Se prestó apoyo por inicio-fondos del Departamento de Farmacología y Biología Química de la Universidad de Pittsburgh Facultad de Medicina. El reconocimiento de los miembros del laboratorio de Jacob que contribuyeron a la presentación de vídeo: Nicholas Graff.

Todos los protocolos descritos a continuación son de acuerdo con la IACUC e IRB juntas de revisión institucional de la Universidad de Pittsburgh Facultad de Medicina. 1. Preparación de los cultivos neuronales del hipocampo en cultivo de tejidos Capucha Nota: Utilice una técnica estéril y reactivos a lo largo Protocolo 1. <ol><li> Preparar poli-D-lisina (0,1 mg / ml en H2O) cubreobjetos de vidrio revestido como un sustrato para el cultivo neuronal. <ol><li> Coloque 4-5 cubreobjetos de vidrio redondos dentro de cada 3,5 cm de tejido placa de cultivo. </li><li> Punto 70 l de poli-D-lisina en cada cubreobjetos de 12 mm. Nota: para imágenes en vivo, un fondo de cristal de 3,5 cm placa de cultivo de tejido se puede utilizar (detectar 200 l de poli-D-lisina en incrustado cubreobjetos de 14 mm). </li><li> Deja los platos preparados en el cultivo de tejidos capó O / N. Para minimizar la poli-D-lisina evaporación y mantener las superficies estériles en la campana de cultivo de tejidos, cerrar la hoja de la ventana y apague ªe soplador de O / N. Nota: Las luces UV no se utilizan durante la incubación O / N. </li><li> A la mañana siguiente, lavar los platos 3 veces con 2 ml de H 2 O. </li><li> Después de quitar el último H2O lavado, añadir 2 ml de medio a cada placa 3,5 cm y dejar los platos en la incubadora hasta que esté listo a la placa de las neuronas en el paso 1.4. </li></ol></li><li> Las neuronas del hipocampo se preparan a partir del día embrionario 18-19 (E18-19) ratas (modificado de Goslin 22). </li><li> Transfectar neuronas recién disociados en el día de cultivo con 1-4 g de ADN de constructo maxiprepped. Nota: típicamente 3 g de ADN se transfecta en 1-2 Millón neuronas, con una viabilidad de 50% y una eficiencia de transfección del 60% (por ejemplo, a partir de 2 millones de neuronas: ((2 x 10 6 neuronas x 0.5) 0.6) proporciona aproximadamente 1 millón de neuronas,.. 600.000 de los cuales son transfectadas 1 cantidades diferentes de construcción de ADN se pueden transfectar la hora de solucionar los posibles problemas desobre-expresión, seguido por la caracterización apropiada de localización y función constructo. </li><li> Placa de las neuronas transfectadas sobre los cubreobjetos recubiertos con poli-D-lisina a una densidad final de aproximadamente 200.000 neuronas por 3,5 cm plato. Nota: para un fondo de cristal plato, placa 40.000-50.000 neuronas en el área del cristal 14 mm. </li><li> Sustituya el soporte 4-24 horas después de la preparación de cultivos neuronales, a continuación, permiten que las neuronas se desarrollan en la incubadora hasta 14-17 días in vitro (DIV) o de la etapa deseada. </li></ol> 2. Ensayo Endocitosis