Name: 3-d imaging and analysis of neurons infected in vivo with toxoplasma gondii
Uploaded: 2014-12-09T15:00:00
Description: Watch this Scientific Journal Video about 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii at JoVE.com

Vi takker hele Koshy laboratorium til nyttige diskussioner. Vi takker Patty Jansma og University of Arizona Neuroscience afdeling for rådgivning og hjælp til billedbehandling. Vi takker også Porreca laboratorium for brug af deres Vibratome. Denne forskning blev støttet af det amerikanske National Institutes of Health (NIH NS065116, AAK).

BEMÆRK: Mus blev avlet og holdt i en temperatur og fugtighed kontrolleret rum med 12 timer vendes lys / mørke cykler med mad og vand tilgængeligt ad libitum på University of Arizona. Eksperimenter blev udført under retningslinier og godkendelse af Institutional Animal Care og brug Udvalg fra University of Arizona. Blev gjort alt for at minimere dyrenes lidelser. Cre-reporter mus er på en C57BL / 6 baggrund 11 og er kommercielt tilgængelige. 1. Mouse Infektion <p ...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+infected+with+low-virulence+strains+of+Toxoplasma+gondii+lose+their+innate+aversion+to+cat+urine,+even+after+extensive+parasite+clearance.">Mice infected with low-virulence strains of Toxoplasma gondii lose their innate aversion to cat urine, even after extensive parasite clearance.</a> PloS one. 8 (9), 75246 (2013).</li><li>Evans, A. K., Strassmann, P. S., Lee, I. -. P., Sapolsky, R. 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(53), (2011).</li><li>Koshy, A., Fouts, A., Lodoen, M., Alkan, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+secreting+Cre+recombinase+for+analysis+of+host-parasite+interactions.">Toxoplasma secreting Cre recombinase for analysis of host-parasite interactions.</a> Nat Methods. 7 (4), 307-309 (2010).</li><li>Koshy, A. a., Dietrich, H. 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=Toxoplasma+co-opts+host+cells+it+does+not+invade.">Toxoplasma co-opts host cells it does not invade.</a> PLoS pathog. 8 (7), (2012).</li><li>Madisen, L., Zwingman, T. 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=A+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain.">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nat Neurosci. 13 (1), 133-140 (2010).</li><li>Caffaro, C. E., Koshy, A. s., Liu, L., Zeiner, G. M., Hirschberg, C. B., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nucleotide+sugar+transporter+involved+in+glycosylation+of+the+Toxoplasma+tissue+cyst+wall+is+required+for+efficient+persistence+of+bradyzoites.">A nucleotide sugar transporter involved in glycosylation of the Toxoplasma tissue cyst wall is required for efficient persistence of bradyzoites.</a> PLoS pathog. 9 (5), (2013).</li><li>Saeij, J. P. J., Boyle, J. P., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+among+the+three+major+strains+of+Toxoplasma+gondii+and+their+specific+interactions+with+the+infected+host.">Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host.</a> Trends in parasitology. 21 (10), 476-481 (2005).</li><li>Dubey, J. P., Lindsay, D. 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Da celleforandringer i inficerede værtsceller har været knyttet til sygdoms resultater i infektioner med andre intracellulære organismer såsom HIV, Rabies, og Chlamydia 18,19, vi udviklet en teknik, der vil give os mulighed for at studere de intime samspil, der opstår mellem CNS værtscelle, og Toxoplasma. Den her beskrevne fremgangsmåde opnår dette mål ved at muliggøre effektiv billeddannelse af kronisk inficerede neuroner. Forud for udviklingen af ​​denne fremgangsmåde, såsom billedd...

