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. Animals 
<ol>
	<li>Use genetically engineered OR-IRES-tauGFP (OR = Olfactory receptor, IRES = Internal ribosome entry site, tauGFP = tau-green fluorescent protein) mice available at the Jackson Laboratory. These mice were developed in Dr. Peter Mombaerts&#39; laboratory in order to analyze axon targeting and development of the olfactory system. For example, the MOR23-IRES-tauGFP line, stock number 006643, bears the official strain name B6;129P2-Olfr16tm2Mom/MomJ; similarly, the SR1-IRES-tauGFP line, stock number 6717 bears the official name B6;129P2-Olfr124tm1Mom/MomJ.</li>
	<li>Regarding the age of the animals: for a better outcome of the protocol, use animals between 2 and 4 weeks of age. In this age group, the dissection is easier (softer bones, firmer olfactory epithelium) and the dendritic knobs are bigger compared to older animals.</li>
</ol>
2. Preparation of Electrodes and Solutions 
<ol>
	<li>For the stimulating pipettes: purchase pre-pulled stimulating pipettes. Otherwise, manually prepare them.
	<ol>
		<li>Using a flame, bend six 1 mm glass pipettes at about 1 cm from the tip. Insert these six pipettes plus a straight 7th in 1.5 cm heat-shrink tubing strengthened by an eyelet.</li>
		<li>Heat shrinks the tubing to maintain the barrels attached together. Attach an additional heat-shrink tubing to the other extremity of the barrels. Pull this stimulating pipette with a multi-barrel puller.</li>
		<li>Add some white liquid glue around the eyelet to strengthen the bent tips. Let dry overnight (O/N).</li>
	</ol>
	</li>
	<li>Prepare 1 or 2 L of normal Ringer&#39;s extracellular solution (in mM: NaCl 124, KCl 3, MgSO4 1.3, CaCl2 2, NaHCO3 26, NaH2PO4 1.25, glucose 15; pH 7.6 and 305 mOsm). Keep at 4 &#176;C until use.</li>
	<li>Prepare intracellular stock solution (in mM): KCl 70, KOH 53, methanesulfonic acid 30, ethylene glycol tetraacetic acid (EGTA) 5, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, sucrose 70; pH 7.2 (KOH) and 310 mOsm. Keep at 4 &#176;C until use. Good for several weeks.</li>
	<li>Prepare intracellular recording solution with nystatin extemporaneously (at the last minute) before experiment.
	<ol>
		<li>Weigh 3 mg of nystatin, add 50 &#181;l of dimethyl sulfoxide (DMSO); vortex 20 sec then sonicate 2-3 min until entirely diluted.</li>
		<li>Add 20 &#181;l of DMSO-nystatin solution in 5 ml of intracellular stock solution. Vortex 20 sec, then sonicate 3 min. Keep this solution at 4 &#176;C and protect from direct light, nystatin is light sensitive. This solution can be used for a few hours. Replace every day.</li>
		<li>Once the olfactory epithelium preparation is under the microscope, take some of this solution in a 1 ml syringe with a flame-elongated yellow tip to fill the electrodes; protect from direct light. Once at room temperature (RT), the nystatin solution is not stable; replace the solution in the 1 ml syringe every hour or keep it in ice.</li>
	</ol>
	</li>
	<li>Pull recording electrodes with a puller to obtain long neck and small tip (~2 &#181;m) with a resistance of 15-20 M&#8486; with the internal nystatin solution.</li>
	<li>Prepare odorant solution at 0.5 M in DMSO under fume hood; aliquot and keep at -20 &#176;C until use. Dilute odorant in Ringer&#39;s solution until the desired concentration. Fill the stimulating pipette.</li>
</ol>
3. Preparation of Olfactory Epithelium
Note: OR-IRES-tauGFP mice express the tauGFP under the control of the OR promoter. In these mice, all neurons expressing the OR of interest are labeled with GFP. This protocol is adapted for ORs expressed in all zones. However, dissections and recordings are easier for ORs expressed in the dorsal zone.
<ol>
	<li>Anesthetize the animal by injecting a mix of ketamine and xylazine (150 mg/kg and 10 mg/kg body weight, respectively). Decapitation can be performed with sharp scissors for young mice or with a properly maintained rodent guillotine for older mice.
