Name: a microbiomechanical system for studying varicosity formation and recovery in central neuron axons
Uploaded: 2018-04-30T13:00:00
Description: Watch this Scientific Journal Video about A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons at JoVE.com

Alla djurförsök har utförts i enlighet med de nationella institut för hälsa djur Använd riktlinjerna. Detta arbete stöds delvis av anslag från National Institutes of Health (R01NS093073 och R21AA024873) till C. Gu.

Alla metoder som beskrivs nedan har godkänts av den institutionella djur vård och användning kommittén (IACUC) av Ohio State University.
1. täckglas förberedelse
<ol><li>Placera en eller flera lådor med 12 mm eller 25 mm coverslips i en glasbägare innehållande 70% salpetersyra och inkubera i coverslips i rumstemperatur över natten. Obs: Tvätta inte den 25 mm coverslips i samma bägaren som den 12 mm coverslips. Det är bättre att tvätta dem separat.</li>
	<li>Överför alla c...

<ol>
	<li>Niki&#263;, I., 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=A+reversible+form+of+axon+damage+in+experimental+autoimmune+encephalomyelitis+and+multiple+sclerosis.">A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis.</a> Nat. Med. 17, 495-499 (2011).</li><li>Yang, J., 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=Regulation+of+axon+degeneration+after+injury+and+in+development+by+the+endogenous+calpain+inhibitor+calpastatin.">Regulation of axon degeneration after injury and in development by the endogenous calpain inhibitor calpastatin.</a> Neuron. 80 (5), 1175-1189 (2013).</li><li>Debanne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+processing+in+the+axon.">Information processing in the axon.</a> Nat. Rev. Neurosci. 5, 304-316 (2004).</li><li>Gu, Y., Jukkola, P., Wang, Q., Esparza, T., Zhao, Y., Brody, D., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+varicosity+initiation+in+central+neuron+mechanosensation.">Polarity of varicosity initiation in central neuron mechanosensation.</a> J Cell Biol. 216 (7), 2179-2199 (2017).</li><li>Chung, R. S., 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=Mild+axonal+stretch+injury+in+vitro+induces+a+progressive+series+of+neurofilament+alterations+ultimately+leading+to+delayed+axotomy.">Mild axonal stretch injury in vitro induces a progressive series of neurofilament alterations ultimately leading to delayed axotomy.</a> J. Neurotrauma. 22 (10), 1081-1091 (2005).</li><li>Staal, J. A., Dickson, T. C., Chung, R. S., Vickers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin-A+treatment+attenuates+delayed+cytoskeletal+alterations+and+secondary+axotomy+following+mild+axonal+stretch+injury.">Cyclosporin-A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury.</a> Dev. Neurobiol. 67 (14), 1831-1842 (2007).</li><li>Tang-Schomer, M. D., Johnson, V. E., Baas, P. W., Stewart, W., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+interruption+of+axonal+transport+due+to+microtubule+breakage+accounts+for+the+formation+of+periodic+varicosities+after+traumatic+axonal+injury.">Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury.</a> Exp. Neurol. 233 (1), 364-372 (2012).</li><li>Donkin, J. J., Vink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cerebral+edema+in+traumatic+brain+injury:+therapeutic+developments.">Mechanisms of cerebral edema in traumatic brain injury: therapeutic developments.</a> Curr Opin Neurol. 23 (3), 293-299 (2010).</li><li>Gardner, A., Jukkola, P., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+of+rodent+hippocampal+neurons+in+culture.">Myelination of rodent hippocampal neurons in culture.</a> Nat Protoc. 7 (10), 1774-1782 (2012).</li><li>Wang, Q., Zhang, X., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+stimulator+for+localized+cell+loading:+fabrication+and+strain+analysis.">Micromechanical stimulator for localized cell loading: fabrication and strain analysis.</a> J. Micromech. Microeng. 23, 015002 (2013).</li><li>Lu, Y. B., 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=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc. Natl. Acad. Sci. USA. 103 (47), 17759-17764 (2006).</li><li>Elkin, B. S., Azeloglu, E. U., Costa, K. D., Morrison, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+heterogeneity+of+the+rat+hippocampus+measured+by+atomic+force+microscope+indentation.">Mechanical heterogeneity of the rat hippocampus measured by atomic force microscope indentation.</a> J. Neurotrauma. 24 (5), 812-822 (2007).</li><li>Franze, K., 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=Neurite+branch+retraction+is+caused+by+a+threshold-dependent+mechanical+impact.">Neurite branch retraction is caused by a threshold-dependent mechanical impact.</a> Biophys. J. 97 (7), 1883-1890 (2009).</li><li>Camp&#224;s, O., 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=Quantifying+cell-generated+mechanical+forces+within+living+embryonic+tissues.">Quantifying cell-generated mechanical forces within living embryonic tissues.</a> Nat. Methods. 11 (2), 183-189 (2014).</li></ol>

