Name: two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation
Uploaded: 2014-05-12T22:00:00
Description: Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

Vi takker James Perna for den grafiske illustrasjonen. Dette arbeidet ble støttet med tilskudd fra National Institute of Mental Health til YZ

Godkjenning må hentes fra hjemmeinstitusjoner før oppstart av kirurgi og avbildning studie. Eksperimenter er beskrevet i dette manuskriptet ble utført i samsvar med de retningslinjer og forskrifter fra University of California, Santa Cruz Institutional Animal Care og bruk komité. En. Kirurgi <ol><li> Autoklaver alle kirurgiske instrumenter og sterilisere arbeidsområdet med 70% alkohol grundig før operasjonen. </li><li> Anesthetize mus ved intraperitoneal (IP) injeksjon av KX anestesi...

<ol>
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For å få et vellykket tynnet-skallen forberedelse, flere trinn i denne protokollen er avgjørende. 1) Tykkelsen av skallen. Den kraniebenet har en sandwichkonstruksjon, med to lag av høy tetthet kompakt ben og et midtre lag av lav-densitet svampaktig ben. Mens høyhastighets mikrobore er egnet for å fjerne de ytre lag av kompakt ben og svampaktig ben, er den mikroblad ideell for tynning det indre laget av kompakt ben. Etter hvert som tykkelsen og stivheten av skallen øker under utvikling, avbildning av voksne mus t...

Pattedyr cortex deltar i mange hjernefunksjoner, fra sanseinntrykk og bevegelseskontroll til abstrakt informasjonsbehandling og kognisjon. Ulike kortikale funksjoner bygger på ulike nevrale kretser, som er laget av forskjellige typer nevroner som kommuniserer og utveksler informasjon på enkelte synapser. Struktur og funksjon av synapser er konsekvent blir endret som følge av erfaringer og patologi. I den modne hjernen, tar synaptisk plastisitet form av både styrke endringer og tillegg / fjerning av synapser, som spiller en viktig rolle i dannelsen og opprettholdelsen av en funksjonell nevrale kretser. Dendrittutløperne er de postsynaptiske komponenter i de fleste eksitatoriske synapser i hjernen hos pattedyr. Den konstante omsetning og morfologiske endringer av pigger er antatt å fungere som en god indikator for endringer i synaptiske forbindelser 1-7. To-foton laserskanning microscopy tilbyr dyp penetrasjon gjennom tykk, ugjennomsiktig forberedelser og lav fototoksisitet, noe som gjør den egnet for live bildebehandling i den intakte hjerne åtte. I kombinasjon med fluorescerende merking, gir to-foton bildebehandling et kraftig verktøy for å kikke inn den levende hjernen og følg strukturell omorganisering ved enkelte synapser med høy romlig og tidsmessig oppløsning. Ulike metoder har blitt brukt til å forberede mus for live bildebehandling 9-13. Her beskriver vi en tynnet-skalle utarbeidelse av in vivo to-foton imaging å undersøke strukturelle plastisitet av postsynaptiske dendrittutløperne i muse cortex. Ved hjelp av denne tilnærmingen, har våre nyere studier avbildet et dynamisk bilde av dendrittiske ryggraden endringer i respons til motoriske læring Med økende tilgjengelighet av transgene dyr med fluorescensmerkede nevronale undergrupper og rask utvikling av in vivo merking teknikker, kan lignende prosedyrer som er beskrevet her også brukes til etterforskningte andre celletyper og kortikale regioner, kombinert med andre manipulasjoner, så vel som benyttes ved sykdomsmodeller 16-23.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ketamine</td>
			<td style="width:181px;">Bioniche Pharma</td>
			<td style="width:108px;">67457-034-10</td>
			<td style="width:775px;">Mixed with xylazine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Xylazine</td>
			<td style="width:181px;">Lloyd laboratories</td>
			<td style="width:108px;">139-236</td>
			<td>Mixed with ketamine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saline</td>
			<td style="width:181px;">Hospira</td>
			<td style="width:108px;">0409-7983-09</td>
			<td>0.9% NaCl for injection and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Razor blades</td>
			<td style="width:181px;">Electron microscopy sciences</td>
			<td style="width:108px;">72000</td>
			<td>Double-edge stainless steel razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Alcohol pads</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">06-669-62</td>
			<td>Sterile alcohol prep pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Eye ointment</td>
			<td style="width:181px;">Henry Schein</td>
			<td style="width:108px;">102-9470</td>
			<td>Petrolatum ophthalmic ointment sterile ocular lubricant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">High-speed micro drill</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">18000-17</td>
			<td>The high-speed micro drill is suitable for thinning the outer layer of compact bone and targeting a small area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Micro drill steel burrs</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">19007-14</td>
			<td>1.4 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Microsurgical blade</td>
			<td style="width:181px;">Surgistar</td>
			<td style="width:108px;">6961</td>
			<td>The microsurgical blade is suitable for thinning the inner layer of compact bone and middler layer of spongy bone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Cyanoacrylate glue</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">NC9062131</td>
			<td>Fix the head plate onto the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Suture</td>
			<td style="width:181px;">Havard Apparatus</td>
			<td style="width:108px;">510461</td>
			<td>Non-absorbale, sterile silk suture, 6-0 monofilament</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Dissecting microscope</td>
			<td>Olympus</td>
			<td style="width:108px;"></td>
			<td>SZ61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">CCD camera</td>
			<td>Infinity</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Two-photon microscope</td>
			<td style="width:181px;">Prairie Technologies</td>
			<td style="width:108px;"></td>
			<td style="width:775px;">Ultima IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">10X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 0.30, air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">60X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 1.1, IR permeable, water immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ti-sapphire laser</td>
			<td style="width:181px;">Spectra-Physics</td>
			<td style="width:108px;"></td>
			<td>Mai Tai HP</td>
		</tr>
	</tbody>
</table>

