1. Descrizione del Microscopio monitoraggio <ol><li> Una piastra di agar è illuminato da una sorgente di luce in fibre e ripreso con una telecamera. Questo sistema è montato un, traduzione Y X stadio. </li><li> Lo stadio è mosso da motori passo-passo standard, che sono collegati ad un controllore motore passo-passo. </li><li> Il controllore e fotocamera sono collegati al computer e controllati da programmi personalizzati scritti in LabVIEW. </li><li> La telecamera le immagini della superficie di una piastra di agar e identifica gli oggetti scuri su sfondo chiaro. </li><li> La qualità dell&#39;immagine viene regolata in modo che il programma di computer può quantificare oggetti in tempo reale. Il guadagno, la luminosità e la velocità dell&#39;otturatore della fotocamera può essere regolato per fornire un oggetto scuro su uno sfondo bianco. </li><li> Un segno di calibrazione per la calibrazione automatica è fatta colpendo la superficie dell&#39;agar con un pick verme. </li><li> Il filtrato, immagine binaria che viene utilizzato dal programma di monitoraggio può essere controllato. </li><li> Il modoftware dispone di una funzione di auto-calibrazione che calcola la matrice di calibrazione con lo spostamento di un oggetto di prova una distanza fissa. </li><li> Distanze in pixel è calibrato per azioni intraprese dal motore passo-passo per la matrice di calibrazione. </li><li> Dopo la calibrazione, il sistema è pronto per andare e non necessita di essere ricalibrata meno che l&#39;ingrandimento viene modificato oppure se la telecamera viene riposizionata. </li></ol> 2. Preparazione di piatti di monitoraggio e C. elegans per il monitoraggio <ol><li> Un anello di rame è usato per all&#39;angolo i vermi e mantenere la loro migrazione verso il bordo della lastra. Il rame fornisce una barriera chimica locale e non influenza il movimento del verme altrimenti per la durata dell&#39;esperimento (&lt;1 ora). Riscaldare il primo anello appoggiandolo su un blocco di calore o equivalente. </li><li> Anello posto su una piastra di agar fresco (1,7% Agar Bacto, 0,25% Bacto-peptone, 0,3% NaCl, 1 mM CaCl 2, 1 mM MgSO 4, 25 mM tampone fosfato di potassio, 5 pg / mlcolesterolo) e premere leggermente verso il basso per incorporare nella superficie dell&#39;agar. </li><li> Molto L4 stadio o vermi adulti giovani su una piastra di agar riempito con qualche tampone NGM (0,3% NaCl, 1 mM CaCl 2, 1 mM MgSO 4, 25 mM tampone fosfato di potassio) per lavarli di residui di cibo. Lasciate che i vermi nuotare per qualche minuto. </li><li> Posizionare con cura un verme solo sulla piastra di inseguimento nei pressi del centro del ring. E poi la piastra sul tracker worm. </li></ol> 3. Worm monitoraggio <ol><li> Eseguire il programma LabVIEW e selezionare le opzioni, se necessario (percorso per immagini, tipi di immagini, misure, impostazioni della fotocamera). </li><li> Utilizzando il joystick spostare il microscopio fino un&#39;immagine del verme è nel campo di visualizzazione (monitor). Premere &quot;traccia&quot; per attivare il programma di monitoraggio. </li><li> Il programma di computer misura effettivamente i movimenti di un immagini binarie filtrati come illustrato e possono effettuare misurazioni su questa immagine in tempo reale </li><li> A poppaer inseguimento una ricostruzione della traiettoria globale può essere fatto dai movimenti del motore passo-passo, mentre le variazioni di forma locali della vite senza fine può essere visto in dettaglio. </li></ol> 4. Analisi dei dati <ol><li> Esegui script skeletonizing (MATLAB) per parametrizzare le forme del worm. (Figura 2) </li><li> Calcolare modi di vibrare dei dati scheletrato. (Figure 3,4) </li><li> Mostra Figura 5. Leggi leggenda figura o risultati rappresentativi. </li></ol>

