Este trabajo fue apoyado por la compañía de seguros de vida &quot;, AG2R-La Mondiale-&quot;, Escuela de Neurociencias de París (PEV), la Agence Nationale de la Recherche &quot;ANR-09-neur-004&quot; en el marco de la &quot;ERA-NET NEURON &quot;del 7 º PM programa por la Comisión Europea y la Fundación Pasteur. Sebastien Wagner fue apoyada por la Fundación Letten.

1. Calibración óptica: LED de medición <ol><li> Adjunte una matriz de LED a un disipador activo refrigerado por un ventilador y poner este mismo aparato LED / disipador de calor a una lente de colimación. </li><li> Cambiar la lámpara utilizada en campo claro de iluminación con LED / disipador / ventilador / aparato de la lente. Este aparato debe ser cuidadosamente colocado de modo que el haz colimado LED viaja en línea recta hacia la óptica de la lente condensadora. Asegúrese de que el disipador / ventilador está correctamente conectado a tierra a un terreno común del sistema. </li><li> Unidad de la matriz de LED con una fuente de alimentación que puede dar pulsos rápidos y de la plaza de la corriente. Esta fuente de alimentación puede ser controlada por un pulso de 5V TTL procedente de un generador de impulsos. </li><li> Centro del haz colimado a lo largo de la trayectoria de la luz se define entre el diafragma de campo y la lente del condensador. Lo ideal sería que el haz LED ligeramente llene excesivamente el diafragma de campo abierto por completo. Por lo general, un haz de luz más bien colimada llenará esta apertura menor, y se produce mortalidade el poder a costa de la uniformidad. En nuestra configuración hemos aumentado uniformidad de la luz por la elección de una lente de colimación que proyecta una imagen un poco más amplia de la matriz de LED en su plano conjugado en el diafragma consenser. </li><li> Alcanzar la iluminación Kohler, centrándose en el condensador para que la imagen del diafragma de campo (el diafragma más cercano a la fuente de luz) se centra en la cámara de corte (fig. 1). Un tejido fino de papel de la lente puede actuar como una pantalla de proyección para visualizar la imagen enfocada del diafragma de campo en otras profundidades. </li><li> Perforar una serie de agujeros de diámetro conocido en un material opaco. Coloque uno de estos pequeños agujeros sobre el sensor de potencia óptica metros. Coloque el medidor de potencia en el escenario de muestras y el centro del medidor de potencia sobre la imagen, centrado en el diafragma de campo con sólo mover el medidor de potencia hasta que se le da una lectura máxima. Colocar el medidor de potencia en esta posición. </li><li> Abra completamente todas las aberturas (diafragma y aperaturecampo de diafragma). Sistemáticamente mover la cámara de corte adjunto / medidor de potencia en relación con el paso de luz óptica y cálculo de la uniformidad de la potencia óptica en el área iluminada. Construcción de la trama la uniformidad de su sistema. Si el microscopio está bien configurada con iluminación Kohler centrado en el foco del objetivo, la potencia máxima debe estar directamente debajo del objetivo, y regiones fuera de este enfoque ahora debe recibir una cantidad conocida de energía de acuerdo con el argumento de uniformidad. </li><li> Para cada tamaño de agujero de alfiler, construir una curva de nivel de potencia óptica frente a la superficie del agujero de alfiler. Mediante el ajuste de la corriente de entrada a la matriz de LED, produce esta curva en varios niveles de potencia y de cada curva de calcular la potencia por mm2. Si el conjunto de LEDs se va a utilizar para la óptica de parches, asegúrese de introducir elementos ópticos necesarios para parchear (condensadores, perforaciones y filtros) para saber la cantidad de luz se transmite bajo la iluminación de parches. </li><li> Dar la vuelta al medidor de potencia parafrente a los objetivos, calcular la intensidad de la iluminación a 470 nm cuando la lámpara de mercurio está encendido. </li><li> Añadir un trozo vivo de la cámara, y la reconstrucción de las curvas de calibración para determinar la potencia de la luz transmitida a través del tejido cerebral de dispersión. </li></ol> 2. Procedimiento de corte y Electrofisiología Parte A: Preparación Cortar <ol><li> Anestesiar (60 mg / kg de ketamina y xilazina 2mg/kg) y decapitar el ratón. Diseccionar el cerebro artificial en el líquido cefalorraquídeo (ACSF, en mM: NaCl 124, KCl 3, 1,3 MgSO 4, 26 NaHCO 3, 1,25 NaHPO 4, 20 glucosa, 2 CaCl2; ~ 310mOsm, pH 7,4, cuando se burbujea con una mezcla de O2 95% y el 5% de CO 2 5,1), teniendo cuidado de no dañar los bulbos olfatorios. Separar los dos hemisferios y el lugar en agar con la superficie ventral, incluso con una ventaja (para las secciones horizontales). </li><li> Pegue el agar y la superficie dorsal de