two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation

I pattedyr cortex, nevroner danner ekstremt kompliserte nettverk og utveksle informasjon ved synapser. Endringer i synaptisk styrke, samt addisjon / fjerning av synapser, forekommer i en opplevelse avhengig måte, og gir den strukturelle grunnlag av nevronal plastisitet. Som postsynaptiske komponenter av de eksitatoriske synapser i cortex, er dendrittutløperne anses å være en god proxy av synapser. Tar fordelene med mus genetikk og fluorescerende merking teknikker, enkelte nerveceller og deres synaptiske strukturer kan merkes i den intakte hjerne. Her introduserer vi en transkranial avbildning protokollen ved hjelp av to-foton laserskanning mikros å følge fluorescensmerkede postsynaptiske dendrittutløperne over tid i vivo. Denne protokoll benytter en tynnet-skallen preparat, som holder hodeskallen intakt og unngår inflammatoriske effekter forårsaket av eksponering i hjernehinnene, og cortex. Derfor kan bildene bli utført umiddelbart etter s.rgery utføres. Den eksperimentelle fremgangsmåten kan utføres gjentatte ganger over forskjellige tidsintervaller som spenner fra timer til år. Anvendelsen av dette preparatet kan også utvides til å undersøke ulike kortikale regioner og lag, så vel som andre celletyper, under fysiologiske og patologiske tilstander.

I YFP-H linjen mus 25, uttrykker gul fluoriserende protein i en undergruppe av lag V pyramidale nevroner, som stikker sine apikale dendritter til de overfladiske lag i cortex. Gjennom tynnet-skallen preparat, kan de fluorescensmerkede dendrittiske segmenter bli gjentatte ganger avbildes i henhold til to-foton mikroskop over forskjellige bildeintervaller, alt fra timer til måneder. Her viser vi et eksempel på en firetids avbildning av de samme dendritter mer enn 8 dager i motor cortex av en 1 måned gamle mu...

Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

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

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

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

Research

JoVE Journal

Neuroscience

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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging av Mouse Spinal Cord Bruke to-foton Mikroskopi

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Påvisning av Microregional Hypoksi i Mus Cerebral Cortex av to-foton Imaging av endogen NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

En polert og forsterket tynnet-skalle Window for langsiktig Imaging av Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualisering og genetisk manipulering av dendritter og pigger i Mus hjernebarken og hippocampus hjelp<em&gt; I fosterlivet</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Tynnet-skull Cortical Window Teknikk for<em&gt; In Vivo</em&gt; Optisk koherens tomografi Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

I Vivo to-foton avbildning av erfaring-avhengige molekylære endringer i kortikale Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

To-foton Imaging av Cellular Dynamics i Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Samtidig To-foton<em&gt; I Vivo</em&gt; Imaging av synaptiske innganger og postsynaptiske mål i Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

I Vivo To-fotonet avbilding av kortikale nevroner i Neonatal mus

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...