Name: ablation of a neuronal population using a two-photon laser and its assessment using calcium imaging and behavioral recording in zebrafish larvae
Uploaded: 2018-06-02T13:00:00
Description: Watch this Scientific Journal Video about Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae at JoVE.com

Disse studiene ble finansiert av tilskudd mottatt fra MEXT, JSP KAKENHI Grant tall JP25290009, JP25650120, JP17K07494 og JP17H05984.

1. ablasjon av en subpopulasjon av Neurons bruker en to-fotonet Laser mikroskopet
Merk: Hvis brukere planlegger å utføre Ca tenkelig følgende ablasjon, bruke UAShspzGCaMP6s linje21. Hvis brukere planlegger utfører atferdsmessige opptak etter ablasjon, bruk UAS:EGFP linjen, ablation EGFP-positive celler er enklere å utføre enn GCaMP6s-uttrykke celler.
<ol><li>Begynn med å sette opp mating Gal4 linjen som merker bestemt neurons studier og UAS:EGF...

<ol>
	<li>Feierstein, C. E., Portugues, R., Orger, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+whole+picture:+A+comprehensive+imaging+approach+to+functional+mapping+of+circuits+in+behaving+zebrafish.">Seeing the whole picture: A comprehensive imaging approach to functional mapping of circuits in behaving zebrafish.</a> Neuroscience. 296, 26-38 (2015).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+Ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nat Biotechnol. 19 (2), 137-141 (2001).</li><li>Muto, A., 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=Genetic+visualization+with+an+improved+GCaMP+calcium+indicator+reveals+spatiotemporal+activation+of+the+spinal+motor+neurons+in+zebrafish.">Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish.</a> Proc Natl Acad Sci U S A. 108 (13), 5425-5430 (2011).</li><li>Lorent, K., Liu, K. S., Fetcho, J. R., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+space+cadet+gene+controls+axonal+pathfinding+of+neurons+that+modulate+fast+turning+movements.">The zebrafish space cadet gene controls axonal pathfinding of neurons that modulate fast turning movements.</a> Development. 128 (11), 2131-2142 (2001).</li><li>Asakawa, K., 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=Genetic+dissection+of+neural+circuits+by+Tol2+transposon-mediated+Gal4+gene+and+enhancer+trapping+in+zebrafish.">Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.</a> Proc Natl Acad Sci U S A. 105 (4), 1255-1260 (2008).</li><li>Sternberg, J. 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H., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+visually+guided+behavior+by+distinct+populations+of+spinal+projection+neurons.">Control of visually guided behavior by distinct populations of spinal projection neurons.</a> Nat Neurosci. 11 (3), 327-333 (2008).</li><li>Huang, K. H., Ahrens, M. B., Dunn, T. W., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+projection+neurons+control+turning+behaviors+in+zebrafish.">Spinal projection neurons control turning behaviors in zebrafish.</a> Curr Biol. 23 (16), 1566-1573 (2013).</li><li>Smear, M. C., 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=Vesicular+glutamate+transport+at+a+central+synapse+limits+the+acuity+of+visual+perception+in+zebrafish.">Vesicular glutamate transport at a central synapse limits the acuity of visual perception in zebrafish.</a> Neuron. 53 (1), 65-77 (2007).</li><li>Bianco, I. H., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visuomotor+transformations+underlying+hunting+behavior+in+zebrafish.">Visuomotor transformations underlying hunting behavior in zebrafish.</a> Curr Biol. 25 (7), 831-846 (2015).</li><li>Trivedi, C. A., Bollmann, J. 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En utmerket romlig oppløsning for å ablate spesielt individuelle neurons har to-fotonet laser bør stor forsiktighet utvises å unngå uønskede skade på hjernevev på grunn av varme. Viktigste i ablasjon eksperimentet er å finne den optimale mengden av laser irradiation. Utilstrekkelig bestråling ikke drepe neurons. For mye bestråling vil varme-skade de omkringliggende vev, som vil resultere i uønskede effekter. Det optimale området av laser irradiation (områder av ROIs, antall gjentakelser og skanning fart) sy...

