patch-clamp recordings from the dendrite of a dopaminergic neuron in a brain slice

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice

In this procedure, transfer a brain slice to the recording chamber. Anchor the slice at the bottom of the recording chamber with a platinum ring, and ensure that the slice is of good quality with a smooth, even surface. Then, select a neuron with a dendrite extending over a long distance in the same plane, and ensure that the dendrite of interest can be followed to a well-defined soma. Next, fill the patch pipette with electrode solution.
To patch the dendrite in cell-attached mode, identify and focus on a portion of the dendrite. Apply positive pressure to the pipette, and lower it using the micromanipulator. Then, position the pipette close to the membrane, and adjust the pressure in order to create a small dimple. Subsequently, release the pressure on the pipette tip and patch the dendrite while controlling the pipette resistance. Try to obtain a seal resistance larger than 1 giga ohm, at best between 3 and 10 giga ohms for cell-attached recordings.
Afterward, retract the pipette away from the membrane by a couple of micrometers to avoid deformation of the dendrite. The action currents seen here represent the spontaneous action potentials of nigral neurons. To suppress the action currents, apply calcium and sodium channel blockers to ACSF. Then, apply a voltage step of negative 90 millivolts from a holding potential of 0 millivolts to evoke the hyperpolarization-activated cation current.

