Name: microfluidics-assisted selective depolarization of axonal mitochondria
Uploaded: 2022-08-04T14:15:00
Description: Watch this Scientific Journal Video about Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria at JoVE.com

Denne undersøgelse blev støttet af den tyske forskningsfond (HA 7728/2-1 og EXC2145 Project ID 390857198) og Max Planck Society.

Alle dyreforsøg blev udført efter de relevante retningslinjer og regler fra regeringen i Oberbayern. De primære neuroner blev fremstillet ud fra E16.5 C57BL/6 vildtype museembryoner af begge køn efter standardmetoder som tidligere beskrevet6.
1. Samling af den mikrofluidiske enhed
<ol><li>Belæg en seks-brønds glasbundet vævskulturplade med en endelig koncentration på 20 μg/ml Poly-D-lysin og 3,4 μg/ml Laminin i PBS (fosfatbufferet saltvand) ...

<ol>
	<li>Murali Mahadevan, H., Hashemiaghdam, A., Ashrafi, G., Harbauer, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+neuronal+health:+from+energy+metabolism+to+Parkinson's+disease.">Mitochondria in neuronal health: from energy metabolism to Parkinson's disease.</a> Advanced Biology. 5 (9), 2100663 (2021).</li><li>Dauer, W., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+mechanisms+and+models.">Parkinson's disease: mechanisms and models.</a> Neuron. 39 (6), 889-909 (2003).</li><li>Moratalla, R., 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=Differential+vulnerability+of+primate+caudate-putamen+and+striosome-matrix+dopamine+systems+to+the+neurotoxic+effects+of+1-methyl-4-phenyl-1,2,3,6-+tetrahydropyridine.">Differential vulnerability of primate caudate-putamen and striosome-matrix dopamine systems to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine.</a> Proceedings of the National Academy of Sciences. 89 (9), 3859-3863 (1992).</li><li>Cheng, H. -. C., Ulane, C. M., Burke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+progression+in+Parkinson+disease+and+the+neurobiology+of+axons.">Clinical progression in Parkinson disease and the neurobiology of axons.</a> Annals of Neurology. 67 (6), 715-725 (2010).</li><li>Taylor, A. 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=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2 (8), 599-605 (2005).</li><li>Harbauer, A. 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=Neuronal+mitochondria+transport+Pink1+mRNA+via+synaptojanin+2+to+support+local+mitophagy.">Neuronal mitochondria transport Pink1 mRNA via synaptojanin 2 to support local mitophagy.</a> Neuron. 110 (9), 1516-1531 (2022).</li><li>Ashrafi, G., Schlehe, J. S., LaVoie, M. J., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+of+damaged+mitochondria+occurs+locally+in+distal+neuronal+axons+and+requires+PINK1+and+Parkin.">Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin.</a> Journal of Cell Biology. 206 (5), 655-670 (2014).</li><li>Shipman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic+acid+(HEPES)+as+a+tissue+culture+buffer.">Evaluation of 4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic acid (HEPES) as a tissue culture buffer.</a> Proceedings of the Society for Experimental Biology and Medicine. 130 (1), 305-310 (1969).</li><li>Harbauer, A. B., Schneider, A., Wohlleber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondria+by+single-organelle+resolution.">Analysis of mitochondria by single-organelle resolution.</a> Annual Review of Analytical Chemistry. 15, 1-16 (2022).</li><li>Taylor, A. 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=Axonal+mRNA+in+uninjured+and+regenerating+cortical+mammalian+axons.">Axonal mRNA in uninjured and regenerating cortical mammalian axons.</a> The Journal of Neuroscience. 29 (15), 4697-4707 (2009).</li><li>Altman, T., 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=Axonal+TDP-43+condensates+drive+neuromuscular+junction+disruption+through+inhibition+of+local+synthesis+of+nuclear+encoded+mitochondrial+proteins.">Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins.</a> Nature Communications. 12 (1), 1-17 (2021).</li></ol>

Den nuværende protokol beskriver en metode til frø og kultur dissocierede hippocampus neuroner i en mikrofluidisk enhed til behandling af aksonale mitokondrier separat. Nytten af denne tilgang med det membranfølsomme farvestof TMRE og den komplekse III-hæmmer Antimycin A (som tidligere demonstreret7) demonstreres her, men denne metode kan let tilpasses andre mitokondriefarvestoffer eller genetisk kodede sensorer af mitokondriefunktioner, der tillader lokale, mikroskopibaserede udlæsninger<sup...

