Name: in vivo neuronal calcium imaging in c. elegans
Uploaded: 2013-04-10T13:00:00
Description: Watch this Scientific Journal Video about In vivo Neuronal Calcium Imaging in C. elegans at JoVE.com

Varias personas contribuyeron al trabajo descrito en este documento. CVG construyó el montaje experimental, y LS, SHC, y CVG realizado los experimentos. CVG y SHC escribió el manuscrito. Todos los autores participaron posteriormente en el proceso de revisión y aprobó la versión final del manuscrito. Damos las gracias a Paul Sternberg para la cepa GCaMP. Algunas cepas de nematodos utilizadas en este trabajo fueron proporcionados por el Centro de Genética Caenorhabditis (CGC), que es financiado por el NIH Centro Nacional para Recursos de Investigación (CNRR). El programa de análisis de imagen MATLAB se adaptó del que se utiliza en 18. Los autores recibieron el apoyo de la Universidad de Boston y el Massachusetts Life Sciences Center.

1. Configuración óptica <ol><li> El uso de un microscopio compuesto estándar con capacidades de imagen de epifluorescencia. Utilizamos una Nikon Eclipse Ti-U microscopio invertido con un iluminador Intensilight HG. </li><li> Para una mejor imagen y calidad de la señal utiliza un gran aumento, objetivo gran apertura numérica. Nos suelen utilizar una Nikon X60 1,4 NA objetivo de inmersión en aceite. En algunos casos es posible usar menor aumento (X40, X20) dependiendo del nivel de expresión del fluoróforo y fuer...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+AFD+neuron+in+C.+elegans+thermotaxis+analyzed+using+femtosecond+laser+ablation.">The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation.</a> BMC Neurosci. 7, 30 (2006).</li><li>Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+dynamic+range+of+fluorescent+indicators+for+Ca(2+)+by+circularly+permuted+yellow+fluorescent+proteins.">Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 10554-10559 (2004).</li><li>Kindt, K. S., Quast, K. B., Giles, A. C., De, S., Hendrey, D., Nicastro, I., Rankin, C. H., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+mediates+context-dependent+modulation+of+sensory+plasticity+in+C.+elegans.">Dopamine mediates context-dependent modulation of sensory plasticity in C. elegans.</a> Neuron. 55, 662-676 (2007).</li><li>Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+a+circuit+for+olfactory+behaviour+in+Caenorhabditis+elegans.">Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans.</a> Nature. 450, 63-70 (2007).</li><li>Clark, D. A., Gabel, C. V., Gabel, H., Samuel, A. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+Cyclic+AMP+Promote+Axonal+Regeneration+in+Caenorhabditis+elegans+and+Require+DLK-1+Kinase.">Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase.</a> Journal of Neuroscience. 30, 3175-3183 (2010).</li><li>Bianchi, L., Gerstbrein, B., Frokjaer-Jensen, C., Royal, D. C., Mukherjee, G., Royal, M. A., Xue, J., Schafer, W. R., Driscoll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotoxic+MEC-4(d)+DEG/ENaC+sodium+channel+conducts+calcium:+implications+for+necrosis+initiation.">The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation.</a> Nat. Neurosci. 7, 1337-1344 (2004).</li></ol>

Genéticamente codificados indicadores de calcio se han utilizado ampliamente en C. elegans neurobiología. Numerosos grupos han empleado estas técnicas para estudiar la respuesta de las neuronas sensoriales primarias a estímulos externos como se demuestra aquí con la respuesta ASJ a un campo eléctrico. Ejemplos destacados incluyen sensación del tacto mecánico, productos químicos específicos, la temperatura y un campo eléctrico 12, 16-19. Actividad de interneuronas y células musculares tamb...