patch-clamp-recordings-from-dendrite-dopaminergic-neuron-brain

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

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 the Solutions
<ol>
	<li>Standard artificial cerebrospinal fluid (ACSF; Table 1)
	<ol>
		<li>Use high-purity salts and clean glass beakers previously rinsed with double-distilled water to prepare a fresh solution. Use high-quality double-distilled water for the preparation of all extra- and intracellular solutions.</li>
		<li>According to Table 1, take a 2 L beaker to dissolve NaHCO3 in 500 mL water and another to dissolve the other salts to prevent precipitation of divalent ions. Clean the spatula systematically with double-distilled water and dry it before taking a salt. Use a magnetic stirrer to homogenize dissolution. Add the solutions from both beakers to an appropriate volumetric flask and bring them to the appropriate volume.</li>
		<li>Ensure that the final extracellular solution is fully transparent and without any trace of precipitation.</li>
		<li>Apply a gas mixture composed of 95% O2 and 5% CO2 (carbogen gas) with a glass microfilter candle (&#216; 13 mm) 20 - 30 min before perfusing the slices. Carefully control the osmolarity (2 - 3 consecutive measurements) of the ACSF with an osmometer (optimal range: 314 - 325 mOsmol/L). Store the remaining solution after preparation at 4 &#176;C and use it within 3 days.</li>
	</ol>
	</li>
	<li>Cutting solution (sucrose-ACSF; Table 2)
	<ol>
		<li>Prepare 3 L of fresh solution. Use 1 L to prepare brain slices from one animal. Store the remaining solution at 4 &#176;C and use it within 3 days. 
		&#8203;NOTE: High Mg2+ and low Ca2+ concentrations are used to decrease synaptic transmission, and some NaCl is substituted with sucrose to preserve the tissue.</li>
	</ol>
	</li>
	<li>Intracellular solutions
	<ol>
		<li>Prepare in advance 100 mL solution for dual whole-cell recordings (Table 3).</li>
		<li>Include methylsulfate to obtain good recovery of cell morphology. Add biocytin (0.1 - 0.4%) to examine the neuron&#39;s morphology subsequently. Include a fluorescent dye (e.g., Alexa 594 or Sulforhodamine 101 41) to follow the extension of dendrites during the recording (optional).</li>
		<li>Prepare in advance 100 mL electrode solution for cell-attached recordings (Table 4). In this case, the internal solution is a high-K+ solution designed to isolate the macroscopic hyperpolarization-activated cation current (Ih) and contains blockers for the other voltage-gated ion channels.</li>
		<li>Store intracellular solutions in 2 mL aliquots at -20 &#176;C. Use a new aliquot for every new recording session. Check the osmolarity of every thawed aliquot carefully with an osmometer. Take a new aliquot if the osmolarity does not correspond to the initial value.</li>
		<li>Transfer the solution from the aliquot into a 3 mL syringe on the top of which a 0.22 &#181;m sterile syringe filter is placed. Keep the syringe on ice during the recording session to limit degradation of its compounds (ATP, GTP or phosphocreatine).</li>
	</ol>
	</li>
</ol>
2. Fabrication and Filling of Patch Pipette
<ol>
	<li>Pulling
	<ol>
		<li>Use a horizontal electrode puller and thick-walled borosilicate glass tubing. For whole-cell and cell-attached recordings, use a 2 mm outer diameter/1 mm inner diameter. Ensure that the glass tubing is absolutely clean.</li>
		<li>If this is not the case, immerse glass capillaries in an organic solvent (e.g., ethanol) first and then in double-distilled water. Briefly heat capillary endings with a Bunsen burner. Alternatively, order glass tubing washed and heated directly from the manufacturer (see Materials list).</li>
		<li>For dual somatodendritic recordings, ensure that the somatic pipette resistance is between 6 and 10 M&#8486; and between 8 and 19 M&#8486; for dendritic pipettes when filled with intracellular solution (Table 3). Standardize pipette resistance for cell-attached recordings to a resistance of 10 M&#8486; to achieve comparable results along the somatodendritic domain of the neuron.</li>
		<li>Prepare fresh pipettes before every recording session (every day) or immediately before patching and use them 5 - 8 h after their fabrication. Store pipettes in a covered glass container to protect them from dust and obstruction of the tip.</li>
	</ol>
	</li>
	<li>Polishing
	<ol>
		<li>Inspect and heat-polish every pipette tip with a microforge to obtain better seals with the membrane. Before using the microforge, melt a tiny piece of glass on the platinum heating filament. Replace this glass coating on the platinum filament daily for constancy (Peter Jonas, personal communication). 
		NOTE: Pipettes used for cell-attached recordings can be coated before the heat-polishing step to reduce background noise. An insulating agent such as molten dental wax or a silicone elastomer can be used to coat the pipettes.</li>
	</ol>
	</li>
