El trabajo fue apoyado por becas de la Fundación Smith Family (BS), la Fundación Dana (BS), John Merck Scholars Program (BS), NINDS (SR1-NS-07100801; BS), NRSA (F32-NS-066698; DPS), Nancy Fundación Lurie Marks (DPS), el NIH (P30-HD-18655; MRDDRC Imaging Core).

<ol>
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P., 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=The+&amp;#34;quad-partite&amp;#34;+synapse:+Microglia-synapse+interactions+in+the+developing+and+mature+CNS.">The &amp;#34;quad-partite&amp;#34; synapse: Microglia-synapse interactions in the developing and mature CNS.</a> Glia. 61 (1), 24-36 (2013).</li><li>Mukhopadhyay, S., 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=Manganese-induced+trafficking+and+turnover+of+the+cis-Golgi+glycoprotein+GPP130.">Manganese-induced trafficking and turnover of the cis-Golgi glycoprotein GPP130.</a> Mol Biol Cell. 21 (7), 1282-1292 (2010).</li><li>Jung, S., 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20, 4106-4114 (2000).</li><li>Knobloch, M., Mansuy, I. 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No hay conflictos de interés declarado.

Con el fin de medir con precisión la fagocitosis, el material engullido debe etiquetarse de tal manera que el investigador puede visualizar una vez se ha producido la degradación lisosomal. Además, se requiere formación de imágenes de alta resolución, seguido por el uso de software que permita el investigador para visualizar el volumen de toda la célula y cuantificar su contenido. En este protocolo, se describe un método altamente fiable y cuantitativa para medir la inmersión de los fagocitos mediada por el uso...

<html> Circuitos sinápticos remodelar largo de la vida de un animal. En el cerebro en desarrollo, las sinapsis se forman en exceso y deben someterse a la poda sináptica que consiste en la eliminación selectiva de un subconjunto de las sinapsis y el mantenimiento y fortalecimiento de esas sinapsis que permanecen 8-10. Este proceso es necesario para lograr la conectividad característica precisa del sistema nervioso adulto. En el adulto, las sinapsis también pueden ser de plástico, en particular en el contexto del aprendizaje y la memoria. Las correlaciones estructurales de esta plasticidad se cree que incluyen la adición y / o eliminación de las espinas dendríticas y presináptica boutons 11-13. Además de estas funciones en el sistema nervioso saludable, remodelación sináptica también está implicada en la enfermedad del sistema nervioso 12,14,15 / lesión. Por ejemplo, después de una lesión de la médula espinal, los axones cortados deben posteriormente remodelar y formar nuevas sinapsis para lograr la recuperación funcional 16-19. nt &quot;&gt; Emergiendo como un aspecto importante de la plasticidad sináptica es el proceso de la fagocitosis o la inmersión de las sinapsis con destino a la eliminación 3,5,20. Recientemente, hemos mostrado este fenómeno en el contexto de la poda sináptica en el sano, el cerebro postnatal ratón 7. Específicamente , microglía, las células inmunes residentes del SNC y los fagocitos, se muestra para engullir entradas presinápticas durante un período de pico y en una región de la poda sináptica de desarrollo, el dorsal posnatal núcleo geniculado lateral (dLGN) del tálamo. bloqueo genética o farmacológica de esta inmersión traducido en déficits sostenidos en la conectividad sináptica. En este protocolo, se describe un ensayo fiable y altamente cuantitativo para medir la inmersión fagocítica mediada de insumos presináptica. A los efectos de este artículo, este ensayo se presentará en el marco del sistema de desarrollo de retinogeniculadas, que incluye las células ganglionares de la retina (CGR) que reside en la retina queproyectar entradas presináptica a la dLGN (Figura 1A). Para empezar, una estrategia de marcaje anterógrado resistente a la degradación lisosomal se describirá, que se utiliza para visualizar entradas presinápticas RGC-específicos en la dLGN (Figura 1) 7,21. Después de esta descripción, una metodología detallada para la imagen y la medición cuantitativa de inmersión utilizando microscopía confocal combinadas con 3 dimensiones (3D) de superficie representación de volumen se le dará. Esta metodología se basa en la preparación del tejido fija, pero también puede ser adaptado para su uso en estudios de formación de imágenes en vivo. Es importante destacar que, mientras que el ensayo ha sido validado en el contexto del sistema de retinogeniculadas saludable, posnatal, se podría aplicar las mismas técnicas para evaluar otras interacciones fagocito-neuronas en todo el cerebro y durante la enfermedad, así como la función fagocítica en otros sistemas de órganos.

