I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1. crudo Dauer Pheromone Producción <ol><li> Crecer OP50 E. coli a partir de una sola colonia O / N a 37 ° C mientras se agita en 100 ml de caldo LB a 200 rpm. NOTA: Este cultivo O / N se puede hacer con antelación y se almacena a 4 ° C. </li><li> Crecer N2 C. elegans en junio 15 a 20 cm placas de Petri con agar NGM (ver receta en la Tabla 1) se sembró con 50 l de E. OP50 coli hasta que las bacterias se ha consumido casi por 19. </li><li> Hacer 5x 250 ml de S medios (Ver receta en la Tabla 1) en 1 L matraces Erlenmeyer de 19. NOTA: Esta solución se puede hacer con antelación. </li><li> Cultivar una cultura de OP50 E. coli en 4 L de caldo LB O / N mediante la inoculación con 1 ml de OP50 en E. coli y agitación a 37 ° C durante 16 horas. </li><li> Lavar los nematodos de las placas (paso 1,2) con 1 ml de solución de S basal (ver receta en la Tabla 1) y nematodos de transferencia 4-5 placas en cada uno de 4 1 matraces Erlenmeyer de L con los medios de comunicación S preparado en la etapa 1.3 (4 5 placaspara cada matraz) 19. </li><li> Centrifugar la OP50 desde el paso 1.4 a 650 xg durante 10 min y retirar el sobrenadante. Resuspender cada sedimento bacteriano en 10 ml de solución de S basal. Transfiera la misma cantidad de bacterias resuspendidas a cada frasco de nematodos. </li><li> Colocar los matraces en un agitador orbital a temperatura ambiente (20 - 25 ° C) y 100 - 150 rpm durante 4 - 5 días hasta que la solución comienza a limpiar, lo que indica un agotamiento de los alimentos. </li><li> Una vez que el alimento se agota, crecer otro cultivo O / N de OP50 E. coli en 4x 1 L de caldo LB. Centrifugar las bacterias a 650 xg durante 10 min y añadir una pastilla de un frasco a cada uno de los matraces S de medios con nematodos. Devolver los frascos S de medios a la agitación, como en el paso 1.7, de 3-4 días hasta que la comida se agota de nuevo. </li><li> Tome un ~ 1 ml alícuota de solución de cada matraz y estimar la concentración de nematodos por sub-muestreo de 1 l de cada alícuota de 1 ml y contando el número de nematodos. NOTA: Lapoblación de nematodos debe ser 50 000 - 75 000 nematodos / ml. A esta concentración la mayoría de los nematodos son dauers. </li><li> Deseche todos los frascos contaminados. NOTA: La contaminación se manifiesta con frecuencia como hifas fúngicas visto durante el paso 1.9. </li><li> Nematodos centrifugar a 4000 xg durante 10 min a 4 ° C. Deseche el sedimento nematodo y retener el sobrenadante. Como alternativa, vea la discusión para el uso de estos animales para hacer una feromona de baja potencia. </li><li> Filtrar el sobrenadante a través de # 1 papel filtro Whatman. NOTA: Un frasco de vacío acelerará este proceso. </li><li> Filtrar aún más el sobrenadante a través de 0,45 micras unidades de filtración de vacío estéril. NOTA: El filtrado se puede mantener O / N a 4 ° C o proceder a 1,14. </li><li> Añadir el filtrado a un vaso de precipitados de 2 l con una barra de agitación. Coloque en una campana extractora en una placa calefactora revuelo. Llevar a ebullición mientras se agita y se evapora hasta aproximadamente 200 ml. Transfiera la solución a un vaso de 500 ml. Continuar la ebullición hasta aproximadamente 50 ml son left. NOTA: Se debe tener cuidado durante este paso, ya que es posible que la solución a hervir. El color de la final de 50 ml es un marrón oscuro. En cualquier momento durante el proceso de cocción, el