</ol>
3. Preparation of Brain Slices
<ol>
	<li>Use healthy Wistar rats aged between 16 - 19 days old. Do not use unhealthy animals or animals suffering from hypothermia or dehydration. Before starting the preparation of slices keep the animal in a safe and silent place. Avoid having any other animal in the room while preparing an animal. Manipulate animals gently.</li>
	<li>Pour sucrose-ACSF into two 400 mL polypropylene (PP) beakers and place them in the -80 &#176;C freezer for 45 min. Mix the solution to get a homogenous ice-cold liquid/frozen solution and place the beakers on ice. Supply the sucrose-ACSF solution with carbogen gas using a microfilter candle (&#216; 13 mm) in each PP beaker.</li>
	<li>Prepare the slicer and the reserve chamber
	<ol>
		<li>Use a high-quality tissue slicer with extremely low vertical vibrations to minimize damage to the superficial layers of slices as much as possible. Fix a fresh and entire razor blade on the slicer.</li>
		<li>Use a new razor blade for each cutting session. Avoid bending of the razor blade. Do not remove the fatty film at the surface of the razor blade (Josef Bischofberger, personal communication).</li>
		<li>If available, check the vertical vibration with a vibroprobe provided by the manufacturer. Alternatively, use a custom-made vibroprobe. Reduce the vertical vibrations of the blade so as to be as close as possible to 0 &#181;m.</li>
		<li>Prepare a 150 mL reserve chamber for slices. Pour standard ACSF into the reserve chamber and place it in a water bath. Supply the reserve chamber solution with carbogen using a microfilter candle (&#216; 6 mm). Ensure that the carbogen bubbles are as tiny as possible. 
		NOTE: Build a new reserve chamber every 2 - 3 months to keep the slice quality constant.</li>
	</ol>
	</li>
	<li>Cut slices
	<ol>
		<li>In a silent room in which the experimenter is not disturbed or stressed, decapitate the animal with surgery scissors (length 150 mm) at the level of the cervical medulla.</li>
		<li>Cut the skin at the top of the animal&#39;s head in the nasal to caudal direction with a scalpel and remove it laterally. Cut the top of the skull from the caudal to the nasal part of the head with fine scissors and remove both parts of the skull laterally. Remove the brain with a thin spatula and drop it carefully into a PP beaker containing ice-cold (0 - 4 &#176;C) sucrose-ACSF equilibrated with carbogen. 
		NOTE: This sequence of steps needs to be done as quickly as possible, but also meticulously (&#60;&#60;1 min, roughly 40 s).</li>
		<li>Keep the brain in the solution of the PP beaker for ~2 to 5 min. Adjust the carbogen pressure to prevent brain movements. If the carbogen bubbles are too large, replace the microfilter candle with a fresh one.</li>
		<li>Place the brain on a 9 cm Petri dish whose inside bottom is covered with a ~0.5 cm thick Sylgard layer. Prepare this petri dish in advance.</li>
		<li>Surround the brain with ice-cold sucrose-ACSF. Ensure that some liquid phase of ACSF-sucrose solution submerges the brain. For coronal slices, cut off the frontal part of the brain in the coronal plane with a scalpel or a razor blade. Remove the cerebellum with a coronal cut.</li>
		<li>Apply cyanoacrylate glue on the specimen tray (area ~1 cm2). Paste the brain block such that the frontal part faces the slicing stage. Carefully drip sucrose-ACSF on top of the pasted brain using the large opening of a glass Pasteur pipette and subsequently slowly submerge the cutting chamber of the slicer. Maneuver the specimen tray so that the cutting surface (i.e., the first tissue to hit the blade) is the ventral surface of the brain.</li>
		<li>Apply carbogen with a bended glass microfilter candle (&#216; 6 mm) to the cutting chamber (optional).</li>
		<li>Adjust the razor blade at an angle of ~15&#176; with reference to the horizontal plane. Cut coronal brain slices of ~500 &#181;m in the caudal to nasal direction before reaching the substantia nigra. Decrease the thickness to cut 300 - 350 &#181;m thick slices containing the region of interest.</li>
		<li>Separate slices from the tissue block with a very thin perfusion needle attached to a syringe parallel to the edge of the razor blade. Bend the needle so as to have an angle of 90&#176; between the needle and the syringe. Ensure that no pressure is applied to the razor blade while removing the slice from the tissue block.</li>
	</ol>
	</li>
	<li>Store slices
	<ol>
		<li>Transfer the slices using the largest opening of a glass Pasteur pipette (attached to a bulb) into the reserve chamber. Once all the slices are transferred into the reserve chamber (Christoph Schmidt-Hieber, personal communication) bring the temperature of the water bath to 34 &#176;C for 0.5 - 1 h.</li>
		<li>After this period switch off the water bath and keep the slices at room temperature. Adjust the carbogen pressure in order to avoid movements of the slices in the reserve chamber. Keep the slices for another 10 to 20 min before starting recordings.</li>
