עבודה במעבדה Hammarlund ממומנת על ידי: NIH מענקים NS066082 R01-01 ו T32GM007223, קרן Beckman, ואת רפואי אליסון קרן.

<ol>
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אין ניגודי אינטרסים הכריז.

מגוון של מערכות לייזר שימשו לחתוך neurites ב C. elegans, וכן מספר מחקרים בחנו את הביצועים שלהם בפירוט 3,7,10,11. הלייזר MicroPoint השתמשו בפרוטוקול שלנו היא מערכת סוהר כי הוא קל להקים ולתחזק, והוא זמין במחיר נמוך לחוקרים לעומת מערכת לייזר TI-Sapphire. לעומת מערכת TI-Sapphire, לעומת זאת, הלייזר MicroPoint צפוי לגרום נזק שטח גדול יותר, אשר עשוי להיות נחות עבור יישומים מסוימים. אם מערכת TI-Sapphire הוא הרצוי, פרוטוקול מעולה על בניית מערכת כזו זמין 12. הפרוטוקול הנוכחי יכול להתבצע על מגוון רחב של הנוירונים ב C. elegans, עם זאת, נציין, כי ההבדלים ביכולות משובי בין סוגים שונים של נוירונים צפויים 13. בנוסף, רקע מהונדס שונים יכולים להשפיע על ההצלחה רגנרטיבית. למרות אחוז התחדשות הנוירונים GABA הוא עקבי למדי בין זנים שונים סמן מהונדס, שציינו עלייה קלה הכוללת התחדשות ב juIs76 14 לעומת 15 oxIs12 תולעים. הבדלים בין סמנים של הנוירונים לגעת גם תוארו 2. אנחנו מעדיפים כדי לשתק את התולעים עם microbeads במקום הרדמה כמו חרוזים את התוצאה מהר immobilization עקבית יותר 16. זהו גם יתרון כאשר אנו מסוגלים להתבונן התחדשות ללא כל תופעות בלבול אפשרי בשל הרדמה 17. שיטה חלופית ללא הרדמה עבור immobilization הוא השימוש התקנים microfluidic. השימוש מיקרופלואידיקה עבור axotomy תוארה בהרחבה 17-22. אנו מוצאים כי עם שימוש עקבי, את הפונקציות לייזר הטוב ביותר כאשר coumarin 440 בתא צבען משתנה פעם בשבוע כל ביצוע ההליך המתואר במדריך MicroPoint. אם יש צורך, המחוון הנחתה ניתן להשתמש כדי להגדיל את כוחו, אבל זה עשוי להיות אינדיקציה צבע ישן או חוסר לייזר. כמו כן, יש לצבוע תאים חיים מוגבל, שבסופו של דבר צריך להיות מחדש או להחליף (ראה פתרון בעיות). זה יכול להיות קשה לתמרן את neurite היעד תחת הכוונת המציינים את המיקוד לייזר, במיוחד אם החיה היא משותקת באופן חלקי. אנו מוצאים כי שלב ידני אינו אופטימלי למטרה זו, למרות שהוא שמיש בהחלט. בשלב ג&#39;ויסטיק שבשליטת ממונע מדויק יותר, אנו מוצאים כי באמצעות תוכנה שתומכת תמונה גרירה עם העכבר כדי להזיז את הבמה ממונע הוא הטוב ביותר. אלמנטים Nikon מספק תכונה זו מתוארת בפרוטוקול זה, אבל את החבילה Micromanager חינם, כמו גם תוכנות הדמיה אחרות, ייתכן פונקציונליות דומה. דרך אחרת של מיקוד בסדר היא להעביר את המיקוד לייזר, ולא החיה. קרן מנגנון היגוי גלונומטר זמין כמו תוספת על הלייזר MicroPoint, אם גישה זו היא המועדפת. מלבד היישום שלה ללימוד התחדשות עצבית, הלייזר יכול לשמש כדי לכרות סוגי תאים אחרים, כגון העור, השריר, או תאים מיוחדים, או לשבש סינפסות עצביות ספציפיות 23-27. יתר על כן, המסלולים המווסתים את התנוונות הנוירונים המלווה פציעה או מחלה יכולה להיחקר עם מערכת זו. ככזה, את השימוש של לייזרים פעמו ימשיכו לשפוך אור על שני גורמים גנטיים השינויים הביולוגיים המאפשרים לתא התחדשות העצבית ותהליכים רלוונטיים אחרים. פתרון בעיות: המתואר כאן כמה בעיות נפוצות ופתרונות הקשורים בהם. <ol type="a" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הלייזר אינו גזירה כראוי. זה צפוי כי גם לייזר הוא מחוץ לפוקוס (ראה סעיף 2), או לצבוע בתא צבע צריך להיות מוחלף (ראה MicroPoint ידנית). לחלופין, התא לצבוע ייתכן שיהיה צורך להחליף או מחדש. </li><li style=";text-align:right;direction:rtl"> התולעים עוברים על agarose כרית. זהו בדרך כלל סימן לכך משטח agarose לח מדי. אנו מוצאים כי רפידות צריך להיות שמאל במשך כ 30 שניות לפני תולעים ממוקמים להם לעקוף את הבעיה הזו. אתה יכול גם לנסות להשתמש בגודל קטן יותר (0.05 מיקרומטר) microbeads. </li><li style=";text-align:right;direction:rtl"> ההתאוששות של תולעים קשה ולא יעיל. זה יכול להיגרם על ידי הסרת coverslip שגוי או על ידי כרית agarose יבש. אם כרית הוא יבש מדי, אתה עשוי להבחין כי האקסונים לפתח מראה חרוזים. במקרים מסוימים, זה בגלל התולעים לא הונחו על משטח די מהר אחרי זה נעשה. לחלופין, המניה הישנה של פתרון agarose אולי ריכוז גבוה מ -3% לאחר ההיתוך חוזרות ונשנות. </li></ol>