Förfarandet i denna microbiomechanical analys är rakt fram. Det kommer att producera tillförlitliga resultat, om alla dess steg utförs noggrant. I området i närheten finns det flera viktiga steg som, om felaktigt utfört, hindrar framgångsrik datainsamling. De kritiska steg börja uppströms i den faktiska tillämpningen av pustande stimulans. Noggrann dissektion, odling och vård av den primära neuron kulturen är avgörande. Om de odlade nervcellerna inte är friska, reagerar de inte konsekvent, eftersom de sku...

Praparat bildandet eller svullnad/beading, längs axoner, är ett framträdande inslag i neurodegeneration observerats i många sjukdomar eller skador i centrala nervsystemet, inklusive multipel skleros, Alzheimers sjukdom, Parkinsons sjukdom, och traumatiska hjärnan skada1,2. Trots de betydande fysiologiska konsekvenserna av axonal varicosities på aktionspotentialens spridning och synaptisk transmission3fortfarande hur varicosities genereras okänd. Nyligen hittade med en nyinrättade microbiomechanical analys på odlade Hippocampus nervceller från gnagare, vi att mekaniska stimuli kan orsaka varicosities i dessa nervceller med mycket spännande funktioner. Först Praparat induktion är snabb (< 10 s) och denna process är oväntat reversibel. Det andra Praparat inledande beror på styrkan pustande tryck: ju högre tryck, desto snabbare inledandet. Det tredje beror Praparat inledande på neuronal ålder. Axoner av yngre nervceller visas mer lyhörda mot mekanisk stress, jämfört med de äldre nervceller. Fjärde, varicosities form längs axoner av hippocampus nervceller, medan dendriter och inledande axon segment av dessa nervceller visar ingen förändring på samma tuffande villkor. Vår studie visade således ett nytt inslag av neuronala polaritet. Dessa fynd med in vitro- system är fysiologiskt relevanta. Använda en in-vivo -modell för mild traumatisk hjärnskada (mTBI), visade vi att axonal varicosities utvecklas i en multi fokal mode i somatosensoriska cortex av mössen omedelbart efter close-skalle effekt, överensstämmer med vår in vitro- resultat4. Det är viktigt att notera att våra färgning och bildbehandling av mTBI möss ger bara ett snap shot av neuronala morfologiska förändringar, sedan utför i vivo time-lapse avbildning av neuronala morfologi under en mekanisk påverkan är ännu inte är möjligt.
Denna vätska-pustande system tillät oss inte bara att fånga unika funktioner i samband med mekanisk stressinducerade Praparat bildandet, men också att fastställa bakomliggande mekanism. Genom att testa olika extracellulära lösningar, blockerare och konservöppnare olika kandidater av mechanosensitive jonkanaler och cellulär elektrofysiologi, identifierat vi att transient receptor potential cationen kanal underfamilj V medlem 4 (TRPV4) kanal som är genomsläppliga för Ca2 + och Na+ och aktiveras av pustande ansvarar huvudsakligen för att upptäcka inledande mekanisk stress under axonal Praparat bildandet4. Detta bekräftades ytterligare med siRNA knockout tillvägagångssätt. Sammantaget skapar detta nya test system har vi utvecklat med Hippocampus neuron kultur, är mycket värdefulla för att studera egenskaperna mikromekaniska av centrala nervceller, särskilt i kombination med andra tekniker.
Detta mikromekaniska system har vi etablerat är unikt och skiljer sig från de tidigare befintliga system i flera viktiga aspekter. Först i detta system uppleva nervceller out-av-plane mekanisk stress i form av komprimering och klippning. Under den mekaniska stötar, neuronala processer sitta kvar på täckglas ytan och flytta inte. Detta skiljer sig från andra experimentella system som främst handlat om böjning och i-plane stretching (eller spänning), exempelvis omläggning av medföljande axoner som flytta strängar5,6 eller stretching axoner som odlas på micropatterned kanaler och töjbart membran7,8. Dessutom även om axonal varicosities kan också induceras i dessa analyser som i våra vätska-pustande system, processen i dessa inställningar tar mycket mer tid (från 10 min till flera timmar6,7,8) och visas irreversibel. Slutligen, vårt system som använder lokala vätska pustande gör granskningen av rumsliga funktioner i Praparat bildandet (t.ex., Dendritutskotten, axonal initiala segment, soma, dendriter, axonal terminaler), förutom dess temporal funktioner. Med detta system, upptäckte vi flera oväntade och unika funktioner av axonal Praparat bildande, särskilt snabbt insättande, långsam reversibilitet och axon-dendrite polaritet.
Det system som vi diskuterar i denna uppsats är kompatibel med många tekniker av molekylära och cellbiologi. Exempelvis för att studera effekterna av mekanisk stress på neuronal morfologi och funktion, kan det användas tillsammans med myelin coculture, time-lapse avbildning av fluorescerande resonans energiöverföringen (bandet) och Totalreflexion fluorescens (Frida), kalcium imaging och patch clamp inspelning. I detta papper fokuserar vi på de centrala delarna av systemet. Hippocampus neuron kultur, vätska-pustande setup, högupplösta time-lapse imaging för axonal transport och kalcium imaging är illustrerade steg för steg nedan.