		<li>Clean the equipment and the microfilter candles carefully yet thoroughly with double-distilled water at the end of the slicing procedure.</li>
	</ol>
	</li>
</ol>
4. Dual Somatic and Somatodendric Recordings in Nigral Neurons and Biocytin Filing
<ol>
	<li>Follow descriptions for the assembly of a patch-clamp setup for slices given in The Axon Guide.</li>
	<li>Prepare recording chamber
	<ol>
		<li>Place 1 - 2 mL of double-distilled water in the recording chamber. Verify that it is not leaky. Place the recording chamber on the shifting x-y table.</li>
		<li>Feed standard ACSF through an oxygen-impermeable perfusion tubing system (polytetrafluoroethylene) to the recording chamber. Stabilize the perfusion flow in the chamber at a rate of 4 - 5 mL min-1. Keep the length of the tubing joining the beaker containing ACSF and the recording chamber as short as possible (1 m).</li>
		<li>Select a brain slice from the reserve chamber and transfer it into the recording chamber using the largest opening of a glass Pasteur pipette. Cover the slice with a platinum ring to anchor it at the bottom of the recording chamber. Use a flat platinum ring (&#216; 1.5 cm) and glue parallel nylon threads (spacing &#62;2 mm) on it.</li>
	</ol>
	</li>
	<li>Adjust and optimize the optics
	<ol>
		<li>Visualize the slices using infrared (IR) video microscopy. Adjust the K&#246;hler illumination 51 and optimize differential interference contrast (DIC) optics or the oblique illumination (Dodt Gradient contrast - DGC).</li>
		<li>Check the quality of the slice. Only keep slices with smooth and even surfaces that are not too strongly contrasted and have only small, dispersed craters.</li>
		<li>Fill 2 fresh and unused patch pipettes with intracellular solution (Table 3). Use a 0.5 mL tube containing the pipette solution to fill the tip of the pipette by capillarity action. To accelerate the tip filling of pipettes without filament, apply negative pressure to the end of the pipette using tubing attached to a syringe.</li>
	</ol>
	</li>
	<li>Position the pipettes above the surface of the slice
	<ol>
		<li>Check the presence of chloride coating on the silver wires.</li>
		<li>Insert the pipettes sequentially into the pipette holders and apply positive pressure (~70 mbar) using a tubing circuit connecting the pipette holder, a three-way tap and a manometer. Lower the pipette into the bath of the recording chamber.</li>
		<li>Check that the pressure value on the manometer is constant for ~1 - 2 min. If the pressure is decreasing, check the tubing or the sealing O-rings inside the pipette holder.</li>
		<li>Place the pipette tip in the middle of the video monitor and ensure that it is not obstructed. Ensure that the pipette tip does not move when applying or releasing pressure to the pipette.</li>
		<li>Check for drift and vibrations of the pipette tip by drawing a small cross at the center of the monitor exactly at the position of the pipette tip and observe for 5 min possible movements of the tip.</li>
		<li>Apply a voltage step (5 mV) to monitor the pipette resistance on an oscilloscope. Set the holding potential of the patch-clamp amplifier to 0 mV. Cancel pipette offset potential such that the DC pipette current reads zero at the amplifier meter.</li>
		<li>Move the pipettes down to the slice and maintain them above the surface.</li>
	</ol>
	</li>
	<li>Select and patch a neuron with long dendrites
	<ol>
		<li>Within the substantia nigra select a healthy neuron with long dendrites that can be followed over a long distance in approximately the same plane (Figure 1A and 1B) using IR-Dodt Gradient Contrast (IR-DGC). Choose a smooth and homogeneous cell body. Avoid the two strongly contrasted cells at the surface of the slice (Figure 1C and 1D), because it is difficult to break in and to preserve stable recording conditions over time (stable series resistance). Select neurons that have their soma 10 - 30 &#181;m underneath the slice surface.</li>
		<li>Move the somatic electrode close to the soma (40 - 50 &#181;m away) and the dendritic electrode close to the dendrite at the same distance. Use a 2X magnification (fourfold changer) to select and patch a portion of a dendrite. Obtain on the monitor an absolute magnification of 2,100X without magnification (1X) and of 5,500X with a 2X&#160; magnification.</li>
		<li>Slightly release the pressure of the somatic pipette by 10 mbar. If necessary, regulate dendritic and somatic pipette pressure to avoid displacement of the cell structure (soma and dendrite) within the slice.</li>
		<li>Position the dendritic pipette close to the membrane in order to see a small dimpling. Release the pressure on the pipette tip and patch the dendrite while controlling the pipette resistance. In ideal conditions, only a very slight suction is sufficient to obtain a good seal.</li>
