eoe sub articles

EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Solutions
<ol>
	<li>Prepare 500 mL of standard artificial cerebrospinal fluid (ACSF) containing the components described in our published work&#160;as listed in&#160;Table 1.
	<ol>
		<li>Use clean glass beakers with deionized water to prepare a fresh ACSF solution. Clean spatulas with deionized water and dry before using to add sodium chloride (NaCl), potassium chloride (KCl), Di-hydrogen sodium phosphate (NaH2PO4), sodium bi-carbonate (NaHCO3), magnesium sulfate (MgSO4), D-glucose, and add the calcium chloride (CaCl2)&#160;after having bubbled the solution with 95% O2&#160;/ 5% CO2&#160;mixture for ~20 min (as detailed in Table 1) to 500 mL of deionized water. Stir until fully dissolved.</li>
		<li>Ensure the ACSF is completely clear without any precipitation. Then, adjust the pH to 7.2-7.4. Bubble with a gas mixture composed of 95% O2&#160;and 5% CO2&#160;for ~20 min.</li>
	</ol>
	</li>
	<li>Prepare 250 mL of slice-cutting solution containing the chemicals&#160;described in&#160;Table 2.
	<ol>
		<li>Add KCl, magnesium chloride (MgCl2), NaH2PO4, NaHCO3, choline&#183;Cl, and D-glucose and add the calcium chloride (CaCl2)&#160;last as indicated in 1.1.1, to 250 mL of deionized water and dissolve as described in 1.1.1 and 1.1.2.</li>
		<li>After bubbling with 95% O2/5% CO2, place the solution in a freezer for 15-20 min so that the cutting solution becomes ice-cold and partially frozen. 
		NOTE: We use this cutting solution when making slices from 1-2 month-old mice; the formula may differ for mice older than 2 months.</li>
		<li>Prepare a lipopolysaccharide (LPS) solution by dissolving it in sterile endotoxin-free isotonic saline (0.9% NaCl). LPS is administered intraperitoneally (0.5 mg/kg) and begins recording 2 h later.</li>
	</ol>
	</li>
</ol>
2. Preparation of Prefrontal Cortex Slice
<ol>
	<li>Take the cutting solution out of the freezer and homogenize to obtain a slush. Place the solution on ice and bubble constantly with 95% O2/5% CO2.</li>
	<li>Using large, sharp scissors, quickly decapitate a mouse as described&#160;and immediately put the head into a beaker filled with ice-cold (0-4&#176;C) cutting solution.</li>
	<li>Cut the top of the skull along the midline in a caudal-to-nasal direction with fine scissors and remove the lateral skull portions. Remove the brain with a fine spatula and drop into ice-cold cutting solution.&#160;&#160; 
	NOTE: Steps 2.2-2.3 must be done rapidly (ideally &#60; 1 min).</li>
	<li>Place the brain on the lid of a 9 cm Petri dish filled with ice. Rinse the brain with ice-cold cutting solution. Cut off the cerebellum and olfactory bulb with a razor blade.</li>
	<li>Apply cyanoacrylate glue to the specimen holder of a vibratome. Carefully place the brain block on the drop of glue, rostral side up, and immediately immerse in ice-cold cutting solution.</li>
	<li>Adjust the blade holder of the vibratome to an angle of 15&#176; with reference to the horizontal plane and prepare 350-&#181;m coronal brain slices (blade vibration frequency = 85 Hz, speed = 0.1 mm/s).</li>
	<li>Using a pipette, transfer the slices into the recovery chamber filled with ACSF, incubate for 1 h (at RT) with constant bubbling of 95% O2/5% CO2.</li>
</ol>
3. Whole-cell Patch Recording
<ol>
	<li>Make glass borosilicate micropipettes for use as recording electrodes or puff micropipettes according to the specific guidelines in the Puller Operation Manual. For recording electrodes, tip resistances are in the range 4-6 M&#8486;, while puff micropipettes have tip diameters in the range 2-5 &#181;m.</li>
	<li>Add Na+&#160;channel blocker (Tetrodotoxin (TTX), 1 &#181;M), to block fast Na+&#160;channel and thus action potentials, to ACSF and maintain a constant flow rate of this solution of 2-3 mL/min in the recording chamber, by peristaltic pump into the chamber and aspiration out of it. Bubble the ACSF with 95% O2/5% CO2&#160;constantly to ensure the viability of the slices.</li>
	<li>Transfer a brain slice into the recording chamber using a modified Pasteur pipette whose fine tip was cut to fit the slice size. Cover the slice with a platinum slice anchor with parallel nylon threads spaced &#8805;2 mm apart to hold the slice on the platform.</li>
	<li>Fill the recording micropipette with internal solution&#160;(Table 3) and fill the puff micropipettes with gamma-aminobutyric acid (GABA) at 10 &#181;M, 50 &#181;M, and 100 &#181;M (dissolved in ACSF), or ACSF as vehicle control.</li>
	<li>To prevent blockage with debris, apply light positive pressure using a 1 mL syringe before immersing the recording electrode in the ACSF. Using a micromanipulator under a microscope (4x objective lens), position the recording electrode and puff micropipette so that the tips appear in the center of the video image in the monitor (Figure 1A).</li>
	<li>Adjust the microscope focus (40x objective lens) while gradually lowering the puff micropipette and place it above the recording neuron at an angle of 45&#176;. Keep the distance between the tip of the puff micropipette and the neuron recorded between 20-40 &#181;m (Figure 1B). Slowly and carefully approach the neuron with the recording electrode and then release the positive pressure. Apply a weak and brief suction through the tubing connected to the electrode holder to create a giga-ohm seal.</li>
