ברצוננו להודות מנואל הצימר סיפק לנו הקובץ הראשוני בעיצוב הותאם לשימוש עם זכרים; פרנק שרדר על סינתזה ועל אספקת ascr #3; רוס Lagoy כדי להפוך את התובנה בסיוע הדמיה וניתוח; לורה Aurilio על הזיוף הבסיס, ומי, לצד כריסטופר המצנח, תרמו הסקירה של כתב היד הזה. מימון עבור עבודה זו סופקה תחת של מכוני הבריאות הלאומיים גרנט 1R01DC016058-01 (ס), המענק הלאומית למדע CBET 1605679 (D.R.A.), ופרס בורוז Wellcome הקריירה ב מדעי ממשק (D.R.A.).

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המחברים אין לחשוף.

השבב חוש הריח הותאם זכר משלבת פנייה לתוך נמל טעינה צר יותר, אשר מאפשר שליטה רבה יותר כיוון ועבור לכידה יעילה של זכר C. elegans. דבר זה מאפשר הפריט החזותי של שני ימינה ושמאלה חברי זוגות דו צדדיים עצביים, ללא צורך z-לערום. עקום זה מוביל אוריינטציה מרחק אנכי 100% של הזמן תולעים שבו זוג הדו-צדדיים ר...

Microfluidic התקני מספקים גישה מוגברת דווקא בסביבות מבוקרות, שבו חיות, כגון תולעים נימיות C. elegans, יכול להיות מרומה השפעול1. מחקרים אלה כוללים מבחני התנהגות, מחקרי הדמיה סידן או הקרנות אפילו על הפנוטיפים ספציפיים, וכתוצאה מכך מדידות מדויקות יותר של תוצאות ניסוי1,2,3,4, 5,6. מיקרופלואידיקה לספק תנאים נוזלי בקנה מידה קטן שדרכו ניסויים מפורטים ניתן להפעיל תוך ניצול כמויות מינימליות של נוגדנים. יש הפקה מתמדת של עיצובים חדשים של המכשיר microfluidic, השימוש של כל משתנה, החל ארנאס המאפשרים טבעי sinusoidal התנועה של C. elegans מבחני התנהגות ולימודים הדמיה עצבית, מלכודת התקנים המשמשים הדמיה עצבית ולימודים חוש הריח, התקנים המאפשרים עבור תפוקה גבוהה פנוטיפי ניתוח גנטי מסכי4,5,6,7. בעקבות ייצור של תבנית בסיס, microfluidic מכשירים זולים לבנות — נתן את שימושית של המאסטר — וקל לשימוש, ומאפשר לדור נתונים מהירה באמצעות מחקרים תפוקה גבוהה. הזיוף של התקנים באמצעות פולימרים כגון polydimethylsiloxane (PDMS) מאפשר היצירה של מכשירים חדשים בתוך שעות.
מחקרי הדמיה סידן להשתמש סידן מקודדים גנטית אינדיקטורים (GECIs) המתבטא התאים היעד כדי למדוד את הדינמיקה עצבית של תאים אלה בזמן אמת8,9,10,11. הטבע שקוף של C. elegans מאפשר ההקלטה של פלורסנט רמות החלבונים האלה בבעלי חיים. באופן מסורתי, GECIs להסתמך על חלבון פלואורסצנטי ירוק (GFP)-מבוסס חיישן ה-GFP-קלמודולין-M13 פפטיד (GCaMP), אך מחקרים מאוחרים יותר הסתגלו חיישנים אלה כדי לאפשר יותר יחס אות לרעש ופרופילים עירור אדום-העביר. בעקבות הפיתוח של GCaMP3, חלבונים עם מפרטים אלה יש מגוונים, כולל חיישנים כגון GCaMP6s ו- GCaMP6f (לאט ומהר פלורסצנטיות חופש-המחירים, בהתאמה), כמו גם RFP-קלמודולין-M13 פפטיד (RCaMP), אשר יש אדום-העביר הפעלת פרופיל. השילוב של אלה GECIs עם C. elegans גנים ספציפיים תא יזם רצפי ניתן לייעד תאים עניין, במיוחד החישה הניריונים12,13,14,15 , 16.