cada hemisferio cortical a la vibrAtome mandril, y poco a poco llenar la bañera con helado de ACSF. Rebanada de la superficie ventral en 300 secciones micras, la transferencia de cada sección para calentar (34-36 ° C) y oxigenada ACSF, que les permita recuperar durante 30-45 minutos. </li></ol> Parte B: Medición parche suelto del Umbral a Spike <ol start="3"><li> Después de llevar los cortes a la temperatura ambiente durante 30 minutos, con cuidado coloque una rebanada en la cámara de grabación de la cámara del microscopio en la perfusión constante de ACSF oxigenada. Corren el riesgo de excesiva estimulación ChR2 neuronas infectadas, la presencia de EYFP-ChR2 se puede confirmar en epifluorescencia (fig. 2a) </li><li> Tire de los electrodos de vidrio en un extractor de pipeta (Sutter P-97). Rellene este electrodo con ACSF. Cuando se coloca en el baño de la resistencia ACSF punta debe estar entre 7.10 MOhms. </li><li> Bajo iluminación fluorescente, localizar en el corte una sana ChR2-EYFP neuronas con morfología madura. También localizar soma esta neurona bajo la óptica de parches. </li><li> Con una ligera presión positiva pasa a través de la punta del electrodo, el electrodo inferior parche hacia la neurona identificado fluorescentes. Contacto con la membrana se hace, de forma rápida liberación de presión positiva y aplique una pequeña cantidad y breve de succión a través de la punta. Un sello de giga-ohm debe hacerse entre la membrana plasmática y las paredes de los electrodos de parche. </li><li> Incluso si un giga-sello no se forma, si el electrodo está suficientemente cerca de una actividad de las neuronas fluorescentes adición debe producir un potencial medible de campo local. Activar ChR2 en esta neurona mediante el parpadeo de las diferentes dosis de la luz. Porque la luz de dosis es una función tanto de LED y la duración, calcular cuánta luz es necesaria para provocar un potencial de acción en múltiples poderes y duración (figura 2b). También se observa la cantidad de adición se produce en el mercurio de iluminación de la lámpara. </li></ol> 3. Los resultados representativos: En nuestro microscopio (Olympus BX51WI), nuestro LED i esn línea con dos aberturas y una lente de condensador, manteniendo así el paso de luz original de la fábrica instalada lámpara de arco. Al cerrar tanto el diafragma de campo y el diafragma aperature, podemos lograr el contraste suficiente para grabaciones de campo claro de patch clamp (fig. 1b). Con todos los diafragmas completamente abierto que exponga el sector a la luz de encendido máximo para canalrodopsina activación (fig. 1a). En nuestro microscopio, esta configuración de parches produce la luz-la densidad que es aproximadamente tres órdenes de magnitud menor que la densidad máxima de campo completo (4,1 mW / mm 2 frente a 6,88 mW / mm 2). Vemos etiquetado robusto de adultos nacidos en gránulos bulbo olfatorio y semanas después de la infección neuronas periglomerular lentiviral de la migración de los neuroblastos en la corriente migratoria rostral (fig. 2a) Una grabación sueltas parche de un solo adulto de origen ChR2-EYFP expresión de células granulares indica que un 5 ms de estimulación en Maximum poder (6,88 mW / mm 2) es suficiente para provocar picos (figura 2b). Dado que el nivel de expresión varía entre las células, la cantidad de luz que pasa el umbral de la punta puede variar y deben ser descritos estadísticamente para cada tipo de célula de interés. <img src="/files/ftp_upload/3379/3379fig1.jpg" alt="Figura 1" /> Figura 1. LED de matriz para la configuración de campo completo de la fotoestimulación y patch-clamp electrofisiología rebanada. Para activar canalrodopsina (ChR2) que proyectan un haz colimado a través de aberturas de atrás abierta y la óptica del condensador (a). Esta configuración se puede cambiar en la óptica de parches de alto contraste por completo cerrar el diafragma de campo y la modulación de la anchura del diafragma de campo (b). Abreviaturas: a. ventilador, disipador de calor b., c. serie LED, d. colimador lentes, espejos e., f. diafragma de campo, el diafragma g. de apertura, la lente del condensador h., i. SAetapa MPLE, j. objetivo. <img src="/files/ftp_upload/3379/3379fig2.jpg" alt="Figura 2" /> Figura 2. Imagen de una sección horizontal de 300μm bulbo olfatorio de patch clamp y fotoestimulación todo el campo (a). Lentivirally adultos infectados nacidos en las células granulares expresar ChR2-EYFP se puede ver irradiando desde el centro del bulbo olfatorio. La luz de dosis necesarias para provocar picos se puede encontrar mediante el aumento de la duración del flash LED (b). El umbral de esta era de células granulares de 5 ms a plena intensidad LED (2.43mW/mm 2). Escala en (a) = 500μm, la escala en (b) = 50 ms.

