אנו מודים וו ריו עצה הדמיה הראשונית שלנו הגדרת, צ&#39;גיתאי על ההצעה להשתמש ImageJ 13 לספור תולעים ג&#39;אשרף לעזרה עם Google SketchUp. CTM הוא חוקר Pew במדעי ביו, חוקר מק &#39;נייט, וכן חוקר קק.

<ol>
	<li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74, 515-515 (1993).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc Natl Acad Sci U S A. 70, 817-817 (1973).</li><li>Bono, M. d. e., 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, 451-451 (2005).</li><li>Mori, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+chemotaxis+and+thermotaxis+in+the+nematode+Caenorhabditis+elegans.">Genetics of chemotaxis and thermotaxis in the nematode Caenorhabditis elegans.</a> Annu Rev Genet. 33, 399-399 (1999).</li><li>Zhang, Y., Lu, H., 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=Pathogenic+bacteria+induce+aversive+olfactory+learning+in+Caenorhabditis+elegans.">Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans.</a> Nature. 438, 179-179 (2005).</li><li>Kauffman, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+signaling+and+dietary+restriction+differentially+influence+the+decline+of+learning+and+memory+with+age.">Insulin signaling and dietary restriction differentially influence the decline of learning and memory with age.</a> PLoS Biol. 8, e1000372-e1000372 (2010).</li><li>Torayama, I., Ishihara, T., Katsura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+integrates+the+signals+of+butanone+and+food+to+enhance+chemotaxis+to+butanone.">Caenorhabditis elegans integrates the signals of butanone and food to enhance chemotaxis to butanone.</a> J Neurosci. 27, 741-741 (2007).</li><li>Silva, A. J., Kogan, J. H., Frankland, P. W., Kida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CREB+and+memory.">CREB and memory.</a> Annu Rev Neurosci. 21, 127-127 (1998).</li><li>Meyer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iterative+image+transformations+for+an+automatic+screening+of+cervical+smears.">Iterative image transformations for an automatic screening of cervical smears.</a> J Histochem Cytochem. 27, 128-128 (1979).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Threshold+Selection+Method+from+Gray-Level+Histograms.">A Threshold Selection Method from Gray-Level Histograms.</a> IEEE Transactions on Systems, Man, and Cybernetics. 9, 62-62 (1979).</li><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, 71-71 (1974).</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, e1000028-e1000028 (2008).</li><li>Abramoff, M. a. g. e. l. h. a. e. s., Ram, P. J., J, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-36 (2004).</li></ol>

אין ניגודי אינטרסים הכריז.