בעוד קלות השימוש C. elegans במחקרים microfluidic הוא לכאורה, כמעט כל המחקרים התמקדו אנדרוגינוסים. למרות זכרים בלבד החשבונאי 0.01-0.02% מהאוכלוסייה פראי סוג, ממצאים שלא יסולא בפז יכול לנבוע האפיון שלהם. ואילו connectome הפיזי של מערכת העצבים אנדרוגינוס מופה באופן מלא במשך עשרות שנים17, connectome זכר נשאר שלם, במיוחד באזור הראש של בעלי חיים18. השימוש של סידן הדמיה של הזכרים יסייע ליצירת הבנה של מערכת העצבים זכר וההבדלים שעולות בין שני המינים. גודל קטן יותר מהזכרים הבוגרים C. elegans מונעת השמנה יעיל ואמין בנמלי טעינת התקנים חוש הריח מסורתי המיועד אנדרוגינוסים גדול יותר. כדי לטפל בבעיה זו, יש את הגירסה שבב חוש הריח Chronis19 פותחה עם יציאת הטעינה צר יותר, ערוץ בגובה נמוך, והופך בנמל הטעינה תולעת (אשר לסובב את החיה), המאפשר החזיית הדו-צדדיים שמאלה/ימינה זוגות עצביים. עיצוב זה מאפשר: (1) השמנה יעיל של זכרים בוגרים צעירים, (2) כיוון יותר אמין של החיה עבור הפריט החזותי של שני בני נוירונים לזווג דו צדדיים, (3) הדמיה מדויקת של פעילות עצבית בנוירונים זכר.
יותר ויותר מחקרים מראים כי גברים C. elegans להגיב באופן שונה מאשר אנדרוגינוסים למגוון רחב של ascarosides (ascr), או תולעים נימיות פרומונים20,21,22,23 ,24. לכן, לפתח הבנה של דינמיקה עצבית, ייצוגים בתוך connectome זכר הפך אפילו יותר רלוונטי. זכר C. elegans מכיל נוירונים ספציפיים סקס 87 לא נוכח אנדרוגינוס25,26, לשנות את connectome כדרכים שטרם נקבע. היכולת תמונה הדינמיקות האלה עצביות ייחודיות יאפשר לנו להבין טוב יותר את תגובות ספציפיות סקס וייצוגי עצבית.
פרוטוקול זה מתאר את השימוש שבב חוש הריח זכר הותאם עבור ההדמיה עצבית של זכר C. elegans chemosensation. הנוירון nociceptive שאש מגיב בצורה אמינה גליצרול 1 מ' אצל הזכרים, בקנה אחד עם הקודם אנדרוגיניות מחקרים27. חשיפה ascarosides עשוי להפיק תגובות כי הם משתנה חיה כדי חיה, הדורשות מספר גדול יותר של בעלי חיים כדי להיבדק. התגובה של הנוירונים צ'ם זכר הספציפי שהוצג בעבר, דרך אלקטרופיזיולוגיה וגם סידן הדמיה מחקרים, להגיב variably ascaroside #323.