<ol>
	<li>Nissant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+promotes+synaptic+plasticity+in+the+olfactory+bulb.">Adult neurogenesis promotes synaptic plasticity in the olfactory bulb.</a> Nature Neuroscience. 12, 728-730 (2009).</li><li>Apicella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+Cells+in+Piriform+Cortex+Receive+Convergent+Input+from+Distinct+Olfactory+Bulb+Glomeruli.">Pyramidal Cells in Piriform Cortex Receive Convergent Input from Distinct Olfactory Bulb Glomeruli.</a> Journal of Neuroscience. 30, 14255-14260 (2010).</li><li>Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=genetically+targeted+optical+control+of+neural+activity.">genetically targeted optical control of neural activity.</a> Nature. 8, 1263-1263 (2005).</li><li>Bardy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb.">where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.</a> The Journal of Neuroscience. 30, 17023-17034 (2010).</li><li>Grubb, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+of+the+first+synapse+in+olfaction:+development+and+adult+neurogenesis.">Functional maturation of the first synapse in olfaction: development and adult neurogenesis.</a> The Journal of neuroscience. 28, 2919-2932 (2008).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit-breakers:+optical+technologies+for+probing+neural+signals+and+systems.">Circuit-breakers: optical technologies for probing neural signals and systems.</a> Nature reviews. Neuroscience. 8, 577-581 (2007).</li><li>Grossman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-site+optical+excitation+using+ChR2+and+micro-LED+array.">Multi-site optical excitation using ChR2 and micro-LED array.</a> Journal of neural engineering. 7, 16004-16004 (2010).</li><li>Dhawale, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-redundant+odor+coding+by+sister+mitral+cells+revealed+by+light+addressable+glomeruli+in+the+mouse.">Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse.</a> Nature neuroscience. 13, 1404-1412 (2010).</li><li>Weick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+control+of+transplantable+human+ESC-derived+neurons+via+optogenetic+targeting.">Functional control of transplantable human ESC-derived neurons via optogenetic targeting.</a> Stem cells. 28, 2008-2016 (2010).</li><li>Toni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+born+in+the+adult+dentate+gyrus+form+functional+synapses+with+target+cells.">Neurons born in the adult dentate gyrus form functional synapses with target cells.</a> Nature Neuroscience. 11, 901-907 (2008).</li><li>Grubb, M. S., Burrone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2+Localised+to+the+Axon+Initial+Segment.">Channelrhodopsin-2 Localised to the Axon Initial Segment.</a> PLoS ONE. 5, e13761-e13761 .</li><li>Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amygdala+circuitry+mediating+reversible+and+bidirectional+control+of+anxiety.">Amygdala circuitry mediating reversible and bidirectional control of anxiety.</a> Nature. 471, 358-362 (2011).</li></ol>

No hay conflictos de interés declarado.

En los últimos años han visto una explosión en la popularidad de las herramientas para la investigación en neurociencias optogenetic 6. Como resultado de ello, es cada vez más importante para reducir la barrera de entrada para los laboratorios que deseen comenzar a utilizar estas nuevas herramientas. A continuación se describe cómo llevar a cabo una adaptación fácil y barato y la calibración de un sistema convencional de patch-clamp plataforma para que pueda hacer todo el campo de la estimulación ?...

