing approximately one-third of the hemisphere), and glue the trimmed side of the brain onto a metallic disk that will be magnetically fixed to the cutting chamber of a vibroslicer to cut sagittal 400 &#181;m sections containing the pedunculopontine nucleus (PPN). Keep PPN slices at RT for 45 minutes prior to whole-cell patch-clamp recordings.</li>
</ol>
4. Recordings of Gamma-band Oscillations in PPN Slices
<ol>
	<li>Preparation of Potassium-gluconate Intracellular Solution (High-K+ Solution)
	<ol>
		<li>Place in ice a clean beaker with 10 ml distilled water. While continuously stirring add: K+-gluconate, 90.68 mg phosphocreatine di Tris salt, 47.66 mg HEPES, 1.5 mg EGTA, 40.58 mg Mg2+-ATP, and 4 mg Na+2-GTP.</li>
		<li>Adjust pH to 7.3 with KOH (100 mM in distilled water). Add distilled water to reach a final volume of 20 mL. If needed, adjust osmolarity with sucrose (100 mM in distilled water) to be 280 - 290 mOsm. After dilution, the final concentration of each compound is 124 mM K+-gluconate, 10 mM phosphocreatine di Tris salt, 10 mM HEPES, 0.2 mM EGTA, 4 mM Mg2+-ATP, and 0.3 mM Na+2-GTP.</li>
		<li>Aliquot intracellular solutions in 1 ml tubes and freeze at -20 &#176;C. Use one aliquot per day and keep at 4 &#176;C during experiments.</li>
	</ol>
	</li>
	<li>Whole-cell Patch Clamp Recordings
	<ol>
		<li>Place slices in an immersion chamber and perfuse them (1.5 ml/min) with oxygenated (95% O2 -&#160; 5% CO2) aCSF containing the following receptor antagonists: selective NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV, 40 &#956;M), competitive AMPA/Kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10&#956;M), glycine receptor antagonist strychnine (STR, 10 &#956;M), the specific GABAA receptor antagonist gabazine (GBZ, 10 &#956;M), and sodium channel blocker tetrodotoxin (TTX, 3&#956;M) 
		&#8203;Fill the recording patch pipettes (Resistance 2-7 M&#937;; made from regular, commercially available thick wall borosilicate glass capillaries of 1.0 mm outer diameter and 0.6 mm inner diameter) with intracellular high-K+ solution using commercially available patch-pipette fillers with a solution filter. Insert the pipette in its amplifier&#39;s holder. Apply a small positive pressure using a 1 ml syringe connected to the pipette holder using a silicon tube connected to a three-way valve. Connect the back of the pipette holder to a patch clamp amplifier.</li>
		<li>Move the recording pipette using a mechanical micromanipulator near the PPN nucleus using a 4X objective combined to near-infrared differential interference contrast optics. 
		NOTE: PPN nucleus can be observed in slices dorsal to the superior cerebellar peduncle (SCP; observable as a thick bundle of axons). Recording pipettes were located in the PPN pars compacta, which is located immediately dorsal to the posterior end of the peduncle.</li>
		<li>Bring the recording pipette in contact with a PPN neuron while visualized using a 40X water immersion lens, and rapidly apply negative suction to form a seal with the cell.</li>
		<li>Use voltage-clamp seal software to monitor pipette resistance during negative suction using the manufacturer&#39;s protocol.
		<ol>
			<li>When negative suction is slowly increased, and resistance values read by the patch-clamp monitor at the tip of the pipette reach 80 - 100 MOhm, rapidly change the holding potential to -50 mV and release the negative pressure. Start continuously applying negative suction until rupturing the neuron&#39;s membrane and electrical access is achieved in the whole-cell configuration.</li>
			<li>If access resistance values measured by the voltage-clamp seal software are 10 MOhm or higher, then continue applying negative suction in smaller amounts.</li>
		</ol>
		</li>
		<li>Compensate capacitance (i.e., slow and fast transients observed after rupturing the membrane of the cell) and series resistance in voltage-clamp mode. Switch recording mode to the current clamp, and rapidly compensate bridge values (e.g., move the amplifier knob or click on the automatic compensation menu using the computer&#180;s mouse).
		<ol>
			<li>Continuously monitor resting membrane potential of PPN neuron being recorded using manufacturer&#39;s protocol. If resting membrane potential shift towards depolarizing or hyperpolarizing values, then use small amounts of direct current (up to 100 pA) to keep the optimum -50 mV final value. 