calor puede ser apagado y el filtrado almacena O / N a temperatura ambiente o 4 ° C. </li><li> Dejar enfriar y centrifugar durante 5 min a 1000 x g. Verter el sobrenadante a un vaso de 100 ml y desechar el sedimento. </li><li> Hervir el sobrenadante a una corteza de color marrón. Retire del fuego y romper la corteza con una espátula. Añadir suficiente etanol prueba 200 para cubrir la corteza. Cubrir el vaso con Parafilm y dejar que repose O / N a temperatura ambiente. Verter el etanol en un vaso de precipitados limpio y poner a un lado. Añadir etanol fresco para cubrir la corteza. Repita extracciones O / N 3 veces. Reunir las fracciones. </li><li> Utilice una bomba de vacío para secar los extractos de etanol. Si una bomba de vacío no está disponible, añadir el extracto de etanol a un vaso de precipitados limpio y cálido a 50 ° C mientras se agita en una campana de humos. NOTA: Una vez que el etanol se ha evaporado un residuo pegajoso de lde color marrón de vuelo debe permanecer. Si se utiliza calentamiento, se debe tener cuidado para no quemar la solución por calentamiento durante demasiado tiempo. </li><li> Disolver residuo en 10 ml de agua destilada estéril. Filtro esterilizar esta solución con un filtro de jeringa de 0,22 micras y se almacenan a -20 ° C en alícuotas de 1 ml. NOTA: 1 ml de agua se utiliza para disolver el residuo por cada 100 ml del cultivo de nematodos líquido original. Por ejemplo, si uno de los matraces de cultivo líquido se descarta debido a la contaminación, a continuación, ajustar la cantidad de agua utilizada para disolver el residuo. </li></ol> 2. Determinación de Pheromone Respuesta a la dosis <ol><li> Prepare feromona Dauer haciendo 6x 5 ml de medio NGM con agar Noble, sin peptona, y se suplementó con 50 mg / ml de sulfato de estreptomicina y una gama de feromona Dauer (véase la receta en la Tabla 1) 4, 19. Mezclar componentes en un 13x100 mm tubo de cultivo de vidrio, hervir sobre un mechero Bunsen, y luego verter en placas de Petri de 3 cm. </li><li> Pesar amtubo icrocentrifuge. Añadir 1 ml de cultivo O / N del E. coli OP50 al tubo y centrifugar a ~ 17.000 xg en un tubo de microcentrífuga. Retire todo el sobrenadante. Pesar el tubo de nuevo y determinar el peso de la pella bacteriana. Resuspender el sedimento con tampón M9 (ver receta en la Tabla 1) para una concentración final de 4% (w / v). Alícuota de 10 l de la suspensión bacteriana en el centro de cada placa de feromona 4. Placas Tienda O / N a temperatura ambiente. </li><li> Escoja 10-15 grávidas N2 hermafroditas de tipo salvaje un día después de la muda hasta la edad adulta en las placas de feromonas 4. Almacenar las placas a 25 ° C durante 3 - 4 horas. </li><li> Escoger de todos los adultos hermafroditas. Anote el número de huevos puestos por cada placa. Sellar las placas con Parafilm y volver a 25 ° C durante 3 días 17. </li><li> Cuente el porcentaje de dauers (Figura 2). Nota: Para ambos métodos es importante tener en cuenta que dauers (y en menor extienda de campaña no dauers) trepará por las paredes y en la tapa de platos de Petri. Por tanto, es importante examinar todas las superficies del plato para los nematodos. </li><li> Ajustar los datos a una curva dosis-respuesta y calcular la concentración EC 90 de feromona (feromona suficiente para inducir 90% de los animales para formar dauers) como se muestra en la Figura 3 Nota:. Normalmente utilizamos un modelo de regresión logística para estimar la CE 