כמה אנשים תרמו לעבודה מתוארת במאמר זה. CVG נבנה מערך הניסוי, וLS, הא&quot;ס, וCVG בצעו את הניסויים. CVG והא&quot;ס כתבו את כתב היד. כל המחברים לאחר מכן לקחו חלק בתהליך השינוי ואשרו את העותק הסופי של כתב היד. אנו מודים לפול סטרנברג למתח GCaMP. כמה זנים נמטודות משמשים בעבודה זו נמסרו על ידי גנטיקת Caenorhabditis המרכז (CGC), אשר ממומן על ידי NIH המרכז הלאומי למשאבי מחקר (NCRR). תכנית ניתוח תמונת MATLAB עובדת מזה המשמש בגיל 18. המחברים נתמכו על ידי אוניברסיטת בוסטון ומסצ&#39;וסטס חי מרכז המדעים.

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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

אינדיקטורים סיד מקודד גנטי כבר נוצלו באופן נרחב בג elegans נוירוביולוגיה. קבוצות רבות שהעסיקו הטכניקות הללו כדי ללמוד תגובה של תאי עצב התחושתי העיקרי לגירויים חיצוניים כפי שמודגם כאן בתגובת ASJ לשדה חשמלי. דוגמאות בולטות כוללות תחושת המגע מכאני, כימיקלים מסוימים, ט?...