<table><tbody><tr><td>Silicon Wafer</td><td>University Wafer</td><td>452</td><td></td></tr><tr><td>SU-8 2035</td><td>MicroChem</td><td>Y111070-0500L1GL</td><td></td></tr><tr><td>Developer</td><td>MicroChem</td><td>Y020100-4000L1PE</td><td></td></tr><tr><td>Wafer Mask</td><td>Cad/Art Services</td><td>-</td><td>Custom order. Printed at 25,000 dpi.</td></tr><tr><td>Sylgard-184</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td></td></tr><tr><td>1.0 mm Dermal Punches</td><td>Acuderm Inc.</td><td>P150</td><td></td></tr><tr><td>Soft Tubing</td><td>Cole-Palmer</td><td>EW-06419-01</td><td></td></tr><tr><td>Hard Tubing</td><td>IDEX Health &amp;amp; Science</td><td>1622</td><td></td></tr><tr><td>Pins</td><td>New England Small Tube</td><td>NE-1027-12</td><td></td></tr><tr><td>Blocking Pins</td><td>New England Small Tube</td><td>0.415/0.425&quot; OD x .500 Long</td><td>Batch PB07027</td></tr><tr><td>3 mL syringes</td><td>BD</td><td>309657</td><td></td></tr><tr><td>30 mL syringes</td><td>Vitality Medical</td><td>302832</td><td>Used as buffer reservoirs.</td></tr><tr><td>Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer</td><td>Component Supply Company</td><td>NE-231PL-50</td><td></td></tr><tr><td>Stopcocks with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile</td><td>Cole-Palmer</td><td>EW-30600-07</td><td></td></tr><tr><td>Fisherfinest Premium Cover Glass</td><td>Fisher Scientific</td><td>12-548-5M</td><td></td></tr><tr><td>Mercator Control System LF-5 Plasma System</td><td>Mercator</td><td>LF-5</td><td></td></tr><tr><td>Scotch Tape</td><td>Scotch</td><td>BSN43575</td><td></td></tr><tr><td>Series 20 Chamber</td><td>Warner Instruments</td><td>P-2</td><td></td></tr><tr><td>Vacuum Desicator</td><td>Bel-Art Scienceware</td><td>420250000</td><td>24 cm inner diameter.</td></tr><tr><td>Weigh Boats</td><td>Cole-Palmer</td><td>EW-01017-27</td><td></td></tr><tr><td>Classic Plus Balance</td><td>Mettler Toledo</td><td>PB1501-S/FACT</td><td></td></tr><tr><td>Glass Pasteur Pipettes</td><td>Cole-Palmer</td><td>EW-25554-06</td><td></td></tr><tr><td>Transfer pipettes</td><td>Genesee Scientific</td><td>30-202</td><td></td></tr><tr><td>Oven</td><td>Sheldon Manufacturing Inc</td><td>9120993</td><td>Model Number: 1500E.</td></tr><tr><td>60 mm, non-vented, sharp edge Petri dishes</td><td>TriTech Research</td><td>T3308</td><td></td></tr><tr><td>Zeiss Axio Observer.A1</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Hammamatsu Orca Flash 4.0 Digital CMOS</td><td>Hammamatsu</td><td>C11440-22CU</td><td></td></tr><tr><td>Blue Fluorescent Light</td><td>Lumencor</td><td>SOLA SM6-LCR-SA</td><td>24-30V/7.9A DC.</td></tr><tr><td>Illumination Adaptor</td><td>Zeiss</td><td>423302-0000</td><td></td></tr><tr><td>Series 1 and 2 Miniature Inert PTFE Isolation Valve</td><td>Parker</td><td>001-0017-900</td><td>3-way valve for controlling flow.</td></tr><tr><td>ValveLink8.2&amp;reg;</td><td>AutoMate Scientific</td><td>01-18</td><td>Flow Switch Controller</td></tr><tr><td>Micro Manager</td><td>Micro-Manager</td><td>-</td><td>Free software, can be downloaded at: https://www.micro-manager.org/wiki/Download_Micro-Manager_Latest_Release</td></tr><tr><td>ImageJ</td><td>ImageJ</td><td>-</td><td>Free software, can be downloaded at: https://imagej.nih.gov/ij/download.html</td></tr><tr><td>Agar, Bacteriological Grade</td><td>Apex</td><td>9012-36-6</td><td></td></tr><tr><td>Peptone</td><td>Apex</td><td>20-260</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>VWR</td><td>BDH0224-1KG</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>230391-1kg</td><td></td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>A11470</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>270741-4L</td><td></td></tr><tr><td>Tetramisole</td><td>Sigma-Aldrich</td><td>L9756-10(G)</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Fluorescein</td><td>Sigma-Aldrich</td><td>FD2000S-250mg</td><td>Light Sensitive. Store in photoprotective vials.</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G6279-1L</td><td></td></tr><tr><td>Ascaroside #3</td><td>-</td><td>-</td><td>Synthesized in the Schroeder Lab (Cornell University).</td></tr><tr><td>NaCl</td><td>Genesee Scientific</td><td>18-215</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>BDH</td><td>BDH9268.25</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3252-025</td><td></td></tr><tr><td>ASH GCaMP3 line</td><td>-</td><td>-</td><td>CX10979 (KyEx2865 [p&lt;em&gt;sra&lt;/em&gt;-6::GCAMP3 @ 100 ng/uL]). Developed in Bargmann lab. Provided from Albrecht Lab library.</td></tr><tr><td>CEM GCaMP6 line</td><td>-</td><td>-</td><td>JSR49 (FkEx98[p&lt;em&gt;pkd&lt;/em&gt;-2::GCaMP::SL2::dsRED + pBX-1]; &lt;em&gt;pha&lt;/em&gt;-1(e2123ts); &lt;em&gt;him&lt;/em&gt;-5(e1490); &lt;em&gt;lite&lt;/em&gt;-1(ce314)). Developed by Robyn Lints. Provided from Srinivasan Lab library.</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; (OP50)</td><td>&lt;em&gt;Caenorhabditis&lt;/em&gt; Genetics Center</td><td>OP50</td><td></td></tr><tr><td>&quot;Reservoir&quot;</td><td>-</td><td>-</td><td>To create a Reservoir: A &quot;30 mL syringe&quot;, is connected to a &quot;Stopcock with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile&quot;, which is connected to a &quot;3 mL syringe&quot; and a &quot;Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer&quot;. The &quot;Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer&quot; is then inserted into &quot;Soft Tubing&quot; approximately 1/3 of the way down the needle.</td></tr></tbody></table>