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			<td height="40" style="height:40px;width:341px;">12 mm coverslips&#160;</td>
			<td style="width:139px;">Warner Instruments&#160;</td>
			<td style="width:119px;">64-0702</td>
			<td style="width:167px;">for 24-well plate&#160;</td>
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			<td height="40" style="height:40px;">25 mm coverslips&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-545-102&#160;</td>
			<td>for 6-well plate&#160;</td>
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			<td height="40" style="height:40px;">Acetic acid</td>
			<td>Fisher Scientific&#160;</td>
			<td>A38-212</td>
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			<td>Fisher Scientific&#160;</td>
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			<td height="40" style="height:40px;">HEPES&#160;</td>
			<td>Fisher Scientific&#160;</td>
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			<td>Fisher Scientific&#160;</td>
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			<td>Gibco&#160;</td>
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			<td height="40" style="height:40px;">MEM Earle&#39;s Salts&#160;</td>
			<td>Gibco&#160;</td>
			<td>11090</td>
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			<td height="40" style="height:40px;">B27 supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504-044</td>
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			<td height="40" style="height:40px;">Neurobasal&#160;</td>
			<td>Gibco&#160;</td>
			<td>21103-049</td>
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			<td height="21" style="height:21px;">Arabinosylcytosine (Ara-C)</td>
			<td>Sigma&#160;</td>
			<td>147-94-4</td>
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			<td height="21" style="height:21px;">Opti-MEM media&#160;</td>
			<td>Gibco&#160;</td>
			<td>31985-070</td>
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			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Invitrogen&#160;</td>
			<td>1854313</td>
			<td>transfection reagent&#160;</td>
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			<td height="21" style="height:21px;">Borosilicate rods&#160;</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td>PG52151-4</td>
			<td>for puffing pipette&#160;</td>
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			<td height="21" style="height:21px;">rubber tubing&#160;</td>
			<td style="width:139px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">14-169-1A</td>
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			<td height="21" style="height:21px;">10cc plastic syringe and plunger</td>
			<td>Becton Dickinson&#160;</td>
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			<td height="21" style="height:21px;">micromanipulator&#160;</td>
			<td>Sutter Instruments&#160;</td>
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			<td height="21" style="height:21px;">NaCl&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>S640-3</td>
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			<td>Fisher Scientific&#160;</td>
			<td>BP366-500</td>
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			<td align="left" height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific&#160;</td>
			<td>C70-500</td>
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			<td height="19" style="height:19px;width:341px;">Cell culture dish (35 mm x 10 mm)</td>
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			<td height="21" style="height:21px;width:341px;">Fluo-4 AM</td>
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			<td style="width:119px;">F14201</td>
			<td style="width:167px;">for calcium imaging&#160;</td>
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			<td height="21" style="height:21px;width:341px;">Mito-YFP construct&#160;</td>
			<td style="width:139px;">Takara Bio Inc.&#160;</td>
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			<td style="width:167px;">for cell transfection&#160;</td>
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			<td height="21" style="height:21px;width:341px;">YFP-N1 construct&#160;</td>
			<td style="width:139px;">Takara Bio Inc.&#160;</td>
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			<td style="width:167px;">for cell transfection&#160;</td>
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			<td height="21" style="height:21px;width:341px;">Model P-1000 Flaming/Brown Micropipette puller&#160;</td>
			<td style="width:139px;">Sutter Instruments&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Eclipse TE2000-U Mcroscope&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:341px;">Plan Fluor ELWD 20x lens&#160;&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">062933</td>
			<td style="width:167px;">objective&#160;</td>
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			<td height="21" style="height:21px;width:341px;">Apo TIRF 100x/1.49 oil lens&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">MRD01991</td>
			<td style="width:167px;">objective&#160;</td>
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	</tbody>
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a microbiomechanical system for studying varicosity formation and recovery in central neuron axons