כאן אנו מציגים שיטות מעשיות לתחום הדמית vivo סיד בג הנוירונים elegans. הפיתוח של fluorophores סיד רגיש המקודד גנטי עם יחס גבוה בין אות לרעש גורם ג elegans מערכת יעילה יחסית פשוטה ועלות למדידת נוירופיזיולוגיה ופעילות. ההדמיה שלנו נעשתה באמצעות מיקרוסקופ מתחם סטנדרטי באמצעות דימות פלואורסצנטי שדה הרחב של fluorophores הזמין בדרך כלל. אנו מציגים כמה טכניקות המעסיקות fluorophores שונה ותכשירים שונים לדוגמה, דנים בנקודתי החוזק והחולשה של כל אחד. נתונים שהוצגו אז משני ניסויים לדוגמה. משאב נוסף מצוין בטכניקות המתוארות כאן ניתן למצוא בWormBook, &quot;הדמיה את הפעילות של תאי עצב ושרירים&quot; על ידי ר &#39;קר, <a href="http://www.wormbook.org" target="_blank">(http://www.wormbook.org</a> 3). שני סוגים עיקריים של genetiכתבי סיד ניאון מקודדים קאלי משמשים בדרך כלל בג elegans: GCaMP ערוץ אחד וCameleon סריג מבוסס. אנו מתארים שיטות ולהראות דוגמאות לנתונים שנוצרו על ידי כל אחד. GCaMP מבוסס על חלבון שונה גרין פלורסנט (GFP), כי הוא רגיש לריכוז הסיד שמסביב. המטרה זו מושגת על ידי שילוב של GFP וcalmodulin חלבון זיקת סידן הגבוה, באופן שמחייב של סידן על ידי calmodulin מביא מולקולת GFP לאישור ניאון יעיל 2. את הפיתוחים האחרונים בfluorophores אלה יוצרים גודל אות יוצא דופן עם גידול של עד 500% בעוצמת קרינה על פני טווח פיסיולוגי של רמות הסידן וקינטיקה במהירות סבירה של זמן ~ 95 אלפיות עלייה ודעיכה ~ 650 אלפית 4. על פני תקופות זמן קצרות יחסית (דקות), האותות הגדולים האלה יכולים לאפשר להדמיה ברזולוציה נמוכה יותר (גדלה נמוכה יותר), וניתנו מחונך initמדידת קו הבסיס IAL, שוללת את הצורך הבסיסי רציפות או מדידות השוואתיות. Cameleon יש את היתרון של להיות fluorophore סריג מבוסס שמייצר מדידת ratiometric השוואה שני ערוצים או אורכי גל 1 עצמאיים. זה מורכב משני fluorophores הנפרד (חלבוני ניאון וצהוב ציאן פולט, וYFP CFP) מקושר על ידי חלבון calmodulin. המתחם מואר באור כחול (440 ננומטר) שמרגש CFP. עקידת הסיד מביאה את fluorophores ביחד קרוב, הגדלת העברת אנרגית תהודת פלואורסצנטי (סריג) מCFP (תורם) לYFP (acceptor) וגורם לפליטת CFP (480 ננומטר) כדי להקטין וYFP הפליטה (535 ננומטר) כדי להגדיל . רמות סידן יחסיות נמדדות כיחס בין עוצמת YFP / CFP. קינטיקה Cameleon היא איטיות יותר מזה של GCaMP, נמדד בגוף חי יש זמן עלייה של ~ 1 שניות וזמן דעיכה של ~ 3 שניות 5. עם זאת,היחס של אותות הפוכים נעים מגדיל את גודל האות ומפצה על מספר החפצים אפשריים עקב שינויים בריכוז fluorophore, תנועה או להיסחף פוקוס וההלבנה. כתבי ניאון מקודד גנטי לשלול הרבה של הכנת המדגם הנדרש עם בדיקות מנוהלות exogenously וג גוף שקוף קטן elegans מאפשר הדמיה תוך שימוש בבעלי החיים בשלמות פלואורסצנטי שדה הרחב פשוט. האתגר הטכני העיקרי בהכנת מדגם לכן, כדי לשתק את החיות בשלום. ישנן מספר טכניקות שונות נפוצים כל אחד עם יתרונות וחסרונות. שימוש תרופתי לשתק את בעלי החיים הוא קל לביצוע ומאפשר הרכבה של חיות מרובות בהכנה אחד (Levamisole, אגוניסט כולינרגית שגורם לרקמת שריר לתפוס הוא בדרך כלל בשימוש 6). ג elegans גם יכול להיות משותק מבחינה פיזית על ידי הרכבתםעל נוקשה 10% agarose 7, 8. השפעה זו מצמצמת בפיזיולוגית בעלי חיים, מאפשרת הדמיה לטווח ארוך (שעות) והתאוששות של בעלי חיים רבים, אבל הוא יותר קשה מבחינה טכנית. שתי הטכניקות האלה להגביל את הגישה פיזית לבעלי החיים (שהם תחת תלוש כיסוי) ולכן יכולים לשמש רק עם גירויים ניסיוניים מסוימים (כמו אור, טמפרטורת שדה, חשמלי או ניזק ליזר). לגירויים שבו גישה פיזית נדרשת, כגון מגע או ממשל של כימיקלים, מחקרים רבים דבוקים בהצלחה ג elegans במקום (באמצעות דבק כיתת וטרינרית) 9. זה יותר מאתגר מבחינה טכנית, הוא תכשיר בהמה אחת ולא מאפשר התאוששות של בעלי חיים. לבסוף, מכשירי microfluidic רבים להיות מועסקים כי פיזי לרסן ג elegans, שימור פיזיולוגית בעלי חיים, המאפשר חשיפה למרבית סוגי גירויים (תלוי בעיצוב המכשיר) ויכול לאפשר חליפין והתאוששות מהירים של בעלי החיים <sup&gt; 10, 11. עם זאת מיקרופלואידיקה דורשת כישורים טכניים נוספים ויכולות בעיצוב, ייצור ויישום. בפעילות וגירוי חיות משותקות תגובה כלל ניתן למדוד בחושי וinterneurons. פעילות של תאי עצב מוטורי דורשת טכניקות מתוחכמות יותר עבור הדמיה בחיות נעות. כאן אנחנו נציג שיטות מפורטות המעסיקות את שתי טכניקות הפשוטות ביותר של השבתה וקיבוע עם agarose הנוקשה תרופתיות. השיטות שהוצגו כאן יכולות לשמש למדידת פעילות עצבית ופיזיולוגיה של תא בג elegans. אנחנו נותנים דוגמה לכל אחד: שימוש GCaMP למדוד את התגובה החושית של ASJ נוירון לשדה חשמלי חיצוני, ובאמצעות Cameleon כדי למדוד את התגובה הפיזיולוגית לסיד ניזק ליזר של תא עצב. דוגמאות אלו מראות את היתרונות וחסרונות של שני סוגים של fluorophores ולהמחיש מה אפשרי עם המערכת.