	<li>Maintain the voltage at 0 mV. After formation of the giga-ohm seal, compensate for fast and slow capacitance manually or automatically. Then apply a brief and strong suction through the tubing mentioned above to get into whole-cell mode.</li>
	<li>Record eIPSC in voltage-clamp mode. Apply low gas pressure (nitrogen, 4 - 6 psi) to the puff micropipette using a Picospritzer controlled by a Master 8 voltage step generator to deliver single or paired GABA puffs (Figure 2).</li>
</ol>
Table 1. Recording ACSF.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration (mM)</td>
			<td>Weight (g/L)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.443</td>
			<td>119</td>
			<td>6.9547</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.551</td>
			<td>2.5</td>
			<td>0.1864</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>137.99</td>
			<td>1</td>
			<td>0.1380</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.007</td>
			<td>26.2</td>
			<td>2.2010</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>120.366</td>
			<td>1.3</td>
			<td>0.1565</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>198.17</td>
			<td>11</td>
			<td>2.1799</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>110.98</td>
			<td>2.5</td>
			<td>0.2775</td>
		</tr>
	</tbody>
</table>
Table 2. Slice cutting solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration (mM)</td>
			<td>Weight (g/L)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.551</td>
			<td>2.5</td>
			<td>0.1864</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>95.211</td>
			<td>7</td>
			<td>0.6665</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>137.99</td>
			<td>1.25</td>
			<td>0.1725</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.007</td>
			<td>25</td>
			<td>2.1002</td>
		</tr>
		<tr>
			<td>Choline&#183;Cl</td>
			<td>139.63</td>
			<td>115</td>
			<td>16.0575</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>198.17</td>
			<td>7</td>
			<td>1.3872</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>110.98</td>
			<td>0.5</td>
			<td>0.0555</td>
		</tr>
	</tbody>
</table>
Table 3. Internal solution for whole-cell patch recordings.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration (mM)</td>
			<td>Weight (g/L)</td>
		</tr>
		<tr>
			<td>Cs gluconate</td>
			<td>328.052</td>
			<td>100</td>
			<td>32.8052</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>168.36</td>
			<td>5</td>
			<td>0.8418</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>238.301</td>
			<td>10</td>
			<td>2.3830</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>95.211</td>
			<td>2</td>
			<td>0.1904</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>110.98</td>
			<td>1</td>
			<td>0.1110</td>
		</tr>
		<tr>
			<td>BAPTA</td>
			<td>476.433</td>
			<td>11</td>
			<td>5.2408</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>551.1</td>
			<td>4</td>
			<td>2.2044</td>
		</tr>
		<tr>
			<td>Na2GTP</td>
			<td>523.2</td>
			<td>0.4</td>
			<td>0.2093</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass Borosilicate micropipettes</td>
			<td>Shutter Instruments</td>
			<td>BF150-86-10</td>
			<td>1.50 mm outer diameter; 0.86 mm inner diameter</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Shutter Instruments</td>
			<td>MODEL P-97&#160;</td>
			<td>Flaming/Brow Micropipette Puller</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Shutter Instruments</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer controlled Amplifier&#160;&#160;&#160;</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Acquisition system&#160;</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Camera</td>
			<td>Nikon&#160;</td>
			<td>2115001</td>
			<td>Inspection equipment</td>
		</tr>
		<tr>
			<td>Microscopy&#160;</td>
			<td>Nikon&#160;</td>
			<td>Eclipse FN1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Master 8</td>
			<td>A.M.P.I.</td>
			<td>Master-8 Pulse stimulator</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome Slicer</td>
			<td>Leica</td>
			<td>VT 1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer <img alt="Equation 1" src="/files/ftp_upload/22712/22712eq01.jpg" style="margin: auto;" /></td>
			<td>Parker Hannifin</td>
			<td></td>
			<td>Pressure Systems for Ejection of Picoliter Volumes in Cell Research</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Gillette</td>
			<td>74-S</td>
			<td>FLYING EAGLE</td>
		</tr>
		<tr>
			<td>Video monitor</td>
			<td>Panasonic</td>
			<td>WV-BM 1410</td>
			<td></td>
		</tr>
		<tr>
			<td>502 Glue</td>
			<td>Deli</td>
			<td>7146</td>
			<td>Cyanoacrylate Glue</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Shanghai JIA PENG Corporation</td>
			<td>BT100-1F</td>
			<td></td>
		</tr>
		<tr>
			<td>Video Camera</td>
			<td>Olympus America Medical</td>
			<td>OLY-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipets</td>
			<td>Biologix</td>
			<td>30-0138A1</td>
			<td></td>
		</tr>
	</tbody>
</table>