PRECAUCIÓN: Nota sobre la α-bungarotoxina y residuos tubocurarina y dirección: <ol><li> Manejo de todas las neurotoxinas de péptidos se recomienda dentro de los lineamientos de Bioseguridad Nivel 2 (BL-2) los protocolos de investigación. Todo el equipo de protección personal adecuado que se debe usar. Polvos liofilizados toxina debe reconstituirse siguiendo las instrucciones del fabricante. Toxina shou residuosld eliminarse siguiendo las normativas de salud y seguridad ambiental directrices de la institución de gobierno. </li><li> Para eliminar posibles agregados de péptidos que se puedan haber formado durante el almacenamiento, alícuotas de α-bungarotoxina deben ser centrifugadas brevemente antes de su uso, y el sobrenadante se deben utilizar para el experimento. </li><li> α-bungarotoxina (Bgt) las existencias se resuspenden a una concentración de 1 mg / ml en H2O estéril y se almacenan en 10 ml de alícuotas a -20 ° C. Existencias Bgt fluorescencia acoplados se utilizan a 3 g / ml. Tubocurarina se utiliza a una concentración final de 150 mM. </li></ol><ol><li> Banco Set top bloque calentador a 16 ° C en cámara frigorífica con aluminio delgado o placa de acero inoxidable en la parte superior. Alternativamente, si está disponible, un alto dispositivo de refrigeración / calefacción banco se puede utilizar para cada paso que requiere 16 ° C. </li><li> Enfriar extracelular solución salina tamponada con Hepes (HBS contienen en mM: 135 NaCl, 4,7 KCl, 10 HEPES, 11 de glucosa, 1,2 MgCl 2 y 2,5CaCl2, pH 7,4) a 16 ° C en un baño de agua. </li><li> Transferencia de los platos de la neurona a la placa de aluminio a 16 ° C y enfriar durante 5 min. </li><li> Retire los medios de comunicación (mantener medios condicionados y reserva en la incubadora para el paso 2.8, endocitosis puntos de tiempo de ensayo) y reemplazar con 1 ml de 16 ° C HBS + tubocurarina (150 M) durante 2 min. </li><li> Eliminar HBS + tubocurarina al contenedor de desperdicio etiquetado y sustituir con 1 ml de 16 ° C HBS + tubocurarina (150 M) + α-bungarotoxina Alexa 594 [3 g / ml], incubando a 16 ° C durante 15 min. </li><li> Eliminar HBS + tubocurarina + α-bungarotoxina Alexa 594 a la etiqueta contenedor de residuos y lavar platos 3 veces con 2 ml de 16 ° C HBS (los lavados se pueden recoger a través de aspiración en un matraz de vacío que contiene 50 ml de 50% de lejía). </li><li> Para los experimentos de series temporales de imágenes en vivo siguientes endocitosis del receptor utilizando un fondo de cristal 3,5 cm placa de cultivo, realice los pasos 2.1 hasta 2.6, a continuación, llevar la cápsula a la etapa de calefacción(Dispositivo Peltier) en el microscopio, y comenzar de imágenes con un microscopio confocal o configuración TIRF. </li><li> La transferencia de T = 0 cubreobjetos de punto de tiempo en un plato de la RT 4% de paraformaldehído / 4% de sacarosa durante 20 minutos, continuando con otros cubreobjetos en