Name: rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection
Uploaded: 2017-06-13T14:00:00
Description: Watch this Scientific Journal Video about Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection at JoVE.com

Vi vil gerne takke Yoichi Miyahara for mange nyttige diskussioner og indsigter. MA og PG anerkender finansiering fra NSERC.

Alle procedurer, der er beskrevet nedenfor, blev godkendt af McGill University&#39;s Animal Care Committee og er i overensstemmelse med retningslinjerne fra det canadiske råd for dyrepleje. 1. Standardisering af neuronale kulturer ved anvendelse af mikrofluidiske enheder: Enhedssamling <ol><li> Vælg en passende mikrofluidisk enhed til det ønskede eksperiment. For at forbinde neuroner inden for samme population, brug Neuro Devices ( Figur 1 ) og til at forbi...

<ol>
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Ved hjælp af standard mikromanipulation og innovative mikrofluidiske indretninger blev der udviklet en ny teknik til hurtigt at indlede, forlænge og netop forbinde nye funktionelle neuronalkredsløb over store afstande. Pipette mikromanipulation er et fælles redskab i de fleste neurovidenskab labs 4 , 13 . Den reelle udfordring for at opnå reproducerbare og pålidelige resultater var standardisering af sunde, nøjagtigt positionerede neuronkulturer i eksperi...

Skader på det voksne centralnervesystem (CNS) kan føre til permanent invaliditet på grund af flere mekanismer, der begrænser axonal genvækst 1 . Efter skader danner mange CNS-axoner ikke en ny vækstkegle og undgår at montere et effektivt regenerativ respons 2 . Desuden hæmmer skader og arvæv omkring CNS læsioner signifikant axonal vækst 1 , 2 , 3 . Nuværende terapier til fremme af CNS-regenerering efter skade har fokuseret på at forøge det skadelige neurons intrinsiske vækstpotentiale og på maskering af inhibitorerne af axonal forlængelse forbundet med myelinrester og glialæren 1 , 3 . På trods heraf forbliver kapaciteten til at regenerere lange axoner til fjerne mål og til at danne passende funktionelle synapser fortsat alvorligt begrænset 4 , </suP&gt; 5 , 6 , 7 . I det nuværende arbejde anvendes mikrobeyler, pipette mikromanipulation og mikrofluidiske indretninger til hurtigt at initiere, forlænge og tilslutte netop nye funktionelle neuronalkredsløb over lange afstande. Tidligere arbejde har vist, at poly-D-lysin-coatede perler (PDL-perler) inducerer membranadhæsion efterfulgt af clustering af synaptiske vesikelkomplekser og dannelse af funktionelle presynaptiske bolte 8 . Det blev også vist, at når PDL-perlen mekanisk trækkes væk efter presynaptisk differentiering, følger den synaptiske proteinklynge perlen, idet en ny neurit 9 initieres. Den følgende fremgangsmåde udnytter denne kendsgerning sammen med evnen til at dyrke embryonale hippocampale neuroner af rotter i organiserede regioner på en dækslip ved anvendelse af polydimethylsiloxan (PDMS) mikrofluidiske indretninger til præcist at omdirigere en neuronalkredsløb. Disse PDMS mikrofluidiske indretninger er ikke-toksiske, optisk transparente og består af to kamre forbundet med et system af mikrokanaler. Når de er monteret på en dæksel, tjener hver enhed som en form til at styre neuronal vækst og opretholde sunde neuronkulturer på præcise mønstre i længere end 4 uger in vitro . Her præsenteres der en ramme for at undersøge grænserne for udvidelse og funktionalitet af den nye neurit. Nye, funktionelle neuritter skabes og positioneres til styrbart (re) wire neuronale netværk. De opnåede forlængelseshastigheder er hurtigere end 20 μm / min over millimeterskalaafstande, og funktionelle forbindelser etableres. Disse resultater viser uventet, at disse neurites indre kapacitet til forlængelse er meget hurtigere end tidligere antaget. Denne foreslåede mekaniske tilgang omgår langsomme kemiske strategier og muliggør styret forbindelse til et specifikt mål. thEr teknik åbner nye veje til in vitro- undersøgelsen af ​​nye terapier for at genoprette neuronal forbindelse efter skade. Det muliggør også manipulation og omlægning af neuronale netværk til at undersøge grundlæggende aspekter af neuronal signalbehandling og neuronal funktion in vitro .

