Name: Auteur in de schijnwerpers: Voortschrijdende ruggenmergstimulatie - Onderzoek naar de cellulaire reacties van motorneuronen door middel van patch-clamp elektrofysiologie
Uploaded: 2023-09-08T14:15:00
Description: Watch this Scientific Journal Video about Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology at JoVE.com

Deze studie werd gefinancierd door de National Natural Science Foundation of China for Young Scholars (52207254 en 82301657) en het China Postdoctoral Science Fund (2022M711833).

De Institutional Animal Care and Use Committee keurde alle dierproeven goed en de onderzoeken werden uitgevoerd in overeenstemming met de relevante dierenwelzijnsvoorschriften.
1. Bereiding van dieren
<ol><li>Dieren<ol><li>Huisvestingsinformatie: Huisvest mannelijke Sprague-Dawley-ratten (postnataal 10-14 dagen, P10-P14) in een specifieke pathogeenvrije omgeving. OPMERKING: De kamercondities werden gehandhaafd op 20 °C ± 2 °C, vochtigheid: 50%-60%, met een licht/donker...

Inzicht in de reacties van motorneuronen op stimulatie van het ruggenmerg

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na(V)1.1+is+essential+for+proprioceptive+signaling+and+motor+behaviors.">Na(V)1.1 is essential for proprioceptive signaling and motor behaviors.</a> Elife. 11, e79917 (2022).</li><li>Romer, S. H., Deardorff, A. S., Fyffe, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+rheostat:+Kv2.1+currents+maintain+or+suppress+repetitive+firing+in+motoneurons.">A molecular rheostat: Kv2.1 currents maintain or suppress repetitive firing in motoneurons.</a> The Journal of Physiology. 597 (14), 3769-3786 (2019).</li><li>Yao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+the+R-type+human+Ca(v)2.3+channel+reveal+conformational+crosstalk+of+the+intracellular+segments.">Structures of the R-type human Ca(v)2.3 channel reveal conformational crosstalk of the intracellular segments.</a> Nature Communications. 13 (1), 7358 (2022).</li><li>Bandres, M. F., Gomes, J., McPherson, J. 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De bewegingsinformatie die door SCS wordt gemoduleerd, wordt uiteindelijk geconvergeerd naar de motorneuronen. Daarom kan het nemen van de motorneuronen als onderzoeksdoel de onderzoeksopzet vereenvoudigen en het neuromodulatiemechanisme van SCS directer onthullen. Om tegelijkertijd verschillende stimuluskenmerken en cellulaire reacties vast te leggen, is een patch-clamp een goede methode om de elektrofysiologische kenmerken op eencellige schaal te bestuderen. Er zijn echter nog steeds enkele moeilijkheden, waaronder hoe...