el paso 2.9. </li><li> Para otros momentos de la hora, sustituya HBS con condicionados equilibró 37 ° C Medios (reservado en el paso 2.4) y volver a la incubadora por endocitosis a 37 ° C. Nota: sugirieron puntos adicionales de tiempo de T = 15, 30 y 60. </li><li> En los puntos de tiempo necesario, tomar los platos de la incubadora, lave rápidamente dos veces con 2 ml de RT DPBS (DPBS sin calcio, magnesio) a continuación, fijar en paraformaldehído al 4% / 4% de sacarosa durante 20 min. </li><li> Después de 20 min de fijación, retire paraformaldehído al 4% / 4% de sacarosa que perder envase y lavar los platos dos veces con 2 ml RT DPBS. </li><li> Incubar cubreobjetos a TA durante 10 min en solución de bloqueo de inmunofluorescencia (solución de bloqueo = 5% de suero de caballo, 0,5% de BSA en DPBS) que contiene 0,2% de Triton X-100 a la permeabilidadize las neuronas y permitir inmunotinción anti GFP de la piscina receptor intracelular y / o etiquetado de las otras proteínas de interés. </li><li> Extraiga el bloque de + 0,2% Triton X-100 y se incuba cubreobjetos en solución de bloqueo inmunofluorescencia sin Triton X-100 durante 20 a 30 min. </li><li> Realice las incubaciones de anticuerpos primarios de cubreobjetos en bloque durante varias horas a temperatura ambiente o O / N a 4 ° C. Nota: la incubación de cubreobjetos con primarios a 4 ° CO / N produce rutinariamente mejores resultados de inmunofluorescencia. </li><li> Lave cubreobjetos 3 veces con DPBS (5 min para cada etapa de lavado). </li><li> Incubar cubreobjetos con anticuerpos secundarios en solución de bloqueo durante 1 hora a TA. </li><li> Lave cubreobjetos 3 veces con DPBS (5 min para cada etapa de lavado). </li><li> Cubreobjetos montaje, manejo cada cubreobjetos de forma individual: eliminar el exceso de líquido de la parte posterior del cubreobjetos con papel de laboratorio, luego invierta cubreobjetos en 4 l de medio de montaje sobre un portaobjetos de vidrio. </li><li> Permita que se sequen a temperatura ambiente cubiertos durante 30 minutos y luego stmineral a 4 ° C hasta el momento de la microscopía. </li><li> Adquisición y análisis de experimentos con microscopía confocal (ciegos a condición experimental) Imagen. 60X petróleo NA 1.49 objetivo de inmersión con los siguientes láseres: gas argón de 488 nm, 561 diode, diodo 640. <ol><li> Utilizando los mismos parámetros de adquisición de imágenes, la obtención de imágenes de sección Z individuales con el cuerpo celular neuronal y varios procesos dendríticas en foco. </li><li> Cuantificar α-bungarotoxina Alexa señal de fluorescencia de GFP y la inmunotinción a lo largo de 20 micras de 3-4 dendritas proximales por neurona, utilizando el mismo umbral para el análisis de todos los datos de endocitosis. </li><li> Analizar los datos de 10 a 12 neuronas para cada punto de tiempo, repitiendo el experimento con varios cultivos neuronales independientes. </li></ol></li></ol>

