עבודה זו מומנה על ידי המרכז לטכנולוגיה ותקשורת עצבית (CNCT), מרכז P41 משאבים הממומן על ידי המכון הלאומי ביו הדמיה Bioengineering (NIBIB, P41 EB002030) ונתמך על ידי המכונים הלאומיים לבריאות (NIH).

<ol>
	<li>Cinar, O., Semiz, O., Can, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microscopic+survey+on+the+efficiency+of+well-known+routine+chemical+fixatives+on+cryosections.">A microscopic survey on the efficiency of well-known routine chemical fixatives on cryosections.</a> Acta. Histochem. 108, 487-496 (2006).</li><li>Fritz, M., Rinaldi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+pressure+measurement+with+the+tail-cuff+method+in+Wistar+and+spontaneously+hypertensive+rats:+Influence+of+adrenergic-+and+nitric+oxide-mediated+vasomotion.">Blood pressure measurement with the tail-cuff method in Wistar and spontaneously hypertensive rats: Influence of adrenergic- and nitric oxide-mediated vasomotion.</a> J. Pharmacol. Toxicol. Methods. 58, 215-221 (2008).</li><li>Ikeda, K., Nara, Y., Yamorii, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+systolic+and+mean+blood+pressure+determination+by+anew+tail+cuff+method+in+spontaneously+hypertensive+rats.">Indirect systolic and mean blood pressure determination by anew tail cuff method in spontaneously hypertensive rats.</a> Laboratory Animals. 25, 26-29 (1991).</li><li>Jacobowitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+discrete+fresh+regions+of+rat+brain.">Removal of discrete fresh regions of rat brain.</a> Brain Res. 80, 111-115 (1974).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> Journal of Neurosci. Meth. 12, 141-149 (1984).</li><li>Jung-Hwa, T. a. o. -. C. h. e. n. g., Gallant, J., Brightman, P. E., Dosemeci, M. W., A, ., Reese, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+at+the+synapse+after+delayed+perfusion+fixation+in+different+regions+of+the+mouse+brain.">Structural changes at the synapse after delayed perfusion fixation in different regions of the mouse brain.</a> J. Comp. Neurol. 501, 731-740 (2007).</li><li>Kasukurthi, R., Brenner, M. J., Morre, A. M., Moradzadeh, A., Wilson, Z. R., Santosa, K. B., Mackinnon, S. E., Hunter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+versus+immersion+fixation+for+assessment+of+peripheral+nerve+regeneration.">Transcardial perfusion versus immersion fixation for assessment of peripheral nerve regeneration.</a> J. Neurosci. Meth. 184, 303-309 (2009).</li><li>Lamberts, R., Goldsmith, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation,+fine+structure,+and+immunostaining+for+neuropeptides:+perfusion+versus+immersion+of+the+neuroendocrine+hypothalamus.">Fixation, fine structure, and immunostaining for neuropeptides: perfusion versus immersion of the neuroendocrine hypothalamus.</a> J. Histochem. Cytochem. 34, 389-398 (1986).</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Aspects+of+Immunohistochemistry.">Technical Aspects of Immunohistochemistry.</a> Vet. Pathol. 42, 405-426 (2005).</li><li>Shi, Z. R., Itzkowitz, S. H., Kim, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+three+immunoperoxidase+techniques+for+antigen+detection+in+colorectal+carcinoma+tissues.">A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues.</a> J. Histochem. Cytochem. 36, 317-322 (1988).</li><li>Walker, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vertebrate dissection. , (1980).</li><li>Zwienenberg, M., Gong, Q., Lee, L. L., Berman, R. F., Lyeth, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.+Neurotrauma.">J. Neurotrauma.</a> Monitoring in the Rat: Comparison of Monitoring in the Ventricle, Brain Parenchyma, and Cisterna Magna. 16, 1095-1102 (1999).</li></ol>

החוקרים אין לי מה לחשוף.

