Name: immunohistochemical visualization of hippocampal neuron activity after spatial learning in a mouse model of neurodevelopmental disorders
Uploaded: 2015-05-12T15:00:00
Description: Watch this Scientific Journal Video about Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders at JoVE.com

Vi vil gerne takke det administrative personale i CIBIO (University of Trento) og CNR Neuroscience Institute for assistance. Giovanni Provenzano understøttes af en ph.d.-stipendium fra Fondazione Veronesi (Milano, Italien). Dette arbejde blev finansieret af det italienske ministerium for universiteter og forskning (PRIN 2008 tilskud # 200894SYW2_002 og PRIN 2010-2011 tilskud # 2010N8PBAA_002 til YB), University of Trento (CIBIO opstart tilskud til YB) og Telethon Foundation (tilskud # GGP13034 til YB).

Alle forsøg blev udført i overensstemmelse med Det Europæiske Fællesskab direktiv 2010/63 / EU, og blev godkendt af det italienske sundhedsministerium. 1. Animal Care, Boliger og behandling <ol><li> Udfør alle forsøgsprotokoller bruger mus i overensstemmelse med retningslinjerne i den respektive institutionelle dyr pleje. </li><li> Oprethold dyr i en 12 timers lys / mørke-cyklus med foder og vand tilgængeligt ad libitum. </li><li> Hus musene i grupper på 2-4, i standard s...

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Her giver vi et frynsegode immunhistokemi protokol for at afsløre neuronal aktivering efter MWM i En2 - / - mus, en musemodel af neurologiske lidelser. Nedsatte niveauer af pERK blev påvist i CA3 underfelt af En2 - / - mutanter i forhold til WT. Til forskel fra, hvad der observeres i CA3 delfelt, en generel stigning på pERK-positive neuroner blev i stedet fundet i både hilus og GCL af En2 - / - mus i forhold til WT. En mulig forklaring på denne modsatte tendens i ERK fosforylering n...