90 , pero diferentes paquetes de software estadísticos pueden tener métodos alternativos que producen estimaciones similares. </li></ol> 3. imágenes en vivo de Dauer IL2 Neuronas <ol><li> Preparar platos de feromonas dauer como en el protocolo 2 utilizando el valor EC 90 determinada en el paso 2.6. </li><li> Inducir dauers utilizando los métodos en el protocolo 2 con una cepa con un reportero transgén integrado IL2 (myIs13 [P klp-6 :: GFP], myIs14 [P klp-6 :: GFP], myIs16 [P <em&gt; klp-6 :: tdTomato] o qIs56 [P desfase-2 :: GFP]) 12, 20, 21. NOTA: Si se utiliza q Is56, expresión de GFP no será visible en los IL2Qs hasta varias horas en el muda Dauer y algunos de los eventos iniciales de remodelación no serán visibles. Además, mientras que la señal fluorescente del P lag-2 :: GFP transgén es en última instancia, más brillante en los IL2s durante dauer que los KLP-6 reporteros fluorescentes P, el P lag-2 :: GFP transgén también expresa débilmente en los músculos de la cabeza que puede enmascarar las ramas finas IL2. </li><li> Después de la puesta de huevos, retire los adultos hermafroditas, sellar las placas con Parafilm y se incuba durante 34 horas a 25 ° C 17. NOTA: La longitud de tiempo para la muda Dauer varía ligeramente entre los individuos. El período más rápido de la arborización dendrítica comienza 4-5 horas después del inicio de la muda dauer (39-44h después de la puesta de huevos a 25 ° C) 12. </li><li> El día antes de formación de imágenes, conforman una solución de 10% (w / v) de agarosa en tampón M9 con feromona Dauer a la concentración CE 90. Alícuota de aproximadamente 100 l de agarosa en un portaobjetos de microscopio y de forma rápida, pero suavemente, coloque otra diapositiva en la parte superior para crear una superficie plana de agarosa. Después de la agarosa se ha solidificado, envuelva las diapositivas en papel film y guardar en una cámara de humedad (caja de puntas de pipeta con toallas de papel húmedas). </li><li> Tras el período de incubación 34 horas, examinar las placas de feromonas para nematodos que han dejado de bombeo de la faringe. Diapositivas separadas de tal manera que sólo una superficie de una diapositiva tiene agarosa en él. Elija uno o más nematodos en una almohadilla de agarosa al 10% y 1 μ l de 0,1 micras perlas de poliestireno 22. NOTA: Con una población bien sincronizado, la mayoría de los nematodos en el plato va a ingresar en la muda de L2d a dauer. Sin embargo, puede haber variación entre ias personas. Por tanto, es importante tener en cuenta características adicionales (oclusión de estoma, la remodelación de la faringe) para estimar la cantidad de tiempo que los animales hayan estado dentro de la muda Dauer (ver resultados representativos, por ejemplo). </li><li> Determine la orientación (izquierda / derecha, dorsal / ventral) del nematodo. Captura imágenes Z-stack de un dendrita IL2Q a 100 aumentos con la intensidad de la fluorescencia bajo posible cada 15 minutos o cuando sea necesario para el experimento específico. Continuar la captura de imágenes hasta que el nematodo se ha separado de la cutícula L2d y la inmovilización es imposible o si el animal se recupera de Dauer (faringe comienza a bombear). Cuando no la formación de imágenes de forma activa, coloque el portaobjetos en una cámara húmeda. NOTA: Nosotros usamos un microscopio de epifluorescencia con un contraste de interferencia diferencial escenario y motorizado (DIC) óptica. </li></ol>