			NOTE: Keep only PPN neurons with a stable resting membrane potential (RMP) of -48 mV or more hyperpolarized (i.e., RMP &#60; -48 mV).</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>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S8501</td>
			<td>C12H22O11, molecular weight = 342.30</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td>NaHCO3, molecular weight = 84.01</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td>KCl, molecular weight = 74.55</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td>MgCl2 &#183; 6H2O, molecular weight = 203.30</td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td>CaCl2 &#183; 2H2O, molecular weight =147.02</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>C6H12O6, molecular weight = 180.16</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5960</td>
			<td>C6H8O6, molecular weight =176.12</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Acros Organics</td>
			<td>327300025</td>
			<td>NaCl, molecular weight = 58.44</td>
		</tr>
		<tr>
			<td>Potassium Gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>G4500</td>
			<td>C6H11KO7, molecular weight = 234.25</td>
		</tr>
		<tr>
			<td>Phosphocreatine di(tris) salt</td>
			<td>Sigma-Aldrich</td>
			<td>P1937</td>
			<td>C4H10N3O5P &#183; 2C4H11NO3, molecular weight = 453.38</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>C8H18N2O4S, molecular weight = 238.30</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E0396</td>
			<td>[-CH2OCH2CH2N(CH2CO2H)2]2, molecular weight = 380.40</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate magnesium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>C10H16N5O13P3 &#183; xMg2+, molecular weight = 507.18</td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate sodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G8877</td>
			<td>C10H16N5O14P3 &#183; xNa+, molecular weight = 523.18</td>
		</tr>
		<tr>
			<td>Tetrodotoxin citrate</td>
			<td>Alomone Labs</td>
			<td>T-550</td>
			<td>C11H17N3O8, molecular weight = 319.27</td>
		</tr>
		<tr>
			<td>DL-2-Amino-5-Phosphonovaleric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5282</td>
			<td>C5H12NO5P, molecular weight = 197.13</td>
		</tr>
		<tr>
			<td>CNQX disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C239</td>
			<td>C9H2N4Na2O4 &#183; xH2O, molecular weight = 276.12</td>
		</tr>
		<tr>
			<td>Strychnine</td>
			<td>Sigma-Aldrich</td>
			<td>S0532</td>
			<td>C21H22N2O2, molecular weight = 334.41</td>
		</tr>
		<tr>
			<td>Mecamylamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>M9020</td>
			<td>C11H21N &#183; HCl, molecular weight = 203.75</td>
		</tr>
		<tr>
			<td>Gabazine (SR-95531)</td>
			<td>Sigma-Aldrich</td>
			<td>S106</td>
			<td>C15H18BrN3O3, molecular weight = 368.23</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Mylan</td>
			<td>67457-001-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>Eclipse E600FN</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>ROE-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heater</td>
			<td>Warner Instruments</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>Cole-Parmer</td>
			<td>Masterflex L/S 7519-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump cartridge</td>
			<td>Cole-Parmer</td>
			<td>Masterflex 7519-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Q-Imaging</td>
			<td>RET-200R-F-M-12-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>Leica VT1200 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigeration system</td>
			<td>Vibratome Instruments</td>
			<td>900R</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Nikon</td>
			<td>Eclipse E600FN</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>ROE-200</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>amplifier</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>heater</td>
			<td>Warner Instruments</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>pump</td>
			<td>Cole-Parmer</td>
			<td>Masterflex L/S 7519-20</td>
			<td></td>
		</tr>
		<tr>
			<td>pump cartridge</td>
			<td>Cole-Parmer</td>
			<td>Masterflex 7519-85</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Q-Imaging</td>
			<td>RET-200R-F-M-12-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