ג למידה אסוציאטיבית elegans הריח מבחני זיכרון המוצגים כאן להבחין בין תהליכים של למידה מרוכזת, למידה במרווחים, הזיכרון לטווח קצר, לטווח ארוך וזיכרון. מבחני אלה מסתמכים על היכולות של תולעים לחוש ריחות מזון כימיים 1,2 סביבתם וליצור אסוציאציות חיוביות בין השניים. 6 לפיכך, מבחני רגישים מאוד רעב באוכלוסייה המוצא של בעלי חיים, טיפול גדול חייב לנקוט על מנת להבטיח כי תולעים ניזונים היטב לפני ואחרי אימון. הכשרה מרוכזת יחיד התשואות לטווח קצר זיכרון אסוציאטיבי. מספר גדל של תקופות מיזוג פרדיגמה אימון במרווחים הוא קריטי להשגת לטווח ארוך זיכרון אסוציאטיבי ב C. elegans כפי שהוא אורגניזמים אחרים. assay 6,8 אולי זו יכולה להיות שונה כדי לייצר זיכרונות ארוכי טווח של אסוציאציות בין גירויים מותנים מותנה שונים. למידה אסוציאטיבית שלנו מבחני זיכרון ניתן להשתמש כדי להשוות את יכולות הלמידה והזיכרון של סוג בעלי חיים בטבע מוטציה גנטית או הזדקנות. לדוגמה, בעוד הן למידה אסוציאטיביים זיכרון לטווח ארוך ויכולות אסוציאטיבי ירידה הזדקנות wild-type תולעים, למידה נשמרת זמן רב יותר עם ​​הגיל אצל בעלי חיים עם איתות אינסולין מופחת, וגם למידה וזיכרון נשמרת זמן רב יותר עם ​​הגיל אצל בעלי חיים מוגבל calorically 6. מבחני אלה גם מקובל RNAi טיפול תרופתי או מניפולציה של C. elegans. לדוגמה, טיפול של תולעים עם daf-2 RNAi משפר קצרי וארוכי טווח זיכרון אסוציאטיבי, לעומת זאת, חסימה של שעתוק הגנים ואת סינתזת החלבון עם תרופות (actinomycin D ו cycloheximide, בהתאמה) במהלך אימון במרווחים משבש לטווח ארוך היווצרות הזיכרון. 6 זיכרון לטווח ארוך הוא תהליך חיוני להישרדות כל בעלי החיים, ונראה כי הרבה של המנגנון המולקולרי המעורב לזכר אגודה הפאבלובית נשמרת מפני תולעים ליונקים. 6 היכולת להבחין ולבדוק למידה אסוציאטיבי, קצר טווח זיכרון לטווח ארוך באמצעות אלה מבחני הריח גורם C. elegans מערכת מעולה למחקר נוסף של זיכרון, עשויה לספק תובנות לגבי תהליכים ניווניות כי להוביל לאובדן של למידה וזיכרון אצל בני אדם.

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<td>Ethyl Alcohol 190 Proof</td>
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<td>InfiniMite Video Lens &#x2013; Alpha Industrial</td>
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<td>Standard Boom Stand</td>
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<td>3/4&#x201D; Post Adaptor for Boom Stands</td>
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<td>Standard Fixed 1/4-20 Mounting Plate</td>
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c. elegans positive butanone learning, short-term, and long-term associative memory assays

הזיכרון של חוויות ומידע נודע הוא קריטי עבור אורגניזמים לקבל החלטות כי הסיוע ההישרדות שלהם. ג elegans מנווט את סביבתו באמצעות זיהוי נוירון ספציפי של ריחות מזון כימיים 1, 2, והוא יכול לשייך מדינות התזונתי עם ריחות כימיים 3, 4 הטמפרטורה, ואת pathogenicity של מקור מזון 5. כאן אנו מתארים מבחני של C. elegans למידה וזיכרון אסוציאטיבי אסוציאטיבי קצר לטווח ארוך. אנו לשנות פרדיגמה למידה הריח מרתיעה 6 במקום כדי לייצר תגובה חיובית; assay כרוך רעב ~ 400 תולעים, ואז להאכיל את התולעים בנוכחות של נוירון-AWC חש butanone chemoattractant נדיף בריכוז זה מעורר מדד chemotactic נמוך (בדומה כדי Toroyama et al. 7). אוכלוסיית תקן chemotaxis assay1 בדיקות משיכה של תולעים odorant מיד או דקות עד שעות לאחר מיזוג. לאחר chemotaxis, מיזוג wild-type &quot;חיות לעליות butanone ~ 0.6 אינדקס Chemotaxis יחידות,&quot; מדד ללמידה &quot;שלה. למידה אסוציאטיבית תלויה בנוכחות מזון והן butanone במהלך אימון. מזון שיוך ו butanone לתקופה מיזוג יחיד (&quot;הכשרה מרוכזת&quot;) מייצרת זיכרון לטווח קצר אסוציאטיבי שנמשך ~ 2 שעות. תקופות מיזוג מרובות עם תקופות מנוחה בין (&quot;אימונים במרווחים&quot;) התשואות לטווח ארוך זיכרון אסוציאטיבי (&lt;40 שעות), והוא תלוי את החלבון cAMP Response Element Binding (CREB), 6 גורם שעתוק נדרש זיכרון לטווח ארוך על פני מינים. 8 פרוטוקול שלנו כולל גם ניתוח התמונה שיטות לקביעת מהירה ומדויקת של מדדי chemotaxis. ניגודיות גבוהה תמונות של בעלי חיים על צלחות chemotaxis assay נלכדים ונותחו באמצעות תוכנת תולעת ספירה ב-Matlab. התוכנה מתקנת על רקע אחיד באמצעות שינוי tophat מורפולוגיים. שיטה 9 של Otsu לאחר מכן נעשה שימוש כדי לקבוע סף תולעים נפרד מן הרקע. 10 חלקיקים קטנים מאוד יוסרו באופן אוטומטי גדול תולעת שאינם אזורים (קצוות צלחת או אגרופים אגר) הם הוסר על ידי בחירה ידנית. התוכנה ואז מעריך את הגודל של התולעת יחיד על ידי התעלמות אזורים שהם מעל גודל מרבי המצוין ולקחת את הגודל החציוני של האזורים הנותרים. מספר תולעים נאמד אז על ידי חלוקת סך כל השטח מזוהה שנכבשו על ידי תולעים על ידי גודל מוערך של תולעת אחת. מצאנו כי לימוד קצר זיכרון לטווח ארוך ניתן להבחין, וכי תהליכים אלה לשתף מולקולות מפתח דומה עם יצורים עילאיים. 6,8 מבחני שלנו יכול במהירות מבחן גנים מועמדים רומן או מולקולות המשפיעות על הלמידה קצר או ארוך זיכרון לטווח ב C. elegans שרלוונטיים פני מינים.

