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

Denna studie stöddes av den tyska forskningsstiftelsen (HA 7728/2-1 och EXC2145 Project ID 390857198) och Max Planck Society.

Alla djurförsök utfördes i enlighet med relevanta riktlinjer och föreskrifter från regeringen i Oberbayern. De primära neuronerna framställdes från E16.5 C57BL/6 musembryon av båda könen enligt standardmetoder som tidigare beskrivits6.
1. Montering av mikrofluidikanordningen
<ol><li>Täck en vävnadsodlingsplatta med sex brunnar med glasbotten med en slutlig koncentration på 20 μg/ml poly-D-lysin och 3,4 μg/ml laminin i PBS (fosfatbuffrad...

<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>

Detta protokoll beskriver en metod för att fröa och odla dissocierade hippocampusneuroner i en mikrofluidisk anordning för att behandla axonala mitokondrier separat. Nyttan av detta tillvägagångssätt med det membrankänsliga färgämnet TMRE och den komplexa III-hämmaren Antimycin A (som tidigare visats7) demonstreras här, men denna metod kan enkelt anpassas till andra mitokondriella färgämnen eller genetiskt kodade sensorer av mitokondriella funktioner som möjliggör lokala, mikroskopi...

Mitokondrier är de viktigaste leverantörerna av ATP (adenosintrifosfat) i neuroner. Eftersom neuronal hälsa är intimt kopplad till mitokondriell funktion är det inte förvånande att dysfunktionell reglering av dessa organeller har associerats med uppkomsten av olika neurodegenerativa sjukdomar, inklusive Parkinsons sjukdom1. Dessutom har mitokondriell förgiftning framgångsrikt använts för att modellera Parkinsons symtom hos djur2. I både djurmodeller och mänsklig sjukdom börjar neurons bortgång vid de distala delarna3,4, vilket antyder att axonala mitokondrier kan vara mer mottagliga för förolämpningar. Mitokondriernas biologi i axoner är emellertid inte väl förstådd på grund av svårigheterna i samband med riktad behandling och analys av axonala mitokondrier utan samtidig störning av cellkroppsprocesser.
De senaste framstegen inom odlingstekniker för dissocierade neuroner in vitro tillåter nu fluidisk separation av axoner och cellkroppar genom mikrofluidiska anordningar5. Som visas i figur 1A har dessa enheter fyra åtkomstbrunnar (a / h och c / i), med två kanaler som förbinder varje par (d och f). De stora kanalerna är förbundna med varandra med en serie av 450 μm långa mikrokanaler (e). Avsiktliga skillnader i fyllningsnivåerna mellan de två kamrarna skapar en vätsketrycksgradient (figur 1B) som förhindrar diffusion av små molekyler från kanalen med en lägre vätskenivå till andra sidan (figur 1C, illustrerad med trypanblått färgämne).
Vi använde nyligen mikrofluidiska enheter för att studera lokala översättningskrav i axonal mitophagi, selektivt avlägsnande av skadade mitokondrier6. I detta protokoll presenteras olika steg för att inducera lokal mitokondriell skada genom selektiv behandling av axoner med användning av mitokondriell komplex III-hämmare 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 är de primära leverantörerna av ATP (adenosintrifosfat) i nervceller. Mitokondriell dysfunktion är en vanlig fenotyp vid många neurodegenerativa sjukdomar. Med tanke på vissa axoners utarbetade arkitektur och extrema längd är det inte förvånande att mitokondrier i axoner kan uppleva olika miljöer jämfört med deras motsvarigheter i cellkroppen. Intressant är att dysfunktion av axonala mitokondrier ofta föregår effekter på cellkroppen. För att modellera axonal mitokondriell dysfunktion in vitro tillåter mikrofluidiska anordningar behandling av axonala mitokondrier utan att påverka de somala mitokondrierna. Den fluidiska tryckgradienten i dessa kamrar förhindrar diffusion av molekyler mot gradienten, vilket möjliggör analys av mitokondriella egenskaper som svar på lokala farmakologiska utmaningar inom axoner. Det nuvarande protokollet beskriver sådd av dissocierade hippocampusneuroner i mikrofluidiska anordningar, färgning med ett membranpotentialkänsligt färgämne, behandling med ett mitokondriellt toxin och den efterföljande mikroskopiska analysen. Denna mångsidiga metod för att studera axonal biologi kan tillämpas på många farmakologiska störningar och avbildningsavläsningar och är lämplig för flera neuronala subtyper.

Primära hippocampusneuroner odlades i mikrofluidiska enheter i 7-8 dagar innan mitokondrier färgades med det membrankänsliga färgämnet (TMRE) i 25 minuter i båda kanalerna. Som visas i figur 2A gav detta homogen färgning av mitokondrier på båda sidor av mikrospåren, men det var otillräckligt för att balansera färgningen i mitten av mikrospåren. Vid tillsats av antimycin A till axonalsidan behöll somala mitokondrier TMRE-signalen (figur 2B och vide...

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

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Detta protokoll beskriver sådd och färgning av neuronala mitokondrier i mikrofluidiska kamrar. Den fluidiska tryckgradienten i dessa kamrar möjliggör selektiv behandling av mitokondrier i axoner för att analysera deras egenskaper som svar på farmakologiska utmaningar utan att påverka cellkroppsfacket.