	<ol>
		<li>Using ring dissecting scissors make a longitudinal medial incision through the dorsal skin. Remove the skin by pulling it apart. Using the scissors, cut the lower jaws at the jaw joint. Remove the upper front teeth by a coronal cut parallel to the teeth.</li>
		<li>Make a coronal cut of the head behind the eyes and keep only the anterior part of the head. Dip it in an ice-cold Ringer solution for the dissection under the scope.</li>
	</ol>
	</li>
	<li>Dissect under the scope: in the ventral side, make a longitudinal cut along the upper jaw/the teeth. Cut the dorsal bones longitudinally, following the dorsolateral side of the nasal cavity. Remove most bones and palate; transfer the septum and the epithelium attached to it in oxygenated Ringer at RT.</li>
	<li>For the final dissection: Right before starting the recording session, peel away the epithelium from the underlying septum with forceps. Detach the epithelium with forceps and with two 4-5 mm scissor cuts (use micro-vannas scissors) at the anterior part of the septum where the adhesion is stronger.
	<ol>
		<li>Carefully remove the vomeronasal organ by cutting it out along its dorsal connection to the septal epithelium. Transfer the epithelium to a recording chamber with the mucus layer facing up; keep it flat in the chamber with a harp.</li>
	</ol>
	</li>
	<li>Install chamber under an upright microscope equipped with fluorescence optics and a sensitive camera. Visualize the preparation on the computer screen at high magnification through a 40X water-immersion objective (numerical aperture 0.8) and an extra 2X or 4X magnification achieved by a magnifying lens. Perfuse continuously with oxygenated Ringer at RT (1-2 ml/min).</li>
</ol>
4. Recording Session
<ol>
	<li>Search for fluorescent cells: excite the preparation at 480 nm for EGFP, which emits light in the 530-550 nm range; target one dendritic knob which is reliably visible in fluorescence and under bright field.</li>
	<li>Fill the electrode with intracellular solution with nystatin; remove bubbles by gently tapping on the electrode.</li>
	<li>Insert electrode on electrode holder, apply positive pressure in the pipette; Resistance should be 15-20 M&#8486;.</li>
	<li>Bring electrode close to the cell; once resistance reaches ~40 M&#8486;, release positive pressure and apply slight negative pressure to reach a gigaseal.</li>
	<li>Once seal is reached, set the membrane potential at about -75 mV;</li>
	<li>Once the cell is opened, proceed with stimulation protocols, pharmacological treatments. To measure the response to a single odorant stimulation, record 200-500 msec of spontaneous activity, stimulate for 500 msec and measure the response of the cell for up to 10 sec. For pharmacological treatments, perfuse the pharmacological agents at the desired concentration.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Heavy equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vibration table with Faraday cage</td>
			<td>TMC</td>
			<td>63-500 SERIES</td>
			<td>required : isolates the recording system from vibrations induced by the environment (movements of experimenter, vibrations of equipment such as fans for cooling computers, etc); can also be purchased with a Faraday cage, or equipped by a custom made Faraday cage; this cage is recommended to avoid electric noise from the environment</td>
		</tr>
		<tr>
			<td>optics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>upright microcope equipped with epifluorescence; fixed or moving stage depending on the user&#39;s preference</td>
		</tr>
		<tr>
			<td>objectives</td>
			<td>Olympus</td>
			<td>LUMPLFL40XW</td>
			<td>at least 2 objectives required: a 4X or 10X for coarse approach to the cell; and a 40X immersion long distance example Olympus LUMPLFL40XW / IR /0,8 / WD:3.3 MM</td>
		</tr>
		<tr>
			<td>magnifier</td>
			<td>Olympus</td>
			<td>U-TVCAC</td>