הערות נוספות לניתוח מוצלח: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר הסרעפת פרוץ, להקדים במהירות היפוקסיה ו hypercapnia תיזום שינויים פיזיולוגיים בלתי הפיכים שעלולים הרודפים את הניתוח שלאחר מכן. </li><li style=";text-align:right;direction:rtl"> כאשר המחט מוחדרת והידק במקום, הלחץ השיורי ידחוף הדם בבסיס המחט (פלאשבק). למעשה, בעלי חיים גדולים יותר עשויים במרץ לגרש דם. זה קריטי, כי דם לפחות להגיע אל הבסיס של המחט, כדי למנוע החדרת בועות אוויר אשר מכשול בפני להשלים זלוף. אם אין דם הוא ציין בבסיס המחט, הוצא את המחט ולמלא עם חיץ על ידי הצמדתו נמל יציאה של המתקן זלוף. לאחר חיץ הופעל באמצעות עצה, המחט אפשר לרחם אל לב. </li><li style=";text-align:right;direction:rtl"> מיקום נכון של המחט בתוך אבי העורקים הוא קריטי, זה לא צריך להגיע עד קשת אבי העורקים. </li><li style=";text-align:right;direction:rtl"> אם perfusions רבים מתקיימים אין צורך לאo הגדרת מההתחלה כל פעם. המשתמש צריך להיות נפח ראוי למאגר וגם מקבע בבקבוק אחד (200mls בעלי חיים / פתרון). הבקבוקים ניתן ומילא אם יש צורך. ההיבט החשוב ביותר שיש להביא בחשבון הוא בפלאשינג את הקו המחבר בין זלוף ההתקנה לבעל החיים (שסתום התפוקה). לאחר השלמת זלוף, קו זה יהיה paraformaldehyde 4% בה. הסר את צינורות מהבקבוק חיץ ולהשתמש מזרק 50 מ&quot;ל מלא מים כדי לשטוף את מקבע. ודא שסתום היציאה ממוקם הגביע בזבוז אוסף לסילוק נאות של paraformaldehyde. חזור על כך שלוש פעמים. מילוי עם המאגר באמצעות אותה שיטה שמוצג 2.3. </li><li style=";text-align:right;direction:rtl"> שיטה זו של קיבעון ניתנת להתאמה מאוד את היכולת לשנות גם חיץ fixatives מקבע אחרים (כגון אלו המשמשים EM) בהתאם לדרישות של הניסוי. </li><li style=";text-align:right;direction:rtl"> המערכת הוא גם משמש לעתים קרובות למיצוי של רקמת חיים. לשם כך1 תהליך פשוט משתמש מסירת חיץ בלבד (בקבוק חיץ תוך עדיין מחוברת המערכת כולה מונחת בתוך דלי קרח בקבוק מקבע הוא כמו תוכנית ההתקנה ריק אבל סגור). זה מבטיח זלוף חיץ קר בלחץ האופטימלי. מכאן שהמערכת יכולה להיות מותאם עירוי של צבעים ניתן לתקן או על ידי הוספת צבע ישירות למאגר (אם הכמות היא לא בעיה) או להשתמש במזרק עם כמות מינימלית של חיץ / פתרון צבע בין למאגר - מקבע צעד. </li><li style=";text-align:right;direction:rtl"> במקרה של יציקת כלי דם, ניטור לחץ צעד חיץ חיוני עם זאת בשל צמיגות וגיבוש אופי הפתרון הליהוק או מזרק 50 מ&quot;ל יש להשתמש למסירה ישירה או משנית מצורף הפנויה למנגנון (שסתום בצינור והחזקה מיכל) עשה להתממשק עם המערכת הנוכחית לשמש. הליהוק של כלי הדם במעבדות שלנו שקדמה עיצוב וייצור של מכשירים זלוף. לכן אנחנו לא יכולים רחובאוכלים כי יש למדוד את הלחץ תהיה מדידה מדויקת באתר המסירה. המספרים אולי צריך להיות מותאם על מנת להשיג ההפצה המוצלחת של נוזלים צמיגה. </li></ol>