<table border="0" cellpadding="0" cellspacing="0" width="533">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Heat pad</td>
			<td style="width:159px;">Vet Equip, Inc.</td>
			<td style="width:168px;">965500&#160;</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:144px;">Warm water source for heat pad</td>
			<td style="width:159px;">Kent Scientific</td>
			<td style="width:168px;">TP-700</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:144px;">Stereo microscope</td>
			<td style="width:159px;">DSC Optical</td>
			<td style="width:168px;">Zeiss Opmi -6 Surgical Microscope</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="40" rowspan="2" style="height:40px;width:144px;">Sliding microtome with freezing stage</td>
			<td rowspan="2" style="width:159px;">Leica</td>
			<td rowspan="2" style="width:168px;">SM2010 R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Microtome blade</td>
			<td style="width:159px;">Leica</td>
			<td style="width:168px;">14021607100</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:144px;">Fluorescent dissecting microscope</td>
			<td style="width:159px;">Nikon</td>
			<td style="width:168px;">SMZ800 with Epi-fluorescence attachment</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:144px;">Spinning disk confocal microscope</td>
			<td style="width:159px;">Perkin Elmer</td>
			<td style="width:168px;">UltraView Vox Spinning Disk Confocal</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">10 &#181;l Hamilton gas tight syringes</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">80030</td>
			<td style="width:144px;">Use a different syringe for each color dye/tracer</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Hamilton needles</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">7803-05, specifications: blunt, 1.5&#34;</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="20">
			<td height="54" rowspan="3" style="height:54px;width:131px;">Alexa-conjugated cholera toxin &#946; subunit (CTB)</td>
			<td rowspan="3" style="width:131px;">Invitrogen</td>
			<td style="width:116px;">488: C22841</td>
			<td rowspan="3" style="width:144px;">Reconstitute in sterile saline, 80 &#181;l (488), 100 &#181;l (594), 20 &#181;l (647)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">594: C22842</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">647: C34778</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Phosphate Buffered Saline&#160;(PBS)</td>
			<td style="width:131px;">Sigma</td>
			<td>P4417-50TAB</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:131px;">Neomycin and Polymyxin B Sulfates and Bacitracin Zinc Ophthalmic Ointment USP (antibiotic ointment)</td>
			<td style="width:131px;">Bausch &#38; Lomb</td>
			<td style="width:116px;">24208-780-55</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">30.5 gauge needle</td>
			<td style="width:131px;">Becton Dickinson</td>
			<td style="width:116px;">305106</td>
			<td rowspan="3" style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Spring scissors</td>
			<td style="width:131px;">Roboz</td>
			<td style="width:116px;">RS-5630</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Cotton-tipped applicator</td>
			<td style="width:131px;">Fisher</td>
			<td style="width:116px;">23-400-125</td>
		</tr>
		<tr height="21">
			<td height="104" rowspan="2" style="height:104px;width:131px;">Paraformaldeyde (PFA)</td>
			<td rowspan="2" style="width:131px;">Electron Microscopy Sciences</td>
			<td rowspan="2" style="width:116px;">15710</td>
			<td rowspan="2" style="width:144px;">Dilute 16%to 4% in PBS. Paraformaldehye is toxic, use&#160; in a fume hood and wear personal protective equipment.</td>
		</tr>
		<tr height="83">
		</tr>
		<tr height="36">
			<td height="138" rowspan="4" style="height:138px;width:131px;">Dissection tools &#8211; scissors, forceps, spatula</td>
			<td style="width:131px;">Small scissors: Fine Science Tools</td>
			<td style="width:116px;">Small scissors:14370-22</td>
			<td rowspan="4" style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Large scissors: Roboz</td>
			<td style="width:116px;">Large scissors: RS-6820</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">#55 forceps: Fine Science Tools</td>
			<td style="width:116px;">#55 forceps: 11255-20</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Spatula: Ted Pella, Inc.</td>
			<td style="width:116px;">Spatula: 13504</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:131px;">Sucrose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S8501-5KG</td>
			<td style="width:144px;">Make 30% sucrose in PBS (weight/vol)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">OCT Compound</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">25608-930</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Weigh boat</td>
			<td style="width:131px;">USA Scientific</td>
			<td style="width:116px;">2347-1426</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">24-well plates</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:116px;">353047</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Sodium phosphate monobasic</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S6566-500G</td>
			<td rowspan="2" style="width:144px;">Make 0.2 M sodium phosphate monobasic (PB-A) in ddH20 and 0.2 M sodium phosphate dibasic (PB-B) in ddH20.&#160; To make 0.1 M PB, combine 19 ml PB-A and 81 ml PB-B, fill to 200 ml with ddH20&#160;&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:131px;">Sodium phosphate dibasic&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td>S5136-500G</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Coverslips, 22 X 50 mm, No. 1.5</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48393 194</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Charged microscope slide</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48311-703</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Vectasheild</td>
			<td style="width:131px;">Vector Laboratories</td>
			<td style="width:116px;">H-1200</td>
			<td style="width:144px;"></td>
		</tr>
	</tbody>
</table>

an engulfment assay: a protocol to assess interactions between cns phagocytes and neurons