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th><th> תגובות </th></tr><tr><td> חרוזי פוליסטירן 0.05 מיקרומטר </td><td> Polysciences, Inc </td><td> 08691 </td><td></td></tr><tr><td> חרוזי פוליסטירן 0.10 מיקרומטר </td><td> Polysciences, Inc </td><td> 00876 </td><td></td></tr><tr><td> Agarose GPG / LE </td><td> האמריקאי Bioanalytical </td><td> 00972 </td><td> אולטרה טהורים </td></tr><tr><td> פלקון 14 מ&quot;ל פוליסטירן Round-Bottom Tube </td><td> BD Biosciences </td><td> 352057 </td><td> 17 x 100 מ&quot;מ סגנון, nonpyrogenic </td></tr><tr><td> Thermo Scientific רגיל שקופיות מיקרוסקופ precleaned </td><td> אירי מדעי החברה </td><td> 420-004T </td><td> 3 &quot;x 1&quot; x 1 מ&quot;מ </td></tr><tr><td> כיסוי החלקות </td><td> VWR </td><td> 48366 205 </td><td> 18 מ&quot;מ x 18 מ&quot;מ מס &#39;1 1 / 2 </td></tr><tr><td> KIMTECH המדע Kimwipes </td><td> קימברלי קלארק </td><td> 34155 </td><td></td></tr><tr><td> BD פלקון 100 x 15mm צלחת פטרי סגנון </td><td> BD Biosciences </td><td> 351029 </td><td></td></tr><tr><td> ריק Tape 3/4w x 500L </td><td> TimeMed </td><td> T-512 </td><td></td></tr><tr><td> טבילה שמן </td><td> ניקון </td><td></td><td> סוג A, nd = 1.515 </td></tr><tr><td> EG1285 או CZ1200 </td><td> סי אלגנס גנטית מרכז </td><td> http://www.cbs.umn.edu/CGC/strains/ </td><td></td></tr><tr><td> OptiScan II </td><td> מדעי מראש </td><td></td><td></td></tr><tr><td> ש-Elements Ar או Br </td><td> ניקון </td><td></td><td></td></tr><tr><td> לייזר MicroPoint אבלציה מערכת </td><td> פוטוניים מדעי </td><td></td><td></td></tr><tr><td> מתחם מיקרוסקופ </td><td> ניקון </td><td> Eclipse 80i </td><td></td></tr><tr><td> Hamamatsu מצלמה </td><td> Hamamatsu Photonics </td><td> דגם C8484-05G01 </td><td></td></tr><tr><td> Dell Precision T3400 PC עם Intel Core 2 Duo </td><td> Dell </td><td></td><td></td></tr><tr><td> Windows XP Professional </td><td></td><td> מיקרוסופט </td><td> גרסת 2002, סדרה Pack 3 </td></tr><tr><td> Dual אמבטיה יבש החממה </td><td> פישר סיינטיפיק </td><td></td><td> אנלוגי שולט </td></tr><tr><td> 4X תוכנית פלואוריד אובייקטיבי </td><td> ניקון </td><td></td><td></td></tr><tr><td> 100x תוכנית Apo VC שמן אובייקטיבי </td><td> ניקון </td><td></td><td></td></tr><tr><td> דומון # 5 / 45 מלקחיים </td><td> דומון </td><td> 11251-35 </td><td> Dumoxel טיפים סטנדרטיים, ניתן גם להשתמש ב # 7 </td></tr><tr><td> הביתור מיקרוסקופ </td><td> ניקון </td><td> SMZ800 </td><td> עם הפנס NI-150 עוצמה גבוהה </td></tr></tbody></table>