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-79 (1974).</li><li>de Bono, M., Maricq, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+substrates+of+complex+behaviors+in+C.+elegans.">Neuronal substrates of complex behaviors in C. elegans.</a> Annu. Rev. Neurosci. (28), 451-501 (2005).</li><li>Pierce-Shimomura, J. T., Morse, T. M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+pirouettes+in+Caenorhabditis+elegans+chemotaxis.">The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis.</a> J. Neurosci. 19 (21), 9557-9569 (1999).</li><li>Gray, J. M., Hill, J. J., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+circuit+for+navigation+in+Caenorhabditis+elegans.">A circuit for navigation in Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (9), 3184-3191 (2005).</li><li>Baek, J. H., Cosman, P., Feng, Z., Silver, J., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+machine+vision+to+analyze+and+classify+Caenorhabditis+elegans+behavioral+phenotypes+quantitatively.">Using machine vision to analyze and classify Caenorhabditis elegans behavioral phenotypes quantitatively.</a> J. Neurosci. Methods. 118 (1), 9-21 (2002).</li><li>Stephens, G. J., Johnson-Kerner, B., Bialek, W., Ryu, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensionality+and+Dynamics+in+the+Behavior+of+C.+elegans.">Dimensionality and Dynamics in the Behavior of C. elegans.</a> PLoS Comput. Biol. 4 (1), e1000028 (2008).</li><li>Stephens, G. J., Johnson-Kerner, B., Bialek, W., Ryu, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+modes+to+movement+in+the+behavior+of+C.+elegans.">From modes to movement in the behavior of C. elegans.</a> PLoS One. 5 (11), e13914 (2010).</li><li>Feng, Z., Cronin, C. J., Wittig, J. H., Sternberg, P. W., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+imaging+system+for+standardized+quantitative+analysis+of+C.+elegans+behavior.">An imaging system for standardized quantitative analysis of C. elegans behavior.</a> BMC Bioinformatics. (5), 115 (2004).</li><li>Ramot, D., Johnson, B. E., Berry, T. L., Carnell, L., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parallel+Worm+Tracker:+A+Platform+for+Measuring+Average+Speed+and+Drug-Induced+Paralysis+in+Nematodes.">The Parallel Worm Tracker: A Platform for Measuring Average Speed and Drug-Induced Paralysis in Nematodes.</a> PLoS One. 3 (5), e2208 (2008).</li><li>Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+behavioral+analysis+in+C.+elegans.">High-throughput behavioral analysis in C. elegans.</a> Nat. Methods. 8 (7), 592-598 (2011).</li><li>Leifer, A. M., Fang-Yen, C., Gershow, M., Alkema, M. J., 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=Optogenetic+manipulation+of+neural+activity+in+freely+moving+Caenorhabditis+elegans.">Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans.</a> Nat. Methods. 8 (2), 147-152 (2011).</li><li>Stirman, J. N., Crane, M. M., Husson, S. J., Wabnig, S., Schultheis, C., Gottschalk, A., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+multimodal+optical+control+of+neurons+and+muscles+in+freely+behaving+Caenorhabditis+elegans.">Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans.</a> Nat. Methods. 8 (2), 153-158 (2011).</li><li>Ben Arous, J., Tanizawa, Y., Rabinowitch, I., Chatenay, D., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+imaging+of+neuronal+activity+in+freely+behaving+Caenorhabditis+elegans.">Automated imaging of neuronal activity in freely behaving Caenorhabditis elegans.</a> J Neurosci Methods. 187 (2), 229-234 (2010).</li><li>Wittenburg, N., Baumeister, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+avoidance+in+Caenorhabditis+elegans:+an+approach+to+the+study+of+nociception.">Thermal avoidance in Caenorhabditis elegans: an approach to the study of nociception.</a> Proc. Natl. Acad. Sci. U.S.A. 96 (18), 10477-10482 (1999).</li></ol>

Nessun conflitto di interessi dichiarati.

Lo studio della locomozione e il comportamento naturale richiede tecniche di rilevamento non invasive nei paesi partner e con le tecniche di riduzione dei dati. Qui abbiamo dimostrato un facile da usare sistema di tracciamento che registra le immagini dettagliate di C. elegans comportamento come striscia sulla superficie di una piastra di agar. La quantità di informazioni contenute in queste immagini è vasto e alto-dimensionali, e così abbiamo anche sviluppato metodi per ridurre la dimensionalità dei dati i...