Det overordnede mål med denne metode er at opnå en høj opløsning, tre-dimensionelle billeder af individuelle neuroner, der er smittet med obligat intracellulær parasit Toxoplasma gondii. Toxoplasma er ofte betragtes som en af de mest succesfulde parasitter på grund af sin store mellemliggende værtsområde, der omfatter mennesker og gnavere. I både mennesker og gnavere, efter akut infektion gennem indtagelse af forurenet mad eller vand, Toxoplasma er i stand til at forårsage en vedvarende infektion i CNS ved at konvertere fra sin hurtige replikerende formen (den tachyzoite) til dens langsomme-replikerende og encysting formular (den bradyzoite ). I immunkompetente individer er denne latent CNS-infektion menes at være relativt symptomfri, men i immunsvækkede personer, såsom AIDS-patienter eller transplanterede patienter, kan genopblussen af parasitten medføre fatal toxoplasmic hjernebetændelse 1,2. Desuden Nylige undersøgelser, have vist, at latent infektion med Toxoplasma kan medføre adfærdsændringer hos gnavere 3,4, selv om mekanismen er stadig ukendt. Overraskende, trods disse data fremhæver betydningen af CNS- Toxoplasma interaktion, relativt lidt om dette forhold, især på det cellulære og molekylære niveau. Evnen til at studere selv simple aspekter af interaktionen hjerne-parasit er blevet hæmmet delvist af Technologic begrænsninger. For eksempel størstedelen af arbejdet viser, at neuroner er de celler, i hvilke cyster resterer er blevet gjort med elektronmikroskopi (EM) 5,6. Selvom EM giver høj opløsning, er det tidskrævende, arbejdskrævende og dyrt. Immunofluorescens (IF) assays er for nylig blevet anvendt i forbindelse med konfokal mikroskopi for at bekræfte arbejde EM 7. Hvis analyser er teknisk let at udføre og relativt billig, men ved anvendelse af disse teknikker til understand det rumlige forhold mellem cyste og den inficerede neuron kræver seriel rekonstruktion, som er tidskrævende, teknisk vanskeligt, og kan føre til tab af værdifulde oplysninger. Vi har således udviklet en metode, der kan anvendes med musemodel for CNS toxoplasmose og tillader os at afbilde hele inficerede neuroner uden EM eller immunhistokemi (IHC). Ved at udvikle en sådan teknik, kan vi begynde at undersøge cellulære forholdet mellem den inficerede celle og cyste i en relativt hurtig og billig måde. Den metode, vi har udviklet kombinerer nyere teknikker til optisk clearing og billedbehandling tykke hjernesnit ved konfokal mikroskopi 8 med et system, der markerer in vivo-celler, der er blevet injiceret med parasit-proteiner 9,10. I dette system, vi inficere Cre-reporter mus, der udtrykker et grønt fluorescerende protein (GFP) efter Cre-medieret rekombination 11 med Toxoplasma </em&gt; Stammer, der udtrykker et rødt fluorescerende protein (RFP) og injicere Cre rekombinase i værtsceller 9. Denne kombination giver os mulighed for at høste den inficerede mus hjernen efter CNS-infektion er etableret, skæres tykke hjernesnit, og hurtigt at identificere relevante områder af billedet ved at finde RFP + cyster. Det er vigtigt at bemærke, at som værtscelle ekspression af GFP alene afhænger injektion af Cre af parasitter, og ikke på infektion, en række af GFP + -celler indeholder ikke parasitter 10. Da målet med denne protokol er at være i stand til at afbilde hele inficerede neuroner er fokus kun på GFP + neuroner, der også indeholder en RFP + cyste, men protokollen kan også bruges til at afbilde GFP + / RFP - neuroner. Når den inficerede hjerne høstes og snit, er afsnittene gøres gennemsigtigt ved glycerol clearing. Passende områder af sektioner derefter afbildes med konfokal mikroskopi, algende hidtil uset visualisering af inficerede værtsceller og de indkapslede parasitter i deres helhed. Her giver vi en komplet protokol til at identificere, optisk clearing, og billedbehandling smittet neuroner.

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			<td height="21" style="height:21px;width:469px;">Name of Reagent/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:1008px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Series 1000 Sectioning System</td>
			<td>Technical Products International, Inc.</td>
			<td></td>
			<td>Other vibratomes are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#1.5 Coverslips</td>
			<td>VWR</td>
			<td>48393 251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamond Scriber</td>
			<td>VWR</td>
			<td>52865-005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss LSM 510 Meta confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketaject&#174; Ketamine HCl Inj., USP 100mg/ml</td>
			<td>Western Medical Supply, Inc.</td>
			<td>4165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnaSed&#174; Injection Xylazine 20mg/ml</td>
			<td>Lloyd Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZsGreen Mice</td>
			<td>Jackson Laboratories</td>
			<td>7906</td>
			<td>B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical equipment</td>
			<td></td>
			<td></td>
			<td>Thumb forceps; Fine scissors-angled to side, sharp-sharp; Sharp-sharp scissors; Kelly hemostats; Mayo scissors; Micro spatula.</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Human Foreskin Fibroblasts (HFF) cells</td>
			<td></td>
			<td></td>
			<td>These are primary cells from human foreskins.&#160; We make these in-house but they may be purchased from outside vendors.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s High Glucose Modified Eagles Medium (DMEM)</td>
			<td>HyClone</td>
			<td>SH30081.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Penicillin Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">200mM L-alanyl-L-glutamine</td>
			<td>Corning</td>
			<td>25-015-Cl</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:469px;">25cm2 Canted neck flask</td>
			<td>Fisher Scientific</td>
			<td>1012639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 1X Without Calcium and Magnesium</td>
			<td>VWR</td>
			<td>45000-446</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline, 10X, USP Sterile Ultra Pure Grade</td>
			<td>amresco</td>
			<td>K813-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Fetal Bovine Serum</td>
			<td style="width:265px;">Gibco</td>
			<td style="width:135px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Bright-Line Hemocytometer</td>
			<td style="width:265px;">Sigma-aldrich</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Brain Slicer Matrix</td>
			<td>Zivic Instruments</td>
			<td>BSMAS005-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td style="width:135px;">BP358-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td style="width:265px;">Sigma-aldrich</td>
			<td style="width:135px;">H3393-100KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Paraformaldehyde</td>
			<td style="width:265px;">Fisher Scientific</td>
			<td style="width:135px;">O4042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">20ml Disposable Scintillation Vials</td>
			<td style="width:265px;">Fisher Scientific</td>
			<td style="width:135px;">FS74500-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol, Ethyl, 95%, 190 Proof</td>
			<td style="width:265px;">In-house</td>
			<td>17212945</td>
			<td>This product is purchased from an in-house stockroom.&#160; Other companies are compatible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Imaris Software</td>
			<td style="width:265px;">Bitplane</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Clear nail polish</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Other brands are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">10ml Syringe with Luer-Lok</td>
			<td style="width:265px;">VWR</td>
			<td>BD309604</td>
			<td>Other syringes are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Three-way Stopcock</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Hypodermic needle</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible - used to pin down mouse.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cell Scraper</td>
			<td style="width:265px;"></td>
			<td style="width:135px;"></td>
			<td>Any brand is compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25G x 12&#34; Tubing, Safety Blood Collection Set, with Luer Adapter</td>
			<td style="width:265px;">Greiner Bio-One</td>
			<td style="width:135px;">450099</td>
			<td>Other brands are compatible</td>
		</tr>
	</tbody>
</table>

3-d imaging and analysis of neurons infected in vivo with toxoplasma gondii

Toxoplasma gondii er en obligat, intracellulær parasit med et bredt værtsområde, herunder mennesker og gnavere. I både mennesker og gnavere, Toxoplasma etablerer en livslang vedvarende infektion i hjernen. Mens denne hjerne infektion er asymptomatisk i de fleste immunkompetente personer, i fosteret eller immunkompromitterede personer, såsom erhvervet immundefektsyndrom (AIDS) patienter denne forkærlighed for og vedholdenhed i hjernen udvikler kan føre til ødelæggende neurologisk sygdom. Således er det klart, at hjerne- Toxoplasma interaktion er kritisk for symptomatisk sygdom forårsaget af Toxoplasma, men vi har lidt forståelse af den cellulære eller molekylære vekselvirkning mellem celler i det centrale nervesystem (CNS) og parasitten. I musemodel for CNS Toxoplasmose det har været kendt i over 30 år, som neuroner er de celler, hvor parasitten varer ved, men få oplysninger om, hvilkedel af neuron er generelt inficeret (soma, dendritceller, axon), og hvis denne cellulære forhold ændrer mellem stammer. I del, denne mangel er sekundær til vanskeligheden ved billedbehandling og visualisering hele inficerede neuroner fra et dyr. Sådanne billeder vil typisk kræve seriel sektionering og syning af væv afbildet ved elektronmikroskopi eller konfokal mikroskopi efter immunfarvning. Ved at kombinere flere teknikker, den her beskrevne fremgangsmåde muliggør anvendelse af tykke sektioner (160 um) for at identificere og billede hele celler, der indeholder cyster, så tredimensional visualisering og analyse af de enkelte, kronisk inficerede neuroner uden behov for immunfarvning, elektronmikroskopi eller seriel sektionering og syning. Ved hjælp af denne teknik, kan vi begynde at forstå den cellulære forhold mellem parasit og den inficerede neuron.

Figur 7 indeholder repræsentative billeder af to cyste indeholdende GFP + neuroner fra to forskellige 160 um tykke sektioner samt en repræsentativ måling af afstanden fra cyste-til-celle-organ for figur 7B. Figur 7 A og B viser, at denne nye protokol giver mulighed for visualisering af det inficerede neuron i sin helhed. Figur 7C viser, at med denne billeddannelse teknik, er det nu muligt at kvantificere afstanden mellem cyste og cellen le...

Watch this Scientific Journal Video about 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii at JoVE.com

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Ved hjælp af denne protokol, var vi i stand til at afbilde 160 um tyk hjerne afsnit fra mus inficeret med parasitten Toxoplasma gondii, som gør det muligt visualisering og analyse af det rumlige forhold mellem encysting parasitten og den inficerede neuron.

3-D Imaging og analyse af neuroner Inficerede<em&gt; In Vivo</em&gt; Med<em&gt; Toxoplasma gondii</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Research

JoVE Journal

Neuroscience

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograd fluorescensmærkning Tillader Målrettet Extracellular Single-unit Optagelse fra Identificerede Neuroner<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Funktionel Neuroimaging Med Ultrasonic blod-hjerne barrieren forstyrrelser og mangan-forstærket MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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

In vivo to-foton billedbehandling af oplevelsesbaseret afhængige molekylære ændringer i kortikal neuroner

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 ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Ændring af et Colliculo thalamus Mouse Brain Slice, Omfattende 3D-print af Afdeling Komponenter og Multi-skala Optisk Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

In Vivo Multimodal billedbehandling og analyse af mus Laser-induceret Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

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

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

In Vivo To-foton billeddannelse af kortikale neuroner 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 ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

Generation af 3-D kollagen-baserede Hydrogels til at analysere Axonal vækst og adfærd under nervesystemet udvikling

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...