Ruggenmergstimulatie (SCS) kan de bewegingsfunctie effectief herstellen na een dwarslaesie (SCI). Andreas Rowald et al. meldden dat SCS de bewegings- en rompfunctie van de onderste ledematen binnen één dag mogelijkmaakt1. Het onderzoeken van het biologische mechanisme van SCS voor locomotorisch herstel is een cruciaal en trending onderzoeksgebied voor het ontwikkelen van een preciezere SCS-strategie. Het team van Grégoire Courtine toonde bijvoorbeeld aan dat excitatoire Vsx2-interneuron en Hoxa10-neuronen in het ruggenmerg de belangrijkste neuronen zijn voor de respons op SCS, en dat celspecifieke neuromodulatie haalbaar is om het loopvermogen van ratten na SCI2 te herstellen. Er zijn echter maar weinig studies die zich richten op het elektrische mechanisme van SCS op eencellige schaal. Hoewel het algemeen bekend is dat de bovendrempelige gelijkstroomstimulus de actiepotentialen (AP's) in het klassieke inktvisexperiment 3,4,5 kan opwekken, is het nog onduidelijk hoe de gepulseerde wisselende elektrische stimulatie, zoals SCS, de motorsignaalgeneratie beïnvloedt.
Gezien de complexiteit van intraspinale neurale circuits, is een geschikte selectie voor celpopulatie belangrijk voor het onderzoeken van het elektrische mechanisme van SCS. Hoewel SCS de motorische functie herstelt door de proprioceptieve route6 te activeren, zijn de motorneuronen de laatste eenheid om het motorcommando uit te voeren, afgeleid van de integratie van proprioceptie-informatie afferente input7. Daarom kan het rechtstreeks bestuderen van de elektrische kenmerken van motorneuronen met SCS ons helpen de onderliggende logica van spinale motormodulatie te begrijpen.
Zoals we weten, is patch-clamp de gouden standaardmethode voor cellulaire elektrofysiologische registratie met extreem hoge spatiotemporele resolutie8. Daarom beschrijft deze studie een methode met behulp van een patchklem om de elektrische reacties van motorneuronen op SCS te bestuderen. Vergeleken met de hersenpleisterklem9 is de ruggenmergklem moeilijker om de volgende redenen: (1) Het ruggenmerg wordt beschermd door het wervelkanaal met een klein volume, wat zeer fijne micromanipulatie en rigoureus ijskoud onderhoud vereist om een betere cellevensvatbaarheid te verkrijgen. (2) Omdat het ruggenmerg te slank is om op de snijplaat te worden vastgezet, moet het worden ondergedompeld in agarose met een laag smeltpunt en na stolling worden bijgesneden.
Daarom biedt deze methode technische details bij het ontleden van het ruggenmerg en het tegelijkertijd handhaven van de levensvatbaarheid van de cel, om het elektrische mechanisme van SCS op motorneuronen soepel te bestuderen en onnodige vallen en opstaan te voorkomen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline Chloride</td>
			<td>Sigma</td>
			<td>C7527</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide tweezers</td>
			<td>VETUS</td>
			<td>36A-SA</td>
			<td>Clip a slice</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>RWD Life Science</td>
			<td>S12006-10</td>
			<td>Cut the diaphragm</td>
		</tr>
		<tr>
			<td>Fluorescence Light Source</td>
			<td>Olympus&#160;</td>
			<td>U-HGLGPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-Gold</td>
			<td>Fluorochrome</td>
			<td>Fluorochrome</td>
			<td>Label the motor neuron</td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate sodium salt hydrate</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>infrared CCD camera</td>
			<td>Dage-MTI</td>
			<td>IR-1000E</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>P1847</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Sigma</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>Sigma</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator&#160;</td>
			<td>Sutter Instrument&#160;</td>
			<td>MP-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter instrument</td>
			<td>P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-scissors&#160;</td>
			<td>Jinzhong</td>
			<td>wa1020</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>Microscope for anatomy</td>
			<td>Olympus&#160;</td>
			<td>SZX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope for ecletrophysiology</td>
			<td>Olympus&#160;</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-toothed tweezers</td>
			<td>RWD Life Science</td>
			<td>F11008-09</td>
			<td>Lift the cut vertebral body</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>V900182</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Phosphocreatine</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens for ecletrophysiology</td>
			<td>Olympus&#160;</td>
			<td>LUMPLFLN60XW</td>
			<td>working distance 2 mm&#160;</td>
		</tr>
		<tr>
			<td>Osmometer&#160;</td>
			<td>Advanced&#160;</td>
			<td>FISKE 210</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp amplifier&#160;</td>
			<td>Axon&#160;</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp digitizer</td>
			<td>Axon&#160;</td>
			<td>Digidata 1550B</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter&#160;</td>
			<td>Mettler Toledo&#160;</td>
			<td>FE28</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice Anchor</td>
			<td>Multichannel system</td>
			<td>SHD-27H</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinal cord stimulatior</td>
			<td>PINS</td>
			<td>T901</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed tweezer</td>
			<td>RWD Life Science</td>
			<td>F13030-10</td>
			<td>Lift the xiphoid</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide band ultraviolet excitation filter</td>
			<td>Olympus&#160;</td>
			<td>U-MF2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo preparation of spinal cord slice for the whole-cell patch-clamp recording in motor neurons during spinal cord stimulation

Ruggenmergstimulatie (SCS) kan de bewegingsfunctie effectief herstellen na een dwarslaesie (SCI). Omdat de motorneuronen de laatste eenheid zijn om sensomotorisch gedrag uit te voeren, kan het direct bestuderen van de elektrische reacties van motorneuronen met SCS ons helpen de onderliggende logica van spinale motorische modulatie te begrijpen. Om tegelijkertijd verschillende stimuluskenmerken en cellulaire reacties vast te leggen, is een patch-clamp een goede methode om de elektrofysiologische kenmerken op eencellige schaal te bestuderen. Er zijn echter nog steeds enkele complexe moeilijkheden om dit doel te bereiken, waaronder het handhaven van de levensvatbaarheid van de cel, het snel scheiden van het ruggenmerg van de benige structuur en het gebruik van de SCS om met succes actiepotentialen te induceren. Hier presenteren we een gedetailleerd protocol met behulp van patch-clamp om de elektrische reacties van motorneuronen op SCS met een hoge spatiotemporele resolutie te bestuderen, wat onderzoekers kan helpen hun vaardigheden te verbeteren bij het scheiden van het ruggenmerg en tegelijkertijd de levensvatbaarheid van de cel te behouden om het elektrische mechanisme van SCS op motorneuron soepel te bestuderen en onnodig vallen en opstaan te voorkomen.