Neurologiske lidelser omfatter en bred og heterogen gruppe af lidelser, såsom Downs syndrom, fragilt X syndrom (FXS), Rett syndrom, neurofibromatosis, knolde sklerose og ASD, hvor udviklingen og modningen af ​​centralnervesystemet (CNS) er forstyrret tidligt under prænatal perioden 1.. Disse udviklingsmæssige hjerne dysfunktioner kan forårsage dybe, livslang effekter på motorik, sprog, indlæring og hukommelse proces. Et væld af genetiske og miljømæssige faktorer er blevet impliceret i patogenesen af neurologiske lidelser i de seneste par år 2,3. Selv om de molekylære mekanismer bag den kliniske fænotype forbliver ukendte, har de ovennævnte konstateringer muliggjorde udviklingen af ​​adskillige musemodeller for disse lidelser. Indlærings- og hukommelsessvigt er blevet identificeret i en række af disse musemodeller såsom TSC1 +/-, TSC2 +/-,NF1 +/- og En2 - / - mus 2,4-7. En vigtig udfordring i forbindelse med forstyrrelser i nervesystemets udvikling er identifikationen af ​​cellulære og molekylære processer bag hukommelse og læring dysfunktion. Udvalgte signalveje aktiveres under indlæring eller hukommelse kan inducere transkription af specifikke gener og endelig føre til de novo-proteinsyntese. Umiddelbare-tidlige gener (IEGs) aktivering og protein-afhængige synaptiske modifikationer hurtigt induceret i hjernens neuroner som reaktion på neuronal aktivitet og adfærdsmæssige uddannelse 8,9. Underskud i signalveje, der involverer neurofibromin er blevet forbundet med nedsat læring i neurologiske lidelser. Neurofibromin er produktet af NF1 genet, hvis mutation forårsager neurofibromatosis type 1, en kompleks genetisk syndrom karakteriseret ved nervesystemet tumorer, adfærdsmæssige og motoriske forsinkelser og kognitiv DISApligtelser 10. Mus heterozygot for NF1 sletning begrænset til hæmmende neuroner viser underskud i den tidlige fase af langsigtet potensering (LTP), samt kompromitteret rumlig indlæring i MWM 5,11,12. Interessant NF1 mangel i denne musemodel fører til en over-aktivering af Ras-signalering i inhibitoriske interneuroner under indlæring, hvilket resulterer i øget ERK phosphorylering og endelig i en unormal forøgelse af GABA-frigivelse fra disse neuroner 5. Baseret på disse resultater, visualiseringen af ​​neuronal aktivitet efter adfærdsmæssige opgaver repræsenterer en måde til at rekonstruere bestemte kredsløb, der er involveret i neurologiske sygdomme. Den immunhistokemi-protokollen beskrevet her har til formål at vurdere og kvantificere hippocampus ERK phosphorylerings- niveauer efter MWM i ASF musemodel med kognitive mangler. MWM er almindeligt anvendt til at undersøge hippocampus afhængige rumlig indlæring og hukommelse hos gnavere 13,14 </sop&gt;. Vi beslutter at bruge ERK fosforylering som molekylær udlæsning af opgaven afhængige hippocampus læring, da ERK blev vist at have en væsentlig rolle i indlæring og hukommelse dannelse 15. Desuden er det nødvendigt for erfaring-afhængig plasticitet i det udviklende visuelle cortex 16 ERK-vejen. Endelig mus, som mangler en af de to ERK isoformer (ERK2) i CNS udviser markante uregelmæssigheder i kognitive, emotionelle og sociale adfærd 17, hvilket indikerer, at ERK-signalering kan spille en kritisk rolle i patogenesen af neurologiske lidelser, såsom ASD. Vi brugte Engrailed 2 knockout (En2 - / -) mus som en model for neurologiske lidelser. En2 - / - mus viser anatomiske og adfærdsmæssige &quot;ASD-lignende&quot; funktioner, herunder tab af forhjernen interneuroner 18, reduceret ekspression af ASD-relaterede gener 19, nedsat selskabelighed, og nedsat kognitiv fleksibilitet 6,7,20. Rumlig learning og hukommelse defekter, såsom dem fundet i MWM, er særligt robust i En2 - / - mus 6,7 og kan være relevante for de kognitive svækkelser observeret i ASD patienter 21. Endvidere viste vi, at svækket rumlig indlæring i MWM er forbundet med reduceret neurofibromin udtryk og øget frynsegode niveauer i hilus af En2 - / - voksne mus 7. Her præsenterer vi den detaljerede protokol for den immunhistokemiske karakterisering af pERK følgende MWM i denne ASD musemodel.

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	<tbody>
		<tr>
			<td>EthoVision XT 8</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td>This software platform is not a requirement &#8211; there are many other behavioral softwares on the market.</td>
		</tr>
		<tr>
			<td>Tempera Paint</td>
			<td>Giotto - Fila Group Company</td>
			<td></td>
			<td>White and liquid, non toxic. Used to prepare opaque water in the Morris water maze.</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>Equivalent models from other companies can be used.</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sigma</td>
			<td>CLS3524</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Fisher Scientific</td>
			<td>A406-20</td>
			<td>Used to make ethanol gradient for dehydration prior to slide mounting.</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>66004-950</td>
			<td>Toxic - to be used under hood. Change xylene every month depending on use.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>&#160;S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Hydrogen Peroxide</td>
			<td>Sigma</td>
			<td>H1009-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Abcam</td>
			<td>G9023-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ABC kit Vectastain&#160;</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>Add in a volume of 5 ml of PBS 2 drops of reagent A, mix and then add 2 drops of reagent B and mix.</td>
		</tr>
		<tr>
			<td>DAB peroxidase substrate</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>Add in a volume of 5 ml ddH2O: 2 drops of buffer stock solution and mix; 4 drops of DAB and mix; 2 drops of H2O2 and mix.</td>
		</tr>
		<tr>
			<td>pERK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>4370</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Biotinylated Goat Anti-Rabbit IgG Antibody</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>Dilution 1:250</td>
		</tr>
		<tr>
			<td>SuperFrost Slides&#160;</td>
			<td>Carl Roth</td>
			<td>1879</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-548-B</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX</td>
			<td>Sigma</td>
			<td>317616</td>
			<td>Mounting medium for slides. Equivalent mounting medium can be used.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>&#160;Zeiss&#160;</td>
			<td>Axio Imager.M2</td>
			<td>Equivalent microscope can be used.</td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe Systems, San Jose, CA</td>
			<td></td>
			<td>To assemble images.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institute of Health</td>
			<td></td>
			<td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>SigmaPlot 11.0</td>
			<td>Systat Software Inc. (USA)</td>
			<td></td>
			<td>Equivalent softwares for statistical analysis can be used.</td>
		</tr>
		<tr>
			<td>Prism 6</td>
			<td>GraphPad Software, Inc. (La Jolla, CA, USA)</td>
			<td></td>
			<td>Equivalent softwares for statistical analysis can be used.</td>
		</tr>
	</tbody>
</table>