		<li>Reduce the amplitude of the pipette capacitance transients as much as possible on top of the current step using a voltage pulse with high gain and little filtering 51 and the oscilloscope. Enter the whole-cell mode with the dendritic pipette.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Double-distillated water</td>
			<td>Millipore</td>
			<td>Super Q</td>
			<td>Resistivity &#62;14 M&#937; cm, ideal 18.2 M&#937; cm at 25&#176;C, filtered (0.22 &#181;m), https://www.merckmillipore.com/BE/en/product/Super-Q-Plus-Water-Purification-System,MM_NF-C1760</td>
		</tr>
		<tr>
			<td>Microfilter candles</td>
			<td>Robu</td>
			<td>Porosity 3, 6 mm or 13 mm diameter</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue slicer with vibroprobe</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>cutting parameters: speed 0.07, amplitude 1.40</td>
		</tr>
		<tr>
			<td>Tissue slicer</td>
			<td>Dosaka</td>
			<td>DTK-1000</td>
			<td>cutting parameters: speed 4, frequency 7</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Gilette</td>
			<td>Super Silver</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection tools</td>
			<td>Braun, Aesculap</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reserve chamber</td>
			<td></td>
			<td></td>
			<td>Custom-made</td>
		</tr>
		<tr>
			<td>PP beckers</td>
			<td>VWR</td>
			<td>213-1725</td>
			<td>400 ml</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Merk Millipore</td>
			<td>Millex - GV 0,22 &#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>UHU</td>
			<td>&#34;Sekunden Kleber&#34;</td>
			<td>liquid glue</td>
		</tr>
		<tr>
			<td>Recording chamber</td>
			<td>Luigs &#38; Neumann</td>
			<td>Slice mini chamber</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal pipette puller</td>
			<td>Sutter Instruments</td>
			<td>Brown-Flaming P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Hilgenberg</td>
			<td>1807524</td>
			<td>2 mm o.d./1 mm i.d. glass capilaries, ends firepolished, washed</td>
		</tr>
		<tr>
			<td>Dental wax</td>
			<td>Colt&#232;ne/Whaledent</td>
			<td></td>
			<td>orthodontic tray wax strips</td>
		</tr>
		<tr>
			<td>Platinum grid</td>
			<td></td>
			<td></td>
			<td>to anchor slices in the recording chamber, custom made with a platinum disk</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-830</td>
			<td>to fire-polish pipette tips</td>
		</tr>
		<tr>
			<td>Potassium methyl sulfate</td>
			<td>MP Biomedicals</td>
			<td>215481</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>Molecular probes</td>
			<td>B1592</td>
			<td></td>
		</tr>
		<tr>
			<td>Manometer</td>
			<td>GREISINGER electronic</td>
			<td>GDH 13 AN</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Dodt Gradient Contrast</td>
			<td>Luigs &#38; Neumann</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulators</td>
			<td>Luigs &#38; Neumann</td>
			<td>LN Mini 25</td>
			<td></td>
		</tr>
		<tr>
			<td>Frame grabber</td>
			<td>The Imaging Source</td>
			<td>DFG/USB2pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>DAGE-MTI</td>
			<td>NC-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Condenser</td>
			<td>Zeiss</td>
			<td></td>
			<td>high numerical aperture condenser working with oil</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Zeiss</td>
			<td>W Plan Apochromat 441470-9900 VIS-IR</td>
			<td>63x magnification, long-distance, high numerical aperture 1.0 water-immersion objective</td>
		</tr>
		<tr>
			<td>Fourfold-changer</td>
			<td>Luigs &#38; Neumann</td>
			<td></td>
			<td>between the camera and the microscope</td>
		</tr>
		<tr>
			<td>Black &#38; white video monitor</td>
			<td>Sony</td>
			<td>SSM-175CE</td>
			<td>16-inch, 850 TV lines, analog</td>
		</tr>
		<tr>
			<td>Sample Bottles, Amber Glass, with Cap</td>
			<td>VWR</td>
			<td>215-2409</td>
			<td>to transfer slices from the setup to histology room</td>
		</tr>
		<tr>
			<td>The Axon Guide</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Book</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>441244</td>
			<td>see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td>
		</tr>
		<tr>
			<td>Well cell culture plate</td>
			<td>Greiner-Bio-One</td>
			<td>662160</td>
			<td>for the staining of fixed slices</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merk</td>
			<td>108643</td>
			<td>see SAFETY DATA SHEET and Dangerous Substances Directive (EU) before use</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade</td>
			<td>Molecular Probes - Thermo Fisher Scientific</td>
			<td>P36961</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Engel, D. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54601">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for patch-clamp recording from the dendritic membrane surface. This helps to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels in dopaminergic neurons.