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Andreas Rowald et al. reported that SCS enables lower limb locomotor and trunk function within a single day1. Exploring the biological mechanism of SCS for locomotor recovery is a critical and trending research field for developing a more precise SCS strategy. For example, Gr&#233;goire Courtine&#39;s team demonstrated that excitatory Vsx2 interneuron and Hoxa10 neurons in the spinal cord are the key neurons to response to SCS, and cell-specific neuromodulation is feasible to restore the rat walking ability after SCI2. However, few studies focus on the electrical mechanism of SCS at a single-cell scale. Although it is well-known that the suprathreshold direct current stimulus can elicit the action potentials (APs) in the classic squid experiment3,4,5, how the pulsed alternating electrical stimulation, such as SCS, affects the motor signal generation is still unclear.
Given the complexity of intraspinal neural circuits, appropriate selection for cell population is important for investigating the electrical mechanism of SCS. Although SCS restores motor function by activating the proprioceptive pathway6, the motor neurons are the final unit to execute the motor command, derived from integrating proprioception information afferent input7. Therefore, directly studying the electrical characteristics of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation.
As we know, patch-clamp is the golden-standard method for cellularly electrophysiological recording with extremely high spatiotemporal resolution8. Therefore, this study describes a method using a patch clamp to study the electrical responses of motor neurons to SCS. Compared with the brain patch-clamp9, the spinal cord patch-clamp is more difficult due to the following reasons: (1) The spinal cord is protected by the vertebral canal with tiny volume, which requires very fine micromanipulation and rigorous ice-cold maintenance to obtain better cell viability. (2) Because the spinal cord is too slender to be secured on the cutting tray, it should be immersed in low-melting point agarose and trimmed after solidification.
Hence, this method provides technical details in dissecting the spinal cord and maintaining the cell viability at the same time so as to smoothly study the electrical mechanism of SCS on motor neurons and avoid unnecessary trials and mistakes.

Auteur in de schijnwerpers: Voortschrijdende ruggenmergstimulatie - Onderzoek naar de cellulaire reacties van motorneuronen door middel van patch-clamp elektrofysiologie 

De ex vivo bereiding van een doorsnede van het ruggenmerg voor de opname van de patchklem in motorneuronen met hele cellen tijdens stimulatie van het ruggenmerg

Spinal cord stimulation can help patients resolve most motion functions after injury. Most neurons are to execute sensory mode behaviors. Studying the electric response of most neurons can aid in understanding the underlying logic of spinal load neuromodulation.A patch clamp is the golden standard method for cellular electrophysiological recordings with the extremely high spatial temporal resolution. Therefore, this study describes a method using a patch clamp to simultaneously record the aortas stimulus characteristics and cellular responses of motor neurons at a single-cell scale. Compared with the brain patch clamp, the spinal cord patch clamp is more difficult, because the spinal cord is well protected by the vertebral canal with tiny volume.Therefore, this protocol provides technical details in quickly micro dissecting the spinal cord and reverse maintenance to obtain better cell viability. Patch clamp allows a unique understanding of the synaptic transmissions and the understanding of action potentials, coding pattern activities by the spinal cord stimulation.

Dankzij het rigoureuze onderhoud bij lage temperaturen tijdens de fijne werking (aanvullende figuur 1, aanvullende figuur 2 en figuur 1) was de levensvatbaarheid van de cel goed genoeg om latere elektrofysiologische opnames uit te voeren. Om het klinische scenario zoveel mogelijk te simuleren, gebruikten we micromanipulatie om de SCS-kathode en anode respectievelijk in de buurt van de dorsale middellijn en DREZ te plaatsen (Figuur 2), wat een ne...

Watch this Scientific Journal Video about Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology at JoVE.com

Dit protocol beschrijft een methode met behulp van een patch-clamp om de elektrische reacties van motorneuronen op ruggenmergstimulatie (SCS) met een hoge spatiotemporele resolutie te bestuderen, wat onderzoekers kan helpen hun vaardigheden te verbeteren bij het scheiden van het ruggenmerg en het tegelijkertijd handhaven van de levensvatbaarheid van de cel.

