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 studien ble støttet av German Research Foundation (HA 7728/2-1 og EXC2145 Project ID 390857198) og Max Planck Society.

Alle dyreforsøk ble utført i henhold til relevante retningslinjer og forskrifter fra regjeringen i Øvre Bayern. De primære nevronene ble fremstilt fra E16.5 C57BL/6 museembryoer av begge kjønn etter standardmetoder som tidligere beskrevet6.
1. Montering av mikrofluidisk enhet
<ol><li>Belegg en seks-brønns glassbunnsvevskulturplate med en endelig konsentrasjon på 20 μg / ml Poly-D-lysin og 3,4 μg / ml Laminin i PBS (fosfatbufret saltvann) (se ...

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	<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 nåværende protokollen beskriver en metode for å frø og dyrke dissosierte hippocampale nevroner i en mikrofluidisk enhet for å behandle aksonale mitokondrier separat. Nytten av denne tilnærmingen med det membranfølsomme fargestoffet TMRE og den komplekse III-hemmeren Antimycin A (som tidligere demonstrert7) er demonstrert her, men denne metoden kan enkelt tilpasses andre mitokondrielle fargestoffer eller genetisk kodede sensorer av mitokondrielle funksjoner som tillater lokale, mikroskop...

Mitokondrier er de viktigste leverandørene av ATP (adenosintrifosfat) i nevroner. Siden nevronhelse er nært knyttet til mitokondriell funksjon, er det ikke overraskende at dysfunksjonell regulering av disse organellene har vært assosiert med utbruddet av ulike nevrodegenerative sykdommer, inkludert Parkinsonssykdom. Videre har mitokondriell forgiftning med hell blitt brukt til å modellere Parkinsonske symptomer hos dyr2. I både dyremodeller og menneskelig sykdom starter bortfallet av nevroner ved de distale delene3,4, noe som antyder at aksonale mitokondrier kan være mer utsatt for fornærmelser. Imidlertid er biologien til mitokondrier i axoner ikke godt forstått på grunn av vanskelighetene forbundet med målrettet behandling og analyse av aksonale mitokondrier uten samtidig forstyrrelse av cellekroppsprosesser.
Nylige fremskritt innen dyrkingsteknikker av dissosierte nevroner in vitro tillater nå fluidisk separasjon av aksoner og cellelegemer gjennom mikrofluidiske enheter5. Som vist i figur 1A, har disse enhetene fire tilgangsbrønner (a/h og c/i), med to kanaler som forbinder hvert par (d og f). De store kanalene er forbundet med hverandre med en serie på 450 μm lange mikrokanaler (e). Tilsiktede forskjeller i fyllingsnivåene mellom de to kamrene skaper en væsketrykkgradient (figur 1B) som forhindrer diffusjon av små molekyler fra kanalen med lavere væskenivå til den andre siden (figur 1C, illustrert med Trypan blått fargestoff).
Vi har nylig brukt mikrofluidiske enheter for å studere lokale oversettelseskrav i aksonal mitophagy, selektiv fjerning av skadede mitokondrier6. I denne protokollen presenteres forskjellige trinn for å indusere lokal mitokondriell skade gjennom selektiv behandling av aksoner ved bruk av mitokondriekomplekset III-hemmeren 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 leverandørene av ATP (adenosintrifosfat) i nevroner. Mitokondriell dysfunksjon er en vanlig fenotype i mange nevrodegenerative sykdommer. Gitt noen aksoners forseggjorte arkitektur og ekstreme lengde, er det ikke overraskende at mitokondrier i aksoner kan oppleve forskjellige miljøer sammenlignet med deres cellekroppsmodeller. Interessant, dysfunksjon av aksonale mitokondrier går ofte foran effekter på cellekroppen. For å modellere aksonal mitokondriell dysfunksjon in vitro, tillater mikrofluidiske enheter behandling av aksonale mitokondrier uten å påvirke de somale mitokondriene. Den fluidiske trykkgradienten i disse kamrene forhindrer diffusjon av molekyler mot gradienten, og muliggjør dermed analyse av mitokondrielle egenskaper som svar på lokale farmakologiske utfordringer i aksoner. Den nåværende protokollen beskriver såing av dissosierte hippocampale nevroner i mikrofluidiske enheter, farging med et membranpotensial følsomt fargestoff, behandling med mitokondrielt toksin og den påfølgende mikroskopiske analysen. Denne allsidige metoden for å studere aksonal biologi kan brukes på mange farmakologiske forstyrrelser og bildeavlesninger, og er egnet for flere neuronale subtyper.

Primære hippocampale nevroner ble dyrket i mikrofluidiske enheter i 7-8 dager før mitokondrier ble farget med det membranfølsomme fargestoffet (TMRE) i 25 minutter i begge kanalene. Som vist i figur 2A ga dette homogen farging av mitokondrier på begge sider av mikrogroovene, men det var likevel ikke tilstrekkelig til å likestille fargingen i midten av mikrogroovene. Ved tillegg av antimycin A til aksonal side beholdt somale mitokondrier TMRE-signalet (figur 2B</stro...

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

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Denne protokollen beskriver såing og farging av nevronale mitokondrier i mikrofluidiske kamre. Den fluidiske trykkgradienten i disse kamrene muliggjør selektiv behandling av mitokondrier i aksoner for å analysere deres egenskaper som svar på farmakologiske utfordringer uten å påvirke cellekroppen.