			<td>ABSOLUTELY REQUIRED: placed in the light path between the objective and the camera; allows to magnify the image on the screen in order to reach precisely the knob with the recording electrode</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td>DP72</td>
			<td>a good camera is required to see the neurons in fluorescence as well as in bright field; the controlling software is simple and allows to take pictures and do live camera image to monitor the approach of the electrode to the cell. An ultrasensitive camera is not necessary</td>
		</tr>
		<tr>
			<td>filters</td>
			<td>Olympus/Chroma</td>
			<td></td>
			<td>depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m</td>
		</tr>
		<tr>
			<td>Recording electrodes/system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>amplifier</td>
			<td>HEKA</td>
			<td>EPC10 USB</td>
			<td>monitors the currents flowing through the recording electrode and also controls the puffing by sending a TTL signal to the spritzer; the EPC10 setup is 
			controled by computer</td>
		</tr>
		<tr>
			<td>software</td>
			<td>HEKA</td>
			<td>Patchmaster</td>
			<td>controls the amplifier during the experiment</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter</td>
			<td>MP225</td>
			<td>precision micromanipulator, allows precise movements down to 1/10th of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift</td>
		</tr>
		<tr>
			<td>electrode puller</td>
			<td>Sutter</td>
			<td>P97</td>
			<td>with a FT345-B wide trough filament; to prepare recording pipets of about 2&#181;m diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>Sutter</td>
			<td>BF120-69-10</td>
			<td>in our recording conditions, this glass is ideal for recording pipets</td>
		</tr>
		<tr>
			<td>recording chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26G</td>
			<td>a chamber is needed to set the preparation under the microscope. To maintain the preparation in the center of the chamber, a net/ anchor should be used.</td>
		</tr>
		<tr>
			<td>Stimulation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td>attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes</td>
		</tr>
		<tr>
			<td>multibarrel puller</td>
			<td>MDI</td>
			<td>PMP-107-Z</td>
			<td>by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels</td>
		</tr>
		<tr>
			<td>precision pressure injector</td>
			<td>Toohey Company</td>
			<td>P/N T25-1-900 Single Channel</td>
			<td>this precision pressure injector controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V TTLs</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>YOU-1</td>
			<td>a coarse manipulator is enough to bring the puffing electrode close to the recording site</td>
		</tr>
		<tr>
			<td>tubings</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>solutions/perfusion/chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vacuum pump</td>
			<td>gardner denver</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>perfusion system</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N3503</td>
			<td>mandatory to perpare internal solution for perforated patch clamp</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>used to disolve nystatin for internal solution for perforated patch</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td>intracellular/extracellular solution</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4 7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>63140</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>EGTA (Ethylene glycol-bis(2- aminoethylether)-N,N,N&#8242;,N&#8242;- tetraacetic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>&#160;internal solution</td>
		</tr>
		<tr>
			<td>Potassium hydroxyde</td>
			<td>Sigma-Aldrich</td>
			<td>P1767</td>
			<td>&#160;internal solution</td>
		</tr>
		<tr>
			<td>Methyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>W387509</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Hepes-Na</td>
			<td>Sigma-Aldrich</td>
			<td>H7006</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>intracellular solution</td>
		</tr>
	</tbody>
</table>