Mitokondrier er de vigtigste leverandører af ATP (adenosintrifosfat) i neuroner. Da neuronal sundhed er tæt forbundet med mitokondriefunktion, er det ikke overraskende, at dysfunktionel regulering af disse organeller har været forbundet med begyndelsen af forskellige neurodegenerative sygdomme, herunder Parkinsons sygdom1. Desuden er mitokondriel forgiftning med succes blevet brugt til at modellere Parkinsons symptomer hos dyr2. I både dyremodeller og menneskelig sygdom starter neuronernes død ved de distale dele3,4, hvilket antyder, at aksonale mitokondrier kan være mere modtagelige for fornærmelser. Imidlertid er mitokondriernes biologi i axoner ikke godt forstået på grund af de vanskeligheder, der er forbundet med målrettet behandling og analyse af aksonale mitokondrier uden samtidig forstyrrelse af cellekropsprocesser.
Nylige fremskridt inden for dyrkningsteknikker af dissocierede neuroner in vitro tillader nu fluidisk adskillelse af axoner og cellelegemer gennem mikrofluidiske enheder5. Som vist i figur 1A har disse enheder fire adgangsbrønde (a/h og c/i), med to kanaler, der forbinder hvert par (d og f). De store kanaler er forbundet med hinanden med en serie på 450 μm lange mikrokanaler (e). Forsætlige forskelle i påfyldningsniveauerne mellem de to kamre skaber en væsketrykgradient (figur 1B), der forhindrer diffusion af små molekyler fra kanalen med et lavere væskeniveau til den anden side (figur 1C, illustreret med Trypan blåt farvestof).
Vi brugte for nylig mikrofluidiske enheder til at studere lokale oversættelseskrav i aksonal mitofagi, selektiv fjernelse af beskadigede mitokondrier6. I den nuværende protokol præsenteres forskellige trin for at inducere lokal mitokondrieskade gennem selektiv behandling af axoner ved anvendelse af mitokondriekomplekset III-hæmmeren Antimycin A 6,7.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well Glass bottom plate</td>
			<td>Cellvis</td>
			<td>P06.1.5H-N</td>
			<td>Silicone device</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS M5000 widefield microscope</td>
			<td>Thermofischer Scientific</td>
			<td>EVOS M5000</td>
			<td>fully integrated digital widefield microscope</td>
		</tr>
		<tr>
			<td>Hibernate E</td>
			<td>BrainBits</td>
			<td>HE500</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted spinning disk confocal</td>
			<td>Nikon</td>
			<td>TI2-E + CSU-W1</td>
			<td>With incubator chamber</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Invitrogen</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic devices</td>
			<td>XONA microfluidics</td>
			<td>RD450</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>TMRE</td>
			<td>Sigma</td>
			<td>87917</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidics-assisted selective depolarization of axonal mitochondria

Mitokondrier er de primære leverandører af ATP (adenosintrifosfat) i neuroner. Mitokondrie dysfunktion er en almindelig fænotype i mange neurodegenerative sygdomme. I betragtning af nogle axoners udførlige arkitektur og ekstreme længde er det ikke overraskende, at mitokondrier i axoner kan opleve forskellige miljøer sammenlignet med deres cellekropsmodstykker. Interessant nok går dysfunktion af aksonale mitokondrier ofte forud for virkninger på cellekroppen. For at modellere aksonal mitokondriel dysfunktion in vitro tillader mikrofluidiske enheder behandling af aksonale mitokondrier uden at påvirke de somale mitokondrier. Den flydende trykgradient i disse kamre forhindrer diffusion af molekyler mod gradienten, hvilket muliggør analyse af mitokondrieegenskaber som reaktion på lokale farmakologiske udfordringer inden for axoner. Den nuværende protokol beskriver såning af dissocierede hippocampale neuroner i mikrofluidiske enheder, farvning med et membranpotentielt følsomt farvestof, behandling med et mitokondrietoksin og den efterfølgende mikroskopiske analyse. Denne alsidige metode til at studere aksonal biologi kan anvendes på mange farmakologiske forstyrrelser og billeddannelsesaflæsninger og er velegnet til flere neuronale undertyper.