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Los autores no tienen nada que revelar.

Las técnicas fijas y basadas BBS descritos aquí pueden ser utilizados para rastrear receptor u otra proteína de membrana de plasma trata de líneas de células, neuronas y otras células primarias. Este método ha sido utilizado con éxito para estudiar inserción en la membrana y la eliminación de canales iónicos activados por ligando y GPCR y evaluar los cambios en el tráfico debido a la presencia de agonistas de los receptores y moduladores. Los aspectos clave incluyen la localización apropiada de la etiqueta ...

El uso de acoplado con fluorescencia α-bungarotoxina para estudiar la localización del receptor y la dinámica fue pionera en los estudios del receptor de acetilcolina nicotínico 13-15, diana endógeno de la toxina. Posteriormente, la incorporación del péptido mínima Bgt de unión (BBS) se ha utilizado para estudiar el tráfico de ambos canales de iones excitatorios e inhibitorios mediados por ligandos y receptores acoplados a proteína G 2,10,16-21. Esta técnica basada en BBS ofrece ventajas a otros enfoques utilizados para estudios de tráfico, como los métodos de biotinilación superficie, el etiquetado de anticuerpos de células vivas con anticuerpos frente a epítopos extracelulares, y la recuperación después de fluorescencia photobleaching (FRAP). Durante superficie celular biotinilación aminas libres están covalentemente modificados, con el potencial de afectar la actividad celular. Los estudios basados ​​en anticuerpos a menudo se han visto obstaculizados por la agrupación antígeno de superficie o de nivelación, lo que puede alterar los eventos de tráfico. Debido a la etapa de blanqueo para FRAP sos estudios, un asunto importante es dañar la estructura celular subyacente. Una ventaja adicional es que BBS etiquetada construcciones también se pueden utilizar para metodologías bioquímicas con el tráfico de biotina acoplada bungarotoxina a asnos del receptor. Esta técnica es fácilmente aplicable a líneas celulares y células primarias. Para uso en células que expresan nicotínicos de acetilcolina (nAChR), los receptores, el antagonista de nAChR tubocurarina debe ser utilizado en todo el protocolo como se indica. Realización de una Bgt etiquetado simple superficie (equivalente al punto de tiempo T = 0 para el protocolo de endocitosis) en las células no transfectadas en ausencia de tubocurarina proporcionará pruebas de nAChR endógeno. Una consideración importante para el uso de esta técnica es la inserción apropiada de la BBS de modo que está presente en una localización extracelular cuando la proteína de interés se entrega a la membrana plasmática. Por ejemplo, los dominios N-terminales de las subunidades GABAAR residen en el lumen vesiculares durante el tráficofico y convertirse extracelular después de la inserción del receptor en la membrana plasmática, lo que permite un etiquetado específico de los receptores de la superficie celular y evaluación de su eliminación de la superficie celular por eventos endocítica. Hemos demostrado anteriormente que la adición de GFP, myc, o BBS epítopos a este dominio de subunidades GABAAR es funcionalmente silenciosa. Los controles estándar se deben realizar para asegurar que la proteína marcada se expresa en niveles similares a una construcción sin etiquetar, que localiza apropiadamente, y que no afecta a la función del receptor. Esta caracterización de las construcciones transfectadas también ayudará en las preocupaciones de sobreexpresión de solución de problemas.

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			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:196px;">Catalog Number</td>
			<td style="width:187px;">Comments/Description</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:227px;">P3 Primary Cell 4D kit&#160;</td>
			<td style="width:201px;">Lonza</td>
			<td style="width:196px;">V4XP-3024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B35450</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 488 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13422</td>
			<td></td>
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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 594 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13423</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:227px;">(+)-Tubocurarine chloride</td>
			<td style="width:201px;">Tocris</td>
			<td style="width:196px;">2820</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:227px;">mounting medium&#160;</td>
			<td>DAKO</td>
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			<td height="20" style="height:20px;">DPBS no calcium,magnesium&#160;</td>
			<td style="width:201px;">Invitrogen&#160;</td>
			<td style="width:196px;">14190-136</td>
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			<td height="20" style="height:20px;width:227px;">poly-D-lysine&#160;</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:196px;">P6407</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">gephyrin antibody</td>
			<td>Synaptic Systems</td>
			<td>147 011</td>
			<td>1:300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">VIAAT/VGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 002</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">anti GFP</td>
			<td style="width:201px;">Life Technologies</td>
			<td>A6455</td>
			<td>1:2000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">glass bottom tissue culture dish</td>
			<td style="width:201px;">MatTek Corporation</td>
			<td colspan="2">P35G-1.5-14-C - Mattek Dishes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">coverslips (round cover glass), #1 thickness, 12 mm</td>
			<td style="width:201px;">Warner Instruments</td>
			<td style="width:196px;">640-0702</td>
			<td></td>
		</tr>
	</tbody>
</table>