Aquí presentamos métodos prácticos para la formación de imágenes in vivo del calcio en C. Elegans neuronas. El desarrollo de las codificadas genéticamente sensibles calcio-fluoróforos con alta relación de señal a ruido hace C. elegans un sistema efectivo comparativamente sencillo y rentable para la medición de la neurofisiología y la actividad. Nuestra formación de imágenes se realiza con un microscopio compuesto estándar utilizando amplio campo de imágenes de fluorescencia de los fluoróforos disponibles comúnmente. Se presentan varias técnicas que emplean diferentes fluoróforos y preparaciones diferentes de la muestra, discutir las fortalezas y debilidades de cada uno. Los datos se presentan a continuación, a partir de dos experimentos de ejemplo. Un excelente recurso adicional sobre las técnicas descritas aquí se pueden encontrar en WormBook, &quot;Imagen de la actividad de las neuronas y los músculos&quot; por R. Kerr, ( <a href="http://www.wormbook.org" target="_blank">http://www.wormbook.org</a> ) 3. Dos clases principales de genéticamentecamente codificados reporteros fluorescentes de calcio se utilizan comúnmente en C. elegans: GCaMP solo canal y cameleon FRET basada. Vamos a describir los métodos y ejemplos para mostrar los datos generados por cada uno. GCaMP se basa en una modificación de la proteína verde fluorescente (GFP) que es sensible a la concentración de calcio circundante. Esto se logra mediante la fusión de GFP y la calmodulina calcio de alta afinidad de proteína, de tal manera que la unión del calcio por la calmodulina lleva la molécula de GFP en una confirmación eficiente fluorescente 2. Los recientes avances en estos fluoróforos generar tamaño de la señal excepcional con hasta 500% de aumento en la intensidad de fluorescencia en un rango fisiológico de los niveles de calcio y la cinética razonablemente rápidos de ~ 95 ms tiempo de subida y el tiempo de decaimiento ~ 650 mseg 4. Durante períodos de tiempo relativamente cortos (minutos), estas señales pueden permitir grandes para obtener imágenes de resolución más baja (menor magnificación) y, dado un buen comportamiento initmedida de referencia ial, niega la necesidad de línea de base continua o mediciones comparativas. Cameleon tiene la ventaja de ser un fluoróforo FRET basada en el que genera una medición radiométrica comparando dos canales independientes o longitudes de onda 1. Se compone de dos fluoróforos diferentes (proteínas fluorescentes cian y amarillo, emisores de PPC y YFP) unidos por una proteína calmodulina. El complejo está iluminado con luz azul (440 nm) que excita la PPC. La unión de calcio trae los fluoróforos más juntos, aumentando la transferencia de energía de resonancia fluorescente (FRET) de la PPC (donante) a la YFP (aceptor) y provocando la emisión PPC (480 nm) para disminuir la emisión y YFP (535 nm) para aumentar . Los niveles relativos de calcio se mide como la relación de la intensidad YFP / CFP. Cinética Cameleon son más lentas que la de GCaMP, medido in vivo para tener un tiempo de subida de ~ 1 sec y un tiempo de desintegración de 3 ~ 5 seg. Sin embargo,la relación de las señales que se mueven opuestamente aumenta el tamaño de la señal y compensa un número de posibles artefactos debido a los cambios en la concentración de fluoróforo, el movimiento o la deriva enfoque y blanqueo. Codificados genéticamente reporteros fluorescentes negar gran parte de la preparación de la muestra requiere exógenamente con sondas administrados y C. elegans pequeño cuerpo transparente permite visualizar imágenes en el animal intacto utilizando fluorescencia sencilla de campo amplio. El principal reto técnico en preparación de la muestra es por lo tanto, para inmovilizar de forma segura a los animales. Hay un número de diferentes técnicas comúnmente utilizadas cada uno con ventajas y desventajas. El uso de un agente farmacológico para paralizar a los animales es fácil de implementar y permite el montaje de múltiples animales en una preparación (Levamisol, un agonista colinérgico que hace que el tejido muscular para aprovechar se utiliza típicamente 6). C. elegans también puede ser físicamente inmovilizada mediante el montaje de losen 10% de agarosa rígido 7, 8. Esto minimiza el impacto en la fisiología del animal, permite a largo plazo de imágenes (horas) y la recuperación de varios animales, pero es más difícil técnicamente. Ambas técnicas restringir el acceso físico a los animales (que están bajo un cubreobjetos) y, por tanto, sólo pueden utilizarse con ciertos estímulos experimentales (tal como luz, temperatura, campo eléctrico o daño láser). Para los estímulos que se requiere acceso físico, como el tacto o la administración de los productos químicos, muchos estudios han pegado con éxito C. elegans en su lugar (el uso de pegamento de grado veterinario) 9. Esto es técnicamente más difícil, es una preparación solo animal y no permite la recuperación de los animales. Por último, numerosos dispositivos de microfluidos han sido empleados que físicamente restringir C. elegans, preservando la fisiología animal, permitiendo la exposición a la mayoría de los tipos de estímulos (dependiendo del diseño del dispositivo) y puede permitir el intercambio rápido y la recuperación de los animales <sup&gt; 10, 11. Sin embargo microfluidos adicionales requieren habilidades técnicas y capacidades en diseño, fabricación e implementación. En actividad inmovilizado animales y estímulo respuesta generalmente se puede medir en sensorial y interneuronas. Actividad de las neuronas motoras requiere de técnicas más sofisticadas para la formación de imágenes en movimiento en los animales. Aquí vamos a presentar métodos detallados que emplean las dos técnicas más sencillas de paralización e inmovilización farmacológica con rigidez de agarosa. Los métodos presentados aquí pueden ser utilizados para medir la actividad neuronal y la fisiología celular en C. elegans. Damos un ejemplo de cada uno de: el uso de GCaMP para medir la respuesta de la neurona sensorial ASJ a un campo eléctrico externo, y utilizando cameleon para medir la respuesta de calcio fisiológica a la lesión con láser de una neurona. Estos ejemplos muestran las ventajas y los inconvenientes de los dos tipos de fluoróforos e ilustran lo que es posible con el sistema.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Eclipse Ti-U inverted 
 microscope</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Intensilight HG Illuminator</td>
 <td>Nikon</td>
 <td>C-HGFI</td>
 <td>Fluorescent light source</td>
 </tr>
 <tr>
 <td>CFI Plan Apo VC 60X Oil</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Optical table or 3'X3' optical 
 grade breadboard</td>
 <td>Thor Labs</td>
 <td>&nbsp;</td>
 <td>If an optical table is not used an 
 optical grade breadboard on a 
 solid laboratory bench should 
 suffice.</td>
 </tr>
 <tr>
 <td>Clara Interline Camera</td>
 <td>Andor 
 Technology</td>
 <td>&nbsp;</td>
 <td>High-sensitivity CCD camera</td>
 </tr>
 <tr>
 <td>wtGFP Longpass Emission</td>
 <td>Chroma Technology 
 Corp.</td>
 <td>41015</td>
 <td>GFP filter set for imaging GCaMP</td>
 </tr>
 <tr>
 <td>Filter 440 +/- 10 nm</td>
 <td>Chroma</td>
 <td>D440/20x EX</td>
 <td>excitation filter for cameleon</td>
 </tr>
 <tr>
 <td>Dichroic mirror &gt; 455 nm 
 longpass</td>
 <td>Chroma</td>
 <td>455DCLP BS</td>
 <td>microscope dichroic for cameleon 
 imaging</td>
 </tr>
 <tr>
 <td>Dichroic mirror &gt; 515 nm 
 longpass</td>
 <td>Chroma</td>
 <td>515DCLP BS</td>
 <td>dichroic mirror for cameleon 
 imaging</td>
 </tr>
 <tr>
 <td>Filter 535 +/- 15 nm</td>
 <td>Chroma</td>
 <td>D535/30m EM</td>
 <td>YFP emission filter</td>
 </tr>
 <tr>
 <td>Filter 480 +/- 20 nm</td>
 <td>Chroma</td>
 <td>D485/40m EM</td>
 <td>CFP emission filter</td>
 </tr>
 <tr>
 <td>Lens, 200 mm, Achromat</td>
 <td>Thor Labs</td>
 <td>AC508-200-A1</td>
 <td>Relay lens for FRET optics (3)</td>
 </tr>
 <tr>
 <td>Silver broadband mirror</td>
 <td>Thor Labs</td>
 <td>ME2S-P01</td>
 <td>FRET optics (2)</td>
 </tr>
 <tr>
 <td>NGM buffer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Levamisole</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polybead Microspheres</td>
 <td>Polysciences, Inc.</td>
 <td>08691-10, 2.5% by 
 volume, 50 nm 
 diameter</td>
 <td>polystyrene nanoparticles for C. 
 elegans immobilization</td>
 </tr>
 <tr>
 <td>Transgenic strain, Strain 
 gpa-9::GCaMP3(in pha-1; 
 him-5 bkg) </td>
 <td>Sternberg Lab</td>
 <td>Strain PS6388</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Transgenic strain, mec- 
 4::YC3.60</td>
 <td>Gabel Lab</td>
 <td>Strain CG1B</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

in vivo neuronal calcium imaging in c. elegans

El gusano nematodo C. elegans es un organismo modelo ideal para relativamente simple, formación de imágenes de bajo coste neuronal in vivo. Su pequeño cuerpo transparente y simple, bien caracterizado sistema nervioso permite la identificación y obtención de imágenes de fluorescencia de cualquier neurona en el animal intacto. Técnicas sencillas de inmovilización con un impacto mínimo sobre la fisiología del animal permitir extendido time-lapse de imágenes. El desarrollo de los fluoróforos de calcio genéticamente codificados sensibles como cameleon 1 y 2 GCaMP permitir la formación de imágenes in vivo de calcio neuronal relacionada tanto fisiología celular y la actividad neuronal. Numerosas cepas transgénicas que expresan estos fluoróforos en neuronas específicas están fácilmente disponibles o se pueden construir utilizando técnicas bien establecidas. A continuación, se describen los procedimientos detallados para medir la dinámica del calcio dentro de una sola neurona in vivo utilizando tanto GCaMP y cameleon. Se discuten las ventajas y disadvantedades de ambos, así como varios métodos de preparación de la muestra (inmovilización animal) y análisis de imagen. Finalmente, se presentan los resultados de dos experimentos: 1) Usando GCaMP para medir la respuesta sensorial de una neurona específica a un campo eléctrico externo y 2) Utilizando cameleon para medir la respuesta de calcio fisiológico de una neurona a daño traumático láser. Técnicas de formación de imágenes de calcio tales como éstos se utilizan ampliamente en C. elegans y se han extendido a las mediciones en los animales que se desplazan libremente, neuronas de forma simultánea y la comparación entre fondos genéticos. C. elegans presenta un sistema robusto y flexible para imágenes in vivo neuronal con ventajas sobre otros sistemas modelo de sencillez técnica y coste.

Aquí se presentan los resultados de dos experimentos separados. El primero emplea GCaMP para medir la respuesta de una neurona sensorial específico a un estímulo externo definido, dando un buen ejemplo de cómo los reporteros fluorescentes de calcio se puede utilizar para controlar ópticamente la actividad neuronal en intacto C. elegans. El segundo emplea cameleon para medir el calcio intracelular transitoria activa dentro de una neurona en respuesta al daño del láser específico, lo que ilustra cómo la ...

Watch this Scientific Journal Video about In vivo Neuronal Calcium Imaging in C. elegans at JoVE.com

In vivo Neuronal Calcium Imaging in C. elegans

Con su pequeño cuerpo transparente y bien documentado neuroanatomía y una serie de técnicas genéticas susceptibles y reactivos,<em&gt; C. elegans</em&gt; Hace que un organismo modelo ideal para<em&gt; In vivo</em&gt; Imagen neuronal utilizando relativamente simples y de bajo costo técnicas. Aquí se describe imagen única neurona dentro de animales adultos intactos utilizando genéticamente codificados indicadores de calcio fluorescente.

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, ...

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Neuroscience

In vivo Neuronal Calcium Imaging in C. elegans

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 ...

Investigación

Neurociencias

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Las respuestas de imagen neuronal en los preparativos de la rebanada del órgano vomeronasal Expresando un sensor de calcio genéticamente codificados

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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 ...

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

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

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 Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

En Vivo Cerebral Funcional Imaging Enfoque Basado en calcio bioluminiscente Indicador GFP-aequorina

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

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 ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Proyección de imagen de calcio de dos fotones en las dendritas neuronales en rebanadas de cerebro

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagen radiométrica calcio de neuronas individuales en comportamiento Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

En proyección de imagen del calcio Vivo de las células de pelo de la línea Lateral en el pez cebra larvas

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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