Primære hippocampale neuroner blev dyrket i mikrofluidiske enheder i 7-8 dage, før mitokondrier blev farvet med det membranfølsomme farvestof (TMRE) i 25 minutter i begge kanaler. Som vist i figur 2A gav dette homogen farvning af mitokondrier på begge sider af mikrorillerne, men det var utilstrækkeligt til at ligevægte farvningen i midten af mikrorillerne. Ved tilsætning af Antimycin A til den aksonale side bevarede somale mitokondrier TMRE-signalet (figur 2B</str...

Watch this Scientific Journal Video about Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria at JoVE.com

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Den nuværende protokol beskriver såning og farvning af neuronale mitokondrier i mikrofluidiske kamre. Den flydende trykgradient i disse kamre muliggør selektiv behandling af mitokondrier i axoner for at analysere deres egenskaber som reaktion på farmakologiske udfordringer uden at påvirke cellelegemets rum.

Mikrofluidik-assisteret selektiv depolarisering af aksonale mitokondrier

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many ...

Mitokondrier er afgørende for neuronal sundhed. I mange neurodegenerative sygdomme er axonale mitokondrier de første, der lider, men det er ikke klart, hvad der gør aksonale mitokondrier så specielle. Den flydende trykgradient i kamrene, der anvendes i denne protokol, muliggør selektiv behandling af mitokondrion axoner.Dette efterlader cellekropsprocesser uforstyrret og giver os mulighed for at fokusere på aksonal biologi. Vi viser her depolariseringen af mitokondrier med et membranpotentielt følsomt farvestof, men denne metode kan anvendes på mange forskellige neuronale celletyper og fluorescerende aflæsninger. Til at begynde med skal du placere de kodede seks brøndplader i en steril hætte og vaske dem to gange med sterilt dobbeltdestilleret vand.Lad pladerne tørre i en vippet position i tre til fem minutter. Fjern overskydende vand ved vakuumsugning eller pipettering. Derefter blødlægges mikrofluidkammeret i 80% ethanol.Tør igen det mikrofluidiske kammer i tre til fem minutter i en vippet position, og fjern overskydende ethanol med en pipettespids. Når det er helt tørt, skal du placere det mikrofluidiske kammer i midten af brønden og pladen. Bank forsigtigt på mikrofluidkammeret ved dets grænser og mikrorillesektionen i midten for korrekt fastgørelse til glaspladen.Først indsamles det ønskede antal dissocierede hippocampale neuroner i et 1, 5 ml reaktionsrør. Centrifuge røret ved 1000 gange G i fire minutter ved stuetemperatur. Kassér supernatanten, og suspender pelleten i otte mikroliter B-27 neuro basalmedier.Dispenser derefter celleopløsningen i kanalindgangen til det mikrofluidiske kammer. Tryk på bagsiden af pladen for at hjælpe strømmen gennem kanalen. Brug en pipette til at aspirere enhver resterende cellesuspension ved udgangen af kanalen.Inkuber det mikrofluidiske kammer med celler i 15 til 20 minutter ved 37 grader Celsius og 5% kuldioxid. Fyld derefter den øverste axonale brønd med 50 mikroliter B-27 neurobasale medier, og tryk på bagsiden af pladen for at hjælpe strømmen. Fyld hver brønd på soma-siden med 150 mikroliter B-27 neurobasale medier, hvilket skaber en spændingsboble ovenpå.Fyld også begge brønde på den aksonale side med 100 mikroliter B-27 neurobasale medier hver. Efter syv til otte dages in vitro-kultur skal du sikre dig, at axonerne er vokset gennem mikrorillerne og forlænget ind i det aksonale rum. Vask enheden ved at fjerne B-27 neurobasalmediet og tilsæt 100 mikroliter forvarmet billeddannelsesmedium til de øverste brønde i både axonal- og somakanalerne.Lad mediet strømme igennem til de nederste