For å forstå hvordan problemet oppstår fra nevrale aktivitet i hjernen, er det nødvendig å identifisere nevrale kretser som er involvert i generasjon adferden. På larvestadiet, sebrafisk gir en ideell dyremodell for studere hjernefunksjon knyttet virkemåten fordi liten, gjennomsiktig hjernen gjør det mulig å undersøke neuronal aktivitet mobilnettet oppløsning i bredt område av den hjernen mens observere atferd1. Bildebehandling neuronal aktivitet i bestemte neurons har blitt mulig gjennom oppfinnelsen av genetisk kodet kalsium (Ca) indikatorer (GECIs) som GCaMP2. GCaMP transgene sebrafisk har vist seg for å være nyttig for å knytte funksjonelle nevrale krets med atferd ved å gjennomføre Ca imaging i oppføre dyr3.
Mens Ca bildebehandling kan vise sammenhenger mellom neuronal aktivitet og atferd, for å vise kausalitet, undertrykkelse av neuronal aktivitet og teste sin consequence(s) oppførsel er viktige skritt. Det finnes ulike måter å oppnå dette: bruk av genetisk mutasjon som endrer bestemte nevrale kretser4, uttrykk for nervegifter i bestemte neurons5,6, bruk av optogenetic verktøy som halorhodopsin7, og laser ablasjon målrettet neurons8,9. Laser ablasjon er spesielt egnet for å fjerne aktiviteten i et relativt lite antall bestemte neurons. Uhelbredelig eliminering av neuronal aktivitet ved å drepe neurons forenkler vurdere atferdsmessige konsekvenser.
En interessant atferd som kan observeres på larvestadiet i sebrafisk er byttedyr fange (figur 1A). Dette visuelt-guidede, målrettet gir en gunstig eksperimentelle system for studier av synsskarphet10, visuomotor transformasjon11,12,13, visuell persepsjon og anerkjennelse av objekter,14,,15,,16,,17,,18, og beslutningsprosesser19. Hvordan bytte er anerkjent av rovdyr og hvordan byttedyr oppdagelsen fører til byttedyr fanger oppførsel har vært et sentralt spørsmål i neuroethology20. I dette papiret, vi fokusere på rollen pretecto-hypothalamus krets dannet av projeksjoner av en kjerne i Pretetto (kjernen pretectalis superficialis pars magnocellularis, heretter, bare kjent som Pretetto) å ILH. Laser-ablasjon av Pretetto ble vist å redusere byttedyr fange aktivitet og avskaffe neuronal aktivitet i ILH som er forbundet med den visuelle byttedyr oppfatning21. Her, laser protokoller for å utføre ablasjon og teste dens effekt ved hjelp av Ca2 + imaging og atferdsmessige opptak i sebrafisk Larvene er beskrevet.

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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">NuSieve GTG Agarose</td>
			<td style="width:223px;">Lonza</td>
			<td style="width:361px;">Cat.#50080</td>
			<td style="width:383px;">low-melting temperature agarose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">6 cm petri dish</td>
			<td>FALCON</td>
			<td>Product#:351007</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">dissecting needle</td>
			<td>AS ONE Corporation</td>
			<td>Cat. No. 2-013-01</td>
			<td>https://keystone-lab.com/en/item/detail/404142</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LSM7MP</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>two-photon laser scanning microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">W Plan-Apochromat 63x/1.0</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>63X objective lens</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Imager.Z1</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>an epi-fluorescence microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ZEN</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Image acquisition software for confocal microscopes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Secure-Seal Hybridization Chamber Gasket, 8 chambers, 9 mm diameter x 0.8 mm depth</td>
			<td>Molecular Probes</td>
			<td>Catalogue # S-24732</td>
			<td>Used as a recording chamber in Ca imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Imageing Chambers</td>
			<td>Grace Bio-Labs</td>
			<td>CoverWell Imaging Chambers PCI-A-2.5</td>
			<td>Used as a behavioral recording chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">surgical knife</td>
			<td>MANI</td>
			<td>Ophthalmic knife MST15</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ORCA-Flash4.0</td>
			<td>Hamamatsu Photonics</td>
			<td>model:C11440-22CU</td>
			<td>a scientific CMOS camera</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:316px;">HCImage</td>
			<td>Hamamatsu Photonics</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">image acuisition software</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Hard Disk Recording module</td>
			<td>Hamamatsu Photonics</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">An software module that enables saving the movie files onto a hard disc drive in a short time</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SZX7</td>
			<td>Olympus</td>
			<td></td>
			<td>stereoscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DF PL 0.5X</td>
			<td>Olympus</td>
			<td></td>
			<td>objective lens for SZX7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Point Grey Grasshopper3 4.1 MP Mono USB3 Visio</td>
			<td>FLIR Systems, Inc.</td>
			<td>Product No. GS3-U3-41C6NIR-C</td>
			<td>CMOS camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XIMEA xiQ camera</td>
			<td>XIMEA</td>
			<td>Product No. MQ042RG-CM</td>
			<td>CMOS camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">a ring LED light</td>
			<td>CCS</td>
			<td>Model: LDR2-100SW2-LA</td>
			<td>White LED</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:316px;"></td>
			<td style="width:223px;"></td>
			<td style="width:361px;"></td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nylon mesh 32&#181;m</td>
			<td>Tokyo Screen</td>
			<td>N-No.380T</td>
			<td>http://www.tokyo-screen.com/cms/sta20347/</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Nylon mesh 13&#181;m</td>
			<td>Tokyo Screen</td>
			<td>N-No. 508T-K</td>
			<td style="width:383px;">http://www.tokyo-screen.com/cms/sta20347/</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:316px;">Metal seive 150 micron aperture</td>
			<td>Tokyo Screen</td>
			<td style="width:361px;"></td>
			<td style="width:383px;">http://www.tokyo-screen.com/cms/sta20341/#ami</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">Metal seive 75 micron aperture</td>
			<td>Tokyo Screen</td>
			<td></td>
			<td>http://www.tokyo-screen.com/cms/sta20341/#ami</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">EBIOS</td>
			<td>Asahi Food &#38; Healthcare, Co. Ltd.</td>
			<td></td>
			<td>dry beer yeast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">LabVIEW</td>
			<td>National Instruments</td>
			<td></td>
			<td>an integrated development environment for programming</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:316px;">Mai-Tai HP</td>
			<td>Spectra Physics&#160;</td>
			<td></td>
			<td>two-photon laser&#160;</td>
		</tr>
	</tbody>
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ablation of a neuronal population using a two-photon laser and its assessment using calcium imaging and behavioral recording in zebrafish larvae