La fagocitosis es un proceso en el que una célula envuelve el material (célula entera, partes de una célula, escombros, etc) en su entorno extracelular que rodea y posteriormente se digiere este material, comúnmente a través de la degradación lisosomal. La microglía son las células inmunes residentes del sistema nervioso central (SNC) cuya función fagocítica se ha descrito en una amplia gama de condiciones de enfermedad neurodegenerativa (por ejemplo, aclaramiento de beta-amiloide en la enfermedad de Alzheimer) para el desarrollo del cerebro sano (por ejemplo, sináptica poda) 1-6. El siguiente protocolo es un ensayo de inmersión desarrollado para visualizar y cuantificar inmersión microglia mediada de insumos presinápticas del sistema retinogeniculadas ratón en desarrollo 7. Mientras que se utilizó este ensayo para evaluar la función microglia en este contexto particular, un enfoque similar se puede utilizar para evaluar otros fagocitos en todo el cerebro (por ejemplo, astrocitos) y el resto del cuerpo(por ejemplo, macrófagos periféricos), así como otros contextos en los que ocurre la remodelación sináptica (por ejemplo, lesión cerebral / enfermedad).

Recientemente, se utilizó este ensayo de inmersión para visualizar y cuantificar inmersión microglia mediada de entradas presinápticas en el sistema retinogeniculadas en desarrollo (Figura 1) 7. CGR de ratones heterocigotos CX3CR1-EGFP fueron anterogradely remontar con CTB-594 y CTB-647 en los ojos izquierdo y derecho, respectivamente. A raíz de esta localización, se obtuvieron imágenes de microglia EGFP-positivas dentro de la dLGN. Estas imágenes fueron posteriormente superficie pres...

Watch this Scientific Journal Video about An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons at JoVE.com

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

La microglía son las células inmunes residentes del sistema nervioso central (SNC) con una alta capacidad de fagocitar o material de envolver en su entorno extracelular. Aquí, se describe un ensayo ampliamente aplicable, fiable, y altamente cuantitativo para visualizar y medir la microglia mediada por inmersión de componentes sinápticas.

Un ensayo de Inmersión en: Un Protocolo para Evaluar Interacciones entre CNS fagocitos y neuronas

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

an-engulfment-assay-protocol-to-assess-interactions-between-cns

Research

JoVE Journal

Neuroscience

18.1K vistas.  Boston Children's Hospital, Harvard Medical School.  La microglía son las células inmunes residentes del sistema nervioso central (SNC) con una alta capacidad de fagocitar o material de envolver en su entorno extracelular. Aquí, se describe un ensayo ampliamente aplicable, fiable, y altamente cuantitativo para visualizar y medir la microglia mediada por inmersión de componentes sinápticas. 

Video: An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural formadoras de colonias ensayo de células: un ensayo para discriminar Bona Fide células troncales nerviosas de células progenitoras neurales

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Investigación

Neurociencias

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

La cartografía y la aplicación de potenciador de la trampa de expresión Flippase en larvas y adultos Drosophila SNC

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Análisis de la morfología espina dendrítica en las neuronas del SNC cultivadas

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

De corte de carga: una herramienta útil para examinar el grado de acoplamiento Marcador brecha unión entre las neuronas retinianas

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Un Enfoque para Mejorar la alineación y la mielinización de las neuronas del ganglio de la raíz dorsal

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Raya de ensayo para estudiar la actividad de atracción o repulsión de un sustrato de proteína usando las neuronas del hipocampo disociadas

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

La indirecta neurona-astrocito Coculture Ensayo: Un<em&gt; In Vitro</em&gt; Puesta en marcha de la investigación detallada de las interacciones neurona-glía

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

Un análisis óptico para el reciclaje de la vesícula sináptica en neuronas cultivadas Overexpressing proteínas presinápticas

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

Un modelo preclínico para evaluar la recuperación cerebral después de un accidente cerebrovascular agudo en ratas

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...