brønde. Fjern mediet fra de nederste brønde og eventuelle rester fra de øverste brønde, og gentag med frisk medium, så brøndene er tomme i slutningen af vasken. Derefter tilsættes 100 mikroliter fem nanomolære TMRE-fortyndinger til både de somatiske og de aksonale topbrønde.Lad mediet strømme gennem begge rum, indtil der er et lige stort volumen i alle brønde. Fyld op med TMRE-indeholdende medium. Vælg et område af interesse i det somatiske rum, og følg det gennem mikrorillerne ind i det aksonale rum.Sørg for, at mikrorillerne afbildet i de somatiske og aksonale rum matches med hinanden. Når du har ændret filnavnet, skal du erhverve røde fluorescensbilleder ved hjælp af 561 nanometer excitation og 625 nanometer emissionsbølgelængder. Sørg for en volumenforskel mellem soma og axonale kamre ved at kontrollere tilstedeværelsen af en spændingsboble på den somatiske side.Fjern derefter billedmediet fra begge brønde på den aksonale side, bortset fra mediet inde i kanalen. For at inducere mitokondriel depolarisering tilsættes 160 mikroliter 20 mikromolær antimycin A fortyndet i billeddannelsesmediet til den øverste axonale brønd. Lad det strømme gennem kanalen, indtil der er et lige stort volumen i begge aksonale brønde.Gentag billeddannelsen, og sørg for, at de samme positioner afbildes som under baseline-anskaffelsen ved at identificere mikrorillerne. Ved hjælp af denne protokol blev primære hippocampale neuroner dyrket i mikrofluidiske enheder efterfulgt af mitokondriefarvning med et membranfølsomt farvestof, TMRE. Homogen farvning af mitokondrier blev observeret på begge sider af mikrorillerne, men ikke i midten.Ved tilsætning af antimycin A til den aksonale side bevarede somale mitokondrier TMRE-signalet, mens fluorescensen gik tabt fra de aksonale mitokondrier Sørg for, at der ikke efterlades fugt i brønden eller på enheden, før du samler enheden. Ellers vil kamrene ikke blive forseglet. Ved hjælp af denne metode kan vi studere virkningerne af tab af mitokondriefunktion i axon- og lokale funktioner samt dens indflydelse på retrograd signalering og global celleoverlevelse.Mitokondriel biogenese var længe blevet antaget at ske udelukkende i cellekroppen. Denne metode gjorde det muligt for os at afsløre, at aksonale mitokondrier skader krævede lokal oversættelse af manglen på protein PINK1.

microfluidics-assisted-selective-depolarization-of-axonal-mitochondria

Research

JoVE Journal

Neuroscience

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

Selektiv Tracing af Auditive fibre i de Avian embryoer Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Subtype-selektive Elektroporation af Kortikal interneuroner

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

Real-time Imaging af axonal Transport af Quantum Dot-mærket BDNF i Primary neuroner

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using &quot;Cargo Mapping&quot; Analysis

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Karakterisere sammensætningen af ​​Molecular Motors på Moving axonal Cargo Brug af &quot;Cargo Mapping&quot; Analyse

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

Tre-dimensionelle billedbehandling og analyse af mitochondrier i menneskelige Intraepidermal nervefibre

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Analyse af Brain mitokondrier Brug Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Visualisering af Axonal Projeksionsmønsteret af Embryoniske Motor Neuroner i<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

In Vivo Elektrofysiologiske målinger af rotte Nervus Nerve med cytoskeletale ophidselse test

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...