For å identifisere rollen som en subpopulasjon av nerveceller i oppførsel, er det viktig å teste konsekvensene av blokkere sin aktivitet i levende dyr. Laser ablasjon neurons er en effektiv metode for dette formålet når neurons er selektivt merket med fluorescerende sonder. Studien, er protokoller for laser ablating en subpopulasjon av neurons bruker to-fotonet mikroskop og testing av funksjonelle og atferdsmessige konsekvensene beskrevet. I denne studien brukes byttedyr fange atferd i sebrafisk larver som studien modell. Pretecto-hypothalamus krets kjent ligger denne visuelt-drevet byttedyr fanger atferd. Sebrafisk Pretetto var laser-ablated, og nevrale aktivitet i dårligere flik av hypothalamus (ILH, målet for pretectal projeksjon) ble undersøkt. Byttedyr fange virkemåte etter pretectal ablasjon ble også testet.

Bestemt neurons var genetisk benevnt med enten EGFP eller GCaMP6s, med uttrykk ble drevet i Gal4 linjer. En Gal4 linje gSAIzGFFM119B ble brukt til å merke en kjerne i pretectal området (magnocellular overfladisk pretectal kjernen), og en subpopulasjon av olfactory pære neurons. En annen Gal4 linje, hspGFFDMC76A, ble brukt til å merke ILH. Vi laser-ablated pretectal neurons bilateralt (figur 2A venstre panel) og også ablated nerveceller i...

Watch this Scientific Journal Video about Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae at JoVE.com

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

Her presenterer vi en protokoll for å ablate en genetisk merket subpopulasjon av neurons av en to-fotonet laser fra sebrafisk larver.

Ablasjon av Neuronal innbyggere med en to-fotonet Laser og sin vurdering ved hjelp av kalsium Imaging og atferdsmessige opptak i sebrafisk larver

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Research

JoVE Journal

Neuroscience

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

Automatiserte høy gjennomstrømming Behavioral Analyser i sebrafisk Larver

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

To-Photon<em&gt; In vivo</em&gt; Imaging av dendrittutløperne i Mouse Cortex Bruke en Tynnet-skalle Forberedelse

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Langvarig Inkubasjon av akutt nervevev for Elektro og kalsium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

To-fotonet kalsium Imaging i Neuronal Dendrites i hjernen skiver

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Kalsiumavbildning i musen dårligere oliven

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Evaluering og manipulering av nevral aktivitet ved bruk av to-foton holografisk mikroskopi

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

In Vivo Wide-Field og Two-Photon Calcium Imaging fra en mus ved hjelp av et stort kranialt vindu

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

Intravital avbildning av fluorescerende proteinuttrykk hos mus med en lukket skalle traumatisk hjerneskade og kranialvindu ved bruk av et to-foton mikroskop

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Advancements in Adult Zebrafish Brain Research (Video) | JoVE

To-foton kalsiumavbildning av forhjerneaktivitet i oppførsel av voksen sebrafisk

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used ...