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The authors have nothing to disclose

Un examen de la neuroplasticidad-dauer específicos y otros cambios morfológicos dauer asociada requiere controlado y formación fiable de dauers ya sea a través de la mutación genética (es decir, mutantes, daf-c) o por la exposición de los animales de tipo salvaje de la feromona. Mientras que el uso de una mutación genética para inducir dauers es conveniente, puede confundir los resultados. Por lo tanto, los datos recogidos con mutaciones daf-c deben confirmarse posteriormente con animales de t...

Plasticidad fenotípica, incluyendo la plasticidad del desarrollo y del comportamiento, es importante para las adaptaciones a las condiciones ambientales adversas y se considera una fuerza impulsora en la evolución 1. C. elegans es un organismo modelo utilizado con frecuencia para estudios en el desarrollo y la neurobiología. En condiciones ideales, C. elegans se desarrolla a través de cuatro estadios larvarios antes de entrar en la etapa reproductiva de adultos. Sin embargo, en condiciones de baja disponibilidad de alimentos y las altas densidades de población, C. elegans puede someterse a un interruptor de desarrollo y entrar en una de larga duración y resistente a la tensión etapa Dauer 2. Dauers muestran varias diferencias morfológicas y de comportamiento de los no dauers que probablemente median esta resistencia al estrés. Las señales ambientales y los mecanismos genéticos que regulan la entrada dauer han sido ampliamente descrito 3, 4, 5, 6, 7. Sin embargo, los mecanismos moleculares que controlan los cambios fenotípicos individuales que ocurren ddauer formación urante son menos conocidos. El examen de los fenotipos dauer específica requiere la producción de animales dauer. Esto se puede lograr de tres maneras: 1) Levantar partir de un cultivo de C. hambre elegans, 2) uso de la formación de dauer constitutiva (daf-c) mutantes, 3) Inducción de la formación de dauer través de feromona purificado. Es relativamente sencillo para recoger dauers de una placa estándar NGM con una C. población elegans que ha agotado su suministro de alimentos-bacteriana. En nuestras manos, una placa estándar NGM originalmente se siembra con 50 l de OP50 Escherichia coli, se dejó crecer durante 3 días y posteriormente inoculadas con 3 - 4 adultos hermafroditas se mantuvo a 25 ° C, se agotará el suministro de alimentos dentro de una semana y producir varios miles dauers dentro de 3 días adicionales (además de numerosos no dauers hambre). Sin embargo, este método es insatisfactorio para examinar procesos que ocurren durante la muda en dauer. Es difícil determinar cuál de los varios miles de animales en un plato típico hambriento están entrando en dauer. Además, el uso de una placa de hambre no ofrece ninguna posibilidad para la sincronización. El uso de mutantes daf-c permite la sincronización y la generación fiable de dauers. Sin embargo, no hay garantía de que una mutación particular daf-c no afectará a otros fenotipos-dauer específica solo o en combinación con mutaciones adicionales. La presencia de una feromona dauer se implicó por primera vez por Cassada y Russell y más tarde demostró por Golden y Riddle. La feromona dauer desde entonces se ha caracterizado químicamente 2, 8, 9, 10. La feromona purificada consiste en una mezcla compleja de ascarosides, que en diferentes formas a regular los procesos conductuales y de desarrollo 9, 11. Uso de un extracto de feromona dauer crudo permite inducción fiable controlada de dauers sincronizados en un fondo de tipo salvaje. </p&gt; El sistema nervioso y las células gliales asociadas se someten a la remodelación durante dauer larva formación 12, 13, 14. Algunos de estos cambios son irreversibles, tales como la remodelación de las células de la glia durante dauer 13, 14. Sin embargo otros eventos de remodelación son reversibles en un retorno a la favorable condiciones ambientales. Por ejemplo, un conjunto de cuatro neuronas IL2Q se someten a la remodelación neuronal rápida y reversible durante la formación de dauer incluyendo: arborización dendrítica, un interruptor de una sola dendríticas a las neuronas y los axones multidendritic remodelación 12. Los métodos de la presente memoria permiten la formación de imágenes in vivo fiable de la neuroplasticidad inducida por estrés en un organismo modelo. Los métodos presentados para la producción y las pruebas de feromona dauer crudo se describen anteriormente y compilados a partir de varias fuentes de 4, 8, 15, 16, 17, 18.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
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			<td height="40" style="height:40px;width:259px;">N2 C. elegans Bristol strain</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td style="width:212px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2660 myIs13[Pklp-6::gfp] III</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2762 myIs14[Pklp-6::gfp] V</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">JK2868 qIs56[Plag-2::gfp] V</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OP50 E. coli</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td style="width:152px;">Tritech Research</td>
			<td>60 mm T3308, 35 mm T3501</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;">NGM agar</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">3 g NaCl, 17 g agar, 2.5 g peptone in 975 mL H2O autoclaved cooled to 55&#176;C followed by 1 mL of 5 mg/ml cholesterol in ethanol, 25 mL of 1 M KPO4 buffer (pH 6.0), 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4</td>
		</tr>
		<tr height="356">
			<td height="356" style="height:356px;">S Media</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">S media consists of S basal solution (1.46 g NaCl, 0.25g K2HPO4, 1.5 g KH2PO4, 0.25 ml of 5 mg/ml cholesterol in ethanol and H2O to 250 mL. Autoclaved.) To the S basal solution add 2.5 mL of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate, H2O to 100 mL, autoclaved), 2.5 mL of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO4&#160;&#8226;7 H2O, 0.2 g MnCl2&#8226;4 H2O, 0.29 g ZnSO4&#160;&#8226;7 H2O, 0.025 g CuSO4&#160;&#8226;5 H2O, H2O to 1 liter. Autoclaved and stored in the dark), 0.75 mL of 1 M CaCl2, and 0.75 mL of 1 M MgSO4.</td>
		</tr>
		<tr height="392">
			<td height="392" style="height:392px;width:259px;">Dauer pheromone plates for dose-response assay</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 0.085 g Noble agar, 0.5 mL of 0.513 M NaCl and 5 &#181;L of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 mL culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H2O to bring the volume to 4.725 mL.&#160; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &#176;C. Add 125 &#181;L 1 M KPO4 buffer (pH 6.0), 50 &#181;l 0.1 M CaCl2, 50 &#181;L 0.1 M MgSO4 and 50 &#181;l of 5 mg/ml streptomycin sulfate to each tube.&#160; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsphere beads 0.1 micron</td>
			<td style="width:152px;">Polysciences Inc.</td>
			<td>00876-15</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">M9 buffer</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 liter. Sterilize by autoclaving</td>
		</tr>
	</tbody>
</table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