Mikrofluidikassisterad selektiv depolarisering av axonala mitokondrier

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

Mitokondrier är viktiga för neuronal hälsa. I många neurodegenerativa sjukdomar är axonala mitokondrier de första som lider, men det är inte klart vad som gör axonala mitokondrier så speciella. Den fluidiska tryckgradienten i kamrarna som används i detta protokoll möjliggör selektiv behandling av mitokondrionaxoner.Detta lämnar cellkroppsprocesser ostörda och låter oss fokusera på axonal biologi. Vi visar här depolariseringen av mitokondrier med ett membranpotentialkänsligt färgämne, men denna metod kan tillämpas på många olika neuronala celltyper och fluorescerande avläsningar. För att börja, placera de kodade sex brunnsplattorna i en steril huva och tvätta dem två gånger med sterilt dubbeldestillerat vatten.Låt plattorna torka i lutat läge i tre till fem minuter. Ta bort överflödigt vatten genom vakuumsugning eller pipettering. Blötlägg sedan mikrofluidikkammaren i 80% etanol.Torka igen den mikrofluidiska kammaren i tre till fem minuter i ett lutat läge och ta bort överskott av etanol med en pipettspets. När den är helt torr, placera den mikrofluidiska kammaren i mitten av brunnen och plattan. Knacka försiktigt på mikrofluidikkammaren vid dess gränser och mikrogroovesektionen i mitten för korrekt fastsättning på glasplattan.Samla först det önskade antalet dissocierade hippocampusneuroner i ett 1, 5 ml reaktionsrör. Centrifugera röret vid 1000 gånger G i fyra minuter vid rumstemperatur. Kassera supernatanten och återsuspendera pelleten i åtta mikroliter B-27 neurobasala medier.Dosera sedan celllösningen i kanalingången till den mikrofluidiska kammaren. Tryck på baksidan av plattan för att hjälpa flödet genom kanalen. Använd en pipett och aspirera eventuell återstående cellsuspension vid kanalens utgång.Inkubera den mikrofluidiska kammaren med celler i 15 till 20 minuter vid 37 grader Celsius och 5% koldioxid. Fyll sedan den övre axonala brunnen med 50 mikroliter B-27 neurobasala medier och tryck på baksidan av plattan för att hjälpa flödet. Fyll varje brunn på soma-sidan med 150 mikroliter B-27 neurobasala medier, vilket skapar en spänningsbubbla ovanpå.Fyll också båda brunnarna på axonalsidan med 100 mikroliter B-27 neurobasala medier vardera. Efter sju till åtta dagars in vitro-odling, se till att axonerna har vuxit genom mikrospåren och sträckts ut i axonalfacket. Tvätta enheten genom att ta bort B-27 neurobasala mediet och tillsätt 100 mikroliter förvärmt bildmedium till de övre brunnarna i både axonal- och somakanalerna.Låt mediet strömma igenom till de nedre brunnarna. Ta bort mediet från de nedre brunnarna och eventuellt kvarvarande medium från de övre brunnarna och upprepa med nytt medium och lämna brunnarna tomma i slutet av tvätten. Tillsätt sedan 100 mikroliter fem nanomolära TMRE-utspädningar till både de somatiska och axonala toppbrunnarna.Låt mediet strömma genom båda facken tills det finns lika stor volym i alla brunnar. Fyll på med TMRE-innehållande medium. Välj en region av intresse i det somatiska facket och följ det genom mikrospåren in i axonalfacket.Se till att mikrospåren som avbildas i de somatiska och axonala facken är matchade med varandra. Efter att ha ändrat filnamnet, skaffa röda fluorescensbilder med 561 nanometer excitation och 625 nanometer emissionsvåglängder. Säkerställ en volymskillnad mellan soma och axonala kamrar genom att kontrollera närvaron av en spänningsbubbla på den somatiska sidan.Ta sedan bort bildmediet från båda brunnarna på axonalsidan, förutom mediet inuti kanalen. För att inducera mitokondriell depolarisering, tillsätt 160 mikroliter 20 mikromolära antimycin A utspädda i bildmediet till den övre axonala brunnen. Låt det strömma genom kanalen tills det finns lika stor volym i båda axonala brunnarna.Upprepa avbildningen och se till att samma positioner avbildas som under baslinjeförvärvet genom att identifiera mikrospåren. Med hjälp av detta protokoll odlades primära hippocampusneuroner i mikrofluidiska anordningar, följt av mitokondriell färgning med ett membrankänsligt färgämne, TMRE. Homogen färgning av mitokondrier observerades på båda sidor av mikrospåren, men inte i mitten.Vid tillsats av antimycin A till axonalsidan behöll somala mitokondrier TMRE-signalen, medan fluorescensen förlorades från axonala mitokondrier Se till att ingen fukt finns kvar i brunnen eller på enheten innan du monterar enheten. Annars kommer kamrarna inte att förseglas. Med hjälp av denna metod kan vi studera effekterna av förlust av mitokondriell funktion i axon- och lokala funktioner, liksom dess inverkan på retrograd signalering och global cellöverlevnad.Mitokondriell biogenes ansågs länge ske uteslutande i cellkroppen. Denna metod gjorde det möjligt för oss att avslöja att axonal mitokondrier skada krävde lokal översättning av kortet av proteinet 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.

Mekanisk Manipulation av nervceller till Control axonal utveckling

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.

Kromatin Immunoprecipitation från dorsalrotsganglier vävnad efter axonal skada

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 spåra Auditiva fibrer i Avian embryonala 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.

Subtypselektiva Elektroporering av 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.

Realtid avbildning av axonal transport av Quantum Dot-märkta BDNF i primära 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.

Karakterisera sammansättning Molecular Motors på Moving axonal Cargo Använda &quot;Cargo Mapping&quot; Analys

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.

Tredimensionell avbildning och analys av mitokondrier inom mänskliga Intraepidermal nervfibrer

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.

Analys av Brain Mitokondrier Använda Serial Block-Face Svepelektronmikroskopi

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 av Axonal Projektionsmönstret av Embryonic Motor Neurons in<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 Elektrofysiologisk mätning av råtta ulnarisnerven med Axonal retbarhet testning

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