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

Flera personer har bidragit till det arbete som beskrivs i detta dokument. CVG byggde experimentuppställning, och LS, SHK, och CVG utfört försöken. CVG och SHK skrev manuskriptet. Alla författare tog därefter del i översynen och godkände den slutliga kopian av manuskriptet. Vi tackar Paul Sternberg för GCaMP stammen. Vissa nematoder stammar som används i detta arbete lämnades av Caenorhabditis Genetics Center (CGC), som finansieras av NIH National Center for Research Resources (NCRR). MATLAB bildanalysprogram anpassades från den som används i 18. Författarna stöddes av Boston University och Massachusetts Life Sciences Center.

1. Optisk inställning <ol><li> Använd en vanlig förening mikroskop med epifluorescens bildfunktioner. Vi använder en Nikon Eclipse Ti-U inverterat mikroskop med en Intensilight HG Illuminator. </li><li> För bästa bild-och signalkvalitet använda hög förstoring, hög numerisk apertur mål. Vi använder vanligtvis en Nikon X60 1,4 NA oljeimmersionsobjektivet. I vissa fall är det möjligt att använda lägre förstoring (X40, X20) beroende på expressionsnivån av fluoroforen och signalstyrkan. </li><li> Använd...

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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=Temporal+activity+patterns+in+thermosensory+neurons+of+freely+moving+Caenorhabditis+elegans+encode+spatial+thermal+gradients.">Temporal activity patterns in thermosensory neurons of freely moving Caenorhabditis elegans encode spatial thermal gradients.</a> J. Neurosci. 27, 6083-6090 (2007).</li><li>Biron, D., Shibuya, M., Gabel, C., Wasserman, S. M., Clark, D. A., Brown, A., Sengupta, P., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+diacylglycerol+kinase+modulates+long-term+thermotactic+behavioral+plasticity+in+C.+elegans.">A diacylglycerol kinase modulates long-term thermotactic behavioral plasticity in C. elegans.</a> Nat. Neurosci. 9, 1499-1505 (2006).</li><li>Ghosh-Roy, A., Wu, Z. L., Goncharov, A., Jin, Y. S., Chisholm, A. 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Genetiskt kodade kalcium indikatorer har allmänt används i C. elegans neurobiologi. Många grupper har använt dessa tekniker för att studera svar av primära sensoriska neuroner på yttre stimuli såsom visas här med ASJ svar på ett elektriskt fält. Framträdande exempel inkluderar känsla av mekanisk beröring, specifika kemikalier, temperatur och ett elektriskt fält 12, 16-19. Aktiviteten av interneuron och muskelceller har också följts både som svar på stimuli och kontroll över djuren...

Här presenterar vi praktiska metoder för in vivo-kalcium avbildning i C. elegans nervceller. Utvecklingen av genetiskt kodade kalcium-känsliga fluoroforer med högt signal-till-brus-förhållande gör C. Elegans en jämförelsevis okomplicerad och kostnadseffektivt system för mätning av neurofysiologi och aktivitet. Vår avbildning görs med en vanlig förening mikroskop med brett fält fluorescens avbildning av allmänt tillgängliga fluoroforer. Vi presenterar flera tekniker som utnyttjar olika fluoroforer och olika preparat prov, diskutera styrkor och svagheter för varje. Data presenteras sedan från två exempel experiment. En utmärkt extra resurs på de tekniker som beskrivs här kan hittas i WormBook, &quot;Imaging aktiviteten hos nervceller och muskler&quot; av R. Kerr, ( <a href="http://www.wormbook.org" target="_blank">http://www.wormbook.org</a> ) 3. Två huvudklasser av genetiskttiskt kodade fluorescerande kalcium reportrar används ofta i C. elegans: en kanal GCaMP och FRET-baserade cameleon. Vi kommer att beskriva metoder och visa exempel på data som genereras av varje. GCaMP baseras på en modifierad grönt fluorescerande protein (GFP) som är känslig för omgivande kalciumkoncentrationen. Detta åstadkommes genom fusion av GFP och den höga kalcium affinitetsprotein kalmodulin, så att bindningen av kalcium från kalmodulin bringar GFP molekylen till en effektiv fluorescerande bekräftelse 2. De senaste framstegen inom dessa fluoroforer genererar exceptionell signal storlek med upp till 500% ökning i fluorescensintensitet över ett fysiologiskt område av kalciumnivåer och rimligt snabb kinetik ~ 95 ms stigtid och ~ 650 ms avklingningstid 4. Under relativt korta tidsperioder (min), kan dessa stora signaler möjliggöra lägre upplösning avbildning (lägre förstoring) och med tanke på en väluppfostrad initIAL referensnivåberäkning, förneka behovet av kontinuerlig baslinje eller jämförande mätningar. Cameleon har fördelen av att vara en FRET-baserad fluorofor som genererar en ratiometrisk mätning jämför två oberoende kanaler eller våglängder 1. Den består av två separata fluoroforer (cyan-och gul-utsläpp fluorescerande proteiner, GFP och YFP) länkade med en calmodulin protein. Komplexet belyses med blått ljus (440 nm) som väcker den gemensamma fiskeripolitiken. Bindning av kalcium ger fluoroforerna närmare varandra, ökar fluorescens resonansenergiöverföring (FRET) från GFP (donator) till YFP (acceptom) och bringar GFP emissionen (480 nm) för att minska och YFP emissionen (535 nm) för att öka . Relativa kalciumnivåer mäts som förhållandet mellan YFP / fiskeripolitiken intensitet. Cameleon kinetik är långsammare än GCaMP, mätt in vivo för att ha en stigtid på ~ 1 sek och en avklingningstid av ~ 3 sekunder 5. Emellertidförhållandet av motsatt rörliga signaler ökar signal storlek och kompenserar för ett antal möjliga artefakter på grund av förändringar i fluorofor koncentration, rörelse eller fokus drift och blekning. Genetiskt kodade fluorescerande reportrar förnekar mycket av provberedning krävs med exogent administrerade sonder och C. elegans liten genomskinlig kropp tillåter avbildning inom den intakta djuret med enkla brett fält fluorescens. Den viktigaste tekniska utmaningen i provberedningen är därför att på ett säkert sätt immobilisera djuren. Det finns ett antal olika vanliga tekniker vardera fördelar och nackdelar. Med hjälp av en farmakologiskt medel att förlama djuren är enkel att implementera och tillåter montering av flera djur på en beredning (Levamisol, en kolinerg agonist som orsakar muskelvävnaden att gripa används typiskt 6). C. elegans kan också fysiskt immobiliseras genom att montera dempå hård 10% agaros 7, 8. Detta minimerar inverkan på djurens fysiologi, medger en långsiktig avbildning (timmar) och återvinning av flera djur men är mer tekniskt svårt. Båda dessa tekniker begränsar fysisk tillgång till djuren (som är under ett täckglas) och kan därför bara användas med vissa experimentella stimuli (t.ex. ljus, temperatur, elektriskt fält eller laser skada). För stimuli där fysisk åtkomst krävs, såsom beröring eller administration av kemikalier, har många studier limmade framgångsrikt C. elegans på plats (med hjälp veterinär klass lim) 9. Detta är tekniskt mer utmanande, är ett enda djur förberedelse och tillåter inte djur återhämtning. Slutligen har många mikrofluidikanordningar använts som fysiskt hindra C. elegans, kan bevara djurens fysiologi, vilket exponering för de flesta typer av stimuli (beroende på enheten design) och möjliggöra snabbt utbyte och återvinning av djuren <sup&gt; 10, 11. Men mikrofluidik kräver ytterligare tekniska kompetens och kapacitet inom design, tillverkning och implementering. I immobiliserade djur aktivitet och stimulans svar kan i allmänhet mätas i sensoriska och interneuronen. Aktivitet av motoriska nervceller kräver mer sofistikerade tekniker för avbildning i rörliga djur. Här kommer vi att lägga fram detaljerade metoder som använder de två mest okomplicerade tekniker för farmakologisk lamslås och immobilisering med hård agaros. De metoder som presenteras här kan användas för att mäta neuronal aktivitet och cellfysiologi i C. elegans. Vi ger ett exempel på varje: med GCaMP att mäta den sensoriska svar ASJ neuron till en extern elektriskt fält, och använder cameleon för att mäta den fysiologiska kalcium svaret på laserskada av en neuron. Dessa exempel visar fördelarna och nackdelarna med de två typerna av fluoroforer och illustrerar vad som är möjligt med systemet.

<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

Den nematod mask C. elegans är en idealisk modell organism för relativt enkla, billiga neuronal avbildning in vivo. Dess små genomskinlig kropp och enkla, gör väl karakteriserade nervsystemet identifiering och fluorescens avbildning av en neuron i det intakta djuret. Enkla immobiliseringstekniker med minimal påverkan på djurets fysiologi tillåter längre tid-lapse avbildning. Utvecklingen av genetiskt kodade kalcium känsliga fluoroforer som cameleon 1 och GCaMP 2 tillåter in vivo avbildning av neuronala kalcium om både cell-fysiologi och nervaktivitet. Talrika transgena stammar som uttrycker dessa fluoroforer i specifika neuroner är lätt tillgängliga eller kan konstrueras med användning av väletablerade tekniker. Här beskriver vi detaljerade förfaranden för att mäta kalcium dynamik i en enda neuron in vivo med både GCaMP och cameleon. Vi diskuterar fördelar och disadvantåldrar såväl samt olika metoder för provberedning (djur immobilisering) och bildanalys. Slutligen, presenterar vi resultaten från två experiment: 1) Använda GCaMP att mäta sensoriska svaret hos en särskild neuron till en extern elektrisk fält och 2) Använda cameleon att mäta den fysiologiska kalcium svaret hos en neuron till traumatisk laserskada. Kalcium avbildningstekniker som dessa används i stor utsträckning i C. elegans och har utvidgats till att mätningar i fritt rörliga djur, flera neuroner samtidigt och jämförelse mellan genetisk bakgrund. C. elegans presenterar ett robust och flexibelt system för in vivo neuronala avbildning med fördelar jämfört med andra modellsystem i teknisk enkelhet och kostnad.

Här presenterar vi resultaten från två separata experiment. Den första utnyttjar GCaMP att mäta svaret hos en särskild sensorisk neuron till en definierad yttre stimulans, vilket ger ett bra exempel på hur fluorescerande kalcium reportrar kan användas för att optiskt övervaka neuronal aktivitet i intakt C. elegans. Den andra använder cameleon att mäta intracellulärt kalcium övergående visas inom en neuron som svar på specifika laserskada, vilket visar hur kalcium fysiologi kan mätas inom en end...

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

Med sin lilla transparent kropp väldokumenterad neuroanatomi och en mängd mottagliga genetiska tekniker och reagens,<em&gt; C. elegans</em&gt; Är en idealisk modell organism för<em&gt; In vivo</em&gt; Neuronal avbildning med relativt enkla, billiga tekniker. Här beskriver vi enda neuron avbildning inom intakta vuxna djur med genetiskt kodade fluorescerande kalcium indikatorer.

In vivo Neuronal kalcium Imaging i 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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JoVE Journal

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 i 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 Neuronal svar i Slice Beredningar av vomeronasala Orgel uttrycker en genetiskt kodade Kalcium 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.

Automatiserad Kvantifiering av synaptisk Fluorescens i<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 avbildning av Dauer specifika Neuronal Remodeling i 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 strategi som bygger på Bioluminescent Kalcium indikator 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 ...

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.

Använda en anpassad mikroflödessystem Olfactory Chip för bildtagning av Neuronal aktivitet som svar på feromoner i manlig C. Elegans huvud nervceller

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.

Två-foton kalcium Imaging i nervcellernas dendriter i hjärnan skivor

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;

Proportionerlig kalcium avbildning av enskilda nervceller i beter sig 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.

I Vivo kalcium avbildning av sidolinje hårcellerna i Larval zebrafiskar

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.