Los mecanismos que controlan el estrés inducido por la plasticidad fenotípica en animales son frecuentemente complejos y difíciles de estudiar in vivo. Un ejemplo clásico de la plasticidad inducida por el estrés es la etapa dauer de C. elegans. Dauers son una etapa larval de desarrollo alternativo formado bajo condiciones de bajas concentraciones de los alimentos bacteriana y altas concentraciones de una feromona Dauer. Dauers muestran amplia plasticidad del desarrollo y del comportamiento. Por ejemplo, un conjunto de cuatro cuadrantes interior-labial (IL2Q), las neuronas se someten a una amplia remodelación reversible durante la formación de Dauer. Utilizando la entrada vías ambientales de regulación Dauer bien conocido, un método previamente establecido para la producción de feromona Dauer crudo a partir de cultivos de nematodos líquido a gran escala se demuestra. Con este método, una concentración de 50.000 - 75.000 nemátodos / ml de cultivo líquido es suficiente para producir una muy potente feromona Dauer crudo. La potencia feromona crudo es determined mediante un bioensayo de respuesta a la dosis. Por último, se describen los métodos utilizados para in vivo time-lapse de los IL2Qs durante la formación de dauer.

La producción de resultados de feromonas dauer crudo en un líquido amarillo (Figura 1) que es tanto el calor como el frío estable. Alícuotas de feromona crudo se almacenan a -20 ° C indefinidamente sin pérdida evidente de la actividad. Después de la producción de feromona, un único bioensayo dosis-respuesta es suficiente para una estimación aproximada de la potencia. Sin embargo, para la mayoría de los experimentos es necesario repetir el bioensayo 2 - 3 veces y tomar un promedio de cada expe...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

En vivo de imágenes de Dauer-específica neuronal Remodelación en C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

in-vivo-imaging-of-dauer-specific-neuronal-remodeling-in-c-elegans

Research

JoVE Journal

Neuroscience

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

11.1K vistas.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

Video: In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

La transfección de células de ratón del ganglio de la retina por In vivo La electroporación

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Investigación

Neurociencias

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

En vivo láser axotomía en C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

La cuantificación automática de la fluorescencia en el Synaptic<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dos registros electrofisiológicos de sinápticamente respuestas evocadas neuronal y astroglial en agudas rodajas de hipocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Formación de imágenes in vivo de calcio neuronal en C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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 ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

Super-resolución de imagen de Neuronales vesículas de núcleo denso

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologías combinadas para definir el papel de la membrana plasmática de entrega Durante Axon ramas y morfogénesis neuronal

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

Usando un Chip de microfluidos adaptado olfativo para la proyección de imagen de la actividad Neuronal en respuesta a las feromonas en las neuronas de cabeza de macho de C. Elegans

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

Enfoques cuantitativos para la puntuación in vivo de la extrusión de agregados neuronales y organelle en vesículas de exoféro grandes en C. elegans