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> ציוד </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td></td><td></td><td></td><td> הרדמה: </td></tr><tr><td> קטמין / xylazine תערובת (הרדמה יכולה להשתנות לפי המעבדה / מוסד) </td><td> Ketaset </td><td> NDC 0856-2013-01 </td><td> 10 מ&quot;ל בבקבוקון </td></tr><tr><td></td><td></td><td></td><td> ניתוח: </td></tr><tr><td> קצה המחט, GA 27 x 1.25 &quot; </td><td> אמצעי שירותים </td><td> 25251 </td><td></td></tr><tr><td> גדולים קהים / בוטה מספריים מעוגלות (~~~HEAD=NNS 14.5 ס&quot;מ) </td><td> מדע כלים עדינים </td><td> 14519-14 </td><td></td></tr><tr><td> ישר איריס מספריים </td><td> מדע כלים עדינים </td><td> 14058-11 </td><td></td></tr><tr><td&gt; תקן פינצטה </td><td> מדע כלים עדינים </td><td> 11027-12 </td><td></td></tr><tr><td> זוג יפה (גרפה) פינצטה </td><td> מדע כלים עדינים </td><td> 11050-10 </td><td></td></tr><tr><td> 1 hemostat מלקחיים גדולים - מעוגלים או ישרים (~~~HEAD=NNS 19 ס&quot;מ) </td><td> surgicaltools.com </td><td> 17.21.51 </td><td></td></tr><tr><td> 2 hemostat מלקחיים רגילים - ישר משוננים (14 ס&quot;מ) </td><td> מדע כלים עדינים </td><td> 13013-14 </td><td></td></tr><tr><td> 1 hemostat שונה (עם חור בקוטר 15 הגיש את קצה) </td><td> מדע כלים עדינים </td><td> 13013-14 </td><td></td></tr><tr><td> 15-מחט מד בוטה או זית שקצהו (מחט זלוף) </td><td> Fisnar </td><td> 5601137 </td><td></td></tr><tr><td></td><td></td><td></td><td> זלוף: </td></tr><tr><td> HyPerfusion מערכת או שווה ערך &lt;/ Td&gt; <td></td><td></td><td></td></tr><tr><td> פוספט pH שנאגרו מלוחים, 7.4 </td><td></td><td></td><td></td></tr><tr><td> Paraformaldehyde 4% במאגר M פוספט 0.1, pH 7.4 </td><td></td><td></td><td></td></tr><tr><td> זכוכית שטוחה או מגש פלסטיק, כ 10 &quot;x 10&quot; </td><td></td><td></td><td></td></tr><tr><td> קרח כתוש </td><td></td><td></td><td></td></tr><tr><td> מי האמבט (37 ° C) </td><td></td><td></td><td></td></tr><tr><td> שעון עצר </td><td></td><td></td><td></td></tr><tr><td> 50 מ&quot;ל במזרק </td><td> Medline תעשיות </td><td> NPMJD50LZ </td><td></td></tr><tr><td></td><td></td><td></td><td> דיסקציה: </td></tr><tr><td> אבאIR של תקן מספריים חדים ישרים / קהה (~~~HEAD=NNS 12 ס&quot;מ) </td><td> מדע כלים עדינים </td><td> 14054-13 </td><td></td></tr><tr><td> Rongeurs מעוגלים או ישרים בינוני (14-16 ס&quot;מ) או הסרת עצם הגולגולת צבת </td><td> מדע כלים עדינים </td><td> 16020-14 </td><td></td></tr><tr><td> ישר איריס מספריים (~~~HEAD=NNS 9 ס&quot;מ) </td><td> מדע כלים עדינים </td><td> 14058-11 </td><td></td></tr><tr><td> מיקרו מרית (כפול 2 &quot;קצוות שטוחים, אחד מעוגל, אחת קוני ל 1/8&quot;) </td><td> מדע כלים עדינים </td><td> 10091-12 </td><td></td></tr><tr><td></td><td></td><td></td><td> לאחר קיבוע &amp; Storage: </td></tr><tr><td> 50 מ&quot;ל בקבוקון זכוכית </td><td></td><td></td><td></td></tr><tr><td> 40 מ&quot;ל HEPES שנאגרו הנקס פתרון (HBHS) עם אזיד הנתרן (90mg / L) </td><td></td><td></td><td></td></tr></tbody&gt; </table>