in vivo laser axotomy in c. elegans

נוירונים לתקשר עם תאים אחרים באמצעות אקסונים דנדריטים, הרחבות קרום דק המכילים התמחויות טרום או פוסט סינפטי. אם נוירון הוא נפגע על ידי פציעה או מחלה, עלול להתחדש. גורמים סלולרי פנימי חיצוני להשפיע על היכולת של תא עצב להתחדש ולשקם את תפקוד. לאחרונה, נמטודות ג elegans התפתחה אורגניזם מודל מצוין לזהות גנים איתות המסלולים המשפיעים על התחדשות של נוירונים 1-6. הדרך העיקרית ליזום התחדשות הנוירונים ב C. elegans היא חיתוך לייזר בתיווך או axotomy. במהלך axotomy, תהליך עצבי fluorescently שכותרתו היא הכרות באמצעות אנרגיה גבוהה פולסים. בתחילה, התחדשות הנוירונים ב C. elegans נבדקה באמצעות לייזר femtosecond מוגבר 5. עם זאת, מחקרים התחדשות לאחר מכן הראו כי לייזר פעמו קונבנציונליים יכולים לשמש במדויק לנתק נוירונים in vivo ו לתגובה משובי דומה 1,3,7. אנו מציגים פרוטוקול לביצוע in vivo לייזר axotomy ב התולעת באמצעות MicroPoint פעמו לייזר, מערכת סוהר זמינה וכי כבר בשימוש נרחב עבור אבלציה תא ממוקד. אנו מתארים יישור לייזר, מקבע את התולעים, חיתוך נוירונים ספציפיים, והערכת התחדשות הבאים. המערכת מספקת את היכולת לחתוך מספר גדול של נוירונים תולעים מרובות במהלך הניסוי אחד. לפיכך, לייזר axotomy כפי שמתואר במסמך זה מערכת יעילה לייזום ניתוח תהליך של התחדשות.

Watch this Scientific Journal Video about In vivo Laser Axotomy in C. elegans at JoVE.com

In vivo Laser Axotomy in C. elegans

פרוטוקול לחתוך נוירונים<em&gt; ג elegans</em&gt; עם לייזר פעמו MicroPoint מוצג. אנו מתארים הקמת המערכת, משתק תולעים ניתוק שכותרתו נוירונים. היתרונות כוללים מערכת יחסית בעלות נמוכה ויכולת לנתק תהליכים עצביים או תאים לכרות<em&gt; In vivo</em&gt;.

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 ...

Research

JoVE Journal

Neuroscience

In vivo Laser Axotomy in C. elegans

18.1K Views.  Yale University School of Medicine. פרוטוקול לחתוך נוירונים ג elegans עם לייזר פעמו MicroPoint מוצג. אנו מתארים הקמת המערכת, משתק תולעים ניתוק שכותרתו נוירונים. היתרונות כוללים מערכת יחסית בעלות נמוכה ויכולת לנתק תהליכים עצביים או תאים לכרות In vivo.

Video: In vivo Laser Axotomy in C. elegans

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfection של תאים עכבר רשתית גנגליון על ידי In vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

הדמייה תפקודית באמצעות שיבוש Ultrasonic הדם למוח הגדר מנגן משופרת MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

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 ...

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 ...

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.

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, ...

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.

חזותי neuroblast cytokinesis במהלך<em&gt; ג elegans</em&gt; עובר

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

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Nanosurgery הלייזר של האקסונים Cerebellar<em&gt; בVivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

בו זמנית<em&gt; Vivo לשעבר</em&gt; בדיקות פונקציונליות של שתי הרשתיות על ידי<em&gt; In vivo</emמערכת electroretinogram&gt;

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...