אנו מודים מיכאל Dübbert, אוליבר הופמן, הנס Scharstein, יאן ואן Sydow Wosnitza לקבלת סיוע טכני מעולה. מחקר זה מומן על ידי מענק DFG Bu 857 / 8,10 אל AB

<ol>
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Physiologie. 70, 425-447 (1970).</li><li>Kirschfeld, K., Zettler, F., Weiler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+resolution+of+lens+and+compound+eyes.">The resolution of lens and compound eyes.</a> Neural Principles in Vision. , 354-370 (1976).</li><li>Kutsch, W., Schwarz, G., Fischer, H., Kautz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wireless+transmission+of+muscle+potentials+during+free+flight+of+a+locust.">Wireless transmission of muscle potentials during free flight of a locust.</a> J Exp Biol. 185, 367-373 (1993).</li><li>Scharstein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+projector+for+optomotor+stimulation.">A universal projector for optomotor stimulation.</a> , (1985).</li><li>Schlegtendal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beitrag+zum+Farbensinn+der+Arthropoden.">Beitrag zum Farbensinn der Arthropoden.</a> Zeitschr. f. vergl. Physiologie. 20, 573-581 .</li><li>Schmidt, J., Grund, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+activity+in+a+motor+axon+induced+by+axotomy.">Rhythmic activity in a motor axon induced by axotomy.</a> Neuroreport. 14, 1267-1271 (2003).</li><li>Shankar, P. N., Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Determination+of+the+Kinematic+Viscosity+of+Glycerol-Water+Mixtures.">Experimental Determination of the Kinematic Viscosity of Glycerol-Water Mixtures.</a> Proc. Mathematical and Physical Sci. 444, 573-581 (1994).</li><li>Tryba, A. K., Ritzmann, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Joint+coordination+during+walking+and+foothold+searching+in+the+Blaberus+cockroach+I.+Kinematics+and+electromyograms.">Multi-Joint coordination during walking and foothold searching in the Blaberus cockroach I. Kinematics and electromyograms.</a> J Neurophysiol. 83, 3323-3336 .</li><li>Tryba, A. K., Ritzmann, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Joint+coordination+during+walking+and+foothold+searching+in+the+Blaberus+cockroach.+II.+Extensor+motor+neuron+pattern.">Multi-Joint coordination during walking and foothold searching in the Blaberus cockroach. II. Extensor motor neuron pattern.</a> J Neurophysiol. 83, 3337-3350 .</li><li>Tsuchida, Y., Hama, N., Takahata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+telemetry+system+for+underwater+recording+of+electromyogram+and+neuronal+activity+from+non-tethered+crayfish.">An optical telemetry system for underwater recording of electromyogram and neuronal activity from non-tethered crayfish.</a> J Neurosci Methods. 137, 103-109 (2004).</li><li>Wang, H., Ando, N., Kanzaki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+control+of+free+flight+maneuvers+in+a+hawkmoth,+Agrius+convolvuli.">Active control of free flight maneuvers in a hawkmoth, Agrius convolvuli.</a> J Exp Biol. 211, 423-432 (2008).</li></ol>

אין ניגודי אינטרסים הכריז.