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

Ruggenmergstimulatie kan patiënten helpen de meeste bewegingsfuncties na een blessure op te lossen. De meeste neuronen zijn bedoeld om sensorisch gedrag uit te voeren. Het bestuderen van de elektrische respons van de meeste neuronen kan helpen bij het begrijpen van de onderliggende logica van neuromodulatie van de wervelkolom.Een patchklem is de gouden standaardmethode voor cellulaire elektrofysiologische opnames met de extreem hoge ruimtelijke temporele resolutie. Daarom beschrijft deze studie een methode met behulp van een patchklem om tegelijkertijd de aorta-stimuluskenmerken en cellulaire reacties van motorneuronen op eencellige schaal vast te leggen. In vergelijking met de klem voor de hersenpleister is de klem voor de pleister van het ruggenmerg moeilijker, omdat het ruggenmerg goed wordt beschermd door het wervelkanaal met een klein volume.Daarom biedt dit protocol technische details voor het snel microontleden van het ruggenmerg en omgekeerd onderhoud om een betere cellevensvatbaarheid te verkrijgen. Patchklem maakt een uniek begrip mogelijk van de synaptische transmissies en het begrip van actiepotentialen, waarbij patroonactiviteiten door de ruggenmergstimulatie worden gecodeerd.

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Research

JoVE Journal

Neuroscience

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.

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.

Spinal Cord Dissection for the Whole-Cell Patch-Clamp Recording in Motor Neurons

After exposing the heart of an anesthetized rat, cut the right atrium using fine scissors and inject 100 milliliters of chilled perfusing fluid at approximately two milliliters per second within one minute. Position the rat in the prone position. Cut the spine at the anterior superior iliac spine and the curvature shifting point of the thoracic column.Immediately place the isolated spine into the oxygenated ice cold perfusing solution to wash off residual blood and fat tissue. Then transfer the isolated spine to the anatomical tray with its dorsal side up and the rostral end near the operator. Fill the tray with 50 milliliters of continuously oxygenated ice cold cutting solution.Under a dissection microscope, cut the vertebral pedicles on both sides from the rostral end using micro scissors. Next, delicately sever the dura mater along its dorsal midline with micro scissors. Lift the rostral part of the spinal cord carefully and cut the nerve root, leaving about one millimeter reserved.After the spinal cord has been separated from the vertebral canal, secure it with the ventral side facing up using two insect pins. Trim any excessive nerve roots, leaving about one millimeter intact. Then separate the lumbar enlargement to a length of six to seven millimeters using a micro scissor.

Ruggenmergdissectie voor de opname van de patch-clamp van de hele cel in motorneuronen

Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons

After dissecting the spinal cord and separating the lumbar enlargement, place the lumbar enlargement on a 35 degree slope with the dorsal side up and the caudal side down. Absorb any excess water on the tissue surface using an absorbent filter paper. Carefully pour the molten agarose gel into the Petri dish containing the lumbar enlargement.Position the Petri dish in the ice water mixture to ensure rapid gel cooling. Shape the gel into a cube of 15 by 10 by 10 millimeters, and mount it onto the specimen disc using super glue. Adjust the vibratome settings to a thickness of 350 micrometers, a speed of 0.14 to 0.16 millimeters per second, an amplitude of one millimeter, and a vibration frequency of 85 hertz.Now, use the cover slide tweezers to clip a slice and place it into the incubation chamber, filled with continuously oxygenated artificial cerebrospinal fluid.

Voorbereiding van een doorsnede van het ruggenmerg voor de opname van de patchklem in motorneuronen met hele cellen

Patch-Clamp Recording to Study the Electrical Responses of Motor Neurons to Spinal Cord Stimulation

To begin, place the prepared spinal cord slice in the recording chamber. Then place the anode of the micro-manipulation system near the dorsal midline in the cathode close to the dorsal root entry zone for proper spinal cord stimulator positioning. Now switch to a 60X objective lens to identify a healthy neuron having a smooth and bright surface without visible nuclei.Slightly decrease the infrared intensity, turn on the fluorescent light source, and adjust the light filter to the wideband ultraviolet excitation option. Now, select an appropriate Fluoro-Gold-positive motor neuron and gradually lower the electrode until it is near the neuron. As the pipette contacts its surface, look for a small membrane indentation at the tip level and release the previously-applied positive pressure.Then apply negative pressure to the pipette using a syringe and wait until the resistance value rises to gigaohms on the software interface. Clamp the membrane potential at minus 70 millivolts. Press the fast capacitance compensation button on the amplifier's software interface, then apply a brief negative pressure to rupture the cell membrane and press the slow capacitance compensation button on the amplifier's software interface.Apply the spinal cord stimulation for one to two seconds keeping the amplitude between one and 10 milliamperes. Determine the motor threshold by gradually increasing the stimulation amplitude until the first action potential is observed. Distinguish delayed and immediate firing motor neurons using a five-second depolarizing current injection around rheobase in the current clamp mode.After turning off the spinal cord stimulation, continue recording the membrane potentials to capture the spontaneous action potentials firing. When the amplitude of spinal cord stimulation was raised, the membrane potential was increased, and the action potentials were triggered every 10 to 20 pulses. After the spinal cord stimulation was turned off, the neuron fired a series of spontaneous action potentials for a short period of time.Then, the resting membrane potential returned to minus 65 millivolts.