Watch this Scientific Journal Video about C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays at JoVE.com

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

כאן אנו מתארים את שיטות הבדיקה C. elegans למידה אסוציאטיבי זיכרון אסוציאטיבי קצר לטווח ארוך. אלה מבחני האוכלוסייה להעסיק את היכולות תולעים chemotax כלפי odorants נדיפים, וליצור אסוציאציות חיוביות על זיווג עם מזון butanone chemoattractant. הגדלת מספר תקופות מיזוג גורם זיכרון לטווח ארוך.

סי אלגנס למידה חיובית Butanone, לטווח קצר, לטווח ארוך מבחני זיכרון אסוציאטיבי

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

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

29.0K Views.  Princeton University. כאן אנו מתארים את שיטות הבדיקה C. elegans למידה אסוציאטיבי זיכרון אסוציאטיבי קצר לטווח ארוך. אלה מבחני האוכלוסייה להעסיק את היכולות תולעים chemotax כלפי odorants נדיפים, וליצור אסוציאציות חיוביות על זיווג עם מזון butanone chemoattractant. הגדלת מספר תקופות מיזוג גורם זיכרון לטווח ארו?...

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

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Assaying של תנועה, למידה, וזיכרון גירעונות תסיסנית מודלים של ניוון מוחיים

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

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.

בשנת vivo לייזר Axotomy ב C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

הקלטה אופטית של פעילות עצבית suprathreshold עם רזולוצית תא בודד ויחיד ספייק

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

מעקב סי אלגנס ומדידת התנהגות

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

למידה אסוציאטיבית הרחה באפטטיבית<em&gt; דרוזופילה</em&gt; זחלים

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

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.

Readout מולקולרי של הסתגלות חוש ריח לטווח הארוך ב<em&gt; ג elegans</em

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

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microchambers agarose לסידן הדמיה לטווח ארוך של<em&gt; Elegans Caenorhabditis</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

הערכת למידה וזיכרון המרחבי קטן קַשׂקַשִׂי זוחל

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

תסיסנית חיזור התניה כמדד למידה וזיכרון

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

הקונפליקט חושי לטווח ארוך באופן חופשי להתנהג עכברים

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...