Mikrofluidikk-assistert selektiv depolarisering av aksonale mitokondrier

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

Mitokondrier er avgjørende for nevronal helse. I mange nevrodegenerative sykdommer er aksonale mitokondrier de første som lider, men det er ikke klart hva som gjør aksonale mitokondrier så spesielle. Den fluidiske trykkgradienten i kamrene som brukes i denne protokollen tillater selektiv behandling av mitokondrion aksoner.Dette etterlater cellekroppens prosesser uforstyrret, og lar oss fokusere på aksonal biologi. Vi viser her depolariseringen av mitokondrier med et membranpotensial følsomt fargestoff, men denne metoden kan brukes på mange forskjellige nevroncelletyper og fluorescerende avlesninger. For å begynne, plasser de kodede seks brønnplatene i en steril hette, og vask dem to ganger med sterilt dobbelt destillert vann.La platene tørke i vippet stilling i tre til fem minutter. Fjern overflødig vann ved vakuumsuging eller pipettering. Deretter suge mikrofluidisk kammer i 80% etanol.Igjen, tørk det mikrofluidiske kammeret i tre til fem minutter i vippet stilling, og fjern overflødig etanol med en pipettespiss. Når det er helt tørt, plasser det mikrofluidiske kammeret i midten av brønnen og platen. Bank forsiktig på mikrofluidkammeret ved grensene og mikrogrooveseksjonen i midten for riktig festing til glassplaten.Først samler du ønsket antall dissosierte hippocampale nevroner i et 1, 5 milliliter reaksjonsrør. Sentrifuger røret ved 1000 ganger G i fire minutter ved romtemperatur. Kast supernatanten, og resuspend pelleten i åtte mikroliter B-27 neuro basale medier.Dispenser deretter celleoppløsningen i kanalinngangen til mikrofluidisk kammer. Trykk på baksiden av platen for å hjelpe strømmen gjennom kanalen. Bruk en pipette, aspirer eventuell gjenværende cellesuspensjon ved utgangen av kanalen.Inkuber mikrofluidkammeret med celler i 15 til 20 minutter ved 37 grader Celsius og 5% karbondioksid. Deretter fyller du den øverste aksonale brønnen med 50 mikroliter B-27 neurobasale medier, og banker på baksiden av platen for å hjelpe strømmen. Fyll hver brønn på soma-siden med 150 mikroliter B-27 neurobasale medier, og skap en spenningsboble på toppen.Fyll også begge brønnene på aksonalsiden med 100 mikroliter B-27 neurobasale medier hver. Etter syv til åtte dager med in vitro-kultur, sørg for at aksonene har vokst gjennom mikrogroovene, og utvidet seg inn i aksonalrommet. Vask enheten ved å fjerne B-27 neurobasal medium, og tilsett 100 mikroliter forvarmet bildebehandlingsmedium til toppbrønnene i både aksonale og somakanaler.La mediet strømme gjennom til de nedre brønnene. Fjern mediet fra de nedre brønnene, og eventuelt gjenværende medium fra de øverste brønnene, og gjenta med friskt medium, og la brønnene være tomme på slutten av vasken. Tilsett deretter 100 mikroliter med fem nanomolar TMRE-fortynning til både de somatiske og aksonale toppbrønnene.La mediet strømme gjennom begge rom, til det er like stort volum i alle brønner. Fyll opp med TMRE-inneholdende medium. Velg et område av interesse i det somatiske rommet, og følg det gjennom mikrogroovene inn i det aksonale rommet.Sørg for at mikrosporene som er avbildet i de somatiske og aksonale rommene, er tilpasset hverandre. Etter å ha endret filnavnet, skaff deg røde fluorescensbilder ved hjelp av 561 nanometer eksitasjon og 625 nanometer utslippsbølgelengder. Sørg for en volumforskjell mellom soma og aksonale kamre ved å kontrollere tilstedeværelsen av en spenningsboble på den somatiske siden.Fjern deretter bildemediet fra begge brønnene på aksonalsiden, bortsett fra mediet inne i kanalen. For å indusere mitokondriell depolarisering, tilsett 160 mikroliter 20 mikromolar antimycin A fortynnet i bildemediet til den øverste aksonale brønnen. La det strømme gjennom kanalen til det er like stort volum i begge aksonale brønner.Gjenta avbildningen, og sørg for at de samme posisjonene er avbildet som under grunnlinjeinnsamlingen, ved å identifisere mikrogroovene. Ved hjelp av denne protokollen ble primære hippocampale nevroner dyrket i mikrofluidiske enheter, etterfulgt av mitokondriell farging med et membranfølsomt fargestoff, TMRE. Homogen farging av mitokondrier ble observert på begge sider av mikrogroovene, men ikke i midten.Ved tilsetning av antimycin A til aksonal side beholdt somale mitokondrier TMRE-signalet, mens fluorescensen gikk tapt fra aksonale mitokondrier Forsikre deg om at det ikke er fuktighet igjen i brønnen eller på enheten før du monterer enheten. Ellers vil kamrene ikke bli forseglet. Ved hjelp av denne metoden kan vi studere effekten av tap av mitokondriell funksjon i akson- og lokale funksjoner, samt dens innflytelse på retrograd signalering og global celleoverlevelse.Mitokondriell biogenese ble lenge antatt å skje utelukkende i cellekroppen. Denne metoden tillot oss å avsløre at aksonale mitokondrier skade krevde lokal oversettelse av kort 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 av auditiv Fibers i 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 ...

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-selektiv Electroporation av Cortical 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 av aksonal Transport av Quantum Dot-merket BDNF i Primær 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.

Karakteriserer Sammensetning for molekylær Motors på Moving aksonale Cargo Ved hjelp av &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.

Tredimensjonale Imaging og analyse av mitokondrier i menneskelig Intraepidermal Nerve Fibre

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 av Brain Mitokondrier Bruke Serial Block-Face Scanning elektronmikroskopi

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 Projeksjonsmønsteret av 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.

I Vivo Elektrofysiologiske måling av rotte Ulnar Nerve med Axonal Excitability Testing

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