תיארנו התקנה המאפשרת את הדור המושרה אופטית של הפיכת התנהגות היתרי במידה רבה שיחררה את הפעילות בהליכה שנוצר neuronally מהשפעות פסיבי שנגרם על ידי עקירה המכני של הגפיים הליכה אחרים על הקרקע. זרימת המידע אפשריים בין הרגליים דרך מערכת העצבים על התגובה לכוחות הקרקע או צרו קשר עם tarsal, מצד שני, עדיין אפשרי ומאפשר הניסוי לבחון את ההשפעה של מידע כזה בהכנת מופחת. היתרונות העיקריים של ההתקנה כוללות משטח חלקלק, כי בעלי החיים מראים נטייה גבוהה מאוד ללכת, ובניגוד הליכה או דריכה על treadwheel, החיה ניתן לבצע סווינג שלב תנועות עמדה בכל ההוראות של הליכה טבעית. בנוסף, דרגות החופש של כל הרגליים לאפשר החיה לבצע הליכה עקומה אם הוא הכנה שלם או חצי שלם. מכיוון הרגליים לא ניתן להעביר באופן פסיבי פשוט על ידי התנועה קדימה של החיה או את התנועה של המצע שמתחת, כל תנועה משקף את הפלט המוטורי של הרגל זה (פך, 1976; גרהם ונדלר, 1981). ההגדרה מתאימה מאוד לחקור את הבסיס העצבי של התנהגויות מסתגלות כגון הפיכת קדימה או אחורה לעומת הליכה, כי ניתן לשלב הקלטות אלקטרו של פעילות מוטורית עם ניתוח של קינמטיקה תנועת הגפיים. השתמשנו בתגובה optomotor החרק של מקל כדי לעורר הליכה. התגובות של בעלי החיים לדפוס פס מסתובב להראות את נכונותם לבצע עקומת הליכה תוך קשור על פני השטח חלקלק. רוב מפתיע אותנו, רגליים אחד, רגל אחת ההכנות איכותית להראות את דפוס נע זהה החיה בשלמותה. ולכן יש לנו סיבה להאמין כי השליטה של ​​הליכה עקומה יכול לתפקד במידה רבה, ללא תיאום סנסורי מהרגליים השכנות. זה יהיה חשוב לבדוק בניסויים נוספים אם את פעילותם של הנוירונים המנוע של הסירה הרגליים מושפעת גם דפוס optomotor. ההתקנה יכול בקלות להיות שונה, כדי לאפשר לימוד של משימות אחרות כגון ישר קדימה ואחורה הליכה ידי הצבת פס יחיד מול החיה או בעדינות מושך את האנטנות. מדידה מדויקת של קשר הקרקע מאפשרת לנו לתאם פעילות שרירים עמדה רגל. ההחלטה הזמן של אות חשמלי זה קשר טוב יותר 1ms, מביאה אותנו אל תובנה חדשה לתוך העיתוי של הבורר מן הנדנדה לשלב עמדה. ההחלטה גרועה עבור העמדה להתנדנד המעבר בשל העיכוב מריחה של חומר סיכה ניצוח וחוסר הצורך להשלים ההמראה במהלך הארכה על משטח חלקלק. עם זאת, הידע על הנדנדה מדויקת המעבר עמדה היא צעד ראשון שימושי במיוחד אם אנחנו רוצים להבין את המנגנונים שריר השליטה העיתוי פעילות מתואמת של שרירי הרגל בהקשרים התנהגותיים שונים (ראה גם: Büschges et al, 2008;. Büschges &amp; Gruhn 2008). כדוגמה, השתמשנו מפשק ו coxae שריר מד של הרגל באמצע בקורלציה בדיוק את פעילותה עם המעבר מן הנדנדה לשלב עמדה בזמן שאנחנו בו זמנית בדק את הקשר ההתנהגותי שבו הרגל היה בשימוש. לשם כך, אנו המושרה הליכה הקליט את פעילות שריר ברציפות. הרגל נתון יכול להיות בתוך או מחוץ רגל, בהתאם לכיוון הפנייה. ברגל באמצע קפיצה, מתנהג כמו רגל בתוך במובן פונקציונלי, ניתן להבחין כי השרירים מפשק ואת מד שניהם יכולים לעבוד כמו שרירי עמדה תפקודית בגלל הרגל לסירוגין מייצרת צעדים לאחור בנוסף צעדים מכוונים קדימה (ראה Fig.4 ). Electromyograms (EMGs) מהשרירים שניהם היו לתקן ו מנורמל הזמן של מטה בנגיעה ואת חביון של קוצים השרירים הראשון היה מחושב. מעניין, latencies של השרירים הן לגבי ההמראה ולגעת למטה תלוי בתפקוד של שריר כמו נדנדה בהתאמה או עמדה שריר (ראה Fig.4) ולא על השריר עצמו, להציג רק שינויים קלים העיתוי של תחילת הפעילות. רוב ההסברים לשנות את המדינה מ - הנדנדה לעמדה להניח כי אותות חושיים של ההדק קשר tarsal ההתחלה של העמדה. השאלה מעניינת של איך latencies קצר בין מטה בנגיעה והפעלת שרירים החרק מקל הם הביאו על מה המידע החושי הם תלויים כעת ניתן לטפל בהגדרת שונה. לסיכום, אנו מראים ההתקנה משטח חלקלק אשר מאפשרת לנו להפיק באופן אמין ללכת ישר עיקול חרקים מקל נייח. קינמטיקה, פעילות שרירים עיתוי tarsal מגע למטה ההמראה ניתן לנטר בקורלציה בשני ג התנהגותיות שונותontexts באותו זמן. זה נותן לנו כלי מצוין כדי לחקור את הקשר בין פעילות מפורט שרירים בהקשר התנהגותיות רגל אחת, כמו גם החיה שלם המנגנונים.

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>2-component epoxy glue</td>
<td>&nbsp;</td>
<td>UHU plus, UHU GmbH, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass screens (diameter 130mm)</td>
<td>&nbsp;</td>
<td>Marata screens, Linos Photonics, G&#246;ttingen, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>dental cement</td>
<td>&nbsp;</td>
<td>ProTemp II, 3M ESPE, Seefeld, Germany</td>
<td>3M Id : 70-2011-0358-0 Catalog Number : 46430</td>
<td>Available through 3M (<a href="http://www.3m.com/">http://www.3m.com/</a>)or dental suppliers</td>
</tr>
<tr>
<td>fluorescent pigments </td>
<td>&nbsp;</td>
<td>Dr. Kremer Farbm&#252;hle, Aichstetten, Germany</td>
<td>Cat.#s: i.e 56200 Fluorescent Pigment Golden Yellow 56350 Fluorescent Pigment Flame Red</td>
<td><a href="http://kremer-pigmente.de/en">http://kremer-pigmente.de/en</a> or <a href="http://www.kremerpigments.com/
">http://www.kremerpigments.com/</a>
</td>
</tr>
<tr>
<td>histoacrylic glue </td>
<td>&nbsp;</td>
<td>3M Vetbond, St.Paul, MN, USA</td>
<td>&nbsp;</td>
<td>supplier: WPI</td>
</tr>
<tr>
<td>coated copper wire</td>
<td>&nbsp;</td>
<td>Elektrisola Eckernhagen</td>
<td>&nbsp;</td>
<td><a href="http://www.elektrisola.com/">http://www.elektrisola.com/</a></td>
</tr>
<tr>
<td>electrode cream </td>
<td>&nbsp;</td>
<td>Marquette Hellige, Freiburg, Germany</td>
<td>&nbsp;</td>
<td>Product is now discontinued, we suggest for example: <a href="www.grasstechnologies.com">www.grasstechnologies.com</a></td>
</tr>
<tr>
<td>pulse generator </td>
<td>&nbsp;</td>
<td>Model MS501, electronics workshop, Zoological Institute, Cologne, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>lock-in-amplifier </td>
<td>&nbsp;</td>
<td>electronics workshop, Zoological Institute, Cologne, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>AD converter </td>
<td>&nbsp;</td>
<td>Micro 1401k II, CED, Cambridge, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>preamplifier </td>
<td>&nbsp;</td>
<td>electronics workshop, Zoological Institute, Cologne, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>high speed video camera </td>
<td>&nbsp;</td>
<td>Marlin F-033C, Allied Vision Technologies, Stadtroda, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>UV LED arrays </td>
<td>&nbsp;</td>
<td>30-50V DC, electronics workshop, Zoological Institute, Cologne, Germany</td>
<td>&nbsp;</td>
<td>&lambda;390-395nm 
Luminance 24cd</td>
</tr>
<tr>
<td>Digitalizing software Spike2 </td>
<td>&nbsp;</td>
<td>Vers.5.05, CED, Cambridge, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>motion tracking software </td>
<td>&nbsp;</td>
<td>(WINanalyze, Vers.1.9, Mikromak service, Berlin, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

studying the neural basis of adaptive locomotor behavior in insects

ללמוד את הבסיס העצבי של הליכה התנהגות, לעתים קרובות בפני את הבעיה כי קשה להפריד בין פלט המיוצר על דריכה neuronally מאלה תנועות הרגל שנובעים כוחות ואינטראקציות פסיבי עם רגליים אחרים באמצעות מגע משותפת עם המצע. אם אנחנו רוצים להבין, איזה חלק של תנועה נתון מופק על ידי פלט מנוע מערכת העצבים, ניתוח kinematic תנועות דריכה, ולכן צריך להיות כהשלמה עם אלקטרו הקלטות של פעילות מוטורית. הקלטה של ​​הפעילות העצבית או שרירית ב חיה מתנהג מוגבל לעתים קרובות על ידי ציוד אלקטרו אשר יכול להגביל את החיה ביכולתה להעביר עם תארים רבים של חופש האפשרי. זה יכול להימנע באמצעות אלקטרודות מושתלת ואחר כך את המהלך החי על גבול ארוך (כלומר Clarac et al, 1987;. Duch &amp; Pflüger, 1995; בוהם et al, 1997;. Gruhn &amp; Rathmayer, 2002) או על ידי שידור את הנתונים באמצעות מכשירים telemetric (Kutsch et al, 1993; פישר et al, 1996;. האמן היפני צוצ&#39;ידה et al 2004;. חמא et al, 2007;.. Wang et al, 2008). שתי שיטות אלה אלגנטי, אשר משמשים בהצלחה פרוקי רגליים גדול יותר, לעתים קרובות קשה להוכיח לחול חרקים קטנים אשר הליכה או בקלות להסתבך לקשור הארוך או הפריע המשקל של המכשיר telemetric וסוללות שלה. בנוסף, בכל המקרים האלה, זה עדיין בלתי אפשרי להבחין בין בסיס עצבי של תנועה טהורה ואת ההשפעות המופעל על ידי צימוד מכני בין הרגליים בהליכה דרך המצע. פתרון אחד לבעיה זו הוא לבצע את הניסויים בבעלי חיים קשור כי הוא חופשי ללכת במקום כי הוא מושעה באופן מקומי, למשל על פני משטח חלקלק, אשר למעשה מסיר מכניקת מגע האדמה ביותר. זה נעשה שימוש כדי ללמוד התגובות לברוח (Camhi ו Nolen, 1981; Camhi ולוי, 1988), מפנה (Tryba ו Ritzman, 2000a, b;. Gruhn et al, 2009a), הליכה לאחור (גרהם אפשטיין, 1985) או שינוי מהירות (Gruhn et al. 2009 ב) והוא מאפשר הנסיין בקלות לשלב התוך תאיים ופיזיולוגיה עם מנתח kinematic (Gruhn et al. 2006). אנו משתמשים ההתקנה משטח חלקלק כדי לחקור את התזמון של שרירי הרגל על ​​החרק מקל מתנהג בכבוד לגעת למטה ולהרים-off תחת פרדיגמות התנהגותיים שונים כמו הליכה קדימה ישר ומעוקל בהכנות שלם מופחת.

Watch this Scientific Journal Video about Studying the Neural Basis of Adaptive Locomotor Behavior in Insects at JoVE.com

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

אנו מתארים שיטה להקליט פעילות מוטורית, בעיתוי לאותת רשמה חשמלית הקשר tarsal ב חרק קשור, הליכה על משטח חלקלק. זה משמש כדי לחקור את הבסיס העצבי של התנהגות אדפטיבית תחת השפעה מופחתת של אינטראקציה מכני בין הרגליים דרך המצע.

ללמוד את הבסיס העצבי של התנהגות מסתגלת של תנועה בתוך חרקים

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

studying-the-neural-basis-of-adaptive-locomotor-behavior-in-insects

Research

JoVE Journal

Neuroscience

12.7K Views.  University of Cologne. אנו מתארים שיטה להקליט פעילות מוטורית, בעיתוי לאותת רשמה חשמלית הקשר tarsal ב חרק קשור, הליכה על משטח חלקלק. זה משמש כדי לחקור את הבסיס העצבי של התנהגות אדפטיבית תחת השפעה מופחתת של אינטראקציה מכני בין הרגליים דרך המצע.

Video: Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Sensillum הקלטות סינגל חרקים תסיסנית melanogaster ו אנופלס gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

שילוב של לימוד למבוגרים, יליד נוירונים

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

ברזולוציה גבוהה הדמיה חיה של התנהגות התא ב Neuroepithelium פיתוח

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

שיתוף ניתוח של מבנה המוח ותפקודו באמצעות fMRI ודיפוזיה משוקללת הדמיה

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In vivo שני פוטוני הדמיה של שינויים מולקולריים ניסיון תלוי בנוירונים בקליפת מוח

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

קביעת הפעילות של תנועה הספונטנית ב<em&gt; Melanogaster דרוזופילה</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

צביעת חרקים למבחני התנהגות: התנהגות חיזור של הרדים<em&gt; דרוזופילה</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

זמן תלויי תגובת הרשת בתגובה לגירוי של תרבויות תאים עצביים על מערכי מיקרו-אלקטרודה

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

ניתוח תפוקה גבוהה של התנהגות גינקולוגיות וזמינותו האי דרוזופילה

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less vulnerable to interference. The neural mechanisms underlying the benefit of sleep for memory can be investigated using polysomnography (PSG). PSG is a combination of physiological recordings including signals from the brain (EEG), eyes (EOG), and muscles (EMG) that are used to classify sleep stages. In this protocol, we describe how PSG can be used in conjunction with behavioral memory assessments, actigraphy, and parent-report to examine sleep-dependent memory consolidation. The focus of this protocol is on early childhood, a period of significance as children transition from biphasic sleep (consisting of a nap and overnight sleep) to monophasic sleep (overnight sleep only). The effects of sleep on memory performance are measured using a visuospatial memory assessment across periods of sleep and wakeful-rest. A combination of actigraphy and parent report is used to assess sleep rhythms (i.e., characterizing children as habitual or non-habitual nappers). Finally, PSG is used to characterize sleep stages and qualities of those stages (such as frequencies and the presence of spindles) during naps. The advantage of using PSG is that it is the only tool currently available to assess sleep quality and sleep architecture, pointing to the relevant brain state that supports memory consolidation. The main limitations of PSG are the length of time it takes to prepare the recording montage and that recordings are typically taken over one sleep bought. These limitations can be overcome by engaging young participants in distracting tasks during application and combining PSG with actigraphy and self/parent-report measures to characterize sleep cycles. Together, this unique combination of methods allows for investigations into how naps support learning in preschool children.

This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy.

מדידת מנגנונים עצביים בבסיס קונסולידציה זיכרון תלויי שינה במהלך התנומה בגיל הרך

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less ...