<table border="0" cellpadding="0" cellspacing="0" width="836">
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			<td>Commercially available at http://www.anandadevices.com</td>
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			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Objective</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;">UIS2, LUCPLFLN 40X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD Camera</td>
			<td style="width:104px;">Photometrics</td>
			<td style="width:119px;">Cascade II: 512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leibovitz&#39;s (1x) L-15 Medium</td>
			<td>Life Technologies</td>
			<td>11415-064</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Typsin-EDTA (0.05%), Phenol red</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (1x) Dulbecco&#39;s Modified Eagle Medium [+4.5 g/L D-Glucose, + L-Glutamine, + 110 mg/L Sodium Pyruvate]</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS (1x) Hank&#39;s Balanced Salt Solution [- Calcium Chloride, - Magnesium Chloride, - Magnesium Sulfate]</td>
			<td>Life Technologies</td>
			<td>14170-112</td>
			<td>Rat Dissection</td>
		</tr>
	</tbody>
</table>

rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection

Hjerne- og rygmarvsskade kan føre til permanent invaliditet og død, fordi det stadig ikke er muligt at regenerere neuroner over lange afstande og nøjagtigt genoprette dem med et passende mål. Her beskrives en procedure for hurtigt at indlede, forlænge og tilslutte netop nye funktionelle neuronalkredsløb over lange afstande. De opnåede forlængelseshastigheder når over 1,2 mm / time, 30-60 gange hurtigere end in vivo- hastighederne for de hurtigst voksende axoner fra det perifere nervesystem (0,02 til 0,04 mm / t) 28 og 10 gange hurtigere end tidligere rapporteret for det samme Neuronaltype på et tidligere udviklingsstadium 4 . For det første dyrkes isolerede populationer af rottehippocampale neuroner i 2-3 uger i mikrofluidiske indretninger for præcist at positionere cellerne, hvilket muliggør nem mikromomanipulation og eksperimentel reproducerbarhed. Derefter anbringes perler overtrukket med poly-D-lysin (PDL) på neuritter for at danne klæbemiddelkontHandlinger og pipette mikromanipulation anvendes til at bevæge det resulterende perle-neurit-kompleks. Efterhånden som perlen er flyttet, trækker den ud en ny neurit, der kan udvides over hundredvis af mikrometer og funktionelt forbundet til en målcelle på mindre end 1 time. Denne proces muliggør eksperimentel reproducerbarhed og nem manipulation, mens man omgår langsommere kemiske strategier til at fremkalde neurit vækst. Preliminære målinger fremlagt her viser en neuronal vækst, der langt overstiger fysiologiske. Kombination af disse innovationer muliggør den nøjagtige etablering af neuronale netværk i kultur med en hidtil uset grad af kontrol. Det er en roman metode, der åbner døren for en overflod af information og indsigt i signaloverførsel og kommunikation inden for neuronalt netværk samt at være en legeplads for at undersøge grænserne for neuronal vækst. De potentielle anvendelser og eksperimenter er udbredt med direkte konsekvenser for terapier, der sigter mod at genoprette neuronaL kredsløb efter traumer eller i neurodegenerative sygdomme.

Hippocampale neuroner af embryonale rotter dyrkes i mikrofluidiske indretninger for at muliggøre præcis positionering af celler, PDL-perler og mikromanipulatorer. Det første skridt er at samle den mikrofluidiske enhed korrekt på en glasdæksel eller skål. Det er vigtigt, at den mikrofluidiske enhed er godt fastgjort til substratet for at undgå, at celler forlader kamrene og bevæger sig under de dele af enheden, som skal forsegles ( figur 1a ). For at ...

Watch this Scientific Journal Video about Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection at JoVE.com

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Denne procedure beskriver, hvordan man hurtigt initierer, udvider og forbinder neuritter organiseret i mikrofluidiske kamre ved anvendelse af poly-D-lysin-coatede perler fastgjort til mikropipetter, som styrer neuritforlængelse.

Rewiring Neuronal Circuits: En Ny Metode til Hurtig Neuritforlængelse og Funktionel Neuronal Forbindelse

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Hurtig Mikro-iontophoresis af glutamat og GABA: et nyttigt redskab til Undersøge Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

A Procedure til implantering Organized Arrays af Microwires for Single-unit optagelser i Vågn op, fungerende dyr

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Inducerende plasticitet astrocytisk receptorer ved manipulation af neuronfyring priser

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Kronisk transkranial elektrisk Stimulation og Intracortical optagelse i rotter

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

Ved at kombinere Optogenetics med kunstige MicroRNA at karakterisere effekter af genet Knockdown på præsynaptiske funktion inden for intakt Neuronal kredsløb

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Optogenetic medrivning af hippocampus Theta svingninger i opfører sig mus

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

En metode til 3D rekonstruktion og Virtual Reality analyse af glial og neuronal celler

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In Vitro Wedge Slice Forberedelse til efterligne In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...