immunohistochemical visualization of hippocampal neuron activity after spatial learning in a mouse model of neurodevelopmental disorders

Induktion af phosphoryleret ekstracellulær-regulerede kinase (pERK) er en pålidelig molekylær udlæsning af læring-afhængig neuronal aktivering. Her beskriver vi et frynsegode immunhistokemi protokol til at studere profilen af ​​hippocampus neuron aktivering efter udsættelse for en rumlig opgave læring i en musemodel præget af kognitive underskud neurologisk oprindelse. Konkret vi brugte pERK immunfarvning at studere neuronal aktivering efter Morris water maze (MWM, en klassisk hippocampus-afhængig læring opgave) i Engrailed-2 knockout (En2 - / -) mus, en model af autisme spektrum forstyrrelser (ASF). I forhold til vildtype (WT) kontroller, En2 - / - mus viste signifikante rumlige læring underskud i MWM. Efter MWM, blev påvist signifikante forskelle i antallet af pERK-positive neuroner i hippocampus specifikke underfelter af En2 - / - mus sammenlignet med WT dyr. Således kan vores protokol håndfast detektere forskelle ipERK-positive neuroner, der er forbundet til hippocampus-afhængig indlæring nyrefunktion i en musemodel af ASD. Mere generelt kan vores protokol anvendes til at undersøge profilen af ​​hippocampale neuron aktivering i genetiske eller farmakologiske musemodeller begge karakteriseret ved kognitive mangler.

Den her beskrevne protokol blev udviklet til at visualisere, ved immunhistokemi, ekspressionen af ​​en specifik markør for hippocampus neuron aktivitet efter MWM i en musemodel af neurologiske lidelser. Alle de eksperimentelle data vist heri, blev taget fra vores seneste arbejde 7. Mandlige og kvindelige WT og En2 - / - aldersmatchede voksne kuld (3-5 måneder gammel, vægt = 25-35 g) opnået fra heterozygote parringer blev anvendt. Toogtyve mus (11 pr genotype) blev anvendt til MWM. Otte mus ...

Watch this Scientific Journal Video about Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders at JoVE.com

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Vi beskriver en immunhistokemisk protokol til at studere profilen af ​​hippocampus neuron aktivering efter udsættelse for en rumlig opgave læring i en musemodel præget af kognitive underskud neurologisk oprindelse. Denne protokol kan anvendes til genetiske eller farmakologiske musemodeller begge karakteriseret ved kognitive underskud.

Immunhistokemisk Visualisering af Hippocampal Neuron aktivitet efter Spatial Læring i en musemodel af forstyrrelser i nervesystemets udvikling

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

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Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Hippocampale Insulin Mikroinjektion og<em&gt; In vivo</em&gt; Mikrodialyse Under Spatial Memory Test

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Kvantitativ analyse af Klatring Defekter i en Drosophila Model af neurodegenerative lidelser

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Sex Forskelle i Mus Hippocampale Astrocytter efter<em&gt; In-vitro</em&gt; Iskæmi

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

High Resolution Kvantitativ Synaptic Proteome profilering Mouse Brain Regioner Efter Auditiv diskrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Low-Density Primary Hippocampal Neuron Culture

Low-Density Primær Hippocampal neuronkultur

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Vurdering Rumlig læring og hukommelse i små Squamate Krybdyr

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Brugen af Trace Eyeblink klassisk konditionering at vurdere hippocampus dysfunktion i en rotte Model af føtale alkohol spektrum forstyrrelser

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 ...