<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

נמטודות התולעת ג elegans הוא אורגניזם מודל אידיאלי להדמיה פשוטה יחסית, בעלות נמוכה עצבית בגוף חי. גופו הקטן השקוף ופשוט, מערכת עצבים היטב מאופיינת מאפשר זיהוי והדמית קרינה של כל נוירון בתוך החיים השלמים. טכניקות קיבוע פשוטות עם השפעה מינימאלית על הפיסיולוגיה של בעלי החיים מאפשרות שיקוף מורחב זמן לשגות. הפיתוח של fluorophores הרגיש סיד הגנטי מקודד כגון 1 Cameleon וGCaMP 2 לאפשר בתחום הדמית vivo של סיד עצבי הנוגע גם פיזיולוגיה של תא ופעילות עצבית. זנים מהונדסים רבים מבטאים fluorophores אלה בתאי עצב ספציפיים הם זמינים או ניתן לבנות תוך שימוש בטכניקות מבוססות היטב. כאן, אנו מתארים הליכים מפורטים למדידת דינמיקת הסידן בתוך תא עצב יחיד בגוף החי הוא באמצעות GCaMP וCameleon. אנו דנים ביתרונות וdisadvantגילים גם כן שיטות שונות של הכנת מדגם (קיבוע בעלי חיים) וניתוח תמונה. לבסוף, אנו מציגים תוצאות משני ניסויים: 1) שימוש GCaMP למדוד את התגובה החושית של נוירון ספציפי לשדה חשמלי חיצוני ו2) שימוש בCameleon כדי למדוד את התגובה הפיזיולוגית של סיד נוירון לניזק ליזר טראומטי. שיטות הדמית סידן כגון אלה נמצאים בשימוש נרחב בג elegans והורחב למדידות בבעלי חיים שנעו בחופשיות, נוירונים מרובים בו זמנית והשוואה על פני רקע גנטי. ג elegans מציג מערכת חזקה וגמישה להדמיה עצבית vivo עם יתרונות על פני מערכות מודל אחרות בפשטות ועלות טכניות.

כאן אנו מציגים תוצאות משני ניסויים נפרדים. 1 מעסיק GCaMP למדוד את התגובה של תא עצב תחושתי ספציפי לגירוי חיצוני מוגדר, נותן דוגמה טובה כיצד ניתן להשתמש בו לכתבי סיד ניאון אופטי כדי לעקוב אחר פעילות עצבית בג השלם elegans. 2 מעסיקים Cameleon למדוד סיד החולף התאי מופעל בתוך...

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

עם גוף מערך עצביה הקטנים שקוף, מתועד היטב ומגוון הרחב של טכניקות גנטיות מתאימים ריאגנטים,<em&gt; ג elegans</em&gt; עושה אורגניזם מודל אידיאלי עבור<em&gt; In vivo</em&gt; הדמיה עצבית תוך שימוש בטכניקות פשוטות יחסית, בעלות נמוכה. כאן אנו מתארים הדמית נוירון בודדה בתוך בעלי חיים מבוגרים ללא פגע באמצעות אינדיקטורים סיד ניאון מקודד גנטי.

בהדמיה עצבית סיד vivo ב C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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JoVE Journal

Neuroscience

In vivo Neuronal Calcium Imaging in C. elegans

24.8K Views.  Boston University School of Medicine. עם גוף מערך עצביה הקטנים שקוף, מתועד היטב ומגוון הרחב של טכניקות גנטיות מתאימים ריאגנטים, ג elegans עושה אורגניזם מודל אידיאלי עבור In vivo הדמיה עצבית תוך שימוש בטכניקות פשוטות יחסית, בעלות נמוכה. כאן אנו מתארים הדמית נוירון בודדה בתוך בעלי חיים מבוגר?...

Video: In vivo Neuronal Calcium Imaging in C. elegans

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.

התגובות הדמיה העצבית ההכנות Slice של איברים Vomeronasal הבעת חיישן סידן מקודד גנטית

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.

כימות אוטומטי של הקרינה סינפטית<em&gt; ג elegans</em

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

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

הדמיה ברזולוציה סופר של שלפוחית ​​צפופות ליבות עצבית

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

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 ההדמיה של דאואר ספציפי עצבי שיפוץ בג 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.

בגישת ההדמיה התפקודי של מוח Vivo בהתבסס על סידן Bioluminescent המחוון 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.

באמצעות שבב חוש הריח של Microfluidic מותאם עבור ההדמיה של פעילות. עצבית בתגובה פרומונים בנוירונים הראש הגברי C. Elegans

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.

שני הפוטונים סידן הדמיה של דנדריטים עצביים במוח פרוסות

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;

סידן רציומטרי הדמיה של נוירונים בודדים ב מתנהג 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.

בתחום ההדמיה סידן Vivo של קו הצד תאים שיער בזחל דג זברה

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.