Axonal varicosities är utvidgade strukturer längs axlarna av axoner med en hög grad av heterogenitet. De finns i hjärnor med neurodegenerativa sjukdomar eller skador, men också i den normala hjärnan. Här beskriver vi ett nyinrättade mikromekaniska system för att snabbt, tillförlitligt och reversibelt inducera axonal varicosities, tillåter oss att förstå de mekanismer som styr Praparat bildandet och heterogena protein sammansättning. Detta system utgör en roman innebär att utvärdera effekterna av komprimering och shear stress på olika subcellulär fack av nervceller, skiljer sig från andra in vitro- system som främst fokuserar på effekten av stretching. Allt på grund av de unika funktionerna i vårt system gjort vi nyligen en roman upptäckten visar att tillämpningen av trycksatta vätskan kan snabbt och reversibelt inducera axonal varicosities genom aktivering av en övergående receptor potentiell kanal. Vårt biomekaniska system kan utnyttjas praktiskt i kombination med drogen perfusion, levande cell imaging, kalcium imaging och patch clamp inspelning. Denna metod kan därför antas för att studera mechanosensitive jonkanaler, axonal transport förordning, axonal cytoskelettet dynamics, calcium signalering och morfologiska förändringar relaterade till traumatisk hjärnskada.

Innan pustande, Visa axoner normalt lite Praparat bildandet. Följande pustande med vårt standardtryck (190 mmH2O höjd), axoner början att utveckla många pärla-liknande varicosities. Bildandet av varicosities är delvis reversibla, som visas av regioner av axon återvänder till sin förväg svälld tillstånd efter en 10 min återhämtningsperiod (figur 2A-B). Efter en längre period av återhämtning (> 20 min), några axone...

Watch this Scientific Journal Video about A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons at JoVE.com

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Det här protokollet beskriver en fysiologiskt relevanta, trycksatt vätske strategi för snabb och vändbar induktion av varicosities i nervceller.

Ett Microbiomechanical System för att studera Praparat bildandet och återhämtning i centrala Neuron axoner

