Name: electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo
Uploaded: 2013-05-29T13:00:00
Description: Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

<p class="jove_content"&gt; Vi takker Dr. Yuval Gottlieb-Dror til elektroporation illustration. Dette arbejde blev støttet af tilskud til DSD fra The National Institute for Psychobiology i Israel og fra Niedersachsen-Israel Research samarbejdsprogram og ved tilskud til AK fra The Israel Science Foundation, The Israel sundhedsministeriet, og The Center of Excellence-Legacy Heritage Biomedical Science partnerskab.</p&gt;

1.. Baghjernen Elektroporation 1.1 Egg håndtering <ol><li> Placer æggene vandret i en befugtet inkubator (37 til 38,5 ° C). Embryoner elektroporeres efter 65-70 timers inkubation når de når 16-17 (HH) etape (25-30 somitter). </li><li> Tag æggene fra rugemaskinen, de forbliver i vandret position. </li></ol> 1.2 Forberedelser <ol><li> Træk glaskapillarer (0,5 mm i diameter). </li><li> Slut bøjede L-formede guld Genetrodes elektroder (3 mm diameter) med en adapte...

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I ovo elektroporation er en realistisk, pålidelig og effektivt redskab til at undersøge celle specifikation og axonal vejledning i chick nervesystem udvikling 20.. I denne protokol beskriver vi en tilstand af elektroporation i chick baghjerne på E2.75 hjælp enhancerelementer som muliggør den betingede mærkning af specifikke interneuroner. Denne strategi er kombineret med det PiggyBac-medierede gennemførelse system til at indsætte fremmede gener i chick genomet, hvilket muliggør sporing af ax...

Baghjerne repræsenterer en vigtig relay hub af nervesystemet ved at kommunikere mellem de centrale og perifere nervesystem via stigende og faldende neuronale netværk. Det regulerer de grundlæggende funktioner, herunder respiration, bevidsthed, hørelse og motorik 1-3. I den tidlige fosterudvikling, er hvirveldyr baghjerne forbigående opdelt langs dens anterior-posterior (AP) akse i gentagne rhombomeres, hvor distinkte neuronale celletyper dannes og generere flere hjernestammen kerner centre 4.. Baghjerne er også opdelt langs dens ryg-ventrale (DV) akse i et basal og Alar plade, hvor diskrete neuronale forfædre bliver specificeret og differentiere forskellige DV steder 3,5,6. Hvordan den tidlige AP og DV-specifikke neuronale mønstre for oprettelse af funktionelle hjernestammen circuitries er stort set ukendt. At få viden om denne grundlæggendeSpørgsmålet er værktøjer der kræves for at mærke specifikke delmængder af neuroner i den tidlige baghjerne og spore deres axonale baner og tilslutningsmuligheder til mere fremskredne stadier. Vi har tidligere anvendt specifikke enhancerelementer, og en Cre / LoxP-baserede betinget ekspressionssystem til sporing axonal bane af dorsale spinale interneuroner i det tidlige hønseembryo 7-9. I den nuværende manuskript, vi har målrettet baghjerne og opgraderet den eksperimentelle paradigme for mærkning sene embryonale baghjernen interneuroner, axoner og deres synaptiske mål, ved hjælp af en modificeret elektroporation strategien og PiggyBac - medieret DNA gennemførelse. Vores nye strategi giver mulighed for mærkning af distinkte neuronale undertyper i den ene side af baghjerne og sporing af deres axonale fremskrivninger og synaptiske sites på forskellige fosterstadiet, fra 2 op til 12 dage efter elektroporation. Baseret på denne metode, mærkede vi den dorsale-de fleste undergruppe af baghjernen interneuroner (dA1/Atoh1 + </sup&gt; celler) og afslørede to kontralaterale opstigende axonale projektion mønstre, der hver stammer fra en anden AP placering og elongates i en særskilt funiculus. DA1 axoner blev fundet projekt og danner synapser i den auditive kerner, midthjernen og i flere lag af cerebellum 10.. Kombinationen af ​​chick elektroporation, genetiske sporing af neuroner og analyse af projektions steder på meget fremskredne stadier af udvikling giver en unik platform for at studere dannelsen af ​​neuronale netværk i hjernen og at belyse molekylære mekanismer, som regulerer kredsløb dannelse.

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electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo

Elektroporation af kylling embryonale neuralrøret har mange fordele, såsom at være hurtig og effektiv for ekspressionen af ​​fremmede gener i neuronale celler. I dette manuskript giver vi en metode, der viser entydigt, hvordan man elektroporere DNA i aviær baghjerne på E2.75 for at specifikt mærke en delmængde af neuronale stamceller og hvordan man følge deres axonale fremskrivninger og synaptiske mål til meget fremskredne stadier af udvikling, op til E14.5. Vi har udnyttet nye genetiske værktøjer, herunder specifikke enhancerelementer, Cre / Lox - baserede plasmider og PiggyBac-medieret DNA gennemførelse system til at drive GFP udtryk i en undertype af baghjernen celler (dorsale mest undergruppe af interneuroner, DA1). Axonale baner og mål for DA1 axoner følges på tidlige og sene embryonale stadier på forskellige hjernestammen regioner. Denne strategi bidrager avancerede teknikker til at målrette celler af interesse i den embryonale baghjerne og trasiering kredsløb dannelse på flere stadier af udvikling.

Denne protokol blev for nylig anvendt til at afdække de axonale mønstre og projektion lokaliteter af DA1 undergruppe af interneuroner i chick baghjerne 10.. Til specifikt mærke disse axoner blev et enhancerelement (Atoh1), der tidligere har været karakteriseret som specifikke for spinal DI1 neuroner 8,12,13, bekræftede at blive udtrykt i baghjernen DA1 celler 10. Elementet blev klonet opstrøms til Cre rekombinase og co-elektroporeres på E2.75 sammen med en Cre afhængig cytoplasma...

Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Hvor neuronale netværk er etableret i embryonale hjernen er et grundlæggende spørgsmål i udviklingspsykologi neurobiologi. Her har vi kombineret en elektroporation teknik med nye genetiske værktøjer, såsom Cre / Lox-plasmider og PiggyBac-medieret DNA gennemførelse system i aviær baghjerne at mærke dorsale interneuroner og spore deres axonale fremskrivninger og synaptiske mål på forskellige udviklingsstadier.

Elektroporation af baghjerne at spore axonale baner og Synaptic Mål i Chick Embryo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

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Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

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Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

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