Table 1: Artificial cerebrospinal fluid (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</td>
			<td>for 1 L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.443</td>
			<td>125 mM</td>
			<td>7.305 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.007</td>
			<td>25 mM</td>
			<td>2.100 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.551</td>
			<td>2.5 mM</td>
			<td>0.186 g</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>137.99</td>
			<td>1.25 mM</td>
			<td>0.172 g</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>198.17</td>
			<td>25 mM</td>
			<td>4.95 g</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M (solution)</td>
			<td>1 mM</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 M (solution)</td>
			<td>2 mM</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity : ~ 310 mOsmol/L (optimal range: 314 &#8211; 325 mOsmol/L) , pH = 7.4</td>
		</tr>
	</tbody>
</table>
Table 2: Sucrose-ACSF to prepare slices
<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</td>
			<td>for 1 L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.443</td>
			<td>87 mM</td>
			<td>5.084 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.007</td>
			<td>25 mM</td>
			<td>2.1001 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>7.551</td>
			<td>2.5 mM</td>
			<td>0.18637 g</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>137.99</td>
			<td>1.25 mM</td>
			<td>0.17248 g</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M (solution)</td>
			<td>7 mM</td>
			<td>7 mL</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>198.17</td>
			<td>10 mM</td>
			<td>1.9817 g</td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>342.29</td>
			<td>75 mM</td>
			<td>25.672 g</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 M (solution)</td>
			<td>0.5 mM</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity : ~ 326 mOsmol/L, pH = 7.4</td>
		</tr>
	</tbody>
</table>
Table 3: Intracellular solution for dual 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</td>
			<td>for 100 mL</td>
		</tr>
		<tr>
			<td>KMeSO4</td>
			<td>150.2</td>
			<td>120 mM</td>
			<td>1.8024 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.56</td>
			<td>20 mM</td>
			<td>0.14912 g</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M (solution)</td>
			<td>2 mM</td>
			<td>200 &#181;l</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>551.1</td>
			<td>2 mM</td>
			<td>0.1102 g</td>
		</tr>
		<tr>
			<td>Na2GTP</td>
			<td>523.2</td>
			<td>0.5 mM</td>
			<td>0.02661 g</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>255.1</td>
			<td>5 mM</td>
			<td>0.1275 g</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>380.4</td>
			<td>0.1 mM</td>
			<td>3.803 mg</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>238.31</td>
			<td>10 mM</td>
			<td>0.23831 g</td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>1 mg/mL</td>
			<td></td>
			<td>0.1 g</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity : ~ 302 mOsmol/L , pH = 7.2 adjusted with KOH</td>
		</tr>
	</tbody>
</table>
Table 4: Electrode solution for cell-attached 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</td>
			<td>for 100 mL</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.56</td>
			<td>120 mM</td>
			<td>0.8947 g</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 M (solution)</td>
			<td>2 mM</td>
			<td>200 &#181;l</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M (solution)</td>
			<td>1 mM</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>238.31</td>
			<td>10 mM</td>
			<td>0.23831</td>
		</tr>
		<tr>
			<td>TEA-Cl</td>
			<td>165.7</td>
			<td>20 mM</td>
			<td>0.3314 g</td>
		</tr>
		<tr>
			<td>4-AP</td>
			<td>94.11</td>
			<td>5 mM</td>
			<td>0.04705 g</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>244.26</td>
			<td>1 mM</td>
			<td>0.02443 g</td>
		</tr>
		<tr>
			<td>CdCl2</td>
			<td>183.32</td>
			<td>0.02 mM</td>
			<td>0.3666 mg</td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>1 mM</td>
			<td>200 nM</td>
			<td>20 &#181;l</td>
		</tr>
		<tr>
			<td colspan="4">Osmolarity: ~ 290 mOsmol/L, adjusted with D-glucose (Ref. 16); pH = 7.4</td>
		</tr>
	</tbody>
</table>
<img alt="Figure 1" src="/files/ftp_upload/22705/22705_Figure1.jpg" /> 
Figure 1. Visualizing the Soma and Proximal Dendrites of Nigral Dopaminergic Neurons Using Infrared Videomicroscopy. (A) IR-DGC image of a nigral DA neuron showing the soma and proximal dendrite and both somatic and dendritic pipettes. A dual somatodendritic recording was successfully performed on this healthy neuron. Observe the low contrast and the smooth surface all over the somatodendritic domain of the cell. (B) Another example of a healthy DA neuron. (C) DA neuron with a rougher and uneven surface and a stronger contrast. While a dual recording has been obtained, the stability was suboptimal, and the recording duration was short (~ 15 min) due to a gradual increase of the access resistance at both pipettes. (D) Second example of a DA neuron showing a strongly contrasted soma and proximal dendrite. In this case, a strong increase of the access resistance was observed shortly after the break in the whole-cell mode. An attempt to decrease the access resistance by negative pressure was unsuccessful. Neurons in panels C and D might have been damaged during the slicing procedure and should be avoided for experiments. (E) Simultaneous double somatic recording in a healthy DA neuron at the beginning of the recording. (F) Same neuron as in panel E with a swollen cell body after ~17 min. Note the full absence of contrast, the ball-like appearance of the soma and the presence of the large nucleus which is not apparent in healthy neurons.