recording gamma band oscillations from pedunculopontine nucleus neurons in a brain slice

Source: Urbano, F. J., et al. <a href="https://app.jove.com/v/54685">Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the method to record gamma-band oscillations in PPN neurons. Using a sagittal brain section with synaptic blockers in aCSF, it employs positive current pulses via a patch pipette to open calcium channels, inducing crucial subthreshold activity for wakefulness.

Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

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 electrophysiological setup with a sagittal brain section submerged in aCSF containing synaptic blockers.
This section includes a neuron-rich PPN region.
The synaptic blockers inhibit various neuronal excitatory and inhibitory receptors and membrane sodium channels, preventing their interference.&#160;
Fill a patch pipette with a potassium-rich solution and insert it into a holder connected to a recording electrode and an amplifier.
Apply positive air pressure to the pipette to prevent clogging, then position it near a PPN neuron.
Apply a negative suction to form a tight seal with the neuron membrane and continue this suction until the membrane breaks.
Deliver the positive current pulses to open the voltage-dependent calcium channels, allowing calcium ions to enter.
This induces gamma-band oscillations, a low-level excitation that is essential for wakefulness without fully exciting the neurons.
Record these signals via the recording pipette to monitor gamma-band oscillation.

recording-gamma-band-oscillations-from-pedunculopontine-nucleus

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

49 Views. Fonte: Urbano, F. J., et al. Registrazione delle oscillazioni della banda gamma nei neuroni del nucleo peduncolopontino. J. Vis. (2016). Il video mostra il metodo per registrare le oscillazioni della banda gamma nei neuroni PPN. Utilizzando una sezione sagittale del cervello con bloccanti sinaptici in aCSF, impiega impulsi di corrente positiva tramite una pipetta di cerotti per aprire i canali del calcio, inducendo un'attività sottosoglia cruciale per la veglia. 

Video: Registrazione delle oscillazioni della banda gamma dai neuroni del nucleo peduncolopontino in una fetta di cervello - Concept

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee prosthesis, and 2) contrast the effects of the proposed technique and that of using intact limb inertial properties on joint kinetic estimates during walking in unilateral, transtibial amputees. An oscillation and reaction board system was validated and shown to be reliable when measuring inertial properties of known geometrical solids. When direct measurements of inertial properties of the prosthesis were used in inverse dynamics modeling of the lower extremity compared with inertial estimates based on an intact shank and foot, joint kinetics at the hip and knee were significantly lower during the swing phase of walking. Differences in joint kinetics during stance, however, were smaller than those observed during swing. Therefore, researchers focusing on the swing phase of walking should consider the impact of prosthesis inertia property estimates on study outcomes. For stance, either one of the two inertial models investigated in our study would likely lead to similar outcomes with an inverse dynamics assessment.

Body segmental inertial properties are required for inverse dynamics modeling. Using an oscillation and reaction board technique, inertial properties of below-knee prostheses were measured. Using direct measures of prosthesis inertia in the inverse dynamics model of the prosthetic leg resulted in lower magnitudes of resultant joint forces and moments.

Oscillazione e di società di reazione tecniche per stimare inerziale proprietà di una protesi sotto il ginocchio

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee ...

Ricerca

JoVE Journal

Bioingegneria

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetica trascinamenti di teta Hippocampal oscillazioni nel comportarsi topi

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Neuroscienze

Employing the Forced Oscillation Technique for the Assessment of Respiratory Mechanics in Adults

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and diseased individuals. FOT, a complementary method to traditional pulmonary function testing, utilizes a range of oscillatory frequencies superimposed on tidal breathing to measure the functional relationship between airway pressure and flow. This passive assessment provides an estimate of respiratory system resistance (Rrs) and reactance (Xrs) that reflect airway caliber and energy storage and dissipation, respectively. Despite the recent increase in popularity and updated Technical Standards, clinical adoption has been slow which relates, in part, to the lack of standardization regarding the acquisition and reporting of FOT data. The goal of this article is to address the lack of standardization across laboratories by providing a comprehensive written protocol for FOT and an accompanying video. To illustrate that this protocol can be utilized irrespective of a particular device, three separate FOT devices have been employed in the case examples and video demonstration. This effort is intended to standardize the use and interpretation of FOT, provide practical suggestions, as well as highlight future questions that need to be addressed.

As the use of forced oscillation technique (FOT) is increasingly utilized to characterize respiratory mechanics, there is a need to standardize methods with respect to nascent technical guidelines and various manufacturer's recommendations. A detailed protocol is provided including FOT assessment and interpretation for two cases to facilitate the standardization of methods.

Utilizzo della tecnica di oscillazione forzata per la valutazione della meccanica respiratoria negli adulti

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and ...

Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

Trascrizione

Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice