Name: imaging neurons within thick brain sections using the golgi-cox method
Uploaded: 2017-04-18T13:00:00
Description: Watch this Scientific Journal Video about Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method at JoVE.com

Questo lavoro è stato sostenuto da un Discovery Grant CDCB dalle scienze naturali e ingegneria Research Council del Canada (NSERC), un John R. Evans Leaders Fund infrastruttura di ricerca concessione di CDCB dal Canada Fondazione per l&#39;innovazione (CFI numero di progetto 30381), e un Discovery Grant NJM da NSERC. ELL è stato sostenuto da una borsa di studio Ontario Laureato e da una borsa di studio OVC dalla Ontario Veterinary College presso l&#39;Università di Guelph. CDS è stato supportato da una ricerca assistentato Undergraduate Student da NSERC. ALM è stato sostenuto da una borsa di studio Alexander Graham Bell da NSERC e da una borsa di studio OVC dalla Ontario Veterinary College presso l&#39;Università di Guelph.

topi CD1-deformazione adulte di sesso femminile sono stati utilizzati in questo studio. colorazione simile può essere realizzato utilizzando entrambi i sessi alle varie età. Animali da esperimento sono stati curati secondo i principi e le linee guida del Consiglio canadese cura degli animali, e il protocollo sperimentale è stato approvato dalla University of Animal Care Comitato Guelph. 1. Golgi-Cox colorazione <ol><li> Golgi-Cox impregnazione di Brains <ol><li> Rendere...

<ol>
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D., 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=Advances+in+thin+tissue+Golgi-Cox+impregnation:+fast,+reliable+methods+for+multi-assay+analyses+in+rodent+and+non-human+primate+brain.">Advances in thin tissue Golgi-Cox impregnation: fast, reliable methods for multi-assay analyses in rodent and non-human primate brain.</a> J Neurosci Methods. 213 (2), 214-227 (2013).</li><li>Ranjan, A., Mallick, B. 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Descriviamo qui un protocollo di colorazione Golgi-Cox insieme a due varianti utili per la visualizzazione neuroni all&#39;interno delle sezioni di cervello spesse. Come mostrato nelle Rappresentante dei risultati, l&#39;uso di un obiettivo ad alta risoluzione che ha una lunga distanza di 800 micron di lavoro permette la visualizzazione affidabile di intere neuroni per tutta la profondità di sezioni di cervello tagliate a 500 micron. Questo studio di sezioni di cervello relativamente spessi aumenta la probabilità che ...

La visualizzazione dei singoli neuroni all&#39;interno di campioni di tessuto consente l&#39;analisi in situ di Neuron caratteristiche morfologiche, che ha notevolmente avanzato la nostra comprensione del cervello e come può essere influenzata dalla malattia endogena o fattori ambientali esogeni. Il metodo di colorazione Golgi-Cox è una soluzione economica, mezzi relativamente semplici di colorando un campione casuale di neuroni nel cervello. Primo sviluppato da Golgi 1 e modificato da Cox 2 nel 1800, i ricercatori hanno ulteriormente perfezionato questa tecnica nel corso degli anni per produrre chiare neuroni, ben colorate, che possono essere utilizzati per visualizzare e quantificare sia albero dendritico morfologia e densità delle spine 3, 4, 5, 6, 7, 8, 9. Una considerazione importante tecnica per la visualizzazione dei neuroni colorati all&#39;interno delle sezioni cerebrali è lo spessore massimo fetta, che è limitata dalla distanza di lavoro di alto ingrandimento / obiettivi del microscopio ad alta risoluzione disponibili. Obiettivi ad immersione in olio comuni nel 60 - 100X raggio forniscono un&#39;eccellente risoluzione, ma sono limitati dalla loro distanze di lavoro che sono in genere non superiore a 200 micron. Sezioni di cervello tagliato al micron tra 200 possono essere sufficienti per visualizzare alcuni tipi di neuroni che possono essere contenuti all&#39;interno di questo spessore di strato, ad esempio neuroni piramidali negli strati superficiali della corteccia cerebrale 10, 11, 12, neuroni piramidali della regione CA1 della ippocampo 13, 14, e cellule granulari del giro dentato dell&#39;ippocampo 15. I neuroni con relativamente più lungoalberi dendritiche, come i neuroni piramidali raggio strati profondi della corteccia cerebrale che per il mouse può estendersi oltre 800 um dal corpo cellulare 16, forniscono una sfida maggiore perché dovrebbero essere sezionati a un angolo perfetto cervello per contenere l&#39;intero albero dendrite entro 200 micron fette. Questo può anche non essere fattibile se un dendrite o uno dei suoi rami si estendono in direzione rostrale o caudale. Mentre è possibile affrontare questa limitazione tracciando un neurone in più sezioni di cervello adiacenti, questo approccio introduce una sfida tecnica significativa allineamento delle sezioni accuratamente per tracciare 17. Un approccio più pratico sarebbe visualizzare intere neuroni contenuti in sezioni di cervello che vengono tagliati a spessore maggiore. Riportiamo qui una tecnica per colorare i neuroni all&#39;interno di 400-500 micron di spessore sezioni del cervello di topi con il metodo di Golgi-Cox, e visualizzare il loro morphology utilizzando un obiettivo ad immersione in olio di silicone ad alta risoluzione che ha una distanza di lavoro di 800 um. L&#39;impregnazione e l&#39;elaborazione del protocollo Golgi-Cox che descriviamo viene modificato da uno dei protocolli moderni più citati nella letteratura 6. Il nostro approccio con sezioni di cervello spesse offre il vantaggio di aumentare la probabilità di identificare neuroni di qualsiasi tipo che sono completamente contenuto all&#39;interno della sezione. Oltre al protocollo principale, abbiamo anche presentare due varianti che forniscono vantaggi unici: (1) colorazione Golgi-Cox con la controcolorante violetto cresolo sulla superficie delle sezioni montate, al fine di definire i confini di regioni cerebrali e identificare strati della corteccia cerebrale, e (2) Golgi-Cox colorazione con una fase di congelamento intermedio per la conservazione a lungo termine di cervelli interi impregnate prima del sezionamento e lavorazione finale.

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">potassium dichromate</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">P188-100</td>
			<td style="width:167px;">Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">potassium chromate</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">P220-100</td>
			<td>Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mercuric chloride</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">S25423</td>
			<td>Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Whatman grade 1 filter paper</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">1001-185</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">isoflurane</td>
			<td style="width:147px;">Pharmaceutical Partners of Canada</td>
			<td style="width:175px;">CP0406V2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">20 mL scintillation vial</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">03-337-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">sucrose</td>
			<td style="width:147px;">Bioshop Canada</td>
			<td style="width:175px;">SUC700.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">sodium phosphate monobasic</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">S5011-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">sodium phosphate dibasic</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">S9390-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">50 mL conical tube</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">12-565-271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">isopentane</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">AC126470010</td>
			<td>also known as 2-methylbutane. Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">agar</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">A1296-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">small weigh dish</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">02-202-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">vibratome</td>
			<td style="width:147px;">Leica</td>
			<td style="width:175px;">VT1000 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">6 well tissue culture plates</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">08-772-1b</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mesh bottom tissue culture inserts</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">07-200-214</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:288px;">paraformadelhyde, 16%</td>
			<td style="width:147px;">Electron Microscope Sciences</td>
			<td style="width:175px;">15710-S</td>
			<td>Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">ammonium hydroxide</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">A669S-500</td>
			<td>Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Kodak Fixative A</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">P7542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">superfrost plus slides</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">CitroSolv clearing agent</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">22-143-975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">anhydrous ethyl alcohol</td>
			<td style="width:147px;">Commercial Alcohols</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">cresyl violet</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">C1791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">permount</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">upright microscope</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:175px;">BX53 model</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">colour camera, 12 bit</td>
			<td style="width:147px;">MBF Biosciences</td>
			<td style="width:175px;">DV-47d</td>
			<td style="width:167px;">QImaging part 01-MBF-2000R-F-CLR-12</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:288px;">three-dimensional motorized microscope stage, controller and enoders</td>
			<td style="width:147px;">MBF Biosciences</td>
			<td style="width:175px;">N/A</td>
			<td>Supplied and integrated with microscope by MBF Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">4x microscope objective</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:175px;">4x 0.16 N.A. UplanSApo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">10x microscope objective</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:175px;">10x 0.3 N.A. UPlan FL N&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">30x microscope objective</td>
			<td style="width:147px;">Olympus</td>
			<td>30x 1.05 N.A. UPlanSApo&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">60x microscope objective</td>
			<td style="width:147px;">Olympus</td>
			<td>60x 1.42 N.A. PlanAPO N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">silicone immersion oil</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:175px;">Z-81114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Neurolucida software</td>
			<td style="width:147px;">MBF Biosciences</td>
			<td style="width:175px;">Version 10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">ImageJ software</td>
			<td style="width:147px;">U. S. National Institutes of Health</td>
			<td style="width:175px;">Current version</td>
			<td>With the OME Bio-Formats plugin installed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Photoshop software</td>
			<td style="width:147px;">Adobe</td>
			<td style="width:175px;">version CS6</td>
			<td></td>
		</tr>
	</tbody>
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imaging neurons within thick brain sections using the golgi-cox method

Il metodo di Golgi-Cox di neurone colorazione è stato impiegato per più di duecento anni per far progredire la nostra comprensione del neurone morfologia all&#39;interno di campioni di cervello istologici. Mentre è preferibile dal punto di vista pratico per preparare sezioni di cervello del spessore maggiore possibile, al fine di aumentare la probabilità di identificare neuroni colorati che sono completamente contenuti all&#39;interno delle singole sezioni, questo approccio è limitato dal punto di vista tecnico dalla distanza di lavoro di alta Ingrandimento obiettivi del microscopio. Riportiamo qui un protocollo per macchiare neuroni utilizzando il metodo Golgi-Cox in sezioni di cervello di topo che vengono tagliati a 500 spessore um, e visualizzare neuroni tutta la profondità di queste sezioni utilizzando un microscopio verticale dotato di alta risoluzione 30X 1,05 NA silicone obiettivo ad immersione in olio, che ha una distanza di lavoro di 800 micron. Si segnala inoltre due varianti utili di questo protocollo che può essere impiegato per colorazione di contrasto alla superficie dimontato sezioni di cervello con il violetto cresolo Nissl macchia, o di congelare cervelli interi per la conservazione a lungo termine prima di sezionamento e lavorazione finale. Il protocollo principale e le sue due varianti producono sezioni di cervello spesse colorate, durante il quale gli alberi pieni neurone e spine dendritiche dendritiche possono essere visualizzati in modo affidabile e quantificate.

Questo protocollo di colorazione Golgi-Cox e le sue due varianti descritte opzionali possono essere impiegati per visualizzare i singoli neuroni a 400 - 500 um di spessore sezioni del cervello. Rappresentativi montaggi di immagini bidimensionali Z-proiezioni catturate con un obiettivo 10X e 5 micron passi nell&#39;asse Z sono mostrati in Figura 1: A1 - C1 per una grande area di sezioni di cervello coronali che include l&#39;area corteccia anteriore cingolata 1 e la corte...

Watch this Scientific Journal Video about Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method at JoVE.com

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

Presentiamo un protocollo secondo il metodo di colorazione Golgi-Cox in sezioni di cervello spesse, per visualizzare neuroni con lunghi alberi dendritiche contenuti in campioni di tessuto singoli. Due varianti di questo protocollo sono anche presentati che coinvolgono di contrasto viola cresile, e il congelamento dei cervelli non trasformati per la conservazione a lungo termine.

I neuroni di imaging all&#39;interno di sezioni di cervello spesse Uso del metodo di Golgi-Cox

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

The overall goal of this procedure is to determine the effective treatment on the morphology of neurons within the rodent brain. This method can help answer key questions in the neuroscience field, such as the normal and experimentally manipulated morphology of neurons within various regions of the rodent brain. This technique has the main advantage of increasing the probability of identifying and visualizing labeled neurons that are fully contained within single histological samples.Begin Golgi-Cox staining by placing a fresh mouse brain into a 20 milliliter glass scintillation vial containing 17 milliliters of Golgi-Cox solution. Protect from light and incubate at room temperature for 25 days. Once the incubation period has elapsed, Cryo protect the brain by placing it into 40 milliliters of sucrose cryoprotectant, and incubate in the dark at 4 degrees celsius for 24 hours.After the incubation in sucrose, the brain can be frozen for long term storage, or immediately blocked for sectioning as shown in the next section of the video. If freezing, immerse the whole brain in 200 milliliters of isopentane that has been precooled on dry ice. Then place the frozen brain into a 50 milliliter conical tube and store in the dark at minus 80 degrees celsius.To begin, remove the brain from the sucrose. And use a razor blade to block it for sectioning by cutting off the cerebellum, leaving a flat edge at the remaining caudal end of the brain. The brain must be blocked at a precise angle to ensure that neurons of interest are fully contained within the resulting sections.For example, we use a coronal plane that is perfectly perpendicular to the rostral caudal axis for cerebral cortex pyramidal neurons. Next, heat agar until it is melted. And let it cool until it is slightly above it's melting point.Then place the brain in a small disposable weigh dish with the caudal end face down. Add melted agar to cover the brain. Once the agar has solidified, trim the excess leaving approximately two to four milliliters surrounding the brain.Then use a small amount of ethyl cyanoacrylate to glue the brain to the stage of a vibratome with it's caudal end face down. Fill the stage area of the vibratome with enough sucrose cryoprotectant to cover the brain. Then section the brain at a slice thickness of 400 to 500 microns depending on the brain region to be examined.Using a small paintbrush, place brain sections into wells of a six well tissue culture plate containing mesh bottom inserts and pre filled with sucrose M phosphate buffer. Protect from light during sectioning. When finished sectioning, incubate sections in the dark at four degrees celsius overnight.Using the mesh bottom inserts, transfer the brain sections into a new well containing five milliliters of paraformaldehyde in phosphate buffer. Incubate on a rocker moving at slow speed at room temperature in the dark for 15 minutes. Wash the sections twice by transferring them to new wells containing five milliliters of water.Protect from light and wash at moderate speed at room temperature for five minutes. Next, transfer the sections into a new well containing five milliliters of ammonium hydroxide. Rock slowly in the dark at room temperature for 15 minutes.Wash the sections twice by transferring them to new wells containing five milliliters of water. Wash on a rocker moving at moderate speed in the dark at room temperature for five minutes. Transfer the sections into a new well containing five milliliters of diluted fixative A.Incubate on a rocker moving at slow speed in the dark at room temperature for 25 minutes.Wash sections in water twice as before. Use a small paintbrush to place the sections onto a microscope slide, then use tweezers and a small tissue to remove excess water and agar. Ensure that all agar is removed before proceeding with the dehydration steps.Allow the sections to air dry at room temperature protected from light. The appropriate drying time is critical, and should be determined for each laboratory. Too short of a time may result in sections falling off of slides during dehydration steps, and too long of a time may result in sections cracking.After drying, first place the slides in clearing agent for five minutes. Place the slides in 100%ethanol for five minutes. Then move through a series of decreasing ethanol concentrations for two minutes each.After the alcohol series, place the slides in water for five minutes. Then stain in 0.5%cresyl violet in water for seven minutes. Following the cresyl violet stain, place the slides in water for two minutes.Then dehydrate the sections through a series of increasing alcohol concentrations for two minutes each. Then two washes in 100%ethanol for five minutes. Place in clearing agent for five minutes.Finally add an anhydrous mounting medium and place a cover slip over the sections. Allow slides to dry horizontally in the dark at room temperature for at least five days. To capture high resolution image stacks of the area containing the neuron of interest, first select a 30X 1.05 NA silicone oil immersion objective on the microscope.Then apply three to four drops of silicone immersion oil to the slide. Place the objective over the slide, ensuring contact between the objective and oil. Focus the image and adjust the camera settings including the exposure and white balance in the imaging software.Next set the upper and lower boundaries for the image stack by focusing to the top of the section, and then selecting set next to top of stack within the image acquisition window. Then focus to the bottom of the section, and select set next to bottom of stack. Set the step distance to one micron by entering one micron next to distance between images within the image acquisition window.Lastly capture the image stack by selecting acquire image stack within the image acquisition window. Repeat the previous steps to capture the entire area of interest making sure that all image stacks overlap by at least 10%in the X and Y axes. This image of the cerebral cortex is a merged Z projection of image stacks acquired from a 500 micron thick section using a 10X objective.The tissue in this image was processed using the main all fresh protocol. The area indicated by the red square was imaged using a 30X silicon oil immersion objective and a higher resolution merged Z projection image stack was acquired. The red arrow shows the two dimensional Z projection of a neuron that was traced from the high resolution 30X image stack.Here the fresh protocol variant with cresyl violet staining was used. This higher resolution image shows the cresyl violet stained section. And this is the neuron tracing obtained.Finally this tissue was processed using the protocol variant in which brains are frozen following Golgi-Cox impregnation. Here the previously frozen tissue is seen at higher resolution. And once again a high quality two dimensional Z projection of the neuron tracing is obtained.This technique can be completed within one month with the processing and imaging steps completed within the final week. Brains can alternatively be frozen following Golgi-Cox impregnation allowing for the processing and imaging to be completed at a later date. Following this procedure, neuroscientists can use the captured images to perform detailed morphological analyses such as dendrite complexity or spine density in order to determine how an experimental manipulation can alter the structure of neurons within the brain.

Research

JoVE Journal

Neuroscience

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A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Ricerca

Neuroscienze

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Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

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Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

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Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

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