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 electrophysiology setup with an immobilized brain slice submerged in an artificial cerebrospinal fluid or aCSF containing different ions.
The setup includes a patch pipette filled with a potassium-rich solution connected to a recording electrode and an amplifier.
Apply positive air pressure to the pipette to prevent its clogging. Then, position it near a dendrite, the signal-receiving extension of a dopaminergic neuron.
The resulting membrane dimpling confirms proximity to the dendrite.
Release the positive pressure to create suction, forming a tight seal between the pipette and the dendritic membrane.
Record the initial ion movement as an action current at the baseline voltage.
Flow-in aCSF with sodium and calcium channel blockers to block the respective channels, preventing ion movement through them.
Apply a negative voltage to open a voltage-gated cation channel.
This channel allows potassium ions to enter the neuron, increasing the action current and suggesting stimulation of the dendrite in a dopaminergic neuron.

24 Views. הקלטות מהדק טלאי מהדנדריט של נוירון דופמינרגי בפרוסת מוח

Video: הקלטות מהדק טלאי מהדנדריט של נוירון דופמינרגי בפרוסת מוח - Experiment

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

הקלטת תיקון מהדק מצורף תא אחד ערוצים

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

JoVE Journal

Biology

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and &#947;-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and &#947;-aminobutyric acid receptors.

מיקרו-טרנספלנטציה של ממברנות סינפטיות להפעלה מחדש של קולטנים סינפטיים אנושיים למחקרים פונקציונליים

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

יישום המדידה Electrophysiology ללמוד את הפעילות של מובילי אלקטרו-נייטרלי

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice

Transcript

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice