נתמך על ידי 2010-65105-20582 USDA-NIFA/AFRI, NSF 0918177 ו התאגדות Bedoukian המחקר.

<html> 1. חרקים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הטבור orangeworm, Amyelois transitella (ווקר) (הפרפרים: Pyralidae) המושבה במעבדה שלנו מקורם עש שנאספו עצי שקד ב Fresno, CA. מושבת חרקים שלנו נשמר בתאי סביבתיים (פרסיבל Inc, IA) בשעה 28 ± 2 ° C, 75 ± 10% לחות יחסית, ותחת 16:08 שעות (אור: חושך) תמונה המשטר. </li><li style=";text-align:right;direction:rtl"> עש מתעוררים נאספים מדי יום, מינית, והועבר קופסאות פלסטיק (669 מ&quot;ל קופסת האוכל, 13 x 13 ס&quot;מ, גובה, 4.5 ס&quot;מ, Rubbermaid) מסופק עם עד 10 שכבות של מגבות נייר ספוג מים (צמאים Ultra סופגים, 27.9 x 27.9 ס&quot;מ ; Safeway). מכסה קופסת הם מחורר, כדי לאפשר זרימת אוויר. הסדר זה מספק לחות יחסית ~ 100%. </li><li style=";text-align:right;direction:rtl"> עבור מדידות אלקטרו אנו משתמשים 2-4 ימים לאחר הופעת עשים הישן, אשר נשמרים תחת לחות גבוהה. </li></ol> 2. אלקטרו הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עשים (זכרים או נקבות) הם דחפו בעדינות לתוך קצה פיפטה (200 μL, ארה&quot;ב מדעי Inc), כי הוא לחתוך מלמעלה יש קוטר טיפ גדול (מ&quot;מ בקירוב 2). עש הוא דחף בעדינות דרך הקצה השני עם בדל רקמה humidified נייר עד אנטנות חלק בראש לבלוט מהקצה. </li><li style=";text-align:right;direction:rtl"> ראש חשופים היא משותקת על ידי כיסוי הראש עם חימר שאינם ייבוש (Claytoon, ואן Aken הבינלאומי, CA) והותירה חלק קטן של עין אחת ושני האנטנה חשוף. הכנה זו ממוקמת על פלטפורמה של Micromanipulator EAG MP-12 (Syntech, גרמניה) בין שני מחזיקי אלקטרודה. </li><li style=";text-align:right;direction:rtl"> אנו משתמשים אלקטרודת הזכוכית עשויה צינורות 1.0 מ&quot;מ נימי בורוסיליקט עם נימה (WPI Inc, פלורידה) ומשך במודל P-97 micropipette חולץ (סאטר מכשירים, CA) להשיג microelectrodes של פחות מ טיפ מיקרומטר. אלקטרודות מלאים בחזרה עם sensillum הלימפה הצלצול (Kaissling ו Thorson 1980). אלקטרודה אחת נועצת את האזור חשוף לעין תחת מיקרוסקופ (לייקה MZ75) ומשמש כנקודת התייחסות, בעוד אלקטרודה הקלטה להכיל את המחושים השניים. כדי להקל על הקשר, דיסטלי מגזרים מחושים 1-2 נחתכים בטרם מוכנס לתוך האלקטרודה ההקלטה. עבור אותות הקלטות sensilla בודדים, אנחנו קצת לשנות את ההגדרה שבה האלקטרודה ההקלטה משופדת תחת מיקרוסקופ (אולימפוס BX51WI; 800x הגדלה). פוטנציאל פעולה נרשמות על ידי החדרת אלקטרודה זכוכית בבסיס sensillum. איתותי הם מוגבר על ידי עכבה גבוהה טרום מגבר (Syntech, גרמניה) מוזן לתוך קו מעל UB-IDAC להיות מנותח עם AUTOSPIKE תוכנה (Syntech, גרמניה). אותות AC הם עוברים סינון הלהקה בין 100 הרץ -10,000 פוטנציאל פעולה מופקים על ידי המחשב באמצעות אלגוריתם AUTOSPIKE שמבדיל אל שוקת לשיאם אמפליטודות מרעש. תגובות של נוירונים בודדים מחושבים להגדיל (או להקטין) בתדירות פוטנציאל פעולה (קוצים לשנייה) על תדר ספונטנית. </li></ol> 3. כימית גירויים פרוטוקול גירוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כימיקלים טוהר הגבוהה ביותר משמשים. המרכיב העיקרי של פרומון למזג את הנקבה מן orangeworm הטבור, (Z, Z) -11,13-hexadecadienal התקבל Bedoukian מחקר Inc, CT. כימיקלים בדילול מלא, w / v, עם הקסאן זכוכית מזוקקים כדי להפוך פתרונות מלאי של 10 דילולים מיקרוגרם / μL ו עשורי נעשים. Aliquot (10 μL) של גירוי כימי מומס הקסאן נטען על מסנן Whatman רצועת נייר (8x40 מ&quot;מ), ממס הוא התאדה ידי רועדות בעדינות למשך 10 שניות מתחת למכסה המנוע קטר ורצועת ממוקם פוליפרופילן 5 מ&quot;ל מזרק (מזרקים BD, NJ) שממנו כרכים שונים נפלטים. הקסאן לבד מזרק ריק לשמש מלאה. </li><li style=";text-align:right;direction:rtl"> ההכנה נערכת בזרם אוויר humidified מועברים על ידי בקר Stimulus Syntech (CS-55 דגם: Syntech, גרמניה) בשעה 20 mL / sec בה דופק גירוי של 4 mL / s נוסף עבור 500 ms. איתותי נרשמות במשך 10 שניות, החל 2 s לפני התחלתה של דופק את הגירוי. הכנה מחושים מגורה עם 500 ms הדופק שבמהלכו CA. 2 מ&quot;ל של אוויר מטוהר מן מזרק פוליפרופילן 5 מ&quot;ל המכיל את גירוי נוסף על זרימת האוויר הראשי. פער של לפחות 1 דקות או יותר, בעקבות התגובות גבוה, מותר בין גירויים. </li></ol> 4. נציג תוצאות האנטנה של עש מאופק (איור 1) מגורה עם רכיב פרומון הנקבה, (Z, Z) -11,13-hexadecadienal ליצור תגובות חזקים תלוי במינון (איור 2). זה מרכיב מרכזי פרומון גם מעורר תלוי במינון התגובות מעוררים ממערכות ארוך trichodea sensilla (איור 3A) מוצג כמו עקבות של הקלטות יחידה אחת (איור 3B). <img src="/files/ftp_upload/2489/2489fig1.jpg" alt="איור 1" /> באיור 1. חיה הטבור orangeworm עש מאופקת אנטנה נחשפים load/2489/2489fig2.jpg &quot;alt =&quot; איור 2 &quot;/&gt; איור 2. תלויה במינון התגובות EAG שהושרו בתגובה (Z, Z) -11,13-hexadecadienal, מרכיב עיקרי פרומון נקבי. <img src="/files/ftp_upload/2489/2489fig3.jpg" alt="איור 3" /> איור 3. Trichoid Sensilla אצל הזכר הטבור orangeworm עש להגיב (Z, Z) -11,13-hexadecadienal באופן תלוי מינון. (א) אנטנה של הזכר הטבור orangeworm עש הוא רב מפולח וכל קטע מעוטר במספר רב של מבנים דמויי שיער, sensilla. מיקרוסקופ אלקטרונים סורק, מראה פרטים על הבלעה (מוטות סולם הם 200 ו - 50 מיקרומטר, בהתאמה). (ב) תאי יחיד יחידת הקלטות sensillum trichoid.

<ol>
	<li>Kaissling, K. -. E., Thorson, J., Sattelle, D. B., Hall, L. M., Hildebrand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+olfactory+sensilla:+structural,+chemical+and+electrical+aspects+of+the+functional+organization.">Insect olfactory sensilla: structural, chemical and electrical aspects of the functional organization.</a> Receptors for Neurotransmitters. Hormones and Pheromones in Insects. , 261-282 (1980).</li></ol>

אין ניגודי אינטרסים הכריז.

זו שיטה חדשה שפותחה עבור הרחקה עש הטבור לחיות orangeworm למדוד אותות הריח הוכיחה להיות חזקה ואמינה מאוד. אנו להעסיק בשיטה זו באופן שגרתי עבור בידוד וזיהוי משיכה רומן מן מצעים לארח טבעי כמו שקדים ופיסטוקים.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> חומר שם </td><td> קטלוג # / דגם </td><td> חברה </td><td> הערה </td></tr><tr><td> מיקרוסקופ </td><td> BX51WI מודל </td><td> אולימפוס, ארה&quot;ב </td><td></td></tr><tr><td> סטריאו מיקרוסקופ </td><td> MZ75 </td><td> לייקה מיקרוסיסטמס ארה&quot;ב </td><td></td></tr><tr><td> 1.0 מ&quot;מ בורוסיליקט צינור נימי עם נימה </td><td> 1B100F-3 </td><td> WPI Inc, פלורידה </td><td></td></tr><tr><td> Micropipette חולץ </td><td> P-97 </td><td> סאטר מכשירים, CA </td><td></td></tr><tr><td> Stimulus בקר </td><td> CS-55 דגם </td><td> Syntech, גרמניה </td><td></td></tr><tr><td> התנגדות גבוהה טרום מגברים (Universal בדיקה שהסתיימה יחיד) </td><td></td><td> Syntech, גרמניה </td><td></td></tr><tr><td> מגבר / מערכת נתונים הרכישה (USB-IDAC) </td><td></td><td> Syntech, גרמניה </td><td></td></tr><tr><td> Micromanipulator EAG MP-12 </td><td></td><td> Syntech, גרמניה </td><td></td></tr><tr><td> (Z, Z) -11,13-hexadecadienal </td><td></td><td> Bedoukian מחקר Inc, CT. </td><td></td></tr><tr><td> Whatman נייר הסינון </td><td> 1001070 </td><td> Whatman ארה&quot;ב </td><td></td></tr><tr><td> 5 מ&quot;ל במזרק פוליפרופילן </td><td> 309633 </td><td> BD מזרקים, NJ </td><td></td></tr><tr><td> פיפטה קצה (200 μL) </td><td> 1111-0806 </td><td> ארה&quot;ב מדעי Inc </td><td></td></tr><tr><td> 669 מ&quot;ל קופסת האוכל, 13 x 13 ס&quot;מ, גובה, 4.5 ס&quot;מ, </td><td></td><td> Rubbermaid </td><td></td></tr><tr><td> צמאים Ultra סופגים, 27.9 x 27.9 ס&quot;מ </td><td></td><td> סייפוויי </td><td></td></tr><tr><td> ללא ייבוש חימר </td><td> 18150 </td><td> Claytoon, ואן Aken הבינלאומי, CA </td><td></td></tr><tr><td> הסביבה קאמרית </td><td> I-30BLL מודל </td><td></td><td></td></tr></tbody></table>

electrophysiological measurements from a moth olfactory system

מערכות חוש הריח חרקים לספק הזדמנויות ייחודיות עבור odorant-Induced התגובות הקלטה בצורות של electroantennograms (EAG) והקלטות sensillum יחיד (SSR), אשר סיכם תגובות מכל קולטן נוירונים odorant (ORNs) הממוקם על האנטנה ועל מאלה שוכנו הפרט sensilla, בהתאמה. גישות אלה נוצלו להשגת הבנה טובה יותר של תקשורת כימית חרקים. גירויים זיהו אז יכול לשמש גם משיכה או דוחי אסטרטגיות ניהול חרקים מזיקים.

Watch this Scientific Journal Video about Electrophysiological Measurements from a Moth Olfactory System at JoVE.com

Electrophysiological Measurements from a Moth Olfactory System

מערכות חוש הריח חרקים לספק הזדמנויות ייחודיות עבור odorant-Induced התגובות הקלטה בצורות של electroantennograms (EAG) והקלטות sensillum יחיד (SSR), אשר סיכם תגובות מכל קולטן נוירונים odorant (ORNs) הממוקם על האנטנה ועל מאלה שוכנו הפרט sensilla, בהתאמה.

מדידות אלקטרו ממערכת עש חוש הריח

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

electrophysiological-measurements-from-a-moth-olfactory-system

Research

JoVE Journal

Neuroscience

13.6K Views.  University of California, Davis. מערכות חוש הריח חרקים לספק הזדמנויות ייחודיות עבור odorant-Induced התגובות הקלטה בצורות של electroantennograms (EAG) והקלטות sensillum יחיד (SSR), אשר סיכם תגובות מכל קולטן נוירונים odorant (ORNs) הממוקם על האנטנה ועל מאלה שוכנו הפרט sensilla, בהתאמה.

Video: Electrophysiological Measurements from a Moth Olfactory System

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

הקלטות אלקטרו מתוך מסלול של סיבים ענק ד melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

בידוד באף גזע תאים חוש הריח של מכרסמים או בני אדם

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

מדידות אלקטרו וניתוח Nociception אצל תינוקות אדם

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

תגובות odorant מושרה הקלטות מתא העצב קולטני ריח באמצעות טכניקת יניקת פיפטה

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

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

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

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

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

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

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

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

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

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

במדידות Vivo אלקטרו על עצבים העכבר השת

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

הקלטת אלקטרו מ<em&gt; דרוזופילה</em&gt; Labellar טעם Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

מערכת חוש הריח כמודל לחקר דפוסי axonal צמיחה ומורפולוגיה<em&gt; In vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

הר תיוג כל צילה במערכת חוש הריח העיקרית של עכברים

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...