<table border="1"><tbody><tr><td> Nome della parte </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Telecamera CCD </td><td> Basler </td><td> A601f </td><td></td></tr><tr><td> Lente </td><td> Edmund Optics </td><td> MMS serie </td><td></td></tr><tr><td> Illuminazione a fibre </td><td> Dolan Jenner </td><td> DC-950H </td><td></td></tr><tr><td> Traduzione fase </td><td> Deltron </td><td> LS3-4 </td><td></td></tr><tr><td> Motore passo-passo </td><td> US digitale </td><td> MS23C </td><td></td></tr><tr><td> Passo-passo motore </td><td> Geco </td><td> G201 </td><td></td></tr><tr><td> Motore passo-passo di controllo </td><td> SimpleStep </td><td> SSXYZ </td><td></td></tr><tr><td></td><td></td><td></td><td> Tutto il codice di programmazione è disponibile. Si prega di inviare una email di richiesta al autore corrispondente. </td></tr></tbody></table>

c. elegans tracking and behavioral measurement

Abbiamo sviluppato tecniche di analisi della strumentazione, elaborazione delle immagini, e dati per quantificare il comportamento motorio di C. elegans come striscia sulla superficie di una piastra di agar. Per lo studio della genetica, basi biochimiche, e neuronali del comportamento, C. elegans è un organismo ideale perché è geneticamente trattabile, suscettibili di microscopia, e mostra un certo numero di comportamenti complessi, compresi i taxi, di apprendimento, e 1,2 interazione sociale. Analisi comportamentale basata sul monitoraggio dei movimenti di vermi che strisciano sulle piastre di agar sono stati particolarmente utili nello studio del comportamento sensoriale 3, locomozione 4, e la fenotipizzazione generale mutazionale 5. Il nostro sistema funziona spostando il sistema di telecamere e illuminazione come i vermi striscia su una piastra di agar stazionaria, che garantisce l&#39;assenza di stimolo meccanico viene trasmessa alla vite senza fine. Sistema di tracciamento è facile da usare e comprende un semi-automatico funzione di calibrazione. Un challenge di video di tutti i sistemi di inseguimento è che genera una quantità enorme di dati che è intrinsecamente dimensionale. Nostra elaborazione delle immagini e programmi di analisi dei dati affrontare questo problema riducendo la forma vermi in un insieme di componenti indipendenti, che globalmente ricostruire il comportamento vermi come funzione di solo dimensioni 3-4 6,7. Come esempio del processo si dimostra che il verme entra ed esce allo stato inversione in maniera specifica fase.

Esempio: Quando foraggiamento, C. transizioni elegans da avanti a indietro movimento, spesso l&#39;esecuzione di un nuovo orientamento (omega giro) prima di tornare allo stato di avanzamento. Quantificare questo passaggio è importante per la comprensione dei modelli di foraggiamento di movimento e anche nel controllo motorio del worm. Il potere di rivelare i dettagli sottili di comportamento locomozione può essere visualizzato utilizzando il nostro dispositivo tracker. A ...

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C. elegans Tracking and Behavioral Measurement

Abbiamo sviluppato un sistema di tassi di video-microscopio di monitoraggio in grado di registrare e quantificare<em&gt; C. elegans</emComportamento&gt; ad alta risoluzione e velocità elevate. Abbiamo anche sviluppato metodi computazionali per ridurre la dimensionalità dei vermi immagini ad un insieme fondamentale di misurazioni che descrivono completamente la forma del verme.

C. elegans Monitoraggio e valutazione del comportamento

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

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Research

JoVE Journal

Neuroscience

C. elegans Tracking and Behavioral Measurement

18.9K Views.  University of Toronto. Abbiamo sviluppato un sistema di tassi di video-microscopio di monitoraggio in grado di registrare e quantificare C. elegans ad alta risoluzione e velocità elevate. Abbiamo anche sviluppato metodi computazionali per ridurre la dimensionalità dei vermi immagini ad un insieme fondamentale di misurazioni che descrivono completamente la forma del verme.

Video: C. elegans Tracking and Behavioral Measurement

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Saggio β-amiloide tossicità utilizzando un transgenici C. elegans Modello

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Ricerca

Neuroscienze

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. elegans Learning Positivo Butanone, a breve termine e lungo termine saggi di memoria associativa

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

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 assotomia 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 ...

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.

Quantificazione automatica di fluorescenza in Synaptic<em&gt; C. elegans</em

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

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

Una lettura molecolare di lungo termine adattamento olfattivo nel<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Insulina Microiniezione ippocampale e<em&gt; In vivo</emMicrodialisi&gt; Durante il test di memoria spaziale

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

In vivo Neuronal Calcium Imaging in C. elegans

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

In vivo Neuronal Calcium Imaging in C. elegans

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

In vivo imaging di calcio neuronale 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, ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

A lungo termine comportamentale inseguimento di Liberamente Nuoto debolmente elettrico Pesce

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Visualizzazione neuroblasti Citocinesi Durante<em&gt; C. elegans</em&gt; Embriogenesi

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

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 Imaging di Dauer-specifica neuronale ritocca 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 ...