whole animal perfusion fixation for rodents

המטרה של קיבעון היא במהירות אחידה לשמר רקמות במצב כמו חיים. תוך שימת רקמות ישירות בעבודות מקבע היטב חתיכות קטנות של רקמות, דגימות גדולות יותר כמו המוח ללא שינוי מהווה בעיה עבור קיבעון טבילה משום מקבע לא מגיע בכל אזורי הרקמה באותו קצב 5,7. לעתים קרובות, שינויים בתגובה היפוקסיה להתחיל לפני רקמה ניתן לשמר 12. היתרון של המרוססת מקבע ישירות דרך מערכת הדם היא כימית יכול להגיע במהירות לכל פינה של האורגניזם באמצעות רשת כלי דם טבעי. על מנת לנצל את מחזור הדם בצורה היעילה ביותר, יש להקפיד להתאים לחצים פיזיולוגיים 3. חשוב לציין כי לחצים פיזיולוגיים תלויים המינים בשימוש. טכניקות קיבוע זלוף משתנים בהתאם רקמות להיות קבוע וכיצד רקמות יעובדו קיבעון הבאה. זה וידאואנו מתארים בעלות נמוכה, בהליך מהיר, קיבעון מבוקר ואחיד באמצעות paraformaldehyde 4% perfused דרך מערכת כלי הדם: דרך הלב של חולדה כדי לקבל את השמירה הטובה ביותר של המוח עבור אימונוהיסטוכימיה. היתרון העיקרי של שיטה זו (לעומת כוח הכבידה-fed מערכות) היא מערכת הדם הוא מנוצל בצורה היעילה ביותר.

Watch this Scientific Journal Video about Whole Animal Perfusion Fixation for Rodents at JoVE.com

Whole Animal Perfusion Fixation for Rodents

כאן אנו מתארים בעלות נמוכה, בהליך מהיר, קיבעון מבוקר ואחיד באמצעות paraformaldehyde 4% perfused דרך מערכת כלי הדם: דרך הלב של חולדה כדי לקבל את השמירה הטובה ביותר של המוח.

בעלי חיים שלמה זלוף קיבוע של מכרסמים

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

whole-animal-perfusion-fixation-for-rodents

Research

JoVE Journal

Neuroscience

378.4K Views.  University of Michigan. כאן אנו מתארים בעלות נמוכה, בהליך מהיר, קיבעון מבוקר ואחיד באמצעות paraformaldehyde 4% perfused דרך מערכת כלי הדם: דרך הלב של חולדה כדי לקבל את השמירה הטובה ביותר של המוח.

Video: Whole Animal Perfusion Fixation for Rodents

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

ניגון אוטומטי של וידאו אינטראקטיבי ללימודי תקשורת של בעלי חיים

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

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 ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

בקנה מידה גדול הקלטה של ​​הנוירונים על ידי בדיקות מטלטלין סיליקון מתנהג מכרסמים

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

הגדרת עלות נמוכה לaudiometry התנהגות במכרסמים

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

גירוי מוחי עמוק עם fMRI סימולטני במכרסמים

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

טכניקת קיבוע אקריליק שרף דפוס מבוסס ראש במכרסמים

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

אסטרטגיה רומן המשלב מערך-CGH, רצף שלם-exome and בתוך הרחם אלקטרופורציה בחולדות לזיהוי גנים סיבתי של מומים המוח

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

טראנס גירוי חשמלי מוחי בחולדות התראה

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

השרשה בצינורית לתוך המלון Cisterna של מכרסמים

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.

Presented is a method for air-inflation with vascular perfusion-fixation of the lungs that preserves the location of cells within airways, alveoli and interstitium for structure-function analyses. Constant airway pressure is maintained with an air-inflation chamber while fixative is perfused via the right ventricle. Lungs are processed for histologic studies.

אוויר-אינפלציה של ריאות מורין עם זילוף וסקולרי-קיבוע

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, ...