<table border="1">
	<tbody>
		<tr>
			<td>Material</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue No.</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>100 mg/ml</td>
			<td>Imalg&egrave;ne 1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>2%</td>
			<td>Rompun</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbps;</td>
			<td>Sigma </td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>P5405</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>M1880</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5761</td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5011</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>C7902</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>A9539</td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>P-97</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>1.5 mm O.D./1.17 mm I.D.</td>
			<td>Harvard Apparatus</td>
			<td>GC150T-10</td>
		</tr>
		<tr>
			<td>LED array</td>
			<td>&nbsp;</td>
			<td>Bridgelux</td>
			<td>BXRA-C2000</td>
		</tr>
		<tr>
			<td>Collimating lens</td>
			<td>40 mm beam diameter</td>
			<td>Thor Labs</td>
			<td>LEDC1</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>2.8 amp</td>
			<td>A1W Electronik</td>
			<td>HKO2800</td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>&nbsp;</td>
			<td>Thor Labs</td>
			<td>PM 100</td>
		</tr>
		<tr>
			<td>Heatsink </td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Fan</td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>&nbsp;</td>
			<td>Leica</td>
			<td>VT1200S</td>
		</tr>
	</tbody>
</table>

using affordable led arrays for photo-stimulation of neurons

Electrofisiología rebanada estándar ha permitido a los investigadores para probar los componentes individuales de los circuitos neuronales mediante el registro de las respuestas eléctricas de las células individuales en respuesta a las manipulaciones de 1,2 eléctrica o farmacológica. Con la invención de métodos para controlar ópticamente genéticamente las neuronas específicas (optogenética), los investigadores ahora tienen un nivel sin precedentes de control sobre grupos específicos de neuronas en la preparación rebanada estándar. En particular, fotosensibles canalrodopsina-2 (ChR2) permite a los investigadores para activar las neuronas con luz 3,4. Mediante la combinación de una cuidadosa calibración de LED basada en la fotoestimulación de ChR2 con electrofisiología rebanada estándar, se podrá analizar con mayor detalle el papel de los adultos nacidos en las interneuronas en el bulbo olfatorio, el primer relevo el centro del sistema olfativo. Utilizando la expresión viral de ChR2-YFP específicamente en los adultos nacidos en las neuronas, que pueden controlar de forma selectiva los adultos jóvenes nacidos en las neuronas en un entorno de mayores unad maduro neuronas. Nuestro control óptico utiliza un sistema sencillo y barato LED, y se muestra cómo este sistema puede ser calibrado para comprender cuánta luz se necesita para provocar picos de actividad de neuronas individuales. Por lo tanto, breves destellos de luz azul puede controlar a distancia el patrón de activación de las células transducidas ChR2-recién nacido.

Watch this Scientific Journal Video about Using Affordable LED Arrays for Photo-Stimulation of Neurons at JoVE.com

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Adultos nacidos en las neuronas que expresan ChR2 puede ser manipulado en los preparativos rebanada electrofisiológicos para examinar su contribución a la función olfativa de los circuitos neuronales.

Utilizando asequible Estructuras de LED para fotos-estimulación de las neuronas

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

using-affordable-led-arrays-for-photo-stimulation-of-neurons

Research

JoVE Journal

Neuroscience

18.5K vistas.  Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). Adultos nacidos en las neuronas que expresan ChR2 puede ser manipulado en los preparativos rebanada electrofisiológicos para examinar su contribución a la función olfativa de los circuitos neuronales.

Video: Using Affordable LED Arrays for Photo-Stimulation of Neurons

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

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Escalable neumático matrices inyector para Viral focalización de los circuitos cerebrales intactas en 3-D

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

Investigación

Neurociencias

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusión lateral y exocitosis de proteínas de membrana en neuronas cultivadas evaluó a través de la recuperación de fluorescencia y la pérdida de fluorescencia photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imaging las espinas dendríticas de las neuronas del hipocampo de rata primaria utilizando Estructurado Iluminación Microscopía

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Producción y aislamiento de los axones de las neuronas sensoriales para el análisis bioquímico Usando porosos Filtros

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

La excitación externa de neuronas usando campos eléctricos y magnéticos en culturas de una y dos dimensiones

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

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 In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Un modelo in vitro para estudiar la agregación de Tau utilizando la transducción de neuronas humanas mediada por lentivirales

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Implantación Crónica de Múltiples Matrices de Electrodos Flexibles de Polímeros

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).