using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

Cada vez es más evidente que los receptores de neurotransmisores, entre ellos los receptores ionotrópicos GABAA (GABAAR), exhibición tráfico altamente dinámico y la movilidad de la superficie celular 1-7. Para estudiar receptor de células localización en la superficie y la endocitosis, la técnica descrita aquí combina el uso de fluorescente α-bungarotoxina con células que expresan construcciones que contienen un-bungarotoxina α (Bgt) sitio de unión (BBS). El BBS (WRYYESSLEPYPD) se basa en la subunidad α del receptor nicotínico de la acetilcolina del músculo, que se une con alta afinidad Bgt 8,9. Incorporación del sitio BBS permite la localización de la superficie y mediciones de la inserción del receptor o la retirada con la aplicación de fluorescente exógeno Bgt, tal como se describe anteriormente en el seguimiento de GABAA y metabotrópicos GABAB receptores 2,10. Además del sitio de BBS, insertamos una GFP sensible al pH (pHGFP 11) entre los aminoácidos 4 y 5 de la subunidad madura GABAAR por m estándarestrategias de biología y de clonación PCR olecular (ver figura 1) 12. El BBS es 3 &#39;de la GFP reportero sensible al pH, separadas por un enlazador alanina / ácido prolina 13-amino. Para el tráfico de los estudios descritos en esta publicación que se basan en muestras fijas, la pHGFP sirve como reportero del total etiquetado GABAAR los niveles de proteína de la subunidad, permitiendo la normalización del etiquetado Bgt población receptora de población total al receptor. Esto minimiza célula a célula Bgt tinción variabilidad de la señal resultante de la mayor o menor expresión basal de las subunidades GABAAR etiquetados. Además, la etiqueta pHGFP permite una fácil identificación de las células que expresan constructos para experimentos de imagen en vivo o fijos.

Caracterización de una construcción de BBS etiquetado incluye controles importantes tales como la determinación de si la proteína expresada se ensambla correctamente (en particular con los receptores de compuestos de múltiples subunidades), trafica a la superficie celular y se localiza apropiadamente. Las sinapsis inhibitorias se componen de GABA A cúmulos superficiales receptor que colocalize con el inhibidor gephyrin andamio de proteínas y están adosados ​​a los terminales presinápticos inhibitorios, iden...

Watch this Scientific Journal Video about Using an Î±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Aquí se demuestra el uso de colorante fluorescente Alexa acoplado a α-bungarotoxina para medir GABA A localización en la superficie del receptor y endocitosis en las neuronas del hipocampo. A través del uso de construcciones que tienen una etiqueta extracelular corta que se une α-bungarotoxina, análisis de membrana de tráfico endocítica de proteínas de plasma se puede lograr.

El uso de un enlace del sitio Tag α-Bungarotoxina para el Estudio de GABA A receptor de membrana localización y el Tráfico

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

using-an-bungarotoxin-binding-site-tag-to-study-gaba-receptor

Research

JoVE Journal

Neuroscience

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

15.7K vistas.  University of Pittsburgh School of Medicine.  Aquí se demuestra el uso de colorante fluorescente Alexa acoplado a α-bungarotoxina para medir GABA A localización en la superficie del receptor y endocitosis en las neuronas del hipocampo. A través del uso de construcciones que tienen una etiqueta extracelular corta que se une α-bungarotoxina, análisis de membrana de tráfico endocítica de proteínas de plasma se puede lograr. 

Video: Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análisis cuantitativo de Reposición Synaptic piscina vesículas en cultivos de neuronas granulares del cerebelo con tintes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Investigación

Neurociencias

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

Utilizando MazeSuite y funcional espectroscopia cercana al infrarrojo para estudiar el aprendizaje de la navegación espacial

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Formación de imágenes pHluorin-etiquetados Inserción del receptor a la membrana plasmática en neuronas primarias de ratón en cultivo

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

La aplicación local de medicamentos para estudiar la función del receptor nicotínico de acetilcolina en rodajas de cerebro de ratón

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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 ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Potencial de Membrana Tinte Imaging de ventromedial del hipotálamo neuronas de ratones adultos para el Estudio de la glucosa Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Cerebro Slice Biotinilación: Una<em&gt; Ex Vivo</em&gt; Enfoque medir tráfico de proteínas específicas Región membrana plasmática en adultos las neuronas

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Localización de Genes Receptor Odorante en Antenas Locustinas por RNA<em&gt; In Situ</em&gt; Hibridación

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Imágenes en vivo de la proteína GLUT4 trata de ratón las neuronas hipotalámica primaria utilizando microscopía de deconvolución

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un enfoque alternativo para estudiar acontecimientos primarios en neurodegeneración con rodajas de cerebro de rata Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...