using an adapted microfluidic olfactory chip for the imaging of neuronal activity in response to pheromones in male c. elegans head neurons

השימוש של סידן אינדיקטורים התעלתה ההבנה שלנו של דינמיקה עצבית ורגולציה. תולעים נימיות Caenorhabditis elegans, עם מערכת העצבים לחלוטין ממופה, אנטומיה שקוף, מציג מודל אידיאלי להבנת הדינמיקה עצבית בזמן אמת באמצעות מחווני סידן. בשילוב עם טכנולוגיות microfluidic עיצובים ניסיוניים, מחקרים סידן-הדמיה באמצעות אינדיקטורים אלה מבוצעות בבעלי חיים חינם-המעבר והן לכוד. עם זאת, רוב המחקרים הקודמים ניצול התקני לכידה, כגון השבב חוש הריח המתוארים Chronis. et al., יש התקנים שנועדו לשימוש ההרמפרודיט נפוץ יותר, כמו הזכר פחות נפוץ הוא שניהם מורפולוגית, מבחינה מבנית שונה. שבב חוש הריח מותאם היה מתוכנן, מפוברק ליעילות מוגברת בתחום ההדמיה עצביים זכר עם באמצעות בעלי חיים למבוגרים צעירים. פנייה סופחה התולעת בטעינת לנמל כדי לסובב את החיות, כדי לאפשר את ההפרדה של הנוירונים בודדים בתוך זוג הדו-צדדיים בתחום ההדמיה 2D. תולעים נחשפים זרימה מבוקרת של odorant בתוך המכשיר microfluidic, כמתואר במחקרים קודמים דו-מיניים. תופעות מעבר סידן ואז ניתוח שימוש בתוכנת קוד פתוח ImageJ. ההליך המתואר במסמך זה אמור לאפשר עבור כמות מוגברת של מבוססי זכר C. elegans סידן הדמיה מחקרים, העמקת ההבנה שלנו על מנגנוני איתות עצביים מין ספציפי.

ניתן לראות דוגמה של הגדרת התקן הכוללת איור 1A-B. איור 1A , מתאר את בניית מאגר תקין וההתקנה. איור 1B מציגה את החיבורים של המאגרים למכשיר microfluidic. איור 1C מתאר התקן microfluidic עם יציאות בודדות המסומנות על בהירו?...

Watch this Scientific Journal Video about Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons at JoVE.com

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

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

השימוש מותאם "צ'יפ חוש הריח" עבור ההדמיה סידן יעיל של C. elegans זכרים מתואר כאן. מחקרים של חשיפה זכר גליצרול פרומון מוצגים גם.

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

using-an-adapted-microfluidic-olfactory-chip-for-imaging-neuronal

Research

JoVE Journal

Neuroscience

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

8.1K Views.  Worcester Polytechnic Institute. השימוש מותאם "צ'יפ חוש הריח" עבור ההדמיה סידן יעיל של C. elegans זכרים מתואר כאן. מחקרים של חשיפה זכר גליצרול פרומון מוצגים גם.

Video: Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

התקני microfluidic פשוטים עבור<em&gt; In vivo</em&gt; הדמיה של<em&gt; ג elegans</em&gt;,<em&gt; דרוזופילה</em&gt; ודג זברה

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Bioengineering

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

סידן הדמיה של ריח עוררו התגובות תסיסנית מחושים האונה

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

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 ...

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 ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

שיטות הקלטה בעל מספר יחידות לאפיין את הפעילות עצבית בארבה (<em&gt; Schistocerca אמריקנה</em&gt;) מעגלי ריח

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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, ...

<meta charset="utf8"></head><body>מיצוי וכימות של תגובות פרומון מין נדיפות ב- C. elegans

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

<meta charset="utf8"></head><body>מחבר זרקור: בחינת השפעת פרומון המין הנדיף על התנהגות גברי C. elegans

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

מיצוי פרומון מין נדיף ובדיקת כימותרפיה ב- Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

Behavior

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

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

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התקני microfluidic פשוטים עבור

סידן הדמיה של ריח עוררו התגובות תסיסנית מחושים האונה

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

Readout מולקולרי של הסתגלות חוש ריח לטווח הארוך ב

שיטות הקלטה בעל מספר יחידות לאפיין את הפעילות עצבית בארבה (

בהדמיה עצבית סיד vivo ב C. elegans

מיצוי פרומון מין נדיף ובדיקת כימותרפיה ב- Caenorhabditis elegans

מיצוי פרומון מין נדיף ובדיקת כימותרפיה ב- Caenorhabditis elegans

מיצוי פרומון מין נדיף ובדיקת כימותרפיה ב- Caenorhabditis elegans

שימוש בהתקן מיקרופלואידיקה גירוי מכני, רזולוציה גבוהה הדמיה של C. elegans