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

The overall goal of this pressurized fluid setup is to produce repeatable and consistent swellings along the axons of cultured neurons. This method can help answer key questions in the brain injury field such as the mechanisms behind subcellular morphological changes in neurons following injury. The main advantage of this technique is that the method produces consistent and repeatable morphological changes that recover over time.This is very important technique we developed in the lab. This is because we can not only use the system to identify molecular mechanism governing mechanical-stress-induced axon morphology change as a new form of neuronal plasticity, but also use the system as a screening tool to identify new drugs and the treatment strategy for mild or traumatic brain injury. To begin this procedure, place one or more boxes of 12-millimeter or 25-millimeter coverslips into a glass beaker containing 70%nitric acid, and incubate the coverslips at room temperature overnight.The next day, transfer all coverslips into a four-liter beaker filled with 2.5 liters of double-distilled water. Shake the beaker containing the coverslips for one hour at 100 RPM, and don't forget to use rubber bands to hold the beaker during shaking. Subsequently, transfer the cleaned coverslips to a dry flask with the metal cover.Bake the coverslips in an oven at 225 degrees Celsius for six hours. Then, allow them to cool to room temperature and store them in a sterilized plastic container until usage. Now, place one coverslip into each well of a 24-well plate.Add 30 microliters of coverslip coating solution onto a 12-millimeter coverslip and swirl. Afterward, add one milliliter of sterile PBS to each well. The plate can be either used immediately or stored at four degrees Celsius in sterile PBS for up to one week.On the day of dissection, remove PBS from the plate. Air dry and place the plate under UV light for two to four hours. To dissociate the hippocampal neurons for culture, thaw the protease enzyme solution and pre-warm the plating medium at 37 degrees Celsius.Next, rinse the dissected hippocampus with SLDS. Then, remove it and add two milliliters of protease enzyme solution and incubate the sample at 37 degrees Celsius for 15 minutes. Afterward, wash the hippocampus explants with five to 10 milliliters of plating medium twice and add five milliliters of plating medium.Dissociate the hippocampal explants into single cells by generating a small vortex using a pipette with a one-milliliter plastic tip. Pipette up and down about 40 times avoiding the formation of oxygen bubbles until the solution becomes cloudy and no large pieces of explant remain. Next, centrifuge the cells at 1, 125 times gravity for three minutes.Remove the supernatant with the cells submerged in medium and re-suspend the cells in 145 milliliters of plating medium. Then, add one milliliter of the cell suspension per well to a 24-well plate. Incubate the cells in the plating medium at 37 degrees Celsius for two to four hours.Afterward, remove the plating medium and replace it with fresh maintenance medium. After two days, replace half of the medium with two micromolar RSC dissolved in maintenance medium to inhibit growth of fibroblast, endothelial and glial cells. Then, after exposing the cells to RSC for two days, replace the medium with fresh maintenance medium.To transfect for the visualization of cell morphology, dilute the construct from stock at one microgram per microliter in Opti-MEM medium and vortex. Then, dilute liposome-mediated transfection reagent in Opti-MEM media and vortex. Subsequently, prepare one construction dilution and one transfection dilution for each well.Next, prepare a one-to-one mixture of the construct and Opti-MEM medium and transfection reagent and Opti-MEM medium and vortex. Incubate the mixture at room temperature for 20 minutes. After 20 minutes, remove half of the medium from each well and transfer this medium into a clean conical tube.Keep the tube in the incubator, then add 100 microliters of the mixture prepared in the previous step to the remaining medium in the well. Allow the cells to incubate at 37 degrees Celsius for 20 to 30 minutes. During incubation, add one volume of fresh maintenance medium to the medium in the conical tube and place the mixture in the same incubator.Following incubation, remove the mediums containing the transfection solution from the wells and replace it with the one-to-one mixture of old and fresh maintenance medium in the conical tube. In this procedure, insert the pipette into the screw top of the micromanipulator to hold it into place. Screw the metal flat-top screw attached to the micromanipulator arm so that it holds the unpulled end of the pipette just above where the rubber tubing is attached.Angle the pipette at a 45 degree angle with the surface of the neuron containing coverslips. Turn on the florescence filter. Open the aperture, and ensure a fluorescent spot can be seen coming through the objective.Using the micromanipulator, move the pipette into a position that lines up the tip with the fluorescent spot and lower the pipette into a position just above the height of the cell culture dish. Once it is in position, turn on the transmitted light and turn off the florescence. Using tweezers, transfer one coverslip with the cells facing toward the pipette from the cell culture plate to the cell culture dish containing 2 milliliters of Hank's buffer at room temperature.Using the fine focus knob and 20 fold objective, focus on a plane about four full turns above the cells. Subsequently, use the micromanipulator to position the pipette tip so that it is focused and in the center left-hand side of the plane as seen through the eyepieces. Once the pipette is in position, focus the microscope on a plane that contains the region of interest.In the imaging of seven div, GFP transfected mouse hippocampal neurons showed an axon pre and post puffing, as well as a following 10 minute recovery period. Here is the Kymograph of time-lapse imaging of the same neuron. Red arrows indicate the varicosities that have formed to following 150 seconds of 190 milliliter H2O puffing, beginning at 30 seconds.Once mastered, this technique can be done in three hours if it is performed properly. Following this procedure, other methods like whole cell recording, time-lapse FRET and TIRF imaging, and calcium imaging can be performed in order to answer additional questions like the impacts of mechanical stress on neural and electrical and calcium signaling, protein trafficking and interactions. After its development, this technique paved the way for researchers in the photo traumatic brain injury to explain molecular mechanisms underlying axonal varicosity formation in living neurons.After watching this video, you should have a good understanding of how to perform the experiments to study mechanosensations of central neurons.

Research

JoVE Journal

Neuroscience

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Systemisk och lokal Drug Delivery för behandling av sjukdomar i centrala nervsystemet i gnagarmodeller

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Synnerven transection: En modell av Vuxen Neuron apoptos i det centrala nervsystemet

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

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Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

I ovo elektroporering i Chick mitthjärnan för att studera genfunktion av dopaminerg Neuron utveckling

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

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Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

En lätt, hörlurar system för att manipulera Auditiv feedback i Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Direkt Intraventrikulär tillförsel av läkemedel till Gnagare Centrala nervsystemet

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistokemisk analys i Rat centrala nervsystemet och perifera lymfkörtelvävnadssnitt

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Laser Nanosurgery of Cerebellar Axons In Vivo

Lasernanosurgery av Cerebellar Axoner<em&gt; In Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo optogenetic Stimulering av gnagare centrala nervsystemet

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...