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

Verschillende mensen hebben bijgedragen aan het werk beschreven in dit document. CVG bouwde de experimentele opstelling, en LS, SHC, en CVG de uitgevoerde experimenten. CVG en SHC schreef het manuscript. Alle auteurs nam vervolgens deel aan het herzieningsproces en keurt de definitieve versie van het manuscript. Wij danken Paul Sternberg voor de GCaMP stam. Sommige nematoden stammen die in dit werk werden verstrekt door de Caenorhabditis Genetics Center (CGC), dat wordt gefinancierd door de NIH National Center for Research Resources (NCRR). De MATLAB beeldanalyse programma werd aangepast van die in 18. De auteurs werden ondersteund door Boston University en het Massachusetts Life Sciences Center.

1. Optische Setup <ol><li> Gebruik een standaard samengestelde microscoop met epifluorescentie imaging-mogelijkheden. We maken gebruik van een Nikon Eclipse Ti-U omgekeerde microscoop met een Intensilight HG Illuminator. </li><li> Voor de beste beeld-en signaalkwaliteit gebruik maken van een hoge vergroting, hoge numerieke apertuur doelstelling. Wij gebruiken meestal een Nikon X60 1,4 NA olie-immersie objectief. In sommige gevallen is het mogelijk om kleinere vergroting gebruiken (X40, X20) afhankelijk van het express...

<ol>
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Genetisch gecodeerd calcium indicatoren zijn op grote schaal gebruikt in C. elegans neurobiologie. Talrijke groepen gebruikt deze technieken reactie van primaire sensorische neuronen externe stimuli bestuderen zoals hier aangetoond met ASJ reactie op een elektrisch veld. Prominente voorbeelden zijn gevoel van mechanische druk, specifieke chemicaliën, temperatuur en een elektrisch veld 12, 16-19. Activiteit van interneuron en spiercellen zijn ook gecontroleerd als respons op stimuli en controle van d...

Hier presenteren we praktische methoden voor in vivo calcium beeldvorming in C. elegans neuronen. De ontwikkeling van genetisch gecodeerde calcium-gevoelige fluoroforen met een hoge signaal-ruisverhouding wordt C. elegans een relatief eenvoudig en kostenbesparend systeem voor het meten van de neurofysiologie en activiteit. Onze beeldvorming wordt gedaan met een standaard samengestelde microscoop met behulp van wide-field fluorescentie beeldvorming van de algemeen beschikbare fluoroforen. We presenteren verschillende technieken inzet van verschillende fluoroforen en verschillende monster voorbereiding, het bespreken van de sterke en zwakke punten van elk. Gegevens worden gepresenteerd op twee voorbeeldexperimenten. Een uitstekende extra bron op de hier beschreven technieken zijn te vinden in WormBook, &quot;Beeldvorming van de activiteit van neuronen en spieren&quot; door R. Kerr, ( <a href="http://www.wormbook.org" target="_blank">http://www.wormbook.org</a> ) 3. Twee belangrijke klassen van genetischautomatisch gecodeerd fluorescerende calcium reporters worden vaak gebruikt in C. elegans: een kanaal GCaMP en FRET-gebaseerde cameleon. Beschrijven we methoden en tonen voorbeelden voor gegevens die zijn gegenereerd door elk. GCaMP is gebaseerd op een gemodificeerd Green Fluorescent Protein (GFP) die gevoelig is voor de omringende calciumconcentratie. Dit wordt bereikt door fusie van GFP en de hoge affiniteit calcium eiwit calmoduline, zodat de binding van calcium door calmodulin de GFP molecuul brengt in een efficiënte fluorescerende bevestiging 2. De recente ontwikkelingen in deze fluoroforen genereren uitzonderlijke signaal formaat met tot 500% toename van de fluorescentie-intensiteit over een fysiologische bereik van calcium niveaus en redelijk snel kinetiek van ~ 95 msec stijgtijd en ~ 650 msec decay tijd 4. Over relatief korte perioden (minuten), kan deze grote signalen zorgen voor lagere resolutie imaging (lagere vergroting) en, gegeven een goed opgevoede initial nulmeting, ontkennen de noodzaak van permanente basislijn of vergelijkende metingen. Cameleon heeft het voordeel dat een FRET-gebaseerde fluorofoor die een ratiometrische meting vergelijken van twee onafhankelijke kanalen of golflengten 1 genereert. Het bestaat uit twee fluoroforen (cyaan-en gele-emitterende fluorescerende eiwitten, CFP en YFP) verbonden door een calmoduline eiwit. Het complex is verlicht met blauw licht (440 nm) dat het GVB prikkelt. Binding van calcium brengt de fluoroforen dichter bij elkaar, waardoor fluorescentie resonantie energie overdracht (FRET) van de CFP (donor) de YFP (acceptor) en waardoor de emissie CFP (480 nm) en neemt de emissie YFP (535 nm) om het . Relatieve calciumgehalte gemeten als de verhouding van de YFP / CFP intensiteit. Cameleon kinetiek langzamer dan GCaMP, gemeten in vivo een stijgtijd van ~ 1 sec en een vervaltijd van 3 sec 5 hebben. Echterde verhouding van tegengesteld bewegende signalen verhoogt de signaalgrootte en compenseert een aantal mogelijke artefacten als gevolg van veranderingen in fluorofoor concentratie beweging of aandacht drift en bleken. Genetisch gecodeerd fluorescerende verslaggevers ontkennen veel van de monstervoorbereiding vereist met exogeen toegediende sondes en C. elegans kleine transparant lichaam maakt beeldvorming binnen de intacte dier met behulp van eenvoudige brede veld fluorescentie. De belangrijkste technische uitdaging in monstervoorbereiding is dan ook om veilig te immobiliseren de dieren. Er zijn een aantal verschillende algemeen gebruikte technieken elk met voor-en nadelen. Met een farmacologisch middel om de dieren verlammen is eenvoudig te implementeren, de bevestiging van meerdere dieren een preparaat (Levamisol, een cholinerge agonist die spierweefsel veroorzaakt grijpen om wordt meestal gebruikt 6). C. elegans kan ook fysiek inactief worden gemaakt door ze aan te koppelenop stijve 10% agarose 7, 8. Dit minimaliseert impact op de dierlijke fysiologie, laat de lange termijn beeldvorming (uur) en het herstel van meerdere dieren, maar is meer technisch moeilijk. Beide technieken de fysieke toegang tot de dieren (die onder een dekglas) en kan daarom alleen worden gebruikt met bepaalde experimentele stimuli (zoals licht, temperatuur, elektrisch veld of laser schade). Voor stimuli waar fysieke toegang nodig is, zoals aanraking of toediening van chemicaliën, hebben vele studies met succes geplakt C. elegans op zijn plaats (met behulp van veterinaire graad lijm) 9. Dit is technisch meer uitdagende, is een enkel dier voorbereiding en staat niet toe dat dierlijke herstel. Ten slotte hebben talloze microfluïdische apparaten in dienst die fysiek C. beperken elegans, kan het behoud van dierfysiologie, waardoor de blootstelling aan de meeste soorten stimuli (afhankelijk van het apparaat ontwerp) en maken een snelle uitwisseling en herstel van de dieren <sup&gt; 10, 11. Maar microfluidics extra technische vaardigheden en mogelijkheden in ontwerp, fabricage en uitvoering. In geïmmobiliseerd dieren activiteit en stimulus respons kan over het algemeen worden gemeten in sensorische en interneuronen. De activiteit van de motorneuronen vereist meer geavanceerde technieken voor beeldvorming bij bewegende dieren. Hier presenteren we gedetailleerde methoden gebruik van de twee meest eenvoudige technieken van farmacologische paralyzation en immobilisatie met stijve agarose. De hier gepresenteerde methoden kunnen worden gebruikt om neuronale activiteit en celfysiologie in C. meten elegans. We geven een voorbeeld van elk: GCaMP met de sensorische respons van de ASJ neuron een extern elektrisch veld meten, en het gebruik Cameleon de fysiologische reactie op calcium laser schade van neuron meten. Deze voorbeelden tonen de voordelen en nadelen van de twee soorten fluoroforen en illustreren wat mogelijk is met het systeem.

<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

De nematode worm C. elegans is een ideaal modelorganisme voor relatief eenvoudige, goedkope neuronale imaging in vivo. Zijn kleine transparante lichaam en eenvoudig, goed gekarakteriseerde zenuwstelsel maakt de identificatie en fluorescentie beeldvorming van een neuron in het intacte dier. Eenvoudige immobilisatie technieken met minimale impact op de fysiologie van het dier kunnen langere time-lapse imaging. De ontwikkeling van genetisch gecodeerde calcium gevoelige fluoroforen zoals cameleon 1 en 2 laten GCaMP in vivo beeldvorming van neuronale calcium worden zowel celfysiologie en neuronale activiteit. Talrijke transgene stammen die deze specifieke fluoroforen in neuronen gemakkelijk verkrijgbaar of kunnen worden geconstrueerd met gevestigde technieken. We beschrijven gedetailleerde procedures voor het meten van calcium dynamiek van een neuron in vivo in zowel GCaMP en cameleon. We bespreken voordelen en disadvantleeftijd waarop en verschillende werkwijzen van monstervoorbereiding (dier immobilisatie) en beeldanalyse. Tenslotte presenteren we de resultaten van twee experimenten: 1) Gebruik GCaMP de sensorische respons van een bepaald neuron aan een extern elektrisch veld en 2) Met Cameleon de fysiologische reactie van een calcium neuron traumatische beschadiging laser meten. Calcium beeldvormingstechnieken zoals deze worden veel gebruikt in C. elegans en zijn uitgebreid tot metingen in vrij bewegende dieren, meerdere neuronen tegelijk en vergelijking tussen genetische achtergronden. C. elegans presenteert een robuust en flexibel systeem voor in vivo neuronale beeldvorming met voordelen ten opzichte van andere modelsystemen in technische eenvoud en kosten.

Wij presenteren resultaten van twee afzonderlijke experimenten. De eerste werk GCaMP de reactie van een specifieke sensorische neuron naar een gedefinieerde externe stimulus te meten, wat een goed voorbeeld van hoe fluorescente calcium reporters kan worden gebruikt om optisch monitoren neuronale activiteit intact C. elegans. De tweede dienst cameleon het meten van de intracellulaire calcium voorbijgaande geactiveerd bij een neuron in antwoord op specifieke laser schade Dit illustreert hoe calcium fysiologie kan...

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

Met zijn kleine transparante lichaam, goed gedocumenteerde neuroanatomie en een groot aantal vatbaar genetische technieken en reagentia,<em&gt; C. elegans</em&gt; Is een ideale modelorganisme voor<em&gt; In vivo</em&gt; Neuronale beeldvorming met relatief eenvoudige, goedkope technieken. Hier beschrijven we enkel neuron imaging binnen intacte volwassen dieren met behulp van genetisch gecodeerd fluorescerende calcium indicatoren.

In vivo neuronale 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, ...

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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.

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 ...

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.

Imaging neuronale responsen bij Slice Bereidingen van Vomeronasal Het uiten van een genetisch gecodeerd Calcium Sensor

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.

Geautomatiseerde Kwantificering van synaptische Fluorescentie in<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.

In vivo beeldvorming van Dauer-specifieke neuronale Verbouwing 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 ...

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.

In vivo Functional Brain Imaging aanpak op basis van Bioluminescent Calcium Indicator GFP-aequorine

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.

Met behulp van een aangepast Microfluidic olfactorische Chip voor de beeldvorming van neuronale activiteit in reactie op de feromonen in mannelijke C. Elegans hoofd neuronen

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.

Twee-foton Calcium Imaging in neuronale Dendrites in hersenen plakjes

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;

Ratiometric Calcium beeldvorming van individuele neuronen in gedragen 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.

In Vivo Calcium beeldvorming van dwarslijn haarcellen in larvale zebravis

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.