perforated patch-clamp recording of olfactory sensory neurons in mice

Source: Jarriault, D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52652">Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor.</a> J. Vis. Exp. (2015).
This video demonstrates electrophysiological recording of olfactory sensory neurons (OSNs) in mice using a perforated patch-clamp technique. The olfactory epithelium, containing OSNs with fluorophore-tagged odor receptors, is isolated from a mouse and secured in a recording chamber. A recording pipette is clamped to the dendritic knob of an OSN, and nystatin within the pipette perforates the membrane to form electrical connectivity with the cell. Finally, another pipette is used to deliver an odor stimulant, and the electrophysiological activity of the patched cell is recorded.

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

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

Take the severed head of a genetically engineered mouse. The olfactory epithelium in its nose contains olfactory sensory neurons, or OSNs, with fluorophore-tagged odor receptors.
Remove the skin and dissect the head to isolate the septum with attached olfactory epithelium.
Separate and secure the epithelium in a recording chamber.
Using a fluorescence microscope, detect the labeled receptors to target the OSNs&#39; dendritic knob.
Bring a recording pipette with an intracellular solution containing nystatin, a membrane-perforating antibiotic.
Apply positive pressure to avoid pipette clogging. Insert the pipette into the epithelium and approach an OSN.
Switch to negative pressure, forming a seal with the cell membrane. Apply current to maintain the membrane at the resting potential.
Nystatin perforates the membrane, allowing measurement of intracellular ionic currents.
Position a pipette containing an odor stimulant near the OSN. Release the stimulant, which binds to the odor receptors, triggering ion channel opening to allow ion influx. Record the resulting electrophysiological signals.

perforated-patch-clamp-recording-of-olfactory-sensory-neurons-in-mice

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

53 visualizações. Fonte: Jarriault, D., et al. Gravação perfurada de neurônios sensoriais olfativos de camundongos em neuroepitélio intacto: análise funcional de neurônios que expressam um receptor de odor identificado. J. Vis. Exp. (2015). Este vídeo demonstra o registro eletrofisiológico de neurônios sensoriais olfativos (OSNs) em camundongos usando uma técnica de patch-clamp perfurada. O epitélio olfatório, contendo OSNs com receptores de odor marcados com fluoróforo, é isolado de um camundongo ...

Vídeo: Registro perfurado de neurônios sensoriais olfativos em camundongos - Conceito

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the clear majority have been performed on dissociated primary cells. To improve our understanding of how teleost pituitary cells, behave in a more biologically relevant environment, this protocol shows how to prepare viable brain-pituitary slices using the small freshwater fish medaka (Oryzias latipes). Making the brain-pituitary slices, pH and osmolality of all solutions were adjusted to values found in body fluids of freshwater fish living at 25 to 28 &#176;C. Following slice preparation, the protocol demonstrates how to conduct electrophysiological recordings using the perforated whole-cell patch-clamp technique. The patch-clamp technique is a powerful tool with unprecedented temporal resolution and sensitivity, allowing investigation of electrical properties from intact whole cells down to single ion channels. Perforated patch is unique in that it keeps the intracellular environment intact preventing regulatory elements in the cytosol from being diluted by the patch pipette electrode solution. In contrast, when performing traditional whole-cell recordings, it was observed that medaka pituitary cells quickly lose their ability to fire action potentials. Among the various perforation techniques available, this protocol demonstrates how to achieve perforation of the patched membrane using the fungicide Amphotericin B.

The article describes an optimized protocol for making viable brain-pituitary tissue slices, using the teleost fish medaka (Oryzias latipes), followed by electrophysiological recordings of pituitary cells using the patch-clamp technique with the perforated patch configuration.

Fatias de cérebro-hipófise saudáveis para investigações eletrofisiológicas das células da hipófise em peixes teleósteos

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the ...

JoVE Journal

Neurociência

Compreendendo as respostas do neurônio motor à estimulação da medula espinhal

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

Autor em destaque: Avançando a estimulação da medula espinhal - explorando as respostas celulares dos neurônios motores por meio da eletrofisiologia do patch-clamp 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

A Preparação Ex Vivo do Corte da Medula Espinhal para o Registro de Patch-Clamp de Células Inteiras em Neurônios Motores Durante a Estimulação da Medula Espinhal

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 &#181;M, 50 &#181;M, 100 &#181;M, and 200 &#181;M) on the Kv channel current (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv showed a dose-dependent trend (no effect for 25 &#181;M, 30% inhibitory rate for 50 &#181;M, 46% inhibitory rate for 100 &#181;M and 54% inhibitory rate for 200 &#181;M). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

Registro de Corrente de Potássio Dependente de Voltagem em Cardiomiócitos H9c2 através da Técnica de Patch-Clamp de Células Inteiras

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ...

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

Transcrição

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice