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

Adskillige mennesker har bidraget til arbejdet beskrevet i dette dokument. CVG byggede forsøgsopstillingen, og LS, SHC, og CVG udførte forsøgene. CVG og SHC skrev manuskriptet. Alle forfatterne efterfølgende deltog i revisionsprocessen og godkendt den endelige kopi af manuskriptet. Vi takker Paul Sternberg for GCaMP stamme. Nogle nematode stammer anvendt i dette arbejde blev leveret af Caenorhabditis Genetics Center (CGC), som er finansieret af NIH National Center for Forskning Ressourcer (NCRR). Det MATLAB billedanalyse programmet blev tilpasset fra den, der anvendes i 18 år. Forfatterne blev støttet af Boston University og Massachusetts Life Sciences Center.

1. Optisk Setup <ol><li> Brug et standard sammensat mikroskop med epifluorescens billedbehandling kapaciteter. Vi bruger et Nikon Eclipse Ti-U omvendt mikroskop med en Intensilight HG illuminator. </li><li> For bedste billedkvalitet og signalkvalitet bruge en stor forstørrelse, høj numerisk apertur mål. Vi bruger typisk en Nikon X60 1,4 NA olieimmersionslinse mål. I nogle tilfælde er det muligt at anvende lavere forstørrelse (X40, X20) afhængigt af ekspressionsniveauet af fluoroforen og signalstyrke. </li><li> ...

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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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Genetisk kodede calcium indikatorer er blevet almindeligt anvendt i C. elegans neurobiologi. Adskillige grupper har anvendt disse teknikker til at studere reaktion af primære sensoriske neuroner på eksterne stimuli som vist her med ASJ reaktion på et elektrisk felt. Fremtrædende eksempler indbefatter fornemmelse af mekanisk kontakt, særlige kemikalier, temperatur og et elektrisk felt 12, 16-19. Aktivitet af interneuron og muskelceller er også overvåges både som respons på stimuli og på kont...

Her præsenterer vi praktiske metoder til in vivo calcium billeddannelse i C. elegans neuroner. Udviklingen af genetisk kodede calcium-følsomme fluoroforer med højt signal-støjforhold gør C. elegans en forholdsvis enkel og omkostningseffektiv system til måling af neurofysiologi og aktivitet. Vores billeddannelse sker med et standard sammensat mikroskop med bred felt fluorescensimagografi af almindeligt tilgængelige fluorophorer. Vi præsenterer flere teknikker anvender forskellige fluoroforer og forskellige sample præparater, diskuterer styrker og svagheder ved hver. Data så præsenteret fra to eksempler eksperimenter. En fremragende supplerende ressource på teknikkerne beskrevet her, kan findes i WormBook, &quot;Imaging aktiviteten af neuroner og muskler&quot; af R. Kerr, ( <a href="http://www.wormbook.org" target="_blank">http://www.wormbook.org</a> ) 3. To store klasser af genetisktisk kodede fluorescerende calcium reportere er almindeligt anvendt i C. elegans: enkelt kanal GCaMP og FRET-baserede Cameleon. Vi vil beskrive metoder og vise eksempler på data genereret af hver. GCaMP er baseret på en modificeret grønt fluorescerende protein (GFP), der er følsom over for det omgivende calciumkoncentration. Dette opnås ved fusion af GFP og den høje calcium affinitet protein calmodulin, således at bindingen af calcium fra calmodulin bringer GFP molekylet i en effektiv fluorescerende bekræftelse 2. Den nylige fremskridt i disse fluorophorer genererer exceptionel signal størrelse med op til 500% stigning i fluorescensintensiteten over et fysiologisk område af calcium og rimeligt hurtig kinetik for ~ 95 msek stigetid og ~ 650 msek henfaldstid 4. Over relativt korte perioder (minutter), kan disse store signaler giver mulighed for lavere opløsning billeddannelse (lavere forstørrelse), og da en artig initial basismåling, ophæve behovet for kontinuerlig baseline eller sammenlignende målinger. Cameleon har den fordel, at et FRET-baseret fluorophor, der genererer en ratiometrisk måling sammenligning af to uafhængige kanaler eller bølgelængder 1. Den består af to separate fluoroforer (cyan-og gul-emitterende fluorescerende proteiner, CFP og YFP) forbundet af en calmodulin-protein. Komplekset belyses med blåt lys (440 nm), der ophidser den fælles fiskeripolitik. Binding af calcium fører fluoroforer tættere på hinanden, hvilket øger fluorescensresonansenergioverførsel (FRET) fra CFP (donor) til YFP (acceptor) og forårsager CFP emission (480 nm) at falde, og YFP emission (535 nm) for at øge . Relative niveauer af calcium måles som forholdet mellem den YFP / CFP intensitet. Cameleon kinetikken er langsommere end GCaMP, målt in vivo til at have en stigetid på ~ 1 sekund og en henfaldstid på ~ 3 sek 5. Imidlertidforholdet mellem modsat bevægelige signaler forøger signalet størrelse og kompenserer for en række mulige artefakter som følge af ændringer i fluorophor koncentration, bevægelse eller fokus drift og blegning. Genetisk kodede fluorescerende reportere ophæver meget af prøven nødvendige forberedelser med exogent administrerede sonder og C. elegans lille gennemsigtig krop giver billeddannelse i det intakte dyr ved hjælp af simple bredt felt fluorescens. Den største tekniske udfordring i prøveforberedelse er derfor for sikkert at immobilisere dyrene. Der findes en række forskellige almindeligt anvendte teknikker hver med fordele og ulemper. Anvendelse af et farmakologisk middel til at lamme dyrene er let at gennemføre og tillader montering af flere dyr på et præparat (Levamisol, en cholinerg agonist, der forårsager muskelvæv at gribe anvendes typisk 6). C. elegans kan også fysisk immobiliseres ved at montere dempå stiv 10% agarose 7, 8. Dette minimerer indvirkning på dyrs fysiologi, tillader langsigtet imaging (timer) og inddrivelse af flere dyr, men er mere teknisk vanskeligt. Begge disse teknikker begrænser fysisk adgang til dyr (som er under et dækglas) og kan derfor kun anvendes sammen med visse eksperimentelle stimuli (såsom lys, temperatur, elektrisk felt eller laser beskadigelse). For stimuli hvor fysisk adgang er påkrævet, såsom berøring eller administration af kemikalier, har mange studier med succes limet C. elegans på plads (ved hjælp af veterinære klasse lim) 9. Dette er teknisk mere udfordrende, er et enkelt dyr forberedelse og tillader ikke dyr opsving. Endelig har adskillige mikrofluidenheder været ansat som fysisk begrænse C. elegans, kan bevare dyrs fysiologi, så udsættelse for de fleste typer af stimuli (afhængigt af enhedens design) og muliggøre hurtig udveksling og nyttiggørelse af dyrene <sup&gt; 10, 11. Men MicroFluidics kræver yderligere tekniske færdigheder og evner inden for design, fremstilling og implementering. I immobiliserede dyr aktivitet og stimulus respons kan generelt måles i sensorisk og interneuroner. Aktivitet af motorneuroner kræver mere sofistikerede teknikker til billeddannelse i bevægelse dyr. Her vil vi præsentere detaljerede fremgangsmåder, der anvender de to mest enkle teknikker til farmakologisk paralyzation og immobilisering med stiv agarose. Fremgangsmåderne præsenteres her kan anvendes til at måle neuronal aktivitet og cellefysiologi i C. elegans. Vi giver et eksempel på hver: hjælp GCaMP at måle den sensoriske respons ASJ neuron til en ekstern elektrisk felt, og ved hjælp Cameleon at måle den fysiologiske calcium respons på laser beskadigelse af en neuron. Disse eksempler viser fordelene og ulemperne ved de to typer af fluoroforer og illustrerer, hvad der er muligt 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

The nematode worm C. elegans er en ideel model organisme for relativt enkle, billige neuronal billeddannelse in vivo. Dens lille gennemsigtig krop og enkle, velkarakteriseret nervesystem tillader identifikation og fluorescensimagografi af enhver neuron i det intakte dyr. Enkle immobiliseringsteknikker med minimal indvirkning på dyrets fysiologi giver udvidet time-lapse imaging. Udviklingen af genetisk kodede calciumfølsomme fluoroforer såsom Cameleon 1 og GCaMP 2 tillader in vivo billeddannelse af neuronal calcium om både celle fysiologi og neuronal aktivitet. Talrige transgene stammer, der udtrykker disse fluoroforer i specifikke neuroner er let tilgængelige eller kan konstrueres ved anvendelse velkendte teknikker. Her beskriver vi nærmere procedurer for måling af calcium dynamik inden for en enkelt neuron in vivo ved hjælp af både GCaMP og Cameleon. Vi diskuterer fordele og disadvantalder såvel såvel som forskellige fremgangsmåder til prøvefremstilling (animalsk immobilisering) og billedanalyse. Endelig præsenterer vi resultater fra to forsøg: 1) Anvendelse GCaMP at måle sensoriske reaktion af en bestemt neuron til et eksternt elektrisk felt og 2) Brug Cameleon at måle den fysiologiske calciumrespons af en neuron for traumatisk laser skader. Calcium billeddiagnostiske teknikker som disse er udbredt i C. elegans og er blevet udvidet til at omfatte målinger i frit bevægelige dyr, flere neuroner samtidigt og sammenligning på tværs af genetiske baggrunde. C. elegans præsenterer en robust og fleksibelt system til in vivo neuronal billeddannelse med fordele frem for andre modelsystemer i teknisk enkelhed og omkostninger.

Her præsenterer vi resultater fra to separate forsøg. Den første anvender GCaMP at måle responsen af en specifik sensorisk neuron til en defineret ekstern stimulus, hvilket giver et godt eksempel på, hvordan fluorescerende calcium reportere kan anvendes til optisk at overvåge neuronal aktivitet i intakt C. elegans. Det andet anvender Cameleon at måle den intracellulære calcium transient udløst i en neuron som reaktion på specifikke laser beskadigelse, hvilket således illustrerer, hvordan calcium fysi...

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 lille transparent legeme, veldokumenteret neuroanatomi og et væld af medgørlige genetiske teknikker og reagenser,<em&gt; C. elegans</em&gt; Gør en ideel model organisme for<em&gt; In vivo</em&gt; Neuronal billeddannelse ved hjælp af relativt enkle, billige teknikker. Her beskriver vi en enkelt neuron billeddannelse inden intakte voksne dyr ved hjælp af genetisk kodede fluorescerende calcium indikatorer.

In vivo Neuronal Calcium 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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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.

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 på Slice Varer af Vomeronasal Organ udtryk for en genetisk kodet 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.

Automatiseret Kvantificering af Synaptic Fluorescens 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 billeddannelse af Dauer-specifik 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 Funktionel Brain Imaging tilgang baseret på Bioluminescent Calcium 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.

Ved hjælp af en tilpasset mikrofluid olfaktoriske Chip til billeddannelse af Neuronal aktivitet som reaktion på feromoner i mandlige C. Elegans hoved neuroner

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.

To-foton Calcium Imaging i Neuronal dendritter i hjernen skiver

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 billeddannelse af enkelte neuroner i opfører 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 Calcium billeddannelse af laterale-line hårcellerne i larve zebrafisk

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.