recording whole-cell currents with gaba puffing in a mouse brain slice

Source: Feng, Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56387">Whole-cell Currents Induced by Puff Application of GABA in Brain Slices.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the recording of whole-cell currents induced by gamma-aminobutyric acid, or GABA, in a mouse prefrontal cortical brain slice. Controlled delivery of GABA opens chloride channels in a postsynaptic neuron, generating inhibitory postsynaptic currents, or IPSCs. These IPSCs increase with higher GABA concentrations, reflecting GABA&#39;s inhibitory activity on synaptic transmission.

<img alt="Figure 1" src="/files/ftp_upload/22712/22712_Figure1.jpg" /> 
Figure 1. Diagram of the puff micropipette and recording electrode.&#160; 
(A)&#160;The distance between the puff and recording micropipettes.&#160;(B)&#160;The puff micropipette (left) and the recording electrode (right) on the surface of a brain slice.&#160;
<img alt="Figure 2" src="/files/ftp_up...

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Solutions

	...

Begin with a secured prefrontal cortical brain slice in an artificial cerebrospinal fluid, or aCSF, in the recording chamber of an electrophysiology setup.
This aCSF contains a sodium ion channel blocker, which inhibits sodium channels in neurons.
Position a puff micropipette filled with gamma-aminobutyric acid, or GABA, above a postsynaptic neuron.
Place an ionic solution-filled recording micropipette with an electrode near the same neuron.
Apply weak suction to create a tight seal with a small patch of the neuronal membrane.
Then, apply strong suction to disrupt the patched membrane, enabling access to the neuron&#39;s interior.
Deliver the GABA, an inhibitory neurotransmitter, which binds with a ligand-gated chloride channel on postsynaptic neurons.
This opens the channel and allows chloride ions to enter the neurons, generating an inhibitory postsynaptic current.
At a constant voltage, record the postsynaptic current. An increase in inhibitory postsynaptic current reflects GABA&#39;s role in neuronal activity inhibition.

recording-whole-cell-currents-with-gaba-puffing-in-a-mouse-brain-slice

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

48 visualizações. Fonte: Feng, Y. et al., Correntes de células inteiras induzidas pela aplicação de GABA em fatias cerebrais.J. Vis. Exp. (2017) Este vídeo demonstra o registro de correntes de células inteiras induzidas pelo ácido gama-aminobutírico, ou GABA, em uma fatia de cérebro cortical pré-frontal de camundongo. A entrega controlada de GABA abre canais de cloreto em um neurônio pós-sináptico, gerando correntes pós-sinápticas inibitórias, ou IPSCs. Essas IPSCs aumentam com concentrações mai...

Vídeo: Registrando correntes de células inteiras com GABA Puffing em uma fatia de cérebro de camundongo - Conceito

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.

Eletrofisiológica e Caracterização Morfológica de Neuronal Microcircuits em aguda fatias do cérebro do Usando Emparelhados patch-clamp Recordings

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

JoVE Journal

Neurociência

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Full-cell Patch-clamp Gravações para determinação eletrofisiológica de Ion Selectivity em Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

Métodos para a descoberta de novos compostos modulando um Receptor do ácido gama - aminobutírico tipo uma neurotransmissão

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Transcrição

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice